Mutations in CNTNAP1 and ADCY6 are responsible for severe arthrogryposis multiplex congenita with...

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© The Author 2013. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]

Mutations in CNTNAP1 and ADCY6 are responsible for severe arthrogryposis multiplex

congenita with axoglial defects

Annie Laquérriere1, Jérome Maluenda2,#,, Adrien Camus2,#,, Laura Fontenas2, Klaus

Dieterich2,3, Flora Nolent2,#, Jié Zhou2, Nicole Monnier4, Philippe Latour5, Damien

Gentil1, Delphine Héron6, Isabelle Desguerres7, Pierre Landrieu8, Claire Beneteau9,

Benoit Delaporte10, Céline Bellesme11, Clarisse Baumann12, Yline Capri12, Alice

Goldenberg13, Stanislas Lyonnet14, Dominique Bonneau15,16, Brigitte Estournet17, Susana

Quijano-Roy17, Christine Francannet18, Sylvie Odent19, Marie-Hélène Saint-Frison20,

Sabine Sigaudy21, Dominique Figarella-Branger22, Antoinette Gelot23, Jean-Marie

Mussini24, Catherine Lacroix25, Valerie Drouin-Garraud13, Marie-Claire Malinge15,

Tania Attié-Bitach14, Bettina Bessieres26, Maryse Bonniere26, Ferechte Encha-Razavi26,

Anne-Marie Beaufrère27, Suonary Khung-Savatovsky28, Marie José Perez29, Alexandre

Vasiljevic30, Sandra Mercier8, Joelle Roume31, Laetitia Trestard32, Pascale Saugier-

Veber13, Marie-Pierre Cordier33, Valérie Layet34, Marine Legendre35, Adeline

Vigouroux-Castera36, Joel Lunardi4, Monica Bayes37, Pierre S. Jouk3, Luc Rigonnot38,

Michèle Granier39, Damien Sternberg40, Josiane Warszawski41, Ivo Gut37, Marie

Gonzales42, Marcel Tawk2, and Judith Melki2,43,*,#

1Pathology Laboratory and NeoVasc Region-Inserm Team ERI28, Institute of Research for

Innovation in Biomedicine, University of Rouen, 76031 Rouen, France

2Unité Mixte de recherche (UMR)-788, Inserm and University Paris 11, 94276 Le Kremlin

Bicêtre, France

3Département de Génétique, CHU Grenoble, Inserm U-836, Institut des Neurosciences, 38043

Grenoble, France

4Laboratoire de Biochimie et Génétique Moléculaire, CHU Grenoble, 38043 Grenoble, France

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5Service de Neurobiologie, CHU de Lyon, 69677 Bron, France

6Département de Génétique, INSERM U-975, CHU Pitié-Salpêtrière, 75013 Paris, France

7Unité de Neuropédiatrie, CHU Necker-Enfants Malades, 75743 Paris, France

8University Paris 11, 94276 Le Kremlin Bicêtre, France

9Service de Génétique Médicale, UMR-1089, Inserm, Université de Nantes, CHU Nantes,

44093 Nantes, France

10Service de Pédiatrie Néonatale, CH Havre, 76083 Le Havre, France

11Service de Neuropédiatrie, CHU Bicêtre, 94276 Le Kremlin-Bicêtre, France

12Département de Génétique, CHU Robert Debré, 75935 Paris, France

13Service de Génétique, CHU Rouen, Inserm U-1079, 76031 Rouen, France

14Université Paris Descartes- Sorbonne Paris Cité, Institut Imagine, INSERM U781, APHP

Necker-Enfants Malades, 75743 Paris, France

15Département de Biochimie et Génétique, CHU Angers, 49933 Angers, France

16UMR CNRS U6214, INSERM U1083, 49933 Angers, France

17Service de Pédiatrie et Réanimation infantile, Hopital R. Poincaré, 92380 Garches, France

18Unité de Génétique Médicale, CHU Clermont-Ferrand, 63003 Clermont-Ferrand, France

19Service de Génétique Clinique, CHU Rennes, UMR6290 IGDR Université Rennes 1, 35203

Rennes, France

20Service d'Anatomie et de Cytologie Pathologiques, Hopital Argenteuil, 95107 Argenteuil,

France

21Département de Génétique Médicale, CHU de La Timone, 13385 Marseille, France

22Service d'Anatomie Pathologique et de Neuropathologie, CHU de la Timone, 13005

Marseille, France

23Service de Neuropathologie, Hopital Trousseau, 75571 Paris, France

24Service d'Anatomie et de Cytologie Pathologiques, CHU de Nantes, 44093 Nantes, France

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25Laboratoire de Neuropathologie, CHU de Bicêtre, 94276 Le Kremlin-Bicêtre, France

26Service d'Histologie-Embryologie-Cytogénétique, CHU Necker-Enfants Malades, 75743

Paris, France

27Service de pathologie, CHU Clermont-Ferrand 63001 Clermont-Ferrand, France

28Unité de Foetopathologie, CHU Robert Debré, 75935 Paris, France

29Département Génétique, Foetopathologie CHU Montpellier, 34295 Montpellier, France

30Centre de Pathologie et Neuropathologie Est, Hospices Civils de Lyon, 69677 Bron, France

31Unité de Génétique Médicale, Hopital de Poissy, 78303 Poissy, France

32Unité de Génétique, Hopital du Belvedère, 76131, Mont-Saint-Aignan, France

33Unité de Génétique Clinique, CHU de Lyon, 69677 Bron, France

34Unité de Génétique Médicale et Cytogénétique, CH Havre, 76083 Le Havre, France

35Service de Génétique Clinique, CHU de Poitiers, 86021 Poitiers, France

36Service de Génétique Médicale, CHU Toulouse, 31059 Toulouse, France

37Centro Nacional de Análisis Genómico, Barcelona, 080028, Spain

38Service d'Obstétrique, Centre Hospitalier Sud-Francilien (CHSF), 91108 Corbeil Essonnes,

France

39Service de Médecine Néonatale, CHSF, 91108 Corbeil Essonnes, France

40Assistance Publique Hôpitaux de Paris, Hôpitaux Universitaires Pitié-Salpêtrière, Service de

Biochimie Métabolique, 75651 Paris, France

41UMR-1018, Inserm et Université Paris 11, Service d’Epidémiologie-Santé Publique, CHU

Bicetre, 94276 Le Kremlin-Bicêtre, France

42Service de Génétique et d'Embryologie Médicales, Université Paris VI, Hopital Trousseau,

75571 Paris, France

43AP-HP, Hôpital Bicêtre, Unité de Génétique Médicale, Le Kremlin-Bicêtre, 94276; CHSF,

91108 Corbeil Essonnes, France

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*Correspondence should be addressed to J. Melki; [email protected]; Tel:

33149595370; Fax: 33149591959, UMR-986, 80 rue du Général Leclerc, 94276, Le Kremlin-

Bicêtre, France

#Present address: UMR-986, Inserm and University Paris 11, 94276 Le Kremlin Bicêtre,

France.

equal contribution

ABSTRACT

Non-syndromic arthrogryposis multiplex congenita (AMC) is characterized by multiple

congenital contractures resulting from reduced fetal mobility. Genetic mapping and whole

exome sequencing were performed in 31 multiplex and/or consanguineous undiagnosed AMC

families. Although this approach identified known AMC genes, we here report pathogenic

mutations in two new genes. Homozygous frameshift mutations in CNTNAP1 were found in

four unrelated families. Patients showed a marked reduction in motor nerve conduction

velocity (<10m/sec) and transmission electron microscopy (TEM) of sciatic nerve in the index

cases revealed severe abnormalities of both nodes of Ranvier width and myelinated axons.

CNTNAP1 encodes CASPR, an essential component of node of Ranvier domains which

underly saltatory conduction of action potentials along myelinated axons, an important

process for neuronal function. A homozygous missense mutation in Adenylate Cyclase 6 gene

(ADCY6) was found in another family characterized by a lack of myelin in the Peripheral

Nervous System (PNS) as determined by TEM. Morpholino knockdown of the zebrafish

orthologs led to severe and specific defects in peripheral myelin in spite of the presence of

Schwann cells. ADCY6 encodes a protein that belongs to adenylate cyclase family responsible

for the synthesis of cAMP. Elevation of cAMP can mimic axonal contact in vitro and

upregulates myelinating signals. Our data indicate an essential and so far unknown role of

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ADCY6 in PNS myelination likely through the cAMP pathway. Mutations of genes encoding

proteins of Ranvier domains or involved in myelination of Schwann cells are responsible for

novel and severe human axoglial diseases.

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INTRODUCTION

Arthrogryposis multiplex congenita (AMC) is characterized by congenital contractures of at

least two distinct joints of the body. The overall incidence is 1 in 3000 of live births (1,2).

Some non genetic factors may cause AMC such as mechanical limitation of fetal movements

or maternal autoimmune myasthenia. A number of genetic syndromes including AMC

phenotype, collectively referred to as syndromic AMC, have been described in several

conditions (3,4). Non-syndromic or isolated AMC are the direct consequence of fetal

akinesia/hypokinesia sequence which may lead, in addition to AMC, to pterygia, lung

hypoplasia, diaphragmatic defect or cleft palate. Isolated AMC are genetically heterogeneous.

Mutations of genes encoding components of the neuromuscular junctions including CHRNG

(MIM 100730), CHRNA1 (MIM 100690), CHRND (MIM 100720), CHRNB1 (MIM 100710),

DOK7 (MIM 610285), RAPSN (MIM 601592) or CHAT (MIM 118490) are responsible for

lethal multiple pterygium syndromes, isolated AMC with neonatal myasthenia or Escobar

syndrome. Fetal motor neuron diseases may also result in lethal congenital contractures

caused by mutations in GLE1 (MIM 603371), PIP5K1C (MIM 606102) or ERBB3 (MIM

190151) genes. AMC also occasionally occurs in type I spinal muscular atrophy (SMA)

caused by mutations of SMN1 (MIM 600354) or congenital SMA linked to TRPV4 mutations

(MIM 605427). More recently, mutations of ECEL1 (MIM 605896) have been reported in

distal AMC. Congenital myopathies associated with distal AMC may be caused by mutations

in TPM2 (MIM 190990), MYH2 (MIM 160740), MYH3 (MIM 160720), MYH8 (MIM

160741), TNNI2 (MIM 191043), TNNT3 (MIM 600692) or MYBPC1 (MIM 160794) genes.

Congenital myotonic dystrophy caused by abnormal triplet expansion of the DMPK (MIM

605377) gene, nemalin myopathy linked to ACTA1 (MIM 102610), TPM2 (MIM 190990) or

NEB (MIM 161650), minicore myopathy linked to RYR1 mutations (MIM 180901) or more

recently SYNE1 mutation (MIM 608441) have all been reported in non syndromic AMC.

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Collectively, non-syndromic AMCs include a large spectrum of diseases of motor neurons,

neuromuscular junctions, or skeletal muscle.

The difficulty in establishing a genetic diagnosis for AMC patients is likely due to the high

genetic heterogeneity and/or to some not yet identified disease causing genes. Moreover, there

is a lack of suitable screening methods of all known AMC genes. In order to gain further

insight into the underlying cause of these diseases, we took advantage of the added value of

whole genome scanning using SNP microarrays alone or combined with whole exome

sequencing (WES) to study a cohort of 31 multiplex and/or consanguineous families with

undiagnosed non-syndromic AMC.

RESULTS

Phenotypic characterization of AMC patients

We enrolled 63 affected fetuses, children, or adults belonging to 31 multiplex and/or

consanguineous families with unexplained non-syndromic AMC (Supplementary Material,

Figure S1). The main clinical criteria were the gestational stage of AMC diagnosis based on

ultrasound examination (weeks' gestation, w.g., the earliest one in multiplex families), the

topography of joint contractures, associated symptoms including reduced fetal mobility,

pterygium, micrognathia and cleft palate, the amount of amniotic fluid, and cystic hygroma

(Table 1). The stage of AMC diagnosis showed a marked heterogeneity of fetal onset starting

from 11 w.g. and up to 32 w.g. Reduced fetal mobility was associated with AMC in 24 out of

28 affected individuals (86%). No information was available in three families. Targeted gene

tests when performed did not lead to a diagnosis (Supplementary Material, Table S1). The

primary defects were established based on gene identification and function (see below) and

were classified as skeletal muscle, neuromuscular junction (NMJ) or axoglial AMC. AMC

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associated with pterygia, both distal and proximal contractures or cystic hygroma are

significantly more frequent in skeletal muscle or NMJ than axoglial classes (P<0.02, Fisher’s

exact test, Figure 1).

Identification of mutations in genes known to be responsible for arthrogryposis

multiplex congenita or neuromuscular disorders

The overall strategy for gene identification is depicted in Supplementary Material, Figure S2.

Candidate loci were identified in each family by multipoint linkage analysis and

homozygosity mapping by using SNP microarrays. When a single known AMC gene was

identified in candidate disease loci, Sanger sequencing of exons and intron-exon junctions

was performed. This was successfully applied in 3 families linked to mutations of RYR1

(family B415), TRPV4 (family B651, Tables 2, S2 and Supplementary Material, Figure S3) or

ECEL1 (family B192, 5). When numerous or none AMC genes were identified in candidate

loci, WES was performed using the DNA sample of one affected individual per family

(n=27). As a first filtering, homozygous or compound heterozygous variants mapping to the

disease loci were selected in recessive and heterozygous variants in dominant forms,

respectively (Tables 2 and S2). Variants were then filtered against a list of genes known to be

involved in AMC or neuromuscular disorders (NMD). Mutations in NEB, RYR1, SYNE1,

TNNT3, TTN or CHRNG genes were successfully identified in 10 out of 31 families (32%,

Table 2 and Figures S3 and S4). Interestingly, homozygous stop gained mutation was found

in exon 136 of SYNE-1 in family K168 which confirms that mutations of SYNE-1 are

responsible for AMC since a single AMC family only was previously reported (6). In family

A642, a non-sense mutation on one allele and a frameshift mutation on the other allele of TTN

were found extending the clinical spectrum of TTN gene mutations. In a consanguineous

family (A659), a homozygous splice mutation was found in RBBP8 (Table 2, Figures S3 and

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S4). Mutations of this gene have been reported in Seckel syndrome type 2 which may

associate microcephaly, holoprosencephaly and arthrogryposis (7) suggesting that the affected

fetus had a syndromic AMC that was not detected during pregnancy. In 16 families, allelic

mutations were not identified using the above criteria. In 4 of them, a single heterozygous

variant in AMC or NMD genes and mapping to candidate loci was identified. Non-covered

exons visualized by the Integrative genomics viewer software (IGV, v1.5.64, 8) were then

sequenced by Sanger method. Allelic mutations were found in ECEL1, RAPSN, RYR1 and

NEB (Table 2 and Supplementary Material, Figure S3). When no mutations were identified

using these filters, the hypothesis of dominant de novo mutation was tested by selecting

variants within AMC or NMD genes without any linkage data filter. A single missense

mutation of the MYH3 gene was identified in the affected fetuses of family A640, the father

showing somatic mosaicism pattern (Table 2 and Supplementary Material, Figure S3).

Identification of mutations in CNTNAP1 encoding CASPR an essential component of

node of Ranvier domains

In two consanguineous families (K182, A641), homozygous frameshift mutations were found

in CNTNAP1 encoding CASPR (Figure 2 and Table 2). In family A641, the three fetuses born

from consanguineous parents carry an homozygous 1bp deletion in CNTNAP1 exon 18

(c.2901_2902del) leading to frameshift and premature stop codon (P967PfsX12). In family

K182, affected patients carry an homozygous 1bp insertion in CNTNAP1 exon 19

(c.3009_3010insT) leading to frameshift (F1003fs). Two additional families with similar

phenotype (see below) were found to carry deleterious mutations in the same gene using

either the combination of both linkage analysis with WES (family K199) or homozygosity

mapping only (B207). In the consanguineous family K199, affected patients carry the same

homozygous 1bp insertion in CNTNAP1 exon 19 (c.3009_3010insT) as the unrelated family

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K182. In family B207, an homozygous frameshift mutation in exon 19 of CNTNAP1 was

found in the patient (intron18-exon19:c.2993-2_2994del, I999WfsX5). In families K182 and

A641, both parents were heterozygous for the mutation as expected. In families K199 and

B207, DNA samples from parents were not available. These mutations were absent in 95

ethnically matched controls. The c.2901_2902del mutation was found at a very low minor

allele frequency (MAF: 0.00016) in the current Exome Variant Server database (EVS,

ESP6500SI-V2). The other mutations were found in neither EVS nor dbSNPv138.

In the four families, the fetal phenotype was quite similar (Table 3) and characterized by a late

onset during pregnancy (from 28 w.g.), polyhydramnios and distal joint contractures

including talipes equinovarus and both proximal and distal interphalangeal joint contractures

of the hands (Figure 3). Proximal joints were not involved. At birth, the patients displayed

severe hypotonia, facial diplegia and a lack of swallowing, autonomous respiratory function

and deep tendon reflexes (Figure 3). Motor nerve conduction velocity was markedly reduced

(<10m/sec). In all patients, death occurred within the first two months of life. Since CASPR is

known to play a key role in the delineation of the axonal domains of myelinated axons in mice

(9), Transmission Electron Microscopy (TEM) of sciatic nerve in the A641 and K182 index

cases was performed and revealed two major abnormalities (Figure 4). First, examination of

longitudinal sections revealed a marked widening of the nodes of Ranvier in patient A641

(3.98+/-0.56; n=5) when compared to control (1.12+/-0.98; n=4, P<0.05; Figure 4). Second,

in transverse sections, myelinated axons of K182 patient nerve (n=50) displayed a significant

reduced surface associated with thinner myelin sheath when compared to an age-matched

control case (n=69, Figure 4, P=0.04 and P<0.0003, respectively, Kruskal–Wallis test).

Similar abnormalities of myelinated axons were found in K199 and B207 patient’s nerve (data

not shown, available on request).

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Identification of mutation in ADCY6 reveals an essential and so far unknown role of

ADCY6 in myelination of Schwann cells

In the consanguineous family A649, a homozygous missense mutation predicted to be

damaging using Polyphen-2 (10) with a high score (1.00) was found in ADCY6 (c.C3346T, p.

R1116C, Figure 2 and Table 2). This mutation was found in neither EVS nor dbSNPv138.

The phenotype of patients was similar to that carrying CNTNAP1 mutations (Table 3). Patient

nerve immunohistochemistry using S100 protein antibody revealed Schwann cells but all

nerve fascicles were negative for MBP antibodies (Supplementary Material, Figure S5). TEM

of nerve revealed no myelinated axons (Figure 4). Some redundant basal lamina of Schwann

cells were also observed, suggesting inability of Schwann cells to properly myelinate axons.

Antisense morpholino oligonucleotides and their corresponding “mismatch” controls were

designed to specifically knockdown the ADCY6 orthologs in zebrafish (adcy6a and adcy6b,

Supplementary Material, Table S3). Knockdown of the ADCY6 orthologs in zebrafish led to a

very similar phenotype as observed in patients (Figure 5) with a loss of mbp expression in the

PNS (showed here is the Posterior Lateral Line nerve, n=36/38) while the central nervous

system mbp expression was comparable to controls. Furthermore, we also found no defects in

Schwann cell migration and axonal growth in the morphants by using transgenic line

foxd3::GFP (11) and acetylated tubulin antibody staining respectively (Figure 5).

DISCUSSION

To our knowledge, this is the first study of a large cohort of undiagnosed AMC

individuals investigated by combining genetic mapping with WES. This strategy markedly

improves the genetic diagnosis of AMC since pathogenic mutations were detected in known

AMC or NMD genes in 19 families (61%). Importantly, this approach allowed identifying

mutations in CNTNAP1 and ADCY6 in 5 families revealing novel axoglial human diseases.

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The fetal phenotype was quite similar and mainly characterized by a late onset during

pregnancy, polyhydramnios (in 4 out of 5 families), distal joint contractures and very severe

motor paralysis at birth leading to death within the first three months of life. A causative role

of mutations in the CNTNAP1 gene encoding CASPR was established by the identification of

distinct homozygous frameshift mutations in four unrelated families. In addition, dramatic

reduction of motor nerve conduction velocities associated with severe abnormalities of the

nodes of Ranvier and myelinated axons were observed. The node of Ranvier, the flanking

paranodal junctions and the juxtaparanodes underlie saltatory conduction of action potentials

along myelinated axons, an essential process for neuronal function. The paranodal junctions

consist of a complex containing the axonal proteins Caspr (12) and contactin (13) and the glial

isoform of neurofascin (14). Mice that lack Caspr exhibit severe motor paresis and most of the

mutant mice die at weaning (P21, 9). In these mice, normal paranodal junctions fail to form

leading to disruption of the paranodal loops. Our data show similar defects of myelinated

axons indicating a critical role for CASPR in the delineation of specific axonal domains and

the axon-glia interactions required for normal saltatory conduction in both mouse and human.

A homozygous missense mutation in ADCY6 has been found in a consanguineous family.

Ultrastructural morphology of patient nerve sample revealed the presence of Schwann cells

but the lack of myelin in the PNS. Although mutation of this gene has been found in a single

family, knock down of the orthologous genes in zebrafish provided evidence for major myelin

defects in the PNS, data similar to those found in patients. These results strongly support an

essential and so far unknown role of ADCY6 in this process. ADCY6 encodes a protein that

belongs to adenylate cyclase family responsible for the synthesis of cAMP (15). During

development, promyelinating Schwann cells associate with one segment of an axon and

differentiate into myelinating Schwann cells to form the myelin sheath. Elevation of cAMP

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can mimic axonal contact in vitro and is thought to upregulate myelinating signals (16-18).

The G protein-coupled receptor Gpr126 has been recently shown to be required in Schwann

cells for myelination (19). Elevation of cAMP in gpr126 zebrafish mutants could restore

myelination suggesting that Gpr126 drives the differentiation of promyelinating Schwann

cells by elevating cAMP levels (19). Our data suggest that ADCY6 is involved in the same

Gpr126- cAMP pathway for myelination of Schwann cells.

Hence, our study extends our knowledge of the pathogenic mechanisms in AMC and indicates

that Caspr and Adcy6 are essential for correct differentiation of peripheral myelinated axons

in human. These genes may be regarded as strong candidates in undiagnosed peripheral

neuropathies.

In spite of some pitfalls, most likely due to the lack of coverage of mutations by WES,

parallel next generation sequencing (NGS) targeting previously known AMC genes, and the

identification of two novel genes in this study should now lead to a molecular diagnosis in

more than 75% of AMC cases since our study concentrated on undiagnosed AMC only,

without previous integration of muscle or nerve morphological studies. Such targeted NGS

approach should avoid the identification of clinically relevant mutations unrelated to AMC.

This will be particularly useful for diagnosis purpose and appropriate management.

PATIENTS AND METHODS

PATIENTS

All cases were evaluated by obstetricians, fetal pathologists, clinical geneticists,

neonatologists or neuropediatricians. The main clinical features of the affected individuals are

summarized in Table 1. The gestational age of AMC diagnosis was based on ultrasound

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examination. Any other defective organ was regarded as exclusion criteria. The parents of all

patients provided written informed consents for genetic analysis of their children or fetuses

and themselves in accordance with the ethical standards of our institutional review boards.

METHODS

Genome-wide linkage analysis

Genomic DNA was isolated from blood or frozen tissue with the use of a QiaAmp DNA midi

or mini Kit respectively (Qiagen). Whole genome SNP scanning was carried out according to

Affymetrix 250K GeneChip Mapping Assay Manual. Multipoint linkage analysis and

homozygosity mapping of SNP data were performed using the Alohomora (20) and Merlin

softwares (21) with the following parameters: autosomal recessive or dominant inheritance,

100% penetrance and disease gene frequency in the population of 1:1000.

Whole exome sequencing (WES)

WES was performed using the DNA sample of one affected individual per family. The

Illumina TruSeq DNA Sample Prep kit v1 and the NimbleGen SeqCap EZ Human Exome

Library v2.0 (targeting 44 Mb, from A631 to A648 DNA samples) or v3.0 (targeting 64 Mb,

from A649 to K199 DNA samples) were used for library preparation and exome enrichment,

respectively as previously described (22). Sequencing was performed on a Genome Analyzer

IIx instrument using paired-end 75 bp reads and following the Illumina’s protocol. The

median coverage of the WES was 33 to 138 (average: 77).

Reads were aligned to the human reference genome sequence (UCSC hg19, NCBI build 37.3)

via the BWA program (23). Variants were selected using the SAMtools (24) then annotated

using Annovar softwares (25). Reads with a coverage of at least 2X were filtered against

dbSNPv131 database. Variants with a minor allele frequency (MAF) less <0.0015 and located

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in coding regions, intron-exon junctions or short coding insertions or deletions were selected.

Then variants mapping to the candidate regions as determined by linkage analysis were

selected. MAF was updated using dbSNPv138 database and EVS (ESP6500SI-V2).

Integrative genomics viewer (IGV, v1.5.64, 8) was used as visualization tool of WES variants.

In silico prediction of the functional effect at the amino acid level was calculated using

Polyphen-2 software (version 2.2.2, 10).

Real time PCR amplification of genomic DNA

Real time PCR amplification was conducted using genomic DNA on a 7300 Real-Time PCR

system (Applied Biosystems) using Power SYBR Green PCR Master Mix (Applied

Biosystems). Albumin gene was used as internal control. Genomic deletion was defined when

the ratio of tested DNA to control DNA was equal to or less than 0.5. Real time PCR

amplification of each sample was performed in duplicate using primers within selected exons

(Supplementary Material, Table S3).

Reverse Transcription-PCR Amplification

Total RNAs were extracted by using TRI Reagent LS method (Sigma). One µg RNA was

used to synthesize cDNA by using random primers following the manufacturer’s manual

(SuperScript III reverse Transcriptase, Invitrogen) in a final volume of 20 µL. PCR

amplification was carried out using 1.5 mM MgCl2, 0.6 U DNA Polymerase (Invitrogen), 0.2

µM each primer and 1 µl cDNA. After an initial cycle of denaturation at 94°C for 5 min., 30

cycles were performed consisting in denaturation at 94°C for 30 sec., annealing at 60°C for 1

min. and extension at 72°C for 1 min., followed by a final extension for 7 min. at 72°C, in a

ABI9700 Thermal Cycler. RT-PCR products were separated by agarose gel electrophoresis

and labelled with ethidium bromide. To determine the effect of mutations closed to intron-

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exon junctions, PCR amplification analysis from single strand cDNA was performed using

primers flanking exons (Supplementary Material, Table S3). As internal control for PCR

amplification, β-Actin cDNA was coamplified (Supplementary Material, Table S3). Sanger

sequencing was performed from the RT-PCR products.

Sanger sequencing

PCR primer pairs were designed from genomic DNA to amplify and sequence each variant

(Supplementary Material, Table S4). PCR amplification was carried out using 1.5 mM MgCl2,

0.6 U DNA Polymerase, 0.25 µM each primer and 50 ng DNA. After an initial cycle of

denaturation at 94°C for 5 min, 30 cycles were performed consisting in denaturation at 95°C

for 30 sec., annealing at 60°C (+/-3°C) for 1 min. and extension at 72°C for 1min., and final

extension 7 min. at 72°C, on an ABI9700 Thermal Cycler. PCR products were then purified

on P100 columns (Bio-Gel P-2 Gel fine, Biorad) sequenced using the forward or reverse

primers and the Big Dye Terminator V3.1 Cycle sequencing kit (Applied Biosystems). The

sequencing reaction products were purified on G50 columns (Sephadex G-50 Superfine, GE

Healthcare) and then migrated on an automated fluorescent DNA sequencer (ABI Prism 3100

Genetic analyzer, Applied Biosystems). The obtained DNA sequences were compared with

published sequences (BLAST, NCBI). Variants fulfilling prioritization criteria were validated

by Sanger sequencing. Sanger sequencing was also performed to establish the genotype of

each family member, and to analyze the segregation of the variants within each family. A

cohort of 95 ethnically matched controls was analyzed for the selected variants when they

were not known to be pathogenic so far.

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Morphology of the neuromuscular system

Muscle and nerve biopsy samples were processed as previously described (26). Nerve

immunohistochemistry was carried out using phosphorylated neurofilament monoclonal

antibody (1:200), myelin basic protein (MBP, 1:200) and S100 protein polyclonal antibodies

(1:2500, Dakopatts). Muscle and nerve ultrastructural studies were carried out according to

standardized protocols. Briefly, tissue samples were fixed in a 2% glutaraldehyde fixative

solution, post-fixed with osmium tetroxide, and embedded in resin epoxy. Semi-thin sections

were stained with toluidine blue. Ultra-thin sections were contrasted with uranyl acetate and

lead citrate, and examined under a PHILIPS CM10 transmission electron microscope.

Knockdown of ADCY6 in zebrafish

Embryos were cared for according to standard protocols (27). Foxd3::GFP transgenic line was

used here (11). Antisense morpholino oligonucleotides (adcy6 MOs) were purchased from

Gene Tools LLC and designed against the two corresponding orthologous zebrafish genes:

adcy6a and adcy6b. The adcy6a MO was designed to target the 5’ UTR of adcy6a mRNA

(XM_001922714, Supplementary Material, Table S3). The corresponding control “mismatch”

morpholino (Supplementary Material, Table S4) had 5 nucleotides altered along its sequence.

The same applies to adcy6b MO targeting adcy6b mRNA (XM_002666490, Supplementary

Material, Table S4). 0.3mM of each of the two morpholinos respectively designed against the

two genes were mixed (either a mix of the 5’UTR MOs or the controls ones; final

concentration: 0.6 mM). 1 nl of morpholino was injected into 1–4 cell stage embryos as

previously described (28).

In situ hybridization. Embryos were fixed in 4% paraformaldehyde and stained as whole

mounts following standard in situ protocols and using mbp probe.

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Immunohistochemistry. For immunostaining, embryos were fixed in 4% paraformaldehyde

and stained as whole mounts. Anti-acetylated tubulin antibody (Sigma) was used at a 1:1000

dilution. Primary antibodies were detected with appropriate secondary antibodies conjugated

to Alexa 568 (Molecular probes) at a 1:200 dilution.

Confocal image analysis. Image acquisition was performed using a Zeiss confocal microscope

and Zeiss LSM imaging software. Image analysis was performed offline using ImageJ and

Adobe photoshop CS6.

SUPPLEMENTARY MATERIAL

Supplementary Material is available at HMG online.

ACKNOWLEDGMENTS

We thank all families for participating in this study. This work was supported by a grant from

the French Ministry of Health (PHRC 2010, AOM10181), the Association Française contre

les Myopathies (AFM, DAJ1891), Inserm and the Alliance Arthrogrypose to J.M and

sponsored by Assistance Publique- Hôpitaux de Paris. L. F is a recipient of the University

Paris 11 scholarship. K.D. was a recipient of the Fédération pour la Recherche Médicale

scholarship and was supported by a grant from the PHRC. The authors would like to thank the

Biomedical Institute of Bicêtre for providing us with Sanger sequencing facilities, the Clinical

Research Unit of Bicêtre Hospital, the Direction of Clinical Research and Development (AP-

HP) and the NHLBI GO Exome Sequencing Project and its ongoing studies that produced and

provided exome variant calls for comparison.

CONFLICT OF INTEREST STATEMENT

The authors declare no competing financial interests

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LEGENDS TO FIGURES

Figure 1: Percentage of AMC patients with associated symptoms and classified upon the

primary tissue targets (Sk Mu: skeletal muscle, NMJ: neuromuscular junction or axoglial). *

indicates a significant difference (P<0.02) between groups using Fisher’s exact test. (P+D)

AMC indicates that contractures involved both proximal and distal joints.

Figure 2: Sanger sequencing of mutations identified in CNTNAP1 and ADCY6 in AMC

families. Arrows indicate mutant nucleotide positions. Open symbols: unaffected; filled

symbols: affected. The nucleotide and amino acid changes are indicated as well as the

genotype (homozygous) in affected individuals.

Figure 3: Clinical features of arthrogryposis associated with mutations of CNTNAP1.

Note the distal involvement of joint retractions and facial diplegia.

Figure 4: TEM analysis of nerve in AMC fetuses carrying deleterious mutations of

CNTNAP1 (A, C) and ADCY6 (E). (A) Longitudinal section of sciatic nerve in AMC fetus

A641 and control (B) of the same gestational age (32 w.g.). Note the marked widening of the

node of Ranvier in AMC fetus (white arrow). (C) Transverse ultrathin section showing the

absence of large myelinated axons associated with thinner myelin sheaths in the AMC fetus

K182 when compared to control (D) of the same gestational age (34 w.g.). (E) Lack of myelin

surrounding axon (white arrow) while Schwann cell morphology appeared normal (black

arrow) in AMC fetus A649 when compared to control of the same age (F, 40 w.g.). Scale

bars: A-B; E-F: 3.8 µm; C-D: 20µm.

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Figure 5: Adcy6a and b genes are essential for PNS myelination. (A, B, E, F, I, J) mbp

mRNA expression by in situ hybridization in WT (A, B), embryos injected with the two

control 5 mismatched Morpholinos (MOs) (0.3 pmole 5 mis adcy6a MO + 0.3 pmole 5 mis

adcy6b MO per embryo, E, F), and embryos injected with adcy6a and adcy6b MOs (0.3

pmole adcy6a MO + 0.3 pmole adcy6b MO per embryo, I, J), respectively. For convenience,

we used adcy6 MO instead of: adcy6a and b MOs. Note that mbp expression is severely

reduced specifically in the PNS in embryos injected with both adcy6a and b MOs (compare I

to A and E) while its expression is normal and comparable to controls in the CNS (compare J

to B and F). Arrows in A and E indicate mbp expression along the Posterior Lateral Line

nerve (PLLn). Arrows in B, F and J indicate the expression of mbp in the hindbrain and spinal

cord while the arrowheads indicate its expression in the PLL ganglion (PLLg). Note the

absence of mbp expression in the PLLg in J. (C, D, G, H, K, L) Schwann cells and PLLn

labeling using the transgenic line foxd3::GFP and acetylated tubulin (ac Tub) antibody

respectively. Note the presence of Schwann cells and PLLn in the adcy6 morphants as

observed in controls (compare K to C and G; L to D and H). Arrows in C, G and K indicate

Schwann cells along the PLLn and in D, H and L indicate the PLLn. Scale bar: 200 m in A,

B, E, F, I and J; 100 m in C, D, G, H, K and L.

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Table 1: Clinical characteristics of AMC families Code Gene Tissue

Target Onset (w.g.)

AMC Hypo/ akinesia

Poly-Hydramnios

Pterygium Micro-gnathia

Cleft palate

Hygroma

A641 CNTNAP1 Axoglial 30 D Y Y - - - -

K182 CNTNAP1 Axoglial 32 D Y Y - - - -

K199 CNTNAP1 Axoglial 30 D unk Y - - - unk

B207 CNTNAP1 Axoglial 28 D unk Y - Y - unk

A649 ADCY6 Axoglial 32 D Y - - - - -

A635 ECEL1 Axoglial 16 P+D Y - - Y - -

B192 ECEL1 Axoglial uk D - - - - - -

B651 TRPV4 Axoglial 19 P+D - - - - - -

A638 CHRNG NMJ 12 P+D Y - Y Y - Y

A651 CHRNG NMJ 11 P+D Y Y Y Y - -

A662 CHRNG NMJ 17 P+D Y - - - - -

A650 RAPSN NMJ 23 P+D Y Y - Y - -

A640 MYH3 Sk Mu 13 P+D Y - Y Y Y Y

A646 NEB Sk Mu 14 P+D Y - Y Y - Y

A631 NEB Sk Mu 13 P+D Y - Y Y - Y

B415 RYR1 Sk Mu 20 P+D Y Y Y Y - Y

A648 RYR1 Sk Mu 13 P+D Y - Y Y Y Y

A636 RYR1 Sk Mu 15 P+D Y Y Y Y Y Y

A656 RYR1 Sk Mu 20 P+D Y - Y Y Y Y

K166 RYR1 Sk Mu 12 P+D Y - Y Y Y Y

K168 SYNE1 Sk Mu 22 D - - - - - -

A663 TNNT3 Sk Mu 20 D - - - - - -

A642 TTN Sk Mu 26 P+D Y Y - Y - -

A659 RBBP8 Other 24 P+D Y - Y Y Y Y

A658 unk 22 D Y Y - - - Y

K180 unk 21 D Y Y - Y - -

K171 unk 14 P+D Y - Y Y Y Y

K169 unk 35 P+D unk unk Y unk - unk

K174 unk 22 P+D Y - - - -

K165 unk 12 P+D Y - Y Y Y Y

A657 unk 14 P+D Y Y - Y Y -

w.g: weeks of gestation; P+D: proximal and distal; D: distal; Y: yes;(-):No; unk: unknown; NMJ: neuromuscular junction; Sk Mu: skeletal muscle

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Table 2: Identified mutations in genes associated with AMC

Code Inheri tance

Gene Transcript Ref.: Nucleotide change MAF (dbSNP)

MAF (EVS)

Genotype Type Protein change

Target

A641 AR* CNTNAP1 NM_003632:exon18:c.2901_2902del 0 0.00016 homozyg. frameshift P967PfsX12 AxoglialK182 AR* CNTNAP1 NM_003632:exon19:c.3009_3010insT 0 0 homozyg. frameshift F1003fs AxoglialK199 AR* CNTNAP1 NM_003632:exon19:c.3009_3010insT 0 0 homozyg. frameshift F1003fs AxoglialB207 AR* CNTNAP1 NM_003632:intron18-exon19:

c.2993-2_2994del 0 0 homozyg. frameshift I999WfsX5 Axoglial

A649 AR* ADCY6 NM_015270:exon20:c.C3346T 0 0 homozyg. missense (d;1) R1116C AxoglialA635 AR ECEL1 NM_004826:exon4:c.925delA 0 0 comp. het. frameshift K309fs AxoglialA635 AR ECEL1 NM_004826:exon2:c.C33G 0 0 comp. het. stop gained Y11X AxoglialB192 AR* ECEL1 NM_004826:c.1685+1G>T 0 0 homozyg. splice K552AfsX33 AxoglialB651 AD TRPV4 NM_021625:exon6:c.G947A 0 0 heterozyg. missense (p) R316H AxoglialA638 AR* CHRNG NM_005199:exon2:c.117_118insC 0 0 homozyg. frameshift P39fs NMJ A651 AR* CHRNG NM_005199:exon7:c.C715T 0 0 homozyg. missense (p) R239C NMJ A662 AR* CHRNG NM_005199:exon7:c.C715T 0 0 homozyg. missense (p) R239C NMJ A650 AR RAPSN NM_032645:exon2:c.C264A 0.0014 0.001 comp. het. missense (p) N88K NMJ A650 AR RAPSN NM_032645:EX1_2del 0 0 comp. het. deletion Ex1_2del NMJ A640 AD MYH3 NM_002470:exon28:c.T3959C 0 0 heterozyg. missense (d;0.99) L1320P Sk Mu

A646 AR* NEB NM_001164508:exon41: c.4858_4866del 0 0

homozyg. frameshift A1620fs Sk Mu

A631 AR* NEB NM_001164508:c.9832-1G>A 0 0 homozyg. splice Y3278MfsX22 Sk Mu B415 AR RYR1 NM_000540:EX70_71del 0 0 comp. het. deletion EX70_71del Sk Mu B415 AR RYR1 NM_000540:exon8:c.G644A 0 0 comp. het. missense (d;1) G215E Sk Mu A648 AR RYR1 NM_000540:exon46:c.G7373A 0 0 comp. het. missense (p) R2458H Sk Mu A648 AR RYR1 NM_000540:c.14364+1G>A 0 0 comp. het. splice W4768CfsX11 Sk Mu A636 AR RYR1 NM_000540:exon46:c.G7373A 0 0 comp. het. missense (p) R2458H Sk Mu A636 AR RYR1 NM_000540:exon90:c.T12580C 0 0 comp. het. missense (d;0.95) F4194L Sk Mu A656 AR* RYR1 NM_000540:exon26:c.G3449A 0 0 homozyg. missense (d;1) C1150Y Sk Mu

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K166 AR* RYR1 NM_000540:exon57:c.C8758T 0 0 homozyg. stop gained R2920X Sk Mu K168 AR* SYNE1 NM_182961:exon136:c.C24577T 0 0 homozyg. stop gained R8193X Sk Mu A663 AD TNNT3 NM_006757:exon10:c.G188A 0 0 heterozyg. missense (p) R63H Sk Mu

A642 AR TTN NM_133378:exon195: c.37862_37863insA 0 0

comp. het. frameshift Y12621_V12622delinsX

Sk Mu

A642 AR TTN NM_133378:exon307:c.C96388T 0 0 comp. het. stop gained R32130X Sk Mu

A659 AR* RBBP8 NM_002894:c.2455-4T>G 0 0.00008 homozyg. splice Y819VfsX33 Other

AR: autosomal recessive; AD: autosomal dominant; *: consanguinity; SNV Chr. Pos. : Chromosome and genomic position; homozyg.: homozygous; comp. het.: compound heterozygous; heterozyg.: heterozygous ; Type: (p)=known pathogenic mutation, (d)= damaging mutation as determined by Polyphen-2 prediction with score; NMJ: neuromuscular junction; Sk Mu: skeletal muscle. Mutations were confirmed by Sanger sequencing or real time PCR

(Supplementary Material, Figure S3) and splice mutations by RNA analysis (Supplementary Material, Figure S4).

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Table 3 : Clinical characteritics of patients carrying CNTNAP1 or ADCY6 mutations

A641 K182 K199 B207 A649

Pregnancy

Age of discovery (w.g.) 30 31 30 28 32

Distal AMC Y Y Y Y Y

Polyhydramnios Y Y Y Y -

Birth

Hypotonia Y Y Y Y Y

Respiratory distress Y Y Y Y Y

Facial diplegia Y unk Y Y Y

Swallowing defect Y Y Y Y Y

Areflexia Y Y Y Y Y

Distal AMC Y Y Y Y Y

Motor NCV (m/Sec.) 7 10 7 7.8 no response

Age of death (postnatal days) 10 33 40 10 80

w.g.: week of gestation; Y: yes; (-): no; unk: unknown.

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ABBREVIATIONS

AMC: arthrogryposis multiplex congenita

TEM: transmission electron microscopy

PNS: peripheral nervous system

cAMP: cyclic adenosine monophosphate

WES: whole exome sequencing

NMJ : neuromuscular junction

NMD: neuromuscular disorders

NGS: next generation sequencing

BWA: Burrows-Wheeler Alignment

MAF: minor allele frequency

EVS: Exome Variant Server

IGV: Integrative genomics viewer

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* * *

0%

20%

40%

60%

80%

100%

120%

Sk Mu (n=12)

NMJ (n=4)

Axoglial (n=8)

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A641: CNTNAP1: c.2901_2902del: p.967_968del; homozygous

K182: CNTNAP1: c.3009_3010insT: F1003fs; homozygous

K199 : CNTNAP1: c.3009_3010insT: F1003fs; homozygous

A649: ADCY6: c.C3346T: R1116C; homozygous

B207: CNTNAP1: intron18-exon19: c.2993-2_2994del: I999WfsX5; homozygous

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K182

K199

B207

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