Abstract functional studies in animal models, and


   Tricuspid Atresia (TA) is a rare life-threatening form of Congenital Heart Defect (CHD). The genetic mechanisms underlying TA is not clearly understood. According
to previous studies, the
endocardial cushioning event as the primary sign of cardiac valvulogenesis is
governed by several overlapping
signaling pathways including Ras/ERK pathway. The RASA1 gene as a regulator of cardiovascular system
development is involved in this pathway. Haploinsufficiency
of RASA1 gene due to heterozygous mutations
has been identified as the etiology underlying autosomal dominant disorder of
Capillary Malformation/Arteriovenous Malformation (CM/AVM). In the current study, using a two-step approach including whole exome
sequencing (WES) and bioinformatics analysis for consanguineous parents with
the history of recurrent abortions and two children with TA along with early
onset CM, we could identify a homozygous RASA1 germline mutation: c.1583A>G (p.Tyr528Cys) which lies
in Pleckstrin Homology (PH) domain of the gene. Patients were carefully
assessed to exclude extra-cardiac anomalies. Parents who were heterozygous for this variant were
presented with CM. In conclusion, on the basis of the bioinformatics-based
evaluation of p.Tyr528Cys mutation, the data from functional studies in
animal models, and previous evidence for involvement of heterozygous RASA1
mutations in CM/AVM associated with multiple forms of CHD, we propose that
phenotypic consequence of homozygous RASA1 p.Tyr528Cys mutation
is more serious than heterozygous one, which could be responsible for the TA
pathogenesis in our patients. We strongly suggest that parents with CM/AVM
should be investigated for RASA1 heterozygous mutations to perform fetal
echocardiography as a precaution in the event of pregnancy.

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Keywords Tricuspid Atresia, Whole Exome Sequencing, RASA1, Pleckstrin
Homology domain


Cardiac valvulogenesis is
known as an embryogenic evolutionary conserved mechanism in all vertebrates. 1 Heart valve formation is described by
the primary formation of Endocardial Cushions (ECs) in the atrioventricular
canal and outflow tract, which starts at embryonic day (E) E31–E35 in human, and
E9.5 in mouse. 2,3 During the
complex endocardial cushioning event, endothelial-mesenchymal transition of a
subgroup of endothelial cells will arise and atrioventricular canal including
mitral valve and tricuspid valve will appear. 4 The critical above stage is governed by overlapping
signaling pathways including VEGF, NFATc1, Notch, Wnt/ beta-catenin,
BMP/TGF-beta, ErbB, EGF, and Ras/ERK (MAPK) pathway. 2,4-6 The interaction between and
relative timing of these signaling pathways are proposed as a signaling network
model for valvulogenesis. 4
Lots of gene disruptions related to these pathways have now been revealed to
influence valve phenotypes. 7 Tricuspid atresia (TA; MIM#605067)
with a prevalence of 1/25000 at live birth is an infrequent form of valvular
Congenital Heart Defects (CHDs) commonly associated with poor prognosis. 1,8,9 Some studies have reported
familial occurrences of TA10-12 however, the genetic mechanisms underlying TA remain unclear 1,13. In this study using Whole Exome Sequencing (WES) approach
as a powerful technique for genetically heterogeneous diseases such as CHD 1,13, we found a germline ‘homozygous’ missense mutation c.1583A>G
p.(Tyr528Cys) in Pleckstrin Homology (PH) domain of RASA1 gene in a consanguineous Iranian family.


       A consanguineous family in which the parents were first
cousins were referred to pediatric cardiology and Neonatal Intensive Care Unit of
Tehran Children Medical Center during prenatal ultrasound screening of CHD for
high risk families. The fetus’s father as well as her paternal uncle were
already diagnosed with TA (currently at the age of 32, and 28 years old
respectively). They were born to healthy consanguineous parents with history of three pregnancy losses in 16-18th week of
gestation. In this family there was also one infant who died at day 11 after
birth with an unknown heart malformation. Although, prenatal ultrasound
screening of CHD for the proband’s fetus appeared normal, this family was
interested in determining genetic etiology underlying the CHD in their family. A signed informed consent form was
taken from all participants after being
informed of the aim of the research study. This research was approved by the ethics committee in
research of the University of Social Welfare and Rehabilitations Sciences of
Tehran, Iran.

      For classical cytogenetics analysis,
5 ml venous blood -collected in the heparinized tube- were handled by cell
culture and harvesting following standard techniques. High-resolution G-banded
lymphocyte culture (520 resolution) was carefully analyzed to exclude
chromosomal abnormality in patients.

      FISH (Fluorescent in Situ Hybridization) analysis was
carried out on suspension of metaphase and interphase cells using KreatechTM
KBI-40103 DiGeorge HIRA (22q11) / 22q13 (SHANK3) probes, according to
manufacturer’s procedure, to exclude 22q11.2 microdeleion.

       Genomic DNA was extracted from 5 ml
venous blood collected in EDTA-containing tube using the standard salting-out
method. An amount of 50?ng of genomic DNA from the fetus’s father (proband) was used for WES by means of Exome Enrichment Kit with
Agilent’s SureSelect Human All Exon V6 capture probes on the Illumina
HiSeq 4000 platform with the average read depth of 100x for the targeted
platform. Sequence alignment and variant
calling were made against the human reference genome GRCh37/hg19 build and wANNOVAR software (http://wannovar.wglab.org/) was used for variant detection and analysis. Several steps were taken to
prioritize the entire high-quality variants as follows. Variants in intergenic,
down/up stream, intronic, and UTR regions and synonymous variations were
excluded. Based on the hypothesis that the
causative mutation for the disease in siblings is rare, SNP variations with
unreported and reported Minor Allele Frequency (MAF) £
0.01 were considered in the following databases including exac,
(http://exac.broadinstitute.org/), the 1000 Genomes project
(www.1000genomes.org), genomAD browser (http://gnomad.broadinstitute.org),
NHLBI Exome Sequencing Project (ESP) (http://evs.gs.washington.edu/EVS/). Moreover, SNP variations
observed in the exomes of 100 unrelated healthy Iranians or Iranians affected
with non-cardiovascular diseases were further excluded.

       In the next step, we classified the rare variants according
to in silico prediction scores in Polyphen2 (http://genetics.bwh.harvard.edu/pph2/), SIFT
(http://sift.bii.astar.edu.sg/), MutationTaster (www.mutationtaster.org),
CADD_phred (cadd.gs.washington.edu/), and GERP (UCSC Genome
Browser). Afterwards, we achieved gene-based arrangements incorporating
conservation scores of the variations using SiPhy_29way_logOdds score.

       Taking into account variations that presented in the homozygous state, X-linked state or that
were compound heterozygous, we finally focused on variants whose genes are involved in biological
pathways related to the cardiovascular system (Figure 1).

        Screening of the candidate variants were further followed by
sanger sequencing for all family members as the gold standard for screening and
verifying genes of interest.


         None of the patients showed
chromosome abnormalities in either karyotype analysis or 22q11.2 microdeletion
using FISH technique. The sanger sequencing verification was performed for
candidate variants found by WES and bioinformatics analysis.



The c.1583A>G (p.Tyr528Cys) variant
for RASA1 gene in homozygous state was the only candidate
variant shared by two patients. Other family members including parents and
proband’s offspring were heterozygote as expected (Figure 2). However, the mutation in RASA1 gene has already been recorded (http://www.hgmd.cf.ac.uk/ac/index.php) in heterozygous form as the cause of autosomal dominant Capillary
Malformation/Arteriovenous Malformation (CM/AVM). 14 Besides, autosomal dominant Hereditary
Hemorrhagic Telangiectasia, which
is also a vascular disorder characterized by skin and nasal telangiectases
along with AVM has also been reported with
same RASA1 gene mutation. 15
Moreover, as several loss of function mutations in this gene have previously
been reported with vascular anomalies in association with multiple forms of CHD
16, we propose that the p.Tyr528Cys homozygous mutation could be
responsible for non-syndromic TA in our family.

evaluated the heterozygous parents more precisely, we noted a unilateral purple-red lesion (2.5 x
3 cm) on the father’s hand and bilateral varicose veins on mother’s legs
signaling CM. Moreover, father had a history of spontaneous subarachnoid
hemorrhage. After the birth of
proband’s offspring, a
pale-pink lesion also appeared in her forehead. None of the parents or
proband’s offspring showed cardiovascular abnormality by echocardiographic
technique. Both patients were thoroughly evaluated by the cardiologists and clinical geneticists
to rule out extra-cardiac malformations. Cardiac phenotypic characterization of
the patients was evaluated with echocardiographic
technique (Table 1).


      In the current
study, we have cosegregated homozygous p.Tyr528Cys germline mutation in RASA1
gene in two patients with isolated TA. RASA1 gene (also known as Ras p21 protein activator
1) is a GTPase activator for
normal RAS p21 but not its oncogenic counterpart. It is the first
described member of Ras GTPase-activating protein (RasGAP) family that encodes p120-RasGAP protein. 17,18 The involvement of
Ras-related signaling pathways in the development of embryonic heart has been
emphasized by the significant contribution of these pathways’ components in the
pathogenesis of Rasopathy disorders. 14,19-21
These molecular components include either RasGAP family members or other
downstream molecules in Ras/Raf/ Mek/ ERK cascade. Compound heterozygous
missense mutation in NFATC1 gene, acting downstream of the Ras/ERK pathway, was also recently
identified for non-syndromic TA in a Lebanese family (Figure 3) 1.

      In spite of syndromic nature
of Rasopathy disorders, heterozygous germline
mutations in RASA1 gene cause disorders of vascular development, without
any developmental defects. 24 RASA1 gene haploinsufficency due to heterozygous mutations has been identified in a subset of the individuals with CM/AVM disorder 18. The CM/AVM is mainly characterized by small multifocal and randomly distributed CM as pink-red to purple lesions, varicosities vein with
or without deep venous anomalies, and fast flow lesions including arteriovenous
malformation (AVM) or an arteriovenous fistula (AVF). 16,20,22,23,25 In our study, the parents and proband’s daughter who
were heterozygous for p.Tyr528Cys mutation were presented with multiple forms of CM/AVM. Two siblings
were also presented with early onset bilateral
varicose veins. Until
now, more than 100 highly penetrant mutations
have been identified across the RASA1 gene 16,22,26; but no genotype–phenotype correlation has
been established. 26 It is noteworthy that in a study performed by Revencu et al., 16 several heterozygous RASA1 mutations (mostly nonsense, frameshift, and
splice-site) have been reported in familial
cases with CM/AVM in association with multiple forms of CHD (Table 2). However, Revencu et al., 16 focused on various forms of vascular
anomalies due to RASA1 gene mutations and they did not consider the
cardiac phenotypes of their patients in detail. This kind of association
indicates that, while there is no previous data to verify homozygous RASA1 mutation
as the cause of more serious phenotype, we can speculate this might be so.

Therefore, high mortality rate in this family along with two children affected
by severely cardiac defects could be the consequence of complete loss of
function of PH domain of RASA1 gene. The importance
of p.Tyr528Cys on PH domain of RASA1 gene is
discussed in more detail as follows. 

p120-RasGAP protein is a monomeric cytoplasmic protein with several domains. 18 Each protein domain is involved in several cellular and developmental processes by either
Ras-dependent or Ras-independent manner (Figure 3). 27 In a
functional study performed on homozygote mice with a point mutation in GAP
domain (Rasa1 R780Q/R780Q  ), the severity of blood vascular abnormalities was
identical to Rasa1-null mice and their cardiovascular manifestations
were also mostly restricted to ECs. This finding suggested that cardiovascular
anomalies are caused by RASA1 inability to control Ras activation in
Ras-dependent manner. 25 In
accordance with this finding, we have focused on the functional importance of
PH domain in Ras-dependent function of p120-RasGAP protein which seems to contribute to the pathogenesis of
cardiovascular phenotype in our family. The
missense substitution p.Tyr528Cys found in our
patients alters cysteine to tyrosine in PH domain. Since this residue is
extremely conserved among human proteins containing PH domains (supplementary
data 1) and among other species (Figure 4), it might be essential for RASA1

Generally, PH domains with structurally conserved motifs, contain about 100 amino acid residues. They are present in several
proteins and contribute
to signal transduction pathways. The PH domain
of p120-RasGAP is located between 474-577 residues and in noncatalytic region
of the protein. However, it binds to catalytic domain (GAP domain) of its own
p120-RasGAP and interferes with the Ras/GAP interaction. 28 According to Hernandez et al., 15 the PH domain of p120-RasGAP
protein  has the ability to bind to
phospholipids subgroups as well as  being
involved in numerous protein–protein interactions (Figure 4). The C-terminal
region of the PH domain (residues 523–591) interacts with Ras and competes with
it for binding to GAP domain. The Tyr528 side chain which is substituted by
cysteine residue in our patients, are exposed on the C-terminal region of the
PH domain. 28,29 This substitution leads to the removal
of the aromatic side chain and creation of a slightly negative charged residue. 15 The
surface exposed position of tyrosine suggests that this substitution may alter
binding of the PH domain to protein partners, or its own GAP domain. 29 Therefore, it seems that complete loss
of function of PH domain in a Ras-dependent manner would lead to RASA1
inability to regulate the Ras molecule, and thereby could have an effect on TA
pathogenesis in our patients.

       Finally, we
considered previous functional studies on murine models deficient for Rasa1
gene. These data indicated the essential role of Rasa1 gene in the
regulation of cardiovascular development. Although heterozygous mice due to
loss of one germline Rasa1 allele had no observable phenotype,
homozygous loss of the gene alleles causes embryonic death at E9.5 to E10.5
which is correlated with the primary formation of EC in the
atrioventricular canal and outflow tract. 2,3,17,30,31 Interestingly, adult mice with
induced homozygous loss of Rasa1 in all tissues have no detectable
spontaneous cardiovascular defect. Therefore Rasa1 seems to be necessary
for the embryonic cardiovascular development and it is not obligatory for
cardiovascular maintenance. 31
These studies also indicated that, while Rasa1 is ubiquitously
expressed, embryonic mortality of mice deficient for Rasa1 is mostly
restricted to ECs. 18

conclusion, on the basis of Ras/ERK pathway contribution in the embryonic
development of heart valves, bioinformatics-based evaluation of p.Tyr528Cys mutation,
and evidence from functional studies in mice models deficient for Rasa1
gene, we suggest that p.Tyr528Cys homozygous mutation in the RASA1 gene
with dominant pattern of inheritance can be responsible for TA phenotype in our
family. However, this hypothetical theory needs to be supported by generating
an animal model carrying p.Tyr528Cys point mutation. We suggest that parents
with CM/AVM should be investigated for RASA1 heterozygous mutation and
if both parents are carrying RASA1 heterozygous mutation, fetal
echocardiography is strongly recommended as a precaution in the event of