Abstract functional studies in animal models, and

Abstract   Tricuspid Atresia (TA) is a rare life-threatening form of Congenital Heart Defect (CHD). The genetic mechanisms underlying TA is not clearly understood. Accordingto previous studies, theendocardial cushioning event as the primary sign of cardiac valvulogenesis isgoverned by several overlappingsignaling pathways including Ras/ERK pathway.

The RASA1 gene as a regulator of cardiovascular systemdevelopment is involved in this pathway. Haploinsufficiencyof RASA1 gene due to heterozygous mutationshas been identified as the etiology underlying autosomal dominant disorder ofCapillary Malformation/Arteriovenous Malformation (CM/AVM). In the current study, using a two-step approach including whole exomesequencing (WES) and bioinformatics analysis for consanguineous parents withthe history of recurrent abortions and two children with TA along with earlyonset CM, we could identify a homozygous RASA1 germline mutation: c.1583A>G (p.Tyr528Cys) which liesin Pleckstrin Homology (PH) domain of the gene. Patients were carefullyassessed to exclude extra-cardiac anomalies. Parents who were heterozygous for this variant werepresented with CM.

In conclusion, on the basis of the bioinformatics-basedevaluation of p.Tyr528Cys mutation, the data from functional studies inanimal models, and previous evidence for involvement of heterozygous RASA1mutations in CM/AVM associated with multiple forms of CHD, we propose thatphenotypic consequence of homozygous RASA1 p.Tyr528Cys mutationis more serious than heterozygous one, which could be responsible for the TApathogenesis in our patients. We strongly suggest that parents with CM/AVMshould be investigated for RASA1 heterozygous mutations to perform fetalechocardiography as a precaution in the event of pregnancy.  Keywords Tricuspid Atresia, Whole Exome Sequencing, RASA1, PleckstrinHomology domain Cardiac valvulogenesis isknown as an embryogenic evolutionary conserved mechanism in all vertebrates. 1 Heart valve formation is described bythe primary formation of Endocardial Cushions (ECs) in the atrioventricularcanal and outflow tract, which starts at embryonic day (E) E31–E35 in human, andE9.5 in mouse. 2,3 During thecomplex endocardial cushioning event, endothelial-mesenchymal transition of asubgroup of endothelial cells will arise and atrioventricular canal includingmitral valve and tricuspid valve will appear.

4 The critical above stage is governed by overlappingsignaling pathways including VEGF, NFATc1, Notch, Wnt/ beta-catenin,BMP/TGF-beta, ErbB, EGF, and Ras/ERK (MAPK) pathway. 2,4-6 The interaction between andrelative timing of these signaling pathways are proposed as a signaling networkmodel for valvulogenesis. 4Lots of gene disruptions related to these pathways have now been revealed toinfluence valve phenotypes.

7 Tricuspid atresia (TA; MIM#605067)with a prevalence of 1/25000 at live birth is an infrequent form of valvularCongenital Heart Defects (CHDs) commonly associated with poor prognosis. 1,8,9 Some studies have reportedfamilial occurrences of TA10-12 however, the genetic mechanisms underlying TA remain unclear 1,13. In this study using Whole Exome Sequencing (WES) approachas a powerful technique for genetically heterogeneous diseases such as CHD 1,13, we found a germline ‘homozygous’ missense mutation c.1583A>Gp.(Tyr528Cys) in Pleckstrin Homology (PH) domain of RASA1 gene in a consanguineous Iranian family. Methods       A consanguineous family in which the parents were firstcousins were referred to pediatric cardiology and Neonatal Intensive Care Unit ofTehran Children Medical Center during prenatal ultrasound screening of CHD forhigh risk families. The fetus’s father as well as her paternal uncle werealready diagnosed with TA (currently at the age of 32, and 28 years oldrespectively). They were born to healthy consanguineous parents with history of three pregnancy losses in 16-18th week ofgestation.

In this family there was also one infant who died at day 11 afterbirth with an unknown heart malformation. Although, prenatal ultrasoundscreening of CHD for the proband’s fetus appeared normal, this family wasinterested in determining genetic etiology underlying the CHD in their family. A signed informed consent form wastaken from all participants after beinginformed of the aim of the research study. This research was approved by the ethics committee inresearch of the University of Social Welfare and Rehabilitations Sciences ofTehran, Iran.

      For classical cytogenetics analysis,5 ml venous blood -collected in the heparinized tube- were handled by cellculture and harvesting following standard techniques. High-resolution G-bandedlymphocyte culture (520 resolution) was carefully analyzed to excludechromosomal abnormality in patients.       FISH (Fluorescent in Situ Hybridization) analysis wascarried out on suspension of metaphase and interphase cells using KreatechTMKBI-40103 DiGeorge HIRA (22q11) / 22q13 (SHANK3) probes, according tomanufacturer’s procedure, to exclude 22q11.2 microdeleion.       Genomic DNA was extracted from 5 mlvenous blood collected in EDTA-containing tube using the standard salting-outmethod. An amount of 50?ng of genomic DNA from the fetus’s father (proband) was used for WES by means of Exome Enrichment Kit withAgilent’s SureSelect Human All Exon V6 capture probes on the IlluminaHiSeq 4000 platform with the average read depth of 100x for the targetedplatform.

Sequence alignment and variantcalling 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 toprioritize the entire high-quality variants as follows. Variants in intergenic,down/up stream, intronic, and UTR regions and synonymous variations wereexcluded.

Based on the hypothesis that thecausative mutation for the disease in siblings is rare, SNP variations withunreported 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 variationsobserved in the exomes of 100 unrelated healthy Iranians or Iranians affectedwith non-cardiovascular diseases were further excluded.       In the next step, we classified the rare variants accordingto 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 GenomeBrowser).

Afterwards, we achieved gene-based arrangements incorporatingconservation scores of the variations using SiPhy_29way_logOdds score.        Taking into account variations that presented in the homozygous state, X-linked state or thatwere compound heterozygous, we finally focused on variants whose genes are involved in biologicalpathways related to the cardiovascular system (Figure 1).        Screening of the candidate variants were further followed bysanger sequencing for all family members as the gold standard for screening andverifying genes of interest.Result         None of the patients showedchromosome abnormalities in either karyotype analysis or 22q11.2 microdeletionusing FISH technique. The sanger sequencing verification was performed forcandidate variants found by WES and bioinformatics analysis.  The c.

1583A>G (p.Tyr528Cys) variant(rs145752649)for RASA1 gene in homozygous state was the only candidatevariant shared by two patients. Other family members including parents andproband’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 CapillaryMalformation/Arteriovenous Malformation (CM/AVM). 14 Besides, autosomal dominant HereditaryHemorrhagic Telangiectasia, whichis also a vascular disorder characterized by skin and nasal telangiectasesalong with AVM has also been reported withsame RASA1 gene mutation. 15Moreover, as several loss of function mutations in this gene have previouslybeen reported with vascular anomalies in association with multiple forms of CHD16, we propose that the p.Tyr528Cys homozygous mutation could beresponsible for non-syndromic TA in our family.      Havingevaluated the heterozygous parents more precisely, we noted a unilateral purple-red lesion (2.5 x3 cm) on the father’s hand and bilateral varicose veins on mother’s legssignaling CM. Moreover, father had a history of spontaneous subarachnoidhemorrhage.

After the birth ofproband’s offspring, apale-pink lesion also appeared in her forehead. None of the parents orproband’s offspring showed cardiovascular abnormality by echocardiographictechnique. Both patients were thoroughly evaluated by the cardiologists and clinical geneticiststo rule out extra-cardiac malformations. Cardiac phenotypic characterization ofthe patients was evaluated with echocardiographictechnique (Table 1).Discussion      In the currentstudy, we have cosegregated homozygous p.Tyr528Cys germline mutation in RASA1gene in two patients with isolated TA.

RASA1 gene (also known as Ras p21 protein activator1) is a GTPase activator fornormal RAS p21 but not its oncogenic counterpart. It is the firstdescribed member of Ras GTPase-activating protein (RasGAP) family that encodes p120-RasGAP protein. 17,18 The involvement ofRas-related signaling pathways in the development of embryonic heart has beenemphasized by the significant contribution of these pathways’ components in thepathogenesis of Rasopathy disorders. 14,19-21These molecular components include either RasGAP family members or otherdownstream molecules in Ras/Raf/ Mek/ ERK cascade. Compound heterozygousmissense mutation in NFATC1 gene, acting downstream of the Ras/ERK pathway, was also recentlyidentified for non-syndromic TA in a Lebanese family (Figure 3) 1.      In spite of syndromic natureof Rasopathy disorders, heterozygous germlinemutations in RASA1 gene cause disorders of vascular development, withoutany 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 withor without deep venous anomalies, and fast flow lesions including arteriovenousmalformation (AVM) or an arteriovenous fistula (AVF). 16,20,22,23,25 In our study, the parents and proband’s daughter whowere heterozygous for p.Tyr528Cys mutation were presented with multiple forms of CM/AVM.

Two siblingswere also presented with early onset bilateralvaricose veins. Untilnow, more than 100 highly penetrant mutationshave been identified across the RASA1 gene 16,22,26; but no genotype–phenotype correlation hasbeen established. 26 It is noteworthy that in a study performed by Revencu et al., 16 several heterozygous RASA1 mutations (mostly nonsense, frameshift, andsplice-site) have been reported in familialcases with CM/AVM in association with multiple forms of CHD (Table 2). However, Revencu et al., 16 focused on various forms of vascularanomalies due to RASA1 gene mutations and they did not consider thecardiac phenotypes of their patients in detail. This kind of associationindicates that, while there is no previous data to verify homozygous RASA1 mutationas the cause of more serious phenotype, we can speculate this might be so.Therefore, high mortality rate in this family along with two children affectedby severely cardiac defects could be the consequence of complete loss offunction of PH domain of RASA1 gene.

The importanceof p.Tyr528Cys on PH domain of RASA1 gene isdiscussed in more detail as follows.         Thep120-RasGAP protein is a monomeric cytoplasmic protein with several domains. 18 Each protein domain is involved in several cellular and developmental processes by eitherRas-dependent or Ras-independent manner (Figure 3). 27 In afunctional study performed on homozygote mice with a point mutation in GAPdomain (Rasa1 R780Q/R780Q  ), the severity of blood vascular abnormalities wasidentical to Rasa1-null mice and their cardiovascular manifestationswere also mostly restricted to ECs. This finding suggested that cardiovascularanomalies are caused by RASA1 inability to control Ras activation inRas-dependent manner. 25 Inaccordance with this finding, we have focused on the functional importance ofPH domain in Ras-dependent function of p120-RasGAP protein which seems to contribute to the pathogenesis ofcardiovascular phenotype in our family.

Themissense substitution p.Tyr528Cys found in ourpatients alters cysteine to tyrosine in PH domain. Since this residue isextremely conserved among human proteins containing PH domains (supplementarydata 1) and among other species (Figure 4), it might be essential for RASA1function.     Generally, PH domains with structurally conserved motifs, contain about 100 amino acid residues. They are present in severalproteins and contributeto signal transduction pathways. The PH domainof p120-RasGAP is located between 474-577 residues and in noncatalytic regionof the protein. However, it binds to catalytic domain (GAP domain) of its ownp120-RasGAP and interferes with the Ras/GAP interaction. 28 According to Hernandez et al.

, 15 the PH domain of p120-RasGAPprotein  has the ability to bind tophospholipids subgroups as well as  beinginvolved in numerous protein–protein interactions (Figure 4). The C-terminalregion of the PH domain (residues 523–591) interacts with Ras and competes withit for binding to GAP domain. The Tyr528 side chain which is substituted bycysteine residue in our patients, are exposed on the C-terminal region of thePH domain. 28,29 This substitution leads to the removalof the aromatic side chain and creation of a slightly negative charged residue.

15 Thesurface exposed position of tyrosine suggests that this substitution may alterbinding of the PH domain to protein partners, or its own GAP domain. 29 Therefore, it seems that complete lossof function of PH domain in a Ras-dependent manner would lead to RASA1inability to regulate the Ras molecule, and thereby could have an effect on TApathogenesis in our patients.        Finally, weconsidered previous functional studies on murine models deficient for Rasa1gene. These data indicated the essential role of Rasa1 gene in theregulation of cardiovascular development. Although heterozygous mice due toloss of one germline Rasa1 allele had no observable phenotype,homozygous loss of the gene alleles causes embryonic death at E9.5 to E10.5which is correlated with the primary formation of EC in theatrioventricular canal and outflow tract.

2,3,17,30,31 Interestingly, adult mice withinduced homozygous loss of Rasa1 in all tissues have no detectablespontaneous cardiovascular defect. Therefore Rasa1 seems to be necessaryfor the embryonic cardiovascular development and it is not obligatory forcardiovascular maintenance. 31These studies also indicated that, while Rasa1 is ubiquitouslyexpressed, embryonic mortality of mice deficient for Rasa1 is mostlyrestricted to ECs.

18         Inconclusion, on the basis of Ras/ERK pathway contribution in the embryonicdevelopment of heart valves, bioinformatics-based evaluation of p.Tyr528Cys mutation,and evidence from functional studies in mice models deficient for Rasa1gene, we suggest that p.Tyr528Cys homozygous mutation in the RASA1 genewith dominant pattern of inheritance can be responsible for TA phenotype in ourfamily. However, this hypothetical theory needs to be supported by generatingan animal model carrying p.Tyr528Cys point mutation. We suggest that parentswith CM/AVM should be investigated for RASA1 heterozygous mutation andif both parents are carrying RASA1 heterozygous mutation, fetalechocardiography is strongly recommended as a precaution in the event ofpregnancy.