Upload
independent
View
0
Download
0
Embed Size (px)
Citation preview
1
© 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
HMG Advance Access published December 6, 2013 by guest on D
ecember 7, 2013
http://hmg.oxfordjournals.org/
Dow
nloaded from
2
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
by guest on Decem
ber 7, 2013http://hm
g.oxfordjournals.org/D
ownloaded from
3
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
by guest on Decem
ber 7, 2013http://hm
g.oxfordjournals.org/D
ownloaded from
4
*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
by guest on Decem
ber 7, 2013http://hm
g.oxfordjournals.org/D
ownloaded from
5
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.
by guest on Decem
ber 7, 2013http://hm
g.oxfordjournals.org/D
ownloaded from
6
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.
by guest on Decem
ber 7, 2013http://hm
g.oxfordjournals.org/D
ownloaded from
7
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
by guest on Decem
ber 7, 2013http://hm
g.oxfordjournals.org/D
ownloaded from
8
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
by guest on Decem
ber 7, 2013http://hm
g.oxfordjournals.org/D
ownloaded from
9
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
by guest on Decem
ber 7, 2013http://hm
g.oxfordjournals.org/D
ownloaded from
10
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).
by guest on Decem
ber 7, 2013http://hm
g.oxfordjournals.org/D
ownloaded from
11
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.
by guest on Decem
ber 7, 2013http://hm
g.oxfordjournals.org/D
ownloaded from
12
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
by guest on Decem
ber 7, 2013http://hm
g.oxfordjournals.org/D
ownloaded from
13
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
by guest on Decem
ber 7, 2013http://hm
g.oxfordjournals.org/D
ownloaded from
14
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
by guest on Decem
ber 7, 2013http://hm
g.oxfordjournals.org/D
ownloaded from
15
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-
by guest on Decem
ber 7, 2013http://hm
g.oxfordjournals.org/D
ownloaded from
16
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.
by guest on Decem
ber 7, 2013http://hm
g.oxfordjournals.org/D
ownloaded from
17
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.
by guest on Decem
ber 7, 2013http://hm
g.oxfordjournals.org/D
ownloaded from
18
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
by guest on Decem
ber 7, 2013http://hm
g.oxfordjournals.org/D
ownloaded from
19
REFERENCES
1. Hall, J.G. Genetic aspects of arthrogryposis. (1985) Clin. Orthop., 194, 44-53.
2. Fahy, M.J. and Hall, J.G. (1990). A retrospective study of pregnancy complications among
828 cases of arthrogryposis. Genet. Couns., 1, 3-11.
3. Bamshad, M., Van Heest, A.E. and Pleasure, D. (2009) Arthrogryposis: a review and
update. J. Bone Joint Surg. Am., 91, 40-46.
4. Navti, O.B., Kinning, E., Vasudevan, P., Barrow, M., Porter, H., Howarth, E., Konje, J. and
Khare, M. (2010) Review of perinatal management of arthrogryposis at a large UK
teaching hospital serving a multiethnic population. Prenat. Diagn., 30, 49-56.
5. Dieterich, K., Quijano-Roy, S., Monnier, N., Zhou, J., Fauré, J., Smirnow, D.A., Carlier,
R., Laroche, C., Marcorelles, P., Mercier, S. et al. (2013) The neuronal endopeptidase
ECEL1 is associated with a distinct form of recessive distal arthrogryposis. Hum. Mol.
Genet., 22, 1483-1492.
6. Attali, R., Warwar, N., Israel, A., Gurt, I., McNally, E., Puckelwartz, M., Glick, B., Nevo,
Y., Ben-Neriah, Z. and Melki, J. (2009) Mutation of SYNE-1, encoding an essential
component of the nuclear lamina, is responsible for autosomal recessive arthrogryposis.
Hum. Mol. Genet., 18, 3462-3469.
7. Sarici, D., Akin, M.A., Kara, A., Doganay, S. and Kurtoglu, S. (2012) Seckel syndrome
accompanied by semilobar holoprosencephaly and arthrogryposis. Pediatr. Neurol., 46,
189-191.
8. Robinson, J.T., Thorvaldsdóttir, H., Winckler, W., Guttman, M., Lander, E.S., Getz, G. and
Mesirov, J.P. (2011) Integrative Genomics Viewer. Nat. Biotechnology, 29, 24-26.
9. Bhat, M.A., Rios, J.C., Lu, Y., Garcia-Fresco, G.P., Ching, W., St Martin, M., Li, J.,
Einheber, S., Chesler, M., Rosenbluth, J. et al. (2001) Axon-glia interactions and the
by guest on Decem
ber 7, 2013http://hm
g.oxfordjournals.org/D
ownloaded from
20
domain organization of myelinated axons requires neurexin IV/Caspr/Paranodin. Neuron,
30, 369-383.
10. Adzhubei, I.A., Schmidt, S., Peshkin, L., Ramensky, V.E., Gerasimova, A., Bork, P.,
Kondrashov, A.S., and Sunyaev S.R. (2010) A method and server for predicting damaging
missense mutations. Nat. Methods, 7, 248-249.
11. Gilmour, D.T., Maischein, H.M. and Nüsslein-Volhard, C. (2002) Migration and function
of a glial subtype in the vertebrate peripheral nervous system. Neuron, 34, 577-588
12. Peles, E., Nativ, M., Lustig, M., Grumet, M., Schilling, J., Martinez, R., Plowman, G.D.
and Schlessinger, J. (1997) Identification of a novel contactin-associated transmembrane
receptor with multiple domains implicated in protein-protein interactions. EMBO J., 16,
978-988.
13. Rios, J.C., Melendez-Vasquez, C.V., Einheber, S., Lustig, M., Grumet, M., Hemperly, J.,
Peles, E. and Salzer, J.L. (2000) Contactin-associated protein (Caspr) and contactin form a
complex that is targeted to the paranodal junctions during myelination. J. Neurosci., 20,
8354-8364.
14. Tait, S., Gunn-Moore, F., Collinson, J.M., Huang, J., Lubetzki, C., Pedraza, L., Sherman,
D.L., Colman, D.R. and Brophy, P.J. (2000) An oligodendrocyte cell adhesion molecule at
the site of assembly of the paranodal axo-glial junction. J. Cell Biol., 150, 657-666.
15. Edelhoff, S., Villacres, E.C., Storm, D.R. and Disteche, C.M. (1995) Mapping of adenylyl
cyclase genes type I, II, III, IV, V, and VI in mouse. Mamm. Genome, 6, 111-113.
16. Morgan, L., Jessen, K.R. and Mirsky, R. (1991) The effects of cAMP on differentiation of
cultured Schwann cells: progression from an early phenotype (04+) to a myelin phenotype
(P0+, GFAP-, N-CAM-, NGF-receptor-) depends on growth inhibition. J. Cell Biol., 112,
457-467.
by guest on Decem
ber 7, 2013http://hm
g.oxfordjournals.org/D
ownloaded from
21
17. Monuki, E.S., Weinmaster, G., Kuhn, R. and Lemke, G. (1989) SCIP: a glial POU domain
gene regulated by cyclic AMP. Neuron, 3, 783-793.
18. Scherer, S.S., Wang, D.Y., Kuhn, R., Lemke, G., Wrabetz, L. and Kamholz, J. (1994)
Axons regulate Schwann cell expression of the POU transcription factor SCIP. J.
Neurosci., 14, 1930-1942.
19. Monk, K.R., Naylor, S.G., Glenn, T.D., Mercurio, S., Perlin, J.R., Dominguez, C., Moens,
C.B. and Talbot, W.S. (2009) A G protein-coupled receptor is essential for Schwann cells
to initiate myelination. Science, 325, 1402-1405.
20. Ruschendorf, F. and Nurnberg, P. (2005) ALOHOMORA: a tool for linkage analysis
using 10K SNP array data. Bioinformatics, 21, 2123-2125.
21. Abecasis, G.R., Cherny, S.S., Cookson, W.O. and Cardon, L.R. (2002) Merlin-rapid
analysis of dense genetic maps using sparse gene flow trees. Nat. Genet., 30, 97-101.
22. Zhou, J., Tawk, M., Tiziano, F.D., Veillet, J., Bayes, M., Nolent, F., Garcia, V., Servidei,
S., Bertini, E., Castro-Giner, F. et al. (2012) Spinal muscular atrophy associated with
progressive myoclonic epilepsy is caused by mutations in ASAH1. Am. J. Hum. Genet., 91,
5-14.
23. Li, H. and Durbin, R. (2009) Fast and accurate short read alignment with Burrows-
Wheeler transform. Bioinformatics, 25, 1754-1760 (a).
24. Li, H., Handsaker, B., Wysoker, A., Fennell, T., Ruan, J., Homer, N., Marth, G., Abecasis,
G. and Durbin, R.; 1000 Genome Project Data Processing Subgroup. (2009) The Sequence
Alignment/Map format and SAMtools. Bioinformatics, 25, 2078-2079 (b).
25. Wang, K., Li, M. and Hakonarson, H. (2010) ANNOVAR: functional annotation of
genetic variants from high-throughput sequencing data. Nucleic Acids Res., 38, e164.
26. Dubowitz, V., Sewry, C.A. and Fitzsimons, R (1985). Muscle biopsy: a practical
approach. 2nd ed. London. Philadelphia: Baillière Tindall.
by guest on Decem
ber 7, 2013http://hm
g.oxfordjournals.org/D
ownloaded from
22
27. Westerfield, M. (1995) The zebrafish book. A guide for the laboratory use of zebrafish
(Danio rerio). EugeneOR, University of Oregon Press.
28. Nasevicius, A. and Ekker, S.C. (2000) Effective targeted gene ‘knockdown’ in zebrafish.
Nat. Genet. 26, 216-220.
by guest on Decem
ber 7, 2013http://hm
g.oxfordjournals.org/D
ownloaded from
23
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.
by guest on Decem
ber 7, 2013http://hm
g.oxfordjournals.org/D
ownloaded from
24
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.
by guest on Decem
ber 7, 2013http://hm
g.oxfordjournals.org/D
ownloaded from
25
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
by guest on Decem
ber 7, 2013http://hm
g.oxfordjournals.org/D
ownloaded from
26
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
by guest on December 7, 2013 http://hmg.oxfordjournals.org/ Downloaded from
27
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).
by guest on December 7, 2013 http://hmg.oxfordjournals.org/ Downloaded from
28
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.
by guest on Decem
ber 7, 2013http://hm
g.oxfordjournals.org/D
ownloaded from
29
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
by guest on Decem
ber 7, 2013http://hm
g.oxfordjournals.org/D
ownloaded from
* * *
0%
20%
40%
60%
80%
100%
120%
Sk Mu (n=12)
NMJ (n=4)
Axoglial (n=8)
by guest on Decem
ber 7, 2013http://hm
g.oxfordjournals.org/D
ownloaded from
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
by guest on Decem
ber 7, 2013http://hm
g.oxfordjournals.org/D
ownloaded from
K182
K199
B207
by guest on Decem
ber 7, 2013http://hm
g.oxfordjournals.org/D
ownloaded from
by guest on Decem
ber 7, 2013http://hm
g.oxfordjournals.org/D
ownloaded from
by guest on Decem
ber 7, 2013http://hm
g.oxfordjournals.org/D
ownloaded from