Mutations in hotspot region of MYH7 gene exon 23 associated with restrictive cardiomyopathy

Restrictive cardiomyopathy (RCM) is characterized by restrictive filling of the ventricles. The association between the variable expressivity and age at onset of disease and disease complexity with double and compound heterozygous state is associated with severity of disease phenotype in recent reports. Sharing of variants of sarcomere genes across cardiomyopathies has implication in clinical expression of different clinical phenotypes. The present study reports Sanger DNA sequencing of MYH7 gene, exon 23 from 30 unrelated RCM patients and 15 primary relatives from sporadic families with the hypothesis that RCM has common etiology with hypertrophic cardiomyopathy (HCM). Rare variant E949K and a de novo compound heterozygous mutation (p.E902K and p.D906N), in two RCM patients with early onset and no ventricular hypertrophy were found. These variants wereabsent in 50 dilated cardiomyopathy, 50 HCM patients and 15 primary relatives screened. The present report of rare and compound heterozygosity cases will further provide basis for the complexity and variable expressivity of phenotypes in patients in such complex diseases. The possible reasons for this phenotypic heterogeneity would be the presence of any other mutations in same chromosome or in different chromosome which is modifying the outcome of the causal mutation.


Introduction
4][5][6][7][8][9] The incidence of sudden cardiac death in pediatric patients with RCM, electrocardiographic evidence of ischemia has been reported as high as 23%. 9rimary cardiomyopathies were first linked to sarcomere genes when mutation in MYH7 and ACTC1 genes were reported to be associated with hypertrophic cardiomyopathy (HCM) 10 and dilated cardiomyopathy (DCM). 11Since then, till date more than 500 mutations were reported in HCM and DCM.In the last decade, restrictive cardiomyopathy was also found to be associated with mutations in sarcomeric genes, suggesting common etiologies among different cardiomyopathies. 12To date all sarcomeric proteins encoding genes are associated with the etiology of RCM with 33-66% incident rate 13,14 with TNNI3 being the first sarcomere gene implicated.However, there are many reports of MYH7 gene to be associated with RCM. 15,16MYH7 gene has a lot of hotspot region access based on the number of mutations found in the exon.][19][20][21][22][23][24][25][26][27] We report two idiopathic RCM patients with mutations in exon 23 of MYH7 gene in which one is rare and other two are novel compound heterozygote mutations.The report of these mutations will further increase knowledge about the genetic basis of restrictive cardiomyopathy and will help in expanding the pathophysiological pathways and therapeutic options.

Clinical evaluation of patients
Thirty patients diagnosed with restrictive cardiomyopathy between 2012 and 2015 by cardiologist at Out-patient Department (OPD), All India Institute of Medical Sciences (AIIMS), Delhi were enrolled for the study.Institutional ethical committee approved the study protocol and informed written consent was taken from all participants including family members.Familial case was considered when patient has any relative diagnosed with RCM and sporadic with unaffected relatives.When genetic variants were found in patients, screening in first-degree family members was done.
All patients and relatives underwentclinical examination, 12-lead electrocardiogram, 2D-echocardiography, cardiac catherisation and endomyocardial biopsy whenever required (Table 1).The diagnosis was based on echocardiographic demonstration of a bi-atrial enlargement, dilated inferior vena cava, restrictive doppler flows, relatively preserved systolic function, mildly reduced LV systolic function with markedly elevated ventricular filling

Sample preparations
Five milliliters of peripheral blood sample was collected in EDTA tubes.DNA was isolated using standardized phenol-chloroform method. 28The quantity and quality suitability for sequencing was checked by spectrophotometrically.

DNA sequencing
Appropriate primers for sequencing of the hotspot region of MYH7 gene i.e. exon 23 were used. 17The PCR product was sequenced by Sanger method (ABI 3730).Screening of available family members was also done.

Clinical assessment
An 11-year old boy was diagnosed with primary RCM in the cardiology OPD at AIIMS, Delhi.The proband showed severe symptoms such as breathlessness on exertion, palpitation, giddiness, fatigability, pedal edema and chest discomfort for 6 months, aggravated for last 10 days.An electrocardiogram (ECG) disclosed sinus rhythm with marked right atrial enlarge-ment, left axis deviation, and nonspecific ST-T wave changes.

Genetic analysis
Genetic testing led to the identification of rare variant leading to the amino acid change, i.e. glutamic acid to lysine on position 949 (c.2845G>A) in MYH7 gene (Figure 1A).This variant was not found in our patient cohort of 29 other RCM and 15 primary relatives, 50 DCM and 50 HCM patients.Genetic and clinical screening was performed in ten available family members.All the family members were unaffected on clinical screening and the variant as well was not found.

Clinical assessment
A 24-year old lady was first listed with a prior complaint like palpitation, shortness of breath due to mild exertion, severe pedal edema and reduced urine output.On physical examination, peripheral edema was present and the jugular venous pressure was increased.On cardiac examination, a holosystolic murmur was present over the apex.
An ECG of a patient had heart rate 83 beats/min with normal sinus rhythm and left axis deviation.On echocardiography examination, the sizes of both ventricles were normal while atria were enlarged.IVC is dilated with <50% variation with respiration.The left ventricle and right ventricle systolic function were found to be normal with 60% ejection fraction.There were no IVS thickening and no pericardial effusion

Genetic analysis
In genetic analysis, Sanger sequencing led to the identification of compound heterozygous mutations E902K and D906N in close proximity and on genetic testing of parents these mutations were found to be de novo (Figure1B).

Discussion
The importance of genetic testing in the etiologies of cardiomyopathies has been recognized over the last decade.RCM constitute nearly 5% of total pediatric cases among different types of cardiomyopathies. 29Due to progression in molecular techniques, mutations in all eight sarcomeric genes are now recognized to be linked with RCM, but the pathophysiological mechanism leading to the final disease phenotype is still unknown.The two utmost implicated genes in this rare cardiomyopathy are TNNI3 and MYH7 genes.However, MYH7 gene has been mostly linked with HCM rather than RCM.Here we report one rare (E949K) and novelcompound heterozygous mutations (E902K and D906N) in exon 23 of β-myosin heavy chain (MYH7).The Exome Aggregation Consortium (EXac) database was searched for these rare and compound heterozygote variant and found E949K is rare variant reported by only one HCM family 30 with MAF of 0.000008236 and the compound heterozygous variants (E902K and D906N) to be novel variants. 31he p.E949K mutation is a rare variant found in RCM patient represents an early age of onset and no ventricular hypertrophy, while by reports, it was implicated with familial HCM suggesting late onset of disease with severe left ventricular hypertrophy earlier reported in patient with European descent, 30 where as present patients are of south Asian, north Indian ethnicity.The important fact is that, the mutation found is located in the extremely conserved region.The mutation in MYH7 is recognized as origin of severe clinical outcomes and early age penetrance with a high frequency of sudden cardiac death 32 which is clearly seen in this patient having a severe form of RCM and early age of onset followed by sudden cardiac death.This mutation has occurred in α-helical segment that connects the myosin head to the backbone of the thick filament.This could affect assembly of the thick filament or stability of protein; the effect may be due to a loss of tensile strength or stiffness and may result in disease phenotype. 33The phenotypic heterogeneity in two patients having the same pathogenic mutation present in two different cardiomyopathies from two different ethnic backgrounds.The other possible reasons for this complexity of the phenotypic heterogeneity would be the presence of any other mutations in same chromosome or in different chromosome or possibility of multiple interactions between genes, or and other modifying factors in the outcome of disease condition.
Compound heterozygote mutations are now frequently seen to be associated with other cardiomyopathies i.e. dilated and hypertrophic cardiomyopathy. 34HCM shows 4% of cases with two mutations. 35,36ecently, many studies also reported cases with RCM associated with compound het-erozygous and double heterozygous mutations. 34,37A 9-year old girl with RCM has been found to have two mutations in TNNI3 gene, i.e. compound heterozygous while Peddy et al. reported double heterozygous mutation in an infant RCM case showing an early age of onset. 34,37Two or more than two mutations in the same gene or different gene are known to cause early age of onset and the severe clinical phenotypes.Patient 2 with compound heterozygous mutation also presented with an early age of onset with severe symptoms leading to NYHA-IV class from II in the span of 1 year and is now a potential case for heart transplant.These both mutations are present on the rod like structure of MYH7 gene and are conserved in all mammalian species, i.e. likely to be pathogenic as they are present on important domain of the protein.
In silico assessment of these rare variants through different bioinformatics tools predicted to have a deleterious effect (Table 2).The causality of the MYH7 p.E949K, p.E902K and p.D906N mutations in the case of RCM is pathogenic since this mutation was observed neither in unaffected

Conclusions
This study reiterates common etiology for RCM and HCM and represents the phenotypic heterogeneity of same rare genetic variant in two ethnically different patients.This also provides basis that expression of disease phenotype may be modified by different mutations in same gene or different modifying factors.From this study, it is advisable to screen all the candidate genes/whole exome to explain the molecular mechanism which leads to possible phenotypic outcomes, and the advent of next generation sequencing (NGS) technologies make it a possible approach.
28 cm]; end-diastolic wall thickness: 0.55 cm [reference range: 0.40-0.67cm].Based on echocardiography and ECG findings a presumptive diagnosis of RCM was made.The proband was subsequently referred for cardiac catheterization.Cardiac catheterization revealed grade III MR, severe TR, severe PAH, mild RV systolic dysfunction with left and right atrial waves measuring 27 mmHg and 26 mmHg respectively, no apical obliteration, no endocardial calcification and fair LV systolic dysfunction.The coronary artery anatomy and flow, as well as the pericardium, were normal.With these evidences, a diagnosis of idiopathic restrictive cardiomyopathy was confirmed.The age of presentation, first symptoms, and rapidity of the progress of the disease were consistent with a particularly severe form of RCM.The proband died due to sudden cardiac death after two years of diagnosis.

Figure 1 .
Figure 1.A) Shows the sequence of exon 23 of MYH7 gene with an arrow pointing to rare G to A changes at codon 949.B) Shows a compound heterozygous variants.Arrows point to the identified alterations G to A changes at codon 902 and 906.

Table 2 . In silico analysis of both compound heterozygous mutations in RCM proband.
present.Left ventricular diastolic function was abnormal with E'<A', E/E'~11 with fraction shortening 30-41%.Doppler study showed mild MR, severe TR and pulmonary stenosis with peak gradient 36 mmHg.On cardiac catherisation examination, the elevation in both side pressures, with a prominent y-descent suggesting a dip and plateau and ventricular concordance was present.