MutationValidation and Co-Segregation AnalysisSanger sequencing in forwardingand reverse directions was performed to validate the candidate variants foundin WES and then segregation analyses were performed in the family.
The primerswere designed by Primer3.0(http://bioinfo.ut.ee/primer3-0.4.0) web-based server (Table 2). We checked out the lack of SNPs in thegenomic region corresponding to the 3? ends of primers by looking through the dbSNP database.
The primers specificity waschecked by the in-silico-PCR tool in UCSCgenome browser and Primer blast of NCBI genome browser and finally, the PCR wasutilized in standard conditions and samples were sent to Sanger sequencing. ResultsClinicalfeaturesWe identified an Iranian family affected by multiple complex phenotypes ranging from CHD, atrial septaldefect (ASD), thyroglossal sinus to refractive errors of the eye and meatalstenosis. The proband (III.1), a 14-year male, affected with ASD andThyroglossal sinus.
Both parents were assessed for the relevant clinicalfeatures but we could not detect any relevant symptoms. Physical examinationdemonstrated ASD in the patient (Table 1). The family history examination clarifiedthat the patient II2 has the samecondition. The II.4 sample, in spite of carrying the mutation, indicated no obvious phenotype implying the reducedpenetrance in this condition. All family members were recruited for further physical examinationand all gathered records have been reported in table 1. GeneticAnalysis It is postulated that the pedigree may represent an autosomaldominant inheritance with reduced penetrance.
To elucidate the underlyinggenetic cause(s), genomic DNA was obtained from the patient and analyzed by whole exome sequencing (WES). The novelmutation was confirmed by Sanger sequencing (Figure 1.B).
The detected SNVs and deletion/insertions were analyzed by severalfiltering methods. 66109 variants were found in the exome of the proband after alignmentand SNV calling. After several exclusion processes by using of dbSNP132, 1000Genomes Project, Exon Sequencing Project (ESP), andExAc databases, thirteen variants were identified and then prioritized by patients’phenotype.
Eventually, tally with the patient’s phenotypes,a novel variant was identified that shared by two affected and one carrierfamily members (II2, III1, II4) but not observed in other healthy parent ornormal control (II5).In the same statement, of the 1187 variants, 13 were ranked usingthree database tools (Provean, Mutation Taster,Sift) and finally, among the thirteen variants,a unique variant was opted as apathogenic mutation of this unique family based on patient’s phenotype by utilizingCentoMD (https://www.centogene.com) and ClinVar.(https://www.ncbi.
nlm.nih.gov/clinvar/) (Figure 1.C).Samples from the available members of the SH1190831 family weresubjected to Sanger sequencing to confirm the candidate variant of MYH6 gene. The polymerase chainreaction (PCR) products were sequenced by ABI 730XL, using the conventionalcapillary system, and then the Sequences were analyzed by Genome Compiler onlinetool to identify the alternations.To find the main cause of CHD in the proband by known geneticmutation(s), based on proband phenotype, we especially focused on the 42 genes thathave critical roles in CHD etiology andrevised our strategies with a filter of pertinent variants in these genes (SupplementaryTable 1).
The single patient analysis excluded the possibility of a knowncausative gene that underlies CHD. DiscussionAtrial septal defects (ASD) belong to a group of CHD thatallow communication between the left and right sides of the heart although thecommunications include several distinct defects in the cardiac terminations ofthe pulmonary veins (sinus venosus andcoronary sinus defects) and in the interatrial septum (atrial septal defects). ASDs,based on the defected gene, have beenclassified into several groups. The mutations in various genes have beenassociated with atrial septal defects, forinstance, mutationsin NKX2-5, GATA4 and TBX5,and MYH6 (14).It has been identified that there are at least 35 classes ofmolecular motors into the myosin superfamily that move along actin filaments (15). Several studies have described various functions for Myosin VIsuch as membrane trafficking, endocytosis,organizing and stabilizing the actin cytoskeleton and playing a material rolein inner-ear hair cells (16-18).
MyosinVI is the merely class of myosin that known to move toward the minus-end ofactin filaments. Intuitively, dimerization of the myosin can expand itsmovement along actin filament but it must be noticed that the Myosin VI does not contain a well-defined coiled-coil dimerizationdomain, suggesting that myosin-VI doesnot form a constitutive dimer on its own. TheMYH6 gene encodes Myosin heavy chain, ? isoform (MHC-?) in human MyosinVI (19). This protein has several important domains such as head domain, IQdomain, cargo-binding domain, tail domainand etc. (Figure 2). The tail domain involves two distinct section: Coiled-coildomain and globular domain. It has been identified that the tail domain has astaple role in interacting with the target, especially uncoated vesicles (20).
NGS and particularly whole exome sequencing techniques have been developedinto a robust and cost-effective tool to identify the new variants or genes forrare Mendelian unknown disorders (21-23). Thistechnique has been used in genetic diagnostics helping to increase the clinicaland mutational spectrum of known and unknown diseases (24, 25). But sometimes it is so difficult to distinguish betweenpathogenic and benign mutations (26, 27).
Several filtering strategies have been developed to excludevariants that are implausible to cause disease.In this study, we utilized the WES technique to identify a novelnonsense mutation at codon 3825 of MYH6 gene. This mutation is locatedat the extremely conserved region in MYH6, Myosin heavy chain-? isoform(MHC-?), and it is presumed to result in a truncatedprotein that is associated with Cardiomyopathy and ASD type 3 (OMIM:614089, 613251). Previously, it has been reported that the mutations in MYH6are associated with late-onset hypertrophic cardiomyopathy, atrial septaldefects and sick sinus syndrome (13, 28). Thereare numerous reports on the association of MYH6 mutations and CHD (29).
In the present study, we identified a novel nonsense variant,c.3835C>T, R1279X, by whole exome sequencing in the coiled-coil region or tail domain of MYH6 gene. This regionmediates interaction with cargo moleculesor other myosin subunits. After several staple filtering and annotationprocesses, to predict whether the novel variant was deleterious or not, weutilized several databases such as SIFT, Mutation Taster, and Provean. We also analyzed intronic,synonymous, nonsense, missense and frameshift indel changes to predict whetherthose changes could affect splicing process by influencing on donor or acceptorsplice sites, with mutation taster and Neutral Network Splice (NNSplice version0.
9). Our result indicates that this nonsense mutation (R1279X) in MYH6might be the genetic cause of congenital heart disease. Our study confirms thatthe MYH6 gene has an important role in heart functions but we recommendthe applying animal modeling to scrutinize the distinctive role of thismutation. ConclusionsFor first time, weidentified a novel nonsense mutation, c.
3835C>T, R1279X, in MYH6 geneas a possible cause of CHD in an Iranian family. This finding will increase ourknowledge about the etiology of this rare condition by effective clarificationof the causative gene mutations and will enhance the mutational spectrum of CHDand should consider in the diagnosis of these diseases.AcknowledgementsWe thank the family for their participation in this study. We areespecially grateful to the staffs of DeNA laboratory for helping us in thisresearch and additionally, we appreciatesupports from Dr. Elika Esmaeilzadeh and Dr.
Farveh Ehya, Tarbiat ModaresUniversity, Tehran, Iran. This researchreceived no specific grant from any funding agency, commercial or not for profit sectors. Conflict of InterestTheauthors report no conflicts of interest.