Present study was designed to evaluate genotype–phenotype correlation in the Index patient and family members harboring a rare mutation in MYH7 (CT at codon 923) gene and suffering from Hypertrophic Obstructive Cardiomyopathy (HOCM).

Total 8 individuals of a family of an index patient were included in this study in which father and youngest son diagnosed with hypertrophic Obstructive cardiomyopathy. All family members were screened with ECG, Echocardiography and clinical evaluation was done by the cardiologists as per the WHO criteria for diagnosis of HCM (1996) [4]. 5mL of intravenous blood was collected along with other physiological and physical parameters and DNA was extracted from blood by Phenol-chloroform method [5]. Suitable primers [6] were used to amplify exon 23 of MYH7 gene, and the PCR product (390bp) was sequenced in an automated sequencer ABI 3700 by Sanger’s method (Figure 1A). The study was ethically approved by institutional committee and informed written consent was taken from all participants.

Figure 1: (A) Sequence region of exon 23 of MYH7 showing heterozygous mutation CT at codon 923. (B) PAGE showing homozygous wild (II) with band size 108bp and 84bp, heterozygous (ID) with band size 108bp, 102bp and 84bp and homozygous mutant 108bp and 102bp. (C) ACE genotyping in 2% Agarose Gel with homozygous wild (II) with band size 490bp, Heterozygous (ID) with band size 490bp and 190bp and homozygous mutant 190bp. (I: Insertion; D: Deletion: L: DNA Ladder).

Screening for other known mutations associated with HCM like E101K in Cardiac muscle Actin (ACTC) gene [7] and P77L in Cardiac Troponin T (TNNT2) gene [8] were done by RFLP method using enzymes Ava I and Sm1I respectively. 5bp deletion polymorphism in intron 3 of TNNT2 gene was also analyzed by RFLP method using Ear I enzyme and genotyped in 8% Polyacrylamide gel (Figure 1B). 287bp Deletion polymorphism of Angiotensin Converting Enzyme (ACE) gene in the intron16 [9] and 25bp Deletion Polymorphism in Cardiac Myosin Binding Protein C (MYBPC3) gene in the intron 32 [10] were analyzed directly by genotyping the PCR product on 2% (Figure 1C) and 3% Agarose gel respectively. To validate MYH7 (Cï?T at codon 923) mutation, 20 HCM families including 21 affected and 46 unaffected family members were screened for exon 23 of MYH7 gene by Sanger sequencing. All sequences were deposited in NCBI data base [GenBank accession nos. JX504729 - JX504803].

The rod region of alpha helical segment connecting myosin head to thick filament results in possible dysfunction in contraction indicating a functional role for variants in MYH7 gene [11]. The rare MYH7 mutation reported in the index family was not found in other 20 HCM families in our study.

The symptoms of HOCM (Hypertrophic obstructive cardiomyopathy) such as dyspnea and angina were observed in the patient at the age of 14 during sports activity performance. Preliminary ECG examination indicated signs of ischemic heart disease which was confirmed as HOCM by the echocardiographic examination.

Screening of all family members established presence of HOCM in father, who had very mild symptoms from the past 3yrs but avoided medical checkup. Figure 2 shows the dominant pattern of inheritance of the MYH7 (C→T at codon 923) mutation. The index patient and one of his elder brothers (20 years) inherited the same mutation from their father. The father’s sisters, mother and his eldest brother was devoid of this mutation. This rare variant had been reported previously in an Indian HCM family and also in European cohort of 1000 genome project (www.ensemble.org).

Figure 2: The Family pedigree showing affected and unaffected family members with genetic profile, present age and age of onset for the affected family members.

The inference from the clinical parameters is depicted in table 1 in consonance with the genotypes. Abnormal ECG and 2D echocardiography revealed hypertrophied LV in index patient and his father only. The Blood Pressure and BMI of the index patient and the father were normal which excludes them from other hypertension which can also lead to hypertrophied LV. It was pertinent to note that the father had late onset of the disease (48yrs) while the youngest son (index patient) had early onset of the disease at the age of 14 years and the elder son (20 yrs) was asymptomatic though all three were having the rare MYH7 mutation.

The Cardiac Troponin T (TNNT2) protein, allows actomyosin interaction and contraction of muscle cell occur in response to Ca²⁺ [12] and ACE converts Angiotensin I to Angiotensin II, an important mediator in secondary myocardial hypertrophy, post-myocardial infarction remodeling and heart failure [13,14].

Screening of other known mutations associated with the HCM was not found in any family members except for the TNNT2 and ACE polymorphism. The inheritance of TNNT2 and ACE polymorphisms revealed that the index patient was distinct from all other family members with respect to genotypes 5bp TNNT2 (II) and 287bp ACE (ID), while the elder brother and the father with the MYH7 mutation (C→T at codon 923) had similar genotypes i.e. 5bp TNNT2 (ID) and 287bp ACE (II) (Figure 2).

Table 1: Clinical findings of the all family members.

The Index patient with rare MYH7 (C→T at codon 923) mutation had severe symptoms and early onset of disease which could be explained due to co-occurrence of ACE (ID) and TNNT2 (II) genotypes. Since his father and elder brother having different ACE (II) and TNNT2 (ID) genotypes harboring same rare MYH7 mutation could have been protective, with father having mild symptoms with late onset of disease and elder brother had no clinical manifestation till now. Also it was reported that ‘D’ allele of 5bpTNNT2 gene polymorphism had higher frequency in Indian populations [15], suggesting it may have some selective advantage. So presence of ‘D’ allele in father and his elder brother could have been beneficial in counteracting pathogenic MYH7 mutation.

The present family study suggested that the combinational effect of two or more variants may modify the phenotypic expression of the pathogenic mutation. In HCM, several private mutations have been reported, but cataloguing the same with clinical data will be a useful exercise for clinical practice with the larger picture of personalized genomic medicine in horizon.

The authors acknowledge University Grants Commission (UGC), India for funding the project on Molecular Genetics of Hypertrophic Cardiomyopathy.