Mutation in forwarding and reverse directions was performed

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Mutation Validation and Co-Segregation Analysis

Sanger sequencing in forwarding and reverse directions was performed to validate the candidate variants found in WES and then segregation analyses were performed in the family. The primers were designed by Primer3.0 ( web-based server (Table 2). We checked out the lack of SNPs in the genomic region corresponding to the 3? ends of primers by looking through the dbSNP database. The primers specificity was checked by the in-silico-PCR tool in UCSC genome browser and Primer blast of NCBI genome browser and finally, the PCR was utilized in standard conditions and samples were sent to Sanger sequencing.



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Clinical features

We identified an Iranian family affected by multiple complex phenotypes ranging from CHD, atrial septal defect (ASD), thyroglossal sinus to refractive errors of the eye and meatal stenosis. The proband (III.1), a 14-year male, affected with ASD and Thyroglossal sinus. Both parents were assessed for the relevant clinical features but we could not detect any relevant symptoms. Physical examination demonstrated ASD in the patient (Table 1). The family history examination clarified that the patient II2 has the same condition. The II.4 sample, in spite of carrying the mutation, indicated no obvious phenotype implying the reduced penetrance in this condition.

All family members were recruited for further physical examination and all gathered records have been reported in table 1.

Genetic Analysis

It is postulated that the pedigree may represent an autosomal dominant inheritance with reduced penetrance. To elucidate the underlying genetic cause(s), genomic DNA was obtained from the patient and analyzed by whole exome sequencing (WES). The novel mutation was confirmed by Sanger sequencing (Figure 1.B).

The detected SNVs and deletion/insertions were analyzed by several filtering methods. 66109 variants were found in the exome of the proband after alignment and SNV calling. After several exclusion processes by using of dbSNP132, 1000 Genomes Project, Exon Sequencing Project (ESP), and ExAc 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 carrier family members (II2, III1, II4) but not observed in other healthy parent or normal control (II5).

In the same statement, of the 1187 variants, 13 were ranked using three database tools (Provean, Mutation Taster, Sift) and finally, among the thirteen variants, a unique variant was opted as a pathogenic mutation of this unique family based on patient’s phenotype by utilizing CentoMD ( and ClinVar. ( (Figure 1.C).

Samples from the available members of the SH1190831 family were subjected to Sanger sequencing to confirm the candidate variant of MYH6 gene. The polymerase chain reaction (PCR) products were sequenced by ABI 730XL, using the conventional capillary system, and then the Sequences were analyzed by Genome Compiler online tool to identify the alternations.

To find the main cause of CHD in the proband by known genetic mutation(s), based on proband phenotype, we especially focused on the 42 genes that have critical roles in CHD etiology and revised our strategies with a filter of pertinent variants in these genes (Supplementary Table 1). The single patient analysis excluded the possibility of a known causative gene that underlies CHD.



Atrial septal defects (ASD) belong to a group of CHD that allow communication between the left and right sides of the heart although the communications include several distinct defects in the cardiac terminations of the pulmonary veins (sinus venosus and coronary sinus defects) and in the interatrial septum (atrial septal defects). ASDs, based on the defected gene, have been classified into several groups. The mutations in various genes have been associated with atrial septal defects, for instance, mutations in NKX2-5, GATA4 and TBX5, and MYH6 (14).

It has been identified that there are at least 35 classes of molecular motors into the myosin superfamily that move along actin filaments (15). Several studies have described various functions for Myosin VI such as membrane trafficking, endocytosis, organizing and stabilizing the actin cytoskeleton and playing a material role in inner-ear hair cells (16-18). Myosin VI is the merely class of myosin that known to move toward the minus-end of actin filaments. Intuitively, dimerization of the myosin can expand its movement along actin filament but it must be noticed that the Myosin VI does not contain a well-defined coiled-coil dimerization domain, suggesting that myosin-VI does not form a constitutive dimer on its own. The MYH6 gene encodes Myosin heavy chain, ? isoform (MHC-?) in human Myosin VI (19). This protein has several important domains such as head domain, IQ domain, cargo-binding domain, tail domain and etc. (Figure 2). The tail domain involves two distinct section: Coiled-coil domain and globular domain. It has been identified that the tail domain has a staple role in interacting with the target, especially uncoated vesicles (20).

NGS and particularly whole exome sequencing techniques have been developed into a robust and cost-effective tool to identify the new variants or genes for rare Mendelian unknown disorders (21-23). This technique has been used in genetic diagnostics helping to increase the clinical and mutational spectrum of known and unknown diseases (24, 25). But sometimes it is so difficult to distinguish between pathogenic and benign mutations (26, 27). Several filtering strategies have been developed to exclude variants that are implausible to cause disease.

In this study, we utilized the WES technique to identify a novel nonsense mutation at codon 3825 of MYH6 gene. This mutation is located at the extremely conserved region in MYH6, Myosin heavy chain-? isoform (MHC-?), and it is presumed to result in a truncated protein that is associated with Cardiomyopathy and ASD type 3 (OMIM: 614089, 613251). Previously, it has been reported that the mutations in MYH6 are associated with late-onset hypertrophic cardiomyopathy, atrial septal defects and sick sinus syndrome (13, 28). There are 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 region mediates interaction with cargo molecules or other myosin subunits. After several staple filtering and annotation processes, to predict whether the novel variant was deleterious or not, we utilized several databases such as SIFT, Mutation Taster, and Provean. We also analyzed intronic, synonymous, nonsense, missense and frameshift indel changes to predict whether those changes could affect splicing process by influencing on donor or acceptor splice sites, with mutation taster and Neutral Network Splice (NNSplice version 0.9).

Our result indicates that this nonsense mutation (R1279X) in MYH6 might be the genetic cause of congenital heart disease. Our study confirms that the MYH6 gene has an important role in heart functions but we recommend the applying animal modeling to scrutinize the distinctive role of this mutation.


For first time,  we identified a novel nonsense mutation, c.3835C>T, R1279X, in MYH6 gene as a possible cause of CHD in an Iranian family. This finding will increase our knowledge about the etiology of this rare condition by effective clarification of the causative gene mutations and will enhance the mutational spectrum of CHD and should consider in the diagnosis of these diseases.


We thank the family for their participation in this study. We are especially grateful to the staffs of DeNA laboratory for helping us in this research and additionally, we appreciate supports from Dr. Elika Esmaeilzadeh and Dr.Farveh Ehya, Tarbiat Modares University, Tehran, Iran. This research received no specific grant from any funding agency, commercial or not for profit sectors.


Conflict of Interest

The authors report no conflicts of interest.

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