Sarcomeric gene mutations in phenotypic positive hypertrophic cardiomyopathic patients in Indian population

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Introduction
Hypertrophic cardiomyopathy (HCM) is a type of cardiac disorder mainly characterized by concentric or symmetric septal hypertrophy with the predominant interventricular septum (1). HCM shows a prevalence of 1:500 in the general population and is inherited in an autosomal dominant fashion. Individuals positive with HCM have a 50% probability of inheriting mutations to offspring. De novo mutations may result in sporadic cases in probands with genotype-negative parents (2). Most of the mutations (90%) present in the MYBPC3 gene affect the physical and functional properties of the concerned proteins (2). These mutations are mainly missense or frameshift mutations affecting several amino acids which results in different products (2). The presence of peak left ventricular obstructive gradient >30 mmHg in HCM patients has a prognostic significance in predicting the risk of sudden cardiac death (SCD) and heart failure (3). HCM probands that are genotypically positive have about 70% of mutations in MYH7 and MYBPC3 genes, while other genes tropomyosin, α-actin, troponin I and troponin T genes account for only 1-5% (3). Genotype penetration and hypertrophic severity are dependent on the type of mutation and gene location e.g. mutations in the MYH7 gene are associated with moderate-severe hypertrophy of left ventricle, onset at a younger age. Mutations in the MYBPC3 gene are related to favorable disease course, less left ventricular hypertrophy, slower development which in turn result in a good prognosis (4). TNNT2 gene mutations are associated with minimal left ventricular hypertrophy (5). Thus this study unravels the mutation spectrum in phenotypically positive HCM patients and the impact of these mutations on the pathophysiology of cardiac functioning.

Materials and methods Patient identification and ethics permission
For the current study, the approval from the Institutional Human Ethics Committee of Savitribai Phule Pune University and Bharati Vidyapeeth deemed university Medical college (Ref: BVDV/MC/44) was granted. 30 HCM patients were recruited from Bharati Hospital and Poona hospital and research centre, Pune. All the patients were screened by 2-D echocardiography and patients showing interventricular septum >13mm were taken as phenotypically positive for the current study (Table 1). Patients showing aortic stenosis and hypertension were excluded from the current study. Control samples (n=30) of the same ethnic background and without a family history of cardiac disorders were recruited for the current study.

Blood collection and DNA extraction
Written informed consent was taken from all the participants. 10ml blood samples were received from HCM patients as well as from control individuals by a trained phlebotomist and samples were immediately processed for DNA extraction. DNA extraction was done by the method of phenol: chloroform: isoamyl alcohol. Briefly, 300µl of blood was used for DNA extraction and mixed with 800µl of 1X SSC (salinesodium citrate) buffer at room temperature. Samples were centrifuged at 10,000 rpm for 2 minutes at room temperature. 20µl of 10% SDS and 10ul proteinase K was also added to the sample and pipetting was done back forth to mix the constituents. After incubation at 55 C for 1 hour, phenol-chloroform isoamyl alcohol was added to the solution and vortexed for 30 sec. 1ml of chilled ethanol was added to the solution and constituents were mixed thoroughly until DNA is visible. DNA quantification was done by Nanodrop (Bio spectrophotometer Basic, Eppendorf, Germany).

Restriction digestion of amplified PCR product
PCR products larger than 300 b.p were digested by diluting (three times that of sample volume) with molecular grade water, buffer 2 µl, and 1µl restriction enzyme was added. Digested products were visualized on 2% agarose gels and documented.

Single-Strand Conformational Polymorphism (SSCP) and PAGE
20µl of Phenol: chloroform: isoamyl alcohol was added to the PCR mixture, vortexed for 30 sec. and centrifuged at 10,000 rpm for 2 min at room temperature. The upper watery layer was aspirated and transferred in a fresh tube. 10µl of formamide was added and pipetted back and forth until all constituents are mixed. Samples were denatured at 95 0 C for 10min. and snap-chilled on ice to prevent renaturation of amplified product. 20µl of the sample was loaded in each well on 8-10% polyacrylamide gels. Each exon was optimized on different gel concentrations to obtain maximum results ( Figure 1) ( Table 2).

Silver staining of polyacrylamide gels
Silver staining was done as per the method of (6). Photography was done by digital camera for future records.

PCR purification and Sanger sequencing
Digested PCR products showing aberrant bands concerning control samples were processed for PCR purification by Sure Clean Plus purification kit (Bioline, India). Samples were visualized on 2% agarose gels and processed for bi-directional Sanger sequencing (Macrogen, South Korea).

In silico Analysis
Chromatograms were visualized on Finch TV 1.4.0 and bases showing quality value >20 were processed further. To check whether the particular variation is "novel" in status, dbSNP, 1000 genomes browser and ExAC browser beta data were cross-verified. Secondary protein structure prediction was done by the PSIPRED tool. To validate the impact of missense mutations on protein functionality, the PolyPhen-2 tool was used. To find the impact of variations on splicing, Human Splicing Finder (HSF-3.1) was used.

Results and discussion
In the present study, of all comorbidities in HCM patients maximum percentage of mitral regurgitation (23.3%), tricuspid valve regurgitation (20%), grade I diastolic dysfunction (13.3%) and left auricle dilated  S236G mutation is shown by a proband of 51 years of age with systole/diastole-120/80mmHg, IVS-24mm, LVEF-60%, LA diameter-30mm, LVPWD-17mm, LVIDs-30mm, LVIDd-51mm. Proband shows slit-like left ventricular cavity, LV cavity obliteration during systole, LV diastolic dysfunction, sclerotic aortic valve, trivial aortic regurgitation, Minimal and tricuspid mitral regurgitation, mild pulmonary hypertension, allergic bronchial asthma, LAD-type III, normal slow flow, RCA-dominant normal, PLVostial plaque and asymmetrical septal hypertrophy without a family history of sudden cardiac death. This mutation results in the breakage of the binding site for SF2/ASF (IgM-BRCA1) and the creation of a new site for 9G8 protein as both are exons splicing enhancer proteins which may affect the splicing process as per Human splicing finder 3.1. This mutation was found as benign type as per Polyphen-2 (0.0) and SIFT scores (1.00) (Figure 3). I736T mutation is present in the proband of 67 years of age showing IVS-14mm, LVEF-60%, LA diameter-22mm, aorta-28mm, LVPWD-14mm, LVIDd-34mm, moderate concentric LVH, Grade I LV diastolic dysfunction, No RWMA, and asymmetrical septal hypertrophy without a family history of sudden cardiac death. A new binding site for SRp40 protein (exon splicing enhancer) upstream from the variation site is created which may alter splicing as per Mutation Taster software. With respect to control, mutated sample show loss of β-strand and replacement by α-helix upstream from mutation site which may have a profound impact on protein tertiary structure ( Figure 4) as predicted by Polyphen-2 tool score (probably damaging-1.00). This mutation is highly conserved across different vertebrate groups as the mutation is present in the conserved region thus, having more impact on protein stability as compared to the non-conserved region ( Figure 5).
Proband of 19 years of age with I1066F mutation shows systolic/diatolic-90/60mmHg, IVS-26mm, LVEF-60%, LA diameter-42mm, LVPWD-12mm, LVIDs-21mm, LVIDd-42mm and hypertrophic obstructive cardiomyopathy with a family history of HCM as his father shows asymmetrical septal hypertrophy. This variation results in breakage of exon splicing enhancer site of 9G8 protein in mutant motif as compared to reference motif and may have a potential alteration of splicing as also predicted by mutation taster software. Polyphen-2 predicts a 'benign' type of mutation (score-0.250) as this mutation is present in a less conserved region as per multiple alignments of amino acids ( Figure 6).  (Figure 8). This mutation is probably damaging (score-0.995) as per Polyphen-2 tool score and SIFT online tool prediction (score 0.000). This mutation site is highly conserved in vertebrates as well as in invertebrates (round worms).
MYBPC3 shows the maximum amount of variations in this study as compared to other sarcomeric gene variations. I736T and K233N mutations were more deleterious thus may have a profound impact on protein structure and stability.
MYBPC3 gene mutations are frequently present in most HCM cases representing 30-40% of all HCM mutations (7). Patients associated with MYBPC3 mutations have decreased penetrance, better prognosis, later disease onset, lifelong expectancy. These mutations mostly affect myosin and titin binding sites, whereas missense mutations preserve these binding sites (8). Most of the MYHC mutations are located in the S1 region in HCM probands and their families. ATP-binding pockets mutations of MYHC protein (Thr124Leu, Phe244Leu) may either alter the water structure or binding of phosphate groups in the active site which in turn decreases the catalytic activity of the S1 fragment (9). Arg403Gln mutation at the actinmyosin interface may impact actin and myosin binding by the closure of the connection between actin and ATP binding sites. Mutations on two reactive thiols (Phe513Cys, Gly584Arg) results in conformational changes during sarcomere contraction or alter the conformation in this region. S2 region mutation Leu908Val may lead to defective force transmission from myosin heads to thick filaments (9). Tropomyosin contains α-helical molecules in a thin filament groove. Asp175Asn and Glu180Gly mutations changes surface charge in TPM1 that has been involved in Ca ++ sensitive TnT binding. Asp175Asn mutated skinned fibers show higher Ca ++ sensitivity as compared to controls (ΔpCa50=0.09) but no significant change in cooperativity, maximum force and maximum shortening velocity (10). Thus α-Tm mutations increase force generations at submaximal Ca 2+ concentrations in HCM patients (11). Mutation dosage in the current study does not show much impact on pathophysiology in clinical output, the presence of two mutations as compared to single mutated HCM probands does not significantly change the clinical output or HCM pathogenesis (12). (13) found that 2.5 fold higher incidence of sudden cardiac death with positive family history, severe cardiac hypertrophy and higher LV wall thickness in patients with multiple mutations. Different studies showed that compound heterozygous, double or homozygous mutations have a higher sudden cardiac death rate, more severe left ventricular hypertrophy and episodes of cardiac arrest in family members (14)(15)(16).

Conclusions
Mitral regurgitation was the most prevalent type of comorbidity in HCM patients in the current study. I736T and K33N mutations were more deleterious in terms of cardio-pathophysiology as compared to other benign mutation types. Other sarcomeric groups including TPM1, MYL2 and MYL3 genes were having the worst cardiac outcome as compared to MYH7 and MYBPC3 group and needs to unravel further. Also, double mutated probands do not show severe complications in terms of cardio-physiology as compared to single mutated and genotypic negative patients in this study. polymorphism), LVPWD (left ventricular posterior wall diameter)  /60  26  60  42  12  21  42  ----------------HOCM  P7  67  F  ---14  60  22