Loss-of-function de novo mutations play an important role in severe human neural tube defects

Abstract

Neural tube defects (NTDs) are very common and severe birth defects that are caused by failure of neural tube closure and that have a complex aetiology. Anencephaly and spina bifida are severe NTDs that affect reproductive fitness and suggest a role for de novo mutations (DNMs) in their aetiology.
We used whole-exome sequencing in 43 sporadic cases affected with myelomeningocele or anencephaly and their unaffected parents to identify DNMs in their exomes.
We identified 42 coding DNMs in 25 cases, of which 6 were loss of function (LoF) showing a higher rate of LoF DNM in our cohort compared with control cohorts. Notably, we identified two protein-truncating DNMs in two independent cases in SHROOM3, previously associated with NTDs only in animal models. We have demonstrated a significant enrichment of LoF DNMs in this gene in NTDs compared with the gene specific DNM rate and to the DNM rate estimated from control cohorts. We also identified one nonsense DNM in PAX3 and two potentially causative missense DNMs in GRHL3 and PTPRS.
Our study demonstrates an important role of LoF DNMs in the development of NTDs and strongly implicates SHROOM3 in its aetiology.
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Full text

SHORT REPORT
Loss-of-function de novo mutations play an
important role in severe human neural tube defects
Philippe Lemay,
1
Marie-Claude Guyot,
1
Élizabeth Tremblay,
1
Alexandre Dionne-Laporte,
2
Dan Spiegelman,
2
Édouard Henrion,
2
Ousmane Diallo,
2
Patrizia De Marco,
3
Elisa Merello,
3
Christine Massicotte,
1
Valérie Désilets,
1
Jacques L Michaud,
1
Guy A Rouleau,
2
Valeria Capra,
3
Zoha Kibar
1
▸Additional material is
published online only. To view
please visit the journal online
(http://dx.doi.org/10.1136/
jmedgenet-2015-103027).
1
CHU Ste-Justine Research
Center, Université de Montréal,
Montréal, Québec, Canada
2
Montreal Neurological
Institute, McGill University,
Montréal, Québec, Canada
3
Istituto Giannina Gaslini,
Genoa, Italy
Correspondence to
Dr Zoha Kibar, Department of
Neurosciences, University of
Montréal, CHU Sainte Justine
Research Center, 3175 Cote-
Ste-Catherine, Room 5999C,
Montreal, Québec,
Canada H3T 1C5;
zoha.kibar@recherche-ste-
justine.qc.ca
Received 16 January 2015
Revised 26 February 2015
Accepted 4 March 2015
Published Online First
24 March 2015
To cite: Lemay P,
Guyot M-C, Tremblay É,
et al.J Med Genet
2015;52:493–497.
ABSTRACT
Background Neural tube defects (NTDs) are very
common and severe birth defects that are caused by
failure of neural tube closure and that have a complex
aetiology. Anencephaly and spina bifida are severe NTDs
that affect reproductive fitness and suggest a role for de
novo mutations (DNMs) in their aetiology.
Methods We used whole-exome sequencing in 43
sporadic cases affected with myelomeningocele or
anencephaly and their unaffected parents to identify
DNMs in their exomes.
Results We identified 42 coding DNMs in 25 cases, of
which 6 were loss of function (LoF) showing a higher
rate of LoF DNM in our cohort compared with control
cohorts. Notably, we identified two protein-truncating
DNMs in two independent cases in SHROOM3,
previously associated with NTDs only in animal models.
We have demonstrated a significant enrichment of LoF
DNMs in this gene in NTDs compared with the gene
specific DNM rate and to the DNM rate estimated from
control cohorts. We also identified one nonsense DNM
in PAX3 and two potentially causative missense DNMs
in GRHL3 and PTPRS.
Conclusions Our study demonstrates an important role
of LoF DNMs in the development of NTDs and strongly
implicates SHROOM3 in its aetiology.
Neural tube defects (NTDs) are a group of con-
genital malformations affecting 1–2 individuals per
1000 births.
1
They are caused by an incomplete
closure of the neural tube during embryogenesis.
1
The most frequent forms of NTDs are anencephaly
and myelomeningocele (MMC) (or spina bifida),
which are caused by a closure defect in the brain
and the spinal cord region, respectively.
1
Children
affected with anencephaly die early in development
or soon after birth, while children affected with
MMC survive but are affected by developmental
physical defects with varying degrees of severity.
Most cases of NTDs are sporadic and non-
syndromic.
2
Periconceptional folic acid intake has
been shown to reduce prevalence of NTDs by 50–
70%,
3
but a large amount of cases remain resistant
to this preventive treatment urging the need for
identification of other causative factors and devel-
opment of novel preventive and counselling
strategies.
NTDs have a strong genetic component with an
estimated heritability of 60%,
1
but so far the
genetics of the disease remains largely unknown.
Previous linkage studies in NTDs have identified
candidate regions on chromosomes 2, 7 and 10 but
failed to identify any causative NTD gene.
4
Few
common variants in folic acid-related genes have
also been shown to be associated with NTDs in
certain populations, but these variants seem to con-
tribute only to a small part of the aetiology of the
disease.
5
Previous gene identification studies in
NTDs have mainly adopted a candidate gene
approach and focused on folate-related genes and
on candidate genes from animal studies.
125
Animal models have demonstrated an important
role of the planar cell polarity pathway in the aeti-
ology of NTDs.
1
Subsequent investigation of genes
of this pathway, including VANGL1 and VANGL2,
in human NTDs has implicated them as risk factors
in a small fraction of patients.
1
Generally, candidate
gene studies in NTDs have faced limited success in
identifying major causative genes predisposing to
NTDs, suggesting the need for novel approaches.
Several recent studies strongly suggest that de novo
mutations (DNMs) represent a common cause of
birth defects and neurodevelopmental diseases.
6
DNM could provide a mechanism by which
early-onset reproductively lethal diseases remain
frequent in the population. Therefore, these var-
iants are strong candidates for causing diseases that
occur sporadically and that have a reduced repro-
ductive fitness.
6
Severe forms of NTDs, such as
anencephaly and MMC, fall in this category and
hence investigation of DNMs may therefore
increase the chance of identifying loss of function
(LoF) DNMs implicated in NTDs.
Forty-three families each composed of one affected
child and two unaffected parents with no family
history of NTDs were recruited through the Montreal
Ste-Justine Hospital Spina Bifida Center, the 3D study
of the Integrated Research Network in Perinatology
of Quebec and Eastern Ontario and the Istituto
Giannina Gaslini in Genoa, Italy. Detailed informa-
tion including folate status, tissue of origin and type
of NTDs of this cohort is summarised in online sup-
plementary table S1. Briefly, all 43 cases were affected
with NTDs including 35 MMC and 8 anencephaly
cases. A total of 21 cases were fetuses and 55.6%
took folate periconceptionally. Tissues from fetuses
were all obtained following induced abortions.
The average maternal and paternal ages were 30.0
±4.8 years and 30.7±5.9 years, respectively.
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Exome data from 43 NTD trios were aligned to the GRCh37
genome reference and called variants were filtered for minimal
quality (genotype quality (GQ) >20, total variant reads >3,
total reads >7) and for absence in parents and public databases
(1000 genome at http://www.1000genomes.org/home and
Exome Variant Server at http://evs.gs.washington.edu/EVS/) to
identify DNMs. Details of the alignment strategy and single-
nucleotide variant (SNV) annotation are included in online sup-
plementary data. Seventy variants were identified and validated
by Sanger sequencing to minimise false negative results. This
approach resulted in the identification of 42 coding mutations
in 25 trios including 3 nonsense, 3 frameshift, 30 missense and
6 synonymous (table 1). The average DNM rate per base pair
was 1.62×10
−8
, which was consistent with published ratios.
78
We identified six LoF DNMs defined as nonsense, frameshift
and splicing variants, in six NTD trios resulting in a per base
DNMs rate of 0.23×10
−8
. This rate was higher than two previ-
ously published per base LoF DNMs rates, 0.05×10
−8
(ref. 8)
and 0.17×10
−8
(ref. 9) but only reached significance (p=0.002)
when compared to the lower rate, suggesting that larger cohorts
Table 1 De novo mutations identified in NTD-affected trios and confirmed by Sanger sequencing.
Family NTD type Chr Position Genes cDNA change Amino acid change Polyphen HDIV*
Loss-of-function de novo mutations (nonsense, frameshift, splicing)
Pr394 MMC 4 77 662 169 SHROOM3 c.2843_2844insG p.L948fs NA
PrZRV Anen 4 77 660 502 SHROOM3 c.1176C>G p.Y392X NA
Pr134 MMC 2 223 161 800 PAX3 c.218C>A p.S73X NA
Pr201 MMC 15 44 116 692 MFAP1 c.69del7 p.K23fs NA
Pr389 MMC X 41 202 544 DDX3X c.620dupA p.Q207fs NA
PrYPT MMC 7 73 279 630 WBSCR28 c.380G>A p.W127X NA
Missense and synonymous de novo mutations
Pr548 MMC 1 24 668 728 GRHL3 c.1171C>T p.R391C 1
Pr548 MMC 19 39 798 985 LRFN1 c.1604C>T p.T535I 1
Pr548 MMC 9 124 751 686 TTLL11 c.1327A>G p.N443D 0.968
Pr125 MMC 19 5 214 591 PTPRS†c.4475G>A p.R1049Q 0.998
Pr125 MMC 2 27 435 209 ATRAID c.138G>A p.A46A NA
Pr122 MMC 12 123 341 629 HIP1R c.1682G>T p.G561V 0.019
Pr122 MMC 17 71 232 301 C17orf80 c.1441C>T p.R481W 0.099
Pr122 MMC X 53 592 096 HUWE1 c.6812G>A p.S2271N 0
Pr134 MMC 14 105 179 874 INF2†c.2971C>T p.R991W 1
Pr191 MMC 8 10 464 772 RP1L1†c.6836C>T p.P2279L 0.053
Pr191 MMC 20 62 371 335 SLC2A4RG c.70C>T p.R24C 0.426
Pr20 MMC 3 38 317 786 SLC22A13 c.1246G>A p.V416M 0.948
Pr20 MMC 16 31 383 022 ITGAX c.2077C>G p.Q693E 0.001
Pr201 MMC 2 47 703 654 MSH2†c.1956A>G p.Q652Q NA
Pr202 MMC 6 30 122 164 TRIM10 c.1028A>T p.D343V 1
Pr202 MMC 7 23 775 208 STK31 c.535A>C p.I179L 0.039
Pr25 MMC 9 134 183 554 PPAPDC3†c.696C>T p.I232I NA
Pr263 MMC 6 1 390 351 FOXF2 c.169G>A p.A57T 0.01
Pr28 MMC 8 124 333 387 ATAD2 c.4160G>A p.S1387N 0.001
Pr282 MMC 19 808 439 PTBP1 c.1233C>A p.N411K 0.013
Pr389 MMC 7 100 285 176 GIGYF1 c.325C>T p.P109S 0.728
Pr402 MMC 17 19 319 353 RNF112 c.1761C>T p.A587A NA
Pr402 MMC 3 49 775 724 IP6K1 c.355C>T p.R119C 1
Pr402 MMC 4 48 424 093 SLAIN2 c.1745G>C p.X582S NA
Pr530 MMC 3 57 616 163 DENND6A c.1605A>C p.E535D 0.997
Pr551 MMC 9 130 279 261 FAM129B c. 848C>T p.A283V 0.004
Pr553 MMC 19 39 329 153 HNRNPL c.1441C>T p.R481W 1
Pr554 Anen 3 142 741 447 U2SURP c.961G>A p.G321S 1
Pr554 Anen 5 93 966 388 ANKRD32 c.371T>C p.F124S 0.999
Pr67 MMC 2 44 566 318 PREPL c.937C>G p.L313V 1
Pr67 MMC 5 176 314 262 HK3 c.1677G>A p.V559V NA
Pr67 MMC 9 33 264 606 BAG1 c.67G>A p.A23T 0.897
PrKKS MMC 15 43 621 819 LCMT2†c.869T>C p.I290T 0.201
PrKKS MMC 7 11 076 097 PHF14 c.1655G>C p.R552P 0.998
PrTVB MMC 22 41 558 745 EP300†c.3690A>G p.Q1230Q NA
PrVWA MMC 9 120 475 791 TLR4 c.1385C>T p.A462V 0.006
*Probably damaging (polyphen HDIV ≥0.957), possibly damaging (0.453 ≤polyphen HDIV ≤0.956); benign (polyphen HDIV ≤0.452).
†Mutation previously reported in the ExAC database (http://exac.broadinstitute.org/). Reported mutations frequencies are PTPRS (frequency: 0.000008368); INF2 ( frequency:
0.000009451); RP1L1 (frequency: 0.0001411); MSH2 ( frequency: 0.0001730), PPAPDC3 (frequency: 00003366); LCMT2 ( frequency: 0.000008281); EP300 (frequency: 0.0000082).
Anen, anencephaly; MMC, myelomeningocele; NA, non-applicable; NTD, neural tube defect.
494 Lemay P, et al.J Med Genet 2015;52:493–497. doi:10.1136/jmedgenet-2015-103027
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of MMC and anencephaly are needed to confirm these initial
findings. This increased rate suggests that LoF DNMs are an
important part of the pathogenicity of NTDs. Details of all stat-
istical analysis can be found in online supplementary data.
Notably, we identified two LoF DNMs in SHROOM3
(NM_020859.3) in two unrelated families: one nonsense
variant c.1176C>G (p.Y392X) and one frameshift variant
c.2843_2844insG leading to a premature stop codon (p.L948fs)
(figure 1B). The c.1176C>G variant was detected in the PrZRV
proband affected with anencephaly with cranial fossa agenesis
and facial dysmorphism (table 1). The mother took folic acid
periconceptionally (see online supplementary table S1). The
c.2843_2844insG variant was detected in the Pr394 proband
affected with a thoracic MMC and Chiari type IV malformation
(table 1). The mother did not take folic acid periconceptionally
(see online supplementary table S1). None of these mutations
were reported in the ExAC database (http://exac.broadinstitute.
org/). SHROOM3 is an actin-binding protein that is known to
be a key regulator of apical constriction, a process by which
cells convert their shape from cuboidal to wedge-like due to a
decrease in their apical area.
9
This function is essential for hin-
gepoint formation and bending of the neural tube during its for-
mation and closure in both vertebrate embryos.
9
Recessive
mutations in SHROOM3 were previously associated to
heterotaxy, which represents a multiple congenital anomaly syn-
drome resulting from abnormalities of the proper specification
of left-right asymmetry during embryonic development.
10
The
LoF DNM in this gene described in our study was detected in
two patients with NTD who showed no sign of heterotaxy, and
hence these NTD mutations might act in a haploinsufficient or a
dominant negative manner. Shroom3 exists in two isoforms that
were demonstrated to have similar functions and expression pat-
terns in the mouse and frog models.
11 12
In the mouse gene
trap mutant, both isoforms are knocked out, leading to exence-
phaly and spina bifida in homozygous embryos at a penetrance
of 100% and 23%, respectively.
11
In our cohort, both truncat-
ing DNMs detected in SHROOM3 seem to affect both isoforms
since they map at positions 1176 and 2843 bp respectively 50or
inside the ASD1 domain (figure 1A). These two DNMs clearly
remove important functional domains and might confer
nonsense-mediated RNA decay and hence they are most likely
LoF mutations. Furthermore, previous studies have demon-
strated the potential of a truncated version of Shroom3 to act in
a dominant negative manner in Xenopus.
12
This supports the
potential pathogenicity of these heterozygous mutations that
could result in limited apical constriction causing the NTD.
The phenotypic variation between the two probands (MMC
and anencephaly) who carry LoF DNM in SHROOM3 could be
Figure 1 De novo mutations (DNMs) identified in SHROOM3 and in PAX3 in trios affected with neural tube defects (NTDs). (A) Protein schematic
representation of the long and predicted short isoforms of SHROOM3 indicating the position of the two DNMs identified in NTD probands.
(B) Chromatograms of the three individuals of family PrZRV and Pr394 ( from top to bottom: proband, mother, father) surrounding the SHROOM3
c.1176C>G nonsense variant and the c.2843_2844insG frameshift variant. (C) Protein schematic representation of the long and predicted short
isoforms of PAX3 indicating the position of the DNM identified in NTD probands. (D) Chromatograms of the three individuals of family Pr134 (from
top to bottom: proband, mother, father) surrounding the PAX3 c.218C>A nonsense variant. HOX, paired-type homeodomain; PAX, paired box
domain.
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caused by many factors including genetic modifiers and environ-
mental factors. The two variants might also have different
pathogenic effects on the protein with different outcomes in the
implicated developmental pathway. These factors could act
alone or in combination to affect the clinical expressivity of the
NTD phenotype.
Considering the estimated LoF DNM rates between
0.05×10
−8
and 0.17×10
−8
SNV per base
78
and the length of
SHROOM3 long isoform (NM_020859.3: 5991 bp), we expect
to find respectively between 0.00026 and 0.00088 LoF DNM
in this gene in our cohort. However, we detected two DNMs in
our cohort of 43 trios, resulting in a frequency of DNM per bp
of 3.88×10
−6
. This represents a significant enrichment of LoF
DNMs in SHROOM3 under the two rates with respective
Bonferroni corrected p values of 6.77×10
−4
and 0.008 using a
two-tailed binomial exact test. A recently published gene-specific
per trio mutation rate for LoF DNMs was also used to test the
significant enrichment of LoF DNMs in SHROOM3.
13
Under
the SHROOM3 gene-specific rate of 1.34×10
−5
, we expected to
find 0.00058 LoF DNMs in out cohort, which was significantly
lower than our observed rate (p Bonferroni=0.003). Details of
all statistical analyses can be found in online supplementary
data.
Also importantly we detected one de novo stopgain in the
NTD-associated gene PAX 3 , c.218C>A ( p.S73X), in the Pr134
case affected with MMC with type II Arnold–Chiari malforma-
tion, hydrocephalus and Waardenburg syndrome (WS)
(figure 1D). The mother did not take folic acid or multivitamins
periconceptionally (see online supplementary table S1). This
mutation was not reported in the ExAC database. PAX3 is a
paired box transcription factor that is a key developmental regu-
lator of the neural crest and its derivatives and that plays an
important role during neurogenesis and myogenesis. It contains
two DNA binding domains, the paired box domain (PAX) and a
paired-type homeodomain (HOX) that interact cooperatively
for DNA binding.
14
The putative truncated protein caused by
the DNM p.S73X lacks all functional domains including both
PAX and HOX (figure 1C) and might confer nonsense-mediated
RNA decay.
In the Splotch mouse model, homozygous LoF mutations of
Pax3 cause spina bifida and other neural crest abnormalities. In
humans, the relative contribution of PAX3 to the overall burden
of NTDs remains unclear.
14
Heterozygous mutations in PAX3
are known to cause WS, an autosomal-dominant condition that
affects neural crest-derived structures and that is occasionally
associated with NTDs. Few individuals with both WS and
NTDs were demonstrated to carry heterozygous PAX3 muta-
tions or deletions.
14
Our report of a new protein truncating
SNV in PAX3 in one patient with NTD provides additional evi-
dence for a pathogenic role of this gene in spina bifida.
While SHROOM3 and PAX3 are by far the strongest candi-
date genes identified in this study, other interesting DNMs have
been identified in our cohort (table 1). We detected three LoF
DNMs, p.W127X in WBSCR28, p.K23fs in MFAP1 and p.
Q207fs in DDX3X, with no previous association to NTDs and
that were not reported in the ExAC database (table 1, see online
supplementary figure S1). The WBSCR28 DNM was detected in
the PrYPT proband affected with lumbosacral MMC and type II
Arnold–Chiari malformation. This gene encodes a putative
transmembrane protein of unknown function that maps to the
region deleted in the Williams–Beuren syndrome (table 1). This
syndrome is characterised by a range of phenotypes including
mental retardation, dysmorphic facies, heart abnormalities,
short stature and infantile hypocalcaemia. WBSCR28 is not the
main candidate gene in this disease.
15
The MFAP1 DNM was
found in the Pr201 proband affected with lumbosacral MMC.
This gene encodes the microfibrillar-associated protein 1 that
represents an uncharacterised protein found in some human
spliceosomal fractions.
16
The DDX3X DNM was found in the
Pr389 proband affected with lumbosacral MMC and hydro-
cephalus. This gene belongs to the DEAD-box proteins, a large
family of ATP-dependent RNA helicases that participate in all
aspects of RNA metabolism.
17
While none of these three genes
represent strong candidates for NTDs based on published data,
the presence of LoF DNMs in these genes still suggests them as
potentially interesting NTD candidates. Additional genetic
studies in larger cohorts and functional studies are needed to
validate their role in NTDs.
Two other interesting missense DNM identified in two NTD
trios implicated genes whose orthologues cause NTD in mice:
c.1171C>T (p.R391C) in GRHL3 (GRAINYHEAD-LIKE 3)
and c.4475G>A (p.R1492Q) in PTPRS (PROTEIN TYROSINE
PHOSPHATASE, RECEPTOR TYPE, S) (see online
supplementary figure S2). The GRHL3 mutation was identified
in the Pr548 proband affected with MMC with type II Arnold–
Chiari malformation and hydromyelia. This mutation was not
previously reported in the ExAC database. Grhl3 is a transcrip-
tion factor that plays an important role in epidermal integrity
and wound healing. Importantly, null alleles at this gene caused
mainly severe spina bifida and occasionally exencephaly. Grhl3
was tightly linked to Curly tail that represents one of the most
well-established mouse models for NTDs.
18
The p.R391C maps
to the DNA binding domain of the GHRL3 protein. The modi-
fied amino acid is highly conserved (see online supplementary
figure S2), and the mutation is defined as probably damaging by
PolyPhen-2 HDIV (table 1). Surprisingly, the same DNM p.
R391C was detected in Van der Woude syndrome, the most
common syndromic form of cleft lip and palate.
19
While this
could point towards a chance finding, we hypothesise that add-
itional genetic and/or environmental factors may modify the
phenotypic expression of the same GRHL3 mutation in differ-
ent individuals. The p.R1492Q in PTPRS was found in the
Pr125 proband affected with lumbosacral MMC with type II
Arnold–Chiari malformation. The mutation was previously
reported on one allele in the ExAC database (http://exac.
broadinstitute.org/), resulting in a frequency of 0.000008368.
This gene is a phosphatase involved in regulating cell prolifer-
ation, cell adhesion and nervous system maturation and causes
exencephaly in mice.
20
PTPRS fibronectin and Ig-like domains
are extracellular and mainly involved in cell interaction, while
the catalytic phosphatase domain is intracellular and mainly
involved in signal transmission. The p.R1049Q variant was
identified in a proband affected with MMC (table 1) and resides
in a conserved region that forms part of the phosphatase
domain (see online supplementary figure S2). It modifies a con-
served arginine to a glutamine, a non-conservative substitution
that removes the positive charge of the amino acid side chain,
changes its size and is predicted to be probably damaging by
PolyPhen-2 (table 1).
Molecular genetic studies of bigger cohorts and careful phe-
notyping are still needed to better understand the mechanism of
action of potential DNMs detected in this study and to assess
their role in the pathogenicity in NTDs. While these DNMs are
most likely highly penetrant, it is possible that they act in
concert with other events to cause the disease. This is consistent
with the two-hit model that was initially proposed as a cancer
mechanism and later suggested as a potential mechanism in
complex diseases.
21
This model was more recently explored in
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explaining variable expressivity in severe developmental
genomic disorders.
21
In this model, a secondary insult is neces-
sary to result in a different or more severe clinical manifestation
of a complex disease. This insult occurs during development
and could be genetic or epigenetic (germ line or somatic) or
environmental. The two ‘hits’could act independently or addi-
tively to each other, resulting in a phenotype that differs from
either hit alone. Alternatively, the two hits could involve gene(s)
from the same or similar biochemical pathway and hence could
interact in an epistatic manner.
The novel approach of whole-exome sequencing has long
been due in the genetic investigation of NTDs. This study uses
this powerful approach in this complex trait and has successfully
identified potential candidate DNMs in novel genes in the
development of human NTDs. We have demonstrated the pres-
ence of LoF variants in five genes, have reported more LoF
DNMs in our cohort than expected and have identified two of
those mutations in orthologues of mouse NTD genes, suggesting
the involvement of those variants in the aetiology of human
NTDs. We have also demonstrated the presence of two inde-
pendent protein truncating variants in SHROOM3 in 43 trios
and presented a statistically significant enrichment of LoF
DNMs in this gene compared with control and gene-specific
DNMs rates. Our data strongly suggest that highly penetrant
pathogenic variants in this gene may account for a significant
part of the genetic aetiology of severe forms of NTD. Further
studies of this gene in a bigger cohort of sporadic cases may
help better assess the significance of these findings.
Acknowledgements We would like to thank all participants in this study.
Contributors PL, JLM, GAR, VC and ZK designed the study. PL, M-CG and ET
validated the mutations. PL, AD-L, DS, EH and OD analysed the genetic data. PL ran
the statistical analysis. PDM, EM, CM, VD, VC and ZK recruited patients and
provided clinical information. PL and ZK wrote the manuscript.
Funding CHU Ste-Justine fundation; Fonds de Recherche du Québec—Santé;
Canadian Institutes of Health Research. This project was conducted as part of the
research programme of the Integrated Research Network in Perinatology of Quebec
and Eastern Ontario (IRNPQEO). This work was supported by the Canadian Institutes
of Health Research (CRI 88413 and MOP 130411).
Competing interests ZK has a salary award from the “Fonds de Recherche du
Québec—Santé”. JLM is a National Scientist of the Fonds de Recherche du
Québec-Santé. PL is supported by “Fondation du CHU Ste-Justine”and “Fonds de
Recherche du Québec—Santé”. Authors have no competing interest.
Patient consent Obtained.
Ethics approval CHU Sainte Justine Hospital (Protocols’numbers: 2598 and
2899) and Istituto Giannina Gaslini, Genoa, Italy (protocol number: 213/2013).
Provenance and peer review Not commissioned; externally peer reviewed.
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