Mutations in PYCR1 cause cutis laxa with progeroid features

Mutations in PYCR1 cause cutis laxa withprogeroid featuresBruno Reversade1,33, Nathalie Escande-Beillard1,33, Aikaterini Dimopoulou2, Bjorn Fischer2, Serene C Chng1,Yun Li3, Mohammad Shboul1, Puay-Yoke Tham1, Hulya Kayserili4, Lihadh Al-Gazali5, Monzer Shahwan6,Francesco Brancati7,8, Hane Lee9, Brian D O’Connor9, Mareen Schmidt-von Kegler2,10, Barry Merriman9,Stanley F Nelson9, Amira Masri11, Fawaz Alkazaleh11, Deanna Guerra12, Paola Ferrari13, Arti Nanda14,Anna Rajab15, David Markie16, Mary Gray16, John Nelson17, Arthur Grix18, Annemarie Sommer19,Ravi Savarirayan20, Andreas R Janecke21, Elisabeth Steichen22, David Sillence23, Ingrid Hau�er24,Birgit Budde25, Gudrun Nurnberg25, Peter Nurnberg25, Petra Seemann10,26, Desiree Kunkel26,Giovanna Zambruno27, Bruno Dallapiccola7, Markus Schuelke28, Stephen Robertson29, Hanan Hamamy6,Bernd Wollnik3,30,31, Lionel Van Maldergem32, Stefan Mundlos2,10,26 & Uwe Kornak2,10

Autosomal recessive cutis laxa (ARCL) describes a groupof syndromal disorders that are often associated with aprogeroid appearance, lax and wrinkled skin, osteopeniaand mental retardation1–3. Homozygosity mapping inseveral kindreds with ARCL identified a candidate regionon chromosome 17q25. By high-throughput sequencing ofthe entire candidate region, we detected disease-causingmutations in the gene PYCR1. We found that the geneproduct, an enzyme involved in proline metabolism, localizesto mitochondria. Altered mitochondrial morphology,membrane potential and increased apoptosis rate uponoxidative stress were evident in fibroblasts from affectedindividuals. Knockdown of the orthologous genes in Xenopusand zebrafish led to epidermal hypoplasia and blistering thatwas accompanied by a massive increase of apoptosis. Our

findings link mutations in PYCR1 to altered mitochondrialfunction and progeroid changes in connective tissues.

Wrinkly skin and bone loss are typical features associated withaging. In some monogenetic diseases these changes occur prema-turely, resulting in a progeroid appearance of affected individuals.As a clinical feature, wrinkly skin or cutis laxa is used as acommon denominator of several overlapping syndromal disordersincluding autosomal dominant cutis laxa (MIM123700), autoso-mal recessive cutis laxa type I (MIM219100), autosomal recessivecutis laxa type II (ARCL2; MIM219200, also called wrinkly skinsyndrome (WSS; MIM278250)), de Barsy syndrome (DBS;MIM219150), and gerodermia osteodysplastica (MIM231070)1–4.Diagnosis is often difficult in these conditions owing to a broadclinical overlap5,6.

Received 15 April; accepted 3 June; published online 2 August 2009; doi:10.1038/ng.413

1Institute of Medical Biology, A*STAR, Singapore, Singapore. 2Institute of Medical Genetics, Charite Universitaetsmedizin Berlin, Germany. 3Center for MolecularMedicine Cologne, University of Cologne, Cologne, Germany. 4Medical Genetics Department, Istanbul Medical Faculty, University of Istanbul, Turkey. 5Department ofPaediatrics, Faculty of Medicine and Health Sciences, United Arab Emirates University, Al Ain, United Arab Emirates. 6National Center for Diabetes, Endocrinology andGenetics, Amman, Jordan. 7Ospedale Casa Sollievo della Sofferenza (CSS), San Giovanni Rotondo and Istituto CSS-Mendel, Rome, Italy. 8Centro Studi Invecchiamento(Ce.S.I.), Department of Biomedical Sciences, Gabriele d’Annunzio University, Chieti, Italy. 9Department of Human Genetics, David Geffen School of Medicine,University of California, Los Angeles, Los Angeles, California, USA. 10Max Planck Institute for Molecular Genetics, Berlin, Germany. 11Departments of Pediatrics,Obstetrics and Gynecology, Faculty of Medicine, The University of Jordan, Amman, Jordan. 12Department of Biomedical Sciences, University of Modena and ReggioEmilia, Modena, Italy. 13Department of Pediatrics, University of Modena and Reggio Emilia, Modena, Italy. 14As’ad Al-Hamad Dermatology Center, Al-Sabah Hospital,Kuwait City, Kuwait. 15Genetic Unit, Directorate General of Health Affairs, Ministry of Health, Muscat, Sultanate of Oman. 16Department of Pathology, University ofOtago, Dunedin, New Zealand. 17Genetic Services of Western Australia, King Edward Memorial Hospital for Women, Perth, Australia. 18The Permanente Medical Group,Sacramento, California, USA. 19The Ohio State University College of Medicine and Nationwide Children’s Hospital, Molecular and Human Genetics, Columbus, Ohio,USA. 20University of Melbourne, Murdoch Childrens Research Institute, Royal Children’s Hospital, and Genetic Health Services Victoria, Parkville, Victoria, Australia.21Division of Clinical Genetics, Innsbruck Medical University, Innsbruck, Austria. 22Klinik fuer Kinder- und Jugendheilkunde, Universitaet Innsbruck, Innsbruck,Austria. 23Department of Clinical Genetics, The Children’s Hospital at Westmead, Sydney, New South Wales, Australia. 24Universitaets-Hautklinik Heidelberg,Heidelberg, Germany. 25Cologne Center for Genomics, Universitat zu Koln, Germany. 26Berlin-Brandenburg Center for Regenerative Therapies, ChariteUniversitaetsmedizin Berlin, Germany. 27Laboratory of Molecular and Cell Biology, Istituto Dermopatico dell’Immacolata - Istituto di Ricovero e Cura a CarattereScientifico, Rome, Italy. 28Department of Neuropediatrics and NeuroCure Clinical Research Center, Charite Universitaetsmedizin Berlin, Germany. 29Department ofPaediatrics and Child Health, University of Otago, Dunedin, New Zealand. 30Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases(CECAD), University of Cologne, Cologne, Germany. 31Institute of Human Genetics, University of Cologne, Cologne, Germany. 32Centre de genetique humaine, CentreHospitalier Universitaire du Sart-Tilman, Universite de Liege, Liege, Belgium. 33These authors contributed equally to this work. Correspondence should be addressed toB.R. ([email protected]) or S.M. ([email protected]).

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Here we describe mutations in PYCR1 associated with a spectrumof disorders characterized by wrinkly skin, osteopenia and progeroidappearance. The clinical features of the 35 affected individuals from 22families included in our study are summarized in Table 1. Theyinclude congenital skin wrinkling, most pronounced on the dorsum ofhands and feet; generalized connective tissue weakness; finger con-tractures; hernias; osteopenia; and a triangular face with a progeroidappearance that is due to lax skin and hypoplasia of the jaw oftenresulting in prognathism (Fig. 1a–d). Ultrastructural investigation ofthe skin showed rarefaction and fragmentation of elastic fibers similarto changes described in ARCL2 (Fig. 1e–h). No glycosylation abnorm-alities were detected by standard diagnostics (data not shown). Avariable degree of mental retardation was observed in all cases but one.In many subjects, agenesis of the corpus callosum was evident.Although most affected individuals were classified as having geroder-mia osteodysplastica or WSS, more severely affected individuals alsoshowed childhood cataracts or dystonic movements and were there-fore diagnosed as having DBS1,3,5–9.

Homozygosity mapping in five consanguineous families revealed aminimal candidate region of 2.8 Mb on chromosome 17q25 betweenmarkers rs8065431 and rs1046896 (Fig. 1i). To screen the 59 candidategenes in this region for disease-causing mutations we combinedconventional sequencing and genomic locus capture followed byhigh-throughput sequencing (H. Lee, B. O’Connor, B. Merriman,N. Homer, Z. Chen et al., unpublished data) (Fig. 1j). The latteridentified 552 genomic mismatches, of which 7 were newly identifiedSNPs that cause non-synonymous changes (Supplementary Table 1).We next cross-examined genes with mutations in four othersequenced samples to evaluate the causality of the observed changes.

Both methods identified mutations in PYCR1, the gene encoding theenzyme D1-pyrroline-5-carboxylate reductase 1 (PYCR1).

In total, we identified eight missense mutations, one frameshiftmutation, five splice-site mutations and one 22-bp deletion encom-passing the exon-intron boundary of exon 5 (Table 1 and Supple-mentary Fig. 1a). Six of these mutations were recurrent, eventhough the affected individuals did not have a common ethnicbackground. Bioinformatic analysis predicted deleterious effects forall changes with the exception of the heterozygous A189V, forwhich we cannot rule out the possibility that it is a rare variant(Supplementary Fig. 1b).

As skin was the most severely affected organ, we isolated skinfibroblasts from probands and their unaffected siblings to examinePYCR1 protein expression (Fig. 2). Strongly reduced PYCR1 proteinlevels were detected in a subject (S.J.) with a homozygous splice sitemutation and in a compound heterozygous subject (D.I.) with amissense and a frameshift mutation (Fig. 2a). The missense mutationsG206W (T.P.) and R251H (E.U.) resulted in a reduction in proteinabundance relative to control cells (Fig. 2b–f). The PYCR1 protein,which has been often described as cytosolic, colocalized with variousmitochondrial markers (data not shown), among them D1-pyrroline-5-carboxylate synthase (P5CS), which also belongs to the proline meta-bolic pathway (Fig. 2b)10,11. We therefore conclude that PYCR1 is amitochondrial protein and that the reported mutations lead to a lossof function.

Pycr1 was most highly expressed in bone and skin, the two tissuesmost severely affected in human subjects (Supplementary Fig. 2a).Two paralogs of PYCR1 exist in human, the highly similar PYCR2and the more distantly related PYCRL. No significant expression

Table 1 Phenotypic characteristics of investigated subjects with ARCL

Phenotypic findings Mutations

Family Origin

No. aff.

ind. IUGR

Lax,

wrinkly

skin

Hip

dislocation Hernias

Osteo-

penia

Abnormal

corpus

callosum

Mental

retardation

Athetoid

movements Cataract

Initial

diagnosis Status cDNA Consequence Exon

Literature

ref. on

phenotype

J.I. Iraq 1 + ++ + � + � � � � WSS Hom 138+1G4A ND 2

T.P. Palestine 1 + + � � ND + + + � WSS Hom 616G4T G206W 5 7

S.P. Palestine 3 + ++ + � + +/� +/� � � GO or WSS Hom 617_633+6del ND 5 5

I.K. Kuwait 2 + ++ ++ ++ ++ � + � � GO or WSS Hom 797G4A R266Q + splicing 6 6

F.S. Kuwait 1 + + ++ ++ ++ � + � � GO or WSS Hom 797G4A R266Q + splicing 6 6

S.J. Jordan 5 + ++ � + ND + + � � WSS Hom 797+2_797

+5del

K215_D319del 6

D.I. Italy 1 + ++ � ++ ++ ND + � � GO Het 11delG G4fsX50 1

Het 355C4G R119G 4

S.O. Oman 1 � ++ + � + � + � � GO Het 356G4A R119H 4

Het 566C4T A189V 5

G.O. Oman 3 ND ++ � � + ND + � � GO Hom 356G4A R119H 4 1

H.B. Bahrain 2 + ++ ++ + � ND + � � GO Hom 535G4A A179T 4

M.A. Austria 1 + + ++ + ++ ND + � � GO Hom 540+1G4A ND 4

M.Q. Qatar 1 ND ++ ++ + ++ ND + � � GO Hom 616G4A G206R 5

H.U. United States 1 + + � � � + + � � GO Hom 633+1G4C ND 5 8

N.A. Australia 1 + ++ + + ND ND + � � GO Het 633+1G4C ND 5

Het 797+1G4T ND 6

A.S. Syria 1 + + � + + + + � � GO Hom 797+2_797+5del K215_D319del 6 5

D.T. Turkey 1 + ++ + � ND ND +/� � � DBS Hom 355C4G R119G 4

M.F. Italy 1 + ++ ND ++ ND ND + + � DBS Het 355C4G R119G 4 3

DBS Het 356G4A R119H 4

S.T. Turkey 2 + + + � ND + + + � DBS Hom 540+1G4A ND 6

E.U. United States 1 + ++ + ++ ND + + � + DBS Hom 752G4A R251H 6

J.A. Australia 3 + ++ � � ND ND ++ + + DBS Hom 769G4A A257T 6 9

K.T. Turkey 1 � + + � ND + + � � DBS Hom 797G4A R266Q + splicing 6

I.T. Turkey 1 + + + � ND � + � � DBS Hom 797+2_797+5del K215_D319del 6

GO, gerodermia osteodysplastica; WSS, wrinkly skin syndrome; DBS, de Barsy syndrome. No. aff. ind., number of affected individuals in family; IUGR, intrauterine growthretardation; Hom, homozygous; Het, heterozygous. ND, not determined; +/�, mild; +, average; ++, strong. For hernias: +, unilateral; ++, bilateral.

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changes in fibroblasts from individuals with PYCR1 mutations wereobserved for PYCR2 or for ALDH18A1, encoding P5CS (Supple-mentary Fig. 2b). As PYCR1 catalyzes the obligatory and final stepin de novo proline synthesis, we first investigated whether affectedindividuals showed hypoprolinemia. Serum proline levels in affectedindividuals were slightly low, but within normal range (Supple-mentary Fig. 3a). Likewise, cultured skin fibroblasts did not showevidence of low proline (Supplementary Fig. 3b) or of reduced pro-liferation rates in medium without proline (Supplementary Fig. 3c),arguing against a proline auxotrophy.

On the basis of the mitochondrial localization of PYCR1, weexamined the mitochondrial network in more detail. Ultrastructuralanalysis revealed abnormal morphology of mitochondria and theircristae (Fig. 3a,b). These differences were exacerbated upon oxidative

stress (Fig. 3c–e). Addition of H2O2 to the cellculture medium induced the collapse of thefilamentous mitochondrial network in cellsfrom affected individuals, whereas almost nochanges were observed in mitochondria ofcontrol cells (Fig. 3e). Under normal cultureconditions, affected and control fibroblastsshowed no differences in their levels of apop-tosis or mitochondrial membrane potential.However, after a short exposure to H2O2 adecreased mitochondrial membrane potentialand a fivefold increase in cell death in affectedfibroblasts compared to controls was observed(Fig. 3f,g). This suggests that PYCR1 isinvolved in the cell’s response to oxidative

stress. In contrast, we detected no significant differences in therespiration rates or reactive oxygen species (ROS) production betweenaffected and control cells (Supplementary Fig. 4).

We next addressed PYCR1’s function during embryogenesis, aspatients show ARCL phenotypes at birth. As PYCR1 is conservedthroughout evolution (Fig. 4a)12, we used a morpholino-based knock-down strategy (Fig. 4b) to model the disease in Xenopus embryos. Bystage 30, Pycr1 morphant embryos showed obvious skin defects andfailed, in a cell-autonomous fashion, to develop eyes and a tail bud(Fig. 4c–f). By the swimming tadpole stage, skin hypoplasia led toregions of ectodermal edema (Fig. 4g,h). Histological sections revealeddisorganization of the skin epithelium, with enlarged cells and nuclei(Fig. 4i,j). We observed a similar phenotype upon morpholinoknockdown of the Pycr1 and Pycr2 genes in zebrafish (Supplementary

Chr. 17

1 25 50 ×Sequencing coverage

78

77

76

75

74

73

q25B

17q2

217

q12

p12

13.1

Chr. 17: 77486276–77486277C>T (7:7:100%[F:6:85.7%][R:1:14.3%])

PYCR1

PYCR1

cDNA 356G>A

Protein R119H

22

222222

22222222

222222

22

222222

22222121

12

11

111111

11

111111111111

111111

12

2222

22222121

11

1111111111

1111

Family T.P.Family S.J.Family I.K.Family H.B.

rs8065431rs1877677rs4890056rs2075722rs7211306rs4789817rs1046896

a b c d

e

i

j

f g h

Figure 1 Phenotypic characteristics and genetic

findings of subjects with PYCR1-related

autosomal recessive cutis laxa with progeroid

appearance. (a) Affected 2-year-old girl from

family T.P., initially diagnosed with wrinkly skin

syndrome. (b) Affected 18-month-old girl from

family D.I. clinically categorized as having

gerodermia osteodysplastica. (c) Newborn boy

from family E.U. diagnosed with de Barsy

syndrome. Typical facial gestalt including

triangular face, malar hypoplasia and dysplastic,

large ears. (d) Skin wrinkling at the dorsum of the

hand and typical adducted thumb in an affected

individual from family H.B. Participants gave

consent for publication of all photos.(e–h) Findings in skin biopsy from affected

individual from family D.I. (e,f) Weigert staining

reveals sparse, thinned and fragmented elastic

fibers in the reticular dermis compared to control

(f); scale bar, 100 mm. (g,h) Ultrastructural

analyses corroborate the markedly reduced elastic

fiber (arrows) size with a reduction in both the

fibrillar and amorphous components in an

affected individual from family D.I. compared to

control (h); scale bar, 500 nm. (i) Homozygosity

mapping resulted in a minimal candidate region

of 2.8 Mb on chromosome (chr.) 17q25 between

markers rs8065431 and rs1046896. Only the

four most informative pedigrees are shown.

(j) Genomic capture followed by next-generation

sequencing of the maximal candidate region

extending from 72.05–78.77 Mb. A sequence

change of C4T was sequenced seven times in asubject from family G.O. This base change

resulted in a single R119H substitution in PYCR1.


L E T T E R S

Fig. 5a–j). Microinjection of human PYCR1 mRNA partially rescuedthe phenotype observed in frogs and fish (Supplementary Figs. 5and 6). Pycr1 morphant frogs had no circulating red blood cells(Supplementary Videos 1 and 2), as validated by the loss of Sclexpression, a marker of primitive hematopoiesis (Fig. 5a,b). To ruleout the possibility that the skin atrophy was caused by anemia,we performed a parabiosis experiment joining control and Pycr1

morphant embryos. Despite shared bloodcirculation (Supplementary Video 3), skinhypoplasia and stunted growth were notrescued in parabiotic Pycr1 morphants eventhough fewer regions of edema were observed(Fig. 5c–e). We conclude that the ancestralPycr1 gene in Xenopus plays dual but inde-pendent roles during blood and skin devel-opment that may be attributed to theparalogous PYCR1 and PYCR2 genes inhuman. Indeed, a role for PYCR2 in humanerythrocytes has been well documented10,11.

The marked increased cell death in thedeveloping skin of frogs and fish, as revealedby acridine orange and TUNEL staining(Fig. 5f–i and Supplementary Fig. 5i,j),underscores the specific requirement forPycr1 in this particular tissue. This parallelsthe data obtained with cells from affectedindividuals. Together these lines of evidencesuggest that PYCR1 mutants have compro-mised mitochondrial function that leads to

apoptosis during embryogenesis and a general inability to thriveduring adult life.

PYCR1 belongs to the NAD(P)H-binding Rossmann-fold domainsuperfamily and catalyzes the conversion of D1-pyrroline-5-carboxy-late to L-proline. Determination of the crystal structure revealedthat the mature enzyme consists of five homodimer subunits arounda peripheral circular groove12. Several heritable disorders have been

S.J.

K215_D319del

D.I.

G4fsX50R119G

R251H

E.U.

T.P.

G206W

Ct

PYCR1 P5CS Merge

40

37Ct T.P. E.U. D.I. S.J.

PYCR1

Actin

a

b

c

d

e

f

Figure 2 Effect of PYCR1 mutations. (a) Immunoblot analysis of PYCR1 protein expression in primary

skin fibroblasts from controls and patients. Actin served as a loading control. PYCR1 expression is

absent in affected individuals D.I. and S.J., whereas E.U. and T.P. show a substantial reduction.

(b–f) Immunofluorescent detection of PYCR1 and P5CS in mitochondria from skin fibroblasts. (b) In

control (Ct) fibroblasts, PYCR1 and the mitochondrial matrix protein P5CS colocalize. In fibroblasts

from subjects D.I. (e) and S.J. (f), immunostaining for PYCR1 is undetectable. In contrast, in fibroblasts

from T.P. (c) and E.U. (d), PYCR1 staining in mitochondria is lower in intensity relative to control.

Mitochondrial network is abnormal in D.I., S.J. and T.P. Scale bar, 20 mm.

104103102101100

Fluorescence intensity



104103102101100

104103102101100

D.I.

S.J.

Control

0

20

40

Cou

nts

6080

100

0

20

40

Cou

nts 60

80

100

0

20

40C

ount

s 6080

100– H2O2+ H2O2

– H2O2

+ H2O2

Aff. (n = 4)Ct (n = 2)Aff. (n = 4)Ct (n = 2)

200 µM H2O2Control

012

4

6

8

Fol

d in

crea

se

FragmentedFragmented Interm.Interm. TubularTubular

Control (n = 3) Affected (n = 4)

P = 0.026

0102030405060708090

100

Affected + H2O2Control + H2O2

AffectedControl

Per

cent

age

of c

ells

P = 0.02

P = 0.05

P = 0.08

P = 10–8

P = 0.003

P = 2 × 10–6

a

c d

be g

f

Figure 3 Loss of PYCR1 causes increased sensitivity to oxidative stress. (a,b) Ultrastructural analysis of mitochondrial morphology. Whereas mitochondria

in control fibroblasts (a) have regularly arranged cristae, fibroblasts from affected individual D.I. (b) show strongly altered cristae and a smaller diameter.

Scale bar, 1 mm. (c,d) Changes in mitochondrial morphology upon oxidative stress. Addition of 500 mM H2O2 to cell culture medium for 10 min leads to

mitochondrial network collapse in fibroblasts from affected individual from family S.J. (d); tubular mitochondrial network remains largely intact in controlcells (c). Scale bar, 20 mm. (e) Quantification of mitochondrial morphology changes upon oxidative stress. Without oxidative stress (gray), most control

(n ¼ 3) and affected (n ¼ 4) cells showed tubular mitochondria. Only a few cells with fragmented or intermediate (interm.) morphologies were seen.

In contrast, under oxidative stress (black) the number of cells with fragmented mitochondria increased much more in affected cells than in control.

(f) Quantification of apoptotic cell death by TUNEL 24 h after oxidative stress. Fibroblasts from control (Ct; n ¼ 2) and affected individuals (Aff; n ¼ 4)

showed no differences in apoptosis under normal culture conditions (control). Fivefold more (P ¼ 0.026) apoptosis was observed in cells from affected

individuals 24 h after a 10 min incubation with 200 mM H2O2. Normalized to Ct cells in control experiment. Data in e,f averaged from three independent

experiments; error bars, s.d. (g) Mitochondrial membrane potential is reduced in fibroblasts from affected individuals S.J. and D.I. after 2 h incubation

with 200 mM H2O2 but unchanged in control cells. Representative results.


L E T T E R S

linked to the proline metabolic pathway13. Most notably, P5CSdeficiency causes a condition of mental retardation, joint hyper-mobility and skin laxity (MIM612652)14–16. As in the subjectsdescribed herein, P5CS deficiencies are not consistently characterizedby low serum proline14,15. Furthermore, fibroblasts from the subjectsdescribed here did not show the proliferation defect describedupon P5CS deficiency16. The phenotype of PYCR1-related diseaseoverlaps with other forms of cutis laxa, mirrored by the fact thataffected individuals from this cohort have been previously describedunder the diagnoses WSS, gerodermia osteodysplastica or DBS1,3,5–9.Whereas gerodermia osteodysplastica caused by mutations in GORAB(formerly known as SCYL1BP1) does not lead to intellectual impair-ment, ARCL2 often includes mild or moderate mental retardationand brain anomalies2,17,18. The mutations we detected cluster in exons4, 5 and 6, which encode the catalytic sites of PYCR1 and arepredominantly missense and splice-site mutations. Although awide range of phenotypes was observed (see Table 1), no cleargenotype-phenotype correlation could be discerned. Mutations in

ATP6V0A2, encoding subunit a2 of the V-ATPase complex thatlocalizes to endosomes and the Golgi apparatus, have been identifiedin ARCL2 (ref. 19). The GORAB gene also encodes a Golgi-localizedprotein18. The phenotypic similarities suggest a common, yet to bedetermined, pathogenetic mechanism in these conditions. Notably,increased apoptosis is also seen in fibroblasts from individuals withARCL2 having ATP6V0A2 defects20.

Besides energy production, mitochondria serve many differentfunctions, among them the generation of potentially harmfulROS21. ROS are involved in the regulation of apoptosis and assuch have become a central focus of aging research22. ROS wereinitially regarded as toxic side products of oxidative phos-phorylation, and their importance for redox-dependent signalingprocesses has emerged only recently22,23. Indeed, proline hasbeen implicated in the regulation of ROS abundance, and PYCR1and PYCR2 have been implicated in the regulation of theNAD(P)H/NAD(P)+ ratio in the cytoplasm and in the mitochon-drion10,11,24. Mitochondrial fragmentation is a known pheno-menon that has been described as impairing cellular function inneurodegenerative disorders25. Notably, changes in mitochondrial

Pycr1 MO

Pycr1 MO

Pycr1 MO

Pycr1 MO

Control

Control Pycr1 MO

Control

×2 D ×2 V

×4

MOMO

MOMO

MOMO

MO

MO

+24Pycr1 MO–1

1 2010–10

Chicken

Human

Zebrafisha

c d

e

g

h

i

j

f

bE. coli

Drosophila

Arabidopsis

C. elegans

Xenopus

X. laevis :

X. tropicalis :

TUNELTUNEL

×2 V

MOMO

Acridine orangeAcridine orange

Parabiosis

Pycr1 MO

Control

TUNEL

Scl Scl

Pycr1 MOControl

a

c

f

h i

g

d

e

b

Figure 4 Phenotype of Pycr1 Xenopus morphants. (a) Pycr1 is evolutionary

conserved among bacteria, plants, insects and vertebrates. (b) Design

of a translation-blocking morpholino (MO) oligonucleotide for Xenopus

Pycr1. (c) Control embryo at stage 30. (d) Pycr1 morphants injected

in each blastomere at the four-cell stage failed to grow a tail and had

underdeveloped eyes. (e) Dorsal (D) MO injections impaired eye but

not tail development. (f) Ventral (V) MO injections impaired tail but not

eye development. Arrowheads, skin wrinkles. (g) Control embryo at the

swimming tadpole stage. (h) Pycr1 morpholino-injected embryos showing

severe growth retardation and large regions of ectodermal edema over the

entire body, which develop from wrinkles seen earlier (e,f). (i) Histological

section of skin from control embryo at stage 45 showing bilayered epidermis

consisting of outer periderm and inner sensorial layer. (j) Disorganized

architecture of epidermis with enlarged cells, enlarged nuclei and cellular

protrusions in Pycr1 morphant.

Figure 5 Skin hypoplasia in Pycr1 morphants is accompanied by

spontaneous apoptosis and is independent of anemia. (a) Scl expression

at stage 25 marks site of primitive hematopoiesis in ventral ectoderm of

control embryo. (b) Scl expression was abrogated in Pycr1 morphants

suffering anemia (Supplementary Videos 1 and 2). (c) Parabiosis performed

at stage 22 between a Pycr1-depleted (MO) tadpole (top) and control

(bottom) did not rescue from apoptosis (inset, stage 33), stunted growth

and skin defects despite shared blood circulation (Supplementary Video 3).

(d) Histological staining. Epidermis of Pycr1 parabiotic morphant embryo

remained disorganized relative to control. (e) Histological section through

epidermis of parabiotic control tadpole at stage 45 showing normal skinarchitecture. (f) Acridine-orange background staining of control embryo

(inset, bright-field image). (g) Acridine-orange labeling of dying cells (intense

green) in Pycr1 morphant embryos injected only in the ventral (V) half

(inset, bright-field image). Only the ventro-posterior half of the embryo,

derived from the injected ventral blastomeres, is undergoing apoptosis.

(h) TUNEL staining in stage 33 tadpole showing few apoptotic cells (inset,

control at stage 25). (i) Pycr1 morphants, with a markedly increased number

of dying cells at stage 33 relative to control (inset, stage 25).


L E T T E R S

fusion have also been shown to trigger cellular senescence26.Although the exact role of PYCR1 in mitochondrial function andintegrity remains to be determined, its orthologs in other species,such as Saccharomyces, Arabidopsis and Drosophila, have beenlinked to stress resistance, which suggests that human PYCR1 aswell may provide adaptive protection against stress by maintaining areservoir of rapidly mobilizable proline and reducing equivalentsin mitochondria10,12.

In summary, mutations in PYCR1 leading to a loss of PYCR1function are a major cause of autosomal recessive cutis laxa,evoking a phenotype that overlaps with gerodermia osteodysplastica,ARCL2 and DBS. The pathophysiological basis appears to be animpaired mitochondrial function leading to developmental defectsthrough increased apoptosis. Our finding highlights the importanceof mitochondrial function in the process of aging and mighthelp to develop new treatment strategies for common age-related disorders22.

METHODSMethods and any associated references are available in the onlineversion of the paper at http://www.nature.com/naturegenetics/.

Accession codes. GenBank RefSeq DNA: Homo sapiens PYCR1isoform A, NM_006907.2; H. sapiens PYCR1 isoform B,NM_153824.1; Xenopus laevis Pycr1, NM_001091619; Danio reriopycr1, BC095354; D. rerio pycr2, BC060905.

Note: Supplementary information is available on the Nature Genetics website.

ACKNOWLEDGMENTSJ. Common and B. Lane for advice and help with primary cultures. J. Gautier forTUNEL protocol. K. Rogers and S. Rogers for their help and advice on histology.This study was supported by the German Federal Ministry of Education andResearch (BMBF) by grant 01GM0623 (SKELNET) to U.K. and S.M. andgrants 01GM0880 and 01GM0801 to B.W., a grant from the DeutscheForschungsgemeinschaft (Collaborative Research Centre 577) to U.K. and P.N.,and by A*STAR and the Branco Weiss Foundation to B.R.

AUTHOR CONTRIBUTIONSB.R., N.E.-B., S.M. and U.K. designed the study. H.K., L.A.-G., M. Shahwan,B.R., F.B., S.F.N., A.M., F.A., D.G., P.F., A.N., A.R., D.M., M.G., J.N., A.G.,A.S., R.S., A.R.J., E.S., D.S., B.D., S.R., H.H., B.W. and L.v.M. made clinicaldiagnoses and collected clinical data and samples. B.R., B.M., A.D., G.N.,B.F., Y.L., B.B., P.N. and B.W. conducted genotyping and linkage analysis.H.L., B.O., B.M. and S.N. performed genomic loci capture. B.R., N.E.-B.,A.D., B.F., D.K., S.C., M.S., P.-Y.T. performed immunoblotting, immunostaining,TUNEL, and proline level and FACS analysis. B.R., N.E.-B. and S.C.performed Xenopus work, ISH and Pycr1 constructs. U.K., B.F., M.S.-v.K.and P.S. performed zebrafish work. B.F., U.K. and M. Schuelke performedrespiratory chain measurements. I.H. and G.Z. were responsible forultrastructural and histological analysis of skin biopsies. B.R., N.E.-B., S.M.and U.K. wrote the manuscript.

Published online at http://www.nature.com/naturegenetics/.

Reprints and permissions information is available online at http://npg.nature.com/

reprintsandpermissions/.

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2. Morava, E. et al. Defining the phenotype in an autosomal recessive cutis laxa syndromewith a combined congenital defect of glycosylation. Eur. J. Hum. Genet. 16, 28–35(2008).

3. Guerra, D. et al. The De Barsy syndrome. J. Cutan. Pathol. 31, 616–624 (2004).4. Zhang, M.C. et al. Cutis laxa arising from frameshift mutations in exon 30 of the elastin

gene (ELN). J. Biol. Chem. 274, 981–986 (1999).5. Al-Gazali, L.I., Sztriha, L., Skaff, F. & Haas, D. Gerodermia osteodysplastica and

wrinkly skin syndrome: are they the same? Am. J. Med. Genet. 101, 213–220 (2001).6. Nanda, A. et al. Gerodermia osteodysplastica/wrinkly skin syndrome: report of three

patients and brief review of the literature. Pediatr. Dermatol. 25, 66–71 (2008).7. Hamamy, H., Masri, A. & Ajlouni, K. Wrinkly skin syndrome. Clin. Exp. Dermatol. 30,

590–592 (2005).8. Sommer, A. Photo essay–geroderma osteodysplastica. Am. J. Med. Genet. C. Semin.

Med. Genet. 145C, 291–292 (2007).9. Kunze, J. et al. De Barsy syndrome–an autosomal recessive, progeroid syndrome. Eur.

J. Pediatr. 144, 348–354 (1985).10. Phang, J.M., Pandhare, J. & Liu, Y. The metabolism of proline as microenvironmental

stress substrate. J. Nutr. 138, 2008S–2015S (2008).11. Hagedorn, C.H. & Phang, J.M. Transfer of reducing equivalents into mitochondria by

the interconversions of proline and delta 1-pyrroline-5-carboxylate. Arch. Biochem.Biophys. 225, 95–101 (1983).

12. Meng, Z. et al. Crystal structure of human pyrroline-5-carboxylate reductase. J. Mol.Biol. 359, 1364–1377 (2006).

13. Mitsubuchi, H., Nakamura, K., Matsumoto, S. & Endo, F. Inborn errors of prolinemetabolism. J. Nutr. 138, 2016S–2020S (2008).

14. Baumgartner, M.R. et al. Delta1-pyrroline-5-carboxylate synthase deficiency: neuro-degeneration, cataracts and connective tissue manifestations combined with hyper-ammonaemia and reduced ornithine, citrulline, arginine and proline. Eur. J. Pediatr.164, 31–36 (2005).

15. Bicknell, L.S. et al. A missense mutation in ALDH18A1, encoding Delta1-pyrroline-5-carboxylate synthase (P5CS), causes an autosomal recessive neurocutaneous syn-drome. Eur. J. Hum. Genet. 16, 1176–1186 (2008).

16. Baumgartner, M.R. et al. Hyperammonemia with reduced ornithine, citrulline, arginineand proline: a new inborn error caused by a mutation in the gene encoding delta(1)-pyrroline-5-carboxylate synthase. Hum. Mol. Genet. 9, 2853–2858 (2000).

17. Van Maldergem, L. et al. Cobblestone-like brain dysgenesis and altered glycosylation incongenital cutis laxa, Debre type. Neurology 71, 1602–1608 (2008).

18. Hennies, H.C. et al. Gerodermia osteodysplastica is caused by mutations inSCYL1BP1, a Rab-6 interacting golgin. Nat. Genet. 40, 1410–1412 (2008).

19. Kornak, U. et al. Impaired glycosylation and cutis laxa caused by mutations in thevesicular H+-ATPase subunit ATP6V0A2. Nat. Genet. 40, 32–34 (2008).

20. Hucthagowder, V. et al. Loss-of-function mutations in ATP6V0A2 impair vesiculartrafficking, tropoelastin secretion, and cell survival. Hum. Mol. Genet. 18, 2149–2165(2009).

21. Wallace, D.C. A mitochondrial paradigm of metabolic and degenerative diseases,aging, and cancer: a dawn for evolutionary medicine. Annu. Rev. Genet. 39,359–407 (2005).

22. Balaban, R.S., Nemoto, S. & Finkel, T. Mitochondria, oxidants, and aging. Cell 120,483–495 (2005).

23. D’Autreaux, B. & Toledano, M.B. ROS as signalling molecules: mechanisms thatgenerate specificity in ROS homeostasis. Nat. Rev. Mol. Cell Biol. 8, 813–824 (2007).

24. Krishnan, N., Dickman, M.B. & Becker, D.F. Proline modulates the intracellular redoxenvironment and protects mammalian cells against oxidative stress. Free Radic. Biol.Med. 44, 671–681 (2008).

25. Knott, A.B., Perkins, G., Schwarzenbacher, R. & Bossy-Wetzel, E. Mitochondrialfragmentation in neurodegeneration. Nat. Rev. Neurosci. 9, 505–518 (2008).

26. Lee, S. et al. Mitochondrial fission and fusion mediators, hFis1 and OPA1, modulatecellular senescence. J. Biol. Chem. 282, 22977–22983 (2007).


L E T T E R S



ONLINE METHODSSubjects. Several of the subjects presented here have already been described in

the literature. All other individuals were recruited based on a clinical diagnosis

of either gerodermia osteodysplastica, autosomal recessive cutis laxa (also called

wrinkly skin syndrome) or de Barsy syndrome. Inclusion criteria were a

progeroid appearance; lax, wrinkly skin at early age; hernias; hip dislocation;

and osteopenia. In some individuals, mental retardation, brain malformations

(abnormality of the corpus callosum), athetoid movements and/or cataracts

were present; these individuals were considered to have de Barsy syndrome.

DNA samples were obtained from 22 families after participants gave their

informed consent and after approval by the local ethics commission (Ethik-

kommission der Charite).

Genotyping and linkage analysis. We performed genome-wide linkage analysis

in eight families using the GeneChip Human Mapping 250K Arrays (Affyme-

trix). Genotypes were called by the GeneChip DNA Analysis Software (v2.0,

Affymetrix). We verified sex by counting heterozygous SNPs on the

X chromosome. Relationship errors were evaluated with the help of the

program Graphical Relationship Representation. The program PedCheck27

was applied to detect mendelian errors and data for SNPs with such errors

were removed from the data set. Nonmendelian errors were identified by using

the program MERLIN28 and genotypes unlikely for related samples were

deleted. Nonparametric linkage analysis using all genotypes of a chromosome

simultaneously was carried out with MERLIN. Parametric linkage analysis was

performed by a modified version of the program GENEHUNTER 2.1 through

stepwise use of a sliding window with sets of 110 or 200 SNPs29. Haplotypes

were reconstructed with GENEHUNTER 2.1 and presented graphically with

HaploPainter30.

Mutation analysis. Positional candidate genes were obtained from the Gen-

Bank and Ensembl databases. Genes were analyzed by direct sequencing of

DNA with primers flanking each exon. Primer sequences were based on the

reference sequences of each gene. The primer sequences for PYCR1 mutation

screening are given in Supplementary Table 2. Sequence analysis was done

with the BigDye Terminator cycle sequencing kit (Applied Biosystems), and

products were run on a 3730 DNA Analyzer (Applied Biosystems).

Genomic locus capture. Briefly, custom arrays (Agilent 244K) were designed to

target every exonic sequence of the genes present in the locus 17q25 between

72048357 and 78774742, excluding the highly repeated regions. DNA libraries

were prepared following the Illumina library generation protocol version 2.3

and were hybridized to the custom arrays according to the Agilent CGH 244K

array protocol, then washed, eluted and amplified. Each sample was submitted

to one channel of an Illumina flow cell and sequenced by Illumina Genome

Analyzer using standard manufacturer’s protocol. We sequenced a total of five

samples from four different families. The image data was processed by the

provided Genome Analyzer Pipeline version 1.0 and all sequences were aligned

to the human reference genome (University of California Santa Cruz, build 18)

by Blat-like Fast Accurate Search Tool (BFAST (see URLs); N. Homer,

B. Merriman and S.F. Nelson, unpublished data). Mismatches were filtered for

those seen two or more times, where the mutation was seen 100% of the time

(homozygous), and did not overlap with a known dbSNP129 entry mismatch.

The open-source SeqWare project, which provides a laboratory information

management system tool for tracking samples (SeqWare laboratory information

management system) and a pipeline for sequence analysis (SeqWare Pipeline)

was used throughout this work.

Cell culture. Human skin fibroblasts were cultivated in DMEM (Lonza)

supplemented with 10% FCS (Lonza), 1% Ultraglutamine (Lonza) and 1%

penicillin/streptomycin (Lonza) at 5% CO2 and 37 1C.

Quantitative PCR. RNA was isolated from organs of postnatal day 4 mice using

the Trizol (Invitrogen) method. One microgram of RNA was reverse-tran-

scribed using the Revert Aid kit (Fermentas) and random hexamers. Quanti-

tative PCR was performed with SYBR green (Applied Biosystems). Primers are

given in Supplementary Table 2.

Immunoblot analysis. Mitochondrial and cytosolic fractions of whole cell

lysates of primary skin fibroblasts were obtained with a mitochondrial isolation

kit for mammalian cells (Thermo Scientific) and resolved by electrophoresis in

SDS polyacrylamide gels. For immunoblot analysis, PVDF membranes were

probed with anti-actin A5060 (Sigma) 1:1,000 and rabbit anti-PYCR1 (Pro-

teintech) 1:500.

Immunofluorescence analysis. For immunofluorescence, cells were washed in

phosphate-buffered saline (PBS) three times. Cells were fixed in 4% (wt/vol)

paraformaldehyde in PBS for 10 min and permeabilized with 0.4% (vol/vol)

Triton-X100 in 3% BSA in PBS for 10 min at 4 1C. Specific antibodies to

PYCR1 (rabbit anti-PYCR1, Proteintech 1:500) and P5CS (mouse anti-P5CS,

Abnova, 1:300) were incubated overnight in 3% BSA at 4 1C. For visualization

an antibody to mouse immunoglobulin G (anti–mouse IgG) Alexa Fluor 555

conjugate (Invitrogen, Molecular Probes) and an anti-rabbit IgG Alexa Fluor

488 (Invitrogen, Molecular Probes) conjugate was applied. DNA was stained by

DAPI and cells were mounted in Fluoromount (Scientific Services). Images

were collected by using an LSM 510 Meta (Carl Zeiss) with a �63 Plan

Apochromat oil immersion objective.

Ultrastructural analysis. All specimens were fixed for at least 2 h at 19–22 1C

in 3% glutaraldehyde solution in 0.1 M cacodylate buffer, pH 7.4, cut into

pieces of B1 mm3, washed in buffer, postfixed for 1 h at 4 1C in 1% osmium

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).

Mitochondrial morphology. Morphology of the mitochondrial network was

investigated as previously described31. Fifty-percent-confluent fibroblasts were

loaded with 100 nM Mito Tracker Red CM-H2XRos (Invitrogen). Cells were

incubated with 500 mM H2O2 for 5 min and then fixed in 4% (wt/vol)

paraformaldehyde in PBS for 10 min at 4 1C. DNA was stained by DAPI and

cells were mounted in Fluoromount (Scientific Services). Pictures of every

sample were collected randomly using a BX60 Olympus microscope.

Apoptosis assay. For apoptosis measurement after hydrogen peroxide (neoLab)

treatment, cells were incubated with 200 mM H2O2 for one hour without FCS

as described32. Subsequently medium with 0.4% FCS was added and cells were

incubated at normal conditions for additional 24 h. Cells were fixed in 4% (wt/

vol) paraformaldehyde in PBS for 10 min and permeabilized with 0.1% (vol/

vol) Triton-X100 in 3% BSA in PBS for 2 min on ice. For apoptosis

determination the TUNEL assay (Roche) was performed according to manu-

facturer specifications. Cells were fixed in 4% (wt/vol) paraformaldehyde in

PBS for 10 min at 4 1C. DNA was stained by DAPI and cells were mounted in

Fluoromount (Scientific Services). Pictures were collected randomly. Each

experiment was performed three times. More than 2,000 cells were analyzed

per assay.

Mitochondrial transmembrane potential measurements. To evaluate mito-

chondrial transmembrane potential, primary fibroblasts were treated with

200 mM H2O2 for 2 h without FCS. After two washes in PBS, 5 � 105 cells

per milliliter were incubated with 40 nM of the fluorescent dye 3,3¢-dihexylox-

acarbocyanine iodide (DiOC6(3); Molecular Probes) for 15 min at 37 1C in

PBS, followed by analysis on a FACSCalibur cytofluorometer (BD Biosciences).

Polarography. Cultured skin fibroblasts of affected (n ¼ 3) and unaffected

(n ¼ 3) subjects were trypsinized and washed in PBS twice. Permeabilization

was done immediately in 50 mg/ml digitonin for 30 s before measurement and

verified by the Trypan blue exclusion test (499% permeabilization was

required). Fibroblasts (7 � 106) were resuspended in reaction buffer and

injected into the reaction chamber with a Clark-type electrode (Hansatech),

and oxygen concentrations were measured in 0.7-ml reaction volume at 37 1C

with substrates and inhibitors according to standard protocols33. The oxygen

consumption is given as fmol O2 per minute per cell. All measurements were

reproduced two to four times. Epstein-Barr virus–immortalized lymphoblas-

toid cell lines from affected subjects (n ¼ 4), heterozygous parents (n ¼ 2) and

NATURE GENETICS doi:10.1038/ng.413

unaffected controls (n ¼ 2) were grown in RPMI (Lonza) suspension

culture, pelleted by centrifugation and washed twice with PBS. Digitonin

permeabilization was done for 60 s, and 2 � 107 cells in 0.7 ml were used

for one experiment as described above.

Reactive oxygen species measurement. Skin fibroblasts were trypsinized,

washed in medium and resuspended in PBS containing 0.2% BSA. The cell

suspension was incubated for 15 min with 20 mM 2,7-dichlorodihydrofluor-

escein diacetate (Molecular Probes) at 19–22 1C with and without 1 mM H2O2.

Fluorescence intensity was determined by FACS using a Canto II (Becton

Dickinson) cell sorter and the analysis software Flowjo (Tree Star). Cell viability

was controlled by DAPI (Molecular Probes) incubation.

Proliferation assay. For the assessment of the proliferation rate, 2.5 � 104 cells

were seeded on glass coverslips. The cells were incubated for 8 h in medium

containing 10 mM bromodeoxyuridine (BrdU) (Roche). Cells were fixed in 4%

(wt/vol) paraformaldehyde in PBS for 10 min after 24, 48 and 72 h and then

incubated in 3 M HCl for 10 min at 19–22 1C. After permeabilization with

0.1% (vol/vol) Triton-X100 in 3% BSA in PBS for 30 min at 4 1C, cells were

incubated with an antibody to BrdU (G3G4, Developmental Studies Hybrido-

ma Bank) 1:500 for 1 h at 19-22 1C. For visualization, an anti–mouse IgG Alexa

Fluor 488 conjugate (Invitrogen, Molecular Probes) was applied. DNA was

stained by DAPI and cells were mounted in Fluoromount (Scientific Services).

The experiment was also performed in medium with 10% dialyzed FCS and in

medium supplemented with 5 mM L-proline (Sigma-Aldrich).

Morpholino oligonucleotides and RNA injections. Morpholino 25-bp anti-

sense oligomers for X. laevis Pycr1, D. rerio pycr1 and D. rerio pycr2 were

obtained from Gene Tools and are described in Supplementary Table 2. The

morpholinos were resuspended in sterile water to a concentration of 1 mM

according to manufacturer’s instructions. Xenopus embryos were injected four

times radially at the two- to four-cell stage with 4 nl (33 ng morpholino per

injection). For zebrafish experiments, morpholinos were injected at the one-cell

stage with 4 nl (33 ng morpholino per injection), or with 2 nl of each

morpholino when co-injected. Synthetic capped mRNAs of human PYCR1

cDNA transcribed in vitro with an SP6 kit (Ambion) was used for rescue

experiments at 200 pg in each blastomere at the four-cell stage for Xenopus

embryos or at 20 pg to 200 pg for zebrafish embryos at the one-cell stage.

Embryological methods. Protocols for fertilization, injections, whole-mount in

situ hybridization and TUNEL34 can be found at our protocols website (see

URLs). Analysis of cell death on live embryos was performed by incubating

tadpoles in the dark for 30 min with acridine orange (2 mg/ml) in Barth

solution35 0.1�. After extensive washes in Barth 0.1�, images of embryos were

captured under fluorescence (excitation/emission, 500/530 nm) with a Leica

DFC 300 FX camera. Parabiosis was performed at stage 22, on dechorionated

embryos joined by ventral ectoderm after small incisions. Parabiotic embryos

were cultured in Barth 1� until healed and transferred into Steinberg solu-

tion35. A stereomicroscope M205 FA equipped with an ICD camera (Leica) was

used to capture images of embryos in successive focal planes. Images were then

merged into one picture with Photoshop CS3 (Adobe).

Statistical analysis. All error bars represent s.d. The significance of pairwise

comparisons was determined by Student’s t-test. Correction for multiple testing

were done according by the Bonferroni method.

URLs. GenBank, http://www.ncbi.nlm.nih.gov/mapview/. Ensembl, http://

www.ensembl.org/. University of California Santa Cruz human reference

genome (build 18, http://genome.ucsc.edu/). Blat-like Fast Accurate Search

Tool (BFAST, http://genome.ucla.edu/bfast/. SeqWare, http://seqware.source-

forge.net/. Unabridged protocols, http://www.reversade.com-a.googlepages.-

com/protocols.

27. O’Connel, J.R. & Weeks, D.E. PedCheck: a program for identification of genotypeincompatibilities in linkage analysis. Am. J. Hum. Genet. 63, 259–266 (1998).

28. Abecasis, G.R., Cherny, S.S., Cookson, W.O. & Cardon, L.R. Merlin—rapid analysisof dense genetic maps using sparse gene flow trees. Nat. Genet. 30, 97–101(2002).

29. Thiele, H. & Nurnberg, P. HaploPainter: a tool for drawing pedigrees with complexhaplotypes. Bioinformatics 21, 1730–1732 (2005).

30. Kruglyak, L., Daly, M.J., Reeve-Daly, M.P. & Lander, E.S. Parametric and nonpara-metric linkage analysis: a unified multipoint approach. Am. J. Hum. Genet. 58,1347–1363 (1996).

31. Duvezin-Caubet, S. et al. Proteolytic processing of OPA1 links mitochondrial dysfunc-tion to alterations in mitochondrial morphology. J. Biol. Chem. 281, 37972–37979(2006).

32. Teramoto, S. et al. Hydrogen peroxide-induced apoptosis and necrosis in humanlung fibroblasts: protective roles of glutathione. Jpn. J. Pharmacol. 79, 33–40(1999).

33. Hofhaus, G., Shakeley, R.M. & Attardi, G. Use of polarography to detect respirationdefects in cell cultures. Methods Enzymol. 264, 476–483 (1996).

34. Hensey, C. & Gautier, J. Programmed cell death during Xenopus development: a spatio-temporal analysis. Dev. Biol. 203, 36–48 (1998).

35. Sive, H., Grainger, R.M. & Harland, R.M. Early Development of Xenopus Laevis: ALaboratory Manual (Cold Spring Harbor Press, New York, 2000).

doi:10.1038/ng.413 NATURE GENETICS

http://www.ncbi.nlm.nih.gov/mapview/

http://www.ensembl.org/

http://www.ensembl.org/

http://genome.ucsc.edu/

http://genome.ucla.edu/bfast/

http://seqware.sourceforge.net/

http://seqware.sourceforge.net/

http://www.reversade.com-a.googlepages.com/protocols

http://www.reversade.com-a.googlepages.com/protocols

5´ UTR3´ UTR

ATG

1

p.Gly206Trp p.Ala257Thr

c.633+1G>C

c.797+1G>A

c.797+2_797+5del

p.Arg119Gly

TGA

p.Arg251Hisp.Ala189Val

p.Ala179Thr

p.Gly4fsX50

c.138+1G>A

p.Gly206Arg

2 3 5 6 7

c.540+1G>A

4

p.Arg119His

c.797G>A; p.Arg266Gln

c.617_633+6del

Missense

Splice Site,

Frameshift

a

b

Supplementary Figure 1a

b

Localization and effect of PYCR1 mutations.( ) Genomic localization. Note that mutations accumulate in exons 4-6 of PYCR1.Red= position of amino acids essential for NAD(P)H binding. Orange= 100% conservedresidues. Yellow= >80% conserved residues. White= spice site and frameshift mutations.( ) Assessment of effect of missense mutations. The computational tools PolyPhen(h t tp : / /gene t i cs .bwh.harvard .edu /pph / re fe rences .h tml ) and Prophy le r(http://www.prophyler.org; http://genome.cshlp.org/content/15/7/978.long) were used toestimate the effects of the PYCR1 missense mutations on protein function. The firstcolumn shows the mutation, columns 2-4 show the PolyPhen predictions, and column 5shows the Prophyler MAPP P value. Low P-values are strong predictions for deleterious-ness, high P-values mean the variant is unlikely to be deleterious. Note that mutationp.Arg266Gln, which is predicted to be benign by both PolyPhen and Prophyler on thebasis of protein characteristics, is predicted to affect splicing by altering the invariabledonor splice site at the 3' end of exon 6. Splicing is not judged by these programs. Themutation p.Ala189Val was also judged to be benign by both programs. In this case, theother allele carries the Arg119His mutation which is predicted to be deleterious. As thep.Ala189Val change was not present in more than 100 control samples, it likely results inreduced residual activity. However, we cannot rule out a rare variant.

Mutation PredictionPrediction

basisSubstitution effect

Prophyler

MAPP P-

value

p.Gly206Trpprobably

damagingstructure

1.1.2: structural effect, buried

site, overpacking

Overpacking at buried site

0.00493

p.Arg266Gln benign alignment N/A 0.8694

p.Arg119Glyprobably

damagingalignment N/A 9.923*E-4

p.Arg119Hisprobably

damagingalignment N/A 0.04909

p.Ala189Val benign alignment N/A 0,2072

p.Ala179Thrprobably

damagingalignment N/A 3.392*E-5

p.Gly206Argprobably

damagingstructure


site, overpacking

Overpacking at buried site

5.365*E-7

p.Arg251Hisprobably

damagingstructure


site, hydrophobicity

Hydrophobicity change at

buried site

0.01093

p.Ala257Thrprobably

damagingstructure


site, hydrophobicity

Hydrophobicity change at

buried site

0.001617

Nature Genetics: doi:10.1038/ng.413

a

b


b

Expression of genes involved in the proline metabolicpathway determined by quantitative PCR. ( ) Expression of genes involved inproline metabolism in tissues from 4 day old mice normalized to expressionlevels in the heart determined by quantitative PCR. Note extremely high Pycr1expression levels in osteoblasts and skin. Pycr2 and Pycrl are also most highlyexpressed in osteoblasts, but otherwise show uniform expression, as does P5cs.( ) Gene expression changes in fibroblasts from individuals with PYCR1mutations and controls. While the Gly206Trp mutation in patient TP does notaffect PYCR1 expression levels the mutations in DI and SJ clearly reducemRNA levels due to nonsense-mediated decay. Expression of PYCR2 andP5CS is not affected.

0

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ve


b

Loss of PYCR1 does not cause hypoprolinemia or a cellproliferation defect. ( ) Proline and ornithine levels in individuals with PYCR1mutations (Pat) compared to controls (Ctr). Note non-significant (p=0,051)reduction of proline concentrations in affected individuals. ( ) Proline levels infibroblast lysates from controls and from individuals with PYCR1 mutation. Nosignificant differenced were detectable. (c) Proliferation rates of fibroblasts fromindividuals with PYCR1 mutations (Pat) and controls (Ctr) grown in medium withdifferent proline concentrations. No significant differences were observed, butmutant cells showed slightly higher proliferation. Representative result of twoindependent experiments. Ctr = control; Pat = patient. Errors are given as s.d..


Supplementary Figure 4 Mitochondrial function in fibroblasts and lymphoblastoid celllines. (a) Respiratory rates (oxygen consumption) were measured in 7 x 10 cultured,digitonin-permeabilized fibroblasts from patients and normal controls. No significantdifferences were found in respiratory chain function. (b)

6

( ) Representative traces showing fluorescenceintensity levels without DCDHF, whith DCDHF and with DCDHF and H O . ( ) Quantitative comparison of mean DCDHF fluorescence intensity in controls and patients with andwithout H O . Representat ive results of one experiment are given.

2

2

2

2

c -

Respiratory rates (oxygen con-sumption) were measured in 2 x 10 cultured,digitonin-permeabilized EBV-immortalizedlymphoblastoid cell lines (LCL) from patients, heterozygous parents and normal controls.No significant differences were found in respiratory chain function between the differentgenotypes. c ROS detection by DCDHF.

7

a

b

0 102

103

104

105

DCDHF

0

200

400

600

800

1000

#C

ells

background

DCDHF only

DCDHF + H2O2

0

5

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40

Ctrl (n=2) Pat (n=4) Ctrl (n=2) Pat (n=4)

Intrinsic ROS level ROS level after treatment with 1mM H2O2

c d


Control

a b c

f

k l m

n o p

Pycr1 Mo Pycr2 Mo

Pycr1+2 Mo Pycr1+2 Mo + PYCR1

Pycr1+2 Mo + PYCR1Control Pycr1+2 Mo

d e

g h

i j

Pycr1+2 Mo + PYCR1Control Pycr1+2 Mo

Supplementary Figure 5a-h

f-h

i,ji j

k-m

n-p

Morpholino (MO)-mediated knockdown of Pycr1 and Pycr2 in zebrafish leadsto malformation, epidermal atrophy and increased apoptosis. ( ) Control, Pycr1 morphant, Pycr2morphant, Pycr1+2 morphant and Pycr1+2 morphant zebrafish injected with 20pg human PYCR1mRNA at stage 24h. ( ) Control, Pycr 1+2 morphant and Pycr1+2 morphant zebrafish injected with20 pg human PYCR1 mRNA at stage 48 hours. Note reduced length of morphant fishes, abnormallybowed tail and loosening of the epidermis around the yolk sac. The severity of the phenotype increaseswhen Pycr1 and Pycr2 are both knocked down. Only subtle changes are seen after rescue of the mor-

phant phenotype with 20pg of human PYCR1 mRNA. ( ) Dermal atrophy in Pycr1+2 morphants. In48 hours-old control fish ( ) a double-layered epidermis surrounds the yolk sac. In Pycr1+2 morphants ( )this epidermis is atrophic. Goldner trichrome staining. ( ) Acridine orange staining of apoptotic cellsat stage 24h. Note significant increased number of apoptotic cells in morphants and almost completereversion of the phenotype by co-injection of the human PYCR1 mRNA. ( ) Acridine staining of apop-totic cells in the tail, higher magnification.


Supplementary Table 1 Results of high‐throughput sequencingStrategy flowchart

5 lanes of Illumina sequence47,880,719 raw reads

Bfast uniquely aligned reads19,271,149 (40%)

Total mismatches to referencein chr17:72048357-787747421,611,834

Mismatches to reference>= 2x coverage, 20-100%71,495

Mismatches to reference >= 2xcoverage, homozygous (80-100%)3,083

Mismatches to referencein dbSNP2,531

Mismatches to referencenot in dbSNP552

Mismatches causing a codingchange in UCSC genes11

Intersection of genes withmutations in other samples


Supplementary Table 1 Results of high‐throughput sequencingList of mismatches on chr17 pos. 72048357‐78774

Chromosome Mismatch position Mismatch description and Coveragechr17 75511361‐ C‐>T(10:10:100%[F:7:70%|R:3:30%])chr17 72653954 G‐>A(15:15:100%[F:13:86.7%|R:2:13.3%])chr17 72831394 C‐>T(8:8:100%[F:7:87.5%|R:1:12.5%])chr17 77628649 G‐>A(2:2:100%[F:0:0%|R:2:100%])chr17 75670643 C‐>T(2:2:100%[F:0:0%|R:2:100%])chr17 78590157 C‐>A(2:2:100%[F:2:100%|R:0:0%])chr17 74705989 C‐>T(7:7:100%[F:6:85.7%|R:1:14.3%])chr17 73027256 T‐>C(2:2:100%[F:2:100%|R:0:0%])chr17 72710115 T‐>G(2:2:100%[F:2:100%|R:0:0%])chr17 72823197 T‐>C(3:3:100%[F:0:0%|R:3:100%])chr17 76341110 T‐>G(16:16:100%[F:3:18.8%|R:13:81.2%])chr17 77948251 G‐>A(5:5:100%[F:4:80%|R:1:20%])chr17 77605614 C‐>T(2:2:100%[F:2:100%|R:0:0%])chr17 74514088 A‐>T(11:11:100%[F:8:72.7%|R:3:27.3%])chr17 75002938 G‐>A(4:4:100%[F:0:0%|R:4:100%])chr17 78088748 A‐>C(2:2:100%[F:0:0%|R:2:100%])chr17 77220760 G‐>A(2:2:100%[F:2:100%|R:0:0%])chr17 75965377 T‐>C(12:12:100%[F:7:58.3%|R:5:41.7%])chr17 72513357 C‐>G(2:2:100%[F:2:100%|R:0:0%])chr17 73627607 G‐>C(2:2:100%[F:0:0%|R:2:100%])chr17 73136581 T‐>G(2:2:100%[F:0:0%|R:2:100%])chr17 77391136 DEL:C‐>‐(5:5:100%[F:1|R:4])chr17 74376893 T‐>C(3:3:100%[F:3:100%|R:0:0%])chr17 74046665 T‐>G(2:2:100%[F:0:0%|R:2:100%])chr17 76162061 G‐>C(25:25:100%[F:11:44%|R:14:56%])chr17 73002342 C‐>T(9:9:100%[F:4:44.4%|R:5:55.6%])chr17 74197247 G‐>A(3:3:100%[F:3:100%|R:0:0%])chr17 75002966 T‐>G(2:2:100%[F:0:0%|R:2:100%])chr17 72312685 T‐>C(2:2:100%[F:0:0%|R:2:100%])chr17 75006248 T‐>C(2:2:100%[F:0:0%|R:2:100%])chr17 77280660 T‐>G(2:2:100%[F:0:0%|R:2:100%])chr17 74715683 A‐>G(7:7:100%[F:5:71.4%|R:2:28.6%])chr17 77712902 C‐>T(20:20:100%[F:10:50%|R:10:50%])chr17 77044169 C‐>G(3:3:100%[F:1:33.3%|R:2:66.7%])chr17 78137702 C‐>G(2:2:100%[F:1:50%|R:1:50%])chr17 74157196 T‐>G(43:43:100%[F:18:41.9%|R:25:58.1%])chr17 73033694 T‐>C(6:6:100%[F:1:16.7%|R:5:83.3%])chr17 75764793 A‐>C(2:2:100%[F:2:100%|R:0:0%])chr17 76975150 T‐>C(2:2:100%[F:1:50%|R:1:50%])chr17 75903648 C‐>G(2:2:100%[F:2:100%|R:0:0%])chr17 72475268 G‐>A(11:11:100%[F:5:45.5%|R:6:54.5%])chr17 75750094 C‐>G(2:2:100%[F:1:50%|R:1:50%])chr17 76549981 C‐>T(55:55:100%[F:4:7.27%|R:51:92.7%])chr17 76071536 C‐>A(11:11:100%[F:3:27.3%|R:8:72.7%])chr17 77014468 C‐>G(3:3:100%[F:0:0%|R:3:100%])chr17 74638390 C‐>T(16:16:100%[F:11:68.8%|R:5:31.2%])


chr17 78590317 G‐>A(11:11:100%[F:6:54.5%|R:5:45.5%])chr17 73484414 A‐>G(2:2:100%[F:0:0%|R:2:100%])chr17 78595757 T‐>G(2:2:100%[F:0:0%|R:2:100%])chr17 75003159 A‐>G(4:4:100%[F:1:25%|R:3:75%])chr17 78256640 DEL:T‐>‐(8:8:100%[F:3|R:5])chr17 74808717 C‐>T(12:12:100%[F:9:75%|R:3:25%])chr17 73065696 T‐>G(6:6:100%[F:2:33.3%|R:4:66.7%])chr17 75841942 G‐>A(39:39:100%[F:22:56.4%|R:17:43.6%])chr17 72084745 T‐>C(8:8:100%[F:0:0%|R:8:100%])chr17 73688790 DEL:GCAC‐>‐‐‐‐(3:3:100%[F:3|R:0])chr17 74679649 A‐>G(4:4:100%[F:4:100%|R:0:0%])chr17 77209759 DEL:T‐>‐(2:2:100%[F:0|R:2])chr17 73365149 T‐>G(2:2:100%[F:1:50%|R:1:50%])chr17 78590158 G‐>A(2:2:100%[F:2:100%|R:0:0%])chr17 75794837 G‐>A(9:9:100%[F:8:88.9%|R:1:11.1%])chr17 73761956 A‐>C(2:2:100%[F:1:50%|R:1:50%])chr17 77302794 G‐>A(2:2:100%[F:1:50%|R:1:50%])chr17 73783732 A‐>G(2:2:100%[F:2:100%|R:0:0%])chr17 78026646 T‐>C(2:2:100%[F:2:100%|R:0:0%])chr17 75708908 A‐>C(2:2:100%[F:0:0%|R:2:100%])chr17 76309188 T‐>G(2:2:100%[F:1:50%|R:1:50%])chr17 74170562 C‐>G(17:17:100%[F:0:0%|R:17:100%])chr17 78592043 C‐>A(2:2:100%[F:0:0%|R:2:100%])chr17 74695004 C‐>T(15:15:100%[F:9:60%|R:6:40%])chr17 73785023 T‐>A(3:3:100%[F:0:0%|R:3:100%])chr17 73785021 T‐>A(2:2:100%[F:0:0%|R:2:100%])chr17 75299398 A‐>C(2:2:100%[F:0:0%|R:2:100%])chr17 74023193 T‐>A(4:4:100%[F:4:100%|R:0:0%])chr17 77139997 G‐>C(4:4:100%[F:3:75%|R:1:25%])chr17 74052703 C‐>G(19:19:100%[F:11:57.9%|R:8:42.1%])chr17 72246962 G‐>T(3:3:100%[F:3:100%|R:0:0%])chr17 74750878 INS:‐‐>T(2:2:100%[F:1|R:0])chr17 77018540 A‐>G(18:18:100%[F:8:44.4%|R:10:55.6%])chr17 74908082 C‐>A(4:4:100%[F:4:100%|R:0:0%])chr17 78330050 T‐>C(2:2:100%[F:0:0%|R:2:100%])chr17 74967014 C‐>T(4:4:100%[F:3:75%|R:1:25%])chr17 76474067 C‐>G(2:2:100%[F:2:100%|R:0:0%])chr17 77715273 C‐>T(2:2:100%[F:2:100%|R:0:0%])chr17 72803793 INS:‐‐>C(2:2:100%[F:1|R:0])chr17 76981869 T‐>A(8:8:100%[F:1:12.5%|R:7:87.5%])chr17 72626853 A‐>T(2:2:100%[F:2:100%|R:0:0%])chr17 75595792 T‐>C(22:22:100%[F:12:54.5%|R:10:45.5%])chr17 74998774 G‐>A(2:2:100%[F:2:100%|R:0:0%])chr17 76733664 INS:‐‐>A(3:3:100%[F:3|R:0])chr17 76303278 C‐>G(2:2:100%[F:0:0%|R:2:100%])chr17 76561434 DEL:A‐>‐(2:2:100%[F:2|R:0])chr17 73380796 DEL:AC‐>‐‐(3:3:100%[F:3|R:0])chr17 72245583 T‐>G(2:2:100%[F:0:0%|R:2:100%])chr17 73826392 C‐>T(16:16:100%[F:8:50%|R:8:50%])chr17 75773469 G‐>A(9:9:100%[F:5:55.6%|R:4:44.4%])


chr17 75730964 C‐>A(2:2:100%[F:0:0%|R:2:100%])chr17 74510205 C‐>T(22:22:100%[F:12:54.5%|R:10:45.5%])chr17 75943880 A‐>G(55:55:100%[F:23:41.8%|R:32:58.2%])chr17 76214749 C‐>A(2:2:100%[F:2:100%|R:0:0%])chr17 76897226 C‐>G(2:2:100%[F:0:0%|R:2:100%])chr17 78511625 C‐>A(12:12:100%[F:9:75%|R:3:25%])chr17 74979237 G‐>A(16:16:100%[F:13:81.2%|R:3:18.8%])chr17 73694942 T‐>G(4:4:100%[F:1:25%|R:3:75%])chr17 74164159 A‐>C(2:2:100%[F:2:100%|R:0:0%])chr17 73631457 T‐>G(2:2:100%[F:2:100%|R:0:0%])chr17 77772072 T‐>C(3:3:100%[F:0:0%|R:3:100%])chr17 75791592 G‐>A(2:2:100%[F:2:100%|R:0:0%])chr17 78159972 T‐>A(2:2:100%[F:0:0%|R:2:100%])chr17 75897895 C‐>G(19:19:100%[F:17:89.5%|R:2:10.5%])chr17 77243855 T‐>G(2:2:100%[F:0:0%|R:2:100%])chr17 75842657 C‐>T(2:2:100%[F:0:0%|R:2:100%])chr17 75635537 C‐>T(3:3:100%[F:3:100%|R:0:0%])chr17 78325523 A‐>G(3:3:100%[F:0:0%|R:3:100%])chr17 76726938 G‐>A(19:19:100%[F:14:73.7%|R:5:26.3%])chr17 75340145 DEL:T‐>‐(2:2:100%[F:0|R:2])chr17 77896821 C‐>T(13:13:100%[F:8:61.5%|R:5:38.5%])chr17 74122893 C‐>A(2:2:100%[F:2:100%|R:0:0%])chr17 74050813 G‐>A(5:5:100%[F:1:20%|R:4:80%])chr17 72468883 G‐>C(3:3:100%[F:1:33.3%|R:2:66.7%])chr17 76655972 INS:‐‐>C(6:6:100%[F:3|R:0])chr17 72473109 A‐>G(15:15:100%[F:3:20%|R:12:80%])chr17 72217940 G‐>A(6:6:100%[F:1:16.7%|R:5:83.3%])chr17 75594573 A‐>T(2:2:100%[F:1:50%|R:1:50%])chr17 78110777 G‐>C(15:15:100%[F:6:40%|R:9:60%])chr17 72754580 A‐>G(2:2:100%[F:2:100%|R:0:0%])chr17 78095650 T‐>C(28:28:100%[F:24:85.7%|R:4:14.3%])chr17 78601168 A‐>C(25:25:100%[F:13:52%|R:12:48%])chr17 78603901 G‐>A(11:11:100%[F:7:63.6%|R:4:36.4%])chr17 73971735 A‐>G(25:25:100%[F:21:84%|R:4:16%])chr17 75596676 C‐>G(26:26:100%[F:12:46.2%|R:14:53.8%])chr17 75601738 A‐>C(4:4:100%[F:2:50%|R:2:50%])chr17 73275280 DEL:CTT‐>‐‐‐(2:2:100%[F:1|R:1])chr17 72472123 G‐>T(2:2:100%[F:1:50%|R:1:50%])chr17 76218675 G‐>A(36:36:100%[F:20:55.6%|R:16:44.4%])chr17 77141624 G‐>T(8:8:100%[F:5:62.5%|R:3:37.5%])chr17 72835012 C‐>A(6:6:100%[F:2:33.3%|R:4:66.7%])chr17 72875407 G‐>C(10:10:100%[F:0:0%|R:10:100%])chr17 72687605 A‐>C(2:2:100%[F:2:100%|R:0:0%])chr17 77154116 DEL:G‐>‐(2:2:100%[F:2|R:0])chr17 73784862 T‐>G(8:8:100%[F:6:75%|R:2:25%])chr17 75991637 T‐>C(2:2:100%[F:0:0%|R:2:100%])chr17 74186129 G‐>A(22:22:100%[F:16:72.7%|R:6:27.3%])chr17 77889325 A‐>C(2:2:100%[F:2:100%|R:0:0%])chr17 73002419 G‐>A(20:20:100%[F:15:75%|R:5:25%])chr17 77715237 G‐>A(3:3:100%[F:0:0%|R:3:100%])


chr17 72915611 A‐>G(2:2:100%[F:0:0%|R:2:100%])chr17 74289181 G‐>A(2:2:100%[F:2:100%|R:0:0%])chr17 72426116 A‐>G(14:14:100%[F:8:57.1%|R:6:42.9%])chr17 72474567 G‐>A(12:12:100%[F:8:66.7%|R:4:33.3%])chr17 75602458 G‐>T(14:14:100%[F:1:7.14%|R:13:92.9%])chr17 77141648 T‐>C(8:8:100%[F:5:62.5%|R:3:37.5%])chr17 76077562 A‐>G(2:2:100%[F:2:100%|R:0:0%])chr17 77453894 A‐>G(2:2:100%[F:1:50%|R:1:50%])chr17 74433472 T‐>G(2:2:100%[F:0:0%|R:2:100%])chr17 73736075 C‐>T(5:5:100%[F:3:60%|R:2:40%])chr17 73289123 T‐>G(3:3:100%[F:1:33.3%|R:2:66.7%])chr17 77149634 T‐>A(8:8:100%[F:0:0%|R:8:100%])chr17 73845925 T‐>G(5:5:100%[F:5:100%|R:0:0%])chr17 74974503 C‐>T(11:11:100%[F:8:72.7%|R:3:27.3%])chr17 72829303 A‐>T(2:2:100%[F:2:100%|R:0:0%])chr17 77102739 G‐>T(2:2:100%[F:0:0%|R:2:100%])chr17 76303280 G‐>T(2:2:100%[F:0:0%|R:2:100%])chr17 74827516 G‐>A(3:3:100%[F:3:100%|R:0:0%])chr17 72471389 C‐>T(13:13:100%[F:9:69.2%|R:4:30.8%])chr17 74566279 A‐>G(2:2:100%[F:0:0%|R:2:100%])chr17 78635226 A‐>G(25:25:100%[F:18:72%|R:7:28%])chr17 74837822 T‐>C(12:12:100%[F:6:50%|R:6:50%])chr17 77140193 G‐>A(3:3:100%[F:1:33.3%|R:2:66.7%])chr17 77728593 C‐>G(2:2:100%[F:0:0%|R:2:100%])chr17 75428166 A‐>G(2:2:100%[F:2:100%|R:0:0%])chr17 72740303 G‐>T(2:2:100%[F:2:100%|R:0:0%])chr17 73893193 T‐>G(2:2:100%[F:0:0%|R:2:100%])chr17 78120035 C‐>T(4:4:100%[F:2:50%|R:2:50%])chr17 76476535 C‐>T(9:9:100%[F:5:55.6%|R:4:44.4%])chr17 74186060 A‐>G(4:4:100%[F:1:25%|R:3:75%])chr17 77171785 T‐>A(10:10:100%[F:9:90%|R:1:10%])chr17 77259954 G‐>A(2:2:100%[F:0:0%|R:2:100%])chr17 78533731 C‐>T(19:19:100%[F:17:89.5%|R:2:10.5%])chr17 77566119 C‐>G(2:2:100%[F:2:100%|R:0:0%])chr17 73265918 T‐>C(23:23:100%[F:11:47.8%|R:12:52.2%])chr17 74094794 A‐>G(2:2:100%[F:2:100%|R:0:0%])chr17 77158837 A‐>C(6:6:100%[F:6:100%|R:0:0%])chr17 75988058 T‐>C(3:3:100%[F:2:66.7%|R:1:33.3%])chr17 74048359 T‐>C(4:4:100%[F:3:75%|R:1:25%])chr17 78121221 C‐>T(2:2:100%[F:2:100%|R:0:0%])chr17 77418760 T‐>C(2:2:100%[F:1:50%|R:1:50%])chr17 73795556 C‐>A(4:4:100%[F:4:100%|R:0:0%])chr17 74513240 G‐>A(10:10:100%[F:6:60%|R:4:40%])chr17 76038179 G‐>A(11:11:100%[F:7:63.6%|R:4:36.4%])chr17 74515399 A‐>G(20:20:100%[F:12:60%|R:8:40%])chr17 77428915 C‐>T(14:14:100%[F:9:64.3%|R:5:35.7%])chr17 77934629 INS:‐‐>C(2:2:100%[F:2|R:0])chr17 72770714 T‐>G(2:2:100%[F:0:0%|R:2:100%])chr17 77285602 T‐>C(2:2:100%[F:2:100%|R:0:0%])chr17 72710117 A‐>T(2:2:100%[F:2:100%|R:0:0%])


chr17 78051018 A‐>T(2:2:100%[F:2:100%|R:0:0%])chr17 73033674 G‐>A(6:6:100%[F:4:66.7%|R:2:33.3%])chr17 78375720 A‐>C(2:2:100%[F:2:100%|R:0:0%])chr17 76270031 T‐>G(2:2:100%[F:2:100%|R:0:0%])chr17 74979306 T‐>G(17:17:100%[F:11:64.7%|R:6:35.3%])chr17 77220761 G‐>T(2:2:100%[F:2:100%|R:0:0%])chr17 72054125 DEL:T‐>‐(2:2:100%[F:0|R:2])chr17 74374798 A‐>G(2:2:100%[F:1:50%|R:1:50%])chr17 74675458 G‐>A(2:2:100%[F:1:50%|R:1:50%])chr17 77158871 A‐>G(28:28:100%[F:17:60.7%|R:11:39.3%])chr17 77497358 T‐>C(2:2:100%[F:0:0%|R:2:100%])chr17 74687850 C‐>T(21:21:100%[F:11:52.4%|R:10:47.6%])chr17 78533650 T‐>G(9:9:100%[F:6:66.7%|R:3:33.3%])chr17 76372432 A‐>G(2:2:100%[F:2:100%|R:0:0%])chr17 72898057 T‐>C(17:17:100%[F:14:82.4%|R:3:17.6%])chr17 78461825 G‐>A(3:3:100%[F:3:100%|R:0:0%])chr17 77679494 C‐>T(8:8:100%[F:5:62.5%|R:3:37.5%])chr17 77217410 DEL:A‐>‐(2:2:100%[F:1|R:1])chr17 72818461 T‐>C(8:8:100%[F:4:50%|R:4:50%])chr17 76281150 C‐>A(2:2:100%[F:1:50%|R:1:50%])chr17 75513785 T‐>C(8:8:100%[F:4:50%|R:4:50%])chr17 76694813 A‐>C(2:2:100%[F:2:100%|R:0:0%])chr17 76392640 A‐>G(2:2:100%[F:1:50%|R:1:50%])chr17 75710302 DEL:A‐>‐(2:2:100%[F:2|R:0])chr17 78280894 INS:‐‐>A(17:17:100%[F:9|R:0])chr17 77014469 C‐>G(2:2:100%[F:0:0%|R:2:100%])chr17 75991633 A‐>T(2:2:100%[F:0:0%|R:2:100%])chr17 72476747 T‐>G(2:2:100%[F:2:100%|R:0:0%])chr17 76309090 G‐>A(4:4:100%[F:2:50%|R:2:50%])chr17 78225302 T‐>C(2:2:100%[F:2:100%|R:0:0%])chr17 74699378 A‐>G(15:15:100%[F:7:46.7%|R:8:53.3%])chr17 76718593 A‐>C(2:2:100%[F:1:50%|R:1:50%])chr17 72755395 C‐>G(2:2:100%[F:0:0%|R:2:100%])chr17 72710120 C‐>T(2:2:100%[F:2:100%|R:0:0%])chr17 78349915 INS:‐‐>T(23:23:100%[F:7|R:0])chr17 74279341 A‐>G(2:2:100%[F:2:100%|R:0:0%])chr17 72308880 C‐>A(2:2:100%[F:0:0%|R:2:100%])chr17 77670315 INS:‐‐>T(5:5:100%[F:5|R:0])chr17 74876886 C‐>T(7:7:100%[F:4:57.1%|R:3:42.9%])chr17 72971392 A‐>G(2:2:100%[F:1:50%|R:1:50%])chr17 75779673 G‐>A(11:11:100%[F:5:45.5%|R:6:54.5%])chr17 77264962 G‐>A(13:13:100%[F:9:69.2%|R:4:30.8%])chr17 76078080 INS:‐‐>G(4:4:100%[F:1|R:0])chr17 73981222 A‐>C(2:2:100%[F:2:100%|R:0:0%])chr17 78218891 T‐>G(28:28:100%[F:12:42.9%|R:16:57.1%])chr17 78486652 C‐>G(9:9:100%[F:2:22.2%|R:7:77.8%])chr17 78231953 T‐>G(2:2:100%[F:0:0%|R:2:100%])chr17 76484839 T‐>C(2:2:100%[F:0:0%|R:2:100%])chr17 73359750 C‐>T(2:2:100%[F:1:50%|R:1:50%])chr17 73289092 C‐>G(13:13:100%[F:5:38.5%|R:8:61.5%])


chr17 78039777 T‐>C(3:3:100%[F:2:66.7%|R:1:33.3%])chr17 72468533 T‐>C(34:34:100%[F:20:58.8%|R:14:41.2%])chr17 77929748 G‐>A(4:4:100%[F:4:100%|R:0:0%])chr17 76151152 INS:‐‐>G(2:2:100%[F:2|R:0])chr17 74754056 C‐>T(14:14:100%[F:9:64.3%|R:5:35.7%])chr17 77788773 C‐>G(2:2:100%[F:1:50%|R:1:50%])chr17 75714235 T‐>G(2:2:100%[F:0:0%|R:2:100%])chr17 77686994 DEL:G‐>‐(2:2:100%[F:0|R:2])chr17 76334575 G‐>T(8:8:100%[F:8:100%|R:0:0%])chr17 77158836 A‐>T(3:3:100%[F:3:100%|R:0:0%])chr17 77678583 C‐>T(24:24:100%[F:13:54.2%|R:11:45.8%])chr17 77445418 DEL:A‐>‐(2:2:100%[F:1|R:1])chr17 77486276 C‐>T(7:7:100%[F:6:85.7%|R:1:14.3%])chr17 74689407 C‐>A(57:57:100%[F:14:24.6%|R:43:75.4%])chr17 75595804 T‐>A(20:20:100%[F:14:70%|R:6:30%])chr17 74218992 C‐>T(2:2:100%[F:2:100%|R:0:0%])chr17 72246963 C‐>A(3:3:100%[F:3:100%|R:0:0%])chr17 78590316 T‐>C(11:11:100%[F:6:54.5%|R:5:45.5%])chr17 76071084 C‐>A(5:5:100%[F:5:100%|R:0:0%])chr17 77936291 G‐>A(9:9:100%[F:6:66.7%|R:3:33.3%])chr17 73787988 G‐>A(4:4:100%[F:0:0%|R:4:100%])chr17 72522632 T‐>G(2:2:100%[F:1:50%|R:1:50%])chr17 76975920 T‐>C(2:2:100%[F:0:0%|R:2:100%])chr17 72468920 C‐>T(2:2:100%[F:1:50%|R:1:50%])chr17 76253726 C‐>T(4:4:100%[F:0:0%|R:4:100%])chr17 78566253 C‐>G(2:2:100%[F:0:0%|R:2:100%])chr17 77569724 T‐>G(3:3:100%[F:2:66.7%|R:1:33.3%])chr17 74182383 A‐>G(2:2:100%[F:1:50%|R:1:50%])chr17 74870036 G‐>A(8:8:100%[F:3:37.5%|R:5:62.5%])chr17 73968295 C‐>T(21:21:100%[F:13:61.9%|R:8:38.1%])chr17 72475923 G‐>C(24:24:100%[F:8:33.3%|R:16:66.7%])chr17 77208891 T‐>C(3:3:100%[F:3:100%|R:0:0%])chr17 72468574 C‐>T(18:18:100%[F:4:22.2%|R:14:77.8%])chr17 76103968 DEL:CTTCTG‐>‐‐‐‐‐‐(2:2:100%[F:2|R:0])chr17 78142588 A‐>T(6:6:100%[F:3:50%|R:3:50%])chr17 74870778 G‐>C(21:21:100%[F:11:52.4%|R:10:47.6%])chr17 78192406 C‐>A(3:3:100%[F:3:100%|R:0:0%])chr17 75002975 T‐>G(2:2:100%[F:0:0%|R:2:100%])chr17 74156376 G‐>C(10:10:100%[F:2:20%|R:8:80%])chr17 75681096 T‐>C(2:2:100%[F:1:50%|R:1:50%])chr17 73336613 T‐>C(8:8:100%[F:1:12.5%|R:7:87.5%])chr17 73334576 C‐>A(2:2:100%[F:1:50%|R:1:50%])chr17 74238619 T‐>C(2:2:100%[F:2:100%|R:0:0%])chr17 73885525 T‐>G(2:2:100%[F:2:100%|R:0:0%])chr17 72820179 INS:‐‐>G(21:21:100%[F:20|R:0])chr17 72470519 A‐>G(16:16:100%[F:13:81.2%|R:3:18.8%])chr17 78192492 G‐>C(27:27:100%[F:20:74.1%|R:7:25.9%])chr17 76506942 A‐>G(7:7:100%[F:3:42.9%|R:4:57.1%])chr17 75002959 C‐>G(3:3:100%[F:0:0%|R:3:100%])chr17 78074970 A‐>G(18:18:100%[F:16:88.9%|R:2:11.1%])


chr17 73313648 T‐>C(6:6:100%[F:0:0%|R:6:100%])chr17 77293653 G‐>A(6:6:100%[F:4:66.7%|R:2:33.3%])chr17 75003327 A‐>G(12:12:100%[F:10:83.3%|R:2:16.7%])chr17 75808893 A‐>C(2:2:100%[F:2:100%|R:0:0%])chr17 74216967 G‐>C(11:11:100%[F:5:45.5%|R:6:54.5%])chr17 73386091 G‐>A(19:19:100%[F:6:31.6%|R:13:68.4%])chr17 76694242 C‐>T(7:7:100%[F:3:42.9%|R:4:57.1%])chr17 77516439 G‐>T(2:2:100%[F:2:100%|R:0:0%])chr17 72234911 A‐>G(2:2:100%[F:2:100%|R:0:0%])chr17 72999208 G‐>T(2:2:100%[F:1:50%|R:1:50%])chr17 73707606 C‐>T(13:13:100%[F:8:61.5%|R:5:38.5%])chr17 75596282 T‐>C(14:14:100%[F:9:64.3%|R:5:35.7%])chr17 73974144 INS:‐‐>A(4:4:100%[F:4|R:0])chr17 75799297 C‐>G(2:2:100%[F:1:50%|R:1:50%])chr17 73785025 T‐>A(3:3:100%[F:0:0%|R:3:100%])chr17 76611370 T‐>C(42:42:100%[F:37:88.1%|R:5:11.9%])chr17 78642427 DEL:TG‐>‐‐(5:5:100%[F:4|R:1])chr17 75962274 G‐>T(2:2:100%[F:2:100%|R:0:0%])chr17 73580181 C‐>G(20:20:100%[F:11:55%|R:9:45%])chr17 73957373 A‐>T(2:2:100%[F:2:100%|R:0:0%])chr17 78641570 G‐>T(3:3:100%[F:2:66.7%|R:1:33.3%])chr17 78595744 A‐>C(2:2:100%[F:0:0%|R:2:100%])chr17 73660427 A‐>T(5:5:100%[F:4:80%|R:1:20%])chr17 74246772 A‐>G(23:23:100%[F:5:21.7%|R:18:78.3%])chr17 74979416 G‐>A(5:5:100%[F:4:80%|R:1:20%])chr17 75549198 A‐>G(2:2:100%[F:0:0%|R:2:100%])chr17 76813266 C‐>G(2:2:100%[F:2:100%|R:0:0%])chr17 76359809 C‐>T(14:14:100%[F:11:78.6%|R:3:21.4%])chr17 72439736 C‐>T(6:6:100%[F:3:50%|R:3:50%])chr17 75587867 G‐>A(53:53:100%[F:47:88.7%|R:6:11.3%])chr17 74234119 A‐>G(34:34:100%[F:16:47.1%|R:18:52.9%])chr17 78592428 A‐>G(3:3:100%[F:2:66.7%|R:1:33.3%])chr17 74045708 T‐>G(2:2:100%[F:1:50%|R:1:50%])chr17 76223269 G‐>T(6:6:100%[F:6:100%|R:0:0%])chr17 73668865 G‐>A(8:8:100%[F:4:50%|R:4:50%])chr17 74470608 A‐>G(2:2:100%[F:2:100%|R:0:0%])chr17 77419218 C‐>T(2:2:100%[F:2:100%|R:0:0%])chr17 76286109 C‐>T(9:9:100%[F:5:55.6%|R:4:44.4%])chr17 73845926 C‐>G(3:3:100%[F:3:100%|R:0:0%])chr17 72347338 T‐>C(2:2:100%[F:0:0%|R:2:100%])chr17 73586446 C‐>A(2:2:100%[F:0:0%|R:2:100%])chr17 77939926 G‐>A(8:8:100%[F:4:50%|R:4:50%])chr17 74468518 DEL:C‐>‐(4:4:100%[F:3|R:1])chr17 76858197 G‐>A(2:2:100%[F:0:0%|R:2:100%])chr17 74842818 G‐>A(17:17:100%[F:6:35.3%|R:11:64.7%])chr17 73484406 T‐>C(2:2:100%[F:0:0%|R:2:100%])chr17 77188595 T‐>G(2:2:100%[F:0:0%|R:2:100%])chr17 78252691 G‐>T(2:2:100%[F:0:0%|R:2:100%])chr17 76839945 A‐>C(2:2:100%[F:2:100%|R:0:0%])chr17 74062213 T‐>A(17:17:100%[F:7:41.2%|R:10:58.8%])


chr17 78597197 A‐>C(2:2:100%[F:2:100%|R:0:0%])chr17 77654997 A‐>C(2:2:100%[F:1:50%|R:1:50%])chr17 78165543 T‐>C(3:3:100%[F:1:33.3%|R:2:66.7%])chr17 75938164 A‐>G(18:18:100%[F:6:33.3%|R:12:66.7%])chr17 74715504 C‐>T(12:12:100%[F:9:75%|R:3:25%])chr17 74185921 C‐>A(23:23:100%[F:12:52.2%|R:11:47.8%])chr17 75732435 G‐>A(42:42:100%[F:31:73.8%|R:11:26.2%])chr17 74967416 A‐>G(13:13:100%[F:6:46.2%|R:7:53.8%])chr17 73689051 T‐>C(2:2:100%[F:1:50%|R:1:50%])chr17 75586741 G‐>T(2:2:100%[F:2:100%|R:0:0%])chr17 73902980 A‐>T(12:12:100%[F:10:83.3%|R:2:16.7%])chr17 75560792 A‐>T(2:2:100%[F:0:0%|R:2:100%])chr17 74642448 C‐>T(4:4:100%[F:3:75%|R:1:25%])chr17 78136867 G‐>T(31:31:100%[F:12:38.7%|R:19:61.3%])chr17 76464389 A‐>G(2:2:100%[F:2:100%|R:0:0%])chr17 72236546 T‐>G(2:2:100%[F:2:100%|R:0:0%])chr17 72591395 C‐>G(8:8:100%[F:1:12.5%|R:7:87.5%])chr17 76064974 A‐>C(3:3:100%[F:0:0%|R:3:100%])chr17 76001147 A‐>C(2:2:100%[F:2:100%|R:0:0%])chr17 72474714 C‐>T(2:2:100%[F:2:100%|R:0:0%])chr17 75457765 C‐>G(6:6:100%[F:5:83.3%|R:1:16.7%])chr17 73940069 DEL:CAG‐>‐‐‐(3:3:100%[F:1|R:2])chr17 75440959 C‐>T(2:2:100%[F:2:100%|R:0:0%])chr17 75860403 T‐>G(2:2:100%[F:1:50%|R:1:50%])chr17 78449802 INS:‐‐>C(7:7:100%[F:0|R:0])chr17 74834157 G‐>C(21:21:100%[F:9:42.9%|R:12:57.1%])chr17 74293857 T‐>A(12:12:100%[F:3:25%|R:9:75%])chr17 78165546 T‐>C(5:5:100%[F:1:20%|R:4:80%])chr17 75836481 C‐>T(7:7:100%[F:3:42.9%|R:4:57.1%])chr17 78443093 G‐>A(19:19:100%[F:11:57.9%|R:8:42.1%])chr17 73839403 G‐>T(2:2:100%[F:2:100%|R:0:0%])chr17 72324053 INS:‐‐>A(2:2:100%[F:2|R:0])chr17 73754072 G‐>A(6:6:100%[F:1:16.7%|R:5:83.3%])chr17 75260782 T‐>A(3:3:100%[F:0:0%|R:3:100%])chr17 76730652 C‐>G(2:2:100%[F:1:50%|R:1:50%])chr17 76190864 G‐>A(4:4:100%[F:2:50%|R:2:50%])chr17 72473858 G‐>A(15:15:100%[F:5:33.3%|R:10:66.7%])chr17 75600362 G‐>C(26:26:100%[F:9:34.6%|R:17:65.4%])chr17 73273584 G‐>A(42:42:100%[F:28:66.7%|R:14:33.3%])chr17 78585948 A‐>C(10:10:100%[F:9:90%|R:1:10%])chr17 73668222 A‐>G(2:2:100%[F:2:100%|R:0:0%])chr17 76195232 G‐>A(41:41:100%[F:25:61%|R:16:39%])chr17 75613225 C‐>T(38:38:100%[F:26:68.4%|R:12:31.6%])chr17 78108814 C‐>T(8:8:100%[F:5:62.5%|R:3:37.5%])chr17 74177073 C‐>T(15:15:100%[F:7:46.7%|R:8:53.3%])chr17 74288857 A‐>G(7:7:100%[F:7:100%|R:0:0%])chr17 74516033 C‐>T(19:19:100%[F:9:47.4%|R:10:52.6%])chr17 74653822 C‐>G(3:3:100%[F:3:100%|R:0:0%])chr17 76643975 T‐>G(3:3:100%[F:0:0%|R:3:100%])chr17 73476126 T‐>G(2:2:100%[F:0:0%|R:2:100%])


chr17 77630838 T‐>C(3:3:100%[F:3:100%|R:0:0%])chr17 74033688 T‐>C(12:12:100%[F:6:50%|R:6:50%])chr17 76793904 INS:‐‐>C(2:2:100%[F:2|R:0])chr17 72774778 C‐>T(9:9:100%[F:7:77.8%|R:2:22.2%])chr17 72845100 C‐>T(25:25:100%[F:14:56%|R:11:44%])chr17 78125723 A‐>T(2:2:100%[F:2:100%|R:0:0%])chr17 72146212 G‐>C(2:2:100%[F:0:0%|R:2:100%])chr17 75710099 C‐>T(3:3:100%[F:0:0%|R:3:100%])chr17 76124328 C‐>A(4:4:100%[F:0:0%|R:4:100%])chr17 76170106 G‐>T(45:45:100%[F:24:53.3%|R:21:46.7%])chr17 74177164 T‐>C(19:19:100%[F:15:78.9%|R:4:21.1%])chr17 78260213 A‐>G(9:9:100%[F:8:88.9%|R:1:11.1%])chr17 74053669 G‐>C(2:2:100%[F:1:50%|R:1:50%])chr17 76354266 G‐>A(25:25:100%[F:8:32%|R:17:68%])chr17 74513074 G‐>A(11:11:100%[F:11:100%|R:0:0%])chr17 74076935 G‐>A(31:31:100%[F:25:80.6%|R:6:19.4%])chr17 76588563 C‐>T(3:3:100%[F:0:0%|R:3:100%])chr17 76934867 A‐>C(2:2:100%[F:0:0%|R:2:100%])chr17 74443240 C‐>G(2:2:100%[F:2:100%|R:0:0%])chr17 76264089 A‐>G(2:2:100%[F:2:100%|R:0:0%])chr17 77889714 T‐>G(2:2:100%[F:2:100%|R:0:0%])chr17 75596902 G‐>A(8:8:100%[F:5:62.5%|R:3:37.5%])chr17 75379806 A‐>C(2:2:100%[F:1:50%|R:1:50%])chr17 74048545 G‐>A(2:2:100%[F:0:0%|R:2:100%])chr17 78125410 G‐>C(24:24:100%[F:16:66.7%|R:8:33.3%])chr17 77939952 G‐>C(6:6:100%[F:3:50%|R:3:50%])chr17 77655423 A‐>T(12:12:100%[F:8:66.7%|R:4:33.3%])chr17 76043229 T‐>C(3:3:100%[F:0:0%|R:3:100%])chr17 76062453 A‐>G(8:8:100%[F:6:75%|R:2:25%])chr17 72093482 C‐>A(2:2:100%[F:0:0%|R:2:100%])chr17 76474068 T‐>A(2:2:100%[F:2:100%|R:0:0%])chr17 72321424 G‐>T(2:2:100%[F:2:100%|R:0:0%])chr17 75595480 C‐>T(14:14:100%[F:11:78.6%|R:3:21.4%])chr17 73784994 A‐>C(3:3:100%[F:2:66.7%|R:1:33.3%])chr17 78224626 DEL:A‐>‐(29:29:100%[F:22|R:7])chr17 76589025 C‐>A(9:9:100%[F:2:22.2%|R:7:77.8%])chr17 76089985 A‐>G(12:12:100%[F:6:50%|R:6:50%])chr17 77947510 T‐>A(2:2:100%[F:0:0%|R:2:100%])chr17 75985128 A‐>C(2:2:100%[F:0:0%|R:2:100%])chr17 74510796 G‐>C(27:27:100%[F:16:59.3%|R:11:40.7%])chr17 75713833 T‐>C(2:2:100%[F:2:100%|R:0:0%])chr17 78009819 G‐>T(9:9:100%[F:2:22.2%|R:7:77.8%])chr17 76484827 A‐>G(4:4:100%[F:2:50%|R:2:50%])chr17 74568923 G‐>T(2:2:100%[F:2:100%|R:0:0%])chr17 74809266 T‐>C(26:26:100%[F:17:65.4%|R:9:34.6%])chr17 77014530 DEL:TT‐>‐‐(2:2:100%[F:2|R:0])chr17 77172281 G‐>A(3:3:100%[F:3:100%|R:0:0%])chr17 77158835 A‐>G(3:3:100%[F:3:100%|R:0:0%])chr17 78089261 C‐>A(2:2:100%[F:0:0%|R:2:100%])chr17 73695367 C‐>T(5:5:100%[F:3:60%|R:2:40%])


chr17 75964682 T‐>C(43:43:100%[F:28:65.1%|R:15:34.9%])chr17 75607833 DEL:C‐>‐(2:2:100%[F:2|R:0])chr17 74025183 C‐>G(23:23:100%[F:15:65.2%|R:8:34.8%])chr17 75791610 C‐>G(2:2:100%[F:2:100%|R:0:0%])chr17 77277518 C‐>T(2:2:100%[F:0:0%|R:2:100%])chr17 74655535 C‐>T(12:12:100%[F:7:58.3%|R:5:41.7%])chr17 76370600 C‐>T(25:25:100%[F:19:76%|R:6:24%])chr17 72468933 T‐>G(18:18:100%[F:15:83.3%|R:3:16.7%])chr17 73247228 G‐>A(2:2:100%[F:0:0%|R:2:100%])chr17 76190863 G‐>A(2:2:100%[F:2:100%|R:0:0%])chr17 76481353 C‐>G(90:90:100%[F:8:8.89%|R:82:91.1%])chr17 74002003 G‐>A(3:3:100%[F:2:66.7%|R:1:33.3%])chr17 77699267 T‐>G(2:2:100%[F:2:100%|R:0:0%])chr17 72508579 G‐>A(2:2:100%[F:2:100%|R:0:0%])chr17 74477660 G‐>C(2:2:100%[F:0:0%|R:2:100%])chr17 74939592 C‐>T(17:17:100%[F:9:52.9%|R:8:47.1%])chr17 74837469 C‐>T(30:30:100%[F:19:63.3%|R:11:36.7%])chr17 78099829 A‐>G(24:24:100%[F:16:66.7%|R:8:33.3%])chr17 78632522 A‐>C(3:3:100%[F:1:33.3%|R:2:66.7%])chr17 77566121 T‐>G(2:2:100%[F:2:100%|R:0:0%])chr17 74053361 C‐>T(20:20:100%[F:10:50%|R:10:50%])chr17 77678571 C‐>T(12:12:100%[F:7:58.3%|R:5:41.7%])chr17 78266171 A‐>C(2:2:100%[F:2:100%|R:0:0%])chr17 76559332 A‐>G(5:5:100%[F:1:20%|R:4:80%])chr17 77515698 G‐>A(12:12:100%[F:8:66.7%|R:4:33.3%])chr17 73250167 G‐>A(34:34:100%[F:14:41.2%|R:20:58.8%])chr17 73067712 INS:‐‐>C(21:21:100%[F:8|R:0])chr17 75977948 C‐>T(30:30:100%[F:14:46.7%|R:16:53.3%])chr17 74512539 T‐>A(2:2:100%[F:2:100%|R:0:0%])chr17 74679645 T‐>A(6:6:100%[F:6:100%|R:0:0%])chr17 72106969 T‐>G(2:2:100%[F:2:100%|R:0:0%])chr17 75922718 G‐>A(20:20:100%[F:10:50%|R:10:50%])chr17 75741814 T‐>C(2:2:100%[F:0:0%|R:2:100%])chr17 76062502 T‐>C(8:8:100%[F:7:87.5%|R:1:12.5%])chr17 77654996 G‐>C(2:2:100%[F:1:50%|R:1:50%])chr17 77686561 G‐>A(10:10:100%[F:5:50%|R:5:50%])chr17 74982650 C‐>T(16:16:100%[F:8:50%|R:8:50%])chr17 75635536 G‐>T(3:3:100%[F:3:100%|R:0:0%])chr17 74808396 A‐>G(16:16:100%[F:6:37.5%|R:10:62.5%])chr17 78575898 T‐>C(6:6:100%[F:6:100%|R:0:0%])chr17 73660231 A‐>C(3:3:100%[F:3:100%|R:0:0%])chr17 78355201 C‐>T(19:19:100%[F:16:84.2%|R:3:15.8%])chr17 74998754 C‐>A(2:2:100%[F:2:100%|R:0:0%])chr17 78507684 A‐>C(2:2:100%[F:0:0%|R:2:100%])chr17 74904565 C‐>T(7:7:100%[F:5:71.4%|R:2:28.6%])chr17 75475208 A‐>G(2:2:100%[F:2:100%|R:0:0%])chr17 74030538 T‐>C(10:10:100%[F:1:10%|R:9:90%])chr17 72468302 G‐>A(10:10:100%[F:5:50%|R:5:50%])chr17 73761952 A‐>C(2:2:100%[F:1:50%|R:1:50%])chr17 78255045 C‐>G(2:2:100%[F:2:100%|R:0:0%])


chr17 78469213 A‐>G(9:9:100%[F:7:77.8%|R:2:22.2%])chr17 74688452 C‐>T(8:8:100%[F:5:62.5%|R:3:37.5%])chr17 76062412 T‐>C(7:7:100%[F:4:57.1%|R:3:42.9%])chr17 78401346 G‐>C(2:2:100%[F:0:0%|R:2:100%])chr17 72467890 A‐>G(2:2:100%[F:2:100%|R:0:0%])chr17 73755877 G‐>C(20:20:100%[F:12:60%|R:8:40%])chr17 76484890 A‐>C(7:7:100%[F:0:0%|R:7:100%])chr17 74246223 A‐>G(16:16:100%[F:10:62.5%|R:6:37.5%])chr17 74832310 C‐>T(20:20:100%[F:13:65%|R:7:35%])chr17 73155330 T‐>C(12:12:100%[F:4:33.3%|R:8:66.7%])chr17 73257593 G‐>A(6:6:100%[F:3:50%|R:3:50%])chr17 73033198 DEL:AGTG‐>‐‐‐‐(5:5:100%[F:2|R:3])chr17 75990785 A‐>C(2:2:100%[F:2:100%|R:0:0%])chr17 73441388 C‐>A(2:2:100%[F:0:0%|R:2:100%])chr17 77287571 A‐>G(2:2:100%[F:0:0%|R:2:100%])chr17 76388148 A‐>C(2:2:100%[F:1:50%|R:1:50%])chr17 73005849 G‐>T(4:4:100%[F:2:50%|R:2:50%])chr17 78640243 INS:‐‐‐>TA(2:2:100%[F:2|R:0])chr17 76838723 G‐>A(4:4:100%[F:2:50%|R:2:50%])chr17 77698608 A‐>G(2:2:100%[F:2:100%|R:0:0%])chr17 76588693 C‐>G(2:2:100%[F:1:50%|R:1:50%])chr17 72831233 C‐>T(5:5:100%[F:4:80%|R:1:20%])chr17 73807188 C‐>T(20:20:100%[F:18:90%|R:2:10%])chr17 72475254 C‐>T(14:14:100%[F:3:21.4%|R:11:78.6%])chr17 75003189 G‐>A(2:2:100%[F:2:100%|R:0:0%])chr17 78472104 C‐>T(8:8:100%[F:2:25%|R:6:75%])chr17 72522961 G‐>A(6:6:100%[F:2:33.3%|R:4:66.7%])chr17 77558076 G‐>A(13:13:100%[F:11:84.6%|R:2:15.4%])chr17 77221731 C‐>T(7:7:100%[F:5:71.4%|R:2:28.6%])chr17 72473287 C‐>T(2:2:100%[F:1:50%|R:1:50%])chr17 75613411 A‐>C(7:7:100%[F:6:85.7%|R:1:14.3%])chr17 76176700 G‐>T(2:2:100%[F:2:100%|R:0:0%])chr17 77925591 T‐>G(2:2:100%[F:1:50%|R:1:50%])chr17 77014467 C‐>T(6:6:100%[F:0:0%|R:6:100%])chr17 74685477 C‐>T(19:19:100%[F:10:52.6%|R:9:47.4%])chr17 77710053 INS:‐‐>G(4:4:100%[F:1|R:0])chr17 77678534 C‐>T(3:3:100%[F:2:66.7%|R:1:33.3%])chr17 77946334 T‐>G(2:2:100%[F:1:50%|R:1:50%])chr17 72992086 T‐>C(10:10:100%[F:5:50%|R:5:50%])chr17 77644945 A‐>C(2:2:100%[F:2:100%|R:0:0%])chr17 75452923 C‐>T(2:2:100%[F:0:0%|R:2:100%])chr17 76032174 A‐>G(19:19:100%[F:9:47.4%|R:10:52.6%])chr17 76253727 C‐>T(4:4:100%[F:0:0%|R:4:100%])chr17 77208892 T‐>C(4:4:100%[F:4:100%|R:0:0%])chr17 75985120 T‐>C(3:3:100%[F:0:0%|R:3:100%])chr17 74260323 C‐>A(2:2:100%[F:1:50%|R:1:50%])chr17 74592794 A‐>C(5:5:100%[F:4:80%|R:1:20%])chr17 74898781 C‐>A(7:7:100%[F:4:57.1%|R:3:42.9%])chr17 77122018 G‐>A(10:10:100%[F:6:60%|R:4:40%])chr17 76875265 G‐>A(9:9:100%[F:3:33.3%|R:6:66.7%])


Supplementary Table 1 Results of high‐throughput sequencingList of novel non‐synonymous sequence changes

Known_Gene Known_Gene_URL Coding_Region Strand Mutation Mutation_Type Mutation_Position Codon_Position Codon_Change AA_ChangeST6GALNAC2 http://genome.ucsc.edu/cgi‐bin/hgGene?hgg_gene=uc002jsg.2 chr17:72073780‐72093485 ‐ A VAR chr17:72093482‐72093483 1 CAT‐>AAT M‐>I

RNF213 http://genome.ucsc.edu/cgi‐bin/hgGene?hgg_gene=uc002jyh.1 chr17:75928543‐75981893 + G VAR chr17:75938164‐75938165 1 ATC‐>GTC I‐>VPYCR1 http://genome.ucsc.edu/cgi‐bin/hgGene?hgg_gene=uc002kcr.1 chr17:77484380‐77487981 ‐ T VAR chr17:77486276‐77486277 2 GCG‐>GTG R‐>HUTS2R http://genome.ucsc.edu/cgi‐bin/hgGene?hgg_gene=uc002ker.1 chr17:77925489‐77926659 + G VAR chr17:77925591‐77925592 1 TCC‐>GCC S‐>ASGSH http://genome.ucsc.edu/cgi‐bin/hgGene?hgg_gene=uc002jxz.2 chr17:75798845‐75808707 ‐ G VAR chr17:75799297‐75799298 3 CTC‐>CTG E‐>Q

C17orf90 http://genome.ucsc.edu/cgi‐bin/hgGene?hgg_gene=uc002kbb.2 chr17:77243733‐77244012 ‐ G VAR chr17:77243855‐77243856 3 GGT‐>GGG T‐>PSLC16A3 http://genome.ucsc.edu/cgi‐bin/hgGene?hgg_gene=uc002kea.1 chr17:77787173‐77790141 + G VAR chr17:77788773‐77788774 2 GCC‐>GGC A‐>G


Supplementary Table 2 Primer and morpholino sequences

Supplementary Table 2.1 Genomic DNA primer sequences

Exon Primer Sequence Product Size [bp]

right GAACCTCCACCCCTCCAGleft CTCCCCATTCCCAGGAAG

right AGCCAAAGCTAGCCATGAAGleft CAGGTCCCAAGAGCAATCAG

right CAGCATTCTCTGTGCCATTCleft TTCTCCTCCTTCCCTTCTGG

right GGTGGAAGAGGACCTGATTGleft ATCTGCTGAGTGCGTGAATG

right TGGTGATGCTGAGCTGATTCleft TTCTCACACGGGAAGGAGAG

Supplementary Table 2.2 Human cDNA primer sequences used for RT-PCR

Gene Primer Sequence Product Size [bp]

right TGCACCACCAACTGCTTAGCleft GGCATGGACTGTGGTCATGAG

right ATGAGCGTGGGCTTCATCleft GGGAGCTAGCCATTATCTTGTG

right TCGGCTCACAAGATAATAGCCleft TTCACCGTCTCCTTGTTGC

right GGCTGATGGCCTTGTATGAGleft TCATGGAAATCCAAATTGGTC

Supplementary Table 2.3 Mouse cDNA primer sequences used for RT-PCR

Gene Primer Sequence Product Size [bp]

right GGGAAGCCCATCACCATCTTleft CGGCCTCACCCCATTTG

right AGACATGGACCAAGCTACGGleft TCACAGCCAGGAAGAGCAC

right GTCGGCTCACAAGATAATAGCCleft TCCTTATTGCTTCGGGTCAG

right GTCTCAGTGGCAGTGGTGTGleft CAGGGTCTGAGCAGCAATG

right GCATTCTTCACCTCCTGACCleft CGGCAGAGATCCTCAACTTC

Supplementary Table 2.4 Morpholino sequencesSpecies Gene

5’-AGCTCCTATGAAGCCCACACTCATG-3’

5’-CAGCTCCGATAAATCCCACACTCAT-3’

5’-CCGCTCCAATGAAGCCCACACTCAT-3’

Morpholino sequence

Xenopus laevis Pycr1

Danio rerio Pycr1

Danio rerio Pycr2

Pycrl 116

P5cs 100

Pycr1 107

Pycr2 99

GAPDH 87

107

85

PYCR2

P5CS

512

PYCR1 103

Gapdh 61

7

596

830

752

815

1

2

3-4

5-6


Mutations in PYCR1 cause cutis laxa with progeroid features

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