Comprehensive clinical and molecular assessment of 32 probands with congenital contractural...

INHERITED ELASTINOPATHIES:

NOVEL CLINICAL AND

ETIOPATHOGENETIC INSIGHTS

Bert L. Callewaert

Promotor: Prof. Dr. A. De Paepe

Co-Promotor: Prof. Dr. B. Loeys

Thesis submitted as partial fulfillment of the requirements for the degree of Doctor in

Medical Science

Center for Medical Genetics

Ghent University Hospital

De Pintelaan 185

B-9000 Ghent, Belgium

+32/9/332 36 03 (phone)

+32/9/332 49 70 (fax)

This thesis is dedicated to:

Maartje, Karel and Amber

and Albert Callewaert (1915-2007), a pioneer in pediatrics.

Thesis submitted as partial fulfillment

of the requirements for the degree of

Doctor in Medical Science

Promotor: Prof. Dr. A. De Paepe

Ghent University Hospital, Belgium

Co-Promotor: Prof. Dr. B. Loeys


Examination committee (*: member of the reading committee).

Prof. Dr. Raoul H.C. Hennekam*

Academisch Medisch Cemtrum, The Netherlands

Institute of Child Health, London, United Kingdom

Prof. Dr. Dirk Matthys *


Prof. Dr. Lut Van Laer *


Prof. Dr. Jo Lambert *


Prof. Dr. Koen Devriendt

UZLeuven, Belgium

Prof. Dr. Johan De Sutter

Hospital “Maria Middelares”, Ghent, Belgium

Prof. Dr. Maryse Bonduelle

UZBrussel, Belgium

Prof. Dr. Johan Vande Walle (chairman of the jury)


The research described in this thesis was conducted at the Center for Medical Genetics,

Ghent University Hospital, Belgium and at Washington University School of Medicine,

St-Louis, USA (Visiting Researcher from 18-02-2008 till 28-08-2009).

From 1-10-2004 until 30-09-2008, Bert Callewaert was a Research assistant of the Fund

for Scientific Research - Flanders.

On the Cover Cover design by Niek van der Laak

Over the last decades, medical science has evolved with dazzling speed. However, we

should always bear in mind that the fundaments of today‟s knowledge were build earlier,

centuries ago, by dedicated scientists, helped by no more than a knife and a stolen corpse.

This cover makes a tribute to those that took a start to unravel the mystery of the circulation

that nourishes the body.

“Throughout the body, the arteries are mingled with veins and veins with arteries, and

both veins and arteries are mingled with nerves and the nerves with these... And of course the

usefulness of such a complete interweaving is very evident, if, that is to say, it is a useful

thing for all parts of the animal to be nourished.” Galenus, 2nd century A.D. The left image

depicts the Galenic vision that held throughout the middle ages. The Galenic tradition

assumed different functions for arteries and veins. Veins would originate from the liver and

contained blood („corporeal fluids that nourish the body‟). Arteries, almost empty on cross-

section, would originate from the heart and contained blood and pneuma („spirit‟),

disseminating vitality to the body.

In the 15th century, the Italian physician Jacopo Berengario Da Carpi was the first to

reveal a close relationship between the arterial and venous system: “there is no artery without

its vein to accompany it. Thus the artery may keep the vein alive, and the vein may give blood

to the artery in its needs, the blood by which the vital spirit is made and the artery itself is

nourished”. This unified physiology was further promoted by Leonardo Da Vinci who wrote

of the circulatory system as “A tree of arteries and veins arises from the heart. The more

removed they are from the heart, the thinner they become and divide into smaller branches.”

This observation is nicely illustrated by Da Vinci himself in the middle image.

In 1543, upon the completion of “On the Fabric of the Human Body”, the Flemish

anatomist Andreas Vesalius still believed in different types of „blood‟ circulating through

arteries and veins. Nevertheless, he was the first to wonder whether the arterial and venous

systems are not more closely related and connected at the heart.

Eventually, a few intermediate observations led to the well-known and remarkably simple

experiments of the English physician William Harvey published in “On the Circulation of

Blood” (1628) that the arteries and veins belong to a single circulatory system connected

through the heart and the lungs. What about the “pneuma” now?

Figure: The experiments of W. Harvey

Nowadays, modern techniques as plastination (patented by the controversial prof. Von

Hagen) or corrosion casting (which is actually based on a principle first described by

Leonardo Da Vinci, who used bees‟ wax to study the ventricles of the brain) can be used to

examine vascular structures in detail as shown in the right image. The latter method is used in

paper 5.

Table of contents VII

Table of Contents

ON THE COVER

TABLE OF CONTENTS .............................................................................................. VII

INTRODUCTION............................................................................................................ 13

I. The Connective Tissue and the Extracellular Matrix ............................................ 13

1. The Connective Tissue ....................................................................................................... 13

2. The Extracellular Matrix .................................................................................................. 14

2.1 Components and assembly of the ECM ..................................................................................... 14

2.1.1 Microfibrils and elastic fibers .............................................................................................. 14

2.1.2 Collagen fibers ..................................................................................................................... 27

2.1.3 Proteoglycans ....................................................................................................................... 28

2.1.4 Glycoproteins ....................................................................................................................... 29

2.2 Function of the connective tissue and extracellular matrix ........................................................ 30

2.2.1 Structural functions .............................................................................................................. 30

2.2.2 Biological functions ............................................................................................................. 30

II. Connective tissue disorders ....................................................................................... 31

1. Introduction ........................................................................................................................ 31

2. Clinical and molecular aspects of the elastinopathies. .................................................... 32

2.1 Marfan syndrome ....................................................................................................................... 32

2.1.1 Epidemiology and diagnosis ................................................................................................ 32

2.1.2 Clinical characteristics ......................................................................................................... 32

2.1.3 Molecular basis and pathogenesis ........................................................................................ 34

2.1.4 Differential diagnosis ........................................................................................................... 38

2.2 The Loeys-Dietz syndrome ........................................................................................................ 42

2.3 Congenital contractural arachnodactyly or Beals-Hecht syndrome ............................................ 43

2.4 Arterial tortuosity syndrome ....................................................................................................... 43

2.5 Cutis Laxa .................................................................................................................................. 44

2.6 Non-syndromic familial thoracic aortic aneurysms and dissections ........................................... 48

2.6.1 Thoracic aortic aneurysm associated with bicuspid aortic valve (BAV/TAA) .................... 48

2.6.2 Familial thoracic aortic aneurym/dissection (FTAAD) ........................................................ 48

2.7 Other diseases characterized by elastic fiber fragmentation ....................................................... 49

VIII Inherited elastinopathies: novel clinical and etiopathogenetic insights

3. General considerations on the management of elastinopathies ..................................... 50

RATIONALE, FOCUS AND AIMS OF THE THESIS ............................................... 53

I. Rationale and General Aims ..................................................................................... 53

II. Focus of the Thesis ..................................................................................................... 53

III. Background and Specific Objectives ........................................................................ 54

1. Ad 1. Marfan syndrome .................................................................................................... 54

1.1 Background ................................................................................................................................ 54

1.2 Specific objectives ...................................................................................................................... 55

2. Ad 2. Congenital contractural arachnodactyly ............................................................... 55

2.1 Background ................................................................................................................................ 55


3. Ad 3. Arterial tortuosity syndrome .................................................................................. 56

3.1 Background ................................................................................................................................ 56


4. Ad 4. Autosomal dominant cutis laxa............................................................................... 57

4.1 Background ................................................................................................................................ 57


MATERIALS AND METHODS .................................................................................... 59

I. Patient populations .................................................................................................... 59

1. Marfan syndrome............................................................................................................... 59

2. Congenital contractural arachnodactyly ......................................................................... 60

3. Arterial tortuosity syndrome ............................................................................................ 60

4. Autosomal dominant cutis laxa ......................................................................................... 60

II. Molecular methods..................................................................................................... 61

1. Polymerase Chain reaction ............................................................................................... 61

2. Direct sequencing ............................................................................................................... 62

3. MLPA .................................................................................................................................. 63

4. Fragment analysis .............................................................................................................. 64

5. Homozygosity mapping ..................................................................................................... 65

6. Quantitative PCR (qPCR) ................................................................................................. 65

III. Biochemical techniques ............................................................................................. 66

Table of contents IX

1. Immunohistochemistry/ immunocytochemistry .............................................................. 66

2. Electron microscopy .......................................................................................................... 66

3. Quantification of insoluble elastin .................................................................................... 67

4. Coacervation assay............................................................................................................. 67

IV. Analysis of mice .......................................................................................................... 68

1. Establishing an ENU based mutated mice model ........................................................... 68

2. Echography ......................................................................................................................... 68

3. Surgical techniques ............................................................................................................ 68

4. Vascular corrosion casting ................................................................................................ 69

RESULTS ......................................................................................................................... 71

I. The Marfan Syndrome. ............................................................................................. 71

Publication 1

Effect of mutation type and location on clinical outcome in 1,013 probands with Marfan

syndrome or related phenotypes and FBN1 mutations: an international study. Faivre L, Collod-

Beroud G, Loeys BL, Child A, Binquet C, Gautier E, Callewaert B, Arbustini E, Mayer K,

Arslan-Kirchner M, Kiotsekoglou A, Comeglio P, Marziliano N, Dietz HC, Halliday D, Beroud

C, Bonithon-Kopp C, Claustres M, Muti C, Plauchu H, Robinson PN, Adès LC, Biggin A, Benetts

B, Brett M, Holman KJ, De Backer J, Coucke P, Francke U, De Paepe A, Jondeau G, Boileau C.

Am J Hum Genet. 2007 Sep;81(3):454-66. Epub 2007 Jul 25 ........................................................... 71

Publication 2

The revised Ghent nosology for the Marfan syndrome. Bart L. Loeys*, Harry C. Dietz

*, Alan C.

Braverman, Bert L. Callewaert, Julie De Backer, Richard B. Devereux, Yvonne Hilhorst-Hofstee,

Guillaume Jondeau, Laurence Faivre, Dianna M. Milewicz, Reed E. Pyeritz, Paul D. Sponseller,

Paul Wordsworth, Anne M. De Paepe. (*: contributed equally) J Med Genet, in press ................ 85

II. Congenital Contractural Arachnodactyly ............................................................... 96

Publication 3

Comprehensive clinical and molecular assessment of 32 probands with congenital contractural

arachnodactyly: report of 14 novel mutations and review of the literature. Callewaert BL, Loeys

BL, Ficcadenti A, Vermeer S, Landgren M, Kroes HY, Yaron Y, Pope M, Foulds N, Boute O,

Galán F, Kingston H, Van der Aa N, Salcedo I, Swinkels ME, Wallgren-Pettersson C, Gabrielli

O, De Backer J, Coucke PJ, De Paepe AM. Hum Mutat. 2009 Mar;30(3):334-41. ........................ 96

III. The Arterial Tortuosity Syndrome......................................................................... 109

X Inherited elastinopathies: novel clinical and etiopathogenetic insights

Publication 4

Mutations in the facilitative glucose transporter GLUT10 alter angiogenesis and cause arterial

tortuosity syndrome. Coucke PJ, Willaert A, Wessels MW, Callewaert B, Zoppi N, De Backer J,

Fox JE, Mancini GM, Kambouris M, Gardella R, Facchetti F, Willems PJ, Forsyth R, Dietz HC,

Barlati S, Colombi M, Loeys B, De Paepe A. Nat Genet. 2006 Apr;38(4):452-7. .......................... 109

Publication 5

Absence of arterial phenotype in mice with homozygous slc2A10 missense substitutions.

Callewaert BL, Loeys BL, Casteleyn C, Willaert A, Dewint P, De Backer J, Sedlmeier R, Simoens

P, De Paepe AM, Coucke PJ. Genesis 2008 Aug;46(8):385-9. ........................................................ 116

Publication 6

Arterial tortuosity syndrome: clinical and molecular findings in 12 newly identified families.

Callewaert BL*, Willaert A*, Kerstjens-Frederikse WS, De Backer J, Devriendt K, Albrecht B,

Ramos-Arroyo MA, Doco-Fenzy M, Hennekam RC, Pyeritz RE, Krogmann ON, Gillessen-

kaesbach G, Wakeling EL, Nik-zainal S, Francannet C, Mauran P, Booth C, Barrow M, Dekens

R, Loeys BL, Coucke PJ, De Paepe AM. (*: joint first authors) Hum Mutat. 2008 Jan;29(1):150-

8. ........................................................................................................................................................... 122

IV. New clinical and pathogenetic insights in autosomal dominant cutis laxa ......... 136

Publication 7

New insights into the pathogenesis of autosomal dominant cutis laxa with report of five

additional ELN mutations Bert Callewaert; Vishwanathan Hucthagowder, Beate Albrecht,

Ingrid Haußer, Edward Blair, Cristina Dias, Alice Albino, Hiroshi Wachi, Fumiaki Sato, Robert

P. Mecham, Bart Loeys, Paul J. Coucke, Anne De Paepe*, Zsolt Urban

* (* joint Last Authors.)

Human mutation, submitted ............................................................................................................... 136

DISCUSSION AND CONCLUSIONS ......................................................................... 171

I. Marfan Syndrome .................................................................................................... 171

1. Genotype – phenotype correlation study ....................................................................... 171

2. Towards a New Nosology for the Marfan Syndrome ................................................... 173

II. Congenital Contractural Arachnodactyly ............................................................. 177

III. Arterial Tortuosity Syndrome ................................................................................ 180

1. Molecular pathogenesis of ATS - Does sugar do the trick? ......................................... 180

2. The search for a representative animal model .............................................................. 182

3. A profound clinical characterization.............................................................................. 183

Table of contents XI

IV. Autosomal Dominant Cutis Laxa ........................................................................... 185

1. The clinical spectrum of ADCL within the cutis laxa syndromes ............................... 185

2. The Molecular Background of ADCL ............................................................................ 186

3. Etiopathogenesis of Cutis Laxa – the emergence of final common pathways? .......... 187

REFERENCES ............................................................................................................... 191

FUTURE PERSPECTIVES .......................................................................................... 211

SUMMARY .................................................................................................................... 215

SAMENVATTING ........................................................................................................ 219

LIST OF ABBREVIATIONS ....................................................................................... 225

CURRICULUM VITAE ................................................................................................ 227

ACKNOWLEDGEMENTS - DANKWOORD ........................................................... 239

XII Inherited elastinopathies: novel clinical and etiopathogenetic insights

Introduction 13

Introduction “Pour ȇtre en bonne santé,

il faut faire un choix judicieux de ses ancȇtres”

Albert Callewaert

I. THE CONNECTIVE TISSUE AND THE EXTRACELLULAR

MATRIX

1. The Connective Tissue

The connective tissue is the most abundant tissue throughout the body and serves

both as a structural support and as a metabolic reservoir for tissue interactions. During

recent years, the historical view on the connective tissue as solely a static „packaging‟

and supporting structure has been abandoned. Literature now bursts from evidence that

the connective tissue actively participates in several important physiological processes

including proliferation, apoptosis, migration, differentiation, cell expression patterns and

development, mainly acting through various cell signaling pathways (see paragraph 2.2).

Three main types can be distinguished: the loose or soft connective tissues (that embed

organs and organ parts), the hard connective tissues (bone and cartilage), and blood. It

consists of an extracellular matrix (ECM) and metabolically active cells that synthesize

and maintain the ECM. Embryologically, it develops mainly from the mesoderm, the

middle layer of the three-layered embryo. In the head and neck region, some of the

connective tissue is derived from the neural crest cells of the epiderm (the upper layer of

the three-layered embryo). Mesodermal cells develop a loosely organized embryonic

connective tissue, called mesenchyme, and differentiate further in connective tissue

specific cell types. These cell types include fibroblasts/fibrocytes, osteoblasts/osteocytes,

chondroblasts/chondrocytes and synthesize/maintain the ECM in respectively the loose

connective tissues, bone, and cartilage, according to tissue specific needs. A special

connective tissue cell type in smooth muscle tissue is the fibromyocyte that combines

characteristics of fibroblasts and the ability to contract like smooth muscle cells.

Inflammatory cells like monocytes migrate from the blood and take part in the

degradation and remodeling of the connective tissue. In bone, these cells fuse into a

14 Inherited elastinopathies: novel clinical and etiopathogenetic insights

special multinuclear cell type, called osteoclasts, that degrades the ECM in order to

remodel the skeletal bones according to different mechanical needs.

2. The Extracellular Matrix

During recent years, the knowledge about the extracellular matrix has greatly

expanded. Nevertheless, many questions remain and the current view on its structure and

assembly still resembles a bit of a jigsaw. A first section (paragraph I.2.1) addresses the

structure and assembly of the extracellular matrix, and focuses on a few important ECM

proteins. Paragraph I.2.2 discusses the current understanding of the functions of the

extracellular matrix.

2.1 Components and assembly of the ECM

The extracellular matrix consists of 4 main components: microfibrils and elastic

fibers, collagen fibers, proteoglycans, and (glyco-) proteins.

2.1.1 Microfibrils and elastic fibers

a. Microfibrils

Microfibrils are rod-like structures with a diameter of approximately 10 nm that exist

as individual structures or in association with elastin to form elastic fibers (1, 2). They

mainly consist of parallel head-to-tail oriented bundles of fibrillin molecules (3, 4) (see

also paragraph II.2.1.1.c - fibrillin). On rotary shadowing electron microscopy

microfibrils show a beads-on-a-string structure with a periodicity ranging from 50 to 150

nm (5, 6) (figure 1a). It is widely accepted that the beads are formed by interaction of the

C- and N-termini of the fibrillin monomers, but it remains subject of discussion how this

arrangement provides extensibility and resilience to the microfibril while retaining

intermolecular interactions (3, 7). Based on ultrastructural findings of the microfibril and

structure characteristics of the fibrillin molecules, 3 models are proposed: i) the pleating

Introduction 15

model in which fibrillin molecules unfold upon stretching (figure 1b) (8, 9) ii) the one

third-staggered model that agrees with the availability of putative transglutaminase

crosslinks (figure 1c) (10, 11) and iii) the half-staggered model that endows the N-

terminal halves of the fibrillin molecules to reside on the outer side mediating interaction

with multiple ECM proteins (figure 1d) (12).

Fig. 1 a) Rotary-shadowed image of a fibrillin microfibril extracted from connective tissue. The bead-to-bead

periodicity is approximately 55 nm. Bar 100 nm. b) The intramolecular pleating model. This model allows for

the extreme extensibility of extracted microfibrils to periodicities of 150 nm. c) The one third-staggered model.

This model better accommodates for the putative transglutaminase crosslinks. d) The half-staggered model. This

model accommodates for ligand-binding sites to the N-terminal halves of fibrillin. Adapted from Ramirez et al,

2009 (13).


The assembly of microfibrils and elastic fibers is a complex multistep process that is

not fully understood. In a first step, profibrillins are processed intracellularly at N- and

C-termini at RX(K/R)X recognition sites by Furin/PACE (Paired basic Amino acid

Cleaving Enzyme) enzymes and secreted as monomers (3, 14). Secreted fibrillin

monomers self-aggregate extracellularly in a head-to-tail organization (15). Discussion is

ongoing on the relative importance of N-terminal / C-terminal interactions versus lateral

interactions (mainly of the proline/glycine rich region – see I.2.1.1.c. fibrillin) in these

early driving forces (13). Also, the importance of heparin sulphate proteoglycans and

fibronectin has been repeatedly demonstrated in correct microfibril assembly (16-19).

Subsequently, reducible disulfide bridging of the unpaired cysteine in the first hybrid

domain (I.2.1.1.c. fibrillin) (20), reshuffling of other cysteine residues by protein

disulfide isomerases, and non-reducible transglutaminase cross-links dispersed

throughout the fibrillin molecule (11, 21) further drive the assembly process and secure

its structure. Tissue culture experiments have implicated a role for many other molecules

including microfibril associated glycoproteins (MAGP – plural MAGPs) (22).

Fibrillin-rich microfibrils interact with many other non-fibrillar components, which

can be integral parts of the microfibrils or may associate with them. These proteins have

a structural role, and/or may be functionally important in microfibril assembly,

elastogenesis, interactions with other ECM proteins, anchoring microfibrils to basement

membranes and cell surfaces, growth factor sequestration, and limb patterning. However,

for many molecules, the exact spatiotemporal relation and function(s) still need to be

addressed. It can be expected that knock-out mice models for these components will shed

new light on this matter. A current highly simplified understanding on the composition

of the microfibril is depicted in figure 2. Table 1 shows a short overview of

microfibrillar (table 1A) and microfibril-associated (table 1B) proteins with their

genomic localization, potential biological role and currently known associated human

diseases. As this thesis will focus primarily on the fibrillins, elastin, and elastogenesis in

general, only some of these proteins will be discussed in detail where relevant.

Introduction 17

Figure 2: Schematic representation of fibrillin ligands (LTBP latent TGFβ-binding protein-1, BMP bone

morphogenetic protein, MAGP microfibril-associated glycoprotein). The proposed model is based on the

microfibril structure model by Kuo et al. (12). Adapted from Ramirez et al, 2009 (13).

Gene

Localization on the

genome

Protein

(Class of

protein)

Potential functions Associated human diseases

FBN1

15q21.1

Fibrillin-1

(Fibrillin-LTBP) Basic structure of microfibrils

TGFβ regulation

MFS (23)

FTAA (24)

MASS (25)

ELS (26)

SGS (27)

AD WMS (28)

Isolated skeletal features (29)

Stiff skin syndrome (30)

FBN2

5q23-q31

Fibrillin-2

(Fibrillin-LTBP) Basic structure of microfibrils

TGFβ regulation?

CCA (31)

FBN3

19p13

Fibrillin-3

(Fibrillin-LTBP) Basic structure of microfibrils during

embryogenesis?

TGFβ regulation?

-

MAGP1 or MFAP2

1p36.1-p35

Microfibril-

associated

glycoprotein-1 or

Microfibril

associated

protein-2

(MAGP)

Tropoelastin deposition

Tropoelastin binding

Binding to fibrillin-1


Complexes with tropoelastin and

biglycan

Binding to type VI collagen

Substrate for transglutaminase

-

MAGP2 or MFAP5

12p12.3-p13.1

Microfibril-

associated Binding to fibrillin-1


-


glycoprotein-2 or

Microfibril

associated

protein-5

(MAGP)

RGD-mediated cell attachment

Interaction with Jagged1

MFAP1

15q15-q21

(Microfibril

associated

protein-1 or

AMP)

MFAP

54 kD protein that is processed to a 36

kD protein

-

MFAP3

5q32-q33.2

Microfibril

associated

protein-3

(MFAP)

40- kD serine-rich protein -

MFAP4

17p11.2

Microfibril

associated

protein or

MAGP-36

(MFAP)

Colocalization to elastic fibers

Role in elastogenesis

-

Table 1A: Overview of known relevant microfibrillar proteins with their genomic localization, function

and currently known associated human diseases. MFS, Marfan syndrome; FTAA, Familial thoracic aortic

aneurysm; MASS, constellation of myopia, mitral valve prolapse, borderline aortic aneurysm; ELS,

ectopia lentis syndrome; SGS, Shprintzen-Goldberg syndrome; AD WMS, autosomal dominant Weill

Marchesani syndrome; kD, kilodalton, TGFβ, transforming growth factor beta.

Gene

Localization on the

genome

(Protein)

Class of Protein Potential Biological role of interaction Associated human diseases

ELN

7q11.2

Elastin

(-) Tropoelastin deposition

Main component of elastic fibers

SVAS (32)

WBS (32)

ADCL (33)

ARCL (34)

LTBP1

2p12

Latent

transforming

growth factor

binding protein-1

(Fibrillin-LTBP)

Sequestering of latent TGFβ

Structural role?

-

LTBP2

14q24

Latent

transforming

growth factor

binding protein-2

(Fibrillin-LTBP)

Structural role? Primary congenital glaucoma

(35)

Ectopia lentis with MFS-like

phenotype (36)

LTBP3

11q12

Latent

transforming

growth factor

binding protein-3

(Fibrillin-LTBP)

Sequestering of latent TGFβ?

Structural role?

Syndromic Oligodontia

associated with short stature

and scoliosis (37)

LTBP4

9q13.1-13.2

Latent

transforming

growth factor

binding protein-4

(Fibrillin-LTBP)

Sequestering of latent TGFβ

Structural role?

Urban-Rifkin-Davis

syndrome (38)

LOX

5q23-31

Lysyl oxidase

(LOX) Crosslinking of collagens

Crosslinking of elastin

(Low copper serum levels

result in functional deficiency

in Menkes disease and

occipital horn syndrome (39))

LOXL1

15q22

Lysyl oxidase

like-1 Crosslinking of collagens


-

Introduction 19

(LOX) Scaffold for elastin polymer deposition

Interacts with fibulin-5

LOXL2

8p21.3-p21.2

Lysyl oxidase

like-2

(LOX)

Crosslinking of collagens


? developmental regulation

Senescence

Tumor suppression

Chemotaxis

-

LOXL3

2p13.3

Lysyl oxidase

like-3

(LOX)



Developmental regulation?

Senescence

Tumor suppression

Chemotaxis

-

LOXL4

10q24

Lysyl oxidase

like-4

(LOX)



Developmental regulation?

Senescence

Tumor suppression

Chemotaxis

Differentiation of chondrogenic cell-

lines?

-

CSPG2

5q13.2

Versican or

Chondroitin

sulphate

proteoglycan 2

(Proteoglycan -

hyalectin)

Link fibrillin-microfibrils to

versican/hyaluronan network

Wagner syndrome (40)

HSPG2

1p36.1

Perlecan or

Heparan sulfate

proteoglycan-2

(Proteoglycan –

hyalectin)

Anchoring microfibrils to basement

membranes and in the biogenesis of

microfibrils

Schwartz-Jampel syndrome,

type I (41)

Dyssegmental dysplasia,

Silverman-Handmaker type

(42)

DCN

12q13.2

Decorin

(Proteoglycan -

Small dermatan

sulfate)

Induction of fibrillin-1

Expression in renal fibroblasts and

mesangial cells

TGFβ regulation

Congenital stromal corneal

dystrophy (43)

BGN

Xq28

Biglycan

(Proteoglycan -

Small dermatan

sulfate)

Role in elastogenesis, binds to

collagen

TGFβ regulation;

Neuronal survival

-

VTN

17q11

Vitronectin

(Glycoprotein) Attachment and spreading of cells

Inhibits complement-mediated

cytolysis

Modulates antithrombin III-thrombin

action

-

FN1

2q34

Fibronectin 1

(Glycoprotein) Adhesive and migratory processes of

cells

Binds C1q

Stimulates endocytosis and promotes

the clearance of particulate material

from the circulation

Immunological role through clearance

of C1q coated material?

-

BIGH3

5q31

Keratoepithelin

or TGFβ

inducible gene

Elastin deposition on MF

Cell adhesion

Corneal dystrophies (44)


h3

(Glycoprotein) Interaction with collagen type 1,

fibronectin and laminin

FBLN1

22q13.2-13.3

Fibulin 1

(Fibulin) Binds tropoelastin

Elastic fiber formation

Enhances proteolysis of Aggrecan

Regulate steroid action?

Role in blood clot formation?

Disrupted in a patient with

complex synpolydactyly

(t11, 22) (45)

Giant platelet syndrome (46)

FBLN2

3p24-p25

Fibulin 2



Mediate/modulate attachment of

fibrillin to tropoelastin

Regulate steroid action?

-

FBLN3

2p16

Fibulin 3



Malattia Leventinese (47)

Doyne honeycomb dystrophy

(47)

FBLN4

11q13

Fibulin 4



ARCL type I with prominent

vascular involvement (48)

FBLN5

14q32.1

Fibulin 5

(Fibulin) “Orchestrator” of elastic fiber

formation

Regulation of the initial deposition of

tropoelastin on to microfibrils

Binds tropoelastin

Tethering of lysyl oxidase to the

elastic fiber

ARCL type I with prominent

pulmonary involvement (49)

Age-related macular

degeneration (50)

FBLN6

1q24-q25

Fibulin 6 or

Hemicentin

(Fibulin)

Cell-cell and cell-matrix interactions Age-related macular

degeneration (51)

FBLN7

2q13

Fibulin 7

(Fibulin) Cell adhesion of odontoblasts and

dental mesenchyme

-

BMP7

20q13.1-q13.3

Bone

morphogenetic

protein-7

(BMP)

Regulation of limb patterning -

EMILIN1

2p23.3-23.2

Elastin

microfibril

interfacer-l

(EMILIN)


Inhibits TGFβ signaling

-

EMILIN2

18p11.3

Elastin

microfibril

interfacer-2

(EMILIN)


-

EMILIN3

20q12

Elastin

microfibril

interfacer-3

(EMILIN)

Differentiation of mesenchymal stem

cells into osteoblastic lineages?

-

Table 1B: Overview of known relevant microfibril-associated proteins with their genomic localization,

function and associated human diseases. SVAS, supravalvular aortic stenosis; WBS, William-Beuren

syndrome; ADCL, autosomal dominant cutis laxa, ARCL, autosomal recessive cutis laxa; MFS -like,

Marfan syndrome-like; kD, kilodalton, TGFβ, transforming growth factor beta.

Introduction 21

b. Elastic fibers

Microfibrils may associate with elastin in a time- and tissue dependant manner to form

elastic fibers (52). The elastic fiber consists of an abundant inner hydrophobic core of elastin

that is surrounded by microfibrils. Elastic fiber formation is characterized by self-

aggregation of tropoelastin molecules in ordered, laterally packed filaments. Studies at

the ultrastructural level give evidence that microfibrils act as a scaffold that guides

elastin assembly and cross-linking (53-55). Fibulins (especially fibulin 5) and matrix-

associated glycoproteins (MAGP-1 and MAGP-2) are essential in the direction of elastin

deposition and act as „bridging‟ molecules between fibrillins, tropoelastin and integrins

(56-59). The carboxy-terminus of tropoelastin is implicated in microfibril associated

glycoprotein binding (60) and interacts with cell-surface glycosaminoglycans (61). However,

recent studies of knock-out mice models for either fibrillin or MAGP-1 showed normal

elastin crosslinking and elastic fiber formation (62, 63). This implies that some

functional redundancy exists within both the fibrillin and MAGP protein families or that

these molecules are important in the preservation and maintenance of the elastic fibers

rather than in the initial assembly process.

The characteristic self-aggregation process can be repeated in vitro by raising the

temperature to physiological conditions and is referred to as coacervation (64, 65). Time-

lapse imaging studies show an initial micro-assembly of tropoelastin monomers into globular

aggregates, followed by a cell-directed, dynamic macro-assembly step that fashions large-

scale fibrillar structures (66, 67). Finally, lysyl oxidase stabilizes the elastin by oxidative

deamination of lysyl residues to form covalent desmosine crosslinks that result into a final

product of insoluble elastin (figure 3). However, recent evidence questions this sequence and

suggests that self-association and oxidation by lysyl oxidase precedes deposition onto the

microfibrillar scaffold (68).

Elastin has a very slow turnover in normal tissues, probably of several years and

approaching the life-time of the organism, even in humans (69). Breakdown is mediated

through elastases, mainly derived from polymorphonuclear leukocytes and monocyte derived

matrix metalloproteinases (70). This process is important in many physiological

circumstances (wound healing, pregnancy, growth and tissue remodeling), but may be

devastating as a pathological mechanism in emphysema, atherosclerosis and aneurysm

formation (71). It also contributes to normal ageing (72).


c e

3 μm

Figure 3: Electron microscopic image of an elastic fiber in a control individual.

Arrow, microfibrils visible at the edge; c, collagen fibers; e, elastin

Courtesy of Dr. I. Haußer.

c. Important microfibril-associated molecules

Fibrillins

Fibrillins are ~350kDa large, cysteine-rich glycoproteins that are evolutionary

conserved from jellyfish to humans (73). Three closely related fibrillins have been

described with a similar structure and both unique and overlapping functions (74).

Fibrillin-2 and -3 are preferentially expressed during early embryological stages before

tissue differentiation. Fibrillin-3 expression is abated first around 20 weeks of gestation

(75). By contrast, fibrillin-1 is increasingly expressed throughout morphogenesis and in

well-differentiated tissues (74, 76, 77). Only in the cardiovascular system, fibrillin-1 is

deposited first (74). It is suggested that fibrillin-3 is prevalent in the brain but it definite

function is yet to be defined (75). In postembryological stages, fibrillin-2 is mainly

expressed in elastic tissues like elastic cartilage, bronchial tissue and the arterial tunica

media, while fibrillin-1 is ubiquitously expressed (78).

Structurally, fibrillins are build-up out of 4 types of motifs (73) (figure 4). A first

important motif is the epidermal growth factor – like (EGF-like) domain which is

repeated 46 or 47 times. Forty-two or 43 of these are calcium binding (cb). These highly

conserved motifs typically start with a D-I-D/N-E sequence (aspartic acid – isoleucine –

aspartic acid/asparagine – glutamic acid) and are found throughout many other ECM

Introduction 23

proteins. The central region of the molecule is composed of a stretch of cbEGF-like

modules and is often referred to as “neonatal” region of the fibrillin-1 molecule. Calcium

binding has been shown to stabilize the molecule in a linear configuration, necessary for

microfibril assembly and protection from enzymatic degradation (79-81). In between, 7

modules of latent transforming growth factor binding protein (LTBP) domains (also

named 8-cys or TB-motif) are dispersed. The 4rd

LTBP module contains an RGD

(arginine, glycine, aspartic acid) cell adhesion sequence that is able to bind α5β1, αVβ3,

and αVβ6 integrins (82-85). The Fib or hybrid motif, a 3rd

domain type, occurs twice and

appears as a fusion product of both an LTBP motif and an EGF-like domain. The first hybrid

motif contains 9 cysteines, thus at least one cysteine is available for intermolecular disulfide

bridging (20).

Finally, a hinge region rich in proline (fibrillin-1), glycine (fibrillin-2) or proline and

glycine (fibrillin-3) next to the first LTBP domain provides the molecule with flexibility and

may be important in the assembly process (3).

Figure 4. The fibrillin-LTBP family. All members of the family display a very similar structure with 4 main

motifs: (cb)EGF-like domains, LTBP domains, a Pro/Gly rich hinge region, and hybrid domains. Adapted from

Robinson et al 2006 (22)


FBN1 and FBN2 span about 235 kb and 280 kb, respectively, of genomic DNA on

chromosome 15q21.1, and have a transcript size of about 9.7 kb and 10.7 kb. The coding

sequence of both genes is spread over 65 exons. With several exceptions, single exons

code for the sequential domains (figure 4).

LTBPs

Latent transforming growth factor beta binding proteins (LTBP, plural LTBPs) are

closely related to the fibrillins. Their structure is very similar with a central stretch of EGF-

like domains (of which most are calcium binding), 8-cys domains and hybrid motifs (figure

4). Some of the 8-cys domains present in the LTBPs sequester TGFβ in an inactive state.

Indeed, TGFβ homodimers are secreted non-covalently bound to their TGFβ latency

associated protein (this is called the small latent complex). This small latent complex is then

anchored through disulfide bounds to the LTBPs (this is called the large latent complex)

which is sequestered in the ECM. The members of the fibrillin-LTBP family are therefore

important players in the bioavailability of TGFβ in the ECM (see below). LTBP-1 and -3

dispose of the strongest TGFβ binding capacity, LTBP-4 binds TGFβ weakly, and LTBP-2

does not bind TGFβ at all (86). Similarly to the fibrillins, the central stretch of cbEGF-like

domains involves in the linear stabilization of the molecule.

Elastin

In contrast to fibrillin, elastin is found exclusively in vertebrates and has evolutionary

adapted to provide resilience and elasticity to tissues subjected to high tensile and cyclic

forces like closed vascular systems (87, 88). Following slight posttranslational modifications

that consist of proline hydroxylation, intracellular trafficking of the 66 kDa precursor protein

is chaperoned by Elastin Binding Protein (EBP) and secreted, after which EBP is recuperated.

Tropoelastin has a characteristic core structure of alternating hydrophobic and crosslinking

domains (89, 90) (figure 5B). The hydrophobic domains consist mainly of glycine, proline,

valine and alanine residues and serve 2 functions: firstly, they promote self-assembly of the

tropoelastin molecules through hydrophobic interactions; secondly, they are essential for the

Introduction 25

elastic properties. Indeed upon stretching, these hydrophobic domains will contact an

aqueous environment. Contraction will occur upon spontaneous return of the elastin molecule

in the lower energy state of hydrophobic interaction (64, 65). The crosslinking domains

(especially those encoded by exon 10, 19 and 25) accommodate the formation of covalent

bounds between tropoelastin molecules to form the insoluble elastin. Lysyl oxidase and lysyl

oxidase-like proteins have been shown to form these lysyl-derived desmosine crosslinks. The

C-terminus of elastin encoded by the conserved exon 34 (bovine exon 36) contains a

negatively charged pocket that may interact with the tyrosine-rich tropoelastin binding region

in MAGP1 (89).

The human ELN gene spans about 42 kb. The 3.4 kb transcript contains 34 exons

(compared with 36 in the bovine gene – exon 34 and 35 are lacking in humans) (figure 5A).

Large introns intersperse the exonic sequences and contain 4 times more Alu sequences than

elsewhere in the human genome, which may indicate that several recombination events may

have contributed to the formation of the elastin molecule (91). Moreover, the gene structure

nicely reflects the protein structure with separate, alternating exons encoding the sequential

hydrophobic and crosslinking domains (92). Elastin expression is the highest during early

development and declines with ageing. Expression is under strict pre- and posttranscriptional

control with regulatory regions in the promoter, intronic and untranslated sequences (93).

This control is influenced by growth factors like transforming growth factor beta 1 (TGFβ1)

and hormones including insulin-like growth factor 1. Posttranscriptional regulation may be

achieved by an open reading frame element at the 5‟ terminus of exon 30 (94) that interacts

with a 50-kD cytosolic protein that induces mRNA decay (94) and feedback autoregulation

whereby tropoelastin accumulation in the ECM inhibits further expression (95). The ELN

mRNA is highly subjected to alternative splicing with exons 22, 23, 24, 26A, 32 and 33 being

most frequently spliced out. The specific function(s) of all these isoforms remains unclear.

Age-related changes in isoform ratios have been observed and some tissue specific

differences exist (96-98).


Figure 5: A: The cDNA structure of tropoelastin. Adapted from Vrhovski et al, 1998 (93). B: The protein

structure of elastin consists of alternating hydrophobic and crosslinking domains. Alternatively spliced domains

are indicated. Adapted from Kielty et al, 2002 (52).

Fibulins

The fibulins, a 7 member family of extracellular glycoproteins, all share a common

organization with 3 structurally different domains (I, II, III) (99). The central portion (domain

II) consists of a variable series of (cb)EGF-like modules. Domain III is a fibulin-type module

that is specific to all fibulins. Several splice forms (A-D) of the fibulin type module exist in

Fibulin 1. The structure of domain I highly varies between the different fibulin members.

Fibulins are divided in 2 groups based on the presence (fibulin-1 and -2) or absence (fibulin-

3, -4, -5, -6, -7) of 3 anaphylatoxin modules in domain I. (figure 6).

Recently, insights arose about the organization of the fibulins in the ECM. Fibulin -1 to -5

bind to tropoelastin and have all been implicated in elastic fiber formation. Increasing

Introduction 27

evidence implies a role for fibulin-5 as an orchestrator both in deposition of tropoelastin,

tethering lysyl oxidases and even elastin degeneration (100-102).

The many binding partners of these versatile proteins, their causal involvement in a

variety of human diseases (table 1B) and the study of knock-out mice clearly denote

important and widespread functions for these molecules. Besides elastic fiber formation,

fibulins have been attributed roles in biological and developmental processes, including cell

proliferation (fibulin-1, -3, -4, -5), malignant transformation (fibulin-1, -4), limb patterning

(fibulin-1), neurite outgrowth (fibulin-1), macular degenerative disease (fibulin-3, -5, -6),

blood coagulation (fibulin-1), vessel patterning (fibulin-4, -5), and vascular endothelial wall

stabilization (fibulin-1) (99).

Figure 6: Domain structures of fibulin family proteins. The seven members of the family display similar modular arrangement, consisting of

domains I, II and III. Domain III at the C terminus contains a fibulin specific motif and domain II at the center consists of EGF-like motifs. These motifs in domains II and III are common to all fibulins. The N-terminal domain I varies in size and motifs among the fibulin family.

After De Vega et al, 2009 (99)

2.1.2 Collagen fibers

Collagens are the most abundant molecules throughout the body and make up to one third

of the total protein mass. All collagen molecules are triple-helical structures, formed by


winding up 3 polypeptide chains, called α-chains, around each-other. These structures can be

homo- or heterotrimeric. Steric requirements for this supercoiled structure of the polypeptide

chains are met by the typical repeating G-X-Y sequence, as only glycine is small enough to

fit the inner portion of the helix. The X and Y positions are frequently occupied by proline

and hydroxyproline to stabilize the helix. Hydroproline residues result from posttranslational

hydroxylation of proline residues and accommodate the formation of intra- and interchain

crosslinks (103).

Based upon structure and function, collagens can roughly be divided into 2 types: fibrillar

and non-fibrillar collagens (104). The most abundant are the fibrillar collagens. Following

extracellular N and C-terminal cleavage, these linear collagen molecules (collagen I, II, III,

V, XI, XXIV, XXVII) are orderly staggered and covalently cross-linked by lysyl oxidase to

form a long, unbranched banded fibril with a typical 67 nm periodicity on electron

microscopy. These fibrils are further packed to strong rope-like fibers. In contrast to the

elastic fiber that endows elasticity to tissues, collagen fibers provide tensile strength. The

second group of collagens, fibril associated collagens with interrupted triple helices (FACIT

– collagen IX, XII, XIV, XVI, XX, XXI, XXII, XXVI) contain one or more non-collagenous

domains that interrupt the helical structure providing a bend to the molecule. They do not

form multimeric aggregates, but attach to pre-existing collagen fibres. They are believed to

regulate collagen fiber formation and accommodate interactions between the fibril and other

components of the extracellular matrix. Other collagen types may form networks (collagen

VIII, X), aggregates (e.g. collagen IV in basement membranes) or have anchoring functions

to cells and basement membranes (VII, XIII, XVII, XXIII, XXV) (104).

2.1.3 Proteoglycans

Proteoglycans (PG – plural PGs) are large complexes that consist of a core protein to

which oligosaccharides and larger carbohydrate polymer chains, called glycosaminoglycans

(GAG), are covalently attached. There is a large variety of PGs based on the structure of the

core protein and the number, types and lengths of the GAG. Some of these proteoglycans

have biochemical characteristics that facilitate the formation of larger aggregates, e.g.

aggrecan molecules are bound to hyaluronan with linking proteins. PGs are highly sulphated

and negatively charged and therefore very hydrated. This results in elastic properties,

compressibility and shock absorbance.

Introduction 29

A special class of PGs are the small leucine rich proteoglycans (SLRP) that interact with

fibrillar collagens among other ECM molecules including fibronectin and elastin. As

demonstrated by the knock-out mice models for several of these molecules, they are directly

involved in the assembly of the ECM and collagen fibrillogenesis specifically. They help

regulate fibril diameter and mediate fibril-fibril and fibril – ECM interactions (105, 106).

Some of these molecules also interfere with TGFβ and bone morphogenic protein (BMP)

signaling (107).

Decorin is the only SLRP linked to a human disorder so far. Frameshift mutations in

decorin cause congenital stromal corneal dystrophy without generalized connective tissue

findings (43). Decorin has a role in fiber assembly and most likely inhibits TGFβ signaling

(107).

Another important PG in the context of elastic fiber assembly is versican, a chondroitin

sulfate proteoglycan that plays a central role in tissue morphogenesis and maintenance. In

addition, versican contributes to the development of a number of pathologic processes

including atherosclerotic vascular diseases, cancer and central nervous system injury. Several

studies have suggested an inverse relationship between versican expression (mainly

controlled by growth factor signaling) and elastic fiber assembly by both inhibiting

tropoelastin secretion and proper elastic fiber assembly (108). Of note, the splice form V3

does not contain GAG side chains and promotes elastic fiber formation (108-110).

2.1.4 Glycoproteins

Finally, a large group of non-collagenous matrix glycoproteins make up a substantial part

of the ECM. Important proteins in this group include trombospondins (TSP), tenascins,

fibronectin, vitronectin, laminin and osteopontin. As „ground substance‟ of the ECM, they

display a variety of functions in tissue morphogenesis and remodeling including cell

adhesion, cell cycle regulation, cell-matrix interactions, and interactions with growth factors

and various other matrix molecules (111). Some of these molecules have been implicated in

disease processes as tumor growth and metastasis. Recently, tenascin X has been linked to a

subtype of classic EDS (112, 113).


2.2 Function of the connective tissue and extracellular matrix

2.2.1 Structural functions

One major function of the connective tissue is to „connect‟ and „embed‟ tissues and

organs in a functionally relevant spatial relationship. Tissue specific mechanical needs

require a large variety of connective tissue architectures. For example, articular cartilage

needs compressibility and renitence and therefore contains high amounts of hydrated

proteoglycans; in arteries, collagen fibers provide strength to resist high pressures while

elastic fibers organized in concentric elastic lamellae provide extensibility and resilience to

normalize blood flow throughout the cardiac cycle; in lung tissue, small, branched elastic

fibers provide extensibility to the alveolae; in the ocular suspensory ligament, parallel

bundles of fibrillin fibers anchor the lens to the ciliary body; the loose meshwork of elastic

fibers in the skin contributes to elasticity, extensibility and pliability.

2.2.2 Biological functions

During recent years it has become clear that with the apparent complexity of the ECM,

many biological functions are associated. The ECM is a dynamic interface for efficient cell-

cell communication, and regulates cell proliferation, cell differentiation, migration,

development and survival. For instance, the diseased ECM in malignant processes contributes

to uncontrolled proliferation, migration (metastasis) and enhanced cell survival. Many of

these processes are controlled through growth factor signaling modulation, as has become

clear from the pathophysiology from Marfan syndrome (114) (see paragraph II.2.1.3).

However, direct contact of cell components with specific ECM components is also important

as illustrated by the pathophysiology of Williams-Beuren and supravalvular aortic aneurysm

syndrome, diseases associated with vascular smooth muscle cell proliferation (115). Finally,

since connective tissue is ubiquitously present, it has important functions in blood

coagulation (116), platelet adhesion (99, 117) and the immunological barrier (118).

Introduction 31

II. CONNECTIVE TISSUE DISORDERS

1. Introduction

Heritable connective tissue disorders (CTD) comprise a heterogeneous group of disorders

that result from genetic defects affecting normal extracellular matrix assembly. This thesis

will focus on diseases of the soft connective tissue, and more specifically on diseases of the

elastic fibers, the so-called elastinopathies, in contrast to the collagenopathies. The clinical

classifications of the soft connective tissue disorders and the criteria describing the different

entities have been challenged repeatedly. Several revisions of these classifications intended to

tackle the well described extensive clinical variability between and within affected families,

the overlap between related CTD that clinicians cope with, and the existing spectrum of CTD

with more common isolated connective tissue manifestations including (familial) thoracic

aortic aneuryms, marfanoid habitus, stroke, osteoarthritis, and osteoporosis. New molecular

advances and insights were often the driving forces for revising the criteria.

Soft connective tissue diseases may involve every organ system, but many are associated

with a significant cardiovascular risk leading to morbidity and mortality in childhood or

young adulthood. Prime examples that represent important genetic models for cardiovascular

pathology are the Marfan syndrome and related disorders. In these conditions, progressive

dilatation of the aortic root leads to aortic dissection, often associated with precocious death.

Over the last decade tremendous progress in clinical and molecular research has changed the

prevailing concept of these syndromes as structural disorders of the connective tissue into

diseases manifesting perturbed cytokine signaling with widespread developmental

abnormalities. These insights opened new and unexpected targets for causally directed drug

treatments for these aneurysm syndromes, and by extent, also for the more common non-

syndromic forms of aneurysm formation, a major cause of morbidity and mortality in the

Western world.


2. Clinical and molecular aspects of the elastinopathies.

2.1 Marfan syndrome

2.1.1 Epidemiology and diagnosis

More than a century ago, the French pediatrician Antoine-Bernard Marfan described a 5

year old girl, Gabrielle, with long slender digits, long bone overgrowth and muscle

hypoplasia (119). Clinical research further delineated this pleiotropic condition, now called

Marfan syndrome (MFS), as an autosomal dominantly inherited systemic disorder of the

connective tissue with severe manifestations in the cardiovascular, ocular and skeletal

system. This impells a multidisciplinary approach for both diagnosis and management. The

estimated prevalence of this rather common disease is 1 per 5000 (120). A set of clinical

criteria for diagnosis of the MFS was defined by expert opinion in the Berlin nosology (121),

which became more stringent in the revised Ghent nosology, incited by the molecular

findings in the fibrillin 1 (FBN1) gene (122). The Ghent nosology defines a set of major and

minor criteria in the skeletal, ocular, cardiovascular, dural, integument, and pulmonary

system and acknowledges the contribution of molecular analysis. The major manifestations

include a combination of 4 out of 8 major skeletal features, ectopia lentis, aortic root

dilatation/dissection and dural ectasia. In an index patient, the diagnosis requires major

involvement of at least 2 different organ systems and minor involvement of a third organ

system, while in the presence of an FBN1 mutation or a positive familial history, one major

and one minor system involvement suffice (122). The Ghent criteria did not miss their goal as

with improving techniques, molecular confirmation of the diagnosis is possible in over 90%

of the Marfan patients (123). Occasionally, FBN1 mutations are detected in patients which do

not strictly meet the criteria as in familial ectopia lentis, isolated aortic aneurysm/dissection

or, rarely, marfanoid habitus (124).

2.1.2 Clinical characteristics

The diagnosis of MFS is often triggered by skeletal features with long bone overgrowth

as one of the most striking observations leading to disproportionately long limbs, anterior

chest deformities and arachnodactyly. Other major manifestations include limited extension

of the elbows, scoliosis or spondylolisthesis, acetabular protrusion (as detected by X-ray) and

Introduction 33

calcaneal displacement resulting in pes planus with hindfoot valgus. Typical facial

characteristics include downslanting palpebral fissures, enophthalmia, retrognathia and a

highly arched palate with dental crowding. Joint hypermobility predisposes to ligamentous

injury, dislocations, chronic joint pain and premature osteoarthritis. Muscle hypoplasia and

myalgia are often under-recognized but may result in disabling fatigue and spinal pain (125).

In the ocular system, lens dislocation of any degree necessitates further assessment for

MFS. It occurs in about half to two thirds of all patients and should be assessed by slit lamp

examination. High myopia, retinal detachment, cataract, and glaucoma occur and may result

in visual impairment or even blindness (126).

While subjective impairment usually results from other systems involved, the

cardiovascular manifestations with aortic root dilatation/dissection remain life-threatening

with the majority of fatal events occurring in early adult life if left untreated. Timely

recognition and appropriate medical and surgical management increased the mean survival

age to 72 years (127). The dilatation, which is generally greatest at the sinuses of Valsalva,

should be normalized to body surface area and age (128). The onset and growth rate of the

aortic dilatation varies highly between individuals. Patients are also prone to more distally

occurring aortic dilatation, especially after aortic root replacement (129). Enlargement of the

pulmonary artery may become apparent before aortic root dilatation, revealing potential

diagnostic value, especially in young children (130). Valvular involvement is confined to

myxomatous changes and mitral and/or aortic insufficiency. At the extreme end of

presentation, infants with a neonatal onset of MFS manifest severely impaired valve

dysfunction leading to congestive heart failure and early death (131). Primary progressive

myocardial dysfunction is rare and usually mild (132).

Dural ectasia is a frequent observation in MFS, with a prevalence up to 92% (133), but

its use in clinical practice is restrained by its limited specificity and accessibility. It rarely

causes discomfort as low back pain, headaches and irradiating leg pains.

Due to a low specificity, skin and pulmonary involvement are only defined as minor

criteria in the Ghent nosology and include striae distensae, inguinal hernia and pulmonary

emphysema or pneumothorax, respectively. Pulmonary emphysema partly results from

alveolar septation defects (114).


2.1.3 Molecular basis and pathogenesis

After almost a century of clinical research on the MFS, a new era was eluded by the

finding of the causal gene, FBN1. Thus far, over 1000 mutations are found dispersed all over

the FBN1 gene and these are often family-specific.

Over 90 percent of patients fulfilling the Ghent criteria are found to carry a FBN1

mutation (134), a number that with more performant sequencing techniques and MLPA

analysis needs to be adjusted to 95% (CMGG, unpublished data). Prior to this work,

suggestions to genotype-phenotype correlations were made (124, 135, 136), but results were

conflicting and the genotype-phenotype issue remained largely unsolved and useless in daily

practice. Therefore, the value of molecular confirmation of the diagnosis has to be weighed

for each patient. In some cases, FBN1 mutation analysis may be a valuable addition to the

diagnosis of the MFS and the identification of individuals at risk. This is especially so in

families with marked intrafamilial variability or in children that present with evolving Marfan

phenotypes (124). Furthermore, FBN1 analysis offers prenatal or pre-implantation diagnosis

to Marfan patients, although the molecular defect does not allow predicting severity in the

offspring (137).

Until recently, it was believed that structural deficiency of the fibrillin-1 protein was the

most important player in the „inherited weakness of the connective tissue‟ that caused MFS,

boding poor for therapeutic options. Whereas this hypothesis, based on microscopic

observations showing rarefaction of the elastic fibers, offers an explanation for aortic

pathology, laxity and lens ectopy, it did not reconcile other clinical features such as long bone

overgrowth, thickening of the cardiac valves or muscle hypoplasia. However, during the last

decade, the creation of fibrillin-1 mutant mouse lines that faithfully recapitulate human MFS

has challenged this hypothesis and showed that structural fibrillin-1 deficiency leads to the

activation of sequestered or latent TGFβ (figure 7) (138). Dysregulation of the TGFβ

pathway, one of the major biological pathways, has been shown to play a pivotal role in the

development of aortic aneurysms, emphysema and muscle hypotonia in MFS and progression

can effectively be blocked with TGFβ antibodies (138-140). In mouse models for MFS,

losartan, an angiotensin II type 1 receptor blocker, has been shown to stop aortic growth and

reconstitute elastic fiber conservation (140). Losartan directly blocks the effect of TGFβ,

diminishes the expression of TGFβ receptors and inhibits the activity of trombospondin, an

extracellular matrix enzyme that enables the release of TGFβ (figure 7B) (141). These data

Introduction 35

changed the concept of MFS as a structural connective tissue disorder into a condition

manifesting perturbed cytokine signaling with widespread developmental abnormalities,

opening new and unexpected targets for drug treatments.

It was suggested that heterozygous loss-of-function mutations in the transforming growth

factor beta receptor type 2 gene (TGFBR2) phenocopy MFS (142). However, it has

subsequently been shown that the phenotype associated with heterozygous mutations in either

TGFBR1 or TGFBR2 is distinct from MFS, but presents some overlap, including aortic

aneurysm, skeletal involvement and dural ectasia (143) (see paragraph II.2.2).

Introduction 37

Figure 7: Pathophysiology in Marfan syndrome. (Legend on next page)


Figure 7: Recent insights in the pathophysiology of Marfan syndrome. (A): Normal situation: Inactive TGFβ is

sequestered in the extracellular matrix in a larger complex, the so-called large latent TGFβ complex, consisting

of a TGFβ homodimer associated with its latency-associated protein and a LTBP molecule. TGFβ is activated

by proteases as trombospondin-1 (TSP-1) and binds to the TGFβ receptor 2, which, after dimerization with the

TGFβ receptor 1, will lead to downstream signaling through the SMAD pathway. (B) In Marfan syndrome,

deficient fibrillin monomers will lead to enhanced activation of TGFβ. Dysregulation of TGFβ signaling will

result in an altered transcription pattern of TGFβ-responsive genes and degeneration of the extracellular matrix.

Losartan effectively blocks TGFβ signaling through (1) reducing the TSP-1 activity, (2) reducing the expression

of TGFβ receptor 2 and (3) inhibition of TGFβ signalling through the angiotensin receptor 1, precipitating

signaling through the angiotensin receptor 2 which antagonizes the effects seen in MFS.

TGFβ, Transforming growth factor beta; LAP, latency associated peptide; LTBP, latent transforming growth

factor binding protein; MF, microfibril; TSP-1, trombospondin-1; TGFβRII, TGFβ receptor II; TGFβRI, TGFβ

receptor I.

2.1.4 Differential diagnosis

The differential diagnosis of MFS is extensive and includes other connective tissue

disorders as well as metabolic diseases with cardiovascular, ocular, and/or skeletal

involvement. Below we present a limited overview of some important entities that should be

differentiated from Marfan syndrome (see overview in table 2).

a. With respect to the cardiovascular manifestations

Many related elastinopathies, including familial thoracic aortic aneurysm and the Loeys-

Dietz syndrome present with aortic dilatation and dissection or rupture and will be discussed

extensively below.

A condition that should always be differentiated upon the finding of vascular involvement

is the Ehlers-Danlos syndrome. The EDS is a heterogeneous group of connective tissue

disorders characterized by joint hypermobility and skin abnormalities including skin

hyperextensibility, atrophic scarring and easy bruising. The most important types are the

classic, hypermobile and vascular type of EDS.

“In classic and hypermobile EDS, aortic root enlargement is rare and non-progressive. In

contrast, life expectancy is clearly reduced in the vascular EDS with the median age of death

being 48 years (144). The latter is caused by deficiency of type III collagen due to mutations

Introduction 39

in the COL3A1 gene. These patients usually have a typical facies with reduced subcutaneous

fat, easy bruising and vascular fragility with ruptures/dissections typically involving the

middle-sized thoracic or abdominal arteries, even without previous aneurysm formation.

Other major complications also include spleen, uterine and bowel rupture. Recently, Arginin

to Cystein mutations in the COL1A1 gene were found in a subset of vascular EDS patients

presenting with aneurysms of the abdominal aorta and iliac arteries (145).”

The kyphoscoliotic type of EDS combines aneurysms/dissections with kyphoscoliosis,

joint and skin manifestations, and is sometimes difficult to differentiate from MFS. This rare

recessive condition is caused by lysyl hydroxylase deficiency, encoded by the PLOD gene

(146).

b. With respect to the ocular manifestations

In contrast to MFS, Weill-Marchesani patients present with a stocky stature,

brachydactyly and stiff joints in conjunction with anterior chamber anomalies including

ectopia lentis. Both recessive and dominant inheritance is described (28, 147).

Homocystinuria is a recessively inherited metabolic disorder characterized by ectopia

lentis, long bone overgrowth, mental retardation and a high predisposition to thrombo-

embolism and coronary artery disease in the absence of aortic root dilatation. Diagnosis is

based on the presence of elevated concentrations of homocysteine in urine or plasma (148).

In several families isolated ectopia lentis has been shown to segregate as a dominant trait

and occasionally, FBN1 mutations are identified (149). Although mild marfanoid skeletal

features may be present, these patients do not meet diagnostic criteria for MFS. However,

lifelong follow-up by echocardiography is advised as in some patients aortic root dilatation

occurs later in life.

Recently, isolated recessive forms have been described with mutations in ADAMTS-Like

4 or Thrombospondin repeat-containing 1 gene (150) and in LTBP2 that also causes a

recessive form of primary congenital glaucoma (35, 151, 152).

Patients with Stickler syndrome may also combine ocular features with a marfanoid

habitus. They typically present with high myopia, vitreal abnormalities and often retinal


detachment. Other features include premature arthrosis due to epiphyseal changes and

hearing loss (153).

c. With respect to the skeletal manifestations

The MASS phenotype depicts a varying constellation of mitral valve prolapse, myopia,

mild non-progressive aortic root dilatation and marfanoid skeletal and skin features in

families not meeting the diagnostic criteria for MFS. This phenotype, for which FBN1

mutations rarely are identified, may segregate as a dominant trait and remain stable over time

(154).

Congenital contractural arachnodactyly (CCA) is an autosomal dominant condition

closely related to MFS, and caused by mutations in the fibrilline 2 gene (FBN2) (31). It will

be discussed in extenso below.

d. Mental retardation syndromes with a marfanoid habitus

If patients with systemic features of MFS present with mental retardation, alternative

diagnoses should be considered, including Lujan-Fryns, Shprintzen-Goldberg, CATSHL

(camptodactyly, tall stature and hearing loss) and even Fragile X-syndrome.

Introduction 41

MFS LDS EDS IV

EDS

VI FTAA TAA/PDA MASS LFS SGS CCA FEL HCU CATSHL

Gene FBN1 TGFBR1

TGFBR2 COL3A1 PLOD

FBN1

TGFBR1/2

ACTA2

MYH11 FBN1

(?)

MED12 /

ZDHHC9

FBN1

(?) FBN2

FBN1 /

ADAMTSL-

4 / LTBP2

cystathio-

nine

beta-

synthase

FGFR3

Cardiovascular

Aortic root aneurysm ++ ++ + + ++ ++ +/- - + (?) +/- +/- - -

Other aneurysm + ++ ++ + - - - - - - - - -

Arterial stenosis - - - - - - - - - - - - -

Arterial tortuosity - ++ - - - - - - - - - - -

Stroke - + ++ - - + - - - - - - -

Integument

Cutis laxa / skin laxity - +/- +/- +/- - - - - - - - - -

Translucent skin - + ++ - - - - - - - - - -

Atrophic scars - + ++ ++ - - - - - - - - -

Lung

Emphysema +/- - - - - - - - - - - - -

Skeletal

Joint laxity + ++ ++ ++ +/- - + + + + +/- - -

Contractures + + - - - - + - + ++ - - +

Marfanoid body habitus ++ ++ - ++ + - ++ ++ ++ ++ +/- ++ +

Hypertelorism - ++ - - - - - - +/- - - - -

Cleft Palate / bifid uvula - ++ - - - - - - - +/- - - -

Craniosynosthosis - + - - - - - - ++ - - - -

Eyes

Ectopia lentis ++ - - - - - - - - - + ++ -

Mental retardation - +/- - - - - - + + - - ++ +

Table 2: Differential diagnosis of Marfan syndrome. MFS, Marfan syndrome; LDS, Loeys-Dietz syndrome; EDS IV, vascular Ehlers-Danlos

syndrome; EDS VI, Ehlers-Danlos syndrome kyphoscoliotic type; FTAA: familial thoracic aortic aneurysm; TAA/PAA, thoracic aortic aneurysm/patent

ductus arteriosus; MASS, myopia, mitral valve prolapse, aortic root dilatation, skeletal and skin features of MFS; LFS, Lujan-Fryns syndrome; SGS,

Shprintzen-Goldberg syndrome; CCA, congenital contractural arachnodactyly; FEL, familial ectopia lentis; HCU, homocystinuria; (?), uncertain feature; ++,

frequently present; +, present; +/- rarely present, -, not present.


2.2 The Loeys-Dietz syndrome

The Loeys-Dietz syndrome (LDS) is an autosomal dominant aortic aneurysm syndrome

characterized by the triad of hypertelorism, bifid uvula/cleft palate, and arterial tortuosity

with ascending aortic aneurysm/dissection, caused by heterozygous transforming growth

factor β receptor 1 or 2 (TGFBR1 or TGFBR2) mutations (143). LDS patients show overlap

with MFS, but rarely satisfy diagnostic criteria for the MFS. Main differences are the absence

of significant dolichostenomelia and lens dislocation. In addition, next to the distinct facial

dysmorphology, multiple other discriminating findings can be present including

craniosynostosis, Chiari malformation, club feet, patent ductus arteriosus, and arterial

tortuosity with aneurysms/dissections throughout the arterial tree. In contrast to this typical

presentation, referred to as LDS type 1, some patients, referred to as LDS type 2, show

phenotypic overlap with the vascular EDS. These patients present with less craniofacial

abnormalities, but have prominent skin manifestations, including a velvety and translucent

skin, easy bruising and widened atrophic scars, joint hypermobility, and uterine rupture,

severe peripartal bleedings, and widespread arterial aneurysm/dissections (155). Importantly,

an in-debt clinical and molecular study also indicated a far more aggressive natural history in

LDS patients with TGFBR1/2 mutations compared to MFS or vascular EDS (mean age at

death, 26 years). Aortic dissections occur in young childhood and/or at smaller aortic

dimensions (<45mm) and pregnancy-related complications are high. However, the

amenability to early and aggressive surgical intervention, clearly distinguishes these patients

from individuals with vascular EDS (155).

The genetic defect in LDS intuitively corroborates the essential role of TGFβ signaling in

the pathogenesis of this disorder. Patients with TGFBR1/2 mutations show increased basic

levels of TGFβ signaling, but preservation of the acute phase response to TGFβ, therefore

indicating paradoxically enhanced TGFβ signaling. These findings strengthen the hypothesis

that upregulation of TGFβ superfamily activity leads to the final common pathway that

results in the vascular phenotype in several genetic conditions (143).

Introduction 43

2.3 Congenital contractural arachnodactyly or Beals-Hecht syndrome

Congenital contractural arachnodactyly (CCA) is an autosomal dominant connective

tissue disorder characterized by crumpled ears, a marfanoid habitus with arachnodactyly,

congenital contractures of small and large joints, and progressive scoliosis (156-158). The

condition was first described in 1971 by Beals and Hecht from whom it received its

eponymous name, Beals-Hecht syndrome (159). In view of the clinical overlap with MFS – it

was suggested that „Gabrielle‟ (the girl initially described by AB Marfan) would have

suffered from CCA, rather than MFS (156) – mutations in CCA patients were sought and

found in the FBN2 gene. Remarkably, so far all mutations cluster in the central stretch of

cbEGF-like domains, a region largely comparable to the middle region –so-called neonatal

region– of the FBN1 gene (160). However, many researchers restricted screening to this

central region. Because of the rarity of the disorder, clinical and molecular data were scarce

prior to this work. Especially uncertainty about the aortic risk troubles many clinicians, with

four probands with FBN2 mutations being reported with aortic root dilatation, previously

thought to be a differentiating finding from Marfan syndrome (MFS) (160, 161).

2.4 Arterial tortuosity syndrome

Arterial tortuosity syndrome (ATS) is a rare autosomal recessive condition characterized

by severe tortuosity, stenosis and aneurysms of the large and middle-sized arteries. Focal

stenoses of the pulmonary and large systemic arteries can be associated. Patients usually

present with characteristic dysmorphic features including an elongated face,

blepharophimosis and downslanting palpebral fissures, a beaked nose, a highly arched palate,

and micrognathia. Other connective tissue manifestations comprise a soft, hyperextensible

skin and skeletal abnormalities such as arachnodactyly, pectus deformity, joint laxity, and

contractures (162-173). Patients are prone to vascular disasters including dissections, and

ischemic events (162, 167, 169, 171, 172, 174, 175). The prognosis is reported to be poor

with mortality rates up to 40% before the age of 5 years (172). Histopathology of affected

vessel walls indicates fragmentation of the inner elastic membrane and the elastic fibers of

the tunica media of the large arteries (163-165, 167, 169-171, 174). Prior to this work,

homozygosity mapping in 21 members of two consanguineous ATS families originating from

Morocco and Italy assigned the gene to chromosome 20q13.12 (176). However, no clear

candidate genes were depicted in this region.


2.5 Cutis Laxa

The term cutis laxa (CL), describing a clinical feature, is used as a common denominator

for a heterogeneous group of rare connective tissue disorders, characterized by an increased

number of inelastic skin folds. Both hereditary and acquired forms can be distinguished.

Acquired forms appear secondary to infections, administration of medications, or as a

paraneoplastic feature. Historically, classification of the hereditary forms is based on clinical

grounds and mode of inheritance. The X-linked (recessive) form, also known as occipital

horn syndrome, includes mild mental retardation, skeletal manifestations including the

pathognomonic occipital horns, and bladder diverticulae (177), and is now classified as a

disorder of copper metabolism (178). Autosomal dominant cutis laxa (ADCL) presents with

generalized lax skin folds resulting in a prematurely aged appearance. It is generally

considered as a mild disease without severe involvement of internal organs (179-182).

However, aortic and pulmonary abnormalities have been occasionally reported in patients

with ADCL (183, 184). Autosomal recessive CL (ARCL) comprises at least three subtypes,

with typically severe emphysema, diverticulosis, disastrous vascular lesions and a short life

expectancy in ARCL type 1 (38, 48, 49), developmental delay, microcephaly and skeletal

abnormalities in ARCL type 2 (185, 186), and corneal clouding, mental retardation and

athetosis in ARCL type 3, also known as de Barsy syndrome (187, 188). In ARCL type 2 N-

and O- glycosylation defects are generally found by iso-electrofocusing of transferrine (185,

186). However, numerous reports describe several related disorders and unclassified forms of

cutis laxa. Wrinkly skin syndrome (WSS) and geroderma osteodysplasticum (GO) are

disorders closely related to ARCL type 2. Patients with these conditions present with a

wrinkled skin (usually localized to hands and feet in GO in contrast to a more generalized

presentation in WSS), deep palmar and plantar creases, short stature, congenital dislocation

of the hip, microcephaly and mental retardation. In WSS, mental retardation and

microcephaly is usually more pronounced and often N- and O-glycosylation defects can be

detected as well. Also, patients with WSS usually present with a large fontanel with delayed

closure, marked hypertelorism, and downslanting of the palpebral fissures and dental

problems (189, 190). Patients with GO usually have pronounced bone fragility. Recently

described cutis laxa entities are a constellation of microcephaly, alopecia, cutis laxa and

scoliosis (MACS syndrome) (191) and a neurocutaneous disorder that overlaps with de Barsy

syndrome and that includes cataract, short stature, congenital hip dislocation, severe mental

retardation with no speech development and motor deterioration. In the latter, metabolic

Introduction 45

abnormalities (hyperammonemia and low citrulline, ornithine and proline) may (192) or may

not (193) be present.

Recently, expanding research has enlightened the molecular background for several of

these conditions. In ADCL, mutations are found in the elastin (ELN) or fibulin 5 (FBLN5)

genes (33, 194). In most cases, patients carry frame shift mutations at the 3‟-end of the elastin

gene predicted to result in an extended elastin protein (183, 184, 195-197). One report

mentioned a complex rearrangement of the 3‟ terminus that results in a larger, secreted

protein (184). Finally, one atypical splice-site mutation in exon 25, encoding an important

crosslinking domain in elastin was suggested to result in a highly variable ADCL phenotype

with severe cutis laxa in the proband, but no symptoms in the affected father (198).

In type I ARCL, mutations in FBLN5, FBLN4 and LTBP4 have been described (38, 48,

49). Patients with FBLN5 mutations encounter pulmonary emphysema as the most prominent

non-dermatological feature, while FBLN4 mutations cause severe tortuosity, aneurysm

formation, and usually less prominent cutis laxa. Marked genitourinary and gastrointestinal

diverticulosis is a prominent feature of a newly described type I ARCL constellation that

includes craniofacial dysmorphism, short stature, severe bronchopulmonary dysplasia and

emphysema. This disorder, eponymously named Urban-Rifkin-Davis syndrome (URDS), is

caused by LTBP4 mutations (38).

Patients with type II recessive cutis laxa, wrinkly skin syndrome and at least one patient with

de Barsy syndrome were shown to harbor mutations in ATP6V0A2 (199, 200), the α2-subunit

of the vesicular ATPase, resulting in a diseased Golgi-lysosome apparatus with tropoelastin

accumulation in the Golgi apparatus, and increased apoptosis of elastogenic cells (201).

Interestingly, in a similar patient population, and in patients with geroderma

osteodysplasticum and de Barsy syndrome, mutations were detected in PYCR1 encoding

pyrroline-5-carboxylate reductase 1 that localizes on the mitochondria (190, 202). This defect

causes mitochondrial dysfunction with decreased (oxidative) stress resistance and

developmental defects through increased apoptosis (190). These observations nicely fit the

observation of Baumgartner et al, 2000 and Bicknell et al, 2008 who found defects in the

ALDH18A1 gene encoding a pyrroline-5-carboxylate synthase in a neurocutaneous syndrome

with cataract, skeletal abnormalities and severe mental retardation (193). Mutations in

SCYL1BP1, encoding a soluble Golgin protein (SCY1-like 1 binding protein 1; NTKL-

binding protein 1) that binds Rab6 were also found in GO (203, 204), while MACS syndrome


is caused by mutation in RIN2, a ubiquitously expressed protein that interacts with Rab5.

Rab5 and Rab6 are RAS-GTPases involved in intracellular trafficking and the endocytic

pathway (191). Finally, a single report describes homozygous ELN mutations to cause a

phenotype varying from isolated skin involvement to an ARCL type 2 phenotype with mental

retardation and skeletal abnormalities. The variation was explained by a polymorphism in the

FBLN5 gene (34). So it seems that the cutis laxa syndromes have taught us that the biology of

connective tissue disorders reaches far beyond the extracellular matrix itself. Indeed where

initially mutations in genes encoding for extracellular matrix proteins (ELN, FBLN-4/5,

LTBP4) emerged, new molecular findings unveil other mechanisms involving mitochondrial

and Golgi functioning, glycosylation, intracellular trafficking and secretory pathways, and

proline metabolism that alter proper extracellular matrix homeostasis.

Table 3: Overview of cutis laxa syndromes. ARCL, autosomal recessive cutis laxa;

URDS, Urban-Rifkin-Davis syndrome; sy, syndrome; MACS, microcephaly, alopecia, cutis

laxa, scoliosis syndrome; PF, palpebral fissures; HES, hyperextensible skin; gen.,

generalized; CL, cutis laxa; SVAS, supravalvular aortic stenosis; ARD, aortic root aneurysm;

BPD, bronchopulmonary dysplasia; CHD, congenital hip dislocation; MR, mental

retardation; MiC, Microcephaly; MaC, Macrocephaly; CNS, central nervous system.

Introduction 47

Table 3: Overview of cutis laxa syndromes. (Legend on previous page)

ARCL type I ARCL type II

Wrinkly Skin

syndrome

Geroderma

osteodysplasticum

ARCL type III

(de Barsy sy) ADCL Neurocutaneous sy MACS

Gene(s) FBLN5 FBLN4 LTBP4 (URDS)

ATP6V0A2

PYCR1

ELN (?)

ATP6V0A2 PYCR1

ATP6V0A2

PYCR1

SCYL1BP1

PYCR1 (ATP6V0A2)

ELN FBLN5

ALDH18A1 RIN2

Orofacial Droopy facies Sunset

phenomenon

Bulbous nose tip

Droopy facies Sagging

cheeks

Droopy facies

Sagging

cheeks

Sparse hair Downslanting PF

Hypertelorism

Dental abnormalities Large lowset

dysplastic ears

Broad prominent forehead

Maxillary hypoplasia

Protruding lowset ears

Broad forehead Sparse hair

Hypertelorism

Large lowset dysplastic ears

Large ears, Sagging cheeks

Long philtrum

Deep, husky voice

Sparse hair Downslanting PF

Puffy eyelids

Full lips Sagging cheeks

Gum hypertrophy,

irregular dentition, High pitched voice

Skin Gen. CL

Hernias

HES

Gen. CL Hernias

HES

Gen. CL Hernias

HES

Gen. CL Hernias

HES

Gen. wrinkles Palmar and plantar creases

Prominent veins

Hernias

HES

Wrinkles (hands and feet)

Hernias

Prominent veins

HES

Gen. CL Hernias

Prominent veins

HES

Gen.CL

Gen. wrinkled and

lax skin.

Soft redundant skin

(facial) Hernias

Cardiovascular Stenosis, SVAS,

Pulmonary artery

hypoplasia

Aneurysms

Dissections

Tortuosity

- - (Septal aneurysm) - - ARD

(carotid

tortuosity)

- -

Pulmonary BPD,

Emphysema

(Emphysema) BPD,

emphysema

- - - - Emphysema - -

Ocular - - - - - - Corneal clouding

Cataract

- (Cataract) -

Urogenital /

Gastrointestinal

Diverticulae - Diverticulae - - - - - - -

Skeletal - Fractures - Short stature

Large fontanel,

Muscle hypotonia Hypermobility

Arachnodactyly

Short stature

Large fontanel,

Muscle hypotonia Hypermobility

CHD

Osteoporosis

Short stature

Muscle hypotonia

Hypermobility CHD

Osteoporosis

Short stature

Muscle Hypotonia

CHD, Small joint

Contractures

- Joint hypermobility,

Hip dislocation,

Mild short stature, Distal muscle

Wasting

Scoliosis

Hypermobility

Neurological - - - MR, MiC

CNS abnormalities

Seizures N- and O-

glycosylation

defects (ATP6V0A2)

MR, MiC

Seizures

( N- and O-glycosylation defects

(ATP6V0A2)

MR, MiC

MR – seizures

athetoid

movements

- Severe MR, MiC

Lower limb

hypertonia, Neonatal seizures

MaC


2.6 Non-syndromic familial thoracic aortic aneurysms and dissections

In thoracic aortic aneurysms/dissections (TAAD), patients present with no or minor

systemic involvement. The clinical presentation is highly variable with regards to age of

onset and degree of progression of the aortic enlargement. Importantly, aortic aneurysms can

also occur distally from the sinuses of Valsalva warranting imaging of the entire aorta. One in

five patients have a familial history showing most often autosomal dominant segregation with

decreased penetrance (205).

2.6.1 Thoracic aortic aneurysm associated with bicuspid aortic valve (BAV/TAA)

With a prevalence of 1-2%, BAV is a common cardiac malformation that is frequently

associated with aortic enlargement/dissection (206). The dilatation often occurs above the

sinuses of Valsalva warranting complete visualization of the ascending aorta. Increasing

evidence supports a common genetic defect for both aberrant valve formation and aortic root

dilatation (207). This condition is genetically heterogeneous (208-210) and accurate

counseling is complicated by non-penetrance.

2.6.2 Familial thoracic aortic aneurym/dissection (FTAAD)

In contrast to syndromic TAAD, the identification of genetic defects in non-syndromic

forms is complicated by large genetic heterogeneity with five causal genes and 2 additional

loci reported so far. Some families do not map to these regions and by consequence other loci

must exist. Therefore, at present, it is not possible to perform routine genetic screening.

However, some clinical characteristics may direct to specific genetic defects.

FBN1 mutations are occasionally identified in patients with TAAD and limited skeletal

marfanoid involvement who probably represent incomplete expression of MFS. A first major

locus maps to 5q13-14 in nine out of fifteen families with autosomal dominantly segregating

FTAAD with reduced penetrance. In a single, large family characterized by a more diffuse

vascular disease, a second locus for familial aortic aneurysms, designated FAA1, mapped to

11q23-24 (211). In a third locus, TAAD2, mapping to 3p24-25, TGFBR2 mutations were

Introduction 49

identified and subsequently confirmed in four out of 80 unrelated families with familial

TAAD (212). The phenotype of this relatively rare cause of FTAAD mainly involves the

ascending aorta, but widespread vascular involvement and other connective tissue findings

are observed. It is currently unknown whether some members of these families have other

features of LDS. One study also found a TGFBR1 mutation in a family with FTAA (213).

Interestingly, two novel gene defects have recently been reported in the context of

FTAAD. MYH11 gene mutations cause a specific phenotype of dominantly segregating

TAAD in conjunction with patent ductus arteriosus (PDA) (214). Focal vascular smooth

muscle hyperplasia was suggested (215). MYH11 encodes myosin heavy chain protein 11, a

specific contractile protein of smooth muscle cells. The structural defect leads to lowered

aortic compliance and elastolysis. Upregulation of insulin growth factor-1 and markers of

angiotensin II related vascular inflammation was observed in patients with MYH11 mutations,

but the precise pathophysiology remains unclear (215). The identification of mutations in

ACTA2 (encoding the smooth muscle cytoskeletal protein ACTIN α2) in families with aortic

root dilatation and focal vascular smooth muscle hyperplasia (216) confirmed the importance

of smooth muscle cell contraction to maintain the structural integrity of the ascending aorta.

2.7 Other diseases characterized by elastic fiber fragmentation

Many other diseases display elastic fiber fragmentation as a main characteristic on

microscopic examination of tissue samples. For instance, patients with Williams-Beuren

syndrome (7q11.23 deletions encompassing the ELN gene) and supravavular aortic stenosis

(ELN mutations) present with typical stenoses of the large arteries, but aneurysms may occur

as well. In both, the elastic fiber abnormalities and vascular presentation is caused by

haploinsufficiency of the ELN gene (32, 217). Other entities include Pseudoxanthoma

elasticum (caused by biallelic ABCC6 mutations, encoding a transporter protein mainly

expressed in liver and kidney), which is characterized by ectopic calcification and fragmented

elastic fibers (218), and the craniofaciocutaneous syndromes and Costello syndrome caused

by defects of the KRAS pathway (219, 220).


3. General considerations on the management of elastinopathies

When suspecting an elastinopathy in a proband, careful multidisciplinary diagnostic

work-up of all systems should be done. Different diagnoses have different consequences for

genetic counseling, follow-up and management. Follow-up and management should be

multidisciplinary as well.

Surgery in patients with connective tissue diseases should be carefully planned and not to

be taken lightly since many patients may risk life-threatening lung and cardiovascular

problems.

For the cardiovascular system, management guidelines are extrapolated from those for

MFS and largely rely on expert opinion (129, 221). Serial aortic imaging should be tailored to

the estimated risk based upon aortic dimensions, the rate of aortic growth and family history.

Beta-adrenergic blockade is a standard treatment to slow aortic growth, mainly by virtue of

its antihypertensive and negative inotropic effects (222). When contra-indicated, angiotensin

converting enzyme inhibitors can be alternatively used. Prophylactic repair of the aorta is

indicated when aortic dimensions exceed 5.0 cm in adults, upon the observation of rapid

progression (>10 mm/year) of the aneurysm, or when significant aortic regurgitation occurs.

Over 10 years of experience favor the valve sparing aortic repair procedure that avoids

livelong anticoagulant therapy, especially in females in their fertile period (223). Subsequent

continuous imaging of the whole aorta is warranted for timely detection of aortic graft

pseudoaneurysms, distally occurring aneurysms and coronary artery aneurysms. Pregnancy

should be managed through a high-risk obstetric clinic with careful preconception counseling,

but should be postponed after surgery if aortic measurements reach 47 mm (221). No clear

guidelines are available for more distally occurring aneurysms and expert opinion is often

warranted.

End-stage emphysema can be treated with long transplantation.

In case of wound healing problems, prevention is of the utmost importance, especially

when planning surgical procedures. Stitches should be made with thin thread and left in place

for a longer period. Surgery for cutis laxa is usually disappointing as the sagging skin easily

comes back.

Introduction 51

For the musculoskeletal system, prevention of chronic pains by avoiding strenuous

exercise and contact sports, while encouraging moderate exercise (cycling, swimming) may

preserve quality of life. Splints may provide temporal relief of joint pain.

The contribution of molecular analysis to patient care is illustrated by the need for a more

aggressive management in patients harboring TGFBR1/2 mutations urging surgery once the

maximal ascending aortic dimension exceeds the 99th percentile in children or approaches 4

cm in adolescents and adults. This practice may not fully eliminate risk of dissection and

death, and earlier intervention based upon family history or the patient's personal assessment

of risk versus benefit may be indicated.

New therapeutic strategies, based on the physiopathology of MFS and LDS, propose

losartan, a commonly used angiotensin type 1 receptor blocker, as an inhibitor of TGFβ

signaling through the angiotensin II type 1 receptor (figure 7). In mouse models for MFS,

losartan has been shown to stop aortic growth and reconstitute elastic fiber conservation

(140). A preliminary trial in pediatric patients with severe aortic root enlargement was

promising (141). Large scale randomized trials in Marfan patients have started to confirm the

efficacy of this treatment in humans before using it on a universal basis (224-226). In a next

step, extrapolation of these results should be verified in other forms of TAAD.

Rationale, focus and aims 53

Rationale, Focus and Aims of the Thesis “It is strange how one feels drawn forward without knowing at first where one is going.”

Gustav Mahler

I. RATIONALE AND GENERAL AIMS

As an ultimate goal, this research on monogenic connective tissue disorders aims to

contribute new knowledge to common disorders that result from diseased connective tissue

functioning, especially in the cardiovascular system. This includes the pathological ageing

process of organ systems and the organism as a whole. Connective tissue diseases encompass

a clinical spectrum ranging from severe neonatal presentations with (often) early morbidity

and mortality to isolated connective tissue findings. In the years prior and during this study,

many new clinical entities, molecular defects and pathophysiological mechanisms have been

described for several connective tissue disorders including MFS, LDS, and several forms of

cutis laxa. These findings have shed new light on connective tissue assembly, maintenance

and function. The study of monogenic connective tissue diseases therefore enables us to

expand our knowledge about the connective tissue in general and elastic fiber formation

specifically. These insights may eventually contribute to a better understanding of more

common „connective tissue‟ diseases including isolated aneurysm formation, stroke,

osteoporosis, and abnormal scarring that cause important morbidity, mortality and a financial

burden in the Western world. Also, new insights about the functioning of the connective

tissue and its place within the general metabolism of the organism might in the end reveal

some aspects of the (pathological) ageing process.

II. FOCUS OF THE THESIS

The unique position of the Center for Medical Genetics as a reference center for

connective tissue diseases enables us to study large series of common phenotypes like MFS

http://www.brainyquote.com/quotes/quotes/g/gustavmahl302013.html


as well as rarer connective tissue phenotypes including CCA, LDS, ATS and cutis laxa. This

thesis focuses on the following connective tissue diseases:

1. The Marfan syndrome

2. Congenital contractural arachnodactyly or Beals-Hecht syndrome

3. Arterial tortuosity syndrome

4. Autosomal dominant cutis laxa

The overall aims of this study are twofold:

1. To better delineate the clinical findings in these disorders.

2. To gain insight in the molecular events underlying these disorders.

III. BACKGROUND AND SPECIFIC OBJECTIVES

1. Ad 1. Marfan syndrome

1.1 Background

Since its initial description in 1895, many clinicians contributed to the delineation of the

full clinical spectrum, resulting in the 1986 Berlin nosology. However, clinical diagnosis has

always been hampered by clinical variability, the evolving phenotype with age, and the co-

existence of related connective tissues. The finding of the causal gene, FBN1, encoding

fibrillin-1 (23) introduced a new era in the diagnosis of MFS enabling clinicians to search for

genotype-phenotype correlations, and even more importantly, to fine-tune their clinical

diagnostics. Indeed, in 1996 the Ghent nosology proposed more stringent criteria to overcome

a trend towards overdiagnosis, especially in family members (122). During past years, new

issues have emerged and need to be taken care of in a new nosology. Among those are the

enormous impact of the diagnosis on the patient‟s life, the lack of evidence-based hinge-

points that indicate an aortic risk, the applicability of some criteria, and the recent emergence

of new, related syndromes with different prognoses.


1.2 Specific objectives

1. In a first part, we focus on a large retrospective international genotype-phenotype

correlation study including 1013 probands with a FBN1 mutation that intends to

reveal new insights on possible genotype-phenotype correlations in MFS and type

I fibrillinopathies.

2. In a second part, we propose a revised nosology for the MFS that meets with the

concerns that have emerged with the Ghent nosology. Specifically, we attempt to

(i) make the criteria more patient-centered by avoiding the diagnosis in absence of

a tangible aortic risk, (ii) make the criteria more evidence-based and applicable,

and (iii) to provide clear therapeutic guidelines and directions to the differential

diagnosis.

2. Ad 2. Congenital contractural arachnodactyly

2.1 Background

Although closely related to Marfan syndrome and caused by mutations in FBN2, a

homologue of FBN1, the knowledge about CCA is still rather poor. This may be partly due to

the low number of patients diagnosed with the disorder and partly because of the rather

benign phenotype of the disease. The phenotype, prognosis and natural history still need

better characterization in patients carrying FBN2 mutations. Concerning the molecular

findings, a particular observation in CCA was the clustering of mutations in exons 24-36.

However, exons outside this region were seldomly explored; hence their contribution to CCA

is unknown. CCA patients with normal FBN2 analysis were previously reported, but no clear

data about mutation uptake rates exist and the question of locus heterogeneity remains open.



1. To provide an indebt study of the CCA phenotype.

2. To define the FBN2 mutation spectrum in CCA and establish a diagnostic strategy

for FBN2 analysis.

3. Determine the mutation uptake rate in CCA patients and address the question of

locus heterogeneity.

3. Ad 3. Arterial tortuosity syndrome

3.1 Background

Arterial tortuosity syndrome has always been an intriguing disorder in which the genetic

defect must play a key role in vascular development in order to have such devastating effects.

The extreme rarity of the disease, its clinical overlap with other connective tissue diseases

including the LDS and cutis laxa, and the absence of any clear candidate genes in a region

identified by Coucke et al (176) only added to its mystery. It was hypothesized that the

elucidation of the molecular defect in ATS would contribute to the general understanding of

connective tissue functioning. Also, a genetic defect for ATS would facilitate the

differentiation of the condition with related disorders, hence enabling a clear delineation of

the phenotype and natural history of the disease.


1. To elucidate the genetic basis of ATS.

2. To gain insight in the pathophysiology of ATS.

3. To delineate the phenotypical spectrum and natural history of the disease.

4. To establish an ATS mouse model.


4. Ad 4. Autosomal dominant cutis laxa.

4.1 Background

Historically, ADCL has been considered benign and confined to the skin. A few reports

though mention internal organ involvement. Due to the rarity of the disease, the phenotype

lacks clear definition. Moreover, the risks associated with the disorder are currently unknown

and the prognosis is uncertain.

ADCL is a prime disorder to study elastic fiber formation, a complex and still poorly

understood process. The process in which elastin self-assembly is a key event is greatly

disturbed due to mutations in the 3‟ end of the elastin gene. So far, a terminal elastin defect

was described in only 7 probands afflicted with this very rare disorder and the disturbed

elastic fiber formation and the molecular sequence underlying has not yet been well-defined.

Moreover, it is unclear how the elastic fiber state connects to the clinical phenotype.


1. To clearly define the clinical phenotype and natural history of this rare condition.

2. To gather new knowledge about elastic fiber formation in this pathological state.

3. To elucidate the molecular link(s) between the diseased elastic fiber state and the

clinical findings

Materials and methods 59

Materials and Methods “I have not failed. I've just found 10,000 ways that won't work.”

Thomas A. Edison.

“Perfection itself is imperfection.”

Vladimir Horowitz

“Without craftsmanship, inspiration is a mere reed shaken in the wind.” Johannes Brahms

I. PATIENT POPULATIONS

The unique position of the Center for Medical Genetics, Ghent, as both a reference health

care and research center for hereditable connective tissue disorders allows the world-wide

collection of patient samples, even for rare diseases. Clinical information was gathered by

means of a detailed checklist and multidisciplinary discussed. When necessary, clinical

pictures were asked. Patients – or a legal representative – were asked to sign a general

informed consent approved by the ethical committee of the Ghent University Hospital.

Specific informed consents were obtained for the publication of clinical pictures or the

maintenance of fibroblast or vascular smooth muscle cell cultures.

1. Marfan syndrome

The Center for Medical Genetics, Ghent contributed molecular and clinical data of 167

probands to a cohort of 1,013 probands harboring FBN1 mutations who had received a

diagnosis of MFS or another type I fibrillinopathy. Patients were identified between 1995 and

2005 via the framework of the UMD-FBN1 (227) and participating centers from 38 countries

on five continents and included upon the following criteria: (1) the presence of a

heterozygous pathogenic FBN1 gene mutation and (2) the availability of clinical information.

The clinical data were collected mainly from standardized questionnaires and a minority of

patient data was recruited from previous publications that reported sufficient clinical data. To

avoid bias as a result of familial clustering, affected family members of a proband were not


included in the analysis. The pathogenic nature of a putative mutation was assessed using

recognized criteria.

2. Congenital contractural arachnodactyly

A group of 34 patients with clinical data suggestive of CCA were selected out of a total of

57 patients sent for FBN2 analysis. All selected patients fulfilled the following inclusion

criteria put forth by consensus: 1) presence of contractures (of small and/or large joints); 2)

crumpled ears or arachnodactyly; and 3) the absence of mental retardation.

3. Arterial tortuosity syndrome

For the mapping studies for ATS, DNA of affected and non-affected members of 6

consanguineous families was obtained for homozygosity mapping. Following the

identification of the causal gene, twelve additional probands were referred for SLC2A10

analysis. Patients and family members were either evaluated at the Center for Medical

Genetics, Ghent, or clinical information was obtained from the clinical geneticist at the

referring center. Imaging of the aorta and the aortic side-branches was performed by

echocardiography, computer assisted tomography, or MRI angiography. Oral glucose

tolerance tests consisted of measuring the serum glucose and insulin levels at time 0, 30, 60,

and 120 minutes after ingestion of a solution containing 75 grams of glucose. In addition,

HbA1c levels were determined at time zero.

4. Autosomal dominant cutis laxa

From a database of 32 cutis laxa patients, 5 probands were selected based on the typical

skin and facial characteristics as described in literature. When available, anatomopathology

results from skin biopsies were asked for and fibroblast were cultured with approval of the

patients or a legal representative by informed consent.


II. MOLECULAR METHODS

Detailed descriptions of the molecular methodology are provided in each paper. FBN1

mutation analysis was carried out by a combination of SSCP and CSGE, dHPLC and direct

sequencing on cDNA and / or gDNA level, and MLPA. gDNA primers were developed to

include exon-intron boundaries in the amplicons.

In CCA patients, FBN2 analysis was carried out using direct gDNA sequencing of all

exons and exon-intron boundaries. In case of a negative result for FBN2 analysis, direct

gDNA sequencing of MAGP-1 analysis was done. gDNA sequencing of the FBN1, TGFBR1

and TGFBR2 genes was additionally performed in CCA patients with cardiovascular

involvement and no mutation in FBN2.

ATS patients were analyzed for SLC2A10 mutations using direct gDNA sequencing. ATS

patients in whom only one SLC2A10 mutation was found were additionally analyzed for large

deletions by qPCR analysis for all exons.

Patients with ADCL were analyzed only for exon 28-34 of the ELN gene.

1. Polymerase Chain reaction

Most DNA based molecular techniques require high amounts of the DNA sequence of

interest. The polymerase chain reaction, described in 1983 by Kary Mullis, is a simple and

efficient technique that cyclic repeats the 3 following steps. The DNA template is first

denatured at 95°, then both the designed 5‟-3‟ forward and 3‟-5‟ reverse primers bind the

template DNA at a reaction specific annealing temperature and are elongated by a thermal

stable Taq polymerase (isolated from Thermophilus aquaticus) at 72° in a third step. As

such, the DNA of interest is exponentially amplified and the number of copies can be

calculated as (2A)n, with A the number of original template DNAs, and n the number of

cycles performed. The PCR reaction is terminated with an extended (10 min) elongation step

to fully elongate incomplete sequences.


2. Direct sequencing

Direct sequencing remains the golden standard in mutation detection, based on the Sanger

Method (228). Patient DNA fragments are amplified by PCR. In a first step, the amplicon is

denatured. Next, a specific primer (usually the same as used for the PCR reaction), hybridizes

to the template and is enzymatically elongated with a mixture of deoxynucleotides and a low

concentration of fluorescently marked dideoxynucleotides that terminate the reaction. This

results in a mixture of single-stranded DNA molecules, varying in length by one base. These

fragments are loaded and electrophoretically separated in polymer-filled capillaries. A laser-

based scanning system translates the fluorescent signals into a graphical representation, called

an electropherogram, which will visualize heterozygous alterations as a double peak, and

homozygous alterations as a single peak of the incorrect nucleotide.

SSCP (Single Stranded Conformation Polymorphism) is based on the conformational

difference between mutant and control wild-type single strand DNA fragments. This

conformational difference will result in a migration shift of the mutant fragment in a non

denaturing polyacrylamide gel (229).

CSGE (Conformation Sensitive Gel Electrophesis) and dHPLC (denaturing High

Performance Liquid Chromatography) detect migration shifts between double stranded DNA

fragments without (homoduplex) or with (heteroduplex) single base mismatches that induce a

conformational change. DNA is denatured and reannealed. As such, in case of heterozygous

mutations, both homoduplexes and heteroduplexes are formed that are separated respectively

on a gel or in a liquid chromatography column. In case of homozygous mutations, DNA from

the patient is mixed with DNA from a control individual. The additional strenghth of dHPLC

relies in the possibility to optimize the temperature and elution buffer for each PCR amplicon

(230, 231).


3. MLPA

Multiplex ligation-dependant probe amplification is a PCR based technique developed to

detect copy number varations of a target sequence, e.g. deletions or duplications within

genes. The MLPA reaction can be divided in five major steps:

1) DNA denaturing. DNA is denatured and hybridized with target specific MLPA probes

containing a stuffer sequence and universal primers X and Y at the loose hanging ends. The

stuffer sequences have a different length for each primer pair. In this way, the mixed

fragments obtained by PCR in the same reaction tube (step 3) can be identified afterwards

using fragment analysis (step 4).

2) Ligation reaction. Only when both probe oligonucleotides are hybridized to their

adjacent targets, they can be ligated, so the number of probe ligation products is a measure

for the number of target sequences in the sample.

3) PCR reaction. Only ligated oligonucleotides can be exponentially amplified during the

PCR reaction using primers developed against the universal primer ends.

4) Separation. The amplicons are separated by electrophoresis.

5) Data analysis. The stuffer sequence will enable us to predict the size of the reaction

product generated by the MLPA reaction. As such, up to 96 reactions can be combined.


Figure 8: Principle of MLPA. Available on www.mrc-holland.com/

4. Fragment analysis

This technique separates PCR products on a capillary according to size. This has many

applications: e.g. assessing different splice forms of messenger RNAs, MLPA (see above),

and linkage analysis. We also used this technique to assess expression of wild type and

mutant alleles in ADCL, since the mutations resulted in gene products with a different length.

http://www.mrc-holland.com/


5. Homozygosity mapping

In order to search a gene for a certain phenotype, one can first search for the genomic

region containing the causal gene by linkage analysis. A region is considered linked with the

phenotype when alleles on this region do not segregate independently from the phenotype at

meiosis. In consanguineous families, one hypothezises a common ancestor for a causal

mutation and recessively inherited phenotypes reveal when regions carrying this mutation

come together in the same individual. Concretely, we analyze highly polymorphic markers

with a known location on the genome in all members of a family. Those markers that are

homozygously present in all patients with the phenotype, but not in healthy individuals may

be linked to the disease. In order to determine the strength of linkage, a LOD score (logarithm

of the ODDS-ratio) is calculated. The higher the LOD score, the higher the chance the region

is linked with the disease.

LOD score = log [P (real linkage) / P (Segregation by chance)]

with “P” defined as „chance on‟

6. Quantitative PCR (qPCR)

By performing a fluorescently labeled PCR reaction, an exponential multiplication curve

can be determined for the studied reaction. This enables us to deduct the initial concentration

of the DNA molecule at the start of the reaction. As such, at gDNA level this technique can

be used to study large deletions or duplicatons as we did in the SCL2A10 gene. At cDNA

level, it indirectly measures the expression level of a certain gene in a certain cell type under

certain conditions and at a certain time point.

Copy or complementary DNA (cDNA) is prepared by performing a reversed transcriptase

reaction on messenger RNA (mRNA) isolated from live cells, in order to get a stable mixture

of DNA molecules in equal amounts as the messenger RNA mixture derived from a specific

cell type under specific conditions.


III. BIOCHEMICAL TECHNIQUES

1. Immunohistochemistry/ immunocytochemistry

Immunohistochemistry/immunocytochemistry localizes antigens in tissues/cell cultures

by means of an antibody based detection technique. Following fixation, the tissue or cells are

incubated with a primary antibody, raised against the molecule of interest, usually in rabbit or

mouse. Following an intensive washing step, the tissue or cells are then incubated with a

second antibody, marked with a fluorochrome and raised (usually in donkey or sheep) against

the antibodies of the species that delivered the primary antibody. After a final intensive

washing step, the slides are mounted in a specific mounting medium and the fluorescent

signal is detected with a fluorescent microscope. For visualization of intracellular molecules,

permeabilisation of the tissue or cells is needed, which is usually obtained through treatment

with Triton-X-100 just before incubation with the first antibody.

2. Electron microscopy

Glutaraldehyde fixed specimens were cut into pieces of ca. 1mm3, washed, postfixed in

1% osmium tetroxide or in 0,5% ruthenium tetroxide, rinsed in water, dehydrated through

graded ethanol solutions, transferred into propylene oxide, and embedded in epoxy resin

(glycidether 100). Semithin and ultrathin sections were cut with an ultramicrotome (Reichert

Ultracut E). Ultrathin sections were treated with uranyl acetate and lead citrate, and examined

with an electron microscope (Philips EM 400).


3. Quantification of insoluble elastin

This protocol is based on the relative „inertness‟ of insoluble elastin and uses a

combination of physical (centrifugation) and chemical methods to isolate it from a cell

fraction. Quantification is done by means of radioactive methods.

For each time point, fibroblasts from patients and controls are plated in 60mm culture

dishes (500,000 cells/dish), grown to confluence and incubated with [3H]-leucine (fresh

media was added at day 4). After the removal of the media, cell layers are washed in 0.1 M

acetic acid, scraped in 0.1 N NaOH and sedimented by centrifugation (10 min at 16000 x g).

After removal of the supernatans, the pellets are boiled (at 97°C) for 45 min in 0.5 ml of 0.1

N NaOH to dissolve all ECM components except the insoluble elastin. After centrifugation

(10 min at 16000 x g) the supernatans is mixed with 2 mL of scintillation fluid and measured

in a scintillation counter. Next, the pellets are boiled in 200 ml of 5.7 N HCl for 1 h to

dissolve the insoluble elastin. The samples are mixed with scintillation fluid and measured in

a scintillation counter (232, 233). Final results were expressed as follows:

%100

)()(

)(

cpmIEcpmECM

cpmIE with IE: insoluble elastin, cpm: counts per minute,

ECM: extracellular matrix proteins except for insoluble elastin.

In this way, insoluble elastin was normalized for total extracellular matrix production.

4. Coacervation assay

Coacervation was assayed by monitoring turbidity using light scattering at 400 nm with a

UV spectrometer. Light scattering was monitored every 0.5 min with the temperature

increasing from 15 °C to 45 °C at a rate of 1 °C/min.


IV. ANALYSIS OF MICE

For all procedures, the “Principles of laboratory animal care” (NIH publication No. 86-

23, revised 1985) were followed. In addition, all procedures were approved by the Ghent

University Committee (ECD 07/20) on laboratory animal tests.

1. Establishing an ENU based mutated mice model

Mice were generated as described previously (234). Mutagenesis in healthy C3HeB/FeJ

male mice was established by feeding ENU containing nutrients. Breeding with healthy

females generated a mutant mice library. Seven slc2a10 mutations that lead to an amino acid

substitution in the glut10 polypeptide chain were found upon screening of this mutant mice

library. Two nucleotide alterations, c.383G>A and c.449C>T, respectively causing

substitutions G128E and S150F, were most likely disrupting protein function and were

selected to generate homozygous mice.

2. Echography

Mice were anesthetized with intraperitoneal injection of Ketamine and Xylazine and

ultrasound of the abdominal aorta was performed using a Vivid i Portable echo system (GE

Healthcare) with a Sector Probe (10S RS 4.5-11.5 MHz).

3. Surgical techniques

Mice were anesthetized with intraperitoneal injection of Ketamine and Xylazine and the

right cervical region was dissected under aseptic conditions to visualize the common and


external carotid artery from the truncus communis till the skull basis. Incisions were sutured

and mice were kept apart for 7 days to ensure efficient wound healing.

4. Vascular corrosion casting

This technique is based on drawings of Leonardo Da Vinci who injected bee‟s wax into

animal cerebral ventricles. A liquid plastic solution (Batson solution) is injected in the

vascular system of sacrificed animals and is hardened in tepid water. Following chemical

maceration of the animal the corrosion cast can be evaluated and photographed.

Results 71

Results “The truth is rarely pure and never simple.”

Oscar Wilde (Lady Windermere's Fan)

I. THE MARFAN SYNDROME.

Publication 1

Effect of mutation type and location on clinical outcome in 1,013

probands with Marfan syndrome or related phenotypes and FBN1

mutations: an international study.

Faivre L, Collod-Beroud G, Loeys BL, Child A, Binquet C, Gautier E,

Callewaert B, Arbustini E, Mayer K, Arslan-Kirchner M, Kiotsekoglou A,

Comeglio P, Marziliano N, Dietz HC, Halliday D, Beroud C, Bonithon-Kopp

C, Claustres M, Muti C, Plauchu H, Robinson PN, Adès LC, Biggin A,

Benetts B, Brett M, Holman KJ, De Backer J, Coucke P, Francke U, De Paepe

A, Jondeau G, Boileau C.

Am J Hum Genet. 2007 Sep;81(3):454-66. Epub 2007 Jul 25

This large international collaborative retrospective study evaluated the effect of

the type and location of the FBN1 mutation on the clinical data in 1013 probands

with MFS or a related fibrillinopathy, gathered from 38 countries on the five

continents. The large number of included patients singificantly raises statistical

power and enables a time – to – event analysis to correct for the evolving

character of the phenotype. The study was performed in an effort to provide sound

evidence for possible genotype-phenotype correlations.


454 The American Journal of Human Genetics Volume 81 September 2007 www.ajhg.org

ARTICLE

Effect of Mutation Type and Location on Clinical Outcomein 1,013 Probands with Marfan Syndrome or Related Phenotypesand FBN1 Mutations: An International StudyL. Faivre, G. Collod-Beroud, B. L. Loeys, A. Child, C. Binquet, E. Gautier, B. Callewaert,E. Arbustini, K. Mayer, M. Arslan-Kirchner, A. Kiotsekoglou, P. Comeglio, N. Marziliano,H. C. Dietz, D. Halliday, C. Beroud, C. Bonithon-Kopp, M. Claustres, C. Muti, H. Plauchu,P. N. Robinson, L. C. Ades, A. Biggin, B. Benetts, M. Brett, K. J. Holman, J. De Backer, P. Coucke,U. Francke, A. De Paepe, G. Jondeau, and C. Boileau

Mutations in the fibrillin-1 (FBN1) gene cause Marfan syndrome (MFS) and have been associated with a wide range ofoverlapping phenotypes. Clinical care is complicated by variable age at onset and the wide range of severity of aorticfeatures. The factors that modulate phenotypical severity, both among and within families, remain to be determined.The availability of international FBN1 mutation Universal Mutation Database (UMD-FBN1) has allowed us to performthe largest collaborative study ever reported, to investigate the correlation between the FBN1 genotype and the natureand severity of the clinical phenotype. A range of qualitative and quantitative clinical parameters (skeletal, cardiovascular,ophthalmologic, skin, pulmonary, and dural) was compared for different classes of mutation (types and locations) in1,013 probands with a pathogenic FBN1 mutation. A higher probability of ectopia lentis was found for patients with amissense mutation substituting or producing a cysteine, when compared with other missense mutations. Patients withan FBN1 premature termination codon had a more severe skeletal and skin phenotype than did patients with an inframemutation. Mutations in exons 24–32 were associated with a more severe and complete phenotype, including youngerage at diagnosis of type I fibrillinopathy and higher probability of developing ectopia lentis, ascending aortic dilatation,aortic surgery, mitral valve abnormalities, scoliosis, and shorter survival; the majority of these results were replicatedeven when cases of neonatal MFS were excluded. These correlations, found between different mutation types and clinicalmanifestations, might be explained by different underlying genetic mechanisms (dominant negative versus haploinsuf-ficiency) and by consideration of the two main physiological functions of fibrillin-1 (structural versus mediator of TGFb

signalling). Exon 24–32 mutations define a high-risk group for cardiac manifestations associated with severe prognosisat all ages.

From the Centre de Genetique, Centre Hospitalier Universitaire (CHU) (L.F.), Centre d’Investigation Clinique–Epidemiologie Clinique/EssaisCliniques,(L.F.; C. Binquet; E.G.; C.B.-K.), and INSERM CIE1 (C. Binquet; E.G.; C.B.-K.), Dijon, France; INSERM U827 (G.C.-B.; C. Beroud; M.C.), University ofMontpellier I (G.C.-B.; C. Beroud; M.C.), and CHU Montpellier, Laboratoire de Genetique Moleculaire, Hopital Arnault de Villeneuve (C. Beroud; M.C.),Montpellier, France; Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium (B.L.L.; B.C.; J.D.B.; P. Coucke; A.D.P.); Institute of GeneticMedicine and the Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore (B.L.L.; H.C.D.); Department of CardiologicalSciences, St. George’s Hospital, London (A.C.; A.K.; P. Comeglio); Centre for Inherited Cardiovascular Diseases, Foundation IRCCS Policlinico San Matteo,Pavia, Italy (E.A.; N.M.); Center for Human Genetics and Laboratory Medicine, Martinsried, Germany (K.M.); Institute of Human Genetics, HannoverMedical School, Hannover (M.A.-K.); Department of Biochemistry, University of Oxford, Oxford, United Kingdom (D.H.); Consultation PluridisciplinaireMarfan (C.M.; C. Boileau) and Laboratoire de Genetique Moleculaire (C. Boileau), Hopital Ambroise Pare, Assistance Publique des Hopitaux de Paris(APHP), Universite Versailles–Saint Quentin en Yvelines, Boulogne, France; Service de Genetique, Hotel Dieu, Lyon (H.P.); Institut fur MedizinischeGenetik, Universitatsmedizin Charite, Berlin (P.N.R.); Marfan Research Group (L.C.A.; A.B.; B.B.; M.B.; K.J.H.) and Departments of Clinical Genetics(L.C.A.) and Molecular Genetics (A.B.; B.B.; M.B.; K.J.H.) and Discipline of Paediatrics and Child Health (L.C.A.), The Children’s Hospital at Westmead,Sydney, Australia; Departments of Genetics and Pediatrics, Stanford University Medical Center, Stanford (U.F.); and Consultation PluridisciplinaireMarfan,Hopital Bichat, APHP, Paris (G.J.)

Received January 17, 2007; accepted for publication May 16, 2007; electronically published July 25, 2007.Address for correspondence and reprints: Pr. L. Faivre, Centre de Genetique, Hopital d’Enfants, 10, boulevard Marechal DeLattre de Tassigny, 21034

Dijon, France. E-mail: [email protected]. J. Hum. Genet. 2007;81:454–466. � 2007 by The American Society of Human Genetics. All rights reserved. 0002-9297/2007/8103-0004$15.00DOI: 10.1086/520125

Marfan syndrome (MFS [MIM 154700]) is a connective-tissue disorder, with autosomal dominant inheritance anda prevalence of 1 in 5,000–10,000 individuals.1 The car-dinal features of MFS involve the ocular, cardiovascular,and skeletal systems.2 The skin, lung, and dura may alsobe involved. MFS is notable for its variability in age atonset, tissue distribution, and severity of clinical mani-festations, both among and within affected families. Be-cause of the high population frequency and the nonspe-cific nature of many of the clinical findings for MFS,

clinical diagnostic criteria for this disorder have been es-tablished,3–4 the latest being the Ghent criteria, which su-perseded the previous so-called Berlin criteria.

Study of the molecular determinants of phenotypicalvariations in MFS has been possible only since the iden-tification of the causative fibrillin-1 (FBN1) gene5 (MIM134797). Fibrillin-1 has a modular structure, with 47 re-peats of six-cysteine epidermal-growth-factor (EGF)–likemotifs, 43 of which are of the calcium-binding (cb) type(cb-EGF).6 Fibrillin-1 monomers associate to form com-

Results 73

www.ajhg.org The American Journal of Human Genetics Volume 81 September 2007 455

Table 1. Number of Patients Recruited to the Study andTheir Laboratory of Origin

OriginNo. of Patients(Laboratories)

Belgium 167 (1)United Kingdom 166 (7)Germany 156 (4)France 154 (3)United States 146 (8)Italy 89 (2)Australia 80 (1)Asia 22 (6)Other European countries 22 (3)Other North and South American countries 11 (2)

Total 1,013 (37)

plex extracellular macroaggregates, termed “microfibrils,”which are important for the integrity and homeostasis ofboth elastic and nonelastic tissues.7–8 The protein also con-tains seven eight-cysteine motifs, which bear homologyto motifs found in the latent transforming-growth-factorbeta–binding proteins (TGFb-BPs), and a proline-rich re-gion. The relationship between FBN1 and TGFb signalinghas been underscored by the identification of mutationsin TGFBR2 (MIM 190182) in patients with MFS and Mar-fan-like conditions9 and in pathological studies in knockinand knockout mice.10 Indeed, fibrillins and TGFb-BPs con-stitute a family of structurally related and interacting pro-teins. In MFS mouse models, deficiency of fibrillin-1 altersmatrix sequestration of the large latent complex of TGFb,rendering the cytokine more prone for activation.10 Re-cently, a specific fibrillin-1 sequence encoded by exons 44–49 has been shown to regulate the bioavailability of en-dogenous TGFb1.11

Two major mutation categories—premature termina-tion codons (PTCs) and inframe mutations—have beenreported in the FBN1 gene.12 A total of 559 pathogenicmutations were reported in the last update of the Uni-versal Marfan Database (UMD)–FBN1,12 and most ofthese are unique to individual families. Two-thirds are mis-sense mutations, the majority of which are cysteinesubstitutions.

Besides classic MFS, mutations in FBN1 have been as-sociated with a broad spectrum of phenotypes, includingneonatal MFS,13 isolated ectopia lentis14 (MIM 129600),isolated ascending aortic aneurysm and dissection,15 iso-lated skeletal features,16,17 and Weill-Marchesani syn-drome18 (MIM 608328). So far, genotype-phenotype cor-relations in FBN1 have been weak except for the clusterof mutations in exons 24–32 reported in neonatalMFS.13,19–22

Indeed, previous reported studies investigating geno-type-phenotype correlations were performed with a max-imum of 101 patients.23–27 Those authors compared pa-tients with mutations leading to a PTC versus patientswith missense mutations, as well as subjects with missensemutations involving a cysteine versus individuals withother missense mutations. Moreover, they focused on ma-jor cardiac, ocular, and skeletal involvement. Differencesbetween the groups, with regard to patients’ age at follow-up, were not taken into account.

Large sets of both clinical and molecular data are neededto study (1) the association between FBN1 mutation typeand severity of the disease and (2) the specificity of organinvolvement in relation to a mutation type. The inter-national UMD-FBN1, set up in 1995, provides these datafor 1,013 probands with known FBN1 mutations who wererecruited from specialized MFS clinics all over the world.We report the results of the statistical analysis of thesedata. These results provide possible clues into pathophys-iological processes.

Patients, Material, and MethodsPatients

A total of 1,191 probands who had received a diagnosis of MFSor another type I fibrillinopathy were identified between 1995and 2005 via the framework of the UMD-FBN112 and participatingcenters. The inclusion criteria in our study were (1) the presenceof a heterozygous pathogenic FBN1 gene mutation and (2) theavailability of clinical information.

Overall, 178 patients (15%) had to be excluded from the study(no clinical data available for 129; insufficient data about cardiac,ocular, or skeletal involvement for 44; two different mutationson the same allele for 4; and compound heterozygosity for FBN1mutations for 1). The 1,013 patients included in our study orig-inated from 38 countries on five continents. The majority (72%)were white Europeans or were of European ancestry, 14% werefrom North and South America, 8% were from Oceania, 4% werefrom Asia, and 2% were from Africa. Table 1 summarizes theparticipation of the different laboratories in the study. Patient ageat inclusion ranged from birth to 72 years. The clinical datawere collected mainly from standardized questionnaires sent tothe referring physician, and a minority were from previouspublications that reported sufficient clinical data. The clinicalinformation included a range of qualitative and quantitative clin-ical parameters, including age at diagnosis of MFS or another typeI fibrillinopathy and the presence or absence of clinical featuresincluding cardiac, ophthalmologic, skeletal, dermal, pulmonary,and dural manifestations. The age at diagnosis and at surgery foraortic dilatation, ectopia lentis, and scoliosis was also noted. Allquestionnaires were collected by one individual (L.F.) to rule outduplication of patients in the study. To avoid bias as a result offamilial clustering, affected family members of a proband werenot included in the analysis.

The pathogenic nature of a putative mutation was assessed us-ing recognized criteria. In brief, all nonsense mutations, all de-letions or insertions (in or out of frame) were considered path-ogenic; for all splice mutations, the wild-type and mutantstrength values of the splice sites were compared using geneticalgorithms,28–30 and only mutations displaying significant devi-ation from the norm were included. Missense mutations wereconsidered pathogenic when at least one of the following featureswas found: (1) de novo missense mutation, (2) missense mutationsubstituting or creating a cysteine, (3) missense mutation in-volving a consensus cb residue,21 (4) substitution of glycines im-plicated in correct domain-domain packing,31 and (5) intrafam-ilial segregation of a missense mutation involving a conserved



Table 2. Frequency of Clinical Features in the Different Systems Involved in MFSand Type I Fibrillinopathies ( )N p 1,013

System and Clinical Feature(s)No. ofEvents

No. ofAvailable Data Percentage

Skeletal:Arachnodactyly 751 969a 78Dolichostenomelia 522 947a 55Joint laxity 600 956a 63Scoliosis 508 965a 53Pectus deformityb 570 962a 59Limited elbow extension 153 974a 16Protrusio acetabulae 69 298a 23Facial dysmorphism 443 913a 49High-arched palate 639 932a 69Dental malocclusion 372 843a 44Pes planus 402 864a 47Orthopedic surgery 113 983a 12Major skeletal involvement 327 1,013 32Minor skeletal involvement 564 1,013 56

Ocular:Ectopia lentis 542 1,013 54Myopia 453 865 52Cataract 39 983 4Retinal detachment 65 980 7Glaucoma 19 905 2Surgery for ectopia lentis 122 910 13Other eye surgeries 43 905 5Major eye involvement 542 1,013 54

Cardiac:Dilatation of the ascending aorta 775 1,013 77Dissection of the ascending aorta 145 1,013 14Dilatation or dissection of the descending or

abdominal aorta before age 50 years 66 1,013 7Mitral valve prolapse 533 983 54Mitral regurgitation 313 959 33Aortic insufficiency 205 975 21Aortic surgery 282 1011 28Isolated valvular surgery 45 1004 4Major cardiac involvement 776 1,013 77Minor cardiac involvement 108 1,013 11

Skin:Striae 444 945 47Herniae 96 988 10Minor skin involvement 480 1,013 47

Lung:Pneumothorax 73 1,002 7Minor lung involvement 73 1,013 7

CNS:Dural ectasia 154 292 53Major CNS involvement 154 1,013 15

a Nineteen patients were classified as having minor, major, or neither minor nor major skeletalfeatures, but details about their skeletal manifestations were not available.

b Includes pectus excavatum, 246 (26%) of 962; pectus carinatum, 288 (30%) of 962; and undefinedpectus malformation.

amino acid. For 38 mutations not displaying one of the abovefeatures, additional data provided by SIFT,32,33 BLOSUM-62,34 andbiochemical values (Kristine Yu’s Web site) were gathered andanalyzed using a new UMD tool (M. Frederic, C. Boileau, D. Ham-roun, M. Lalande, M. Claustres, C. Beroud, G. Collod-Beroud,unpublished data).

Involvement of Different Organ Systems

The proportion of each specific clinical feature or system involvedwas compared in the different groups of mutation types or lo-

cations. The following were each considered as a system: the skel-eton, the eye, the heart, the skin, and the dura. The clinical fea-tures of each system are listed in table 2. No attempts were madeto incorporate dilatation of the pulmonary artery, calcification ofthe mitral valve annulus, apical blebs, flat cornea and iris, orciliary muscle hypoplasia in the analyses, since those phenotypeswere rarely evaluated. Similarly, the axial globe length had rarelybeen measured, and the definition of myopia varied widely fromcenter to center. For this reason, myopia of any degree was in-cluded. In consequence, the presence or absence of minor oph-

Results 75


thalmological involvement could not be assessed. The ages atdiagnosis and at surgery were collected for scoliosis, ectopia lentis,and aortic dilatation or dissection. The probability of surgery forectopia lentis was studied only in the group of patients withectopia lentis. Similarly, the probability of aortic dissection andsurgery for aortic dilatation or dissection was studied only in thegroup of patients with aortic dilatation. The number of systemsinvolved was also assessed according to the Ghent nosology.4

Patients were classified as having MFS if the diagnostic Ghentnosology criteria were met, including the presence of an FBN1mutation as a major feature and, in a second step, excluding thepresence of an FBN1 mutation as a major feature. All other pa-tients were considered as displaying a type I fibrillinopathy. Forthe purpose of this study and in the absence of consensus di-agnostic features, patients were classified as having neonatal MFSwhen severe features of MFS, including severe valvular insuffi-ciencies, were present before age 4 wk.

Mutation Screening

Mutation screening, with the consent of the patient or a guardian,was performed in the 38 different laboratories by use of SSCPanalysis, denaturing high-performance liquid chromatography,heteroduplex analysis, long-range RT-PCR, or direct sequencingof RNA extracted from cell lines or of genomic DNA from pe-ripheral-blood samples. PTC mutations were classified as thosethat would be likely to produce no or a truncated FBN1 protein(frameshifts, stop codons, and out-of-frame splice mutations),whereas inframe mutations were classified as missense muta-tions, inframe deletions/duplications, or inframe splice muta-tions. Twenty-nine splice mutations could not be classified andwere therefore excluded from the analyses that compared typesof mutations.

Statistical Analysis

The frequency of many features of MFS increases with age. Sincethe patients had different lengths of follow-up, x2 tests are notappropriate for comparing clinical features between groups. Thus,we used a time-to-event analysis technique to estimate a reliablecumulative probability of observing the different manifestationsof MFS. This technique could be applied for the following events:diagnosis of type I fibrillinopathy and diagnosis of scoliosis, ec-topia lentis, and/or aortic dilatation or dissection, as well as sur-gery for these different manifestations, for which the ages atdiagnosis were systematically collected. In all time-to-event anal-yses, the baseline date (time zero) was the date of birth. The time-to-event diagnosis was defined as the interval between the base-line date and the date of first observation of the event. Subjectswho did not manifest the studied event during the follow-upcourse were censored at their last follow-up. Subjects for whomthe age at diagnosis of a specific manifestation was not availablewere excluded from these analyses (a maximum of 4% of pa-tients). The number of observations for each clinical feature isindicated in table 2. The Kaplan-Meier method35 was used to es-timate the cumulative probabilities of clinical manifestations ofthe disease at ages 10, 25, and 40 years, to describe the diagnosisof clinical features over time. Clinical differences among the dif-ferent mutation groups (different locations or types of mutation)were tested using the nonparametric log-rank test. Overall sur-vival was also described and compared, using the same method,according to the type/location of the mutation. To underline the

importance of taking into account the time to diagnosis of clinicalfeatures, we compared the results obtained for aortic dilatationusing a x2 test and the time-to-event technique.

For the other features (skeletal features other than scoliosis,skin, lung, and dural involvement) for which the age at diagnosiswas not collected, age at last follow-up was the only informationavailable about the time of observation of clinical features. Toindirectly take into account the patient’s length of follow-up evenin this situation, we adjusted all comparisons of MFS manifes-tation proportions for the age at last follow-up, categorized into10-year age groups. These adjusted comparisons were performedusing the Mantel-Haenszel (MH) test.36 Since this test is appro-priate only if the relationship between the mutation type andthe clinical manifestation is similar in the different strata of ageat last follow-up, we checked the homogeneity between stratausing the Breslow-Day x2 test of homogeneity.37 If an interactionwas observed, results were presented for each category of age atlast follow-up. In both analyses, if no information was availablefor a patient’s given clinical feature, he or she was excluded fromthe analysis of that specific lesion.

To study the effect of mutation types, we compared (1) patientswith a PTC with patients with an inframe mutation, (2) patientswith a particular subtype of mutation (nonsense, frameshift,splicing, missense, or inframe deletions/insertions) 2 by 2, and(3) patients with missense mutations eliminating or creating acysteine. There was no recurrent mutation frequent enough toallow a correlation study regarding the other FBN1 mutations. Tostudy the consequences of mutations in different structural andfunctional domains, we compared (1) patients with a mutationlocated in an EGF domain (cb and non-cb) with those with amutation located in a TGFb-BP domain; (2) patients with a mu-tation at the 5′ end of the FBN1 gene (exon 1–21, inclusive) withthose with a mutation at the 3′ end of the gene (exon 43–65,inclusive), to take into account the regions involved in the pro-cessing of the protein; (3) patients with a mutation located withinthe so-called neonatal region (exons 24–32) with those with amutation located in other exons; and (4) patients with a mutationlocated within the TGFb1 regulating sequence (exons 44–49) withthose with a mutation located in other exons. When locationsof mutations were compared, studies were performed with alltypes of mutations and with missense mutations alone, to excludea bias due to different types of mutations. Mutations located inexons 24–32 were compared with mutations located elsewhere,with and without the inclusion of neonatal MFS.

SAS software version 9.2 and Stata software version 8 were usedfor all statistical analyses. Only P values !.001 were consideredsignificant, since multiple tests were performed.

ResultsMutations

A total of 803 pathogenic mutations were found in 1,013probands (including 114 recurrent mutations in 324probands). The distribution of mutations is presented infigure 1. Of the missense mutations, 61% (348 of 573)involved a cysteine (284 replacing and 64 creating a cys-teine). Sixty-eight percent (665 of 984) were inframe,whereas 32% (319 of 984) generated a PTC (29 splicingmutations were not classified, since the consequence atthe mRNA level could not be determined unambiguously).The majority of mutations were located in an EGF domain



Figure 1. Types of FBN1 mutations included in the study. Of1,013, 573 (56%) could be classified as missense mutations, 170(17%) as frameshift mutations, 137 (14%) as nonsense mutations,110 (11%) as splicing mutations, and 23 (2%) as inframe deletionsor insertions.

(74% [747 of 1,013])—701 of which were located in a cb-EGF domain—and 15% (153 of 1,013) were located in aTGFb-BP domain. Twenty-nine percent (293 of 1,013) ofmutations were found in the 5′ region of the gene, and379 (37%) were found in the 3′ region of the gene. Twentypercent (198 of 1,013) of mutations were located in exons24–32. Ten percent (102 of 1,013) of mutations were lo-cated in exons 44–49. No major differences in mutation-type categories were detected between laboratories.

Phenotype in the Overall Patient Cohort

Fifty-four percent of patients were males and 46% werefemales. A family history of MFS was found in 52% ofcases. The median age at last follow-up was 29 years (in-terquartile range [IQR] 15–40 years), including 322 pa-tients aged !18 years (32%). The median age at diagnosisof type I fibrillinopathy was 20 years (IQR 9–34 years).Five percent of patients ( ) had a neonatal presen-n p 48tation. Overall, at the time of analysis, 61 (6%) had died,31 (51%) in the context of neonatal MFS, 19 of aorticdissection, 10 during or after aortic surgery, and 1 from acause unrelated to MFS.

The frequency of manifestations of each organ systemin the full cohort of patients is listed in table 2. In par-ticular, of the 1,013 patients, 145 (14%) had dissection ofthe ascending aorta, 43 (4%) had dissection of the de-scending aorta, and 30 (3%) had dissection of the abdom-inal aorta. Protrusio acetabulae and dural ectasia—al-though they are included in the Ghent nosology—were

rarely looked for in our patients ( and ,n p 298 n p 292respectively). The majority (89%) of patients could be clas-sified, according to Ghent nosology, as having MFS at theirlast follow-up, when the presence of an FBN1 mutationwas considered a major feature (72% when the presenceof an FBN1 mutation was not considered a major feature).Phenotypic differences depending on the sex of the pro-band were studied for all clinical parameters. Significantdifferences were found only for the cumulative probabilityof aortic surgery for patients with aortic dilatation. Indeed,46% of males had surgery for aortic dilatation before orat age 40 years (99.9% CI 38%–55%) compared with 34%of females (99.9% CI 26%–50%) ( ). A marginallyP p .0002significant result was found for the cumulative probabilityof ascending aortic dilatation, with a probability of 80%before or at age 40 years in males (99.9% CI 5%–84%)compared with 70% in females (99.9% CI 64%–76%)( ).P p .0036

Types of Mutations

Missense mutations substituting or creating a cysteine versusother missense mutations.—The probability of ectopia lentiswas significantly higher with missense mutations involv-ing a cysteine when compared with other missense mu-tations (log-rank test ) (fig. 2). The cumulativeP ! .0001probability of ectopia lentis diagnosed before or at age 25years was 59% (99.9% CI 50%–68%) for patients with mis-sense mutations involving a cysteine compared with 32%(99.9% CI 22%–44%) for patients with other missense mu-tations. Consequently, the percentage of patients withpositive Ghent criteria was higher in the group of patientswith missense mutations involving a cysteine when com-pared with other missense mutations (76% vs. 63% whenthe presence of an FBN1 mutation was not considered asa major feature; MH test ).P p .0003

PTC versus inframe mutations.—Patients with a PTC mu-tation more frequently had major skeletal involvement(40% vs. 28%; MH test ) with a higher propor-P p .0008tion of arachnodactyly, dolichostenomelia, joint hyper-laxity, pectus deformity, high-arched palate, and pesplanus. Moreover, a higher frequency of striae distensae(64% vs. 40%; MH test ) was observed in patientsP ! .0001with a PTC mutation (fig. 3). The cumulative probabilityof a diagnosis of ascending aortic dilatation before or atage 40 years was 77% (99.9% CI 8%–85%) for patientswith PTC mutations compared with 74% (99.9% CI 67%–80%) for patients with an inframe mutation (log-rank test

). Conversely, the cumulative probability of aP p .7791diagnosis of ectopia lentis and ophthalmologic surgerywas significantly lower for patients with PTC mutationscompared with patients with an inframe mutation (log-rank test and , respectively) (table 3P ! .0001 P p .0001and figure 2). These results became insignificant for theage at diagnosis of ectopia lentis and ophthalmologicsurgery when patients with PTC mutations were com-pared with patients with missense mutations not involv-

Results 77


Figure 2. Kaplan-Meier analyses for the probability of ectopia lentis diagnoses for patients with different types of mutations. A,Probability of ectopia lentis in PTC versus inframe mutations. The cumulative probability of diagnosis of ectopia lentis before or at age25 years was 23% (99.9% CI 15%–32%) for patients with PTC mutations (thin line) compared with 50% (99.9% CI 43%–57%) forpatients with inframe mutations (thick line) (log-rank test ). B, Probability of ectopia lentis for patients with missense mutationP ! .0001involving a cysteine versus other missense mutations. The cumulative probability of diagnosis of ectopia lentis before or at age 25years was 59% (99.9% CI 50%–68%) for patients with missense mutations involving a cysteine (thin line) compared with 32% (99.9%CI 22%–44%) for patients with other missense mutations (thick line) (log-rank test ).P ! .0001

ing a cysteine (log-rank test and ,P p .0424 P p .1020respectively).

Other mutation subtypes.—As expected for the results ofPTC mutations versus inframe mutations, a lower prob-ability of ectopia lentis was found when nonsense orframeshift mutations were compared with missense mu-tations (log-rank test ). No significant differenceP ! .0001was found for any manifestation when patients with non-sense mutations were compared with those with frame-shift mutations or when patients with missense mutationswere compared with those with splicing mutations. Ahigher probability of ascending aortic dilatation (log-ranktest ) and mitral valve prolapse (log-rank testP ! .0001

) and a higher frequency of arachnodactyly andP p .0007joint laxity (MH test and , respec-P p .0002 P p .0006tively) were found in patients with a mutation eliminatinga cysteine than in patients with a mutation creating acysteine.

Location of Mutations

Exons 24–32 versus other exons.—A neonatal onset of thedisease was found in 22% ( ) of patients with a mu-n p 42tation in exons 24–32, compared with 0.6% ( ) ofn p 4patients with a mutation in other exons (x2 test P !

). When patients with mutations within exons 24–.000132 were compared with patients with mutations in theother exons, significant differences were found for jointlimitations (34% vs. 11%; MH test ), scoliosis,P ! .0001ectopia lentis, ascending aortic dilatation, aortic surgery,mitral valve abnormalities (mitral valve prolapse, mitralregurgitation, and/or mitral surgery), younger age at di-agnosis of type I fibrillinopathy, and a shorter overall sur-vival (log-rank test ) (table 3 and fig. 4). Indeed,P ! .001

76% of patients with mutations in exons 24–32 were aliveat age 40 years (99.9% CI 61%–87%) compared with 98%(99.9% CI 93%–99%) of patients with mutations locatedin other exons (log-rank test ). Moreover, the cu-P ! .0001mulative probability of ascending aortic dilatation diag-nosed before or at age 40 years was 87% (99.9% CI 77%–95%) for patients with a mutation in exons 24–32 com-pared with 72% (99.9% CI 67%–78%) for patients with amutation in other exons (log-rank test ), and theP ! .0001cumulative probability of aortic surgery before or at age40 years was 55% (99.9% CI 35%–77%) for patients witha mutation in exons 24–32 compared with 38% (99.9%CI 30%–48%) for patients with a mutation in other exons(log-rank test ). Apart from ectopia lentis, the re-P ! .0001sults were similar when patients with a neonatal onsetwere excluded. Results were also similar when all types ofmutations were included in the analysis and when onlymissense mutations (except for ectopia lentis) or only mis-sense mutations involving a cysteine were included.

The distribution of types of mutations was significantlydifferent in exons 24–32 from the distribution in the otherexons, with an overrepresentation of missense mutationsand an underrepresentation of nonsense mutations (table4) (Fischer test ). PTC mutations within exonsP p .000224–32 were rarely associated with a neonatal MFS presen-tation (2 [5%] of 43) compared with inframe mutationswithin this region (41 [95%] of 43). A higher frequencyof neonatal presentations was found for mutations inexon 25 when compared with mutations distributed inexons 24–32 (x2 test ).P ! .0001

Exons 44–49 versus other exons.—No significant differencewas found for any clinical parameter for patients with amutation located in the specific sequence that regulates



Figure 3. Frequency of skeletal, skin, pulmonary, and dural phenotypes in study participants with PTC mutations (gray bars), comparedwith those with inframe mutations (black bars). An asterisk (*) indicates that differences between groups were statistically significant(MH test ).P ! .001

the bioavailability of endogenous TGFb1, compared withthose with a mutation located elsewhere.

EGF/TGF b-BP domains.—No significant difference wasfound for any clinical parameter for patients with a mu-tation located in an EGF domain compared with thosewith a mutation in a TGFb-BP domain, nor between pa-tients with a mutation in a cb-EGF domain and those witha mutation in a non–cb-EGF domain. Similar results werefound when all types of mutations or only missense mu-tations were included in the analysis.

5′ versus 3′ mutations.—Patients with a mutation locatedin the 5′ region of the gene had a higher probability ofectopia lentis (log-rank test ). This result wasP p .001highly significant when only missense mutations were in-cluded in the analysis (log-rank test ), whereasP ! .0001all the other results remained nonsignificant.

Discussion

FBN1 mutations have been associated with a broad spec-trum of phenotypes now often called “type I fibrillino-pathies,” ranging from single connective-tissue manifes-tations, such as isolated ectopia lentis, to MFS and lethalneonatal MFS. Every patient with an FBN1 mutation is atrisk for developing severe cardiovascular, skeletal, andophthalmologic complications (L. Faivre, G. Collod-Be-roud, B. Loeys, A. Child, C. Binquet, E. Gautier, B. Cal-lewaert, E. Arbustini, K. Mayer, M. Arslan-Kirchner, A.Kiotsekoglou, P. Comeglio, N. Marziliano, D. Halliday, C.Beroud, C. Bonithon-Kopp, M. Claustres, H. Plauchu, P.N. Robinson, L. Ades, J. De Backer, P. Coucke, U. Francke,A. De Paepe, C. Boileau, G. Jondeau, unpublished data).Here, we aimed at identifying the type or location of agiven FBN1 mutation that could be associated with thepresence of a clinical feature, severity, and/or age at onset.Although no specific manifestation or set of features ispathognomonic for a particular subtype of FBN1 muta-

tion, the occurrence of specific organ involvement differedsignificantly in some instances.

The mechanism by which mutations in FBN1 result indisease is unclear, since the biochemical pathway of fi-brillin-1 assembly into microfibrils is still poorly under-stood and since the role of fibrillin-1 in TGFb signalinghas only recently emerged. A dominant negative modelwas first proposed,38–39 in which the mutant monomer dis-rupts assembly of normal fibrillin-1 into microfibrils or isitself misincorporated into the microfibril. Recent studieshave given evidence of a critical contribution of haploin-sufficiency in the pathogenesis of MFS.40 Different effectson trafficking have also been demonstrated, with somemutations acting as dominant negative and others as hap-loinsufficient.41 Here, our data suggest that both geneticmechanisms are involved and that their tissue distributionmay differ.

The first striking result of this study is the strong cor-relation found between ectopia lentis and the presence ofa mutation affecting a cysteine residue, confirming earlierconclusions on a smaller sample.24–27,42 It is noteworthythat phenotypes strongly overlapping with type I fibril-linopathies are associated with mutations in TGFBR1/2and thus altered TGFb signaling. However, the main fea-ture of these phenotypes, as compared with type I fibril-linopathies, is the almost consistent absence of ocularinvolvement.43 Thus, it could be speculated that the func-tional aspect of fibrillin-1 that is altered in patients withectopia lentis is not involved in TGFb signaling but is astructural function in the extracellular matrix. Our datasuggest that correct cysteine localization and disulfidebonding play an important role in the structural integrityof the suspensory ligaments of the lens, itself relying onthe structural function of fibrillin-1 in this organ.44 Also,in the subgroup of patients with a mutation affecting acysteine residue, we found a significantly higher proba-bility of ascending aortic dilatation and mitral valve pro-

Results 79

Tabl

e3.

Com

pari

son

ofPr

obab

ilit

ies

ofCl

inic

al-F

eatu

reDi

agno

sis

ata

Spec

ific

Age

for

Pati

ents

wit

hM

FSan

dOt

her

Type

IFi

bril

lino

path

ies,

Acco

rdin

gto

the

Type

orLo

cati

onof

FBN

1M

utat

ions

Freq

uenc

y

Prob

abili

ties

ofCl

inic

alFe

atur

esDi

agno

sis

(%)

atAg

eFr

eque

ncy

Prob

abili

ties

ofCl

inic

alFe

atur

esDi

agno

sis

(%)

atAg

e

n(%

)10

2540

n(%

)10

2540

Clin

ical

Feat

ure

PTC

Mut

atio

nsIn

fram

eM

utat

ions

Pa

Age

atdi

agno

sis

ofty

peI

fibri

llino

path

y31

910

0.0

20.4

51.7

85.2

665

100.

032

.361

.286

.9.0

103

Asce

ndin

gao

rtic

dila

tati

on25

981

.210

.638

.776

.949

173

.820

.243

.573

.8.7

791

Aort

icdi

ssec

tion

inth

epo

pula

tion

pres

enti

ngw

ith

asce

ndin

gao

rtic

dila

tati

on65

25.1

.03.

631

.178

15.9

.25.

122

.9.2

014

Aort

icsu

rger

yin

the

popu

lati

onpr

esen

ting

wit

has

cend

ing

aort

icdi

lata

tion

102

39.4

.09.

442

.014

729

.91.

812

.240

.4.5

301

Surv

ival

303

95.0

99.7

98.8

95.1

436.

595

.594

.493

.2.2

311

Mis

sens

eM

utat

ions

Invo

lvin

ga

Cyst

eine

Othe

rM

isse

nse

Mut

atio

ns

Age

atdi

agno

sis

ofty

peI

fibri

llino

path

y34

810

0.0

32.8

62.6

89.1

225

100.

028

.957

.882

.2.0

983

Asce

ndin

gao

rtic

dila

tati

on26

275

.321

.346

.375

.615

870

.217

.637

.469

.4.0

797

Aort

icdi

ssec

tion

inth

epo

pula

tion

pres

enti

ngw

ith

asce

ndin

gao

rtic

dila

tati

on41

15.6

.04.

629

.925

15.8

.75.

210

.8.4

249

Aort

icsu

rger

yin

the

popu

lati

onpr

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ting

wit

has

cend

ing

aort

icdi

lata

tion

8030

.51.

314

.047

.449

31.0

2.7

10.4

29.0

.271

2Su

rviv

al33

094

.896

.596

.194

.514

6.2

96.0

93.5

93.5

.678

5

Exon

s24

–32

Othe

rEx

ons

Age

atdi

agno

sis

ofty

peI

fibri

llino

path

y19

810

0.0

51.0

75.8

91.9

815

100.

023

.153

.785

.2!.0

001

Asce

ndin

gao

rtic

dila

tati

on16

482

.842

.565

.787

.360

974

.711

.336

.472

.4!.0

001

Aort

icdi

ssec

tion

inth

epo

pula

tion

pres

enti

ngw

ith

asce

ndin

gao

rtic

dila

tati

on21

12.8

.75.

828

.512

420

.4.2

4.3

25.7

.306

4Ao

rtic

surg

ery

inth

epo

pula

tion

pres

enti

ngw

ith

asce

ndin

gao

rtic

dila

tati

on52

31.7

4.7

17.5

55.2

201

33.0

.59.

938

.4!.0

001

Surv

ival

159

80.3

84.5

81.1

76.1

222.

799

.699

.197

.5!.0

001

NOTE

.—Al

lag

esar

ein

year

s.Re

sult

sar

eKa

plan

-Mei

eres

tim

ates

.a

Log-

rank

test

Pva

lues

wer

efo

rPT

Cm

utat

ions

vers

usin

fram

em

utat

ions

,fo

rm

isse

nse

mut

atio

nsin

volv

ing

acy

stei

neve

rsus

othe

rm

isse

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mut

atio

ns,

orfo

rm

utat

ions

wit

hin

exon

s24

–32

vers

usm

utat

ions

inot

her

exon

s.

Results 81


Table 4. Types of Mutations Found in Exons24–32, Compared with Mutations in the Other Exons

Mutation

No. (%) of Mutations in

Exons 24–32( )n p 198

Other Exons( )n p 815

Nonsense 13 (6.6) 124 (15.2)Frameshift 27 (13.6) 143 (17.5)Splicing 15 (7.6) 95 (11.7)Missense 139 (70.2a) 434 (53.3b)Inframe deletion/insertion 4 (2.0) 19 (2.3)

NOTE.—Global Fischer exact test for differences was used,according to the location of mutations. .P p .0002

a 54% involving a cysteine.b 63% involving a cysteine.

Figure 4. Kaplan-Meier analyses for the probability of MFS clinical-features diagnosis for patients with different locations of mutations.A, Age at diagnosis of type I fibrillinopathy with a mutation in exons 24–32 versus in other exons. Fifty percent of patients with amutation in exons 24–32 (thin line) received a diagnosis at age 9 years (IQR 1–24 years) versus age 24 years (IQR 12–35 years) ofpatients with a mutation in other exons (thick line) (log-rank test ). B, Survival of patients with mutations in exons 24–32P ! .0001versus in other exons. Seventy-six percent of patients with mutations within exons 24–32 (thin line) were alive at age 40 years (99.9%CI61%–87% years) compared with 98% (99.9% CI 93%–99%) of patients with mutations located in other exons (thick line) (log-ranktest ). C, Probability of diagnosing a dilatation of the ascending aorta for patients with mutations in exons 24–32 versus inP ! .0001other exons. The cumulative probability of diagnosis of ascending aortic dilatation before or at age 40 years was 87% (99.9% CI 77%–95%) for patients with mutations in exons 24–32 (thin line) compared with 72% (99.9% CI 67%–78%) for patients with mutations inother exons (thick line) (log-rank test ). D, Probability of aortic surgery for patients with mutations in exons 24–32 versus inP ! .0001other exons. The cumulative probability of aortic surgery before or at age 40 years was 55% (99.9% CI 35%–77%) for patients withmutations in exons 24–32 (thin line) compared with 38% (99.9% CI 30%–48%) for patients with mutations in other exons (thick line)(log-rank test ). E, Probability of ectopia lentis for patients with mutations in exons 24–32 versus in other exons. TheP ! .0001cumulative probability of ectopia lentis diagnosis before or at age 25 years was 53% (99.9% CI 39%–67%) for patients with mutationsin exons 24–32 (thin line) compared with 38% (99.9% CI 33%–44%) for patients with mutations in other exons (thick line) (log-ranktest ). F, Probability of scoliosis for patients with mutations in exons 24–32 versus in other exons. The cumulative probabilityP p .0003of scoliosis diagnosis before or at age 25 years was 61% (99.9% CI 47%–75%) for patients with mutations in exons 24–32 (thin line)compared with 44% (99.9% CI 38%–51%) for patients with mutations in other exons (thick line) (log-rank test ).P ! .0001

lapse and a higher frequency of arachnodactyly and jointlaxity, when comparing patients with a mutation elimi-nating a cysteine with those with a mutation creating acysteine. Therefore, it seems that the disappearance of aconserved cysteine residue implicated in a disulfide bondleads to a more severe disorganization of a given modulethan does the introduction of a new and supernumerarycysteine residue.

The second striking result of our study is the strongcorrelation between FBN1 PTC mutation and severe skel-etal and skin phenotypes. Contrary to what is describedin the preceding paragraph, mutations in TGFBR1/2 areassociated with skeletal and sometimes skin alterationshighly comparable to those found in patients with FBN1.Thus, it is expected that a function or pathway commonto fibrillin-1 and the TGFb type 1/2 receptors is altered inthese patients. It could be speculated that haploinsuffi-ciency for fibrillin-1 in bone and skin has a stronger effecton the TGFb signaling function of the protein than on itsstructural function—and thus that, in bone growth, fi-brillin-1 acts as a mediator of TGFb signaling. The differentcorrelations found for skeletal and skin manifestations onthe one hand versus the ocular system on the other handmight then be explained by differences in the compositionand function of fibrillin-rich microfibrils in different or-gans and further underscore the complexity of the com-position of microfibrils and their interactions within tis-sues. Patients with a mutation in exons 24–32 had a moresevere and complete phenotype, including younger age atdiagnosis and higher probability of scoliosis, ectopia len-tis, ascending aortic dilatation, aortic surgery, mitral valveabnormalities, and shorter survival. However, patientswith aortic dilatation and a mutation in exons 24–32 didnot present a higher probability of aortic dissection thandid patients with aortic dilatation and a mutation locatedelsewhere. These data can be explained in part by a higherprobability of aortic surgery in such patients. It can also

be postulated that, because of the general severity of thephenotype, type I fibrillinopathy was diagnosed before theoccurrence of aortic dissection in patients with a mutationin exons 24–32. Since the majority of these results werereplicated even when neonatal MFS cases were excluded,we conclude that patients with a mutation in exons 24–32 have a poorer prognosis, with earlier onset of morbiditythan in patients with a mutation located elsewhere. Thepresence of a mutation in this region appears to be thebest indicator of early-onset aortic risk. More than a “neo-natal region,” it should be considered a “severe region.”This result can be explained by the role of these exons inthe central stretch of contiguous EGF-like domains andtheir importance for alignment and stability of the 10-nmmicrofibrils in the extracellular matrix.45 The distributionof the mutation types in exons 24–32 is different fromthe distribution found in other exons of the gene. Mu-tations leading to PTC are significantly underrepresented,contrasting with an overrepresentation of inframe mu-tations. In particular, nonsense mutations have never toour knowledge been described in association with neo-natal MFS, and this observation may be of importance



in elucidating the pathogenetic role of the exon 24–32region.

In this study, we sought to avoid the main bias inherentin our study design. Clinical/molecular correlations arecomplicated by a wide age range in individuals. In youngerpatients, the clinical phenotype and symptoms may notbe fully developed. Indeed, incorrect significant resultscan be obtained when x2 tests are used, and the use of theKaplan-Meier approach allowed us to take into accountthe heterogeneity of the length of follow-up amonggroups, as well as the young median age of the patientsin the cohort. Since age at lesion onset cannot be assessedfor MFS, notably for aortic dilatation, we used the ages atwhich each main clinical manifestation was discovered.We considered only probands, to minimize the possibleinfluence of early medical interventions in relatives (ear-lier monitoring and earlier preventive therapy with b-blockers) and to avoid overrepresentation of a mutation.Finally, we also excluded patients (4%) for whom no in-formation about one of the major systems of MFS (cardiac,ocular, and skeletal system) was available. The low numberof excluded patients had no significant impact on ourresults but provided a homogeneous study population,whatever the clinical feature investigated. However, itshould be noted that the majority of patients were of Eu-ropean origin, thus the conclusions may not be totallyapplicable to all ethnic groups. Another aspect of the pro-band collection is the high frequency of de novo muta-tions. It is now well documented that the yield in mu-tation screening is highest in probands displaying an MFSphenotype diagnosed using the Ghent nosology.24 Fur-thermore, molecular confirmation of an apparent de novoevent is important for genetic counseling in at-risk rela-tives. Therefore, the important number of de novo mu-tations found in the probands does not reveal a higher-than-reported mutation rate but reflects practices andscreening results from the contributing centers worldwide.

A few studies have addressed the question of a genotype-phenotype correlation in FBN1 carriers. Only five includeda sufficient number of patients (101, 93, 57, 81 and 76patients)23–27 to draw conclusions. A simple x2 approachwas used, and probands as well as their affected familymembers were considered in two of the five series. Authorsmainly compared the different phenotypes related to PTCmutations with those of cysteine substitutions. A signifi-cantly higher frequency of ectopia lentis associated withcysteine substitutions when compared with PTC muta-tions was a consistent finding. A tendency toward a moresevere skeletal phenotype23–24 and a more severe cardiacphenotype23,27 in the PTC group was discussed, but withinconsistency in significant results. This could be ex-plained partly by the small sample sizes of the populationsstudied. The protein phenotypes were studied only bySchrijver et al.23 A preponderance of probands with PTCmutations had markedly reduced extracellular fibrillin de-position with reduced synthesis, whereas individuals withcysteine substitutions had normal levels of fibrillin syn-

thesis and markedly reduced matrix deposition. Geno-type-phenotype correlations in type I fibrillinopathieshave also been complicated by clinical heterogeneityamong individuals with the same mutation, within andamong families.46–48 The type or location of a mutationalone cannot explain these variations. The existence ofgenetic or environmental modifiers, as well as the suscep-tibility of microfibrillar matrices to proteolytic degrada-tion, or intrafamilial variation in FBN1 expression havebeen postulated.40,49

In conclusion, our results show a strong correlation be-tween ectopia lentis and the presence of a mutation af-fecting a cysteine residue, whatever its location within theprotein. Conversely, PTC mutations are associated withsevere skeletal and skin phenotypes. These correlationsfound between different mutation types and clinical man-ifestations may indicate different underlying pathophys-iologic mechanisms, both genetic (dominant negative vs.haploinsufficiency) and functional (structural function offibrillin-1 vs. mediator of TGFb signaling). Finally, weshow that the location of a mutation within the exon 24–32 region is associated with a severe prognosis, not onlyin newborns but at all ages. However, we believe that theseresults cannot be used for individual prognosis but showthat aortic monitoring is warranted in every patient withan FBN1 mutation.

Acknowledgments

We thank I. Kaitila (Helsinki), P. Khau Van Kien (Montpellier), S.Davies (Cardiff), and T. Uyeda (Irosaki, Japan) for their partici-pation in the study. We also thank C. Bonaıti (Villejuif, France)for her helpful comments in the statistical-analyses design. Thiswork was supported by a grant from the French ministry of health(PHRC 2004), GIS maladies rares 2004, Bourse de la Societe Fran-caise de Cardiologie, Federation Francaise de Cardiologie 2005,and ANR-05-PCOD-014 from the Agence Nationale pour la Re-cherche. B.C. and B.L.L. are, respectively, a research fellow and asenior clinical investigator of the Fund for Scientific Research–Flanders.

Web Resources

The URLs for data presented herein are as follows:

Kristine Yu’s Web site, http://cmgm.stanford.edu/biochem218/Projects%202001/Yu.pdf (for paper entitled “Theoretical De-termination of Amino Acid Substitution Groups Based on Qul-aitiative Physicochemical Properties”)

Online Mendelian Inheritance in Man (OMIM), http://www.ncbi.nlm.nih.gov/Omim/ (for MFS, FBN1, TGFBR2, isolated ectopialentis, and Weill-Marchesani syndrome)

UMD, http://www.umd.be/

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Publication 2

The revised Ghent nosology for the Marfan syndrome.

Bart L. Loeys*, Harry C. Dietz

*, Alan C. Braverman, Bert L. Callewaert, Julie De

Backer, Richard B. Devereux, Yvonne Hilhorst-Hofstee, Guillaume Jondeau,

Laurence Faivre, Dianna M. Milewicz, Reed E. Pyeritz, Paul D. Sponseller, Paul

Wordsworth, Anne M. De Paepe. (*: contributed equally)

J Med Genet, in press

The Ghent nosology has clearly proven his usefulness in the assessment of patients

suspected with MFS. Over the last 15 years some issues arose about the applicability

and evidence-based value of some criteria. Moreover, due to changing sociological

circumstances, there is need for a more patient-centered approach that avoids

overdiagnosis in the absence of a tangible aortic risk. Recently, newly described

related conditions with different prognoses may complicate the diagnostic process and

should also be addressed in a new nosology. This study reflects the conclusions of an

expert assembly that convened to address the drawbacks of the Ghent nosology.


The revised Ghent nosology for the Marfan syndrome

Bart L Loeys,1 Harry C Dietz,2 Alan C Braverman,3 Bert L Callewaert,1

Julie De Backer,1 Richard B Devereux,4 Yvonne Hilhorst-Hofstee,5

Guillaume Jondeau,6 Laurence Faivre,7 Dianna M Milewicz,8 Reed E Pyeritz,9

Paul D Sponseller,10 Paul Wordsworth,11 Anne M De Paepe1

ABSTRACTThe diagnosis of Marfan syndrome (MFS) relies ondefined clinical criteria (Ghent nosology), outlined byinternational expert opinion to facilitate accuraterecognition of this genetic aneurysm syndrome and toimprove patient management and counselling. TheseGhent criteria, comprising a set of major and minormanifestations in different body systems, have proven towork well since with improving molecular techniques,confirmation of the diagnosis is possible in over 95% ofpatients. However, concerns with the current nosologyare that some of the diagnostic criteria have not beensufficiently validated, are not applicable in children ornecessitate expensive and specialised investigations.The recognition of variable clinical expression and therecently extended differential diagnosis furtherconfound accurate diagnostic decision making.Moreover, the diagnosis of MFSdwhether or notestablished correctlydcan be stigmatising, hampercareer aspirations, restrict life insurance opportunities,and cause psychosocial burden. An international expertpanel has established a revised Ghent nosology, whichputs more weight on the cardiovascular manifestationsand in which aortic root aneurysm and ectopia lentis arethe cardinal clinical features. In the absence of any familyhistory, the presence of these two manifestations issufficient for the unequivocal diagnosis of MFS. Inabsence of either of these two, the presence ofa bonafide FBN1 mutation or a combination of systemicmanifestations is required. For the latter a new scoringsystem has been designed. In this revised nosology,FBN1 testing, although not mandatory, has greaterweight in the diagnostic assessment. Specialconsiderations are given to the diagnosis of MFS inchildren and alternative diagnoses in adults. Weanticipate that these new guidelines may delaya definitive diagnosis of MFS but will decrease the risk ofpremature or misdiagnosis and facilitate worldwidediscussion of risk and follow-up/management guidelines.

INTRODUCTIONSince Antoine-Bernard Marfan described the 5-year-old Gabrielle with skeletal manifestations of thedisease that now bears his name,1 importantprogress has been made in the delineation of theMarfan syndrome (MFS) and recognition of asso-ciated risks. The main features of this autosomaldominant disorder include disproportionate longbone overgrowth, ectopia lentis and aortic rootaneurysm. In 1955, Victor McKusick first estab-

lished a classification of connective tissue disorders,which resulted in the publication of his monograph‘Heritable connective tissue disorders’.2 3 In 1986,an international panel of experts defined a set ofclinical criteria (Berlin nosology) for the diagnosisof MFS4 with the aim of facilitating accuratecommunication about the condition betweenhealthcare providers, researchers and patients. Itwas felt that this would improve proper patientmanagement and effective patient counselling.Following the identification of FBN1 (encoding

fibrillin-1) as the causal gene for MFS,5 it wasrecognised that the Berlin criteria falsely alloweda diagnosis of MFS in individuals with a positivefamily history of MFS, who had only non-specificconnective tissue findings themselves and who didnot carry the mutation present in more typicallyaffected family members. New diagnostic criteriawere therefore put forth in 1996, referred to as theGhent nosology.6 These Ghent criteria were morestringent than the Berlin criteria, mitigating over-diagnosis of MFS and providing better guidelines todifferentiate MFS from related, ‘overlapping’conditions such as the MASS phenotype (myopia,mitral valve prolapse, borderline and non-progres-sive aortic root dilatation, skeletal findings andstriae) and mitral valve prolapse syndrome (MVPS).Since physicians associate the diagnosis of

‘Marfan syndrome’, above all else, with risk foraortic aneurysm/dissection, it can be detrimental todiagnose MFS in patients without tangible evidenceof such risk. Avoidable consequences associatedwith misdiagnosis of MFS include: restriction ofcareer aspirations or access to insurance benefits;additional financial burden associated withfrequent medical care; anxiety or situationaldepression; unfounded marital or reproductivedecisions; loss of health benefits or psychosocialstigmatisation associated with exercise restriction,a particularly important issue during childhood.The challenge is to balance such concerns with theparamount need to maintain good health throughproper counselling and application of sound antic-ipatory medical practices. Towards this objective, itis also important to avoid the diagnosis of MFSwhen clinical or molecular observations couldreveal alternative (and often more severe) diagnosesthat mandate specialised counselling or manage-ment protocols.The Ghent nosology employs a set of ‘major ’ and

‘minor ’ manifestations in numerous tissuesincluding the skeletal, ocular, cardiovascular, andpulmonary systems and the dura, skin and integu-ment.6 Major manifestations include ectopia lentis,

1Center for Medical Genetics,Ghent University Hospital,Ghent, Belgium2McKusick-Nathans Institute forGenetic Medicine, JohnsHopkins University and HowardHughes Medical Institute,Baltimore, USA3Department of Cardiology,Washington University School ofMedicine, Saint-Louis, USA4Weill Cornell Medical College,New York, USA5Center for Human and ClinicalGenetics, Leiden UniversityMedical Center, Leiden, theNetherlands6Centre de Reference pour leSyndrome de Marfan etapparantes, Hopital Bichat,Paris, France7Center for Genetics, Children’sHospital, Dijon, France8Department of MedicalGenetics, University of TexasMedical School, Houston, USA9Department of MedicalGenetics, University ofPennsylvania, Philadelphia, USA10Department of Orthopedics,Johns Hopkins University,Baltimore, USA11Clinical Rheumatology,Nuffield Orthopeadic Center,Oxford, UK

Correspondence toProfessor Bart Loeys, Center forMedical Genetics, GhentUniversity Hospital, BuildingOK5, De Pintelaan 185, 9000Gent, Belgium;[email protected]

BLL and HCD contributedequally to the manuscript.

Received 26 August 2009Revised 16 December 2009Accepted 17 December 2009

Loeys BL, Dietz HC, Braverman AC, et al. J Med Genet (2010). doi:10.1136/jmg.2009.072785 1 of 10

Original article

Results 87

aortic root dilatation/dissection, dural ectasia or a combinationof $4 out of eight major skeletal features. The diagnosis of MFSin an index patient requires major involvement of at least twoorgan systems with minor involvement of a third organ system.In the presence of an FBN1 mutation known to cause MFS ora first degree relative who was unequivocally diagnosed basedupon Ghent nosology, the presence of one major and one minormanifestation in different organ systems is sufficient to makethe diagnosis.

Current status of the Ghent nosologyThe Ghent criteria have found worldwide application in helpingphysicians to diagnose MFS appropriately. New moleculartechniques allow the detection of FBN1 mutations in up to 97%of Marfan patients who fulfil the Ghent criteria.7 8 This suggeststhat the current Ghent criteria have excellent specificity toidentify patients with FBN1 mutations. Consideration ofsensitivity is highly complex due to varying definitions of the‘target’ population and competing clinical priorities. Forexample, the current criteria have been criticised for takinginsufficient account of the age dependent nature of some clinicalmanifestations (making the diagnosis in children more difficult)9

and for including some rather non-specific physical manifesta-tions or poorly validated diagnostic thresholds. Although theassignment of major and minor criteria within the Ghentnosology has contributed to its utility, several of those criteria arenot intuitive when considered from the perspective of thedifferential diagnosis or patient management. Consideration ofthe diagnosis of familial ectopia lentis is particularly illustrativeof the prevailing issues. This diagnostic category has beenwidely applied for individuals and families that show lensdislocation and skeletal features of MFS but do not show aorticenlargement or dissection. FBN1 mutations are seen in familialectopia lentis and are not easily distinguished from thosecausing MFS on the basis of character or location within thegenedsuggesting either occult phenotypeegenotype correla-tions or the influence of modifiers.

The Ghent nosology clearly attempted to accommodate thefact that some people with ectopia lentis, skeletal findings andeven FBN1mutation have less cardiovascular risk (ie, risk to theaortic root) than seen in classic MFS, by allowing the diagnosisof familial ectopia lentis in the absence of a second majorMarfan manifestation. However, inadequate data were avail-able to evaluate the critical issue of whether cardiovascular riskcould be predicted by the presence of non-cardiac features, suchas dural ectasia or major versus minor skeletal involvement. Atthe other extreme, is it justified not to diagnose MFS insomeone with typical lens dislocation and aortic root enlarge-ment simply because they lack minor skeletal or skin findings?To address some of these issues, an international panel (seeacknowledgement) of experts in the diagnosis and manage-ment of MFS was convened in Brussels, Belgium by theNational Marfan Foundation (USA) and charged with consid-ering modifications to the Ghent criteria. Other factors underconsideration included the specialised nature, availability andcost of diagnostic tests for selected manifestations (eg, duralectasia), the need to define certain diagnostic categories better(eg, familial ectopia lentis, MASS phenotype10 and MVPS), todefine features that should trigger alternative diagnoses anda desire to complement diagnostic criteria with follow-up, andmanagement guidelines for various patient groups includingchildren who do not yet fulfil the diagnostic criteria but may doso in the future.

Proposal for new nosologyThis proposal for a revised nosology (box 1) was based on criticalreview of clinical characteristics in large published patientcohorts,7 8 11 12 and expert opinions of the panel members withextensive experience in applying the current criteria, the differ-ential diagnosis of MFS, and the strengths and limitations ofmolecular genetic testing. Several guiding principles werefollowed: maximal use of evidence based decision making;attention to practical (patient centric) implications; a focus onfeatures and criteria that distinguish MFS from other disorders;and definition of purposeful thresholds for diagnosis. As a result,five major changes in the diagnostic guidelines are proposed.First, more weight is given to two cardinal features of MFS,

aortic root aneurysm/dissection and ectopia lentis. In theabsence of findings that are not expected in MFS, the combi-nation of ectopia lentis and aortic root enlargement/dissectionshould be sufficient to make the diagnosis. All other cardiovas-cular and ocular manifestations of MFS and findings in otherorgan systems, such as the skeleton, dura, skin and lungs,contribute to a ‘systemic score’ (box 2) that guides diagnosiswhen aortic disease is present but ectopia lentis is not.Second, a more prominent role is assigned to molecular

genetic testing of FBN1 and other relevant genes (eg, TGFBR1and 2), as well as other genes indicated in table 1. In practice,this does not make FBN1 testing a formal requirement (which

Box 1 Revised Ghent criteria for diagnosis of Marfansyndrome and related conditions

In the absence of family history:(1) Ao (Z $2) AND EL¼MFS*(2) Ao (Z $2) AND FBN1¼MFS(3) Ao (Z $2) AND Syst ($7pts)¼MFS*(4) EL AND FBN1 with known Ao¼MFS

EL with or without Syst AND with an FBN1 not known with Ao orno FBN1¼ELSAo (Z < 2) AND Syst ($5 with at least one skeletal feature)without EL¼MASSMVP AND Ao (Z <2) AND Syst (<5) without EL¼MVPS

In the presence of family history:(5) EL AND FH of MFS (as defined above)¼MFS(6) Syst ($7 pts) AND FH of MFS (as defined above)¼MFS*(7) Ao (Z$2 above 20 years old, $3 below 20 years) +FH ofMFS (as defined above)¼MFS*

* Caveat: without discriminating features of SGS, LDS or vEDS(as defined in table 1) AND after TGFBR1/2, collagen biochem-istry, COL3A1 testing if indicated. Other conditions/genes willemerge with time. Ao, aortic diameter at the sinuses of Valsalvaabove indicated Z-score or aortic root dissection; EL, ectopialentis; ELS, ectopia lentis syndrome; FBN1, fibrillin-1 mutation (asdefined in box 3); FBN1 not known with Ao, FBN1 mutation thathas not previously been associated aortic root aneurysm/dissection; FBN1 with known Ao, FBN1 mutation that has beenidentified in an individual with aortic aneurysm; MASS, myopia,mitral valve prolapse, borderline (Z<2) aortic root dilatation,striae, skeletal findings phenotype; MFS, Marfan syndrome;MVPS, mitral valve prolapse syndrome; Syst, systemic score(see box 2); and Z, Z-score.

2 of 10 Loeys BL, Dietz HC, Braverman AC, et al. J Med Genet (2010). doi:10.1136/jmg.2009.072785

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imposes financial burden in some countries, and does not yethave 100% sensitivity and specificity), but allows its appropriateuse when available.

Third, some of the less specific manifestations of MFS wereeither removed or made less influential in the diagnostic evalu-ation of patients. This avoids the use of obligate thresholds thatlack clear validation or general availability.

Fourth, the new criteria formalise the concept that additionaldiagnostic considerations and testing are required if a patient hassufficient findings to satisfy the criteria for MFS but also showsunexpected findings, particularly if they segregate with diseasein the family or if they are suggestive of a specific alternativediagnosis. Particular emphasis is placed on SphrintzeneGoldbergsyndrome (SGS), LoeyseDietz syndrome (LDS), and thevascular form of EhlerseDanlos syndrome (vEDS). SGS and LDShave substantial overlap with MFS, including the potential forsimilar involvement of the skeleton, aortic root, skin and dura(table 1). Occasionally, vEDS shows overlap in the vascularsystem, dura, skin and skeleton. It is essential to consider

discriminating features (table 1) because each of these conditionshas a unique risk profile and management protocol.Finally, this nosology should help to allay concerns regarding

delayed or ambiguous diagnoses by providing context specificrecommendations for patient counselling and follow-up.In the revised nosology, new diagnostic criteria have been

defined for a sporadic patient and for an index patient witha positive family history (box 1). In the absence of a conclusivefamily history of MFS, the diagnosis can be established in fourdistinct scenarios:1. The presence of aortic root dilatation (Z-score $2 when

standardised to age and body size) or dissection13 and ectopialentis allows the unequivocal diagnosis of MFS, irrespectiveof the presence or absence of systemic features except wherethese are indicative of SGS, LDS or vEDS (table 1).

2. The presence of aortic root dilatation (Z$2) or dissection andthe identification of a bona fide FBN1 mutation (box 3) issufficient to establish the diagnosis even when ectopia lentisis absent. An overview of criteria that enhance confidence inthe pathogenetic potential for MFS of particular FBN1mutations is provided in box 3. These include missensemutations that substitute or create cysteine residues, alterone of the conserved residues important for calcium bindingin epidermal growth factor-like (EGF) domains, createa premature termination codon (nonsense mutations),delete or insert coding sequence, or disrupt the consensussequence for pre-mRNA splicing. Evidence for pathogenicityof other types of missense mutations would include itsabsence in at least 400 ethnically matched control chromo-somes and co-segregation with disease in the family, or denovo occurrence in a sporadic case (with confirmation ofpaternity). Definitive evidence of linkage to a predisposingFBN1 haplotype can substitute for an FBN1 mutation fordiagnostic purposes, but this linkage analysis requires at leastsix informative meioses in the patient’s family to confirm theMFS associated FBN1 allele. The absence of a mutation in theFBN1 gene despite complete screening is possible in MFS.

3. Where aortic root dilatation (Z $2) or dissection is presentbut ectopia lentis is absent and the FBN1 status is eitherunknown or negative, an MFS diagnosis is confirmed by thepresence of sufficient systemic findings ($7 points, accordingto a new scoring system) (box 2). However, featuressuggestive of SGS, LDS or vEDS must be excluded andappropriate alternative genetic testing (TGFBR1/2, collagen

Box 2 Scoring of systemic features

< Wrist AND thumb sign e 3 (wrist OR thumb sign e 1)< Pectus carinatum deformity e 2 (pectus excavatum or chest

asymmetry e 1)< Hindfoot deformity e 2 (plain pes planus e 1)< Pneumothorax e 2< Dural ectasia e 2< Protrusio acetabuli e 2< Reduced US/LS AND increased arm/height AND no severe

scoliosis e 1< Scoliosis or thoracolumbar kyphosis e 1< Reduced elbow extension e 1< Facial features (3/5) e 1 (dolichocephaly, enophthalmos,

downslanting palpebral fissures, malar hypoplasia, retrogna-thia)

< Skin striae e 1< Myopia > 3 diopters - 1< Mitral valve prolapse (all types) e 1Maximum total: 20 points; score $7 indicates systemicinvolvement; US/LS, upper segment/lower segment ratio.

Table 1 Features of differential diagnosis

Differential diagnosis Gene Discriminating features

LoeyseDietz syndrome (LDS) TGFBR1/2 Bifid uvula/cleft palate, arterial tortuosity, hypertelorism, diffuseaortic and arterial aneurysms, craniosynostosis, clubfoot,cervical spine instability, thin and velvety skin, easy bruising

ShprintzeneGoldberg syndrome (SGS) FBN1 and other Craniosynostosis, mental retardation

Congenital contractural arachnodactyly (CCA) FBN2 Crumpled ears, contractures

WeilleMarchesani syndrome (WMS) FBN1 and ADAMTS10 Microspherophakia, brachydactyly, joint stiffness

Ectopia lentis syndrome (ELS) FBN1LTBP2ADAMTSL4

Lack of aortic root dilatation

Homocystinuria CBS Thrombosis, mental retardation

Familial thoracic aortic aneurysm syndrome (FTAA) TGFBR1/2, ACTA2 Lack of Marfanoid skeletal features, levido reticularis, irisflocculiFTAA with bicupid aortic valve (BAV)

FTAA with patent ductus arteriosus (PDA) MYH11

Arterial tortuosity syndrome (ATS) SLC2A10 Generalised arterial tortuosity, arterial stenosis, facialdysmorphism

EhlerseDanlos syndromes (vascular, valvular, kyphoscoliotictype)

COL3A1, COL1A2, PLOD1 Middle sized artery aneurysm, severe valvular insufficiency,translucent skin, dystrophic scars, facial characteristics


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Results 89

biochemistry, COL3A1, and other relevant genetic testingwhen indicated and available upon the discovery of othergenes) should be performed.

4. In the presence of ectopia lentis but absence of aortic rootdilatation/dissection, the identification of an FBN1 mutationpreviously associated with aortic disease is required beforemaking the diagnosis of MFS. If the FBN1 mutation is notunequivocally associated with cardiovascular disease in eithera related or unrelated proband, the patient should be classifiedas ‘ectopia lentis syndrome’ (see differential diagnosis).In an individual with a positive family history of MFS (where

a family member has been independently diagnosed using theabove criteria), the diagnosis can be established in the presenceof ectopia lentis, or a systemic score $7 points or aortic rootdilatation with Z $2 in adults ($20 years old) or Z $3 inindividuals <20 years old.

Special consideration should be given to young individuals(<20 years old). In sporadic cases, these children may not fit inone of the four proposed scenarios. If insufficient systemicfeatures (<7) and/or borderline aortic root measurements (Z <3)are present (without FBN1 mutation), we suggest to use theterm ‘non-specific connective tissue disorder ’ until follow-upechocardiographic evaluation shows aortic root dilation (Z $3).If an FBN1mutation is identified in sporadic or familial cases butaortic root measurements are still below Z¼3, we propose to usethe term ‘potential MFS’ until the aorta reaches threshold.Neonatal MFS is not considered as a separate category, butrather represents the severe end of the MFS spectrum.

In adults (>20 years), we define three main categories ofalternative diagnoses: ectopia lentis syndrome (ELS), MASSphenotype (myopia, mitral valve prolapse, borderline (Z<2)aortic root enlargement, skin and skeletal findings), and mitralvalve prolapse syndrome (MVPS) (see differential diagnosis).

Finally, we recognise that some patients will remain difficultto classify due to overlap of phenotypes from different entities,the evolving nature of these connective tissue diseases, absenceof mutation after screening of the appropriate genes, or diver-gence between the phenotype and the genotype. However, thesepatients should be uncommon and will hopefully benefit frombetter definition of still unrecognised phenotypes in the future.

ORGAN SYSTEM SPECIFIC CONSIDERATIONSCardiovascular criteriaA key diagnostic criterion in the new nosology is aortic rootaneurysm or dissection. Aortic root aneurysm is defined asenlargement of the aortic root at the level of the sinuses ofValsalva. Aortic root measurements should be done parallel tothe plane of the aortic valve and perpendicular to the axis ofblood flow. The largest correctly measured root diameterobtained from at least three transthoracic images should becorrected for age and body size and interpreted as a Z-score.There are varying practices regarding whether root measure-ments should be done in systole or diastole and whether thethickness of one aortic wall should be included (ie, the leadingedge to leading edge method). The method employed mustmatch that used to generate the normative data for Z-scores tobe valid. For echocardiographic measurements made from innerwall to inner wall during systole in individuals #25 years,a convenient Z-score calculator can be found at http://www.marfan.org. For echocardiographic measurements made fromleading edge to leading edge in diastole in all age groups, refer-ence graphs and Z-score equations are available13. If trans-thoracic echocardiographic evaluations do not allow precisevisualisation of the proximal aorta, transoesophageal echocar-diography or CTor MRI imaging should be applied, with specialattention to using double-oblique images to obtain correctdiameter measurements and use of the same nomograms.14

Mitral valve prolapse is also a common finding in MFS and isincluded as a feature in the systemic score. Mitral valve prolapseshould be defined by echocardiography as protrusion of one orboth of the mitral valve leaflets across the plane of the mitralannulus during systole. This is best detected in parasternal longaxis or apical long axis three-chamber or two-chamber views.There are no special criteria for diagnosing MVP in MFS andstandard practices should be applied.15

Pulmonary artery (PA) dilation (eg, main PA diameter>23 mm in adults)16 is often seen in MFS, but it is not specificto this diagnosis. In addition, complications of pulmonary arterydisease occur rarely. PA dilation was not therefore included inthe systemic score because further research is needed regardingthresholds and the diagnostic utility of this finding.Patients withMFS can develop aortic enlargement or dissection

at segments distant from the aortic root. The frequency of thisfinding (particularly at the proximal descending thoracic aorta andin the abdomen) appears to be increasing with the prolongedsurvival due to improvedmanagement of disease at the aortic root.While descending aortic aneurysm or dissection in the absence ofaortic root enlargement can occur inMarfan syndrome,17 18 this israre and given the low specificity of this finding for MFS, thisfinding is not included in the diagnostic criteria. Intermittentimaging of the descending thoracic aorta is indicated in adultpatients where there is a clinical suspicion ofMarfan syndrome inthe absence of aortic root enlargement. Widespread vasculardisease is more common with other conditions in the differentialdiagnosis, such as vascular EDS and LDS. For example, systemicvascular imaging (head to pelvis) is recommended if there isa suspicion of LDS because of the high frequency of tortuosity,aneurysms and dissections throughout the vascular tree.

Ocular criteriaThe most prominent ocular features of MFS are myopia andectopia lentis. The diagnosis of ectopia lentis is based on slit-lamp examination after maximal dilatation of the pupil. Ectopialentis reflects failure of supporting structures called ciliaryzonules. Dislocation of the lens in MFS is most typically upward

Box 3 Criteria for causal FBN1 mutation

< Mutation previously shown to segregate in Marfan family< De novo (with proven paternity and absence of disease in

parents) mutation (one of the five following categories)< Nonsense mutation< Inframe and out of frame deletion/insertion< Splice site mutations affecting canonical splice sequence or

shown to alter splicing on mRNA/cDNA level< Missense affecting/creating cysteine residues< Missense affecting conserved residues of the EGF consensus

sequence ((D/N)X(D/N)(E/Q)Xm(D/N)Xn(Y/F) with m and nrepresenting variable number of residues; D aspartic acid, Nasparagine, E glutamic acid, Q glutamine, Y tyrosine, Fphenylalanine)

< Other missense mutations: segregation in family if possible +absence in 400 ethnically matched control chromosomes, ifno family history absence in 400 ethnically matched controlchromosomes

< Linkage of haplotype for n$6 meioses to the FBN1 locus


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and temporal, but deviation in any direction may occur. If lenssubluxation is deemed equivocal or minimal, manifesting only asa scalloped or ruffled lens margin at extremes of gaze, the eyeexam should be repeated later before a definitive diagnosis ofectopia lentis can be made (such findings can occur outside thecontext of MFS, eg, in individuals with high myopia). Increasedglobe length and corneal flattening are seen in MFS, but theyhave unclear specificity and are not routinely measured byophthalmologists. Given that myopia is very common in MFS, isroutinely monitored, and tends to show early onset, highseverity and rapid progression, myopia of >3 diopters contrib-utes to the systemic score for diagnosis. However, since myopiais quite a common finding in the general population we haveonly attributed one point to it in the systemic score.

Systemic criteriaClinical manifestations of MFS in other organ systems werecritically evaluated for their specificity and diagnostic utilitybased on expert opinion and the available literature. Several ofthe ‘minor ’ criteria from the old Ghent nosology were elimi-nated, but the most selective systemic features were included inthe ‘systemic score’.

Three points are assigned to the combination of wrist andthumb signs. The thumb sign is positive when the entire distalphalanx of the adducted thumb extends beyond the ulnar borderof the palm with or without the assistance of the patient orexaminer to achieve maximal adduction. The wrist sign ispositive when the tip of the thumb covers the entire fingernail ofthe fifth finger when wrapped around the contralateral wrist. Ifeither of the two signs is absent, only one point is assigned.

Two points were assigned to each of five other specificsystemic manifestations including anterior chest deformity,hindfoot deformity, spontaneous pneumothorax, dural ectasiaand acetabular protrusion. Pectus carinatum is believed to bemore specific for MFS than pectus excavatum and is assignedtwo points. Subjective qualifiers in the original Ghent criteriasuch as ‘requiring surgery’ have been eliminated, but theexaminer should be confident that a positive finding (pectusexcavatum or chest wall asymmetry) extends beyond normalvariation of chest contour in the general population beforeassigning one point. Hindfoot valgus19 (two points) in combi-nation with forefoot abduction and lowering of the midfoot(previously referred to as medial rotation of the medialmalleolus) should be evaluated from anterior and posterior view.The examiner should distinguish this from the more common‘flat foot’ (one point) without significant hindfoot valgus. As inthe past, any spontaneously occurring pneumothorax remainsa diagnostic feature. For the detection of lumbosacral duralectasia, no preferred method (CTor MRI) or uniformly acceptedcut-offs have emerged from the literature20e23 and local stan-dards should apply. Dural ectasia is a sensitive but not a specificsign of MFS and, as such, is no longer considered on equalfooting with lens dislocation or aortic root enlargement. It iscommonly seen in LDS and has been described in mutationproven vEDS. Finally, an additional technical exam for detectionof acetabular protrusion24 can be helpful but is not mandatory:classical x-ray, CT or MRI can be used. On an x-ray ante-rioreposterior pelvis angle, the medial protrusion of theacetabulum at least 3 mm beyond the ilio-ischial (Kohler) line isdiagnostic. Criteria on CT or MRI are not precisely defined butinvolve loss of the normal oval shape of the pelvic inlet at thelevel of the acetabulum.

One point is assigned to eight other manifestations, onecardiovascular (mitral valve prolapse), one ocular (myopia, $3

diopters) and six features from other organ systems. These areconsidered less specific features for MFS and can be observed inother connective tissue disorders or as normal variation in thegeneral population.18

The combined presence of reduced upper segment to lowersegment (US/LS) ratio (for white adults <0.85; <0.78 in blackadults; no data have been assessed in Asians) and increased armspan to height ratio (for adults >1.05) in the absence of signif-icant scoliosis contributes one point to the systemic score. InAsians the incidence of an enlarged arm span to height ratio inMarfan patients was noted to be lower25 and prior studies ofAsian (and also Afro-Caribbean) populations demonstrateddifferent distributions of arm span and height, so one shouldconsider these ethnic differences when using cut-off values.26

For the US/LS ratio in children, abnormal ratios are US/LS <1(for age 0e5 years), US/LS <0.95 (for 6e7 years), US/LS <0.9(8e9 years old) and <0.85 (above age 10 years). The lowersegment is defined as the distance from the top of the symphysispubis to the floor in the standing position, and the uppersegment is the height minus the lower segment. Importantly,neither of these ratios provides an accurate measurement ofbone overgrowth in the presence of severe scoliosis or kyphosis.Scoliosis27 can be diagnosed either clinically if, upon bendingforward, a vertical difference of least 1.5 cm between the ribs ofthe left and right hemithorax is observed or if a Cobb’s angle(angle between a line drawn along the superior end plate of thesuperior end vertebra and a second line drawn along the inferiorend plate of the inferior end vertebra of the scoliosis measuredon anterioreposterior view of the spine) of at least 208 is seen onradiographs. In the absence of scoliosis, one point can becontributed by the presence of an exaggerated thoracolumbarkyphosis. Elbow extension is considered reduced if the anglebetween the upper and lower arm measures 1708 or less uponfull extension. One point can be assigned based upon facialcharacteristics if the patient shows at least three of the fivetypical facial characteristics including dolichocephaly, down-ward slanting palpebral fissures, enophthalmos, retrognathiaand malar hypoplasia. Striae atrophicae are considered signifi-cant as a diagnostic feature if they are not associated withpronounced weight changes (or pregnancy) and if they have anuncommon location such as the mid back, lumbar region, theupper arm, axillary region or thigh.The following criteria were removed from the current

nosology because of lack of perceived specificity: joint hyper-mobility, highly arched palate, and recurrent or incisionalherniae.18

Differential diagnosisSeveral conditions have been recognised which present over-lapping clinical manifestations with MFS in the cardiovascular,ocular or skeletal systems. These include conditions with aorticaneurysms (LDS, bicuspid aortic valve, familial thoracic aorticaneurysm, vEDS, arterial tortuosity syndrome), ectopia lentis(ectopia lentis syndrome, WeileMarchesani syndrome, homo-cystinuria, Stickler syndrome) or systemic manifestations ofMFS (ShprintzeneGoldberg syndrome, congenital contracturalarachnodactyly, LDS, MASS phenotype and MVPS (table 1).

Conditions with cardiovascular features of MFSHistorically the terms MASS phenotype and MVPS have beenused but several issues about the use of these terms have arisen.First, the definition of the MASS phenotype is not unequivocallyapplicable as it required at least two, but preferably three, of thefollowing manifestations: myopia, mitral valve prolapse,


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borderline aortic root enlargement, skin and minor skeletalfeatures (insufficient to fulfil the major skeletal criterion of theoriginal Ghent nosology).6 This definition indirectly alsoassumes a non-progressive nature of the aortic root dilatation,but it is currently unknown to what proportion of patients thisapplies. Third, FBN1 mutations have been found occasionally inMASS phenotype patients,18 28 but the precise risk for thedevelopment of aortic aneurysm and progression for thesepatients is poorly studied. Analogous to the ectopia lentissyndrome, the spirit of the definition of MASS phenotype aimsto avoid the diagnosis of MFS without documented risk foraortic root aneurysm development. The diagnosis of MASS ismade in individuals with an aortic root size below Z¼2, at leastone skeletal feature and a systemic score $5. The presence ofectopia lentis precludes this diagnosis. If an FBN1 mutation isidentified in a MASS patient, this patient has the potential toevolve into MFS, but it is currently unknown how often andwhich factors predict this transition over time.

Alternatively, when mitral valve prolapse is present in asso-ciation with limited systemic features (score <5), we suggest useof the term mitral valve prolapse syndrome (MVPS). MVPS isa common condition usually inherited in autosomal dominantmode29 with several candidate gene loci,30 but with evidence forrare X-linked inheritance31 which affects w1.5% of the popu-lation. In addition to prolapse of the mitral leaflets, MVPScommonly includes pectus excavatum, scoliosis and mildarachnodactyly.32 However, aortic enlargement and ectopialentis preclude this diagnosis.

LoeyseDietz syndrome (LDS) is an autosomal dominantaortic aneurysm syndrome characterised by the triad of hyper-telorism, bifid uvula/cleft palate, and/or arterial tortuosity withascending aortic aneurysm/dissection. It is caused by heterozy-gous mutations in the genes encoding the type 1 or 2 subunit ofthe transforming growth factor-b receptor (TGFBR1 orTGFBR2).33 Other more variable clinical features that distin-guish LDS from MFS include craniosynostosis, Chiari malfor-mation, clubfoot deformity, congenital heart disease, cervicalspine instability, easy bruising, dystrophic scarring, translucentskin and, most importantly, a high risk of aneurysm anddissection throughout the arterial tree. Patients with LDS arenot typically inappropriately tall and do not exhibit dispropor-tionally long extremities, although arachnodactyly is observed.Some patients with TGFBR1/2 mutations lack overt craniofacialfeatures despite an equal or greater severity of vascular orsystemic findings. Importantly, the natural history of patientswith LDS tends to be more aggressive than those with MFS orvEDS. In LDS, aortic dissections often occur at a younger age orat smaller aortic dimensions (<40 mm) compared to MFS, andthe incidence of pregnancy related complications is particularlyhigh.34 As with FBN1 mutations, the phenotype associated withTGFBR1/2 mutations can be variable, even within families, andcan be associated with skeletal features of MFS leading tooverlapping phenotypes in the old Ghent nosology.35e37 In orderto avoid persistent ambiguity even under the proposed criteria,molecular testing should be strongly considered because itinfluences the clinical management.34 It has been proposed thatpatients with TGFBR1/2 mutations who lack outward discrim-inating features of LDS should be designated LDS2, highlightingthe potential for more aggressive vascular disease than seen inMarfan syndrome (MIM 190181 and 190182).

With a population prevalence of up to 1%,38 39 bicuspid aorticvalve (BAV) is the most common congenital cardiac malforma-tion. A subset of individuals with BAV present with ascendingaortic aneurysm; however, such patients usually lack ocular or

other systemic findings that contribute strongly to MFS diag-nosis. Skeletal findings such as pectus deformity and scoliosiscan be observed in these families. BAV and aortic aneurysm canoccur together in some family members but independently inothers, indicating that they can be variably penetrant conse-quences of a common underlying genetic defect.40 41 UnlikeMFS, this condition commonly shows maximal or exclusivedilatation in the ascending aorta above the sinotubular junc-tion.42 Mutations have been identified in the NOTCH1 andKCNJ2 genes, but these account for only a small fraction of BAVpatients, who may have prominent valve calcification or asso-ciated forms of congenital heart disease. Linkage analysis revealsgenetic heterogeneity with putative loci on chromosomes 18q,5q and 13q.43

Familial thoracic aortic aneurysm and dissection syndrome(FTAAD) is a clinically and genetically heterogeneous group ofdisorders where thoracic aortic disease predominates. The age ofonset and rate of progression of aortic dilatation is highly vari-able and conditions that include variable or subtle systemicmanifestations of a connective tissue disorder have beenincluded in this designation. It is anticipated that future strati-fication of patients by genetic aetiology will help to refinephenotypic descriptions and inform patient counselling andmanagement. To date, there are five genes and two additionalloci44 45 associated with FTAAD. Mutations have been identifiedin FBN1, TGFBR1/2, MYH11, and ACTA2, the latter twoencoding components in the smooth muscle cell contractileapparatus. Mutations inMYH11 associate aortic root aneurysmswith patent ductus arteriosus (PDA).46 Mutations in ACTA2,accounting for up to 16% of FTAAD, associate aortic aneurysmwith other variable features including iris flocculi, livedo retic-ularis, cerebral aneurysm, BAV and PDA.47 In addition tothoracic aortic aneurysms and dissections, patients with ACTA2mutations can present with vascular disease in the cerebrovas-cular system (premature ischaemic strokes, Moyamoya diseaseand cerebral aneurysms) or premature coronary artery disease.48

The vascular type of EDS (previously EDS IV), is caused bymutations in COL3A1, the gene encoding type III collagen; it ischaracterised by vascular and tissue fragility. Cardinal featuresdistinguishing vEDS from MFS include translucent skin, easybruising, dystrophic scarring and a tendency for intestinal oruterine rupture. Typically, dissection or rupture occurs inmedium sized arteries in vEDS, although aortic involvement issometimes observed. There is no particular predisposition at theaortic root. About half of the aneurysms/dissections occur inthoracic or abdominal branch arteries; arteries of the head, neckand limbs are less frequently involved.49

Three other rare types of EDS have been associated withvascular problems. The kyphoscoliotic type (previously type VIEDS) is characterised by kyphoscoliosis, joint laxity, and musclehypotonia. This autosomal recessive condition is caused bydefects in the enzymatic activity of lysyl hydroxylase, encodedby the PLOD1 gene. Aortic dilation/dissection and rupture ofmedium sized arteries have been observed.50 Patients with theso-called ‘cardiac valvular subtype of EDS’, which associatessevere cardiac valvular problems and features of the classic typeof EDS (atrophic scars, skin hyperelasticiy and joint hypermo-bility), were found to have a complete deficiency of the proa2-chain of type I collagen (COL1A2).51 Most recently, patientswith arginine to cysteine substitutions in the proa1-chain oftype I collagen (COL1A1) displayed classic EDS but evolved toa vascular EDS-like phenotype later in life, with increased riskfor spontaneous arterial rupture, most prominently affecting thefemoral and iliac arteries.52


Original article


Arterial tortuosity syndrome (ATS) is a rare autosomalrecessive connective tissue disorder, characterised by severetortuosity, stenosis, and aneurysms of the aorta and mediumsized arteries.53 Skeletal and skin involvement is common. Theunderlying genetic defect is homozygosity or compoundheterozygosity for loss-of-function mutations in SLC2A10, thegene encoding the facilitative glucose transporter GLUT10.54

The condition is lethal in infancy in a subset of patients, butsome survive into adulthood and seem to do well.55

Conditions with ectopia lentisPatients with familial ectopia lentis typically have some skeletalfeatures of MFS and an FBN1 mutation. While lack of aorticdisease is a defining feature of this condition, it may be difficult todistinguish from emerging MFS in the absence of other affectedfamily members or at a young age. Even within extended pedi-grees with familial ectopia lentis, later onset aortic aneurysm maybe observed. In order to better highlight the systemic nature ofthis condition and to emphasise the need for assessmentof features outside the ocular system, we propose the designationectopia lentis syndrome (ELS). The presence of a personal orfamily history of aortic aneurysm, or the identification of an FBN1mutation previously associated with aortic aneurysm, would besufficient to transition the diagnosis to MFS, independently of thenumber or distribution of systemic features. To ensure thatadequate vigilance of other organ systems is maintained, thediagnosis of ELS cannot be formally invoked before the age of20 years. The disorder is genetically heterogeneous, with auto-somal dominant inheritance caused by FBN1 mutations56 andrecessive forms caused by LTBP2 and ADAMTSL4 mutations.57 58

Importantly, in ELS patients with FBN1mutations, cardiovascularfollow-up by imaging should be maintained throughout life.

Ectopia lentis can be present as a component of other rareconditions. Ectopia lentis et pupillae is an autosomal recessivecondition in which remnants of the pupillary membrane arepresent. However, it is not associated with cardiovascular orskeletal features of MFS.

In WeilleMarchesani syndrome (WMS), the lens dislocation istypically associated with microspherophakia (small, roundedand thickened crystalline lens) and a shallow anterior eyechamber. WMS patients are short with brachydactyly and jointstiffness. Both autosomal dominant and recessive forms of WMShave been described and are caused by FBN1 mutations59 60 ormutations in the ADAMTS10 gene,61 respectively. Homo-cystinuria is often easily differentiated from MFS by the pres-ence of mental retardation and thrombosis, and can be excludedby urine amino acid analysis in the absence of pyridoxinesupplementation. In homocystinuria, the lens usually dislocatesdownward due to complete loss of support by ciliary zonules. InStickler syndrome, patients can present with a Marfanoidhabitus. Typical ocular signs include vitreal degeneration, retinaldetachment, myopia and open angle glaucoma. Early cataractsare common, but lens subluxation is not. Other potentialdiscriminating features from MFS include cleft palate, hearingloss and epiphysial changes of the bones.

Conditions with overlapping systemic featuresShprintzeneGoldberg syndrome (SGS) is a rare craniosynostosissyndrome characterised by some of the systemic features foundin MFS (pectus abnormalities, scoliosis, arachnodactyly),craniofacial dysmorphism (exophthalmos, hypertelorism,downslanting palpebral fissures, maxillary and mandibularhypoplasia, high arched palate and low set ears) and develop-mental delay. So far, only two SGS patients have shown an

FBN1 mutation.62 63 Another patient reported by Kosaki et al63

as SGS was felt to have LDS based on arterial tortuosity and thepresence of a bifid uvula.64 Other important distinguishingfeatures between SGS and either LDS or MFS are the highincidence of cognitive impairment and the low frequency ofvascular disease in the former.Congenital contractural arachnodactyly (CCA) is an auto-

somal dominant disorder characterised by a Marfan-like bodyhabitus and arachnodactyly.65 Most affected individuals have‘crumpled’ ears that present as a folded upper helix, andcontractures of major joints (knees and ankles) at birth. Theproximal interphalangeal joints of the fingers and toes haveflexion contractures (camptodactyly). Kyphosis/scoliosis ispresent in about half of affected individuals. Mild enlargementof the sinuses of Valsalva has been reported, but there is noevidence that the aortic dilatation progresses to dissection orrupture.66 CCA is caused by mutations in FBN2, the geneencoding the extracellular matrix protein fibrillin-2.67

MANAGEMENTManagement guidelines for MFS patientsAortic root dilatation in MFS is usually progressive. Thereforeabsence of aortic root enlargement on initial clinical examinationdoes not necessarily exclude the diagnosis, even in adulthood.All individuals who meet the criteria for MFS should initially

have at least yearly echocardiograms. More frequent imagingshould be performed if the aortic diameter is approachinga surgical threshold ($4.5 cm in adults; less well defined inchildren) or shows rapid change ($0.5 cm/year) or withconcerns regarding heart or valve function. Individuals under age20 with systemic findings suggestive of MFS but no cardiovas-cular involvement should have annual echocardiograms due tothe potential for rapid evolution of the phenotype. Adults withrepeatedly normal aortic root measurements can be seen atintervals of 2e3 years.Although several alternative medical treatments have been

proposed (angiotensin converting enzyme (ACE) inhibitors,calcium channel antagonists), the standard of care in mostcentres for the prevention of aortic complications in MFSremains b-blockade.68 More data are required before ACEinhibitor therapy can be considered standard treatment.69 b-blockade should be considered in all patients with MFS,including children and those with aortic root diameters <4 cm,unless contraindicated. The b-blocker should be titrated toeffect, aimed at a heart rate after submaximal exercise (eg,running up and down two flights of stairs) <100 beats/min inindividuals over 5 years of age. Angiotensin receptor blockers(ARBs) have shown the ability to prevent aortic enlargement ina mouse model of Marfan syndrome,70 and encouraging resultswere observed in a pilot experience in children with severeMFS.71 Several multicentre trials of losartan versus or on top ofatenolol in MFS are currently underway.72 If b-blockers arecontraindicated or not tolerated, other classes of antihyperten-sive agents can be used, but there is not definitive evidence thatthey will afford protection in people with MFS.Management of acute dissection of the ascending aorta (type

A dissection) is emergency surgery. Consideration of prophy-lactic surgery is recommended when the diameter at the sinusesof Valsalva approaches 5.0 cm. Other factors that inform thetiming of surgery include a family history of early dissection, therate of aortic root growth, the severity of aortic valve regurgi-tation, associated mitral valve disease, ventricular dysfunction,pregnancy planning in women, and the desire for a valve sparingoperation.


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Type B dissection (originating in the thoracic descendingaorta) accounts for about 10% of all dissections in MFS. Possibleindications for surgery include intractable pain, limb or organischaemia, an aortic diameter exceeding 5.5 cm, or a rapidincrease in the aortic diameter. Open surgery is still preferred asexperience with intravascular stenting in MFS is very limited,and the pressure endovascular stents need to apply against thewall of adjacent normal sized aortic segments to remain wellseated may not be tolerated by weakened connective tissue, orthe adjacent aorta may also be dilated. Regular imaging of theentire aorta is encouraged after root surgery and in adulthood.

Mitral valve repair or replacement is advised for severe mitralvalve regurgitation with associated symptoms or progressive leftventricular dilatation or dysfunction. Repair should be consid-ered, especially in patients undergoing aortic valve sparing rootreplacement. If a mechanical aortic valve prosthesis is chosen,mitral valve replacement may be considered, although preser-vation of left ventricular function may be better with mitralvalve repair. After isolated mitral valve repair, one should care-fully monitor aortic root size as increased rates of enlargementhave been observed.

Decisions regarding exercise restriction should always be madeon an individual basis. Recommendations from the NationalMarfan Foundation (http://www.marfan.org) and guidelinesfrom the American Heart Association/American College ofCardiology task forces73 are useful templates. In general,patients with MFS should avoid contact sports, exercise toexhaustion and especially isometric activities involvinga Valsalva manoeuvre. Most patients can and should participatein aerobic activities performed in moderation.

Pregnancy in MFS women is associated with increasedcardiovascular risk, with the majority of aortic complications(progressive dilatation and dissection) occurring in the thirdtrimester or in the early postpartum period. The risk of aorticroot complication is increased when the aortic root diameter isabove 4.0 cm at the start of the pregnancy.74

Annual ophthalmological evaluation for the detection ofectopia lentis, cataract, glaucoma and retinal detachment isessential. Early monitoring and aggressive refraction is requiredfor children with MFS to prevent amblyopia. Indications forsurgical lens extraction include lens opacity with poor visualfunction, anisometropia or refractive error not amenable tooptical correction, impending complete luxation, and lensinduced glaucoma or uveitis.

Skeletal manifestations such as scoliosis and pectus deformityshould be treated according to standard orthopaedic manage-ment rules.

Management guidelines for related conditionsRegular follow-up including annual cardiovascular imaging andophthalmological evaluation is advised in MASS, MVPS and ELSto monitor aortic size, and the degree of mitral regurgitation,over time. Counselling for patients with either ELS or MASSphenotype should include the risk of a more severe presentationin their offspring, including aortic enlargement.

Careful cardiovascular and ophthalmological follow-up isstrongly indicated in children with potential MFS or non-specificconnective tissue disorders.

CONCLUSIONThe diagnostic evaluation for MFS is unavoidably complex dueto the highly variable presentation of affected individuals, theage dependent nature of many of its manifestations, the absenceof gold standards, and its extensive differential diagnosis. While

diagnostic criteria should emphasise simplicity of use and thedesire for early diagnosis, accuracy receives highest priority inorder to avoid the deleterious and often irreversible consequencesof ungrounded or erroneous assignment. While the increasedfocus on vascular disease for the diagnosis of MFS in thisproposal will likely prove controversial, it is responsive to thepractical burden faced both by patients and physicians and doesnot represent a true departure from the spirit of prior diagnosticguidelines. Ongoing concerns about delayed diagnosis and/or theuse of diagnostic categories that may prove provisional shouldbe offset by additional discussion of ongoing risk and the defi-nition of follow-up and management principles. A comparativeanalysis on different retrospective datasets has shown w90%concordance between the old and revised Ghent nosology. The10% discordance was generally beneficial by facilitating earlierdiagnosis in young children with a convincing clinical pheno-type and delayed diagnosis in individuals without clear cardio-vascular risk. The current proposal will benefit froma prospective analysis, leading to further refinement. A webbased diagnostic tool for the application of these criteria can beaccessed at http://www.marfan.org.

Acknowledgements The expert panel meeting took place in February 2007 withsupport of the National Marfan Foundation and consisted of Bart Loeys, Harry Dietz,Alan Braverman, Richard Devereux, Guillaume Jondeau, Laurence Faivre, DiannaMilewicz, Reed Pyeritz, Paul Sponseller, Paul Wordsworth and Anne De Paepe.

Competing interests None.

Provenance and peer review Not commissioned; externally peer reviewed.

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65. Callewaert BL, Loeys BL, Ficcadenti A, Vermeer S, Landgren M, Kroes HY, Yaron Y,Pope M, Foulds N, Boute O, Galan F, Kingston H, Van der Aa N, Salcedo I, SwinkelsME, Wallgren-Pettersson C, Gabrielli O, De Backer J, Coucke PJ, De Paepe AM.Comprehensive clinical and molecular assessment of 32 probands with congenital

contractural arachnodactyly: report of 14 novel mutations and review of theliterature. Hum Mutat 2009;30:334e41.

66. Gupta PA, Putnam EA, Carmical SG, Kaitila I, Steinmann B, Child A, Danesino C,Metcalfe K, Berry SA, Chen E, Delorme CV, Thong MK, Ades LC, Milewicz DM. Tennovel FBN2 mutations in congenital contractural arachnodactyly: delineation of themolecular pathogenesis and clinical phenotype. Hum Mutat 2002;19:39e48.

67. Putnam EA, Zhang H, Ramirez F, Milewicz DM. Fibrillin-2 (FBN2) mutations result inthe Marfan-like disorder, congenital contractural arachnodactyly. Nat Genet1995;11:456e8.

68. Shores J, Berger KR, Murphy EA, Pyeritz RE. Progression of aortic dilatation and thebenefit of long-term beta-adrenergic blockade in Marfan’s syndrome. N Engl J Med1994;330:1335e41.

69. Ahimastos AA, Aggarwal A, D’Orsa KM, Formosa MF, White AJ, Savarirayan R,Dart AM, Kingwell BA. Effect of perindopril on large artery stiffness and aortic rootdiameter in patients with Marfan syndrome: a randomized controlled trial. JAMA2007;298:1539e47.

70. Habashi JP, Judge DP, Holm TM, Cohn RD, Loeys BL, Cooper TK, Myers L, Klein EC,Liu G, Calvi C, Podowski M, Neptune ER, Halushka MK, Bedja D, Gabrielson K, RifkinDB, Carta L, Ramirez F, Huso DL, Dietz HC. Losartan, an AT1 antagonist, preventsaortic aneurysm in a mouse model of Marfan syndrome. Science 2006;312:117e21.

71. Brooke BS, Habashi JP, Judge DP, Patel N, Loeys B, Dietz HC 3rd. Angiotensin IIblockade and aortic-root dilation in Marfan’s syndrome. N Engl J Med2008;358:2787e95.

72. Lacro RV, Dietz HC, Wruck LM, Bradley TJ, Colan SD, Devereux RB, Klein GL, Li JS,Minich LL, Paridon SM, Pearson GD, Printz BF, Pyeritz RE, Radojewski E, Roman MJ,Saul JP, Stylianou MP, Mahony L. Rationale and design of a randomized clinical trialof beta-blocker therapy (atenolol) versus angiotensin II receptor blocker therapy(losartan) in individuals with Marfan syndrome. Am Heart J 2007;154:624e31.

73. Maron BJ, Chaitman BR, Ackerman MJ, Bayes de Luna A, Corrado D, Crosson JE,Deal BJ, Driscoll DJ, Estes NA 3rd, Araujo CG, Liang DH, Mitten MJ, Myerburg RJ,Pelliccia A, Thompson PD, Towbin JA, Van Camp SP. Recommendations for physicalactivity and recreational sports participation for young patients with geneticcardiovascular diseases. Circulation 2004;109:2807e16.

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Original article


II. CONGENITAL CONTRACTURAL ARACHNODACTYLY

Publication 3

Comprehensive clinical and molecular assessment of 32 probands with

congenital contractural arachnodactyly: report of 14 novel mutations and

review of the literature.

Callewaert BL, Loeys BL, Ficcadenti A, Vermeer S, Landgren M, Kroes HY,

Yaron Y, Pope M, Foulds N, Boute O, Galán F, Kingston H, Van der Aa N,

Salcedo I, Swinkels ME, Wallgren-Pettersson C, Gabrielli O, De Backer J,

Coucke PJ, De Paepe AM.

Hum Mutat. 2009 Mar;30(3):334-41.

This paper presents the clinical and molecular findings in 32 patients diagnosed with

CCA. The phenotype is further characterized with special attention to the

cardiovascular manifestations. Data are compared to a review of the literature and

represented in a comprehensive overview. In our cohort, we screened the complete

FBN2 gene on gDNA level in order to evaluate the relative contribution of mutations

inside or outside the central region of FBN2 and to address the question of locus

heterogeneity.

Results 97

Human MutationRESEARCH ARTICLE

Comprehensive Clinical and Molecular Assessment of 32Probands With Congenital Contractural Arachnodactyly:Report of 14 Novel Mutations and Review of the Literature

Bert L. Callewaert,1 Bart L. Loeys,1 Anna Ficcadenti,2 Sascha Vermeer,3 Magnus Landgren,4 Hester Y. Kroes,5 Yuval Yaron,6

Michael Pope,7,8 Nicola Foulds,9 Odile Boute,10 Francisco Galan,11 Helen Kingston,12 Nathalie Van der Aa,13

Iratxe Salcedo,14 Marielle E. Swinkels,5 Carina Wallgren-Pettersson,15,16 Orazio Gabrielli,2 Julie De Backer,1

Paul J. Coucke,1 and Anne M. De Paepe1�

1Center for Medical Genetics, Ghent University Hospital, Belgium2Paediatric Department, Salesi Children Hospital, Ancona, Italy3Department of Human Genetics, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands4Department of Paediatrics, Skaraborg Hospital, Skovde, Sweden5Department of Biomedical Genetics, University Medical Center, Utrecht, The Netherlands6Genetic Institute, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel7Institute of Medical Genetics, University Hospital of Wales, Cardiff, UK8Department of Pathology, Cambridge University, Cambridge, UK9Wessex Clinical Genetics Service, Princess Anne Hospital, Southampton, UK10Service de Genetique Clinique, Centre Hospitalier Regional Universitaire de Lille, Lille, France11Centro de Genetica Humana, Universidad de Alicante, Alicante, Spain12Regional Genetic Service, St-Mary’s Hospital, Manchester, UK13Center for Medical Genetics, University Hospital of Antwerp, Antwerp, Belgium14Center for Medical Genetics, Hospital Comarcal Santiago Apostol, Miranda De Ebro, Burgos, Spain15Department of Medical Genetics, University of Helsinki, Helsinki, Finland16The Folkhalsan Institute of Genetics, Helsinki, Finland

Communicated by Reed E. Pyeritz

Received 26 February 2008; accepted revised manuscript 1 June 2008.

Published online 12 November 2008 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/humu.20854

ABSTRACT: Beals-Hecht syndrome or congenital contrac-tural arachnodactyly (CCA) is a rare, autosomaldominant connective tissue disorder characterized bycrumpled ears, arachnodactyly, contractures, and scolio-sis. Recent reports also mention aortic root dilatation, afinding previously thought to differentiate the conditionfrom Marfan syndrome (MFS). In many cases, thecondition is caused by mutations in the fibrillin 2 gene(FBN2) with 26 mutations reported so far, all located inthe middle region of the gene (exons 23–34). We directlysequenced the entire FBN2 gene in 32 probandsclinically diagnosed with CCA. In 14 probands, wefound 13 new and one previously described FBN2mutation including a mutation in exon 17, expandingthe region in which FBN2 mutations occur in CCA.Review of the literature showed that the phenotype of theFBN2 positive patients was comparable to all previouslypublished FBN2-positive patients. In our FBN2-positive

patients, cardiovascular involvement included mitralvalve prolapse in two adult patients and aortic rootenlargement in three patients. Whereas the dilatationregressed in one proband, it remained marked in a childproband (z-score: 4.09) and his father (z-score: 2.94),warranting echocardiographic follow-up. We confirmparadoxical patellar laxity and report keratoconus,shoulder muscle hypoplasia, and pyeloureteral junctionstenosis as new features. In addition, we illustrate largeintrafamilial variability. Finally, the FBN2-negative pa-tients in this cohort were clinically indistinguishable fromall published FBN2-positive patients harboring a FBN2mutation, suggesting locus heterogeneity.Hum Mutat 30, 334–341, 2009.& 2008 Wiley-Liss, Inc.

KEY WORDS: congenital contractural arachnodactyly;CCA; FBN2; fibrillin 2; genotype-phenotype

Introduction

Congenital contractural arachnodactyly (CCA; MIM] 121050)is an autosomal dominant connective tissue disorder characterizedby crumpled ears, a marfanoid habitus with arachnodactyly,

OFFICIAL JOURNAL

www.hgvs.org

& 2008 WILEY-LISS, INC.

Additional Supporting Information may be found in the online version of this article.

Contract grant sponsor: Fund for Scientific Research, Flanders.

�Correspondence to: Anne De Paepe, Center for Medical Genetics, Ghent

University Hospital, De Pintelaan 185, 9000 Gent, Belgium.

E-mail: [email protected]


congenital contractures of small and large joints that usuallyimprove over time, and progressive scoliosis [Hecht and Beals,1972; Ramos Arroyo et al., 1985; Viljoen, 1994]. Importantly,reports on four probands also mention aortic root dilatation, afeature previously thought to be a differentiating finding fromMarfan syndrome (MFS; MIM] 154700) [Gupta et al., 2002,2004].

MFS and CCA are caused by mutations in the fibrillin 1 (FBN1)(MIM] 134797) and FBN2 (MIM] 121050) genes, respectively,encoding fibrillin 1 and 2 [Dietz et al., 1991; Putnam et al., 1995].Fibrillins are cysteine-rich glycoproteins that polymerize extra-cellularly as parallel bundles of head-to-tail monomers and formmacroaggregates, called microfibrils, in association with otherproteins such as latent transforming growth factor beta bindingproteins (LTBPs), elastin microfibril interface located proteins(EMILINs), microfibril-associated glycoproteins (MAGPs), mi-crofibril-associated proteins (MFAPs), and fibulins [Ramirez et al.,2004]. Microfibrils provide force-bearing structural support andcan associate with elastin to form elastic fibers, providing elasticityin a time- and tissue-dependent manner [Ramirez et al., 2004;Robinson et al., 2006]. They also sequester transforming growthfactor beta (TGFb) and control its release. Perturbation of TGFbsignaling was recently recognized as a major disease-causingmechanism in MFS [Dietz et al., 2005].

While FBN1 knockout mice phenocopy the human MFS, so far,no FBN2 knockout animal models fully recapitulate the clinicalcharacteristics of CCA in humans. Homozygous knockout mousemodels demonstrate syndactyly and transitory neonatal contrac-tures but no external ear deformities, arachnodactyly, or spineanomalies [Chaudhry et al., 2001]. In addition, deafness wasreported in the sy mouse strain (shaker-with-syndactylism),caused by a contiguous deletion involving the SCL12A2 (MIM]600840) gene [Dixon et al., 1999]. On the other hand, FBN2hemizygous mice fail to exhibit any abnormal phenotype[Chaudhry et al., 2001]. Therefore, it remains important tocollect and compare the clinical and molecular data of all patientsaffected by this ‘‘orphan disease’’ [Tuncbilek and Alanay, 2006].

In contrast to MFS, with over 1,000 reported mutationsdispersed throughout the FBN1 gene, CCA is a rare disorder, withonly 26 FBN2 mutations published to date, all located in the middleregion (exon 23–35) of the gene, the so-called neonatal region,encoding a stretch of calcium-binding epidermal growth factor(cbEGF)-like domains. However, only four groups explicitly statedthat they had screened the entire FBN2 gene [Belleh et al., 2000;Maslen et al., 1997; Nishimura et al., 2007; Park et al., 1998], andCCA patients without FBN2 mutations were reported [Gupta et al.,2002]. Moreover, a premature truncating mutation was reported inCCA [Gupta et al., 2002] and could occur in other regions of thegene as well. It therefore remains unclear whether mutationsoutside the middle region of the FBN2 gene might cause CCA.

To answer this question, we screened all exons of the FBN2 genein 32 probands clinically diagnosed with CCA. We reviewed allFBN2-positive patients reported to date and compared them tothe FBN2-negative patients in our cohort.

Materials and Methods

Patients

Patients included in the study were diagnosed with CCA by aclinical geneticist at the referring center. For all patients, clinicaldata were collected by means of a detailed checklist. Inclusioncriteria were as follows: 1) presence of contractures (of small and/

or large joints); 2) crumpled ears or arachnodactyly; and 3) theabsence of mental retardation. A blood or DNA sample was sentfor mutation analysis of the FBN2 gene to confirm the diagnosis ofCCA. Statistical analysis was performed using the two-tailedFisher’s exact test. Significance was determined at P level 0.05.

Molecular Analysis

Genomic DNA was extracted from blood samples by standardprocedures. Touchdown PCR amplification of all exons of theFBN2 gene was performed using forward and reverse primerslocated in the flanking introns (primer sequences available uponrequest), followed by direct sequencing on the ABI PRISM 3730automated sequencer (Applied Biosystems, Foster City, CA) usingthe BigDye terminator cycle sequencing chemistry (AppliedBiosystems). Sequences were compared to the wild-type sequenceas submitted to GenBank (accession number NM_001999.3). Thenucleotides were numbered starting from the first base of the startcodon (ATG) of the cDNA reference sequence. Amino acidresidues are numbered from the first methionine residue,according to the protein accession number NP_001990.2. Theannotation has changed recently, introducing three nucleotides inexon 5 and all previously reported mutations have been adapted tothis new reference sequence. All previously unpublished mutationswere verified in a panel of at least 200 control alleles. When aorticroot dilatation or other cardiovascular malformations werepresent and FBN2 analysis remained negative, fibrillin 1 (FBN1)and TGFb receptor 1 and 2 (TGFBR1 and TGFBR2) analysis wascarried out as well. All FBN2-negative patients were screened formutations in microfibril associated protein 2 (MFAP2; MIM]156790, RefSeq NM_002403.2). Primer sequences are available onrequest.

Splice-site prediction was performed using freely-availablesoftware (www.fruitfly.org/seq_tools/splice.html).

Results

Clinical Characteristics of All Probands

A total of 30 probands originated from all over Europe and twoprobands from the Middle East (Table 1). Mean age at diagnosiswas 10.6 years. External ear anomalies (92%), arachnodactyly(91%), camptodactyly (94%), large joint contractures (69%), andgeneralized muscle weakness (76%) were commonly observed.Contractures generally improved over time (82%). More thanone-half of all probands (55%) had scoliosis or kyphosis thattended to be progressive. High arched palate (71%) and pectusdeformity (31%) were frequently associated. Cardiovascularmalformations consisted of nonprogressive aortic root dilatation(13%), mitral valve prolapse (10%), and septum defects (6%).Two patients had a interrupted aortic arch or aortic coarctation,respectively, and were consequently diagnosed with severe/lethalCCA. One individual had a cleft palate. Fisher’s exact test revealedno significant differences between our cohort and all probandsand patients previously reported with an FBN2 mutation in theliterature (data not shown).

Molecular Data

A total of 13 novel and one previously described FBN2mutation were identified (Table 2) in 14 out of the 32 probands.If parental DNA was available, mutations were verified to be denovo (Families A, D, F, G, and J) or to segregate with the

HUMAN MUTATION, Vol. 30, No. 3, 334–341, 2009 335

Results 99

Table 1. Summary of the Clinical Findings of the FBN2-Positive and Negative Patients in Our Cohort and the FBN2-Positive PatientsPreviously Published�

Our seriesa Literaturea Totala

All

probands

FBN2–

probands

FBN21

probands

FBN2–compared

to FBN21

probands

(P value)

FBN21

patientsbPro-

bands Patients

FBN21 patients

(our series and

literature (%))

FBN2 –probands

compared

to all FBN21

patients

(P value)

Family history 9/27 1/13 8/14 0.012 13/24 21/38 (53) 0.003

Tall stature 17/26 9/13 8/13 NS 13/24 9/13 18/32 31/56 (55) NS

External ear

anomalies

26/30 14/17 12/13 NS 21/25 20/24 40/52 61/77 (79) NS

Highly arched

palate

15/21 8/12 7/12 NS 12/20 10/15 22/50 34/70 (49) NS

Cleft palate/bifid

uvula

1/32 1/18 0/14 NS 0/27 1/14 2/50 2/77 (3) NS

Arachnodactyly 29/32 16/18 13/14 NS 23/24 19/23 36/49 59/73 (81) NS

Camptodactyly 30/32 17/18 13/14 NS 23/26 22/24 42/51 65/77 (84) NS

Contractures of

large joints

22/32 11/18 11/14 NS 19/25 19/24 43/50 62/75 (83) NS

Contractural

improvement

18/22 10/11 8/11 NS 12/17 10/15 30/35 42/52(81) NS

(Kypho-)scoliosis 16/30 8/16 8/14 NS 16/27 18/22 33/50 49/77 (64) NS

Pectus deformity 9/29 5/15 4/14 NS 8/25 11/20 24/50 32/75 (43) NS

Dolichostenomelia 6/19 3/8 3/11 NS 9/22 4/23 14/36 23/58 (40) NS

Septal defects 2/31 1/18 1/13 NS 1/19 2/20 2/50 3/69 (4) NS

Aort root dilation 4/31 2/18 2/13 NS 3/19 4/21 5/50 8/69 (12) NS

Progressive 0/4 0/2 0/2 NS 0/3 0/2 0/5 0/8 (0) NS

Mitral valve

prolapse

3/31 3/18 0/13 NS 2/19 1/20 2/50 4/69 (6) NS

Interrupted aortic

arch

2/32c 2/18c 0/14 NS 0/19 1/23 1/50 1/69 (1,4) NS

Generalised muscle

weakness

19/25 9/12 10/13 NS 16/24 7/10 21/25 37/49 (76) NS

�The clinical characteristics of the FBN2-negative probands in our cohort are compared with the FBN2-positive probands in our cohort and with all thus far published FBN2-

positive patients in literature.aNumber varies per criterion according to the number of patients for whom clinical data are available.bOnly patients with defined molecular status and sufficient clinical data are available.cOne patient had aortic coarctation.

NS not significant.

Table 2. Results of FBN2 Analysis of the respective Probands/Families�

Family �CDNA Protein Exon Functional domain

A c.4222G4Aa p.Asp1408Asn 33 23th cbEGF-like/vWA matrilllin domain,

consensus sequence DLDE

B c.4273T4Ca p.Cys1425Arg 33 23th cbEGF-like

B c.3437A4Ga p.Tyr1146Cys 26 16th cbEGF-like

D c.422214_422215delAGa exon 32del 32 22th cbEGF-like

E c.3467G4Ta p.Cys1156Phe 26 16th cbEGF-like

F c.3272A4Ga p.Asn1091Ser 25 15th cbEGF-like

G c.3343G4C p.Asp1115His_ex25del 26 15–16th cbEGF-like, consensus sequence DIDE

H c.3364T4Ca p.Ser1122Pro 26 16th cbEGF-like

I c.2260G4Aa p.Gly754Ser 17 2nd 8-cys domain

J c.3737G4Ta p.Cys1246phe 29 19th cbEGF-like

K c.4151G4Ta p.Cys1384Phe 32 22th cbEGF-like

L c.4151G4Aa p.Cys1384Tyr 32 22th cbEGF-like

M c.3424T4Ca p.Cys1142Arg 26 16th EGF-like

N c.3481G4Aa p.Glu1161Lys 27 17 th cbEGF-like/VWA matrillin domain

consensus sequence DLDE

�Sequences were compared to the wild-type sequence as submitted to GenBank accession number NM_001999.3. the nucleotides were numbered starting from the first base of

the start codon (ATG) of the cDNA reference sequence. Amino acid residues are numbered from the first methionine residue, according to the protein accession number

NP_001990.2.aPreviously unreported mutation.

336 HUMAN MUTATION, Vol. 30, No. 3, 334–341, 2009


phenotype (Families B, E, H, K, L, M, and N). The mutationfound in the three siblings of Family C was not found in themother’s leukocytes; no DNA was available from the father. Noparental DNA was available for Family I.

A total of 13 out of 14 mutations were found in the middle regionof the gene (exons 24–36). Three mutations alter the highly-conserved calcium binding consensus sequences (DLDE or DIDE)of cbEGF-like domains (p.Asp1408Asn, p.Asp1115His, andp.Glu1161Lys). Seven mutations alter or produce a cysteine residue(p.Cys1425Arg, p.Tyr1146Cys, p.Cys1156Phe, p.Cys1246Phe,p.Cys1384Phe, p.Cys1384Tyr, and p.Cys1142Arg). One mutation isa de novo splice site mutation (c.422214_422215delAG) leading toan exon 32 skip. Of the eight missense mutations, two (c.4222G4Aand c.3343G4C) are also predicted to influence splicing.

One mutation was found outside the neonatal region in exon 17(p.Gly754Ser). This mutation is likely to be causal, as: 1) it issituated in a functionally important domain (second 8-cysdomain); 2) Gly754 is evolutionary conserved in dog, monkey,cattle, and mouse; 3) it replaces a small hydrophobic glycineresidue by a polar serine residue; 4) it was absent in a set of 200control chromosomes in our laboratory and 98 chromosomesscreened by Sakai et al. [2006].

Additional sequencing of FBN1, TGFBR1, and TGFBR2 inprobands with aortic involvement or cardiovascular malforma-tions did not reveal any mutations. Also, sequencing of thecandidate gene MFAP2 in all FBN2-negative patients did notreveal causalmutations.

Clinical Characteristics of the 14 FBN2-Positive Probandsand Thirteen Affected Relatives

Clinical findings in FBN2-positive probands and their familymembers are represented in Table 3 and Supplementary Figure S1(available online at http://www.interscience.wiley.com/jpages/1059-7794/suppmat). The mean age of all FBN2-positive pro-bands was 11.6 years. Sixteen patients were male, eleven werefemale. Five probands presented with all four typical features,including external ear anomalies, small and/or large jointcontractures, arachnodactyly and (kypho-)scoliosis; four pro-bands had external ear anomalies, joint contractures, andarachnodactyly, but were too young (aged 4 years or less) to beevaluated for scoliosis. Five probands had at least 3 out of 4features (incomplete clinical information in one proband). Whilecontractures and external ear deformities tended to improve overtime (with or without physiotherapy initiated), scoliosis (8/14probands) was usually progressive. Additionally, a highly archedpalate (7/12) was a common finding. Other skeletal features,including pectus deformity (4/14) and dolichostenomelia (3/11),were less frequently encountered. Generalized muscle hypoplasia isoften observed (10/13). Two probands had patellar laxity withsubsequent quadriceps atrophy and disability, requiring a walkingstick in one. Eight out of 13 probands were tall (490thpercentile), and 3 out of the 5 probands not reaching this limithad moderate to severe scoliosis. These findings were largelycomparable for the affected family members. A positive familyhistory was found in 8 out of 14 probands (Table 3).

Six patients had cardiovascular involvement. Aortic rootenlargement was found in three patients. The dilatation normal-ized in one proband (Patient D-II:1) after the age of 2 years, butremained marked in a child proband (Patient H-III:12; z-score4.09) and his father (Patient H-II:8; z-score 2.94). Two adults hadmitral valve prolapse. One proband had a small atrial septumdefect, type II. Ocular involvement was confined to myopia and

keratoconus. Two probands manifested a rather thin skin. Newfindings were localized shoulder girdle atrophy, a findingsegregating with the mutation in Family C, and keratoconus andpyeloureteral junction stenosis, both diagnosed in two probandseach (Patients I-II:1 and G-II:1, and Patients L-II:1 and M-III:1,respectively).

Statistical analysis did not provide evidence for any majordifferences between all FBN2-positive probands from our seriesand all previously published FBN2-positive probands or betweenall FBN2-positive patients from our series and all previouslypublished FBN2-positive patients (data not shown).

Clinical Characteristics of the 18 FBN2-Negative Probands

Clinical findings in FBN2-negative probands and their familymembers are represented in Supplementary Table S1 and Supple-mentary Figure S2. The mean age of all FBN2-negative probandswas 9.9 years. Five out of 18 probands had all four typical features,including external ear anomalies, small and/or large joint contrac-tures, arachnodactyly, and (kypho-)scoliosis; seven probands hadexternal ear anomalies, joint contractures, and arachnodactyly butwere too young (aged 4 years or less) to be evaluated for scoliosis.Five probands had at least 3 out of 4 features (incomplete clinicalinformation in one proband); one proband (individual 8) had onlytwo typical features (arachnodactyly and contractures). Interestingly,one male neonate (individual 16) had a type-b interrupted aorticarch and a large ventricular septal defect (VSD), associated withcrumpled ears, arachnodactyly, and contractures of small and largejoints; a clinical constellation very suggestive of lethal CCA.Similarly, no defect could be found in a female patient (individual4) with coarctation of the aorta associated with external earanomalies, arachnocamptodactyly and scoliosis. In both patients,karyotyping was normal and velocardiofacial syndrome was ruledout by 22q11 fluorescence in situ hybridization (FISH) analysis.Importantly, Fisher’s exact test, comparing these FBN2-negativeprobands with all previously published probands or patients,including those reported in this study, was highly significant forthe absence of a family history (P 5 0.003), but could notdemonstrate any major differences with regard to other phenotypiccharacteristics (Fig. 2; Table 1).

Genotype-Phenotype Correlations

High intrafamilial variability (Table 3) and incomplete penetrance(Fig. 1) was demonstrated for ear anomalies, arachnodactyly,contractures, and especially for scoliosis. The severity of scoliosisranged from absent to disabling, so far requiring surgery in fivepatients in our cohort (Table 3). Previously, intrafamilial variabilitywas only explicitly reported in the context of splicing mutations. Wenow have confirmed this phenomenon in families with missensemutations (Families C, E, H, and M).

Genotype-phenotype correlation studies comparing splice-sitemutations, cysteine substitutions, and other missense substitu-tions revealed no major differences, although pectus deformityoccurred more frequently in patients with splice-site alterations(P 5 0.018).

Discussion

In contrast to MFS, with mutations throughout the FBN1 gene,it is not known whether mutations outside the middle region ofthe FBN2 gene (exons 23–34) cause CCA. For the first time wereport a mutation outside this region in a patient with classical


Results 101

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CCA. Interestingly, this mutation is situated in exon 17, a regionpreviously found to bind microfibril-associated glycoprotein 1[Werneck et al., 2004]. Therefore, this region might be importantin interactions with other components of the extracellular matrixessential for proper microfibril assembly. Because the mutationdetection rate is very low outside the central stretch of cbEGF-likedomains, we recommend a two-step screening, beginning withexons 24–36. In total we identified 13 novel and one previouslydescribed FBN2 mutation in a cohort of 32 patients presentingwith CCA. Since we directly sequenced the FBN2 gene, it isunlikely that technical imperfection would account for this lowmutation detection rate (44%). However, intron mutations andwhole-exon deletions might be missed, but usually do notrepresent the majority of the mutations. Chromosomal deletions

around FBN2 were not formally excluded by comparative genomichybridization array (CGH), but are unlikely in the absence ofmental retardation, a feature not present in classic CCA [Viljoen,1994]. It is striking that, with the exception of one nonsensemutation [Gupta et al., 2002], all currently reported human FBN2mutations result in missense substitutions or in-frame exondeletions/duplications, which may suggest a gain-of-functioneffect. This could explain why no comparable phenotype isobserved in FBN2 mice models that all harbor loss-of-functionmutations [Chaudhry et al., 2001]. Alternatively, it has beenobserved that fibrillin-1 expression starts earlier in mice and couldcompensate for altered fibrillin-2 function.

We subsequently compared the clinical characteristics of theFBN2-negative probands with those of all currently published

Figure 1. Family trees of all families. Families are indicated as in the text. Square: male; round: female; arrow: proband; upper left quarterfilled: crumpled ears; upper right quarter filled: arachnodactyly; lower left quarter filled: contractures; lower right quarter filled: scoliosis;question mark: insufficient clinical information; slash line: died.


Results 103

FBN2-positive probands or patients. Only a positive family historywas significantly associated with the presence of an FBN2 mutation(P 5 0.003). However, as the clinical characteristics tend to improvewith age, affected parents may remain unrecognized if notexamined carefully. We could not demonstrate any other clinicaldifferences between both groups, and therefore, genetic locusheterogeneity is likely in CCA. We found microfibril-associatedprotein 2 (also known as microfibril-associated glycoprotein 1) tobe a good candidate gene, as it has a very similar spatiotemporalexpression pattern compared to FBN2 [Henderson et al., 1996] andit interacts with the central region of FBN2 [Werneck et al., 2004].However, we did not identify any causal aberrations uponsequencing of MFAP2 in our FBN2-negative cohort.

Review of the data on all current and previously publishedFBN2-positive patients (Table 1) reveals a largely comparablephenotype. The main features are external ear anomalies (79%),arachnodactyly (81%), contractures of small (84%) and large(83%) joints, sometimes causing an isolated motor delay, and(kypho-)scoliosis (64%). Scoliosis is usually progressive andrequires surgery in about one-third of patients. In contrast, jointcontractures ameliorate in most cases (81%), with a potentialbenefit of physiotherapy. Also, the ear anomalies, frequentlydescribed as ‘‘crumpled’’ ears, become less pronounced over time,although the helical crus remains prominent, giving a ‘‘tramtrack’’ appearance of the helical and antihelical cruri (Supple-mentary Fig. S1, Patients B:II-2, H:III-12, K-III:2, K-IV:2, L:II-1,M:III-1, M:II-2, L:I-2, L:II-1, L:II-2, and L:II-3). Paradoxically,laxity of joints can be present, especially of the patella. Otheranomalies are a highly arched palate (49%), pectus deformity(43%), and dolichostenomelia (40%). In contrast to FBN2knockout mice (F. Ramirez, personal communication ), patientstend to be tall (490th percentile), from birth onward, in theabsence of scoliosis. Cleft palate is a sporadic finding. In addition,we add keratoconus, shoulder girdle atrophy, and pyeloureteraljunction stenosis as new clinical features. The latter, found in twoprobands, is unlikely to be coincidental, as the populationfrequency for pyeloureteral junction stenosis is about 2 to 3 in1,000 [Kim et al., 1996].

Severe cardiovascular and gastrointestinal anomalies are seen ina condition termed lethal CCA [Currarino and Friedman, 1986;Lipson et al., 1974; Macnab et al., 1991]. Only one patient withthis condition was reported harboring a splice-site mutation

causing skipping of exon 34 [Wang et al., 1996]. FBN2 sequencingin two patients in our cohort with a similar phenotype (proband16 and proband 4) did not reveal a causal mutation. Interestingly,proband 16 was subsequently found to have phenylketonuria. Hismother was only an asymptomatic heterozygote carrier excludingprenatal hyperphenylalaninemia as a cause for the cardiovasculardefects. It therefore remains unresolved whether lethal CCArepresents the extreme end of the phenotypic spectrum or whetherit is caused by disruption of other genes.

Our data confirm that aortic root dilation occurs in FBN2-positive patients with CCA [Gupta et al., 2002, 2004; Park et al.,1998]. One proband (Patient D-II:1) was found to have anenlargement (z-score 5 2.5) at the age of 2 years that subsequentlyregressed, while another proband (Patient H-III:12) in this studyhad a severe dilatation at the age of 9 years (z-score 5 4.1). Hisaffected father also had a mildly dilated aorta (z-score 5 2.94).Therefore, prospective studies are needed to assess the aortic riskin this condition. The population frequencies of minor cardiacdefects, including mitral valve prolapse (6%) and septum defects(4%), equal the population frequencies in FBN2-positive patientswith CCA, in contrast to reports published before molecularanalysis became available [Viljoen, 1994].

Somatic mosaicism with a consistently less severe phenotypecompared to the full mutation was previously reported in twofamilies [Putnam et al., 1997; Wang et al., 1996]. In Family C, themutation detected in the children was not found in the leukocytesof the mother, who presented with isolated camptodactyly. Apaternal sister and grandfather were described to have a marfanoidhabitus with arachnodactyly. Because no DNA from the clinicallyunaffected father was available, it is unclear whether thisrepresents a third case of mosaicism or a first case ofnonpenetrance, but both hypotheses have their importance ingenetic counseling.

Intrafamilial variability, mainly with regard to scoliosis andcontractures, was thus far only described in three families[Babcock et al., 1998; Maslen et al., 1997], with splicingmutations. We now illustrate this phenomenon in families withmissense mutations (Families C, E, H, and M) (Fig. 1, Table 3). Asfibrillin 1 and 2 are both important scaffolds of microfibrils, onecould speculate that phenotypic variability in CCA results fromsimilar mechanisms as in MFS, combining dominant negativeeffects, loss-of-function, and TGF-b upregulation [Dietz et al.,2005], rather than from mutant allele expression only [Babcocket al., 1998; Maslen et al., 1997].

Finally, we could not demonstrate any clear genotype-phenotypecorrelations. Although pectus deformity occurred more frequently inpatients with splice-site mutations (P 5 0.018), no other parametersof long bone overgrowth followed this trend, even when analyzed ina subgroup of adult patients, and we therefore presume that this wasa coincidental association.

In conclusion, we report 13 new and one previously reportedFBN2 mutations in 32 probands with CCA. Although locusheterogeneity is likely, we advocate complementing a negativescreening of the middle region of the gene with analysis of theremaining exons. Follow-up with echocardiography is recom-mended, as aortic dilatation is documented, although the risk fordissection is not yet established. Genetic counseling should takeinto account somatic mosaicism or even nonpenetrance.

Acknowledgments

We are indebted to all families for their interest and cooperation. We thank

P. Van Acker and H. Vandevoorde for technical assistance and P. Willems

Figure 2. Comparison of FBN2-negative patients with all FBN2-positive probands (from our cohort) and all FBN2-positive patientsreported in the literature. Significance was only reached for thepresence of a family history (Fisher’s exact test; p 5 0.0003). [Colorfigure can be viewed in the online issue, which is available atwww.interscience.wiley.com.]



for accommodating referral of the patient samples. B.C. and B.L. are

research fellow and senior clinical investigator, respectively, and are

supported by the Fund for Scientific Research, Flanders.

References

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Results 105

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; HA

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, Cle

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ity; D

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Results 107

Cal

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l., H

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Mut

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Cal

lew

aert

et a

l., H

uman

Mut

atio

n 4

Results 109

III. THE ARTERIAL TORTUOSITY SYNDROME

Publication 4

Mutations in the facilitative glucose transporter GLUT10 alter angiogenesis

and cause arterial tortuosity syndrome.

Coucke PJ, Willaert A, Wessels MW, Callewaert B, Zoppi N, De Backer J, Fox

JE, Mancini GM, Kambouris M, Gardella R, Facchetti F, Willems PJ, Forsyth R,

Dietz HC, Barlati S, Colombi M, Loeys B, De Paepe A.

Nat Genet. 2006 Apr;38(4):452-7.

This paper describes the search for the genetic cause of ATS using a homozygosity

mapping strategy and subsequent sequencing of the genes in the candidate region.

Based on basic research on limited patient material, we propose a pathophysiological

model that connects the genetic defect with the altered vasculature in ATS.


Mutations in the facilitative glucose transporter GLUT10alter angiogenesis and cause arterial tortuosity syndromePaul J Coucke1, Andy Willaert1, Marja W Wessels2, Bert Callewaert1, Nicoletta Zoppi3, Julie De Backer1,Joyce E Fox4, Grazia M S Mancini2, Marios Kambouris5, Rita Gardella3, Fabio Facchetti6, Patrick J Willems7,Ramses Forsyth8, Harry C Dietz9, Sergio Barlati3, Marina Colombi3, Bart Loeys1 & Anne De Paepe1

Arterial tortuosity syndrome (ATS) is an autosomal recessivedisorder characterized by tortuosity, elongation, stenosis andaneurysm formation in the major arteries owing to disruptionof elastic fibers in the medial layer of the arterial wall1.Previously, we used homozygosity mapping to map a candidatelocus in a 4.1-Mb region on chromosome 20q13.1 (ref. 2).Here, we narrowed the candidate region to 1.2 Mb containingseven genes. Mutations in one of these genes, SLC2A10,encoding the facilitative glucose transporter GLUT10, wereidentified in six ATS families. GLUT10 deficiency is associatedwith upregulation of the TGFb pathway in the arterial wall, afinding also observed in Loeys-Dietz syndrome, in which aorticaneurysms associate with arterial tortuosity3. The identificationof a glucose transporter gene responsible for altered arterialmorphogenesis is notable in light of the previously suggestedlink between GLUT10 and type 2 diabetes4,5. Our datacould provide new insight on the mechanisms causingmicroangiopathic changes associated with diabetes andsuggest that therapeutic compounds intervening withTGFb signaling represent a new treatment strategy.

Facilitative glucose transporters (GLUTs), encoded by a family ofSCL2A genes, are responsible for the uptake of several monosacchar-ides, including glucose, fructose, mannose, galactose and glucosamine.So far, mutations in two of these genes have been linked to geneticdisorders with intuitive relevance to altered glucose metabolism.Heterozygous mutations in SLC2A1 cause a defect of glucose transportinto the brain, resulting in an epileptic encephalopathy with lowspinal-fluid glucose levels6. Homozygous mutations in SLC2A2 havebeen shown to cause Fanconi-Bickel syndrome, characterized byhepatorenal glycogen accumulation, nephropathy and diarrhea7,whereas heterozygous mutations in this gene result in non–insulindependent diabetes mellitus8,9.

We report that loss-of-function mutations in a third member of theSLC2A family, SLC2A10, cause arterial tortuosity syndrome (ATS;OMIM 208050), an autosomal recessive condition1 characterized bytortuosity of the large and medium-sized arteries (Fig. 1a), oftenresulting in death at young age. Other typical features includeaneurysms of large arteries and stenosis of the pulmonary artery, inassociation with facial features (Fig. 1b) and several connective tissuemanifestations. Histopathological findings include fragmentation ofthe elastic fibers in the tunica media of the large arteries (Fig. 1c)10–13.Previously, homozygosity mapping in 21 members of two consangui-neous families with ATS originating from Morocco (family 1) andItaly (family 4; Fig. 1d) assigned the gene to chromosome 20q13.1(ref. 2). Subsequently, this localization was confirmed in four smallerfamilies originating from Morocco (families 2 and 3) and the MiddleEast (family 5 and 6). Key recombinants delineated a candidate linkageinterval of 4.1 Mb between markers D20S836 and D20S109. Weperformed further fine mapping in three families (families 1–3)originating from the same region in Morocco, under the assumptionthat one recessive ancestral mutation might have caused ATS in thesefamilies. Families 1 and 2, but not family 3, shared haplotypes betweenmarkers D20S888 and mSAT11 (Fig. 1e), a region of 1.2 Mb contain-ing seven genes (SLC13A3, TP53RK, SLC2A10, EYA2, PRKCBP1,NCOA3, SULF2) and one pseudogene (RPL35AP). We sequencedthese genes directly and identified homozygous mutations (deletion,nonsense, missense) in the SLC2A10 gene in all six families (Fig. 2a,b).In families 1 and 2, we found a homozygous nonsense mutation510G-A (W170X) in all clinically affected individuals. All affectedindividuals in families 3 and 4 were homozygous for frameshiftmutations 961delG (V321fsX391) and 1334delG (G445fsX484),respectively. Both mutations result in a premature stop codon. Theaffected individuals from families 5 and 6 shared the same homo-zygous missense mutation, 243C-G (S81R). Both families had acommon haplotype between markers mSAT1 and mSAT7, indicating a

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Received 13 October 2005; accepted 13 February 2006; published online 19 March 2006; doi:10.1038/ng1764

1Center for Medical Genetics, Ghent University, B-9000 Ghent, Belgium. 2Department of Clinical Genetics, Erasmus University Medical Center, 3015 GD Rotterdam,The Netherlands. 3Division of Biology and Genetics, Department of Biomedical Sciences and Biotechnology, University of Brescia, Brescia 25123, Italy. 4Departmentof Pediatrics, North Shore University Hospital, Manhasset, New York 11030, USA. 5Yale University School of Medicine, New Haven, Connecticut 06510, USA.6Department of Pathology, University of Brescia, Brescia, Italy. 7GENDIA, B-2000 Antwerp, Belgium. 8Department of Pathology, Ghent University, B-9000 Ghent,Belgium. 9McKusick-Nathans Institute of Genetic Medicine and Howard Hughes Medical Institute, Johns Hopkins School of Medicine, Baltimore 21205, Maryland,USA. Correspondence should be addressed to P.J.C. ([email protected]).

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Results 111

founder mutation in these families (data not shown). We assume thatthe latter mutation causes disease on the basis of the followingarguments: (i) Ser81 is evolutionarily strictly conserved in GLUT10(Fig. 2b); (ii) an uncharged amino acid is changed to a positivelycharged amino acid in the third transmembrane domain and (iii) themutation was absent in 200 control chromosomes. All parents ofaffected individuals (in families 1–6) were heterozygous for therespective mutations.

The presence of homozygous loss-of-function mutations in at leastfour ATS families identifies SLC2A10 as the gene responsible for ATS.The gene contains five exons and encodes GLUT10, a 541-residueglucose transporter14–16. Human GLUT10 has been shown to facilitateD-glucose, D-galactose and 2-deoxy-D-glucose transport whenexpressed in Xenopus laevis oocytes4. GLUT10 is an outlier withinthe GLUT family because of its longer exofacial loop and differencesin motif characteristics for glucose transporters, suggesting thatGLUT10 may have additional functions, different from other GLUTfamily members4,5.

Tissue expression analysis has uncovered a widespread distributionof SLC2A10 mRNA, mainly in liver, pancreas and adipose tissue4,5,17.We studied mRNA and protein expression of GLUT10 in culturedskin fibroblasts and vascular smooth muscle cells (VSMCs) from

individuals affected with ATS and from controls. Quantitative PCR(Q-PCR) of samples derived from individuals with ATS homozygousfor premature stop codon mutations demonstrated a near-absence ofSLC2A10 mRNA in VSMCs as well as in fibroblasts (Fig. 2c), asexpected by virtue of clearance of mutant transcripts by the nonsense-mediated mRNA decay (NMD) pathway. We observed normalSLC2A10 mRNA expression in samples derived from an individualwith ATS homozygous for the 243C-G missense mutation. Weobserved (peri)nuclear localization of GLUT10 in normal individuals,but there was no detectable GLUT10 signal in individuals with ATS,as shown by immunofluorescence analysis of cultured skin fibro-blasts and VSMCs (Fig. 2d). An additional argument to suggesta nuclear localization of GLUT10 is the low dissociation constant(Km ¼ 0.3 mM), which is compatible with the glucose concentrationin the cytoplasm4.

The SLC2A10 gene has previously been considered as a candidategene for diabetes because of its function in glucose transport and itsmap position, which coincides with a type 2 diabetes locus4,5.However, a causal role for SLC2A10 in diabetes has not been demon-strated18,19. Theoretically, homozygous mutations in SLC2A10 couldlead to ATS, whereas heterozygous mutations could lead to diabetes,analogous to the situation where homozygous mutations in SLC2A2

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20p13

20p12.320p12.2

20p12.1

20p11.2320p11.2220p11.2120p11.1

4.1

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D20S888µSAT1µSAT2µSAT3µSAT4µSAT5µSAT6µSAT7µSAT8

µSAT9µSAT10

µSAT11

D20S891

D20S109

D20S197

20q11.120q11.2120p11.2220q11.23

20q12

20q13.1120q13.12

20q13.13

20q13.220q13.3120q13.3220q13.33

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Figure 1 Clinical anomalies in ATS and pedigrees. (a) MR angiography showing typical arterial tortuosity of the cerebral arteries in an individual with

ATS (IV:4 in family 1) in comparison with a healthy aged-matched control. (b) Typical facial phenotype with micrognathia, elongated face, down-slanting

palpebral fissures, blepharophimosis and a beaked nose (individual IV:4 in family 1). (c) Organization of elastic fibers in a control and in an individual with

ATS, as shown by orcein staining. Aorta elastic laminae in the media of an individual with ATS are coarser, less abundant and more disorganized than in

control aorta. Magnification: 400�. (d) Pedigree structure of the six ATS families. Symbols: circle, female; square, male; open symbol, unaffected; filled

symbol, affected; slash line, deceased; double relationship line, consanguinity. Asterisks indicate that DNA, fibroblasts or both are available. (e) Ideogram

of chromosome 20 showing the initial linkage interval, the final candidate region and haplotypes for chromosome 20q13.1 markers in the candidate region

in families 1–3.

2 ADVANCE ONLINE PUBLICATION NATURE GENETICS

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lead to Fanconi-Bickel syndrome7 and heterozygous mutations todiabetes8. However, this hypothesis is unlikely, given that we did notobserve an increased frequency of diabetes in the heterozygotes fromthe ATS families.

There is substantial phenotypic overlap between ATS and a newlyidentified genetic condition called Loeys-Dietz syndrome (LDS;OMIM 609192) that associates arterial tortuosity with aneurysmformation3. Other findings in common between the two conditionsinclude arachnodactyly, joint laxity or contractions, microretro-gnathia, hypertelorism, cleft palate and/or bifid uvula (Table 1).LDS is caused by heterozygous loss-of-function mutations in thegenes encoding the type 1 or type 2 TGFb receptors (TGFBR1 orTGFBR2). This leads to a paradoxical increase in TGFb signalingin the arterial wall, as evidenced by increased phosphorylationand nuclear translocation of Smad2 (pSmad2), a downstream effectorof the TGFb signaling pathway, and increased expression of down-stream targets of TGFb such as connective tissue growth factor(CTGF) and collagens3. Because of the clinical overlap with LDS,we investigated whether the TGFb pathway is involved in thepathogenesis of ATS.

Immunostaining for pSmad2 and CTGF (Fig. 3a) in the arterialwall of an individual with ATS demonstrated increased signal intensitycompared with control specimens, similar to the increase observed inindividuals with LDS3. In agreement with the in vivo observations, Q-PCR measurements showed a significantly higher steady-state mRNAexpression level for CTGF (Fig. 3b) in cultured VSMCs of theindividual with ATS compared with controls (P o 0.05), indicativeof upregulation of TGFb signaling.

The mechanisms by which mutations in SLC2A10 lead to TGFbactivation are unclear. Notably, the expression of decorin, a proteogly-can inhibitor of TGFb signaling20, is regulated by a defined glucoseresponse element in its gene promoter21. Therefore, we studied theexpression of decorin in cultured VSMCs of individuals with LDS,individuals with ATS and controls (Fig. 4). Decorin expression wasseverely reduced in cultured VSMCs of individuals with ATS as com-pared with controls, as shown by immunofluorescence staining(Fig. 4a). Q-PCR experiments using VSMCs confirmed the reducedexpression of decorin mRNA in individuals with ATS (Fig. 4b). Thespecific decrease of decorin expression in individuals with ATS, incontrast to individuals with LDS, might indicate divergent mechanisms

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Family 1 (IV:4)510G→A (W170X)

Family 2 (V:3)510G→A (W170X)

Family 3 (IV:3)961delG (V321fs)

Family 4 (IV:5)1334delG (G445fs)

Family 5 (IV:5)243C→G (S81R)

Family 6 (II:1)243C→G (S81R)

Membrane

Exofacial

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H. sapiens SLC2A10C. familiaris slc2a10M. musculus slc2a10R. norvegicus slc2a10G. gallus slc2a10X. tropicalis slc2a10D. rerio slc2a10T. negroviridis slc2a10

VSMCs

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4.III:2 4.III:3 4.III:4

Control 4.IV:1 Control 5.IV:5 1.IV:4

a

c

d

b

Figure 2 SLC2A10 (GLUT10) mutation and expression data. (a) SLC2A10 mutations identified in families 1–6. (b) Location of GLUT10 mutations at a

schematic representation of the GLUT10 protein. GLUT10 contains 12 hydrophobic transmembrane domains (ovals) with a hydrophilic endofacial loop

between transmembrane domains 6 and 7 and a large exofacial loop containing a potential N-linked glycosylation site between transmembrane domains

9 and 10. Evolutionary conservation of the substituted amino acid observed in families 5 and 6 in GLUT10 is shown. (c,d) SLC2A10 (GLUT10) expression

in the control and in individuals with ATS. (c) mRNA expression of SLC2A10 as determined by Q-PCR in VSMCs and skin fibroblasts. In VSMCs in

individuals with ATS, the level of mRNA was severely reduced. In skin fibroblasts, the individual carrying a homozygous nonsense mutation also showed

a significant reduction (P o 0.05) compared with the control, but the individual homozygous for a missense mutation did not show any reduction. Bars

indicate the 95% confidence interval of the mean expression level. (d) Immunofluorescence analysis of GLUT10 in VSMCs and skin fibroblasts. Expression

of GLUT10 was nearly absent in VSMCs and fibroblasts from individuals with ATS, as compared with the control. The fluorescence signal in heterozygous

individuals was approximately half that of the controls. Magnification: 1,000�.


LET TERS

Results 113

for upregulation of TGFb signaling in these two conditions. Notably,we did not observe any differences in expression between fibroblasts ofindividuals with ATS and those of healthy controls (data not shown).Given the (peri)nuclear localization of GLUT10, a decrease in intra-cellular glucose and failure of glucose-mediated transcriptional upre-gulation of the decorin promoter seems to be unique to ATS. Incontrast, primary alterations in the TGFb receptors lead to increasedTGFb signaling in LDS. In order to determine the specificity ofthe decorin response, we monitored the expression of versican, a

proteoglycan that is known not to be strongly regulated by glucose butwhose expression is driven by TGFb22. As predicted from ourpathogenetic model, samples from patients with either ATS or LDSshowed increased expression of versican (Fig. 4c). An inhibitory roleof versican on elastic fiber assembly has been proposed23, perhapsproviding a mechanism for failed elastogenesis in both ATS and LDS.

Although TGFb signaling is disturbed in cells and connective tissuederived from individuals with ATS, other mechanisms leading toabnormal matrix deposition cannot be excluded. Impaired uptake ortransport of other monosaccharides could hinder glycosylation eventsimportant for the production of mature glycoproteins and proteogly-cans, essential structural components of the arterial wall and con-nective tissue in general.

We were surprised to identify a glucose transporter gene responsiblefor a connective tissue disorder. No other connective tissue disorder,with the exception of pseudoxanthoma elasticum (caused by a muta-tion in ABCC6), is known to be caused by a transporter proteindefect24. No clear function of the ABCC6 transporter has been identi-fied, and the underlying pathogenic mechanism leading to disturbedelastin homeostasis in PXE is unknown. Insights derived from the studyof ATS may prove relevant to other disorders related to failed intra-cellular transport of glucose. Indeed, the microangiopathic changes andfibrosis seen in diabetic retinopathy, nephropathy and peripheralvascular disease correlate with increased TGFb signaling25. Diabetes-associated arteriolar tortuosity is seen in tissues undergoing postnatalangiogenesis, including the retinal and coronary microcirculations,perhaps recapitulating events occurring on a broader scale duringembryogenesis in ATS. These data suggest that antagonism of TGFbsignaling may contribute to therapeutic advances in a wide variety ofgenetically determined and acquired disorders, including ATS.

METHODSPatients. Appropriate informed consent, including specific consent to publish

the photos in Figure 1b, was obtained from all patients involved in the study.

Detailed clinical descriptions of five ATS families, except for family 6, have been

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ATS patient 5.IV:5 Control 1 Control 2 Control 3

pSmad2

CTGF

5

4

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2

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0Control 1 Control 2 ATS

patient 4.IV:1

Exp

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a

b

Figure 3 Immunostaining and Q-PCR analysis for phosphorylated Smad2and CTGF in arterial tissue. (a) Three controls and individual 5.IV:5 with

ATS were examined. Note the increased intensity of nuclear phosphorylated

Smad2 and extracellular CTGF. Scale bars, 10 mm. (b) Expression levels of

CTGF in VSMCs of affected individual IV:1 of family 4 and two controls in

steady-state conditions, as measured by Q-PCR. CTGF shows fourfold higher

expression in the individual with ATS. Bars indicate the 95% confidence

interval of the mean expression level.

Table 1 Clinical comparison of individuals with ATS and individuals

with LDS

Symptoms ATS LDS

Arterial anomalies

Tortuosity +++ +++

Aneurysms + +++

Stenosis a. pulmonalis ++ 0

Aneurysm a. pulmonalis + ++

Skin laxity +++ +

Skeletal anomalies

Contractures ++ ++

Pectus deformity + ++

Joint laxity +++ +++

Arachnodactyly + ++

Facial anomalies

Hypertelorism + +++

Cleft palate, bifid uvula ++ +++

Microretrognathia +++ ++

0, not described; +, present; ++, common; +++, typical

454035302520151050

Control 1

Control 1

8

7

6

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Control 2 LDS

Control 2 LDS ATS 4.IV:1

ATS 4.IV:1

Control

4.IV:1

ba

c

Figure 4 Immunofluorescence and Q-PCR analysis of decorin and versican

in VSMC. (a) In individual 4.IV:1 with ATS, expression of decorin is nearlyabsent, as compared with control VSMCs. Magnification: 1,000�.

(b,c) Q-PCR analysis for (b) decorin and (c) versican in VSMCs of an

individual with ATS compared with an individual with LDS and two controls.

The expression of decorin mRNA in the individual with ATS is significantly

lower (P o 0.05) than in the LDS patient and the controls, whereas

the expression of versican is significantly higher (P o 0.05) in the

individual with ATS and the individual with LDS compared with controls.

Bars indicate the 95% confidence interval of the mean expression level.


LET TERS


published previously1,12,26. DNA was extracted from peripheral blood and/or

cultured skin fibroblasts. Skin fibroblasts were cultured in Dulbecco’s modified

Eagle’s medium (DMEM) supplemented with 15% fetal bovine serum (FBS) in

the presence of antibiotics. Vascular smooth muscle cells (VSMCs) were

obtained from the aortic media from individual IV:1 with ATS in family 4

and from an individual with LDS (TGFBR1 mutation, R487P)3 and were

cultured in smooth muscle basal medium supplemented 5% FBS, 0.2%

fibroblast growth factor (FGF), 0.1% insulin, 0.1% epidermal growth factor

(EGF), and 0.1% gentamicin sulfate/amphotericin. Paraffin-embedded aortic

tissue was available only from individual IV:5 in family 5.

Microsatellite and sequence analysis. Microsatellite markers in the ATS

linkage region on chromosome 20q13.1 (ref. 2) were taken from the Marshfield

map or designed based on the simple tandem repeat finder in the University of

California Santa Cruz genome browser (mSAT1-11). We carried out genotyping

on an Applied Biosystems Prism 3100 Genetic Analyzer (Applied Biosystems).

The data were processed using Genescan software (Applied Biosystems).

We amplified all coding exons from all seven genes in the ATS linkage

region (SLC13A3, TP53RK, SLC2A10, EYA2, PRKCBP1, NCOA3, SULF2) by

PCR using intronic primers and additional exonic primers for larger

exons (Supplementary Table 1 online). Sequencing was performed using the

BigDye v3.1 ET terminator cycle sequencing kit (Applied Biosystems).

Sequencing reactions were loaded onto an Applied Biosystems Prism 3100

Genetic Analyzer.

Immunostaining. For GLUT10 and decorin analysis in VSMCs and skin

fibroblasts by immunofluorescence microscopy, we grew 1 � 105 VSMCs or

skin fibroblasts from controls and from individuals with ATS for 72 h in

complete medium. To analyze GLUT10, the cells were washed in PBS,

permeabilized in 0.5% Triton X-100 and 3% paraformaldehyde for 2 min,

fixed for 20 min in 3% paraformaldehyde, incubated for 30 min at room

temperature (21 1C) with 5% BSA/PBS and incubated overnight at 4 1C with

20 mg ml–1 polyclonal antibody to GLUT10 (Alpha Diagnostic). To analyze

decorin levels, the cells were fixed in 3% paraformaldehyde for 10 min, washed

twice for 5 min in PBS and incubated for 40 min at room temperature (21 1C)

with 20 mg ml�1 monoclonal antibody to decorin (clone 115402, R&D

Systems). Next, the VSMCs and fibroblasts were incubated for 1 h at room

temperature (21 1C) with rhodamine-conjugated anti-rabbit IgG (1:50 in 1%

BSA/PBS) and anti-mouse IgG (1:100 in 1% BSA/PBS), respectively;

washed in PBS; mounted in 1:1 PBS-glycerol solution on glass slides and

photographed with a Zeiss Axiovert 10S/H fluorescence microscope. Quanti-

tative evaluation of the fluorescence was performed as previously reported27.

For GLUT10, quantitative evaluation was repeated on 20 randomly selected

cells for each cell strain. Images were digitized to measure the cell and the

nuclear areas and the integrated optical density (IOD) corresponding to the

fluorescence signals.

For immunohistochemical staining for CTGF and pSmad2 in arterial tissue,

we selected representative specimens of formalin-fixed, paraffin-embedded

arterial media of three healthy control individuals and one individual with

ATS. From these specimens, 5-mm thick paraffin sections were cut, deparaffi-

nized and rehydrated. These tissues were pretreated with a protease-1 enzy-

matic solution (Ventana). For immunohistochemical analysis, we used

antibodies directed against pSmad2 and CTGF (Alpha Diagnostic, Cell Signal-

ing Technology and Abcam, respectively) and previously described methods3.

Light microscopy was performed on an Olympus BX45 microscope.

Q-PCR. RNA was isolated using the RNeasy Mini Kit (Qiagen), and cDNA was

synthesized using SuperScript II Reverse Transcriptase Kit with random

hexamer primers (Invitrogen) in a total volume of 20 ml. Two microliters of

cDNA (1:10 dilution) and 250 nM gene-specific primers were used with the

Q-PCR Core Kit for SYBR Green I (Eurogentec) for Q-PCR on a GeneAmp

5700 Sequence Detector (Applied Biosystems). The Q-PCR program consisted

of 40 cycles with 15 s at 95 1C and 1 min at 60 1C, followed by a dissociation

run to determine melting curves. We carried out all reactions in duplicate and

normalized them to the geometric mean of three reference genes (GAPDH,

HPRT1 and YWHAZ). We used fibroblasts from controls and individuals with

ATS and VSMCs, obtained from aortic media from two controls, from

individual IV:1 with ATS (from family 4) and from an individual with LDS3.

We grew cells in 6-cm dishes to 80% confluence. Expression levels were

determined in three independent experiments for each cell line. Differential

gene expression was considered significant when the difference was at least 50%

and the 95% confidence interval of the mean expression levels did not overlap

(equivalent to P o 0.05).

Accession codes. GenBank: SLC2A10 cDNA, NM_030777; SLC2A10 coding

region, NT_011362.

Note: Supplementary information is available on the Nature Genetics website.

ACKNOWLEDGMENTSWe are indebted to the families and patients for their interest and cooperation.We thank C.E. Catsman-Berrevoets for referring family 1. We thank P. Van Ackerand L.A. Myers for technical assistance. This study was supported by the Fundfor Scientific Research, Flanders (Belgium); Ghent University; the Ministerodell’Istruzione, dell’Universita e della Ricerca, Fondo per gli Investimenti dellaRicerca di Base 2001; Fondazione Cariplo; Fondazione Berlucchi; the WilliamSmilow Center for Marfan Syndrome Research; the Howard Hughes MedicalInstitute and the US National Institutes of Health. B.C. and B.L. are researchassistant and senior clinical investigator, respectively, and are supported bythe Fund for Scientific Research, Flanders. J.D.B. is a research fellow fromGhent University.

COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests.

Published online at http://www.nature.com/naturegenetics

Reprints and permissions information is available online at http://npg.nature.com/

reprintsandpermissions/

1. Wessels, M.W. et al. Three new families with arterial tortuosity syndrome. Am. J. Med.Genet. A 131, 134–143 (2004).

2. Coucke, P.J. et al. Homozygosity mapping of a gene for arterial tortuosity syndrome tochromosome 20q13. J. Med. Genet. 40, 747–751 (2003).

3. Loeys, B.L. et al. A syndrome of altered cardiovascular, craniofacial, neurocognitive andskeletal development caused by mutations in TGFBR1 or TGFBR2. Nat. Genet. 37,275–281 (2005).

4. Dawson, P.A. et al. Sequence and functional analysis of GLUT10: a glucose transporterin the Type 2 diabetes-linked region of chromosome 20q12–13.1. Mol. Genet. Metab.74, 186–199 (2001).

5. McVie-Wylie, A.J., Lamson, D.R. & Chen, Y.T. Molecular cloning of a novel memberof the GLUT family of transporters, SLC2a10 (GLUT10), localized on chromo-some 20q13.1: a candidate gene for NIDDM susceptibility. Genomics 72, 113–117(2001).

6. Seidner, G. et al. GLUT-1 deficiency syndrome caused by haploinsufficiency of theblood-brain barrier hexose carrier. Nat. Genet. 18, 188–191 (1998).

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10. Ertugrul, A. Diffuse tortuosity and lengthening of the arteries. Circulation 36,400–407 (1967).

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14. Barrett, M.P., Walmsley, A.R. & Gould, G.W. Structure and function of facilitative sugartransporters. Curr. Opin. Cell Biol. 11, 496–502 (1999).

15. Joost, H.G. & Thorens, B. The extended GLUT-family of sugar/polyol transport facil-itators: nomenclature, sequence characteristics, and potential function of its novelmembers (review). Mol. Membr. Biol. 18, 247–256 (2001).

16. Scheepers, A., Joost, H.G. & Schurmann, A. The glucose transporter families SGLT andGLUT: molecular basis of normal and aberrant function. JPEN J. Parenter. EnteralNutr. 28, 364–371 (2004).

17. Wood, I.S., Hunter, L. & Trayhurn, P. Expression of Class III facilitative glucosetransporter genes (GLUT-10 and GLUT-12) in mouse and human adipose tissues.Biochem. Biophys. Res. Commun. 308, 43–49 (2003).

18. Andersen, G. et al. Genetic variation of the GLUT10 glucose transporter (SLC2A10)and relationships to type 2 diabetes and intermediary traits. Diabetes 52, 2445–2448(2003).

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Publication 5

Absence of arterial phenotype in mice with homozygous slc2A10 missense

substitutions.

Callewaert BL, Loeys BL, Casteleyn C, Willaert A, Dewint P, De Backer J,

Sedlmeier R, Simoens P, De Paepe AM, Coucke PJ.

Genesis 2008 Aug;46(8):385-9.

The goal of this study was to create and characterize a mouse model for ATS. Due to

the limited availability of human ATS tissues, an established mouse model would be a

great help in the study of the pathophysiology of the disease.

Results 117

LETTER

Absence of Arterial Phenotype in Mice WithHomozygous slc2A10 Missense SubstitutionsB.L. Callewaert,1* B.L. Loeys,1 C. Casteleyn,2 A. Willaert,1 P. Dewint,3 J. De Backer,1

R. Sedlmeier,4 P. Simoens,2 A.M. De Paepe,1 and P.J. Coucke11Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium2Department of Morphology, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium3Department of Rheumatology, Ghent University Hospital, Ghent, Belgium4Ingenium Pharmaceuticals AG, Martinsried, Germany

Received 18 April 2008; Revised 23 May 2008; Accepted 26 May 2008

Summary: Arterial tortuosity syndrome (ATS, MIM#208050) is a rare autosomal recessive connective tissuedisease, mainly characterized by widespread arterialinvolvement with elongation, tortuosity, and aneurysmsof the large and middle-sized arteries (Callewaert et al.,2008, Hum Mutat 29:150–158). Recently, mutations wereidentified in the SLC2A10 gene encoding the facilitativeglucose transporter GLUT10 (Coucke et al., 2006, NatGenet 38:452–457). It was hypothesized that loss-of-func-tion of the transporter results in upregulation of the trans-forming growth factor beta (TGFb) signaling pathway(Coucke et al., 2006, Nat Genet 38:452–457). We antici-pated that a mouse model would help to gain moreinsight in the complex pathophysiological mechanism ofhuman ATS. Here, we report that two mouse models,homozygous respectively for G128E and S150F missensesubstitutions in glut10 do not present any of the vascular,anatomical, or immunohistological abnormalities asencountered in human ATS patients. We conclude thatthese mouse strains do not phenocopy human ATS andcannot help the further elucidation of pathogenetic mech-anisms underlying this disease. genesis 46:385–389,2008. VVC 2008 Wiley-Liss, Inc.

Key words: arterial tortuosity syndrome; mouse model;slc2a10; glut10; vascular corrosion casting

Genetically modified animal models are highly effectivetools to study gene function, molecular biologic distur-bances, clinical course, and therapeutic strategiesin vivo and in vitro (Capecchi, 2005). They become ofparticular interest when the human disease is rare, rele-vant patient tissues scarce, the gene product unex-pected, and the pathogenesis obscure. Specifically, inthese circumstances, mouse models have proven to playa key role in unraveling the pathogenic mechanisms inmany diseases. A prominent example in the field of con-nective tissue diseases are the Marfan mouse models (Pe-reira et al., 1997, 1999). Fbn1 knock-out and hypomor-phic mice models faithfully recapitulate the human Mar-fan syndrome phenotype (MFS, MIM#154700) (Pereira

et al., 1997, 1999) and have led to a better understand-ing of extracellular matrix organization and function. Amajor breakthrough was realized when these models ledto insights in the essential role of transforming growthfactor beta (TGFb) signaling in vascular tissues. Thisresulted in new therapeutic strategies for aortic dilation(Habashi et al., 2006).

Arterial tortuosity syndrome (ATS, MIM#208050) is arare autosomal recessive connective tissue disorder,characterized by severe tortuosity, stenosis, and aneur-ysms of the aorta and middle-sized arteries (Callewaertet al., 2008; Wessels et al., 2004). Recently, the underly-ing genetic defect has been identified as bi-allelic loss-of-function mutations in the SLC2A10 gene, encoding thefacilitative glucose transporter GLUT10 (Coucke et al.,2006). Although it was a surprising finding that a glucosetransporter defect is responsible for this rare connectivetissue disorder, additional studies indicated that upregu-lation of the TGFb signaling pathway, like in MFS, has akey role in the vascular malformations in this condition(Coucke et al., 2006). Immunofluorescence staining sug-gests that GLUT10 is residing on the nuclear membrane.It is hypothesized that loss-of-function of the transporterresults in diminished glucose responsive transcription ofdecorin, a known inhibitor of the TGFb signaling path-way leading to an enhanced expression of TGFb respon-sive elements, such as connective tissue growth factorand versican (Coucke et al., 2006).

Current evidence supports the idea that upregulationof the TGFb signaling pathway is a common observation

*Correspondence to: Bert L. Callewaert, Center for Medical Genetics,

Ghent University Hospital, Medical Research Building, De Pintelaan 185,

B-9000 Ghent, Belgium.

E-mail: [email protected]

Contract grant sponsor: Scientific Research-Flanders; Contract grant num-

ber: G.0094.06; Contract grant sponsor: Ghent University; Contract grant

number: GOA 12051203Published online 8 August 2008 in

Wiley InterScience (www.interscience.wiley.com).

DOI: 10.1002/dvg.20409

' 2008 Wiley-Liss, Inc. genesis 46:385–389 (2008)


in different aortic aneurysm diseases such as Marfan syn-drome (Neptune et al., 2003), Loeys-Dietz syndrome(LDS, MIM#609192) (Loeys et al., 2005), and Fibulin 4(FBLN4) deficiency (Hanada et al., 2007). LDS combinesarterial aneurysms and tortuosity with widespread sys-temic involvement and is caused by mutations in thegenes encoding the TGFb receptors 1 and 2 (TGFBR1and TGFBR2) (Loeys et al., 2006). FBLN4-deficient statesin humans and mice lead to arterial aneursyms and tortu-osity as well as other connective tissue abnormalities(Hucthagowder et al., 2006; McLaughlin et al., 2006).We created ATS mouse models as the next logical step inthe study of the role of glut10 in the homeostasis of theextracellular matrix and in arterial development.

We generated heterozygous and homozygous mice onthe C3HeB/FeJ genetic background, which carry aG128E or S150F substitution in slc2a10, respectively.The causal nature of these mutations is predicted by thefollowing findings: (1) both induce major amino acidalterations: the G128E substitution replaces a small,uncharged glycine with a bulky negatively charged glu-tamic acid residue in the fourth transmembrane domainof glut10 (Grantham score 87) (Grantham, 1974),whereas the S150F substitution replaces a small, polarserine residue by a bulky, hydrophobic phenylalanineresidue in the fifth transmembrane domain of glut10(Grantham score 155) (Grantham, 1974); (2) both muta-tions are predicted to be deleterious in their respectivepositions according to slc2a10 analysis with SIFT soft-ware (http://blocks.fhcrc.org/sift/SIFT.html) (Ng andHenikoff, 2003); (3) both affected amino acid residuesare conserved through evolution; (4) both mutations areabsent in 200 human control chromosomes; and (5) andfinally, a G246E mutation, which is located in a trans-membrane region, similar to the G128E alteration, hasbeen reported in a patient with ATS (Callewaert et al.,2008).

All comparative studies on wild-type, heterozygous,and homozygous mice were done on litter mates of eachstrain. Mice were born with expected Mendelian fre-quency and grew without lethality or significant morbid-ity to at least 5 months of age. All experiments were car-ried out on two animals per genotype at 5 months ofage. The gross appearance and skin features were similarin wild-type (WT), heterozygous, and homozygous micefor both the G128E and S150F substitution (data notshown). In vivo, ultrasound of the abdominal aortashowed straight aortas of normal caliber (Fig. 1a) (WT:0.45 mm, G128E: 0.35 mm; S150P: 0.33 mm). Local sur-gical exploration of the right cervical region in thehomozygous mice demonstrated a normally developedtruncus brachiocephalicus and carotid artery withouttortuosity, dilation, stenoses, or abnormal branchingfrom the aorta (data not shown).

We subsequently applied vascular corrosion casting inorder to evaluate the complete arterial tree. Unlikehuman subjects harboring bi-allelic SLC2A10 mutationswho all demonstrate kinking or tortuosity of the aortaand large arteries (Callewaert et al., 2008; Wessels et al.,

2004), no arterial abnormalities could be demonstratedwith this technique comparing wild-type, heterozygous,and homozygous mice (Fig. 1b). Finally, elastin stainingof the tail and popliteal artery in both mice strains didnot reveal any fragmentation of elastic fibers in the lam-ina elastica interna or the tunica media in contrast to aor-tic histology described in human ATS (Coucke et al.,2006; Pletcher et al., 1996) (Fig. 2).

Mice harboring homozygous G128E and S150F substi-tutions in glut10 therefore do not recapitulate thehuman ATS phenotype and fail to represent a goodworking model to study the disorder. Although we veri-fied the absence of these variants in 200 human controlchromosomes and in silico predictions do indicate afunctional effect, it might still be possible that they onlyrepresent rare polymorphisms. However, it is not un-precedented that mouse models do not recapitulatehuman phenotypes. For instance, fbn2 knock-outs donot present with the typical clinical characteristics ofcongenital contractural arachnodactyly (Chaudhry et al.,2001), a rare connective tissue disorder in humans, char-acterized by external ear anomalies, arachnodactyly, con-tractures, and scoliosis. Several mechanisms couldexplain the lack of an arterial phenotype in glut10 defi-cient mice: (1) the close relationship between facilitativeglucose transporters may result in functional redun-dancy (Scheepers et al., 2004); (2) the genetic back-ground of the C3HeB/FeJ strain used in these experi-ments may decrease the penetrance of the phenotype(Banbury Conference on genetic background in mice,1997); (3) vasculogenesis in mice might be regulated bymechanisms independent of normal glut10 function.

In conclusion, G128E and S150F substitutions inglut10 do not mimic the human ATS phenotype in miceand fail to represent a good animal model to study thehuman disorder.

METHODS

Mice

Mice were generated as described previously (Augustinet al., 2005). In brief, seven slc2a10 mutations that leadto an amino acid substitution in the polypeptide chainwere found upon screening of a mutant mice library gen-erated following N��ethyl��N��nitrosurea (ENU) muta-genesis in healthy C3HeB/FeJ males. Two nucleotidealterations, c.383G>A and c.449C>T, respectively caus-ing substitutions G128E and S150F, were most likely dis-rupting protein function and were selected to generatehomozygous mice.

In vivo Analysis

Mice were anesthetized with intraperitoneal injectionof 2 mg Ketamine and 0.1 mg Xylazine using a tuberculinsyringe (1 ml) and a 25 gauche needle. Ultrasound of theabdominal aorta was performed using a Vivid i Portableecho system (GE Healthcare) with a Sector Probe (10SRS 4.5–11.5 MHz). Next, the right cervical region was

386 CALLEWAERT ET AL.

Results 119

dissected under aseptic conditions and the common andexternal carotid artery was visualized from the truncuscommunis till the skull basis. Incisions were sutured andmice were kept apart for 7 days to ensure thoroughwound healing.

Vascular Corrosion Casting

Following 24 h food restriction, mice were sacrificedby CO2 asphyxiation. The abdominal aorta was dissectedthrough a large median incision and catheterized with a26 gauche catheter (Terumo reference SR1DU2619PX).For each cast, fresh Batson solution was prepared usingthe Batson’s #17 corrosion kit (Omnilabo International,Aartselaar, Belgium), and consisted of 5 ml base mono-mer, 0.75 ml catalyst, two drops of promoter, and a traceof red pigment. Two ml Batson solution was injectedthrough the catheter using 1 ml tuberculin syringes.Mouse bodies were immersed for 30 min in tepid water

to ensure polymerization and macerated overnight in25% potassium hydroxide (KOH). The vascular corrosioncasts were rinsed gently for 3 h with a warm stream ofwater. Blood vessels were evaluated and photographedwith a dissecting microscope (Wild M7A, Wild Heer-brugg, Switzerland) equipped with a charge-coupled de-vice camera (Olympus DP50, Olympus Belgium).

Histologic Examination

For histology, after sacrificing the mice, the tail andleft hind paw were fixed for 24 h in 4% paraformalde-hyde in 0.1 M phosphate buffer (pH 7.2), dehydrated,and embedded in paraffin wax. Eight micrometer sec-tions were stained with hematoxylin (Merck KGaA, Ger-many) and eosin (Eosine yellow, VWR international, Bel-gium), orcein (Acetic orcein, Klinipath, The Nether-lands), and van Gieson (VWR International, Belgium) toevaluate the general conformation, elastic fibers, and

FIG. 1. (a) Representative ultra-sound images of the abdominalaorta of wild type mice and micehomozygous for the G128E andS150F mutation. Diameters of theabdominal aorta (at the level ofthe renal arteries) are indicated.(b) Vascular corrosion casting ofwild-type mice and mice homozy-gous for the G128E and S150Fmutation. Representative imagesof the aortic arch and its side-branches, the circulus of Willisand the retinal arteries are shown.

387ABSENCE OF ARTERIAL PHENOTYPE IN MICE


connective and smooth muscular tissue of the arteries.Tissue sections were examined with a motorized micro-scope (Olympus BX 61, Olympus Belgium) linked to adigital camera (Olympus DP 50, Olympus Belgium).

For all procedures, the ‘‘Principles of laboratory ani-mal care’’ (NIH publication No. 86-23, revised 1985)were followed. In addition, all procedures wereapproved by the Ghent University Committee (ECD 07/20) on laboratory animal tests.

ACKNOWLEDGMENTS

The authors are indebted to L. De Bels, L. Standaert enJ. Weytens for technical assistance. BC and BL are respec-tively a research fellow and senior clinical investigator ofthe Fund for Scientific Research, Flanders.

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FIG. 2. Hematoxylin and eosin and orcein staining of the tail and popliteal artery of wild type mice and mice homozygous for the G128Eand S150F mutation. No fragmentation of elastic fibers in the media or inner elastic membrane was present.

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Results 121

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389ABSENCE OF ARTERIAL PHENOTYPE IN MICE


Publication 6

Arterial tortuosity syndrome: clinical and molecular findings in 12 newly

identified families.

Callewaert BL*, Willaert A*, Kerstjens-Frederikse WS, De Backer J, Devriendt

K, Albrecht B, Ramos-Arroyo MA, Doco-Fenzy M, Hennekam RC, Pyeritz RE,

Krogmann ON, Gillessen-kaesbach G, Wakeling EL, Nik-zainal S, Francannet C,

Mauran P, Booth C, Barrow M, Dekens R, Loeys BL, Coucke PJ, De Paepe AM.

(*: joint first authors)

Hum Mutat. 2008 Jan;29(1):150-8.

This paper expands the clinical and molecular spectrum in ATS. Special attention is

paid to the initial presentation and the natural history of the disorder. In view of the

genetic defect and the proposed pathophysiology we investigated the role of GLUT10

in serum glucose homeostasis, and whether heterozygote carriers may present with

mild cardiovascular abnormalities.

Results 123

HUMANMUTATION 29(1),150^158,2008

RESEARCH ARTICLE

Arterial Tortuosity Syndrome: Clinicaland Molecular Findings in 12 NewlyIdentified Families

B.L. Callewaert,1 A. Willaert,1 W.S. Kerstjens-Frederikse,2,3 J. De Backer,1 K. Devriendt,4 B. Albrecht,5

M.A. Ramos-Arroyo,6 M. Doco-Fenzy,7 R.C.M. Hennekam,8,9 R.E. Pyeritz,10 O.N. Krogmann,11

G. Gillessen-kaesbach,12 E.L. Wakeling,13 S. Nik-zainal,14 C. Francannet,15 P. Mauran,16 C. Booth,17

M. Barrow,18 R. Dekens,1 B.L. Loeys,1 P.J. Coucke,1 and A.M. De Paepe1�

1Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium; 2Department of Medical Genetics, University Medical CenterGroningen, Groningen, The Netherlands; 3Department of Medical Genetics, University of Groningen, Groningen, The Netherlands; 4Center forHuman Genetics, University of Leuven, Leuven, Belgium; 5Institut fur Humangenetik, Universitatsklinikum Essen, Essen, Germany; 6S. GeneticaMedica, Hospital Virgen del Camino, Pamplona, Spain; 7Service de Genetique, CHRU Reims, UFR de medecine, Reims, France; 8Clinical andMolecular Genetics Unit, Institute of Child Health, London, United Kingdom; 9Department of Clinical Genetics, Academisch Medisch Centrum,Amsterdam, The Netherlands; 10Division of Medical Genetics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania;11Klinik fur Kinderkardiologie-Angeborene Herzfehler, Herzzentrum, Duisburg, Germany; 12Institut fur Humangenetik, UniversitatsklinikumSchleswig-Holstein, Lubeck, Germany; 13NW Thames Regional Genetics Service, Kennedy Galton Centre, Northwick Park and St Mark’sHospitals, Harrow, United Kingdom; 14Department of Medical Genetics, Addenbrooke’s Hospital NHS Trust, Cambridge, United Kingdom;15Service de Genetique Medicale, CHRU Clermont-ferrand, Clermont-ferrand, France; 16Service de Cardiologie Pediatrique, American MemorialHospital, CHU Reims, Reims, France; 17Genetics Department, Lutheran General Children’s Hospital, Park Ridge, Illinois; 18Departmentof Clinical Genetics, University Hospital of Leicester, NHS Trust, Leicester, United Kingdom

Communicated by Garry R. Cutting

Arterial tortuosity syndrome (ATS) is a rare autosomal recessive connective tissue disease, characterized bywidespread arterial involvement with elongation, tortuosity, and aneurysms of the large and middle-sized arteries.Recently, SLC2A10 mutations were identified in this condition. This gene encodes the glucose transporter GLUT10and was previously suggested as a candidate gene for diabetes mellitus type 2. A total of 12 newly identified ATSfamilies with 16 affected individuals were clinically and molecularly characterized. In addition, extensivecardiovascular imaging and glucose tolerance tests were performed in both patients and heterozygous carriers. All16 patients harbor biallelic SLC2A10 mutations of which nine are novel (six missense, three truncating mutations,including a large deletion). Haplotype analysis suggests founder effects for all five recurrent mutations. Remarkably,patients were significantly older than those previously reported in the literature (P 5 0.04). Only one affected relativedied, most likely of an unrelated cause. Although the natural history of ATS in this series was less severe thanpreviously reported, it does indicate a risk for ischemic events. Two patients initially presented with stroke,respectively at age 8 months and 23 years. Tortuosity of the aorta or large arteries was invariably present. Two adultprobands (aged 23 and 35 years) had aortic root dilation, seven patients had localized arterial stenoses, and five hadlong stenotic stretches of the aorta. Heterozygous carriers did not show any vascular anomalies. Glucose metabolismwas normal in six patients and eight heterozygous individuals of five families. As such, overt diabetes is not related toSLC2A10 mutations associated with ATS. Hum Mutat 29(1), 150–158, 2008. rr 2007 Wiley-Liss, Inc.

KEY WORDS: arterial tortuosity syndrome; SLC2A10; GLUT10; genotype–phenotype

INTRODUCTION

Arterial tortuosity syndrome (ATS; MIM] 208050) is a rare,autosomal recessive connective tissue disorder characterized byelongation, tortuosity, and aneurysms of the large and middle-sizedarteries. Focal stenoses of the pulmonary arteries and aorta can beassociated. Patients usually present with characteristic dysmorphicfeatures including an elongated face, blepharophimosis anddownslanting palpebral fissures, a beaked nose, a highly archedpalate, and micrognathia. Other connective tissue manifestationscomprise a soft, hyperextensible skin and skeletal abnormalitiessuch as arachnodactyly, pectus deformity, joint laxity, and

Published online 12 October 2007 in Wiley InterScience (www.interscience.wiley.com).

DOI10.1002/humu.20623

The Supplementary Material referred to in this article canbe accessed at http://www.interscience.wiley.com/jpages/1059-7794/suppmat.

Received 29 April 2007; accepted revised manuscript 9 July 2007.

Grant sponsor: Fund for Scienti¢c Research, Flanders.B.L. Callewaert and A.Willaert contributed equally to this work.

�Correspondence to: Anne De Paepe, MD, PhD,Center for MedicalGenetics, Ghent University Hospital, De Pintelaan 185, B-9000Ghent, Belgium. E-mail: [email protected]

rr 2007 WILEY-LISS, INC.


contractures [Abdul Wahab et al., 2003; Beuren et al., 1969;Ertugrul, 1967; Franceschini et al., 2000; Gardella et al., 2004;Lees et al., 1969; Meyer et al., 2005; Pletcher et al., 1996; Riveraet al., 2000; Welch et al., 1971; Wessels et al., 2004; Zaidi et al.,2005]. Patients are prone to aneurysm formation, dissections, andischemic events [Abdul Wahab et al., 2003; Ades et al., 1996; AlFadley et al., 2000; Lees et al., 1969; Pletcher et al., 1996; Welchet al., 1971; Wessels et al., 2004]. The prognosis is reported to bepoor with mortality rates up to 40% before the age of 5 years[Wessels et al., 2004].

Histopathology of affected vessel walls indicates fragmentationof the inner elastic membrane and the elastic fibers of the tunicamedia of the large arteries [Ades et al., 1996; Beuren et al., 1969;Ertugrul, 1967; Franceschini et al., 2000; Lees et al., 1969;Pletcher et al., 1996; Rivera et al., 2000; Welch et al., 1971].

Recently, the causal gene has been identified as the SLC2A10gene (MIM] 606145), which encodes the facilitative glucosetransporter GLUT10 [Coucke et al., 2006]. It is proposed thatloss-of-function of the transporter results in diminished glucoseresponsive transcription of decorin, a known inhibitor of thetransforming growth factor beta (TGFb) signaling pathway. Thisleads to upregulation of the TGFb responsive elements,connective tissue growth factor, and versican [Coucke et al.,2006], inhibiting proper extracellular matrix formation, inparticular elastogenesis [Huang et al., 2006].

There is considerable clinical and histological overlap with otherconditions associated with arterial tortuosity and arterial aneurysmsincluding the Loeys-Dietz syndrome (LDS; MIM] 609192) [Loeyset al., 2005, 2006] and some forms of recessive cutis laxa (MIM]219100) caused by mutations in the genes encoding Fibulin 4 and 5(FBLN4 (OMIM �604633) and FBLN5 (MIM�604580) [Huctha-gowder et al., 2006; Loeys et al., 2002]. Moreover, pathophysiologyseems comparable between LDS and ATS, as both evoke disturbedTGFb signaling [Coucke et al., 2006; Loeys et al., 2005].

Previously, a region on chromosome 20q13 containingSLC2A10 has been linked to type 2 diabetes mellitus [Ghoshet al., 1999; Klupa et al., 2000], and SLC2A10 was suggested as agood candidate gene [Andersen et al., 2003; Bento et al., 2005;Lin et al., 2006; McVie-Wylie et al., 2001; Mohlke et al., 2005;Rose et al., 2005]. Evaluation of glucose metabolism in patientsharboring pathogenic SCL2A10 mutations could elucidatewhether this association with diabetes mellitus exists.

We present the clinical characteristics and molecular findingsfrom 12 newly identified SLC2A10 families. We provide furtherinsights into the phenotype in both patients and carriers, andevaluate glucose metabolism.

MATERIALSANDMETHODSPatients and Additional Examinations

All patients were evaluated by a clinical geneticist at thereferring center. Imaging of the aorta and the aortic side-brancheswas performed by echocardiography, computer assisted tomogra-phy, or MRI angiography.

Oral glucose tolerance tests consisted of measuring the serumglucose and insulin levels at time 0, 30, 60, and 120 minutes afteringestion of a solution containing 75 grams of glucose. In addition,HbA1c levels were determined at time zero.

Informed consent was obtained from all subjects.

MolecularAnalysis

Genomic DNA was extracted from blood samples by standardprocedures, followed by touchdown PCR amplification of all exons of

the SLC2A10 gene using forward and reverse primers located in theflanking introns (primer sequences and PCR conditions are availableupon request). All fragments were directly sequenced on an ABIPRISM 3730 automated sequencer (Applied Biosystems, Foster City,CA) using the BigDye terminator cycle sequencing chemistry. Thesesequences were compared to the wild-type sequence as submitted toGenBank Accession number NM_030777.3. The nucleotides werenumbered starting from the first base of the start codon (ATG) of thecDNA reference sequence. Amino acids were numbered from thefirst methionine residue. All missense mutations were checked in atleast 200 control chromosomes. Heterozygous mutations were verifiedto be in the trans position by either sequencing both parents or bycloning if parents were not available. PCR fragments comprising bothheterozygote mutations were cloned using a TOPO TA Clonings

system (Invitrogen, Carlsbad, CA) following the instructions of theOne Shots Chemical Transformation Protocol supplied by themanufacturer.

Detection of SLC2A10 exon deletions was performed using real-time quantitative PCR (Q-PCR) as described previously[Hoebeeck et al., 2005]. Specific primer pairs were designed foreach of the five exons of the SLC2A10 gene. PCR reaction mixturescontained Roche LightCycler SYBR Green Master Mix (RocheDiagnostics, Basel, Switzerland), 5mM of each forward and reverseprimer, and 10 ng template DNA. The cycling conditions were asfollows: 5 min at 951C, 45 cycles at 951C for 10 s, 601C for 30 s, and721C for 1 s. After PCR amplification a melting curve (60–951C) wasgenerated for every amplicon to check PCR reaction specificities. Q-PCR analysis was performed on a LightCycler 480 (Roche) deviceand calculation of the gene copy number was performed using qBasesoftware (http://medgen.ugent.be/qbase). Two reference genes,ZNF80 and GPR15, were used for normalization of the relativequantities [Hoebeeck et al., 2005].

Deletions were confirmed with a long-range PCR using theiProof kit (Bio-Rad Laboratories, Hercules, CA) according to themanufacturer’s instructions. Amplicons, generated using existingprimers from the SLC2A10 screening, were analyzed by ethidiumbromide gel electrophoresis to estimate the size of the deletion.Subsequently, new internal primers were developed to sequencethe region around the breakpoints.

HaplotypeAnalysis

Haplotype analysis with previously described markers [Couckeet al., 2006] was performed in parents and patients harboringidentical mutations. In addition, 12 single nucleotide polymorph-isms (SNPs) were analyzed to confirm the similarity of thehaplotypes (rs3091788, rs2425908, rs3091822, rs1136540,rs1136544, rs3092706, rs6012026, rs6018015, rs6094451,rs11699610, rs11699667, and rs6066059). Primers and PCRconditions are available on request.

Statistical Analysis

Statistical analysis comparing ages of our cohort to ages reportedin the literature was done using the Student’s t-test. Aneurysmformation and death was compared using chi-squared analysis.

Z-scores, used for the interpretation of aortic root dilation,indicate the number of standard deviations (SDs) to the mean,normalized for age and body surface area.

RESULTSDemographic Features

Pedigrees of the 12 newly identified ATS families are shown inFigure 1. Eight families originate from Europe [(the Netherlands

HUMANMUTATION 29(1),150^158,2008 151

Human Mutation DOI 10.1002/humu

Results 125

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(Families H and K), Belgium (Family A), England (Family I),France (Families F and L), Spain (Family G)), and Poland (FamilyC)], two families are American with German (Family B) andPolish/Sicilian (Family E) ancestry, one family has Turkish roots(Family D) and one family originates from Sudan (Family J). Threefamilies (Families A, D, and J) are known to be consanguineous.The male to female ratio of affected individuals is 11 to 5. Thecurrent age of the probands ranges from 16 months to 39 years(mean 12.9 years, median 9 years); mean age at diagnosis is 5.3years (median 12 months).

Our series is remarkable for the fact that 10 of the 16 patientspassed the age of 5 years. Moreover, half of these patients are olderthan 19 years, the age of the thus far oldest reported patient in theliterature [Bottio et al., 2007]. All patients are alive except PatientG:II-2 who suddenly died at the age of 33 years. Autopsy revealedsevere tortuosity of the arterial system, but no obvious vascularcause of death. He was also known with a tachyarrhythmia thatsegregated independently from the ATS phenotype.

Clinical Presentation

The clinical characteristics of all patients are presented inSupplementary Table S1 (available online at http://www.inters-cience.wiley.com/jpages/1059-7794/suppmat). Six probands initi-ally presented with a cardiac murmur before the age of 15 months(Families A, C, D, F, I, and L) and subsequently arterial tortuositywas diagnosed by echocardiography. Some of them had associatedfindings: Patient D:II-1 manifested generalized cutis laxa andPatient C:II-1 was evaluated in a preoperative setting for adiaphragmatic hernia. Three other patients had cyanosis as thepresenting symptom in the neonatal period (Patients K:II-1 andF:II-6) or during hernia surgery (Patient H:II-1). Cardiovascularworkup showed severe arterial tortuosity but no obvious cause forthe cyanosis. In Patient E:II-1 preoperative assessment for aninguinal hernia at 4 years of age revealed arterial tortuosity, but thefinal diagnosis of ATS was not made until his 30 s. The probandand affected sibs of Family G were evaluated for striking pulsatilecarotid arteries at ages 8, 13, and 25 years, respectively. Finally,two probands (Patients B:II-2 and J:II-1) came to medicalattention following a stroke with long-term neurological disability.Patient B:II-2 was known to have cutis laxa and recurrent inguinalhernias, but the diagnosis of ATS was not made until age 23 years,when he developed sudden pain in the right eye, with subsequentdiplopia. Neurological workup confirmed marked cerebral arterialtortuosity. The proband of Family J presented with right-sidedhemiplegia at the age of 8 months. At that time, wrinkling of theskin of hands and feet was noted. Vascular imaging showed aninternal left carotid artery dissection. His affected sibling presentedwith a pulmonary artery stenosis in the neonatal period. Those twoobservations in the same family evoked the diagnosis of ATS.

Overall, at diagnosis, patients presented with at least fourtypical craniofacial features including a long and slender face,sagging cheeks, downslanting and short palpebral fissures withperiorbital fullness, large protruding ears, a beaked nose, malarhypoplasia, a highly arched palate, and micrognathia (Fig. 2A). Inthe current series, six out of 16 patients had mild hypertelorism,but none had a cleft palate or bifid uvula. About half of patientshad an aged appearance. Other skin involvement ranged from asoft/thin skin to marked cutis laxa. Inguinal (8/15) anddiaphragmatic hernias (three anatomical, four sliding hernias)were commonly observed. Surgery for hernias was performed inseven patients without major wound healing or bleeding problemsand inguinal hernias were recurrent in Patients B:II-2 and E:II-2.

The most striking skeletal manifestation is joint laxity (12/14),with recurrent subluxations of the elbow (Patients G:II-1, K:II-1,and L:II-2) and temporomandibular joints (Patients G:II-1) as isoften seen in the Ehlers-Danlos syndrome [De Coster et al., 2005].Arachnodactyly (6/15), pectus deformity (5/15), and scoliosis(3/15) were less frequent, although these features may appear withgrowth. Ocular involvement was confined to myopia (6/13) andkeratoconus/keratoglobus (3/13). Five patients manifested vaso-motor instability with Raynaud phenomenon (Patient A:VI-2),cyanosis (Patients F:II-6, H:II-1, and L:II-2), or vagal syncopes(Patient F:II-3).

Miscellaneous features in this cohort are macrocephaly (PatientsB:II-2 and K:II-1), abnormalities of the trachea (tracheomegaly inPatient B:II-2 and tracheomalacia in Patient F:II-3), and urinarytract (duplication of the right collecting system in Patient B:II-2and an ectopic left kidney and bladder diverticula in Patient F:II-3).

Vascular Imaging

Patients. Vascular imaging was performed in all patients,except in Patient J:II-2 (Supplementary Table S1). Tortuosity ofthe aorta or large arteries was invariably present. Five patients(Patients C:II-1, F:II-3, I:II-1, J-II-1, and K:II-1) presentedaberrant origins of the aortic side-branches involving the truncusbrachiocephalicus (brachiocephalic trunk) and the left carotid andsubclavian artery. Aortic root dilation at the sinus of Valsalva wasdiagnosed in Patients B:II-2 and E:II-1, respectively, at age 23years (41 mm, z-score 3.9) and 35 years (38 mm, z-score 3.0). InPatient B:II-2 aortic root measurements remained stable in theupper normal range (35 mm) up to the age of 21 years butsubsequently increased to 41 mm in 2 years’ time. In Patient E:II-1a rapid progression to 49 mm (aortic root, z-score 7.5) wasdocumented over the last year in conjunction with an ascendingaortic aneurysm of 55 mm. Patient B:II-2 also manifests aborderline mean pulmonary artery dilation (23 mm). Two patients(Patients C:II-1 and D:II-1) had aortic coarctation, needingsurgical correction in the latter. MRI angiography showed longstenotic stretches along the aortic arch (Patient L:II-1), extendinginto the descending aorta in four patients (Patients B:II-2, G:II-1,G:II-3, and K:II-1). Patients B:II-2 and K:II-1 had a focal stenosisof the left carotid artery and left subclavian artery, respectively.Peripheral pulmonary artery stenosis was present in four patients(Patients D:II-1, I:II-1; J:II-2, and K:II-1). Remarkably, retinaltortuosity was absent in all patients evaluated by fundoscopy(Patients E:II-1, G:II-1, G:II-3, H:II-1, I:II-1, and K:II-1).

Carriers. MRI angiographies in eight proven SLC2A10mutation carriers of Families A, C, and G did not reveal anyarterial abnormalities (Fig. 2B).

Oral GlucoseToleranceTests in Patients and Carriers

We performed HbA1c dosage and oral glucose tolerance tests inthe patients and carriers of Families A, C, and F and the probandsof Families H and K. In all patients and carriers HbA1c levels(r5.5%), glucose and insulin profiles were normal except for the41-year-old father of Family A who had mild hyperinsulinemiafollowing a recent weight gain (10 kg in 2 years) and mildhypertension.

MolecularAnalysis

Mutations in all probands are represented in Table 1. A total of11 mutations were found, of which nine are previously unreported.All missense mutations involve conserved residues throughoutevolution (Mus musculus, Canis familiaris, Macaca mulatta, and

HUMANMUTATION 29(1),150^158,2008 153


Results 127

FIGURE 2. Clinical characteristics. A:Typical facial characteristics in six probands. Patient A:VI-2: aged 12 years; Patient C:II-1: at age 15months (left) and 3 months (right); Patient E:II-1 in infancy (left) and aged 35 years (right); Patient H:II-1aged 2 years; Patient I:II-1, aged6 years; andPatientK:II-1, aged11years. Note long faces (PatientsA:VI-2,C:II-1, E:II-1, H:II-1, I:II-1, andK:II-1), aged appearance (PatientsE:II-1, I:II-1, andK:II-1), sagging cheeks (PatientsC:II-1, E:II-1, H:II-1, I:II-1, andK:II-1), large ears (Patients A:VI-2,C:II-1, H:II-1, andK:II-1),mild hypertelorism (Patients A:VI-2,C:II-1, E:II-1, I:II-1, andK:II-1), short palpebral ¢ssures with periorbital fullness (Patients A:VI-2, C:II-1,andK:II-1), andmicrognathia (PatientsA:VI-2,C:II-1, andK:II-1).B:Theclinical andvascular phenotype inpatient andcarriers of FamilyA.3Dvascular imaging inpatients andcarriers of FamilyA (posterior view). From left to right: father, brother, mother, andproband.

154 HUMANMUTATION 29(1),150^158,2008



Xenopus tropicalis). Moreover, amino acids p.Arg132, p.Gly426,p.Glu437, and p.Gly246 are conserved through the facilitativeglucose transporter family. The first three amino acids are essentialfor sugar transport function [McVie-Wylie et al., 2001], whereasthe latter is the last amino acid in the Q242QLTG sequence motifcharacteristic of human glucose transporters [Dawson et al.,2001]. Two patients (Patients E:II-1 and I-II-1) have an exon 4deletion with identical breakpoints, due to an unequal crossing-over event between two highly homologous Alu repeat elements(AluSx and AluY in intron 3 and 4, respectively) (SupplementaryFig. S1).

Taken together, from the current and previously reportedmolecular data [Coucke et al., 2006; Drera et al., 2007], SLC2A10mutations have been identified in 19 probands of which 10 areknown to be consanguineous. However, only 15 differentmutations (eight missense, seven truncating) were found(Fig. 3A). This is explained by the presence of five recurrentmutations in our cohort. Both Dutch patients (Patients H:II-2 andK-II:1) share the same haplotype for all markers tested and mightbe closely related although genealogical studies did not revealcommon ancestors after 1700. Four families (Families B, F, G, andI) with European roots harbor the same c.1334delG mutation as apreviously reported Italian family [Coucke et al., 2006]. Haplotypeanalysis revealed a minimal shared region of 229 kB (Fig. 3B).Patients sharing the same c.1309G4A, c.1276G4T, orc.394C4T mutation also had similar haplotypes around theSLC2A10 gene (Fig. 3B). Both patients (Patients E:II-1 and I:II-1)with an identical exon 4 deletion shared a small haplotype,suggesting a founder effect rather than a mutational hotspot.

DISCUSSION

We present the clinical and molecular data of 12 newlyidentified ATS families. Patients were geographically widespread.

This series suggested a male preponderance but these data balancewhen taking into account previously reported data [Coucke et al.,2006] with a total male to female ratio of 19 to 20. Clinicalpresentation in all 16 patients was strikingly similar. Facialresemblance was obvious and all patients presented with variableinvolvement of the skin and skeleton. We observed macrocephalyand large, protruding ears as new features. Involvement of thepulmonary and urinary system with tracheomalacia and bladderdiverticula was suggested before [Abdul Wahab et al., 2003;Wessels et al., 2004] and may be due to insufficiency of the elasticcomponent. In addition, we found renal ectopy and duplication ofthe collecting system, suggesting that GLUT10 also has a role inthe patterning of the urinary tract. We confirm the increasedfrequency of cyanosis [Pletcher et al., 1996; Wessels et al., 2004]indicating that arterial elongation and tortuosity might beassociated with vasomotor instability.

Tortuosity of the aorta and large arteries was invariably present.For the first time, we report aberrant origins of the aortic side-branches and long stenotic stretches of the aorta (25% in ourcohort). Focal stenoses of the systemic circulation (25%) also seemto be more common than suggested from sporadic reports [Leeset al., 1969; Pletcher et al., 1996; Rasooly et al., 1988]. This mightbe due to differences in vascular assessment. In contrast,pulmonary artery stenosis occurred less frequently (28%) thanreported in literature (60%) [Wessels et al., 2004].

In ATS, the natural history with regard to aneurysm formationand dissection has not been well established but is of the highestconcern. A recent review by Wessels et al. [2004] mentions a 40%mortality rate before the age of 5 years. The thus far oldestreported patient was 19 years old [Bottio et al., 2007]. Causes ofdeath are variable, including pulmonary infection [Wessels et al.,2004], myocarditis [Ades et al., 1996], and tissue infarctions[Beuren et al., 1969; Pletcher et al., 1996; Wessels et al., 2004],but are frequently unknown. The most striking observation in thiscohort is that mean age is older than previously reported

TABLE 1. SCL2A10 Mutations Found inThis Study�

FamilyExoncDNA cDNA Protein level Functional domain

A Exon 2 c.685C4T (homozygous)a p.Arg229X Endofacial loop betweenTMD6 andTMD7B Exon 2 c.1276G4Ta p.Gly426Trp TMD10

Exon 3 c.1334delGb p.Gly445fs Endofacial loop betweenTMD10 andTMD11C Exon 2 c.730_733delCTAAa p.Leu244fs TMD7/Q242QLTG sequencemotif characteristic of human glucose

transportersExon 3 c.1309G4Ab p.Glu437Lys Endofacial loop betweenTMD10 andTMD11

D Exon 2 c.737G4A (homozygous)a p.Gly246Glu TMD7/Q242QLTG sequencemotif characteristic of human glucosetransporters

E Exon 3 c.1309G4Ab p.Glu437Lys Endofacial loop betweenTMD10 andTMD11Exon 4 c.14111480_c.15471

299delap.Gly471_Arg515delXfs Endofacial loop betweenTMD11andTMD12,TMD12,COOH terminus

F Exon 2 c.394C4Ta p.Arg132Trp Endofacial loop betweenTMD4 andTMD5Exon 3 c.1334delGb p.Gly445fs Endofacial loop betweenTMD10 andTMD11

G Exon 2 c.692G4Aa p.Arg231Gln Endofacial loop betweenTMD6 andTMD7Exon 3 c.1334delGb p.Gly445fs Endofacial loop betweenTMD10 andTMD11

H Exon 2 c.394C4Ta,b p.Arg132Trp Endofacial loop betweenTMD4 andTDM5Exon 2 c.1276G4Ta,b p.Gly426Trp TDM10

I Exon 3 c.1334delGb p.Gly445fs Endofacial loop betweenTMD10 andTMD11Exon 4 c.14111480_c.15471

299dela,bp.Gly471_Arg515delXfs Endofacial loop betweenTMD11andTMD12,TMD12,COOH terminus

J Exon 2 c.425G4T (homozygous)a p.Gly142Val TMD5K Exon 2 c.394C4Ta,b p.Arg132Trp Endofacial loop betweenTMD4 andTMD5

Exon 2 c.1276G4Ta,b p.Gly426Trp TMD10L Exon 3 c.1334G4A (homozygous)a p.Gly445Gln Endofacial loop betweenTMD10 andTMD11

�Sequences were compared to the wild-type sequence as submitted toGenBankAccession number NM_030777.3.The nucleotides were numbered starting from the ¢rst base ofthe start codon (ATG) of the cDNA reference sequence. Amino acids were numbered from the ¢rst methionine.aPreviously unreportedmutation.bRecurrent mutation.TMD, transmembrane domain.

HUMAN MUTATION 29(1),150^158,2008 155


Results 129

(P 5 0.04). Only one patient died, most likely due to an unrelatedcause (P 5 0.016). Aneurysms tend to be less frequent thanreported in literature (18.8% vs. 31%, P 5 0.15) [Wessels et al.,2004]. At diagnosis, only two adult patients had moderate,although rapidly progressive, aortic root dilation. Two patients hada stroke, due to an arterial dissection in one case. Overall, theclinical implications of the vascular phenotype in this cohort seemto be less severe than previously reported in the literature,

pointing to a high clinical variability. We did not observe anycorrelation between the nature of the mutations and the clinicalseverity. Moreover, we previously demonstrated that missensemutations also result in loss-of-function as no protein was detectedon immunofluorescence microscopy [Coucke et al., 2006].Although we acknowledge a possible ascertainment bias in thecurrent series, a publication bias toward the most severe cases andthe prior inability to distinguish ATS from other disorders with

FIGURE 3. Molecular data. A: Location of all currently identi¢ed GLUT10 mutations. GLUT10 contains 12 hydrophobic transmem-brane domains (ovals) and a hydrophilic endofacial loop between transmembrane domains 6 and 7 and a large hydrophilic loopcontaining a potential N-linked glycosylation site between transmembrane domains 9 and 10. �, mutation described by Couckeet al. [2006]; z, mutationdescribedbyDrera et al. [2007].B:Haplotype analysis. Haplotypes for all patientswith recurrentmutationsfor chromosome 20q13.1 markers around SLC2A10 are shown. SNP haplotype 1 comprises rs3091788, rs2425908, rs3091822,rs1136540, rs1136544, and rs3092706. SNP haplotype 2 comprises rs6012026, rs6018015, rs6094451, rs11699610, rs11699667,and rs6066059.

156 HUMANMUTATION 29(1),150^158,2008



arterial tortuosity and aneurysms formation as LDS [Loeys et al.,2006] and type I autosomal recessive cutis laxa [Hucthagowderet al., 2006] might have distorted the reported prognosis. Anassociation between arterial tortuosity and stroke has beenpreviously reported [Cartwright et al., 2006] and autopsy in threeATS cases revealed cardiac, gastrointestinal, spleen, liver, orkidney infarctions [Beuren et al., 1969; Pletcher et al., 1996;Wessels et al., 2004]. Although the exact mechanism remains tobe elucidated, tortuosity may result in a higher shear stress,inducing endothelial lesions that might become thrombogenic inthemselves or predispose to arterial dissection [Cartwright et al.,2006; Golomb and Fullerton, 2006]. With regard to clinicalmanagement, follow-up should be tailored for the individualpatient based on vascular imaging results, the rate of progression ofvascular diameters in the family history, and the patient’sperception of his own risk. A baseline whole-body vascularimaging study is mandated.

Importantly, we showed in three different families thatheterozygous carriers, harboring nonsense, frameshift, or missensemutations, did not demonstrate tortuosity of any degree excludingboth haploinsufficient and dominant negative effects. These datawill need long-term confirmation, but suggest that cardiovascularfollow-up is not necessary in carriers.

SLC2A10 is situated within a region showing linkage withdiabetes mellitus type 2. Thus far no associations betweenmutations or polymorphisms in GLUT10 and insulin resistancewere demonstrated [Andersen et al., 2003; Bento et al., 2005; Linet al., 2006; Mohlke et al., 2005; Rose et al., 2005]. Based onglucose tolerance tests in patients and carriers with pathogenicmutations in SLC2A10, we excluded the presence of early insulinresistance in both. It is therefore unlikely that proper GLUT10function is needed to maintain a proper glucose gradient across theplasma membrane, but rather has a role in intracellular substratetransport as suggested previously [Coucke et al., 2006].

All patients diagnosed with ATS harbored homozygous orcompound heterozygous SCL2A10 mutations. All missensemutations are located in the transmembrane or endofacialdomains of the protein. Therefore, these domains seem morecritical in substrate transport than the exofacial regions. Inaddition to the recurrent c.243C4G mutation, reported byCoucke et al. [2006], we found five new recurrent mutations inthis cohort. Haplotype analysis identified small shared chromoso-mal regions for all recurrent mutations, suggesting ancient foundereffects. Moreover, several patients sharing a same mutation couldbe traced back to the same geographical roots.

In conclusion, the cardiovascular prognosis in ATS seems lesssevere than previously reported. Follow-up will further determinethe risk for vascular complications and differentiate the phenotypicspectrum from other syndromes with arterial tortuosity. Carriers donot exhibit arterial tortuosity and seem not at risk for vascularcatastrophes. Pathogenic mutations in SLC2A10 in the homo- orheterozygous state do not lead to overt diabetes mellitus. Themolecular data suggest founder effects in SLC2A10.

ACKNOWLEDGMENTS

We are indebted to the families and patients for their interestand cooperation. We thank D. Devos and B. Schweiger forradiological support and Y. Hellenbroich for accommodating thereferral of patient samples. B.C. and B.L. are research assistant andsenior clinical investigator, respectively, and are supported by theFund for Scientific Research, Flanders. J.D.B. is a research fellowfrom Ghent University.

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158 HUMANMUTATION 29(1),150^158,2008



Supplementary Table S1 (part 1, 2, 3): Clinical characteristics of all ATS patients Abbreviations: y, years; mo, months; wk(s), week(s); M, male; F, female; MPA, mean pulmonary artery; GER, gastro-esophageal reflux; AI, aortic insufficiency; TA, tachyarrythmia; HT, hypertension; CoA: coarctatio aortae; RD: retinal degeneration (1) reduced diameter of the ascending aorta, aortic arch, descending aorta and common iliac arteries (2) reduced diameter of the aortic arch *: deceased

Supplementary Table S1: Clinical characteristics - part 1Family A Family B Family C Family D Family E

Ancestry Belgian American German Turkish American (German) (Polish/Sicilian)

Consanguinity Yes No No Yes No

Individual VI-2 II-2 II-1 II-1 II-1Age 12 y 23 y 15 mo 4 y 35 ySex M M M F M

Diagnosis

First presentation Cardiac murmur

Cutis laxa; stroke

Cardiac murmur,

diaphragmatic hernia

Cardiac murmur

(CoA); cutis laxa

Tortuosity noticed on pre-

operative assessment

Age at diagnosis 15 mo 23 y 1 w 3 mo 30 y

CraniofacialLong face + + Mild +Hypertelorism Mild - Mild - +Short palpebral fissures + + + -Downslanting palpebral fissures - + + + +Beaked nose + - - +Malar hypoplasia - + + -Cleft palate/bifid uvula - - - - -High arched palate - + +Micrognathia + - + -Sagging cheeks - - + + +Large ears (>p75) + - + + -Aged appearance - - - + (face) ++

OcularKeratoconus / -globus - + - +Astigmatism - - - + +Myopia - + - - +

IntegumentVelvety skin - - + + -Thin skin + - + -Hyperextensible skin + + Mild + -Cutis laxa - + - + -Improvement of the cutis laxa +Inguinal hernia + + - - +Diaphragmatic hernia - Anatomical Anatomical - -

SkeletalPectus deformity + - + - -Scoliosis - - - - -Arachnodactyly - - + - -Clinodactyly + - - - -Contractures of large joints - - - - -Joint laxity (Beighton score) + (5/9) + (8/9) - + - (0/9)

CardiovascularAortic tortuosity + + + + +Tortuosity of other arteries + + + + +Abberant origin

of the aortic branchesAortic root aneurysm - + (41 mm) - - + (38 mm)Other arterial aneurysms - MPA - - -Arterial dissections - - - - -Stenosis of the pulmonary arteries - - - + -Aortic stenosis - (1) CoA CoA -Other stenoses - Carotis art. - - -Vasomotor instability Raynaud - - -

Other manifestationsTracheal abnormalities Tracheomegaly

Urinary tract abnormalitiesDuplication right collecting system

- -- + -

Results 133

Supplementary Table S1: Clinical characteristics - part 2Family G Family H

Ancestry Dutch

Consanguinity No

Individual II-3 II-1 II-2 II-3 II-1Age 16 y 39 y 33 y * 30 2 y, 8 moSex M F M F M

Diagnosis

First presentation Cardiac murmur

Marked carotid artery

pulsations


pulsations


pulsations

Cyanosis during surgery

Age at diagnosis 5 mo 8 13 25 6 wks

CraniofacialLong face + + + + +Hypertelorism - - - - -Short palpebral fissures + + - - -Downslanting palpebral fissures - + + - -Beaked nose - + + Surgery -Malar hypoplasia + + + + +Cleft palate/bifid uvula - - - - -High arched palate - + + - +Micrognathia - - - - +Sagging cheeks - + + +Large ears (>p75) + - - - +Aged appearance - + + -

OcularKeratoconus / -globus - - - - -Astigmatism + - - - -Myopia + + Mild Mild -

IntegumentVelvety skin + - - - +Thin skin - + + -Hyperextensible skin + - - -Cutis laxa - + + -Improvement of the cutis laxa +Inguinal hernia - + + - +Diaphragmatic hernia Sliding Anatomical - - -

SkeletalPectus deformity - - - - +Scoliosis + - Mild -Arachnodactyly + + + + -Clinodactyly - - - - -Contractures of large joints - - - - -Joint laxity (Beighton score) + (9/9) + (8/9) + + (6/9) + (8/9)

CardiovascularAortic tortuosity + + + - +Tortuosity of other arteries + + + + +Abberant origin

of the aortic branchesAortic root aneurysm - - - - -Other arterial aneurysms - - - - -Arterial dissections - - - - -Stenosis of the pulmonary arteries Transient - - - -Aortic stenosis - (1) - (1) -Other stenoses - - - - -Vasomotor instability Vagal - - - Cyanosis

Other manifestationsTracheal abnormalities

Urinary tract abnormalities

Miscellaneous findings Hearing loss, delayed teeth

eruptionTA, HT, RD TA TA, RD, AI

Tracheomalacia

+

-

--

+ (6/9)

+

-Mild

+-

Sliding

-

--

-

--

---

---

--+-

II-67 y, 5 mo

F

-

Neonatal cyanosis; Cardiac murmur

1 wk

+-

Mild

Family FFrench

No

+

-

Spanish

No

-

Delayed teeth eruption

- -+

----

Cyanosis

Ectopic left kidney, bladder

diverticula


Supplementary Table S1: Clinical characteristics - part 3Family I Family K Family L Total

Ancestry English/Italian Dutch French

Consanguinity No No No 3/10

Individual II-1 II-1 II-2 II-1 II-1Age (years) 6 y 2 y, 6 mo 13 mo 11 y, 8 mo 16 moSex M M M M F

Diagnosis

First presentation Cardiac murmur Stroke Pulmonary

artery stenosis

Neonatal oxigen

requirement

Cardiac murmur

Age at diagnosis 1 y 8 mo birth 8 mo 1 wk

CraniofacialLong face + + + + 14/14Hypertelorism Mild - + + 6/16Short palpebral fissures + - + + 9/14Downslanting palpebral fissures + - - - 8/15Beaked nose - + - + 8/14Malar hypoplasia - + + - 9/14Cleft palate/bifid uvula - - - - - 0/16High arched palate - - - - 5/13Micrognathia - + - + 5/14Sagging cheeks + + + + 10/14Large ears (>p75) - + + - 7/15Aged appearance + + + - 7/14

OcularKeratoconus / -globus - - + - 3/14Astigmatism + - + - 5/15Myopia - - - - 6/15

IntegumentVelvety skin - + + + 7/15Thin skin - - - - 4/13Hyperextensible skin + - - + 7/14Cutis laxa - Wrinkling - - 5/14Improvement of the cutis laxa 2/5Inguinal hernia - + - + - 8/15Diaphragmatic hernia - Sliding Sliding - - 7/15

SkeletalPectus deformity - - - + - 5/15Scoliosis - - - + - 3/15Arachnodactyly - - - - 6/15Clinodactyly - - - 2/15Contractures of large joints - - Ankles - Ankles 2/16Joint laxity (Beighton score) + (8/9) + (6/9) + (7/9) 12/14

CardiovascularAortic tortuosity + + + + 14/15Tortuosity of other arteries + + + + 15/15Abberant origin

of the aortic branchesAortic root aneurysm - - - - - 2/16Other arterial aneurysms - - - - - 1/16Arterial dissections - Left carotid art. - - - 1/16Stenosis of the pulmonary arteries + - + + - 5/16Aortic stenosis - - - (1) (2) 7/15Other stenoses - - - Subclavia art. - 2/15Vasomotor instability - - Cyanosis - 5/14

Other manifestationsTracheal abnormalities

Urinary tract abnormalities

Miscellaneous findings Gastric volvulus Crumpled ears

Macrocephaly, convergent strabismus

5/16+ +

Family J

+- -

Sudanese

Yes

Results 135

Intron 3 Intron 4

Intron 3 Intron 4

AluSx Exon 4 AluY

BreakpointIntron 3 - AluSx Intron 4 - AluY

T T G G G G A G G G G G G G G G G G C C C C C C C C C C C C C C C C C C C C C C C C C C A A A A A A A

Patient I.II:1

Patient E.II:1

Mutant allele

UnequalCrossover

Wild type allele

Supplementary Figure S1: Breakpoint analysis of the exon 4 deletion (c.1411+480_1547+299del). Schematic representation of the unequal crossing over event between highly homologous AluSx and AluY elements, represented by red and blue colored boxes, respectively. The electropherograms of the recombinant sequence in patients E:II-1 and I:II-1 are represented. The breakpoint region is indicated by a grey bar.


IV. NEW CLINICAL AND PATHOGENETIC INSIGHTS IN

AUTOSOMAL DOMINANT CUTIS LAXA

Publication 7

New insights into the pathogenesis of autosomal dominant cutis laxa with

report of five additional ELN mutations

Bert Callewaert; Vishwanathan Hucthagowder, Beate Albrecht, Ingrid Haußer,

Edward Blair, Cristina Dias, Alice Albino, Hiroshi Wachi, Fumiaki Sato, Robert

P. Mecham, Bart Loeys, Paul J. Coucke, Anne De Paepe*, Zsolt Urban

* (* joint

Last Authors.)

Human mutation, submitted

This study presents an in depth clinical and molecular study of ADCL. We describe 5

additional probands with this very rare disease and focus on the natural history with

internal organ involvement. We provide new insights in the diseased elastic fiber

formation in ADCL and relate the repercussions of these findings to the clinical

phenotype.

Results 137

1

New insights into the pathogenesis of autosomal dominant cutis

laxa with report of five additional ELN mutations

Bert Callewaert1,2

; Vishwanathan Hucthagowder2, Beate Albrecht

3, Ingrid Haußer

4,

Edward Blair5, Cristina Dias

6, Alice Albino

7, Hiroshi Wachi

8, Fumiaki Sato

8, Robert P.

Mecham9, Bart Loeys

1, Paul J. Coucke

1, Anne De Paepe

1,†, Zsolt Urban

2,10,†,*

1Center for Medical Genetics, Ghent University Hospital, B-9000 Ghent, Belgium

2Department of Pediatrics, Washington University School of Medicine, St. Louis, MO

63110, USA 3Institut für Humangenetik, Universitätsklinikum Essen, D-45122 Essen, Germany

4Department of Dermatology, University of Heidelberg, D-69115 Heidelberg, Germany

5Department of Clinical Genetics, Churchill Hospital, Oxford OX3 9DU, UK

6Centro de Genética Médica Doutor Jacinto Magalhães - INSARJ, 4050-111 Porto,

Portugal 7Department of Pediatrics, Hospital de Crianças Maria Pia, Centro Hospitalar do Porto,

4050-111 Porto, Portugal 8Department of Clinical Chemistry, Hoshi University School of Pharmacy and

Pharmaceutical Sciences, Tokyo 142-8501, Japan 9Department of Cell Biology and Physiology, Washington University School of

Medicine, St. Louis, MO 63110, USA 10

Department of Human Genetics, Graduate School of Public Health, University of

Pittsburgh, Pittsburgh, PA 15261, USA

† Joint Last Authors

*To whom correspondence should be addressed. Department of Human Genetics,

Graduate School of Public Health, University of Pittsburgh, 130 DeSoto Street, Crabtree

Hall A300, Pittsburgh, PA 15261, Phone: (314) 648-8269, Fax: (412) 624-3020,

Email: [email protected]


2

Abstract

Autosomal dominant cutis laxa (ADCL) is characterized by a typical facial appearance and

generalized loose skin folds, occasionally associated with aortic root dilatation and emphysema.

We sequenced exons 28-34 of the ELN gene in 5 probands with ADCL features and found 5 de

novo heterozygous mutations: c.2296_2299dupGCAG (CL-1), c.2333delC (CL-2), c.2137delG

(CL-3), c.2262delA (monozygotic twin CL-4 and CL-5) and c.2105del26insG (CL-6). Four

probands (CL-1, -2,-3, -6) presented with progressive aortic root dilatation. CL-2 and CL-3 also

had bicuspid aortic valves. CL-2 presented with severe emphysema. Electron microscopy

revealed elastic fiber fragmentation and diminished dermal elastin deposition. RT-PCR studies

showed stable mutant mRNA in all patients and increased exon 32 skipping in patients with exon

32 mutations explaining a milder phenotype. Mutant protein expression in fibroblast cultures

impaired deposition of tropoelastin onto microfibril-containing fibers, but enhanced tropoelastin

coacervation and globule formation leading to lower amounts of mature, insoluble elastin.

Mutation-specific effects also included endoplasmic reticulum (ER) stress and increased

apoptosis. Increased pSMAD2 staining in ADCL fibroblasts indicated enhanced transforming

growth factor beta (TGFβ) signaling. We conclude that ADCL is a systemic disease with

cardiovascular and pulmonary complications, associated with increased TGFβ signaling and

mutation-specific differences in ER stress and apoptosis.

Key Words: mutation, connective tissue, skin, aneurysm, emphysema

Results 139

3

Introduction

Cutis laxa (CL) comprises a heterogeneous group of acquired and inherited connective tissue

disorders characterized by loose, redundant skin folds. The syndromic forms of CL include X-

linked, autosomal dominant and recessive forms, which differ in severity and spectrum of

associated clinical manifestations (de Schepper, et al., 2003; Milewicz, et al., 2000). Many

related disorders are described and several forms of cutis laxa remain unclassified.

The X-linked form, also known as occipital horn syndrome, includes mild mental retardation,

skeletal manifestations including the pathognomonic occipital horns, and bladder diverticulae

(Tsukahara, et al., 1994). This condition is allelic with Menkes disease and is caused by

hypomorphic mutations in the ATP7A gene encoding a copper transporter (Kaler, et al., 1994).

Autosomal recessive CL (ARCL) comprise at least three subtypes, with typically severe

emphysema and life threatening vascular lesions in ARCL type I, developmental delay,

microcephaly and skeletal abnormalities in ARCL type II and corneal abnormalities in ARCL

type III, also known as de Barsy syndrome. In type I, both mutations in FBLN5 and FBLN4 have

been described (Hucthagowder, et al., 2006; Loeys, et al., 2002). Mutations in ATP6V0A2 have

been shown to cause type II recessive cutis laxa with microcephaly and mental retardation and

wrinkly skin syndrome (Kornak, et al., 2008). Interestingly, mutations in PYCR1 encoding the

mitochondrial pyrroline-5-carboxylate reductase 1 were identified in a similar patient population

as well as in patients with geroderma osteodysplasticum and the Barsy syndrome (Guernsey, et

al., 2009; Reversade, et al., 2009). The recently defined Urban-Rifkin-Davis syndrome describes

a constellation of craniofacial dysmorphism, cutis laxa, short stature, lung dysplasia, and

genitourinary and gastrointestinal diverticulosis caused by mutations in the gene for latent

transforming growth factor beta-binding protein 4 (LTBP4) (Urban, et al., 2009).

Autosomal dominant cutis laxa (ADCL) presents with generalized lax skin folds resulting in a

prematurely aged appearance. It is generally considered as a milder disease without the severe

involvement of internal organs (de Schepper, et al., 2003; Marchase, et al., 1980; Nahas, et al.,

1999; Sarkar, et al., 2002). However, aortic and pulmonary abnormalities have been occasionally

reported in patients with ADCL (Szabo, et al., 2006; Urban, et al., 2005). In most cases, patients

carry frame shift mutations at the 3’-end of the elastin gene (ELN), predicted to result in a mutant

tropoelastin protein with an extended carboxy-terminal missense peptide sequence (Rodriguez-

Revenga, et al., 2004; Szabo, et al., 2006; Tassabehji, et al., 1998; Urban, et al., 2005; Zhang, et

al., 1999).

When tropoelastin is secreted, it is deposited on the microfibrillar scaffold and becomes highly

cross-linked to form insoluble elastin. These structures consisting of microfibrils with an

amorphous elastin core provide tissues with elasticity and resilience in a developmental stage

and tissue dependent manner (Kielty, et al., 2002). Consequently, histopathology in ADCL is


4

characterized by disorganized and often shortened elastic fibers that are predicted to result in a

weakened and less elastic connective tissue (Szabo, et al., 2006).

Little is known concerning the molecular mechanisms causing ADCL, in part because of the

scarcity of patient tissue samples. Previously, both dominant negative effects and gain-of-

function with enhanced proteolysis have been suggested for elastin mutations (Urban, et al.,

2005). Molecular insights into this rare disease could be relevant for far more common diseases

that are also characterized by elastic fiber fragmentation or impaired elastic fiber repair,

associated with aortic and pulmonary damage such as familial thoracic aortic aneurysms and

emphysema (Robinson, et al., 2006; Shifren and Mecham, 2006). Conversely, more common

monogenic diseases with degeneration of the elastic fibers, like Marfan syndrome (MFS), caused

by mutations in fibrillin-1 (Dietz, et al., 1991), a main component of the microfibrils, could

provide helpful hints towards pathogenesis in cutis laxa. Recently, enhanced transforming

growth factor beta (TGFβ) signaling has been reported as a major disease-causing mechanism in

MFS (Neptune, et al., 2003), a finding with direct therapeutic consequences (Habashi, et al.,

2006). We report 5 new probands with de novo ELN mutations in the 3’ terminus of which four

presented with internal organ involvement. We show enhanced TGFβ signaling as a common

mechanism, similarly to other elastic fibers diseases, including MFS. We also demonstrate that

mutation-specific differences in intracellular mutant protein processing cause different levels of

ER stress and apoptosis.

Results 141

5

Materials and Methods

Patients and samples

All patients were evaluated by a clinical geneticist at the referring center. Skin specimens were

obtained from patients CL-1, CL-2, CL-3 and CL-4. Part of the skin specimens were prepared for

electron microscopy (see below). DNA was extracted from peripheral blood (patient CL-2, CL-5,

CL-6) and/or cultured skin fibroblasts (CL-1, CL-3, CL-4). No fibroblast cultures were available

for CL-2, CL-5 and CL-6. Skin fibroblasts were cultured in Dulbecco’s modified Eagle’s

Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) in the presence of

antibiotics and antimycotics. Appropriate informed consent, including specific consent to publish

clinical pictures (Fig. 1) was obtained from all patients involved in the study.

Molecular analysis

Genomic DNA was extracted from blood samples by standard procedures, followed by

touchdown PCR amplification of exons 28 through 34 of the ELN gene using forward and

reverse primers located in the flanking introns (primer sequences available upon request). The

PCR products were analyzed by gel electrophoresis and visualized by ethidium bromide staining

on 2% agarose gels. All amplimers were directly sequenced with an ABI PRISM 3730

automated sequencer (Applied Biosystems, Foster City, CA) using the BigDye terminator cycle

sequencing chemistry. These sequences were compared to the wild type sequence as submitted to

Ensembl Accession number ENST0000358929. The nucleotides were numbered starting from

the first base of the start codon (ATG) of the cDNA reference sequence. Amino acid residues are

numbered from the first methionine residue (reference sequence ENSP00000351807). All

mutations were confirmed to be de novo and absent in a panel of 100 normal control individuals.

Expression studies

For the analysis of allelic expression, RNA was isolated from fibroblasts using the RNeasy Mini

Kit (Qiagen, Valencia, CA, USA), and cDNA was synthesized using Superscript II Reverse

Transcriptase Kit with hexamer primers (Invitrogen, Carlsbad, CA) in a total volume of 20 µl.

First strand cDNA (1:10 dilution) was amplified using a oligonucleotide sense and antisense

primers complementary to exon 29 and the 3’ untranslated region (3’UTR), respectively (Szabo,

et al., 2006). The sense primer was fluorescently labeled using a M13 primer tail. Fragments

were run on an Applied Biosystems Prism 3130 genetic analyzer (Applied Biosystems). The data

were processed using a Genescan software (Applied Biosystems).

Elastin staining

Semi-thin sections (1 micrometer thick) of the resin embedded skin biopsies were incubated with

1% aqueous methylene blue for 2-3 min at 80 degree Celsius (on a hot plate), rinsed with water

and dried. Using this method, elastic material is stained darker blue than collagen bundles.


6

Electron microscopy

All specimens were fixed for at least 2h at room temperature in 3% glutaraldehyde solution in

0.1M cacodylate buffer pH 7.4, cut into pieces of ca. 1mm3, washed in buffer, postfixed for 1 h

at 4o in 1% osmium tetroxide or in 0,5% ruthenium tetroxide, rinsed in water, dehydrated

through graded ethanol solutions, transferred into propylene oxide, and embedded in epoxy resin

(glycidether 100). Semithin and ultrathin sections were cut with an ultramicrotome (Reichert

Ultracut E). Ultrathin sections were treated with uranyl acetate and lead citrate, and examined

with an electron microscope (Philips EM 400).

Generation of an fmTE specific antibody

To generate an antibody specific to fmTE we chose a peptide antigen encoded by exon 34 and

the 3’-UTR of ELN as a result of an upstream –1 frameshift. The peptide,

VGPAWGKLVAGRENELPRTPDSRPHQRQC, was synthesized on an ABI431 peptide

synthesizer using FMOC chemistry and the sequence was confirmed by mass spectrometry. The

antibody was generated in rabbits immunized with the peptide coupled to rabbit serum albumin.

Immunostaining

Cells were grown for 3 weeks postconfluency for staining with elastin/fibrillin-1, mutant

elastin/fibrillin-1 and pSMAD2. Staining for elastin/BIP, peIF2α and Caspase-3 was performed

at 1 week postconfluency. Details about the antibodies used in this study are represented in Table

2. In brief, confluent cultures were washed with PBS once and fixed in 4% paraformaldehyde in

PBS at 4°C for 30 min. After several washes in PBS, cell cultures were incubated in 3% bovine

serum albumin (BSA) in either Tris-buffered saline (TBS, 20 mM Tris/HCl, pH 7.4, and 0.15 M

NaCl) for non-permeabilized cells or TBS/0.05% Tween for permeabilized cells for 1 h. Cell

cultures were then incubated overnight at 4°C with primary antibody in TBS or TBS/0.05%

Tween. After several washes, cell cultures were incubated with secondary antibody and nuclear

stain (Hoechst 33258) in PBS or PBS/Tween 0.05% for 1 hour at room temperature in the dark.

Cell cultures were washed several times, mounted with Gel/Mount (Biomeda, Foster City, CA,

USA) and visualized with an Axioskop BX60 microscope (Olympus, Center Valley, PA, USA).

Insoluble elastin assay

The insoluble elastin protocol was carried out as described previously (Urban, et al., 2002) with

slight modifications. In brief, for each time point (4 and 8 days), fibroblasts from 3 patients and 3

age- and sex-matched controls were plated in 60mm culture dishes (500,000 cells/dish) and were

grown to confluence. Then, 20 µCi of [3H]-leucine was added to each dish, along with fresh

media and cultures were either incubated for the next 4 days or maintained in a time course as

long as 8 days in the presence of [3H]-leucine (fresh media was added at day 4). At each time

point of the course, triplicate cultures belonging to each experimental group were terminated,

and insoluble elastin was assessed. After the removal of the media, cell layers were washed in

0.1 M acetic acid, scraped in 0.1 N NaOH and sedimented by centrifugation (10 min at 16000 x

Results 143

7

g). After removal of the supernatans, the pellets were boiled (at 97°C) for 45 min in 0.5 ml of 0.1

N NaOH to dissolve all ECM components except the insoluble elastin. After centrifugation (10

min at 16000 x g) the supernatans were mixed with 2 mL of scintillation fluid and measured in a

Beckman Coulter LS6500 scintillation counter. Next, the pellets were boiled in 200 ml of 5.7 N

HCl for 1 h to dissolve the insoluble elastin. The samples were mixed with scintillation fluid and

measured in a scintillation counter (Hinek and Rabinovitch, 1994; Hinek, et al., 1993). Final

results were expressed as follows:

%100

)()(

)(

cpmIEcpmECM

cpmIE with IE: insoluble elastin, cpm: counts per minute, ECM:

extracellular matrix proteins except insoluble elastin. In this way, insoluble elastin was

normalized for total extracellular matrix production.

Purification of recombinant human tropoelastin

A recombinant full-length human tropoelastin (TE) and a mutant elastin cDNA with a -1

frameshift in exon 32 (c.2262delA, fmTE), which was extended with a 72-base pair sequence at

the C-terminus of elastin, were prepared as previously described (Wachi, et al., 2005). Each

construct was inserted into a bacterial expression pTrcHis-TOPO vector (Invitrogen, Carlsbad,

CA). The bacteria were grown to the mid-log phase at 37ºC, and IPTG (isopropyl-(-D-

thiogalactoside) was added to the culture to 1 mM in order to induce expression. Purified TE or

fmTE was resuspended in SDS-PAGE sample buffer (62.5 mM, Tris, pH 6.8, 0.4% (w/v) SDS,

10% (v/v) glycerol, and 0.003% (w/v) bromophenol blue) including 100 mM DTT. Samples

were run on SDS-PAGE gels and subjected to Western blot analysis to verify the purity of the

recombinant proteins.

Coacervation assay

Coacervation was assayed by monitoring turbidity using light scattering at 400 nm with a UV

spectrometer and JASCO V-500 software (Nihonbunkou, Tokyo, Japan). The cuvette holder was

connected to a re-circulating water bath in order to control the temperature of samples. Light

scattering by each solution was monitored every 0.5 min while the temperature was increased

from 15 °C to 45 °C at a rate of 1 °C/min. Both TE and the fmTE were used at concentrations of

6.25 μM and 12.5 μM in PBS, and as a mixture at a concentration of 6.25 μM each in PBS.

Reference samples of pure recombinant TE and fmTE proteins were studied at concentrations of

12.5 μM in PBS.

Statistics

Immunostaining data were quantified for each coverslip by counting all positive cells in 10 high

power fields containing at least a total of 300 nuclei. This number was divided by the total

number of nuclei counted. Patients were compared to age, sex and passage matched controls

using the Fisher’s exact test. For the insoluble elastin assay, results were obtained as described

above and analyzed using the paired, one-tailed student t-test.


8

Results

Clinical reports

Patient CL-1

Patient 1 is a 4 year old Caucasian boy. He was born to unrelated, healthy parents after an

uneventful pregnancy at 42 weeks. Birth weight, length and head circumference were 4150g

(75th

percentile), 56 cm (97th

percentile) and 36 cm (75th

percentile), respectively. Cutis laxa was

noted immediately at birth. At the age of three months, he presented with generalized cutis laxa

with marked skin folds on face, neck, trunk, abdomen and limbs, but not on palms and soles. He

had an aged appearance with loose redundant skin folds of the cheeks and chin, a high forehead,

a long philtrum and large ear lobes. External genitals were normal. Growth and neuromotor

development were appropriate. There was a large right-sided direct inguinal hernia, which

needed surgical correction. Wound healing was normal. Pulmonary evaluation and

echocardiography were within normal limits.

At age 3 years 5 months (Fig. 1A), he measured 100 cm (75-90th

percentile) and weighed 14.8 kg

(90th

percentile). Clinical examination was unchanged, but a hoarse voice was noted. During

exercise he experienced mild dyspnoea. Echocardiography revealed borderline enlarged sinuses

of Valsalva and minimal aortic and mitral valve insufficiency. At age 6, the dilation of the aortic

root progressed to a Z score of 4.

Patient CL-2

Patient CL-2 was born to unrelated, healthy parents after an uneventful pregnancy at 40 weeks.

Birth weight was 3500 g (50th

percentile), length of 50 cm (25-50th

percentile). Marked cutis laxa

was immediately observed. Loose redundant skin folds were generalized but more prominent in

the face, neck and abdomen (Fig. 1B). She had an aged appearance with a coarse face, a long

philtrum and large dysplastic ear lobes. She had bilateral inguinal hernias.

Growth and neuromotor development were normal. A slight strabismus was noted.

At the age of 12, the skin folds became less pronounced but gave a strikingly aged appearance.

Plastic surgery (face-lift) was planned but cancelled because preoperative assessment revealed

severe emphysema (total lung capacity 118%, residual volume 209%, FEV1/FVC 42,9 %

predicted) and a dilated aortic root. In addition, MRI-angiography showed a bicuspid aortic

valve, a dilated aortic root of 41 mm (Z score = 7) at the sinuses of Valsalva compressing the left

atrium and the right ventricular outflow tract without significant hemodynamic changes. The

diameter of the sinotubular junction was normal (25 mm) but the ascending aorta was enlarged

up to 32-33 mm at the pulmonary bifurcation. The aortic arch was somewhat tortuous, but not

enlarged. Treatment with β-blocking agents was initiated to limit aortic aneurysm growth.

Surgery is planned, but declined by the patient because of the high anesthetic risk.

Results 145

9

Patient CL-3

Patient 3 is a 17-year old female patient born to unrelated, healthy parents. She was reported

previously at the age of 6 years (Jung, et al., 1996). She was noted to have generalized, loose,

redundant skin folds at birth. During infancy, she suffered from several lung infections. At the

age of six years, she presented with marked joint hypermobility and prominent facial

characteristics with an aged appearance, large ears, beaked nose, and long philtrum. She had a

deep, husky voice attributed to slack vocal chords observed by laryngoscopy. Blood and urine

analysis including serum copper, ceruloplasmin and α1-antitrypsin were in normal ranges (Jung,

et al., 1996). The karyotype was normal. Chest X-ray revealed a dilated aortic root. During

further follow-up, she developed a progressive dilation of the aortic root and of the ascending

aorta (29 mm) associated with a bicuspid aortic valve. At age 17, the Z score of the aortic root

was 4.3. At that time a mitral valve prolapse was noted. She had several cosmetic surgeries to

correct skin folds and ears. Wound healing was normal but the skin folds rapidly recurred (Fig.

1C).

Patient CL-4 and CL-5

Patients CL-4 and CL-5 are 3-year old identical twins born at term to unrelated healthy parents.

They presented at birth with generalized skin folds and typical facial characteristics including

large ears, a coarse face and a long philtrum. Both had a deep and husky voice. Until the last

evaluation at the age of 3 years, development and growth were normal (Fig. 1D). To date (at the

age of 6), neither of them has developed any cardiovascular or pulmonary complications.

Patient CL-6

This 7 year old Caucasian boy was the single child of unrelated parents. He was born following

an uneventful pregnancy with Caesarean section at 39 weeks of gestation. Birth weight was 3570

g (25th

percentile), length 50 cm (10th

percentile), head circumference 35 cm (25th

percentile).

Besides a mild, transient neonatal jaundice treated with phototherapy the postnatal period was

uneventful. Growth and psychomotor development was adequate. Skin laxity was first noticed at

the age of seven months. Subsequent follow-up showed recurrent but uncomplicated upper

airway infections and recurrent balanitis. At age 2, abdominal, renal and pelvic ultrasound were

normal. At age 5 (Fig. 1E, F), a cystourethrogram and chest X-ray were normal, but an

echocardiography showed dilatation of the aortic root (Z score = 4.7).

Electron microscopy shows diminished and disorganized elastin deposition onto the

microfibril bundles

Light microscopic examination of skin biopsies of patients CL-1 CL-2 and CL-3 showed severe

rarefaction of elastic tissue (Fig. 2B, C, D) compared to control (Fig. 2A). Electron microscopic

findings in the same patients (Fig. 2G, H, I) revealed a reduced number of elastic fibers with

diminished amounts of amorphous elastic material due to reduced and disorganized elastin

deposition onto microfibrillar bundles, compared to a control (Fig. 2F). The amorphous


10

component of the elastic fibers showed extensive branching and fragmentation and was not

properly associated with the microfibrils. A particular finding was the increasing electron density

from inner to outer regions of the elastic material and the separate elastic globules. Patent CL-4

showed milder abnormalities both by light (Fig. 2E) and by electron microscopy (Fig. 2J).

COOH terminal ELN mutations in ADCL

Upon direct sequencing, frameshift mutations were found in exon 30 (CL-3 and CL-6), exon 32

(CL-4, CL-5) and in exon 33 (CL-1, CL-2) representing 5 different mutations, as patients CL-4

and CL-5 were twins and had the same mutation (Fig. 3A). All mutations were found to be de

novo upon sequencing of the parents. Four mutations were novel but one, c. 2262delA, was

identical to a previously published mutation (Tassabehji, et al., 1998) identifying c. 2262delA as

a recurrent mutation.

Four of the five mutations c.2123del26insG (p.G708fsX), c.2137delG (p.A713fsX), c.2262delA

(p.L754fsX), c.2333delC (p.P778fsX) were -1 frameshifts, whereas one was a +1 frameshift

(c.2296_2299dupGCAG, p.A766fsX)). Irrespective of the location or type, each mutation was

predicted to result in the replacement of the normal C-terminus of tropoelastin (TE) with a

missense peptide sequence (Fig. 3B), similar to previously published mutations (Rodriguez-

Revenga, et al., 2004; Szabo, et al., 2006; Tassabehji, et al., 1998; Urban, et al., 2005; Zhang, et

al., 1999).

In patients CL-1, CL-2, CL-3 and CL-4 the presence of coding region and splice site mutations

was excluded by sequence analysis of the fibulin-4 and 5 (FBLN4 and FBLN5) genes. In patients

CL-2 and CL-3, α1-antitrypsin deficiency was excluded to be a predisposing factor for the

development of severe emphysema by the finding of an M/M genotype and phenotype through

direct sequencing of the SERPINA1 gene, and normal serum α1-antitrypsin measurement,

respectively (Jung, et al., 1996).

Stable mutant transcripts in ADCL fibroblasts

A complex interplay of the location of the mutation, naturally occurring alternative splicing of

exon 32, mutation-induced exon skipping and nonsense-mediated decay (NMD) can influence

the outcome of frameshift mutations in at the 3’-end of ELN (Szabo, et al., 2006). Therefore, we

studied the expression of wild type and mutant alleles by RT-PCR and semi-quantitative analysis

of amplification products by fluorescent labeling and capillary gel electrophoresis. Fibroblast

cultures were available for patients CL-1 (mutation c.2296_2299dupGCAG in exon 33), CL-3

(mutation c.2137delG in exon 30), and CL-4 (mutation c.2262delA in exon 32) (Fig. 3B).

Removal of exon 32 by alternative splicing was observed in control fibroblasts in about 55% of

the transcripts (peak at 249 base pairs).

Results 147

11

Patient CL-3 with a mutation in exon 30 (c.2137delG) had two abundant mutant mRNA species.

Inclusion of exon 32 yielded a premature termination codon and a truncated open reading frame

in 9% of ELN mRNA (Fig 3B, CL-3 MT). When exon 32 was spliced out, the transcript

contained an extended C-terminal missense sequence of 61 amino acids (AA) with a read-

through into the 3’-UTR and yielding an additional 25 AA (Fig. 3B, CL-3 MT-e32). This mutant

transcript represented 60% of ELN mRNA. Interestingly, CL-3 fibroblasts showed marked

overexpression of the mutant allele producing 69% of ELN mRNA, whereas the wildtype (WT)

allele accounted for only 31%. However, the abundance of the mutant full length (exon 32

containing) message was lower than wild type, presumably because frame shift mutation

c.2137delG introduced a premature termination codon introduced into exon 32 leading to NMD

of the mutant mRNA. Nevertheless, both mutant and wild type mRNA isoforms with exon 32

continued to represent a relatively minor fraction of total elastin mRNA.

In patient CL-4 (mutation c.2262delA in exon 32), the -1 frameshift mutation resulted in C-

terminal altered sequence of 28 AA and extension with 25 AA in the full length transcript

accounting for 44% of ELN mRNA (Fig. 3B). When exon 32 was spliced out, a normal elastin

protein isoform was produced, indistinguishable from the –e32 splice product of the wildtype

allele. CL-4 fibroblasts also showed significant overexpression of the mutant allele.

Patient CL-1 had a duplication of 4 nucleotides in exon 33 (c.2296_2299dupGCAG). This

frameshift mutation resulted in a C-terminal missense sequence of 26 AA and an extension of 23

AA irrespective of exon 32 splicing. Analysis of mRNA also revealed preferential expression of

the mutant allele (Fig. 3B). Irrespective of the inter-individual differences among patients in the

splicing frequency of exon 32 and the precise composition of the different mutant and splice

isoforms, we found high expression of the mutant allele in each case, suggesting that the

expression of a stable mutant elastin mRNA may be an important feature of ADCL.

Mutant tropoelastin is deposited in the extracellular matrix and results in clumping of

elastin

We performed colocalization immunofluorescent staining for fibrillin-1 and elastin on cultured

fibroblasts from CL-1, CL-3 and CL-4 and matching controls. In patient fibroblasts, elastin was

less densely deposited in elastic fibers fibrils composed of elastin and fibrillin-1, but showed

enhanced deposition in globular deposits consisting of elastin only (patients CI95%: 2.28 ± 0.64

gobules/nucleus versus control CI95%: 1.35 ± 0.21 globules/nucleus). Representative images are

shown for patient CL-4 and a matching control. Co-localization experiments of fibrillin-1 and

mutant elastin with a polyclonal antibody raised against the C-terminal -1 frameshift tropoelastin

sequence (fmTE) showed more prominent intracellular and globular extracellular staining and

less robust extracellular fibrillar staining in both patient CL-3 and CL-4 (Fig. 4B).


12

Elastic fiber formation is believed to depend on the polymerization of tropoelastin and on its

association with fibrillin microfibrils. In order to elucidate the reason of enhanced tropoelastin

globule formation, we analyzed the self-association properties of normal tropoelastin (TE) and

mutant tropoelastin with a -1 frameshift in exon 32 (fmTE) in vitro by monitoring the

temperature-dependent coacervation. Despite lower overall hydropathy (which generally inhibits

coacervation) of fmTE, its coacervation temperature was lower than that of TE (Fig. 5A),

consistent with increased self-association of mutant tropoelastin yielding globular aggregates.

Moreover, the coacervation temperature of an equimolar mixture of TE and fmTE were close to

fmTE alone, showing a dominant effect of fmTE on TE coacervation (Fig. 5B).

ADCL fibroblasts deposit less insoluble elastin

To test if abnormal deposition of elastin observed by immunostaining also affects the amount of

mature, crosslinked elastin in the ECM, we metabolically labeled the cell cultures, biochemically

isolated insoluble elastin and analyzed this compound relative to the amount of other intra- and

extracellular proteins (as a measure for cell metabolism). This revealed a significantly lower

amount of insoluble elastin in ADCL cells compared to controls after 4 and 8 days (Fig. 6).

Elastic fiber disorganization in ADCL results in upregulation of TGFβ signaling

Elastic fibers constitute of an elastic core embedded in a microfibrillar structure. These

microfibrils also serve as important extracellular storage sites for TGFβ. We therefore tested

whether the disruption of the elastic fibers influences TGFβ signaling. Staining for pSMAD2 in

fibroblast cultures of all patients showed significant upregulation of the TGFβ signaling pathway

(Fig. 7A, B).

Intracellular retention of fmTE and unfolded protein response

Recently, a transgenic mouse model for ADCL generated using a human elastin minigene with a

-1 frameshift in exon 30, showed intracellular retention of fmTE, induction of unfolded protein

response (UPR) and initiation of the apoptotic cascade in lung tissue (Hu et al, submitted).

Therefore, we investigated the UPR pathway in our ADCL fibroblast lines. The chaperone

binding protein (BiP) was upregulated in varying degrees in fibroblast cell cultures of patients

CL-1 (mutation in exon 33) (p=0.14), CL-3 (mutation in exon 30) (p<0.01) and CL-4 (mutation

in exon 32) (p<0.001), (Fig. 7C, Fig. 1S). BiP co-localized with TE in the ER, indicating

misfolding (Fig. 1S). We then assessed phosphorylated eukaryotic translation initiation factor

(peIF2α), a marker for ER stress, which was only significantly upregulated in patient CL-3 (Fig.

7D). This latter observation paralleled the findings for Caspase-3, a marker for apoptosis (Fig.

7E).

Results 149

13

Discussion

To date, cutis laxa syndromes have been classified mainly on both clinical grounds and mode of

inheritance (de Schepper, et al., 2003). Clinical overlap and related, unclassified disorders

illustrate many shortcomings of this classification. The recent progress in the molecular

elucidation of cutis laxa subtypes will improve the clinical definition of these disorders and will

facilitate genotype-phenotype correlations. In this study, we demonstrate that in ADCL, formerly

considered as a more benign disease, patients are also at risk for severe aortic and pulmonary

complications (including aortic root dilatation and emphysema), blurring the clinical distinction

from type I recessive cutis laxa. The typical facial characteristics were the most distinguishing

and ubiquitously present feature in all patients, with a long philtrum, large ears, a husky voice

and often a beaked nose. Generally, patients had severe, generalized and congenital cutis laxa,

but we also documented progression of an initially milder phenotype in one proband.

Molecular heterogeneity has been suggested in ADCL with most patients carrying a -1

frameshift in the 3’terminus of the ELN gene (Rodriguez-Revenga, et al., 2004; Szabo, et al.,

2006; Tassabehji, et al., 1998; Zhang, et al., 1999), and a family having a partial tandem

duplication (Urban, et al., 2005), all resulting in an extended protein. However, 2 reports

described a splice site mutation in exon 25 of ELN (Graul-Neumann, et al., 2008), a region

important in elastin crosslinking and a tandem duplication in the fibulin 5 gene (Markova, et al.,

2003), respectively. Our data now suggest that, at least in the majority of cases, ADCL can be

attributed to a mutant tropoelastin in which the C-terminus is replaced by an extended missense

peptide sequence as a result of a frameshift (fmTE). The observation of a first +1 frameshift (CL-

1) in a patient with a similar ADCL phenotype suggests that the molecular mechanisms act

largely independent of the amino acid composition at the 3’ terminus.

The mechanisms by which an extended elastin protein disrupts elastic fiber assembly and leads

to the observed phenotype have been a subject of discussion. Matrix deposition of mutant

tropoelastin was shown for a partial tandem duplication in the elastin gene (Urban, et al., 2005),

and confirmed in this report for the more common frameshift mutations. This carboxy-terminus

of TE has been implicated in microfibril associated glycoprotein binding (Brown-Augsburger, et

al., 1994) and interaction with cell-surface glycosaminoglycans (Broekelmann, et al., 2005), both

important in elastic fiber formation.

Time-lapse imaging studies showed that the assembly of elastic fibers was a complex process

with initial micro-assembly of tropoelastin monomers into globular aggregates, followed by a

cell-directed, dynamic macro-assembly step that fashioned large-scale fibrillar structures

(Czirok, et al., 2006; Kozel, et al., 2006). We observe an accumulation of globular elastin

aggregates in fibroblast cultures from patients with ADCL, suggesting that fmTE disrupts later

stages of the elastin assembly process. Decreased deposition of insoluble elastin by ADCL cells


14

provides further evidence for this notion. Finally, our electron microscopic results demonstrate

the abnormal elastin assembly, characterized by poor integration of elastin with microfibrils.

Our studies also clarify the exact mechanism by which fmTE disrupts elastin assembly. We

demonstrate that fmTE has increased self-association properties manifested by a lower

coacervation temperature resulting in enhanced globule formation. Moreover, fmTE had a

dominant effect on lowering the coacervation temperature in mixtures of fmTE and nTE

providing a molecular basis for the autosomal dominant inheritance of ADCL. Other studies

focusing on fmTE showed reduced binding to fibrillin-1 and fibulin-5 and, as a result, impaired

elastin accumulation on microfibrils in cell based assays (Sato, et al., 2006). Taken together,

these results suggest that a combination of increased aggregation of fmTE and its decreased

binding to microfibrils leads to a poor integration of the elastin and microfibrils in the elastic

fibers of patients with ADCL. Importantly, impaired association of tropoelastin with microfibrils

and abnormal aggregation of tropoelastin have been shown to be part of the molecular

mechanisms leading to multiple forms of cutis laxa, including ARCL1 caused by mutations in

FBLN5 (Hu, et al., 2006), and ARCL2 caused by mutations in ATP6V0A2 (Hucthagowder, et al.,

2009). These studies, together with the present report demonstrate shared disease mechanisms in

multiple CL syndromes.

A recently established transgenic mouse model, generated by pronuclear injection of a minigene

encoding a human elastin gene with a -1 frameshift in exon 30, showed intracellular retention

and initiation of the apoptotic cascade in lung tissue (Hu et al., submitted). In this report, we

confirmed increased ER stress and enhanced apoptosis in patient CL-3 with a similar mutation.

ER stress and apoptosis were not apparent in cells from of patients CL-1 (exon 33 mutation) and

CL-4 (exon 32 mutation), indicating that both the intrinsic characteristics of the protein

determining its subsequent folding and the amount of protein retained are important in this

mechanism. Apoptosis in these cells might manifest only when subjected to metabolic stress, or

under conditions of high TE synthesis. Developmental defects through increased apoptosis was

also shown in ARCL type II, caused both by ATP6V0A2 and PYCR1 mutations (Hucthagowder,

et al., 2009; Reversade, et al., 2009) and may therefore represent another common mechanism in

cutis laxa. However, the observed allele-specific differences in the extent of ER stress alone do

not explain the observed clinical variability in pulmonary and aortic involvement among

patients.

In MFS, another disease of the elastic fibers with aortic dilatation as a main clinical

manifestation, disruption of fibrillin-1 results in enhanced release of active TGFβ from the

extracellular matrix, inducing disorganization of the arterial wall (Habashi, et al., 2006).

Pulmonary emphysema in MFS is due to disruption of the pulmonary alveolae, also caused by

enhanced TGFβ signaling (Neptune, et al., 2003). Both the histological aberrations and the

clinical manifestation of aortic dilatation are prevented by antagonizing TGFβ signaling.

Results 151

15

Because of the close relationship between elastin and microfibrils in the elastic fibers, we

assessed TGFβ signaling in the fibroblast cultures of ADCL patients and found highly increased

levels of pSMAD2 staining consistent with increased TGFβ activity. TGFβ is released by

stretching the extracellular matrix and upon traction from myofibroblasts (Wipff, et al., 2007). In

further support of these data, a transgene mouse model of ADCL that develops emphysema

displays decreased lung stiffness, increased stretch at physiological pressures and increased

TGFβ signaling (Hu et al., submitted). Therefore, increased TGFβ activity likely accounts for

aortic root dilatation and emphysema and opens perspectives for treatment with losartan

(Habashi, et al., 2006). Also, different matrix characteristics may induce different levels of active

TGFβ with parallel clinical consequences for the respective tissues. It should be noted that

patients with exon 32 deletions have less severe internal organ involvement (p < 0.005, Table 1).

Indeed, exon 32 skipping results in a partial rescue and better elastic fiber formation (Fig. 2, Fig.

4). Hence, this extracellular matrix might preserve its resilient characteristics better in lung and

aortic tissues reducing the amount of activated TGFβ upon stretching.

In conclusion, these data provide new insights in the clinical and molecular findings in ADCL.

Partly retained mutant elastin may predispose to ER stress and apoptosis, while partly secreted

elastin results in impaired elastic fiber formation and reduced elastin quality. Enhanced TGFβ

release from this extracellular matrix clinically manifests in aortic dilatation and pulmonary

emphysema. Therefore these data may also be relevant for more common diseases of the elastic

fiber including isolated aortic aneurysms and emphysema.


16

Acknowledgements

We thank the families and patients for their interest and cooperation in this research. BC

is a research fellow and BL is a senior researcher of the Fund of Scientific Research –

Flanders. This work was supported by the National Institutes of Health [HL084944 to

Z.U. and R.P.M] and a Methusalem grant from the Flemmish Government and the Ghent

University (01M01108).

Results 153

17

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Tables:

Table 1. Aortic involvement in all published ADCL patients to date with an N-

terminal ELN mutation

ARD No ARD

Ex. 32

mutations 0 4 4

Mutations

outside ex. 32 9 1 10

9 5 14

ARD: Aortic root dilatation.

Fisher’s exact test: p = 0.005

3 patients with unknown clinical data and 3 patients with a chromosomal rearrangement

were excluded from the analysis.


22

Table 2: Antibodies used in this study

Antigen Host (type) label Conc provider Cat #

TE/elastin

Rabbit

(polyclonal) - 1:200 Dr. Mecham

fmTE

Rabbit

(polyclonal) - 1:200 Dr. Mecham

Fibrillin-1

Mouse

(monoclonal) - 1:500

Chemicon,

Billerica, MA MAB1919

BiP

Mouse


BD

Bioscience,

Franklin

Lakes, NJ 610978

peIF2α

Rabbit

(polyclonal) - 1:500

Cell Signaling

Technology,

Danvers, MA 3597

Caspase-3

Rabbit


Cell Signaling

Technology 9664

pSMAD2

Rabbit

(polyclonal) - 1:500

Cell Signaling

Technology 3101S

rabbit IgG

Donkey

(monoclonal)

AlexaFluor

488 (red) 1:500

Invitrogen,

Carlsbad, CA A21203

Mouse IgG

Donkey

(monoclonal)

AlexaFluor

594 (green) 1:500

Invitrogen,

Carlsbad, CA A21206

Results 159

23

Figure legends

Figure 1.

Clinical presentation of patients A: CL-1; B: CL-2; C: CL-3; D: CL-4 and 5, E, F: CL-6.

Note the variable severity of generalized cutis laxa, and the typical facial characteristics

including a premature aged appearance and a long philtrum (C, D) and large ears (D).

Figure 2.

Light microscopic images of semi-thin methylene blue-stained sections of skin biopsies

from A: a control (female, 15 years old); B: CL-1; C: CL-2; D: CL-3; E: CL-4. Using

this method, elastic fibers are stained darker than collagen bundles. Arrows indicate

elastic fibers (dark grey). Elastic fibers are scarce in ADCL samples (B - E).

Electron microscopic images of F: a control sample; G: CL-1; H: CL-2; I: CL-3; J: CL-

4 ; c, collagen fibers; e, elastin; arrow, microfibrils not embedded in elastin. Patients

show elastic fibers with diminished amounts and abnormal morphology of amorphous

elastin, including extensive branching and fragmentation and a lack of proper association

with the microfibrils. The electron density increases from inner to outer regions of the

elastic material and there are separate elastic globules. In patient CL-4 (J), similar, but

milder abnormalities are present.


24

Figure 3.

A: Schematic representation of the 3’-end of ELN with the location of the mutations

identified in each patient. The coding exons are drawn to scale but the 3’-untranslated

region (3’-UTR, hatched) and the introns are not.

B: Schematic representation of the structure of wildtype (WT) and mutant (MT) mRNA

products with semi-quantitative analysis. The expression of ELN transcripts in ADCL and

control fibroblasts was studied using a oligonucleotide sense and antisense primer

complementary to exon 29 and the 3’-UTR, respectively (Szabo, et al., 2006). M13

fluorescently labeled PCR-based amplicons were run on a genetic analyzer and data were

processed using Genescan software (Applied Biosystems). Electropherograms are shown

next to schematic representations of mRNA species identified in the control and in each

patient. Cross-hatched bars indicate sequences coding for missense or read-through

peptides generated by each frame shift mutation. Normal ELN sequence is shown by open

bars. For patients only the products of the mutant allele are drawn, but electropherograms

show both WT and MT products. Identities, sizes and % abundance relative to total ELN

mRNA in each sample are shown next to each peak. In control fibroblasts exon 32 is

spliced out (-e32) in about 55% of the transcripts (peak at 249 nucleotides). In CL-3 there

is marked overexpression of the mutant allele in transcripts lacking exon 32. Full

transcripts of the wild type and mutant allele are present at low quantities. In patient CL-4

total expression of the full length mRNA equals the transcript without exon 32 with

preferential expression of the mutant allele in the full-length transcript. In patient CL-1,

preferential expression of the 4 nucleotides larger mutant allele is observed.

Figure 4.

A: Representative colocalization immunofluorescent staining for fibrillin-1 (red) and TE

(green) of non-permeabilized cell cultures of patient CL-4 and a matching control (Co).

In the patient, elastin was less densely deposited in fibers, and showed significantly

enhanced aggregation in clumps. Nuclei were stained blue. Scale bar: 25 µm.

B: Colocalization immunofluorescent staining for fibrillin-1 (red) and fmTE (green) with

a polyclonal antibody against a C-terminal epitope of -1 frameshift mutant elastin in

permeabilized fibroblast cultures from both patients CL-3 and CL-4 showed more

incorporation of mutant elastin into globules and less deposition into elastic fibers than

TE. Arrows: clumps of elastin; arrowhead: elastic fiber. Nuclei were stained blue. Scale

bar: 25 µm.

Figure 5.

A: Coacervation of TE and fmTE. TE (triangles) and fmTE (circles) were diluted to a

concentration of 6.25 μM (open symbols) and 12.5 μM (closed symbols) in PBS. Light

scattering was monitored every 0.5 min while raising the temperature form 15 °C to 45

°C.

Results 161

25

B: Coacervation of a mixture of TE and fmTE was studied at a concentration of 6.25 μM

each in PBS. As reference, pure TE and fmTE protein solutions were evaluated at

concentration of 12.5 μM in PBS.


26

Figure 6.

Biochemically isolated insoluble elastin was measured form metabolically labeled cell

cultures and normalized to all intra- and extracellular proteins in the cell layer (as a

measure for cell metabolism) and cell count. Bars represent the averaged values of

insoluble elastin in all ADCL and matched control fibroblasts. There was a significant

reduction of mature, crosslinked, insoluble elastin after 4 and 8 days in ADCL patients.

*p value < 0.01 using the paired one-tailed student T-test. Error bars show the standard

deviation of the mean.

Figure 7.

A: p-SMAD2 staining in fibroblast cultures. All patients (CL-1, CL-3, and CL-4) showed

increased proportion of p-SMAD2 positive cells (p<0.001) indicating enhanced TGFβ

signaling compared to matching controls (Co-1, Co-3 and Co-4). Scale bar: 50 µm.

B: Quantitative morphometry of pSMAD2 staining in fibroblasts of ADCL patients and

their matching controls.

Quantitative morphometry of C: BiP; D: peIF2α; E: caspase-3 staining in fibroblasts of

ADCL patients and matching controls. Patients CL-3 and CL-4, but not patient CL-1,

showed upregulation of the chaperone BiP co-localizing with TE in the ER. peIF2α and

caspase-3 are significantly upregulated in patient CL-3, but not in CL-1 or CL-4. **:

p<0.001; *: p<0.01; NS: not significant.

Results 163

27

Figure 1

Figure 2


28

Results 165

29

Figure 3


30

Figure 4

Results 167

31

Figure 5


32

Figure 6

Results 169

33

Figure 7


Supplemental Data

Figure S1.

Immunofluorescent staining for elastin (green) and BiP (red) of permeabilized of patients

(CL-1, CL-3, and CL-4) and matching controls (Co-1, Co-3, Co-4). Arrowheads show

double positive cells in merged images. Nuclei were counterstained blue.

Discussion and conclusions 171

Discussion and Conclusions “The thing I hate about an argument is that it always interrupts a discussion”

Gilbert K. Chesterton

I. MARFAN SYNDROME

1. Genotype – phenotype correlation study

FBN1 mutations cause Marfan syndrome as well as a wide range of related connective

tissue disorders including FTAA, isolated EL, MASS, marfanoid habitus, SGS, and stiff skin

syndrome. Genotype – phenotype correlation studies have been hampered by the large intra-

and interfamilial variability as well as the progressive nature of the disease. Associations

made in previous studies are rather weak due to the small patient groups, the inclusion of

family members, and statistical methods that did not consider the age at which different

manifestations occurred (124, 135, 136, 235, 236). To overcome these problems, we focused

on a large set of 1013 probands only (to exclude intrafamilial variability as a confounding

variable) and used a time-to-event analysis to reliably estimate the cumulative probability of

observing the different manifestations of the type I fibrillinopathy.

This method enables us to establish some conclusions about the phenoype-genotype

correlations in type I fibrillinopathies. In congruence with previous findings (124, 135, 136,

235, 236), we show sound evidence for the association of cysteine alterations with an

increased prevalence of EL, and a more severe skeletal phenotype with underlying premature

truncation of the mutant allele. The incidence and severity of aortic disease seems to be

unrelated to the type of mutation. Only when comparing mutations eliminating a cysteine

residue versus introducing a cysteine residue, ARD is more frequently observed in the

former.

When addressing the location of the mutations, exon 24-32 mutations cause more severe

aortic, skeletal, and eye manifestations. A higher incidence of EL is observed in probands

with 5‟ mutations versus 3‟ mutations, but no differences are found when comparing

probands with mutations in exon 44-49, a region important in the regulation of the


bioavailability of TGFβ1 (237) versus other regions, or mutations in EGF-like domains

versus LTBP domains. There is concern that these results might be inflicted by report bias.

Indeed, in the beginning of FBN1 analysis only patients with convincing phenotypes were

molecularly examined and several laboratories only looked at this region, especially in cases

of neonatal MFS. However, a reanalysis of the data after exclusion of patients with a neonatal

onset reproduced, apart from EL, the same results at all ages. Moreover, there is a clear

overrepresentation of missense mutations in this region and premature truncation mutations

are never associated with a neonatal presentation. This is further underscored by a detailed

subanalysis of patients with mutations in exon 24-32 (238). These patients are in general

more severely affected, but the overall effects of the type of mutation type are alike.

This phenotype-genotype study never aimed at predicting severity or complications

associated with certain types of mutations in specific regions of the FBN1 gene. Indeed, many

exceptions to these indicative results are documented in literature (124, 154, 239, 240) and in

the expertise of the Center for Medical Genetics. By contrast, this study provides some

mechanistic insights in the pathogenesis of the type I fibrillinopathies. These results argue for

a tissue-specific consideration of both structural defects of the microfibril and functional

consequences of fibrillin-1 deficiency. Indeed, clinical impact is likely to result from a

combination of haploinsufficiency and dominant negative effects acting within the context of

sequestered TGFβ. Fibrillin-1 deficiency related distortion of TGFβ signaling is likely

dependant on the amount of tissue specific TGFβ stocks. For instance, the predominating

dominant negative effect of incorrect cysteine localization likely has a detrimental effect on

zonular microfibril structure and implicates a higher risk for the development of lens

luxation, unrelated to TGFβ signaling. By contrast, the association of a more severe skeletal

phenotype with premature truncation mutations implicates a critical contribution of

haploinsufficiency to long bone overgrowth. This finding subscribes the previously suggested

hypothesis, based on knowledge of studies in mice, that haploinsufficiency-imposed

mechanisms may be sufficient to reach clinical significance, whilst dominant negative

mechanisms need a critical context of half normal production of wild type protein (241).

However, for aortic pathology no major differences between mutation types can be

demonstrated. This is in line with previous evidence from studies in mice that show that both

dominant negative effects and haplo-insufficiency-imposed reduction in microfibrils result in

enhanced TGFβ signaling in the aorta (25, 242-246). The above findings also comply with

the pathophysiology in LDS. TGFBR1 or TGFBR2 mutations implicate altered TGFβ


signaling rather than structural abnormalities of the microfibril (247). LDS patients show

considerable clinical overlap with MFS for the skeletal and cardiovascular system, but do not

have lens dislocation. Thus, the tissue specific organization of normal and mutant fibrillin

microfibrils has its implications whether FBN1 mutations may become clinically relevant and

through which mechanism(s). Finally, the finding of a more severe and complete phenotype

in association with mutations in the central region (exons 24-32) confirms the important role

of this region in the stabilization of the fibrillin molecule with obvious consequences towards

structure and function.

The absence of overt genotype-phenotype correlations together with well-documented

clinical variability within families argues for the existence of modifier genes. Recent findings

suggest that some of these modifier genes might be found in both the canonical (Smad2/3

signaling cascade) and non-canonical (MAPK: Jnk, Erk, p38) pathways of the TGFβ

signaling pathways (248). Moreover elevated TGFβ1 serum concentrations were found in

MFS and may serve as both a prognostic and therapeutic marker. Indeed, TGFβ1 serum

levels decreased upon effective treatment with losartan (249).

2. Towards a New Nosology for the Marfan Syndrome

Diagnostic criteria for diseases are defined to improve communication between health

care providers, to provide easy and integrated access to healthcare for patients, and to

facilitate communication between researchers. From this point of view a few drawbacks of

the Ghent nosology need to be addressed:

1. The high specificity of the 1996 revised Ghent nosology has been demonstrated with

molecular confirmation of the diagnosis in over 90% of the Marfan patients (123).

Evaluation of sensitivity is more complex and depending on the „target‟ population, as

underscored by studies in child probands and adults not fulfilling the Ghent criteria

(149, 250). Within this context, it should be noted that molecular testing is not always

available or feasible.

2. While eye and skeletal manifestations can be disabling (251), the main concern in

patients with MFS remains the aortic risk. In our and other‟s experience, it is that risk

that limits personal and professional aspirations and endangers psychological well-


being (251). Discrimination by employers and insurance companies is based on this

manifestation that may shorten life expectancy and jeopardize the ability to work. In

this context, it is of concern that in the absence of aortic dilatation, patients may carry

the diagnosis rather as a social stigma than as a guarantee for optimal medical care.

3. Some criteria are difficult to assess (e.g. dural ectasia, acetabular protrusion, increased

globe length of the eye), making them impractical and often not documented (252).

Moreover, several hinge-points within the Ghent nosology have not been validated for

sensitivity and specificity in an evidence-based manner (e.g. dural ectasia) and do not

necessarily make a fundamental difference in the management of the patient as they

do not independently increase the risk for aortic root dilatation.

4. Recently, confusion arose about the definition and molecular basis of MFS and

related syndromes, and the aortic risks they infer. The cost, availability, and

sensitivity of molecular analysis may differ geographically. Therefore the criteria

should be complemented with clinical triggers that urge further assessment of the

patient towards related disorders, in order to provide correct management for the

patient.

5. Finally, the diagnostic criteria should be complemented with management and follow-

up guidelines for various patient groups, including children that do not yet fulfill the

criteria. This will facilitate communication between health care providers.

The revised criteria put more weight on the 2 main manifestations of MFS: aortic root

dilatation and ectopia lentis. In a proband, the presence of ARD and EL are sufficient to make

the diagnosis of MFS, irrespective of systemic involvement, as no other disease described so

far combines both manifestations. When ARD is present in the absence of EL, the finding of

a sufficient systemic score (≥ 7 points) or a pathogenic FBN1 mutation completes the

diagnosis.

The use of the systemic score avoids obligate thresholds that lack clear validation and will

hopefully result in a more evidence-based assessment. The systemic score comprises all

relevant clinical findings in MFS (besides ARD and EL), that have sufficiently been

validated. Some of the findings, however, are made less influential (dural ectasia), are

abandoned (pulmonary artery dilatation and descending aortic dilatation) or are simplified


(flat cornea and increased globe length). Dural ectasia is a sensitive, but non-specific finding

and therefore cannot be attributed the same value as ARD or EL. Pulmonary artery dilatation

is often seen in MFS, even before ARD becomes apparent, but clear thresholds still need

validation (130). Descending aortic dissection is rare in the absence of ARD and is a highly

non-specific finding. Increased globe length and flat cornea are sensitive parameters in MFS

but are seldom documented (252) as assessment is impractical. Therefore, we propose to

attribute one point to severe myopia (> -3 diopters) as a sensitive but non-specific

manifestation.

From a therapeutic point of view aortic dilatation caused by FBN1 mutations should be

followed and treated similarly irrespective of systemic or eye involvement. This concept puts

more weight on the molecular screening of the FBN1 gene, which has become feasible with

the increased performance of screening techniques (134). Moreover, data based on the Ghent

criteria show a molecular contribution to the diagnosis in over 12% of patients (252), and up

to 29 % when considering child probands only (250). However, it is stressed that a negative

FBN1 analysis, even after thorough screening, does not exclude MFS.

In case a proband presents with EL without ARD, the presence of an FBN1 previously

associated with ARD (in a related or unrelated proband) is required to diagnose MFS. In case

of a family history of MFS, the presence of ARD, EL or a systemic score of ≥ 7 points suffice

to establish the diagnosis.

These criteria introduced new important philosophies in the diagnostic process of Marfan

syndrome. Firstly, in the absence of a clear indication of aortic risk, the diagnosis of MFS is

not established, but the patient is subjected to strict follow-up. Possible scenarios in adults

include ectopia lentis syndrome, mitral valve prolapse syndrome and the MASS phenotype

(myopia, mitral valve prolapse, aorta, skin and skeletal findings). In patients below the age of

20 years old, we prefer to use the terms „non-specific connective tissue disorder‟ when

insufficient systemic features (< 7) and/or borderline aortic root measurements (Z < 3) are

present (without FBN1 mutation) or „potential MFS‟ in case an FBN1 mutation is found and

aortic dimensions are below the threshold. This may delay the diagnosis in some instances

but will avoid overdiagnosis of a disease that is primarily associated with aortic risk in the


absence of tangible evidence of such risk. Indeed, the diagnosis may be stigmatizing, create a

psychosocial burden and has an important impact on the patient personal aspirations, career

development, life-insurance and family planning. This will hopefully result in a more patient-

centered approach to the diagnosis. Indications of aortic risk include the presence of aortic

dilatation in a family member or the presence of an FBN1 mutation that has previously been

associated with aortic root dilatation. Indeed, large studies suggest that clinical variability is

determined by modifier genes or that genotype-phenotype correlations are subtle (252). As

such the aortic risk is unpredictable based on the nature and location of the mutations alone.

Secondly, these criteria introduced the concept that diagnostic perseverance is needed when

patients satisfy the criteria for MFS, but do present with unexpected findings, that may point

to related disorders, including LDS, vascular EDS, SGS. This is not trivial since related

disorders often have a unique risk profile and warrant different follow-up and management

guidelines. Screening of relevant genes is therefore advised if available and feasible.

We recognize that the diagnostic evaluation remains complex due to the high

interindividual variability and evolving character of connective tissue diseases, the extensive

differential diagnosis and the absence of truly validated standards. Some patients will remain

difficult to classify but these patients should be rare and will hopefully benefit from better

definition of still unrecognized phenotypes in the future. As simplicity is subjected to

accuracy, concerns about delayed diagnosis and additional diagnostic categories will

hopefully be countered by the focus on vascular disease - avoiding deleterious and often

irreversible consequences - and the addition of clear follow-up and management guidelines. It

is our hope that this revision of the criteria will make them more patient-centered and

evidence-based. A comparative retrospective analysis on the Ghent dataset has shown ~90%

concordance between the new nosology and the Ghent nosology. The 10% discordance was

generally protective with earlier diagnosis in children with a clinically convincing phenotype

and delayed diagnosis in the absence of a clear cardiovascular risk. Moreover, sensitivity and

specificity were further increased to 98% and 93% (CMGG, unpublished data).


II. CONGENITAL CONTRACTURAL ARACHNODACTYLY

Described for the first time in 1971 by Beals and Hecht (159), congenital contractural

arachnodactyly or distal arthrogryposis type 9 still remains understudied both on the clinical

and molecular level. In 1994, a large review by Viljoen et al (158) comprehensively reported

the clinical findings in all thus far published patients with CCA, but these results might have

been mitigated by the unavailability to confirm the diagnosis on a molecular level.

In 1995, the clinical resemblance with Marfan syndrome led to the discovery of the causal

gene FBN2, encoding fibrillin-2 (31), but in contrast to Marfan syndrome, patients and

mutations reported remained confined to small series and case reports with a total of 26

reported mutations (31, 160, 161, 253-258). Remarkably, all of these mutations situate in the

central stretch of cbEGF-like domains of the molecule and it has been suggested that

mutations outside this region do not relate to CCA (160, 256). However, only a minority of

researchers screened the whole gene (254-256, 259) and in some CCA patients, no mutation

in the central region of the FBN2 gene was found (160). Also, one patient was reported with a

premature truncating mutation (160) that corroborates a contribution of haploinsufficiency in

the etiopathogenesis as well.

Prior to this work, the phenotype was not fully characterized. Our cohort provides an in-

depth study of the clinical presentation. All probands show at least 3 main characteristics of

CCA (arachnodactyly, contractures, crumpled ears and scoliosis), and most of them even all

4. Besides a detailed description of known CCA features, this report expands the phenotype

with new features. Pyelo-ureteral junction stenosis in CCA patients adds to the knowledge

that connective tissue diseases may affect the urinary tract, as seen in some forms of cutis

laxa and ATS (38, 260). Keratoconus, a rather non-specific finding, was found in two

patients. Lens ectopy, still considered a CCA feature in 1994 (158), is never documented in a

patient with an FBN2 mutation. Most importantly, this work assesses the cardiovascular

findings in detail. ARD, a finding previously thought to differentiate from MFS, was present

in 3 probands, in addition to 4 previously published probands (160, 161). Although aortic

dilatation has not been unequivocally shown progressive to dissection, it should warrant

aortic follow-up at regular intervals. Minor cardiovascular anomalies including septal defects

and mitral valve prolapse are rare and converge with population frequencies (261).


This study also presents the largest molecular study ever reported on CCA. In 32

carefully diagnosed probands, we detected 14 mutations of which 13 were novel. We are the

first to report a mutation outside the central stretch of cbEGF-like domains. One patient

harbored an exon 17 mutation affecting a conserved residue in the 2nd

8-cys domain. We

therefore propose a two-step strategy for FBN2 screening, starting with exons 24-36. Our

molecular data show that, like in MFS, many mutations affect conserved cysteins, the

calcium binding consensus sequence DID/NE, or correct splicing (252). In contrast to FBN1

mutations, nonsense mutations are rare in the FBN2 gene. This may be due to the fact that

mutations are only sought in the central region of the gene. Indeed, when considering the

neonatal region only in the FBN1 gene, there is a clear underrepresentation of premature

truncation mutations as well (238, 252). For similar genes, the difference in number of

reported mutations in the FBN1 and FBN2 genes is surprising. Moreover, de novo mutations

represent up to 30% in FBN1 gene (252), which contrasts with the low number of CCA

patients mutations without a family history. Since FBN1 and FBN2 have overlapping

functions, and FBN1 expression takes over from FBN2 expression during later development,

FBN1 probably puts more weight on proper microfibril function (62, 77, 78, 262). This

hypothesis is supported by the age – related improving of the CCA phenotype. An interesting

observation in this cohort is the absence of FBN2 mutations in 2 patients with a severe/lethal

form of CCA. Previously one patient with a segregating FBN2 mutation was found to have an

interrupted aortic arch and a large VSD (258). Although the lethal phenotype might still

represent the extreme end of CCA, it is more likely caused by disruption of other genes.

Indeed, we recently found a complex 5p rearrangement in a patient with arachnodactyly,

interrupted aortic arch, contractures, and genital abnormalities (CMGG, unpublished results).

The question of locus heterogeneity emerges from this study. In analogy with the

mutational spectrum in the FBN1 gene, direct sequencing of all exons and exon-intron

boundaries should be able to detect most mutations. Therefore, the homogeneity of the FBN2

positive and FBN2 negative group contrasts with the mutation uptake rate of only 44%, a

number that situates within the range of previously reported series (27% – 75%) (160, 255).

MAGP1 seemed a good candidate gene because its spatiotemporal expression pattern

parallels FBN2 (263), it is a binding partner of fibrillin-2 (264) and it has a role in microfibril

assembly, but no MAGP1 mutations were identified in the FBN2 negative probands. Other

candidates can be sought in proteins important in microfibril assembly, genes causing related


syndromes including FGFR3 mutations that cause a syndrome of contractures,

arachnodactyly, tall stature and hearing loss (CATSHL) (265), and the family of distal

arthrogryposes (265).

Like in many connective tissue disorders, inter- and intrafamilial variability poses a major

counseling issue to the physician. An illustrative example is the difference in number of

probands with and without an FBN2 mutation and a positive family history. Assessment of

family members in retrospect usually occurs more carefully once a mutation is found. Some

authors speculate that variability parallels mutant allele expression (254, 258) and that

overexpression of the mutant allele is necessary to produce a CCA phenotype (257). This

hypothesis is refuted by others (253), as levels of mutant transcript as low as 25% were found

penetrant at the clinical level (254). In MFS, similar hypotheses have been postulated and

refuted (25, 242, 244-246). Alternatively, it cannot be excluded that fibroblast expression

levels introduce artifacts, since FBN2 is only expressed at low levels in these systems.

Moreover, ex vivo systems may certainly not reflect expression during fetal development.

Even in control individuals, preferential expression of one allele is sometimes observed

(257). A discussion on the relative contribution of haplo-insufficiency and dominant negative

effects will be difficult to strengthen with evidence in the absence of relevant patient tissues

or mice models that faithfully represent the disorder. This discussion becomes even more

difficult in view of the recent findings connecting enhanced TGFβ signaling with disturbed

microfibril functioning (114). Also, genotype-phenotype correlations are subtle in MFS and

corroborate a role for modifier genes in the clinical expression of the disease (252). Similar

mechanisms are to be expected in CCA. Also important for genetic counseling, we confirmed

somatic mosaicism in a third family (257, 258), although non-penetrance could not be fully

excluded.


III. ARTERIAL TORTUOSITY SYNDROME

1. Molecular pathogenesis of ATS - Does sugar do the trick?

In arterial tortuosity syndrome the extreme vascular developmental defects in association

with craniofacial, skin and skeletal connective tissue findings is highly reminiscent of a

connective tissue disease related to cutis laxa, MFS and Loeys-Dietz syndrome (162, 165,

174, 266). In the search for the molecular defect, obvious functional candidate genes

encoding extracellular matrix proteins were firstly analyzed and excluded (166). Moreover, a

previously mapped region did not include prominent candidate genes (176). In the absence of

high-throughput sequencing techniques, we used a fine-mapping strategy based upon the

assumption that the families originating from a same region in Morocco would possess a

same founder mutation. Sequencing of the remaining 7 genes revealed mutations in

SLC2A10, encoding a facilitative glucose transporter (266).

This surprising finding corroborates a link with glucose transport and broadens the view

on extracellular matrix homeostasis (267). Being an outlier within the group of facilitating

glucose transporters (268-270), GLUT10 still remains a poorly characterized protein. It lacks

the 4th

sugar transport motif and is characterized by the presence of a large extracellular loop

between the 9th

and 10th

transmembrane domain (268-270). Our immunocytochemistry data

indicate an intense perinuclear staining of GLUT10. However, it remains unsolved whether

the main function of GLUT10 is to mediate glucose transport and if this transport regulates

nuclear glucose levels. Till now, glucose was always considered to diffuse through the

nuclear pores and no measurable gradients are measured when analyzing glucose

concentrations in the nucleus and cytoplasm upon exposition of COS-7 cells to increasing

external glucose levels (271). However, some observations favor transnuclear glucose

transport; (i) Xenopous oocyte experiments do demonstrate high affinity transport of glucose

and other hexoses (268) that can mediate glucose transport away from the low glucose

concentrated cytosol. (ii) Normal glucose homeostasis in ATS patients precludes a major role

for GLUT10 in glucose transport across the plasma membrane (260). (iii) The distinct

structural features of GLUT10 suggest some differences towards function or location

compared to other, better characterized glucose transporters. Still, in this early stage,


precaution should be used to determine the definite localization of the transporter, especially

as some authors localize GLUT10 on the mitochondria (272, 273).

In congruence with LDS (155), our findings show enhanced TGFβ signaling in smooth

muscle cells from ATS patients. Impaired glucose-dependant regulation of decorin

expression, a slrp TGFβ inhibitor (107, 274, 275), elegantly links nuclear glucose transport,

TGFβ signaling and vasculopathy. An intriguing question that arises from this research is

whether altered TGFβ signaling is important in the pathophysiology of diabetic vasculopathy.

Indeed, the pathophysiology in diabetic nephropathy involves increased TGFβ signaling

(276-279), and long term effects can be prevented by TGFβ antagonism in mice (280). It is

known that glucose levels have a direct impact on extracellular matrix proteins that influence

TGFβ signaling including TGFβ1 (281), TSP1 (282), matrix metalloproteinases, and

proteoglycans. Also, end-stage glycosylation products may enhance active TGFβ release

through a stiffer extracellular matrix (283). Our data now suggest that glucose levels may

directly influence the TGFβ signaling pathway and induce vascular changes in diabetics. In

congruence with this, glucose-induced cell hypertrophy is mediated through upregulation of

the TGFβ receptors 1 and 2 and increased activation of TGFβ by matrix metalloproteinases,

and can be prevented by blocking the kinase activity of the TGFβ receptor 1 (284). In view of

the different vascular anatomy of ATS and advanced diabetes, it should be noted that the

effect and impact of TGFβ signaling on vascular patterning might be highly different

according to the stage of vascular development. Indeed, MFS is primarily characterized by

aneurysm formation, in contrast to tortuosity. Marfan mouse models have shown that

fibrillin-1 is necessary to preserve, rather than to assemble the elastic fibers so enhanced

TGFβ signaling occurs only after elastic fiber completion (285, 286). In LDS and ATS both

tortuosity and aneurysms are found. Therefore, one might hypothesize that altered TGFβ

signaling before elastic fibers are composed induces aberrant patterning and tortuosity, while

malfunctioning elastic fibers, whether due to early degeneration (like in diabetes and aging)

or structural insufficiency of the elastic fiber (like in MFS), will increase TGFβ signaling

only afterwards and result in aneurysm formation and vascular degeneration. Indeed, in

diabetic retinopathy, microaneurysms occur on the vessels in the non-proliferative stage,

while retinal neo-angiogenesis in the proliferative stage shows tortuosity and underscores this

hypothesis. Therefore, the molecular finding in ATS might be highly relevant for the

pathogenesis in diabetes mellitus.


2. The search for a representative animal model

The poor availability of appropriate tissues from patients with ATS precludes a thorough

understanding of the pathogenetic molecular mechanisms connecting the genetic defect with

the clinical presentation of the disease. In first instance, we established a knock-in mouse

model carrying homozygous G128E and S150F missense mutations, with highly probable

pathogenicity. The mice did not show any macroscopic vascular anomalies as shown by

abdominal ultrasound, surgical exploration, and vascular corrosion casting. In addition,

microscopy revealed normal elastic fibers and wall thickness of both the tail and femoral

arteries. The absence or low-grade expression of a phenotype, or a clinically unrelated

presentation in mice carrying a same genetic defect as their human counterparts has been

recognized before. FBN2 deficient mice show syndactyly, and are small but do not show

contractures, scoliosis or ear abnormalities (287). Mice knock-out for ABCC6 show ectopic

calcification but not the typical clinical manifestations of Pseudoxanthoma elasticum (288).

Decorin targeted mice show an EDS like phenotype (289), while in humans decorin

deficiency results in congenital stromal corneal dystrophy (43). A subsequent publication in

exactly the same GLUT10 mutant mouse strain, did report some thickening and irregularities

of the vessel wall due to increased elastic fibers and some endothelial hypertrophy of the

intima (290). Because no robust quantification of the lesions was performed, and because

these findings were only reported present in older mice the significance of these findings is

unclear. Moreover, all mice had a normal lifespan (>18 months). We cannot exclude that

slight arterial aberrations occur with age. However, the vascular lesions in these mice strains

are not at all comparable to the vascular aberrations seen in human ATS patients, nor by

anatomy, time of presentation, progression (260), or histology. We therefore need another

approach to further examine the molecular consequences of GLUT10 deficiency.

A Zebrafish morpholino approach does show a wavy notochord and cardiovascular

abnormalities with a reduced heart rate and blood flow, and incomplete and irregular

patterning of stenotic vasculature, especially in the tail (291). This parallels the findings in

the human correlate of stenotic arteries and aberrant origins of aortic side-branches.

Comparative array analysis for cDNA shows marked alterations in both the TGFβ signaling

pathway, that can be copied by pharmacological inhibition of TGFβ Receptor 1 transduction


(291), concordant with data found by Wu et al (284). Interestingly, GLUT10 knockdown (but

not TGFβ receptor 1 inhibition) also induced severe alterations in mitochondrial functioning,

which is not surprising in view of recent data in patients with PYCR1 mutations (190). It is

therefore possible that GLUT10 also has a function in transmitochondrial glucose transport,

as previously suggested (272, 273).

3. A profound clinical characterization

Recently, arterial tortuosity has been described in several syndromes that show

considerable clinical overlap including the LDS (247), and recessive cutis laxa due to FBLN4

mutations (48). In view of this, the ATS phenotype still needed profound characterization.

We were able to study 16 ATS patients clinically, radiologically, metabolically, and

molecularly into detail. Upon clinical presentation, the most distinguishing features with

related disorders is the typical craniofacial presentation with an elongated face,

blepharophimosis, large ears and sagging cheeks that becomes more pronounced with age. In

most cases, a cardiovascular symptom (cardiac murmur, cyanosis, stroke, visible artery

pulsations) brings the patient to medical attention. A few times, an inguinal or diaphragmatic

hernia was the reason for searching medical aid. Other system involvement is diverse and

variable, and includes the skeletal, skin, eye, respiratory and urological system.

Extensive tortuosity of large and middle-sized arteries is present in all patients and

aberrant origins of the aortic side-branches are frequently documented. Smaller arteries are

relatively spared as tortuosity was absent in the retinal arteries. This implies a role for

SLC2A10 especially in the developmental patterning of the large arteries. It is difficult to

differentiate ATS from related disorders based on the tortuosity alone. More generalized

tortuosity, aberrant origins of the aortic side-branches, pulmonary stenoses, focal stenoses of

the large arteries and long stenotic stretches of the aorta are rather suggestive of ATS. Also,

in our series, patients seem to have a better prognosis than previously reported, both for

aneurysm formation and survival. Only one patient died to an unrelated cause and the

majority passed the age of five with ages ranging from 16 months to 39 years, in contrast to a

review that mentions a mortality rate of 40% before the age of five (172). Only two patients

were diagnosed with an aneurysm of the aortic root. These observations were confirmed in

subsequent reports (292-294). The previously reported prognosis might have been mitigated


by a publication bias towards the more severe phenotypes, but also because ATS was difficult

to distinguish from related disorders in the absence of molecular diagnostics. Indeed, some

patients with a clinical phenotype suggestive of ATS, now are confirmed harboring FBLN4

mutations (295) or Loeys-Dietz syndrome (CMGG, unpublished results). Recent case reports

also illustrate the surgical amenability of both aortic aneurysms and pulmonary hypertension

due to peripheral stenoses (296, 297). One report mentioned a prosperous pregnancy outcome

following caesarean section (298). A main issue though remains the stroke risk. Previously,

patients have been reported with ischemic events (163, 169, 172) and our data confirm this

risk. At this point it remains unclear which events precede the strokes. We could document

dissection in one patient, but not in another. Other mechanisms may include trombotic

processes following increased shear stress and intima injury in tortuous or stenotic vessels or

even severe vasoconstriction. Indeed, vasomotor instability was a frequent complaint in ATS

patients. This distinction is not irrelevant as depending on the cause of the ischemic event,

salicylate therapy may be indicated or not.

Some recessive disorders present with a mild phenotype in unaffected carriers. MR

angiographies in several carriers of the different mutation types (missense, nonsense and

frameshift) excluded anatomical vascular abnormalities. This might not fully eliminate a

small cardiovascular risk at long term.

Finally, reviewing the molecular findings in ATS patients reveals two clinically relevant

observations. Firstly, many mutations are recurrent and on several occasions we were able to

track them down to a founder mutation after haplotyping. In one mutation, where unequal

crossing over between 2 Alu elements resulted in a deletion of exon 4, it was unclear whether

this might represent a mutational hotspot or a founder effect since only a small overlapping

region was demonstrated. Secondly, all non-truncating mutations identified to date, are

localized on the endofacial site or transmembrane parts of the protein. It could be that the

exofacial loop characteristic of GLUT10, that could be important for specific features, is not

necessary for proper basic functioning of the protein. Mutation directed targeting experiments

could reveal clearer insights into the real function of this domain.


IV. AUTOSOMAL DOMINANT CUTIS LAXA

1. The clinical spectrum of ADCL within the cutis laxa

syndromes

With recent breakthroughs in the elucidation of the genetic background of cutis laxa

syndromes (PYCR1, LTBP4 (38), ATP6V0A2 (200)), molecular findings can be coupled back

to the clinical presentation. As such, it became clear that several clinical entities fit the same

spectrum. For example, PYCR1 defects are shared by patients initially classified as having

ARCL type 2, GO, WSS, and de Barsy syndrome, previously considered distinct entities

(190, 199, 202). Alternatively, similar clinical entities can be caused by different genes. For

instance, GO patients may harbor SCYL1BP1, PYCR1, or ATP60VA2 defects (190, 200, 204).

Careful consideration of both molecular causes and clinical presentation are now important to

reevaluate the previous classification and attributed clinical and molecular spectrum of the

different cutis laxa entities.

ADCL has historically been considered a benign disease confined to the skin. The

integration of our molecular and clinical data has now made clear that these patients do have

an increased risk for severe cardiovascular and pulmonary complications. Indeed, 4 out of 5

probands did have mild to severe aortic root dilatation. Progression was shown in two

patients and in one of them aortic surgery was canceled because of severe pulmonary

emphysema. Aortic complications, including aortic dissection, were previously noted in

adults with dominant cutis laxa (299). Another report also mentioned pulmonary emphysema

requiring lung transplantation in a 50 year old female with additional risk factors including

smoking and hemizygosity for α1-antitrypsine deficiency (184). These risk factors were

absent in our patient series.

Our data demonstrate that the differential diagnosis between type 1 recessive cutis laxa

and dominant cutis laxa is no longer clear-cut, especially in the absence of a family history.

At least in two patients a recessive form of cutis laxa was initially suspected and one patient


was published as such in the past (300), both based on clinical grounds and pathology.

Nevertheless, in retrospect several clinical findings hint towards the diagnosis. For instance,

patients with ADCL typically present with distinct craniofacial features including a long

philtrum, large ears, and a husky voice. The generalized cutis laxa seems more severe than in

patients with FBLN4 mutations (48, 295), but comparable to patients with FBLN5 mutations

(49). Indeed, FBLN4 mutations usually result in a hyperextensible, soft skin, without obvious

cutis laxa. In contrast to patients with FBLN4 mutations (48, 295), arterial tortuosity is

usually not seen in ADCL, or very mild (197). Most importantly, there still remains a

difference in survival between ADCL and ARCL type 1 (38, 49, 299). Therefore, the use of

molecular genetics has an important place in the diagnosis and management of cutis laxa

patients.

We also document the aberrant histology and electron microscopy in ADCL in detail.

Histology shows sparse and fragmented elastic fibers. On electron microscopy these elastic

fibers appear as globular elastin deposits on the microfibrils, while in normal skin the elastin

forms a continuous, smooth and solid core embedded in the microfibrillar scaffold. Despite

these distinct abnormalities on light and electron microscopy, they do not seem specific

enough to enable a definite diagnosis of ADCL, without integrating them with the clinical

and molecular findings. Indeed, in patients with FBLN5 mutations the elastic fiber appears

very similar both on light and electronmicroscopy (301, 302). In contrast, in LTBP4 mutants,

the globular aggregates appear more smoothened and seem to position more on the outer side

of the microfibrillar bundles (38).

2. The Molecular Background of ADCL

Since the first report of a 3‟ terminal ELN mutation in a patient with ADCL (33), only 6

additional mutations were published (33, 184, 195, 197, 299). We gathered 5 additional

probands with generalized cutis laxa and typical craniofacial features reminiscent of ADCL

and were able to find 3‟ terminal ELN mutations in all. This brings the total number of 3‟

terminal ELN mutations reported to 12 and implicates them as the major cause of ADCL.

Moreover, we confirm that an altered and extended C-terminal sequence is the most

important determinant for disease. We show that there are no obvious differences in clinical


findings or pathology data between -1 and +1 frameshift mutations. Likewise, Urban et al

(184) showed earlier that a complex karyotypic rearrangement at the ELN 3‟ terminus also

induces an ADCL phenotype. Molecular heterogeneity has been reported in 2 cases. One

patient carried a tandem duplication in the FBLN5 gene expected to result in a dominant

negative mechanism (194). Although plausible, the causal implication of this duplication has

not been proven. Indeed, (1) no DNA was available from the father to confirm that the

mutation occurred de novo; (2) the duplication resulted in a secreted stable mutant fibulin 5

product, but no evidence of a dominant negative mechanism was given. More recently a

splice site mutation in exon 25 was found in both a normal father and his affected son (198).

Although the exon 25 mutation is situated in the lysyl crosslinking domain, an important

region for the maturation of tropoelastin into insoluble elastin, it is troubling that no evidence

of disease whatsoever was found in the father. Overall, we conclude that in ADCL, molecular

analysis should start with the C-terminal ELN exons.

Mutations in different exons of the ELN gene induce different transcripts and tropoelastin

proteins that vary both in composition and size of the C terminal end. The low number of

mutations published does not yet allow clear phenotype-genotype correlation. However, we

observed that no internal organ involvement occurred in all 4 patients with exon 32 mutations

(p = 0.005). This can be explained by the fact that exon 32 is spliced out in skin fibroblasts in

about two thirds of the transcripts and will generate a normal protein in these instances. No

data about the exon 32 splicing frequency in vascular smooth muscle cells and lung tissue are

available.

3. Etiopathogenesis of Cutis Laxa – the emergence of final

common pathways?

The recent advances in the molecular understanding of cutis laxa syndromes reveal an

even more complex heterogeneity than expected. Mutations in diverse genes with seemingly

unrelated functions result in disorganized elastic fiber assembly and similar clinical

presentations. It is likely that somehow, these genetic defects are linked in one or more final

common pathways.


Our data show mutant tropoelastin deposition in the ECM with clump formation resulting

in reduced elastin quality as shown in fibroblast cultures and by electron microscopy. In a

patient with an exon 30 mutation, we also demonstrate endoplasmatic reticulum stress,

parallel with increased apoptosis, probably due to significant amounts of retained misfolded

elastin protein. Evidence for increased endoplasmatic reticulum stress and apoptosis is also

found in other types of cutis laxa. Fibroblast cultures of patients with mutations in the PYCR1

or ATP6V0A2 genes show impaired response to oxidative stress and increased apoptosis. It

might be interesting to evaluate the response to oxidative stress in ADCL as well. It is

plausible that in view of increased endoplasmatic reticulum stress, elastogenic cells in ADCL

are prone to early degeneration, oxidative stress and ageing resulting in improper homeostasis

of the ECM. This hypothesis might shed new light on acquired forms of cutis laxa. Indeed, at

least one patient with cutis laxa secondary to infection with Toxocara canis, had an

underlying defect in the elastin gene (303).

In several connective tissue diseases, including MFS (114), LDS (247), ATS (266), and

cutis laxa due to FBLN4 mutations (295), enhanced TGFβ signaling is shown to be a major

player in the pathogenesis of aortic dilatation. In MFS, aortic dilatation, pulmonary

emphysema and muscle hypotonia are successfully stabilized and even reversed by

antagonism of the TGFβ signaling pathway (114, 139, 140). In our patient series with ADCL,

fibroblast cultures clearly show enhanced TGFβ signaling. It is likely that the amount of

TGFβ signaling in aortic and pulmonary tissue in vivo, influenced by interindividual and

mutation specific differences, is a determinant of complications in these respective tissues. It

remains to be elucidated if this hypothesis also yields for other forms of cutis laxa.

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305. Popplewell, L.J., Adkin, C., Arechavala-Gomeza, V., Aartsma-Rus, A., de Winter, C.L., Wilton, S.D., Morgan, J.E., Muntoni, F., Graham, I.R. and Dickson, G. Comparative analysis of antisense oligonucleotide sequences targeting exon 53 of the human DMD gene: Implications for future clinical trials. Neuromuscul Disord, 20, 102-10.

306. Sazani, P., Weller, D.L. and Shrewsbury, S.B. Safety Pharmacology and Genotoxicity Evaluation of AVI-4658. Int J Toxicol.

References 209

Future perspectives 211

Future perspectives Wat de ploeg was voor de landbouw,

wat het wiel was voor de mechanica,

dat is de wc voor het idee (…) Eurekaka!

Dimitri Verhulst

This work aims to contribute to the clinical and molecular dissection of heritable

disorders of the connecting tissue that show structural abnormalities of the large arteries

impelling aortic and arterial risks. Examples of these disorders include the Marfan syndrome

(mutations in the FBN1 gene), Loeys-Dietz syndrome (mutations in the TGFBR1 and

TGFBR2 genes) and the vascular form of Ehlers-Danlos syndrome (mutations in the COL3A1

gene). The clinical characterization and molecular knowledge of these disorders has been

successfully implemented in the diagnosis and management of affected patients.

In many related connective tissue disorders that associate with arterial aneurysms,

stenosis, dissections, fragility and cardiac valve abnormalities, the clinical and molecular

picture was still insufficiently elucidated. In this work we specifically aim at a thorough

clinical and molecular dissection of some of these phenotypes. This thesis specifically

focuses on 4 major diseases:

1) The Marfan syndrome

2) Congenital contractural arachnodactyly or Beals (- Hecht) syndrome

3) The arterial tortuosity syndrome

4) Autosomal dominant cutis laxa

It is a familiar feeling that in research, answers create more questions than they resolve.

In this final part of this thesis, we engage to put forward new questions that will guide further

investigations.

Ad 1)

In Marfan syndrome, a common disease with significant morbidity and mortality,

previous characterization successfully focused on the timely diagnosis and prevention of

complications. In the 21st century, evidence-based and patient-centered care becomes a most


forefront issue. For the patient, the diagnosis remains a devastating event with detrimental

effects on personal, social and professional functioning. For the physician, the diagnosis is

primarily associated with unpredictable aortic risk. Moreover, the recent description of

related syndromes with different risks and prognoses may create confusion. We therefore put

forth a new set of diagnostic criteria to overcome the risk of misdiagnosis and make the

criteria more evidence-based. Prospective evaluation of these criteria in practice, preferably

in an international context, will provide final proof of the applicability, manageability, and

improved patient care. In first place, they should be evaluated for early recognition of an

aortic risk without overdiagnosing. They should also correctly trigger the search for

alternative diagnoses. Finally, easy and correct use in the hands of clinicians less experienced

in the diagnosis of MFS will provide final proof of the applicability. Establishment of a

mutation/SNP database that includes clinical data will ease patient evaluation.

Phenotype-genotype correlation in MFS has been a head breaker for many research

groups in the field of connective tissues. We contributed to an elaborate international

genotype-phenotype correlation study that found several statistical associations, but the

conclusions did not provide a platform for individualized patient care. It is likely that

modifier genes largely contribute to clinical expression. To investigate this hypothesis, our

center took off with a large SNP analysis platform in order to correlate SNP genotypes of

genes that influence TGFβ signaling with phenotypic severity of aortic and skeletal

manifestations in MFS patients.

Ad 2)

In rare disorders, pooling of patient data is of utmost importance to overview all aspects

of a phenotype. Our large cohort of patients with congenital contractural arachnodactyly

enriched the literature data on the clinical delineation of the phenotype of patients harboring

FBN2 mutations. More specifically, our data strengthen the belief that precaution is

warranted concerning aortic risk, although progression of the dilatation remains unclear.

Prospective evaluation the aortic risk in these and newly diagnosed CCA patients will

provide more definite prognostic conclusions.

Molecular findings in the Ghent cohort showed that mutations may occasionally occur

outside the central stretch of cbEGF-like domains and suggested molecular heterogeneity.

Future perspectives 213

Further research focusing on the molecular background of the disorder may identify

additional genes in CCA and CCA-related phenotypes. Candidate genes should be sought in

microfibril components and associated proteins, or in genes related to clinically overlapping

disorders including CATSHL syndrome and the family of arthrogryposes. These findings

may shed new light on microfibril assembly and functioning. Indeed, fibrillin-2 is a far less

studied protein compared to fibrillin-1, and its specific functions and spatiotemporal

relationship in the microfibrils and connective tissue are not yet fully understood. A recently

described zebrafish model carrying a disrupted FBN2 gene showed impairment of notochord

development (304) and offers new research possibilities. It allows studying the effect of

FBN2 disruption on early embryogenesis and on microfibril assembly. Moreover, a

phenotype screen of a mutant zebrafish library for similar phenotypes might identify new

candidate genes.

Ad 3)

Before this work was started, only a few sporadic reports existed on arterial tortuosity

syndrome. With the discovery of the genetic basis of the syndrome, we were able to clearly

define the phenotype and natural history, and differentiate this entity from related syndromes

that have previously been confused. Still, due to the rarity of the disease, the total number of

reported patients remains low. Further refinement can result from additional clinical reports.

The most intriguing question remains the exact function of GLUT10 and the molecular

events that connect this transporter with TGFβ signaling resulting in abnormal vascular

patterning and distorded extracellular matrix organization. The main mysteries remain the

exact localization of GLUT10 and the elucidation of the substrate of this transporter. To this

purpose, we created a zebrafish model for the disorder. This enables expression and

pharmacological studies that could reveal new etiopathogenetic secrets. Preliminary results

suggest improper mitochondrial functioning. These results will hopefully have a direct impact

on treatment possibilities in ATS patients.


Ad 4)

We refuted the historical belief that ADCL is a benign disease solely confined to skin

manifestations. Further prospective follow-up of this cohort will document the exact risks for

progression of aortic dilatation in ADCL. We also shed new light on the mechanism by which

an altered C-terminus leads to improper elastic fiber formation. Further examination of the

effect of the mutant C-terminus in correct elastic fiber assembly (interaction partners) will

introduce new insights on the spatial relationships and interactions of extracellular matrix

proteins. Also, our results indicate endoplasmatic reticulum stress and apoptosis in at least

one ADCL fibroblast cell line. It will be interesting to investigate if superimposed metabolic

stress also results in cellular decompensation in the other cell lines and by which

mechanisms. Our observations also favor a role for increased TGFβ signaling in the

pathogenesis. These data need to be confirmed by pharmacological studies in mouse models

first. If confirmed, and if the extrapolation of TGFβ modification by losartan from mice to

humans holds for other diseases like Marfan syndrome, treatment of ADCL patients with

losartan can be envisioned. Another attractive option is to evaluate a mouse model for

treatment with morpholinos. This might (partially) rescue the phenotype, as seen in Duchenne

muscular dystrophy (305, 306).

As a general conclusion, our work has contributed to new clinical and molecular insights

in MFS, CCA, ATS and ADCL and has significantly increased our knowledge about the

cardiovascular risks in these entities. Our investigations of the pathogenetic mechanisms in

these disorders have broadened the existing understanding of extracellular matrix

homeostasis. The unexpected finding of GLUT10 deficiency in ATS provides a nice example

on how rare disorders of the connective tissue connect with widespread conditions, such as

diabetes mellitus. The recurrent link between enhanced TGFβ signaling and vascular

complications already offered new medical treatment options in severe forms of MFS.

Moreover, this unmasked relationship might include other benefits for patient care. Indeed,

measurement of circulating TGFβ levels offers a practical tool for the diagnosis and risk

assessment (249). As such, extrapolation and verification of (subsets of) these data to more

common diseases of the cardiovascular system, may be triggered. In the near future more

patients afflicted with a risk for aortic aneurysms/dissections may benefit from these

discoveries.

Summary 215

Summary

Aortic aneurysms and dissections are an important cause of morbidity and mortality in the

Western World. Many monogenic disorders of the connective tissue present with arterial

aneurysms and dissections and their understanding may increase our knowledge about

connective tissue functioning and isolated forms of arterial disease. Examples include the

Marfan, Loeys-Dietz, and some forms of the Ehlers-Danlos syndrome. The well-

characterized molecular background of these disorders has been successfully implemented in

patient care. Moreover, insights in their pathophysiology have opened perspectives for new

treatment options. Many related disorders that associate with arterial tortuosity, aneurysms

and dissections, or stenoses still need profound clinical and molecular characterization. This

work aims to contribute to the clinical characterization of some of these entities and to

unravel their genetic basis and underlying molecular cascades. This thesis focuses on the

clinical and molecular aspects of

1. Marfan syndrome,

2. Congenital contractural arachnodactyly,

3. Arterial tortuosity syndrome, and

4. Autosomal dominant cutis laxa.

Ad 1: Marfan syndrome

In a first part, we contribute to a large, international collaborative genotype-phenotype

correlation study on Marfan syndrome and related disorders including 1013 probands with an

FBN1 mutation. This study provides clear associations between type or location of the FBN1

mutation and some clinical manifestations. Creation or substitution of a cysteine residue is

associated with a higher probability of lens ectopy, premature truncation mutations result in a

more severe skeletal phenotype and exon 24-32 mutations lead to a more severe presentation

in all age groups with shorter survival. Although these associations do not provide a platform

for individualized patient care, they provide some mechanistic insights into the pathogenesis

of Marfan syndrome. In particular, this study nicely demonstrates that both structural defects

of the microfibril and functional consequences of fibrillin-1 deficiency should be considered

tissue-specifically.


In a second part, we propose a revised nosology for the Marfan syndrome. The 1996

Ghent nosology did prove his accuracy and usefulness in clinical practice, but during recent

years new issues arose and urged for a revision. The proposal puts more weight on the two

main features of Marfan syndrome -aortic root dilatation and ectopia lentis- and on the

contribution of molecular genetics. Other manifestations were combined in a „systemic

score‟. This systemic score avoids the use of criteria that lack clear validation or criteria that

are difficult to assess. Since Marfan syndrome is primarily associated with an aortic risk that

may restrict the patient‟s personal aspirations, the revised nosology avoids the diagnosis in

the absence of tangible evidence of such risk. The subsequent diagnostic gaps in adults not

entirely fulfilling the new criteria are filled by clearly defined categories as ectopia lentis

syndrome, mitral valve prolapse syndrome, and MASS syndrome (a constellation of mitral

valve prolapse, myopia, borderline aortic dilatation, skeletal, and skin manifestions). Strict

follow-up and management guidelines accommodate for a possible delay in diagnosis in

some children and adolescents and will facilitate communication between health care

providers. Another renewing concept in the revised nosology is that diagnostic perseverance

is warranted when patients fulfill the diagnostic criteria for Marfan syndrome, but do present

with other manifestations suggestive of a related disorder.

Ad 2: Congenital contractural arachnodactyly

This part carefully characterizes 32 probands with this rare Marfan-like disorder, both

clinically and molecularly. This study expands the knowledge on the clinical presentation in

this disorder, especially on the cardiovascular features. We show that patients do have an

aortic risk for aortic dilatation, but the evolution to rupture or dissection remains

unconfirmed. Literature data suggest that all FBN2 mutations cluster in the central region of

the gene, but our molecular data formally denote the first mutation outside exons 24-36.

Moreover, a mutation uptake rate of 44% argues for locus heterogeneity. The absence of

FBN2 mutations in patients with the severe, lethal type of congenital contractural

arachnodactyly favors the disruption of other genes in this condition.

Summary 217

Ad 3: Arterial tortuosity syndrome

In a first part, using a homozygosity mapping strategy with sequencing of the genes in the

remaining candidate region we identify mutations in the facilitative glucose transporter

GLUT10 (encoded by SLC2A10) as the genetic cause underlying the arterial tortuosity

syndrome. Immunohistochemistry data suggest that the transporter resides on the nuclear

membrane. Furthermore, similarly as in the clinically related Loeys-Dietz syndrome, vascular

smooth muscle cells of arterial tortuosity syndrome patients show enhanced TGFβ signaling.

Decreased glucose-responsive transcription of decorin, a TGFβ inhibitor, elegantly links

GLUT10 dysfunction with TGFβ dysregulation. These surprising findings broaden the view

on extracellular matrix homeostasis and may even be relevant for common diseases as

diabetes mellitus.

In a second part, we establish a mice model for arterial tortuosity syndrome. Two mice

strains, selected from an ENU-mutated mice library, with homozygous p.G128E and p.S150F

are characterized, but no clinical or vascular presentation whatsoever was found on surgical

exploration, abdominal ultrasound, or vascular corrosion casting. Moreover,

immunohistology of arterial tissues shows normal elastic fibers. As such, this model does not

enable further studies into the pathogenesis of arterial tortuosity syndrome.

In a third part, we define the clinical spectrum in 16 patients from 12 newly identified

families with molecularly proven arterial tortuosity syndrome. The phenotype is clearly

recognizable, with a typical facial dysmorphology and manifest, generalized tortuosity. The

most important observation is that patients have a far better prognosis and survival than

expected on the basis of literature data. However stroke remains a significant risk and

progressive aortic root dilatation may occur. Furthermore, we confirm normal serum glucose

homeostasis in patients and carriers, and we exclude that carriers might present with a mild

phenotype. The molecular data show many recurrent mutations due to founder effects.

Ad 4: Autosomal dominant cutis laxa

This final part of the thesis contributes new and important clinical and molecular data to

this rare and still insufficiently characterized disorder. Previously, the condition was

considered benign and confined to the skin. We carefully describe the clinical presentation in

5 new probands and unequivocally demonstrate an aortic risk. Patients may also show severe


emphysema. All patients in our cohort harbor C-terminal elastin mutations resulting in an

elongated protein. These mutated proteins are secreted and show enhanced coacervation,

resulting in globular aggregates and less insoluble elastin. Partial retention of mutated elastin

may lead to increased apoptosis. Finally, we demonstrate increased TGFβ signaling in

fibroblast cultures and hypothesize that tissue-specific alterations in TGFβ signaling

contribute to the varying internal organ manifestations in autosomal dominant cutis laxa.

Moreover, patients with exon 32 mutations may be relatively spared from severe

complications since alternative splicing of exon 32 frequently occurs and may partially rescue

the phenotype.

In conclusion, this thesis contributes new clinical and molecular insights in several

elastinopathies, including Marfan syndrome, congenital contractural arachnodactyly, arterial

tortuosity syndrome and autosomal dominant cutis laxa. Patient care will benefit from the

detailed clinical and molecular characterization in this work. Our data corroborate a role for

enhanced TGFβ signaling in several of these disorders, boding for new therapeutic options.

Finally, this work broadens our understanding of extracellular matrix homeostasis and

provides an interesting basis for further research that may link to more common forms of

arterial aneurysms and dissections.

Samenvatting 219

Samenvatting

Aorta-aneurysma‟s en –dissecties zijn een belangrijke oorzaak van morbiditeit en

mortaliteit in de westerse wereld. Vele monogenische bindweefselziekten gaan eveneens

gepaard met arteriële afwijkingen waaronder aneurysma‟s en dissecties. Een grotere kennis

hierover kan bijdragen tot een beter begrip van de frequentere, geïsoleerdere vormen.

Voorbeelden hiervan zijn het Marfan, Loeys-Dietz, en sommige vormen van het Ehlers-

Danlos syndroom. Deze aandoeningen zijn moleculair reeds grondig gekarakteriseerd en de

kennis ervan werd geïmplementeerd in de kliniek. Nieuwe inzichten in de pathofysiologie

van deze ziekten openden nieuwe perspectieven voor behandeling. Vele gerelateerde

bindweefselaandoeningen gaan eveneens gepaard met arteriële afwijkingen zoals tortuositeit,

aneurysma‟s en dissecties of stenoses, maar moeten nog ten gronde gekarakteriseerd worden,

zowel klinisch, moleculair genetisch, alsook naar de onderliggende moleculaire cascade toe.

Deze thesis spitst zich toe op de klinische en moleculaire aspecten van

1. Marfan syndroom,

2. Congenitale contracturele arachnodactylie,

3. Het arteriële tortuositeitssyndroom, en

4. Autosomaal dominante cutis laxa.

Ad 1: Marfan syndroom

In een eerste deel dragen we bij tot een grote, internationale multicentrische genotype-

fenotype correlatie studie met inclusie van 1013 probands met een FBN1 mutatie. Deze toont

enkele duidelijke associaties tussen type en locatie van de mutatie enerzijds en bepaalde

klinische manifestaties anderzijds. Zo werd een verband gevonden tussen de vorming of het

verdwijnen van een cysteine en een grotere kans op lensluxatie, tussen mutaties die leiden tot

een verkort proteïne en een ernstiger skeletaal fenotype, en tussen mutaties in exonen 24-32

en een ernstiger verloop in alle leeftijdscategorieën met een kortere overleving. Hoewel deze

associaties niet kunnen bijdragen tot de geïndividualiseerde patiëntenzorg, leiden deze

resultaten tot een beter begrip van de pathogenese van het Marfan syndroom. Meer bepaald

toont deze studie aan dat de gevolgen van de structurele defecten van de microfibrillen en de


functionele gevolgen van fibrilline-1 deficiëntie in hun weefselspecifieke context moeten

beschouwd worden.

In een tweede luik rond het Marfan syndroom stellen we een hernieuwde ziekteleer van

deze aandoening voor. Ondanks hun accuraatheid en bewezen nut in de dagdagelijkse

praktijk, toonden de in 1996 opgestelde criteria voor MFS doorheen de jaren toch enkele

tekortkomingen. In de gereviseerde ziekteleer wegen de hoofdmanifestaties –aortadilatatie en

lensluxatie– zwaarder door en wint ook de moleculaire analyse aan belang. De overige

manifestaties werden samengebracht in een „systemische score‟. Deze systemische score

vermijdt het gebruik van onvoldoende onderbouwde criteria of criteria die in de praktijk

moeilijk te evalueren zijn. De associatie van Marfan syndroom met voornamelijk

aortadilatatie en -dissectie belemmert de patiënt vaak in zijn persoonlijke en

maatschappelijke ontplooiing. Daarom zijn de nieuwe criteria terughoudend om de diagnose

te stellen wanneer er geen aantoonbaar risico is op aortadilatatie. De zo ontstane

diagnostische lacunes bij volwassenen die niet volledig voldoen aan de nieuwe criteria

worden hierbij opgevangen door duidelijk omschreven entiteiten als ectopia lentissyndroom,

mitraalklepprolapssyndroom, of MASS fenotype (mitraalklepprolaps, myopie, milde

aortadilatatie, huid en skelet afwijkingen). Strikte adviezen rond opvolging en behandeling

vangen hierbij het risico van een laattijdige diagnose op bij kinderen en adolescenten, en

zullen de communicatie tussen zorgverleners vergemakkelijken. Een laatste hernieuwend

concept van de nieuwe criteria is dat verdere diagnostische uitwerking nodig is wanneer

patiënten voldoen aan de criteria voor Marfan syndroom, maar toch manifestaties vertonen

die suggestief zijn voor een verwante aandoening.

Ad 2: Congenitale contracturele arachnodactylie

Dit gedeelte van dit onderzoeksproject karakteriseert nauwgezet, zowel klinisch als

moleculair, 32 indexpatiënten met deze zeldzame aandoening die nauw verwant is aan het

Marfan syndroom. Deze studie breidt de kennis van de klinische presentatie in deze

aandoening uit, voornamelijk met betrekking tot de cardiovasculaire afwijkingen. We tonen

aan dat deze patiënten weldegelijk een risico vertonen op aortadilatatie, maar de evolutie naar

dissectie of ruptuur blijft onbevestigd. Data uit de literatuur suggereren dat alle FBN2

mutaties in de centrale regio van het gen clusteren. Onze moleculaire bevindingen tonen

echter formeel een mutatie aan buiten deze exonen. De mutatiedetectieratio van 44%

Samenvatting 221

suggereert tevens sterk dat ook andere genen gemuteerd kunnen zijn in deze ziekte. Tot slot is

de afwezigheid van FBN2 mutaties in patiënten met het ernstige, letale type van congenitale

contracturele arachnodactylie er suggestief voor dat afwijkingen in andere genen aan de basis

van dit subtype liggen.

Ad 3: het arteriële tortuositeitssyndroom

In een eerste luik tonen we met behulp van homozygositeitsanalyse, gevolgd door het

sequeneren van de genen in de resterende regio, aan dat mutaties in de facilitatieve

glucosetransporter GLUT10 (gecodeerd door SLC2A10) aan de basis van deze aandoening

liggen. Immunocytochemie suggereert dat deze transporter zich op de kernmembraan

bevindt. Ook vonden we, net als in het klinisch verwante Loeys-Dietz syndroom, verhoogde

TGFβ signalisatie in de vasculaire gladde spiercellen van patiënten met het arteriële

tortuositeitssyndroom. Verminderde glucosegereguleerde expressie van Decorine, een TGFβ

modulator, linkt GLUT10 dysfunctie met verhoogde TGFβ signalisatie. Deze verrassende

bevindingen verbreden onze kijk op de extracellulaire matrix homeostase en zijn wellicht

relevant voor frequente aandoeningen als diabetes mellitus.

In een tweede luik creëren we een muismodel voor het arteriële tortuositeitssyndroom.

We evalueren twee muismodellen, geselecteerd uit een ENU-gemuteerde muizenbibliotheek,

met respectievelijk een homozygote p.G128E en p.S150F mutatie, door middel van

chirurgische exploratie, abdominale echografie, en vasculaire afgietsels, maar kunnen geen

klinische of vasculaire afwijkingen weerhouden. Daarenboven blijkt op histologisch

onderzoek van de arteriële vaatwand dat de elastische vezels normaal aangelegd zijn. We

besluiten dat dit muismodel daarom niet kan bijdragen tot de verdere studie van de

pathogenese in het arteriële tortuositeitssyndroom.

In een laatste luik beschrijven we het klinische spectrum in 16 patiënten uit 12 nieuw

geïdentificeerde families met moleculair bewezen arteriële tortuositeitssyndroom. Het

fenotype blijkt duidelijk herkenbaar met een typische dysmorphologie en manifeste,

gegeneraliseerde tortuositeit. De belangrijkste observatie is dat patiënten een veel betere

prognose en overleving kennen dan verwacht op basis van literatuurgegevens. Toch blijft er

een belangrijk risico op (hersen)infarcten en kan er aortadilatatie optreden. Verder bevestigen

we dat de serum glucose homeostase bij patiënten en niet-aangetaste dragers normaal is. We


sluiten tevens uit dat dragers een mild fenotype zouden vertonen. De moleculaire data tonen

meerdere recurrente mutaties, veelal te wijten aan een mutatie bij een gemeenschappelijke

voorouder.

Ad 4: Autosomaal dominante cutis laxa

Dit laatste deel van de thesis levert een belangrijke klinische en moleculaire bijdrage tot

dit zeldzame en nog onvoldoende gekarakteriseerde fenotype. Voorheen werd deze

aandoening als goedaardig beschouwd met enkel betrokkenheid van de huid. Deze studie

beschrijft nauwgezet de klinische presentatie in 5 nieuwe indexpatiënten en toont

ontegensprekelijk een risico op aortadilatatie aan. Sommige patiënten vertonen ook

emfyseem. Alle patiënten in deze cohorte hebben een C-terminale elastine mutatie die

aanleiding geeft tot een verlengd proteïne. Deze gemuteerde proteïnes worden gesecreteerd

en klitten versneld samen tot bolvormige aggregaten met uiteindelijk een verminderde

hoeveelheid matuur elastine tot gevolg. De afwijkende elastinemoleculen worden ook

gedeeltelijk in de cel weerhouden en kunnen leiden tot een verhoogde celdood. Tot slot tonen

we ook verhoogde TGFβ signalisatie aan in fibroblastculturen van patiënten met autosomal

dominante cutis laxa. Onze hypothese is dan ook dat weefselspecifieke veranderingen in

TGFβ signalisatie aan de basis liggen van afwijkingen in de bloedvaten en de longen bij

patiënten met autosomaal dominante cutis laxa. Zo worden patiënten met exon 32 mutaties

relatief gespaard van ernstige complicaties van de interne organen, aangezien dit exon

frequent verwijderd wordt uit het transcript waardoor een normaal proteïne ontstaat en de

elastische vezelvorming minder verstoord wordt.

Afsluitend kunnen we stellen dat dit werk nieuwe klinische en moleculaire inzichten

bijbrengt in verscheidene elastinopathieën, waaronder het Marfan syndroom, congenitale

contracturele arachnodactylie, het arteriële tortuositeitssyndroom en autosomaal dominante

cutis laxa. De hernieuwde klinische inzichten in dit werk dragen bij tot een betere

patiëntenzorg in deze zeldzame aandoeningen. Daarenboven tonen we aan dat verhoogde

TGFβ signalisatie een belangrijke rol heeft in verscheidene van deze aandoeningen. Dit biedt

nieuwe aangrijpingspunten voor therapeutische interventie. Tenslotte verruimt dit werk onze

kennis omtrent de homeostase van de extracellulaire matrix en biedt een interessante basis

Samenvatting 223

voor verder onderzoek naar meer voorkomende vormen van arteriële aneurysma‟s en

dissecties.

List of abbreviations 225

List of Abbreviations

(cb)EGF (Calcium binding) epidermal growth factor

(F)TAAD (Familial) thoracic aortic aneurysm/dissection

ADCL Autosomal dominant cutis laxa

ARCL Autosomal recessive cutis laxa

ATS Arterial tortuosity syndrome

BAV Bicuspid aortic valve

BAV/TAA Bicuspid aortic valve with thoracic aortic aneurysm

BiP Binding protein

BMP Bone morphogenetic protein

CCA Congenital contractural arachnodactyly

cDNA Copy DNA or complementary DNA

CL Cutis laxa

CSGE Conformation sensitive gel electrophoresis

CTD Connective tissue disorder

C-terminus COOH-terminus or Carboxyterminus

dHPLC Denaturing high performance liquid chromatography

DNA Deoxyribonucleic acid

ECM: Extracellular matrix

EDS Ehlers-Danlos syndrome

ENU N-ethyl-N-nitrosurea

FACIT Fibril-associated collagen with interrupted triple helix

GAG Glycosaminoglycan

gDNA Genomic DNA

GO Geroderma osteodysplasticum

kb Kilobases

kDa Kilodalton

LDS Loeys-Dietz syndrome

LTBP Latent transforming growth factor beta binding protein

MACS Macrocephaly, alopecia, cutis laxa, scoliosis syndrome

MAGP Microfibril associated glycoprotein


MASS Myopia, mitral valve prolapse, borderline aortic dilatation, skin and

skeletal manifestations

MFAP Microfibril associated protein

MFS Marfan syndrome

MMP‟s Matrix metalloproteinase

mRNA Messenger RNA

PCR Polymerase chain reaction

PG Proteoglycan

PTC Premature termination codon

RNA Ribonucleic acid

SLRP Small leucine-rich proteoglycan

SSCP Single stranded conformation polymorphism

SVAS Supravalvular aortic stenosis

TGFβ Transforming growth factor beta

TSP-1 Trombospondin-1

URDS Urban-Rifkin-Davis syndrome

WSS Wrinkly skin syndrome

Curriculum vitae 227

Curriculum Vitae

PERSONALIA

Last name: Callewaert

First names: Bert Louis Jos Marlies

Date of Birth: April 7th

, 1979

Place of birth: Sint-Niklaas, Belgium

Citizenship: Belgian

Marital status: married with Maartje van der Laak

Children: Karel Callewaert and Amber Callewaert

Office address: Department of Paediatrics and Genetics, De Pintelaan 185, 9000 Ghent,

Phone: +32/9/240 5026, Fax: +32/9/240 4970, E-mail: [email protected]

DEGREES

2003 Certificate “electrocardiography”

Faculty of Medicine and Medical Sciences, Ghent University, Belgium

2004 Medical doctor, summa cum laude

Faculty of Medicine and Medical Sciences, Ghent University, Belgium

2008 Certificate “Laboratory animal science - FELASA C attestation”

Faculty of Veterinary Medicine, Ghent University, Belgium

2009 Certificate “European Pediatric Life Support (EPLS)”


PROFESSIONAL EXPERIENCE - RESEARCH ACTIVITIES

October 1997 – July 2004:

Medical training and internship, Ghent University Hospital

March – May 2004:

Medical internship, CHU de Toulouse, France (Hôpital de Rangueil et Hôpital de

Purpan) – „Erasmus - exchange‟

July 2000 - October 2003

Scientific collaborator at the laboratory for experimental cancerology of Prof. Dr. M.

Mareel, Department of Radiotherapy, Ghent University Hospital

September 2004 – present

Residency in Pediatrics, Ghent University Hospital

October 2004 – September 2008

Research fellowship of the Fund for Scientific Research, Flanders (FWO).

Center for Medical Genetics, Ghent University Hospital

Promotor Prof. Dr. A. De Paepe / co-promotor Prof. Dr. B. Loeys

February 2008 – August 2008

Visiting Researcher at Zsolt Urban‟s Laboratory, Department of Pediatrics and

Genetics, Washington University School of Medicine, 660 Euclid Avenue, 63110 St-

Louis, MO

October 2008 - present: Scientific collaborator at the Center for Medical Genetics,

Ghent University Hospital


AWARDS

Laureate of the Pfizer Price, Ghent University, Belgium, Faculty of Medicine,

Promotion year 2004

Presentation award Belgian Society of Paediatrics of Nestlé Belgium:

Callewaert B, Coucke PJ, Willaert A, Wessels MW, Zoppi N, De Backer J, Fox JE,

Mancini GM, Kambouris M, Gardella R, Facchetti F, Willems PJ, Forsyth R, Dietz

HC, Barlati S, Colombi M, Loeys B, De Paepe A. Mutations in SLC2A10/GLUT10, a

facilitative glucose transporter, cause arterial tortuosity syndrome. Bruges, Belgium.

March 17-18, 2006.

Best Poster Presentation Award. First World Congress on Genodermatology,

Maastricht November 7-10th

, 2007. B. Callewaert, B. Albrecht, B. Loeys, I. Haußer, S.

Demuth, P. Coucke, Z. Urban, A. De Paepe. Four novel mutations in the ELN gene in

patients with autosomal dominant cutis laxa and variable systemic manifestations.

CHAPTERS IN BOOKS

Coucke P.; Loeys B; Callewaert B and De Paepe A. Discovery of genes in thoracic

aortic aneurysms. In: Aortic Aneurysms : New insights into an old problem. Editions

de L‟Université de Liege. Editor Natzi Sakalihasan and co, 2008

PUBLICATIONS

Peer-reviewed articles in international journals

1. Lauwaet T, Oliveira MJ, Callewaert B, De Bruyne G, Saelens X, Ankri S,

Vandenabeele P, Mirelman D, Mareel M, and Leroy A. Proteolysis of enteric cell

villin by Entamoeba histolytica cysteine proteinases. J Biol Chem. 2003 Jun

20;278(25):22650-6.

2. Lauwaet T, Oliveira MJ, Callewaert B, De Bruyne G, Mareel M, Leroy A. Proteinase

inhibitors TPCK and TLCK prevent Entamoeba histolytica induced disturbance of

tight junctions and microvilli in enteric cell layers in vitro. Int J Parasitol. 2004

Jun;34(7):785-94.

3. Coucke PJ, Willaert A, Wessels MW, Callewaert B, Zoppi N, De Backer J, Fox JE,


HC, Barlati S, Colombi M, Loeys B, De Paepe A. Mutations in the facilitative glucose

transporter GLUT10 alter angiogenesis and cause arterial tortuosity syndrome. Nat

Genet. 2006 Apr;38(4):452-7.

4. Loeys BL, Schwarze U, Holm T, Callewaert BL, Thomas GH, Pannu H, De Backer

JF, Oswald GL, Symoens, Manouvrier S, Roberts A, Faravelli F, Greco MA, Pyeritz

RE, Milewicz DM, Coucke PJ, Braverman AC, Byers PH, De Paepe AM, Dietz HC.

Presentation, natural history and management of aneurysm syndromes caused by

mutations in TGFBR1 or TGFBR2 encoding for transforming growth factor-

receptors N Engl J Med. 2006 August;355:788-98

5. Effect of mutation type and location on clinical outcome in 1013 probands with

Marfan syndrome or related phenotypes with FBN1 mutations: an international study.


Faivre L, Collod-Beroud G., Loeys BL., Child A., Binquet C., Gautier E., Callewaert

B., Arbustini E., Mayer K., Arslan-Kirchner M., Kiotsekoglou A., Comeglio P.,

Marziliano N., Dietz HC., Halliday D., Beroud C., Bonithon-Kopp C., Claustres M.,

Muti C., Plauchu H., Robinson PN., Adès LC., Biggin A., Benetts B., Brett M.,

Holman KJ., De Backer J., Coucke P., Francke U., De Paepe A., Jondeau G., Boileau

C. American Journal of Human Genetics – 2007 Sep;81(3):454-66.

6. Arterial tortuosity syndrome: clinical and molecular findings in 12 newly identified

families. B. L. Callewaert, A. Willaert, W. S. Kerstjens-Frederikse, J. De Backer, K.

Devriendt, B. Albrecht, M. A. Ramos-Arroyo, M. Doco-Fenzy, R. C. M. Hennekam,

R. E. Pyeritz, O.N. Krogmann, G. Gillessen-Kaesbach, E. L. Wakeling, S. Nik-Zainal,

C. Francannet, P. Mauran, C. Booth, M. Barrow, R. Dekens, B. L. Loeys, P. J.

Coucke , A. M. De Paepe. Human mutation, 2007 Oct 12;29(1):150-158

7. New insights in the pathogenesis and treatment of arterial aneurysms and dissections.

Review. Bert L. Callewaert, Anne M. De Paepe, Bart L. Loeys. Current

Cardiovascular Risk Reports. 2007, 1:401-409.

8. Marfan syndrome and related disorders: from clinic to bench to clinic. “Fortschritt in

der Pathogenese des Marfan-Syndroms und verwandter Krankheiten - Von der Klinik

zum Labor und zurück”. Review. Bert L. Callewaert, Anne M. De Paepe,

Medizinische Genetik. 2008;20(1):6-17.

9. The Ehlers-Danlos syndromes and the Marfan syndrome. Bert Callewaert, Fransiska

Malfait, Bart Loeys, Anne De Paepe, MD, PhD. Best Pract. Res. Clin. Rheumatol.

2008 Mar; 22(1):165-89.

10. Contribution of molecular analyses in diagnosing Marfan syndrome and type I

fibrillinopathies: an international study of 1009 probands Faivre L, Collod-Beroud G,

Child A, Callewaert B, Loeys BL, Binquet C, Gautier E, Arbustini E, Mayer K,

Arslan-Kirchner M, Stheneur C, Kiotsekoglou A, Comeglio P, Marziliano N, Halliday

D, Beroud C, Bonithon-Kopp C, Claustres M, Plauchu H, Robinson PN, Adès L, De

Backer J, Coucke P, Francke U, De Paepe A, Boileau C, Jondeau G. J Med Genet.

2008 Jun; 45(6):384-90

11. Absence of arterial phenotype in mice with homozygous slc2A10 missense

substitutions. B. L. Callewaert, B. L. Loeys, C. Casteleyn, A. Willaert, P. Dewint, J.

De Backer, R. Sedlmeier, P. Simoens, A. M. De Paepe and P. J. Coucke. Genesis.

2008 Aug; 46(8):385-9.

12. Clinical and mutation-type analysis from an international series of 198 probands with

a pathogenic FBN1 exons 24-32 mutation.Faivre L, Collod-Beroud G, Callewaert B,

Child A, Binquet C, Gautier E, Loeys BL, Arbustini E, Mayer K, Arslan-Kirchner M,

Stheneur C, Kiotsekoglou A, Comeglio P, Marziliano N, Wolf JE, Bouchot O, Khau-

Van-Kien P, Beroud C, Claustres M, Bonithon-Kopp C, Robinson PN, Adès L, De

Backer J, Coucke P, Francke U, De Paepe A, Jondeau G, Boileau C. Eur J Hum

Genet. 2008 Nov 12. [Epub ahead of print]

13. Comprehensive clinical and molecular assessment of 32 probands with congenital

contractural arachnodactyly: report of 14 novel mutations and review of the literature.


Bert L. Callewaert, Bart L. Loeys, Anna Ficcadenti, Sascha Vermeer, Magnus

Landgren, Hester Y. Kroes, Yuval Yaron, Michael Pope, Nicola Foulds, Odile Boute,

Francisco Galán, Helen Kingston, Nathalie Van der Aa; Iratxe Salcedo; Marielle E.

Swinkels; Carina Wallgren-Pettersson, Orazio Gabrielli, Julie De Backer, Paul J.

Coucke, Anne M. De Paepe. Human Mutation. 2009 Mar; 30(3):334-41.

14. Unusual 8p inverted duplication deletion with telomere capture from 8q. Buysse K,

Antonacci F, Callewaert B, Loeys B, Fränkel U, Siu V, Mortier G, Speleman F,

Menten B. Eur J Med Genet. 2009 Jan-Feb; 52(1):31-6.

15. Clinical and molecular study of 320 children with Marfan syndrome and related type I

fibrillinopathies in a series of 1009 probands with pathogenic FBN1 mutations. Faivre

L, Masurel-Paulet A, Collod-Béroud G, Callewaert BL, Child AH, Stheneur C,

Binquet C, Gautier E, Chevallier B, Huet F, Loeys BL, Arbustini E, Mayer K, Arslan-

Kirchner M, Kiotsekoglou A, Comeglio P, Grasso M, Halliday DJ, Béroud C,

Bonithon-Kopp C, Claustres M, Robinson PN, Adès L, De Backer J, Coucke P,

Francke U, De Paepe A, Boileau C, Jondeau G. Pediatrics. 2009 Jan;123(1):391-8.

16. Pathogenic FBN1 mutations in 146 adults not meeting clinical diagnostic criteria for

Marfan syndrome: further delineation of type 1 fibrillinopathies and focus on patients

with an isolated major criterion. Faivre L, Collod-Beroud G, Callewaert B, Child A,

Loeys BL, Binquet C, Gautier E, Arbustini E, Mayer K, Arslan-Kirchner M,

Kiotsekoglou A, Comeglio P, Grasso M, Beroud C, Bonithon-Kopp C, Claustres M,

Stheneur C, Bouchot O, Wolf JE, Robinson PN, Adès L, De Backer J, Coucke P,

Francke U, De Paepe A, Boileau C, Jondeau G. Am J Med Genet A. 2009

May;149A(5):854-60.

17. Short stature, severe aortic root dilation, skin hyperextensibility, extreme joint laxity

and craniofacial dysmorphic features: a probable new syndrome. Elke Verstraeten,

Sofie Symoens, Marjolijn Renard, Bert Callewaert, Kristof Vandekerckhove, Julie De

Backer, Fransiska Malfait, Luc Marks, Paul Coucke, Anne De Paepe, Bart Loeys.

Clinical dysmorphology. In press.

18. The revised Ghent nosology for the Marfan syndrome. Bart L. Loeys, Harry C. Dietz,

Alan C. Braverman3, Bert L. Callewaert

1, Julie De Backer

1, Richard B. Devereux,

Yvonne Hilhorst-Hofstee, Guillaume Jondeau, Laurence Faivre, Dianna M. Milewicz,

Reed E. Pyeritz, Paul D. Sponseller, Paul Wordsworth, Anne M. De Paepe. Journal of

Medical Genetics. In press.

19. Major cardiovascular involvement in autosomal recessive cutis laxa patients confirms

fibulin-4 as a key player in vascular elastic fiber formation. Marjolijn Renard, Tammy

Holm, Bert L. Callewaert, Lesley C. Adès, Osman Baspinar, Angela Pickart, Majed

Dasouki, Anita Rauch, Paul J. Coucke, Harry C. Dietz, Anne M. De Paepe, Bart L.

Loeys. Eur J Hum Genet. In Press.

20. New insights into the pathogenesis of autosomal dominant cutis laxa with report of

five additional ELN mutations. Bert Callewaert, Vishwanathan Hucthagowder, Beate

Albrecht, Ingrid Haußer, Edward Blair, Cristina Dias, Alice Albino, Hiroshi Wachi,

Fumiaki Sato, Robert P. Mecham, Bart Loeys, Paul J. Coucke, Anne De Paepe*, Zsolt

Urban*. Human Mutation, submitted.


21. A zebrafish model for ATS reveals that GLUT10 plays a role in mitochondrial

functioning and regulates vascular and notochord development in early

embryogenesis by modifying TGFbeta signaling. Andy Willaert*, Bert Callewaert*,

Paul J Coucke, Seth Crosby, Bart Loeys, Anne De Paepe, Zsolt Urban. *: these

authors equally contributed to this work. In preparation.

Peer-reviewed articles in national journals

1. Een Belgische patiënt met Arterial Tortuosity Syndrome. Callewaert B, Loeys B, De

Backer J, Willaert A, Devos D, Gewillig M, Coucke P, Devriendt K, De Paepe A.

Tijdschrift van de Belgische Kinderarts. 2007 July; 9(2):6-8

ORAL PRESENTATIONS

1. B. Callewaert. Imprinting. Ghent University Hospital, department of Paediatrics and

Gynaecology. 20 December 2005.

2. B. Callewaert. Genetics of Arterial Tortuosity and Aortic Aneurysms – Research in

progress. Center for medical genetics, Ghent University Hospital. Februari 7, 2006

3. Callewaert B, Coucke PJ, Willaert A, Wessels MW, , Zoppi N, De Backer J, Fox JE,


HC, Barlati S, Colombi M, Loeys B, De Paepe A. Mutations in SLC2A10/GLUT10, a

facilitative glucose transporter, cause arterial tortuosity syndrome. Oud Sint-Jan,

Bruges, Belgium. March 17-18, 2006.

4. B. Callewaert. Genetics of Arterial Tortuosity and Aortic Aneurysms. Scientific staff

meeting, Department of Pediatrics. Ghent University Hospital, June 20, 2006

5. B. Callewaert. Clinical and Molecular Findings Associated with Cutis Laxa

Syndromes. Geneskin Workshop – Connective tissue diseases. Center for medical

genetics, 05/12/2006

6. B. Callewaert. Genetics of Arterial Tortuosity and Aortic Aneurysms. Research in

progress. Center for medical genetics, Ghent University Hospital. January 9, 2007

7. B. Callewaert. Genetics of Arterial Tortuosity and Aortic Aneurysms. Research in

progress. Center for medical genetics, Ghent University Hospital. December 4th

, 2007

8. B. Callewaert. Comprehensive clinical and molecular assessment of 32 probands with

congenital contractural arachnodactyly: report of 14 novel mutations and review of

the literature. Dysmorphology meeting – UZ Brussel, Jette, Belgium, December 7th

,

2007

9. B. Callewaert. The Arterial Tortuosity Syndrome. Washington University School of

Medicine, Department of Pediatrics and Genetics, St-Louis, MO, USA February 25,

2008


10. B. Callewaert. Do I have cutis laxa? – Genetic disorders similar to cutis laxa. 1st

American symposium on cutis laxa. Washington University School of Medicine,

Department of Pediatrics and Genetics, St-Louis, MO, USA June 20, 2008.

11. B. Callewaert. Arterial tortuosity syndrome: from fish to men: a morpholino

approach. Scientific staff meeting, Department of paediatrics, Ghent University

Hospital, Belgium. January 20, 2009.

12. B. Callewaert. Comprehensive clinical and molecular assessment of 32 probands with

congenital contractural arachnodactyly: report of 14 novel mutations and

review of the literature. Neonatology staff meeting, Ghent University Hospital, June

11, 2009

ABSTRACTS

1. Lack of TGFBR1/2 mutations in patients with classic Marfan syndrome, familial

thoracic aortic aneurysm syndrome and Shprintzen-Goldberg syndrome. Callewaert

B, De Backer J, Loeys B, Coucke PJ, De Paepe A. 7th International symposium on

the Marfan syndrome, Ghent, 13-17 september 2005

2. Evaluation of the Ghent nosology in a series of 1081 patients with a pathogenic FBN1

mutation: an international study. Faivre L., Collod-Beroud G., Child A., Callewaert B,

Binquet C, Arbustini E, Kiotsekoglou A, Gauthier E, Bonithon-Kopp C, Comeglio P,

Halliday D, Muti C, Plauchu H, Robinson P, Ades L, De Backer J, Coucke P, Francke

U, De Paepe A., Jondeau G., Boileau C. 7th

International symposium on the Marfan

syndrome, Ghent, 13-17 September 2005

3. Effect of mutation type and location on clinical outcome in patients with a FBN1

mutation: an international study. Faivre L, Collod-Beroud G, Child A, Callewaert B,

Binquet C, Arbustini E, Kiotsekoglou A, Gauthier E, Bonithon-Kopp C, Comeglio P,

Halliday D, Muti C, Plauchu H, Robinson P, Ades L, De Backer J, Coucke PJ,

Francke U, De Paepe A, Boileau C, Jondeau G. 7th International symposium on the

Marfan syndrome, Ghent, 13-17 September 2005

4. Clinical and mutation type analysis in patients with isolated ectopia lentis issued from

an international series of 820 probands with a pathogenic FBN1 mutation. Faivre L,

Collod-Beroud G, Child A, Callewaert B, Binquet C, Arbustini E, Kiotsekoglou A,

Gauthier E, Bonithon-Kopp C, Comeglio P, Halliday D, Muti C, Plauchu H, Robinson

P, Ades L, De Backer J, Coucke PJ, Francke U, De Paepe A, Jondeau G, Boileau C.

7th International symposium on the Marfan syndrome, Ghent, 13-17 September 2005

5. Clinical and mutation type analysis in patients with neonatal Marfan syndrome

phenotype issued from an international series of 820 probands with a pathogenic

FBN1 mutation. Faivre L, Collod-Beroud G, Child A, Callewaert B, Binquet C,

Arbustini E, Kiotsekoglou A, Gauthier E, Bonithon-Kopp C, Comeglio P, Halliday D,

Muti C, Plauchu H, Robinson P, Ades L, De Backer J, Coucke P, Francke U, De

Paepe A, Boileau C, Jondeau G. 7th International symposium on the Marfan

syndrome, Ghent, 13-17 September 2005


6. Mutations in the facilitative glucose transporter GLUT10 alter angiogenesis and cause

Arterial Tortuosity Syndrome. Coucke PJ, Willaert A, Wessels MW, Callewaert B,

Zoppi N, De Backer J, Fox JE, Mancini GM, Kambouris M, Gardella R, Facchetti F,

Willems PJ, Forsyth R, Dietz HC, Barlati S, Colombi M, Loeys B, De Paepe A.

American Society of Human Genetics, Salt Lake City, Utah, 25-29 October 2005





Belgian society of Human Genetics, Antwerp, 17 February 2006

8. Lack of TGFBR1/2 mutations in patients with classic Marfan syndrome, familial

thoracic aortic aneurysm syndrome and Shprintzen-Goldberg syndrome. Callewaert

B, De Backer J, Loeys B, Coucke PJ, De Paepe A. Belgian society of Human

Genetics, Antwerp, 17 February 2006

9. Autosomal dominant cutis laxa with aortic root dilatation: two novel mutations in the

ELN gene. Callewaert B, Loeys B, Vanakker O, Coucke PJ, De Paepe A. 34th

Annual

Society of Belgian paediatricians, Bruges 17-18 March 2006


Arterial Tortuosity Syndrome. Callewaert B, Coucke PJ, Willaert A, De Backer J,

Forsyth R, Loeys B, De Paepe A.34th

Annual Society of Belgian paediatricians,

Bruges, 17-18 March 2006

11. Two novel mutations in the ELN gene in patients with autosomal dominant cutis laxa

and systemic manifestations. Callewaert B, Loeys B, Vanakker O, Coucke P, De

Paepe A. Wetenschapsdag Ghent University, Het Pand, Ghent, March 30 2006





Wetenschapsdag Ghent University, Het Pand, Ghent, March 30 2006





European Conference of Human Genetics, Amsterdam, 6-9 May, 2006

14. Presentation, natural history and management of aneurysm syndromes caused by

mutations in TGFBR1 or TGFBR2 encoding for transforming growth factor-

receptors. Loeys BL, Schwarze U, Holm T, Callewaert BL, Thomas GH, Pannu H, De

Backer JF, Oswald GL, Symoens, Manouvrier S, Roberts A, Faravelli F, Greco MA,

Pyeritz RE, Milewicz DM, Coucke PJ, Braverman AC, Byers PH, De Paepe AM,

Dietz HC. European Conference of Human Genetics, Amsterdam, May 6-9, 2006



and systemic manifestations. Callewaert B, Albrecht B, Loeys B, Gillessen-Kaesbach

G, Haußer I, Vanakker O, Coucke PJ, Urban Z, De Paepe A. European Conference of

Human Genetics, Amsterdam, May 6-9, 2006

16. The diverse phenotypic spectrum associated with fibulin-4 mutations. Loeys B, Ades

L, Wettinck K, De Backer J, Callewaert B, Coucke PJ, De Paepe A. 4th

European

Meeting on Elastin, Lyon, 9-12 July 2006


and systemic manifestations. Callewaert B, Loeys B, Albrecht B, Gillessen-Kaesbach

G, Haußer I, Vanakker O, Coucke P, Urban Z, De Paepe A. 4th

European Meeting on

Elastin, Lyon, July 9-12, 2006

18. Clinical and molecular study of 348 children with Marfan syndrome and related type I

fibrillinopathies out of a series of 1057 probands with a pathogenic fbn1 mutation. L.

Faivre, C. Stheuner, G. Collod-beroud, P. Chevallier, A. Child, B. Callewaert, C.

Binquet, E. Gautier, E. Arbustini, K. Mayer, A. Kiotsekoglou, P. Comeglio, B. Loeys,

J. De Backer, P. Coucke, U. Francke, L. Ades, A. De Paepe, C. Boileau, G. Jondeau.

American Society of Human Genetics, New Orleans, October 9-13, 2006

19. Array CGH reveals complex chromosome 8 rearrangement in a patient with

supravalvular pulmonary stenosis. Buysse K, Antonacci F, Menten B, Callewaert B,

Loeys B, De Paepe A, Mortier G, Speleman F. American Society of Human Genetics,

New Orleans, October 9-13, 2006

20. A critical evaluation of phenotypes associated with mutations in the TGFβ receptor

genes Loeys BL, Schwarze U, Holm T, Callewaert BL, Thomas GH, Pannu H, De

Backer JF, Oswald GL, Symoens S, Manouvrier S, Roberts A, Faravelli F, Greco

MA, Pyeritz RE, Milewicz DM, Coucke PJ, Braverman AC, Byers PH, De Paepe

AM, Dietz HC. American Society of Human Genetics, New Orleans, October 9-13,

2006

21. Das „Arterial Tortuosity Syndrome“ Fallbeschreibung und molekulargenetische

Diagnostik. Th. Menke, K. Walter, Y. Hellenbroich, B. Albrecht, G. Gillessen-

Kaesbach, B. Callewaert, P.J. Coucke, O.N. Krogmann. Meeting of the German

Society of Paediatric Cardiology (October 7-10, 2006)

22. Arterial tortuosity syndrome: clinical and molecular characterization of 12 newly

identified families. Bert Callewaert, Andy Willaert, Julie De Backer, Koen Devriendt,

Renée Dekens, Paul J. Coucke, Bart Loeys & Anne De Paepe. 7th

BeSHG annual

meeting, Marcinelle, April 20, 2007


and systemic manifestations. Bert Callewaert, Beate Albrecht, Bart Loeys, Gabriele

Gillessen-Kaesbach, Ingrid Haußer, Olivier Vanakker, Zsolt Urban, Paul J. Coucke &

Anne De Paepe. 7th

BeSHG annual meeting, Marcinelle, April 20, 2007


families B. L. Callewaert, A. Willaert, W. S. Kerstjens-Frederikse, J. De Backer, K.



R. E. Pyeritz, G. Gillessen-Kaesbach, E. L. Wakeling, S. Nik- Zainal, C. Francannet,

P. Mauran, C. Booth, M. Barrow, P. J. Coucke, B. L. Loeys, A. M. De Paepe. P0024.

European Human Genetics Conference. Nice, France, June 16-19th

, 2007 Eur J Hum

Genet: 15, S1 (June 2007), p.41

25. Clinical factors associated with the occurrence of an aortic dilatation within a cohort

of 1013 patients with Marfan syndrome or another type I fibrillinopathy. L. Faivre, G.

Collod-Beroud, B. Loeys, A. Child, C. Binquet, G. Elodie, B. Callewaert, E.

Arbustini, K. Mayer, M. Arslan-Krichner, P. Comeglio, C. Beroud, C. Bonithon-

Kopp, M. Claustres, L. Ades, J. De Backer, P. Coucke, U. Francke, A. De Paepe, C.

Boileau, G. Jondeau. P0162. European Human Genetics Conference. Nice, France,

June 16-19th

, 2007. Eur J Hum Genet: 15, S1 (June 2007), p.71


families B. L. Callewaert, A. Willaert, W. S. Kerstjens-Frederikse, J. De Backer, K.



P. Mauran, C. Booth, M. Barrow, P. J. Coucke, B. L. Loeys, A. M. De Paepe. Gordon

Research Conference on Elastin & Elastic Fibers. University of New England,

Biddeford, ME. July 29th

– August 3rd

, 2007

27. Short toes as a useful diagnostic sign in two skeletal disorders: Czech dysplasia and

fibrodysplasia ossificans progressiva. G. Mortier, K. Hoornaert, B. Callewaert, F.

Caveye, K. Verstraete, B. Loeys. XVIIIth European Meeting on Dysmorphology,

Strasbourg. September 6 - 7, 2007





P. Mauran, C. Booth, M. Barrow, P. J. Coucke, B. L. Loeys, A. M. De Paepe.

American Society of Human Genetics, San Diego, October 23-27, 2007

29. Four novel mutations in the ELN gene in patients with autosomal dominant cutis laxa

and variable systemic manifestations. B. Callewaert, B. Albrecht, B. Loeys, I. Haußer,

S. Demuth, P. Coucke, Z. Urban, A. De Paepe. First World Congress on

Genodermatology, Maastricht November 7-10th

, 2007.

30. Arterial Tortuosity phenotype not present in mice homozygous for G128E and S150F

substitutions in GLUT10 P. J. Coucke1, B. L. Callewaert

1, C. Casteleyn

2, A. Willaert

1,

P. Dewint3, J. De Backer

1, P. Simoens

2, A. M. De Paepe

1 and B. L. Loeys

1. Keystone

meeting, January 6-11th

, 2008, Colorado, USA?





P. Mauran, C. Booth, M. Barrow, P. J. Coucke, B. L. Loeys, A. M. De Paepe.

Wetenschapsdag Universiteit Ghent, March 18th

, 2008. Het Pand, Ghent, Belgium.


32. Comprehensive clinical and molecular assessment of 32 probands with congenital

contractural arachnodactyly: report of 14 novel mutations and review of the literature.

Bert L. Callewaert, Bart L. Loeys, Anna Ficcadenti, Sascha Vermeer, Magnus

Landgren, Hester Y. Kroes, Yuval Yaron, Michael Pope, Nicola Foulds, Odile Boute,

Francisco Galán, Helen Kingston, Nathalie Van der Aa; Iratxe Salcedo; Marielle E.

Swinkels; Carina Wallgren-Pettersson, Orazio Gabrielli, Julie De Backer, Paul J.

Coucke, Anne M. De Paepe. European Society of Human Genetics. Barcelona, Spain

May 31 – June 3, 2008

33. Study of human disease associated with FBLN4 mutations confirms fibulin-4 as a key

player in vascular elastic fiber formation. Marjolijn Renard, Lesley Ades, Pamela

Trapane, Bert Callewaert, Karen Wettinck, Paul Coucke, Anne De Paepe and Bart

Loeys. XXIst Meeting of the Federation of the European Connective Tissue Societies

(FECTS). Marseille - France. July 9-13, 2008.

34. Towards a revised Ghent nosology for the Marfan syndrome. B. Loeys, B. Callewaert,

J. De Backer, L. Faivre, G. Jondeau, R. Devereux, R. Pyeritz, P. Sponseller, P.

Wordsworth, D. Milewicz, H. Dietz, A. De Paepe. American Society for Human

Genetics. Philadelphia, Pennsylvania. November 11-15, 2008.

ATTENDED MEETINGS

1. 7th

International symposium on the Marfan syndrome, Ghent, 13-17 september 2005

2. Belgian society of Human Genetics, Antwerp, 17 February 2006

3. 34th

Annual Society of Belgian paediatricians, Bruges 17-18 March 2006

4. Wetenschapsdag Ghent University, Het Pand, Ghent, March 30 2006

5. European Conference of Human Genetics, Amsterdam, 6-9 May, 2006

6. Wetenschapsdag Ghent University, Het Pand, Ghent, March 14, 2007

7. Joined meeting with Dutch and Belgian Marfan clinics, Ghent University Hospital,

March 15th

, 2007

8. 7th

BeSHG annual meeting, Marcinelle, April 20, 2007

9. Gordon Research Conference on Elastin & Elastic Fibers. University of New England

in Biddeford, ME. July 29th

– August 3rd

, 2007

10. 1st American symposium on cutis laxa. Washington University School of Medicine,

Department of Pediatrics and Genetics, St-Louis, MO, USA June 20, 2008.


VARIA

Counseler for the medical school theses:

1. Congenital contractural arachnodactyly. Supervisor (promoter: Prof. Dr. A. De Paepe)

of thesis submitted by Arif Karakaya. 2006-2007.

2. Genetics of aortic aneurysms. UGent, 2e Bachelor, Supervisor (promoter: Dr. B.

Loeys) of thesis submitted by Anneleen Geerts 2006-2007.

3. Genetisch advies bij consanguiniteit. UGent, 2e Bachelor, Supervisor (promotor Prof.

Dr. A. De Paepe) of thesis submitted by Cedric Goes 2006-2007

4. Cerebrale aneurysmata: genetica, fysiopathologie en zin van familiale screening.

UGent, 2e Master, Supervisor (promotor Prof. Dr. A. De Paepe) of thesis to be

submitted by Annelies Capiau 2007-2009

Dankwoord 239

Acknowledgements - Dankwoord

Aan een doctoraat beginnen is een sprong in het diepe, een reis waarbij je niet weet wat je

zult tegenkomen. Het enige houvast zijn de vakkundige mensen die je richting kunnen geven

en bijstaan. Zonder hen lukt het niet, vandaar dit woord van dank!

Prof. De Paepe, in de eerste plaats gaat mijn dank en erkenning naar u uit. U werd me

aangekondigd als iemand die erg gedreven, en manu militare een project steeds tot een goed

einde brengt. Inderdaad, als mijn promotor, waren uw visie en stuurkunsten essentieel om het

einddoel te bereiken. Uw enthousiasme en steun om mijn onderzoekshonger uit te breiden tot

de USA, heb ik enorm geapprecieerd en waren essentieel bij het welslagen van deze thesis.

Doorheen de jaren gaf u me ook de kans een ruime klinische ervaring op te doen en u leerde

me dat in alles wat we doen, zowel in de kliniek als in het onderzoek, de patiënt centraal blijft

staan.

Prof. Bart Loeys, beste Bart, met grote trots mag ik vermelden dat ik de eerste

doktoraatsstudent ben die onder uw promotorschap een doktoraat aflever. Het mag gezegd

worden: “you did a great job!”. Al vanaf je terugkomst uit Baltimore was het duidelijk dat er

een herbronnende wind doorheen het CMGG waaide. Je eeuwig enthousiasme, je

gedrevenheid en doorzicht in het onderzoek, je streven naar perfectie in het schrijven van

artikels en je klinische vaardigheden hebben een diepe indruk op me nagelaten. Dit alles

combineerde je steeds met een luisterend oor. Altijd staat de deur van je bureau open, een

geruststellend gevoel!

Prof. Zsolt Urban, dear Zsolt, thank you so much for the opportunity you gave me to

complete my research in your lab! I will never forget our open and thoughtful discussions at

the lab bench raising new insights in elastic fiber dynamics. I am also very appreciative of

your help outside the lab, especially when my car did not start (again). Also, I want to thank

Andrew Maxfield, John Gansner, and Vichwanathan Hucthagowder, for their kind and

encouraging help when I was struggling with the zebrafishes.

Prof. Coucke, beste Paul, ook door u werd ik in het bindweefsellabo warm ontvangen. Na een

turbulente start van dit doktoraat, ben ik je relativeringsvermogen erg gaan appreciëren. Dank

ook voor het nodige tegengewicht bij de vaak zware werklast: de onderhoudende discussies,

de hulp bij de organizatie van het praktische werk, de jaarlijkse „bindweefselparties‟ en

barbecues, en de rondzwervingen langs de Amsterdamse grachten...

Prof. Matthys, beste Dirk, in een drukke klinische discipline als pediatrie, valt het onderzoek

vaak als eerste weg. Daarom ben ik u erg dankbaar voor uw strijd voor meer onderzoek, ook

al kiest u daarbij niet voor de gemakkelijkste weg.

Niets is mogelijk zonder de gedreven handen van de laboranten van het bindweefsellabo.

Petra, Karen, Jozefien, Els, Renée, Inge, Chantal, Hendrik en alle anderen, dank je wel voor

de geduldige begeleiding van mijn eerste schuchtere pasjes in het labo, en het meedenken

wanneer de boel mislukte! Nooit was een opdracht te zwaar, nooit een diepe zucht wanneer ik

opnieuw een vragende blik opwierp!


Beste Andy, van harte dank voor de aangename en intense samenwerking rond ATS. Met

vereende krachten konden we prachtige data generen in dit project. Sofie, Sophie, Marjolein

en Jan, dank voor de nodige (last minute) hulp in het labo, het helpen doorploeteren van

stapels dossiers, en vooral de aangename momenten zowel op het werk, als tot in de late en

soms vroege uurtjes nadien…

Julie en Fransiska, van jullie kon ik veel klinische ervaring opdoen. Julie, voor jou nog een

extra woord van dank voor je advies en hulp in mijn onderzoek, en de morele ondersteuning

waar nodig. Aan de overige clinici van het CMG, prof. Geert Mortier, prof. Jules Leroy, Prof.

Bart Leroy, en Dr. Sandra Janssens, Dr. Barbara Delle Chiaie en Prof. Bruce Poppe, dank

voor het delen van jullie uitgebreide kennis! Virginie, bedankt voor het onmisbare „poli-

geregel‟.

Kristien en Olivier, na de nodige omzwervingen door de vergeten gangen van de K5, zijn we

uiteindelijk samen in één bureau beland. Steeds was het daar aangenaam vertoeven in onze

„mini-supermarkt‟ met op tijd en stond een humorische noot.

Ook aan de mensen van het secretariaat, Katia, Mieke, Melissa, Isabelle, Nathalie en Leen:

dank voor de administratieve bijstand doorheen de voorbije jaren.

Maar vooral een woord van dank voor…

Mijn vrienden, ook al heb ik jullie noodgedwongen wat verwaarloosd, toch was één

telefoontje genoeg om steeds het nodige enthousiasme weer op te wekken! Maar er komt

verandering: Bert is back in town!

Freddy en Marcella, voor het onvoorwaardelijk bijspringen waar nodig, het babysitten, het

onderhoud van ons huis tijdens ons avontuur in de States, de bordjes met een heerlijke

maaltijd tijdens mijn eenzame schrijfmarathons, en de vele gezellige momenten.

Mijn schoonouders, Anja en Henk, voor jullie interesse en de enorme praktische hulp bij de

uitdagingen van de voorbije jaren. Nooit was een vraag te veel, zelfs al ging het om een

retourtje St-Louis. Mijn schoonbroer, Niek, voor het last minute cover design.

Mijn ouders, mama, papa, voor een onvoorwaardelijke steun, jullie interesse en alle kansen

die jullie me gegeven hebben. Steeds was er de nodige „pep-talk‟ wanneer die het meest

nodig was.

Mijn „bollekes‟, Karel en Amber, voor jullie enthousiasme en lach telkens wanneer papa na

een lange dag (en vaak nacht) doodop thuiskwam. Hoe zwaar het ook was, jullie maakten

alles goed en zetten alles weer in het juiste perspectief!

Mijn vrouw, Maartje. Wat ik de voorbije jaren van jou gevraagd heb, is enorm. Vaak stond je

er helemaal alleen voor om het gezin draaiende te houden en ondertussen je eigen werk uit te

bouwen. Maar steeds kon ik rekenen op je steun en sloegen we ons door alle moeilijke

momenten. Je onvoorwaardelijke inzet, liefde en tederheid apprecieer ik eindeloos. Zonder

jou was het schip allang vergaan… Let the future be bright!

Lovendegem, 28 februari 2010.

Comprehensive clinical and molecular assessment of 32 probands with congenital contractural...

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