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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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.


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

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


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

authors report no conflicts of interest.