Introduction mutations in MYH6 (12). It has not


Congenital Heart Defects (CHDs) are one of the major causes of
death due to congenital malformations and show some of the more preponderant malformations
among live births. It has been revealed that
both familial and sporadic forms of CHDs result
from mutations in several genes based on human cases and animal models (1). Based on targeted deletions studies in mice, it has been
suggested that there are more than five hundred genes involved in heart
disorders (Mouse Genome Informatics ( (2). CHDs treat greatly as a complex trait and to date, the number of
familial cases has distinguished by the Mendelian segregation of single-gene mutations are
so few (3).

Both inherited and non-inherited factors account for congenital
heart disease (CHD). The incidence of CHD approximately is 0.4-0.6% live births
and real prevalence is about 4% (4, 5). Our knowledge about CHD’s causes and mechanisms remains
restricted in spite of the advances in diagnosis and interventions. With development
of whole exome/genome sequencing more CHD causing genes possibly will be
clarified which will increase our insight into
the genetic causes of CHD.

Numerous epidemiological studies have suggested a genetic
component of CHD etiology. Approximately,
25 percent of CHD cases occur as a complex trait with related defects in other organs
as a sporadic malformative association,
Mendelian syndrome or chromosomal abnormality (6). The rest of cases occur as isolated defects and both sporadic and
familial cases that showing Mendelian patterns of inheritance, have been
reported (7, 8). To date, several diseases associated with MYH6 mutations
such as hypertrophic (HCM), dilated cardiomyopathy (DCM) and atrial septal
defect (ASD) have been reported (9). ASD is categorized as the second most common CHD and accounts
for 10% of all cardiac malformations (10). Around 80% of persistent small
ASDs close spontaneously during infancy or childhood, but the large one could cause serious defects such as
congestive heart failure, pulmonary vascular disease and etc. (11). There are various types of ASD. ASD type 3 is caused by
mutations in MYH6 (12).  It has not been
identified any correlation between the nonsynonymous mutation of MYH6
and ASD3 but in the present study, we
could detect this correlation.

In the present study, we checked out a clinically characterized
family with a history of congenital heart disease. In this family, we observed an obvious
autosomal-dominant inheritance with reduced penetrance (K=50%). We identified a novel nonsense mutation in MYH6, NM_002471.3
c.3835C>T; R1279X, by WES of the patient and his parents in the SH1190831 family and then this mutation was confirmed
by Sanger sequencing.


Material and Methods

Patients and
clinical evaluations

The study protocol was approved by the local medical ethics
committee of Tarbiat Modares University, Tehran, Iran. Informed consents were obtained from
all individuals. All of the patient’s clinical information and the medical
histories were collected at the Department of Medical Genetics, DeNA
Laboratory, Tehran, Iran.

We enrolled 5 members of this family in our study (two affected, two
unaffected and one carrier) (Table 1). Subjects were adjusted by meticulous
medical records including a complete physical examination, a 12-lead
Echocardiogram (ECG), Ultrasonic cardiogram (UCG)
and other relevant features such as PR, QRS interval, QT, QTc duration and QRS
axis were measured. QRS axis was presumed as normal when its value was measured
between-30? and +90? and was classified abnormal when out
of this range. The normal range of ECG was performed based on the individual ages.
For adults, a PR interval above 210 ms and an increased above 100 ms of QRS interval
were thought-out prolonged (13).



DNA was isolated from peripheral blood of the family members by the ROCHE
DNA Extraction Kit (Cat. No.



Exome capturing and high throughput sequencing (HTS) was performed
on the proband (III:1) and his parents. The Nextera Rapid Capture Exome kit
with 340,000 probes designed against the human genome was utilized to enrich the approximately 37 Mb (214,405 exons) of
the Consensus Coding Sequences (CCS) from fragmented genomic DNA. Due to
limitations of the method, not all exons
were fully covered and all of the pathogenic
variants cannot be totally excluded. An overall coverage of 98.19% was
achieved, with 2188 missing base pairs (a coding
region including ± 2bp). At the next step, an end to end in-house bioinformatics pipelines including
base calling, primary filtering of low-quality
reads and probable artifacts, and annotation of variants were applied.

The reads were aligned to the NCBI human reference genome
(gh19/NCBI37.1) with SNP & Variation Suite version 8.0 (SVS v8.0) and
DNASTAR Lasergene12 (DNASTAR Inc., Madison,
Wisconsin USA). Small indel detection was used with the Unified
Genotyper tool from GATK tools in Galaxy online database
( The missense, nonsense, silent, and indel mutations
rates were estimated by Galaxy online tool and finally were confirmed by
Ivariantguide® (

Several filtering steps were applied to prioritize all variants:
1) Variants in dbSNP132
( and 1000 Genomes
( with allele frequencies more than 1%
were excluded. 2) The rest of variants underwent further exclusion in Exome
Sequencing Project (ESP) ( and Exome
Aggregation Consortium (ExAC) database.
3) The intragenic, intronic, UTRs regions
and synonymous variants were excluded from later analysis. 4) The SIFT (,
Provean (
and Mutation Taster
( were used to predict variants
pathogenicity (Table 3).

All suspected pathogenic variants were checked
out in HGMD (
and ClinVar ( Finally, based on family
pedigree, autosomal dominant inheritance pattern and clinical information were
used to evaluate identified variants. Based on the clinical information,
specific attention has been paid to the
42 genes known for Arrhythmogenic Cardiomyopathy, HCM, and DCM. Eventually, we identified a novel nonsense mutation in MYH6
(NM_002471.3, c.3835C>T, R1279X) that can play a destructive role in the
function of the tail domain in Myosin VI
protein. Also, ConSurf (
was applied to provide evolutionary conservation profile for Myosin VII protein
and showing the staple role of the novel mutation (Figure 2.C).