Novel Mutations in ß-MYH7 Gene in Indian Patients With Dilated Cardiomyopathy.

BACKGROUND: Heart failure is a hallmark of severe hypertrophic cardiomyopathy and dilated cardiomyopathy (DCM). Several mutations in the ß-MYH7 gene lead to hypertrophic cardiomyopathy. Recently, causative mutations in the ß-MYH7 gene have also been detected in DCM from different populations. METHODS: Here, we sequenced the ß-MYH7 gene in 137 Indian DCM patients and 167 ethnically matched healthy controls to detect the frequency of mutations and their association. RESULTS: Our study revealed 27 variations, of which 7 mutations (8.0%) were detected exclusively in Indian DCM patients for the first time. These included 4 missense mutations-Arg723His, Phe510Leu, His358Leu, and Ser384Tyr (2.9%); a frameshift mutation-Asn676_T-del (1.5%); and 2 splice-site mutations (IVS17+2T) T>G and (IVS19-1G) G>A (3.6%). Remarkably, all 4 missense mutations altered evolutionarily conserved amino acids. All 4 missense mutations were predicted to be pathogenic by 2 bioinformatics tools-polymorphism phenotyping v2 (PolyPhen-2) and sorting intolerant from tolerant (SIFT). In addition, the 4 homology models of ß-MYH7-p.Leu358, p.Tyr384, p.Leu510, and p.His723-displayed root-mean-square deviations of ∼2.55 Å, ∼1.24 Å, ∼3.36 Å, and ∼3.86 Å, respectively. CONCLUSIONS: In the present study, we detected numerous novel, unique, and rare mutations in the ß-MYH7 gene exclusively in Indian DCM patients (8.0%). Here, we demonstrated how each mutant (missense) uniquely disrupts a critical network of non-bonding interactions at the mutation site (molecular level) and may contribute to development of dilated cardiomyopathy (DCM). Therefore, our findings may provide insight into the understanding of the molecular bases of disease and into diagnosis along with promoting novel therapeutic strategies (through personalized medicine).

INTRODUCTION: L'insuffisance cardiaque est une caractéristique de la cardiomyopathie hypertrophique grave et de la cardiomyopathie dilatée (CMD). Plusieurs mutations dans le gène ß-MYH7 conduisent à la cardiomyopathie hypertrophique. Récemment, les mutations causales dans le gène ß-MYH7 ont également été détectées au sein de différentes populations atteintes de CMD. MÉTHODES: Ici, nous avons séquencé le gène ß-MYH7 de 137 patients indiens atteints de CMD et de 167 témoins sains appariés selon l'origine ethnique pour détecter la fréquence des mutations et leur association. RÉSULTATS: L'étude nous a permis de révéler 27 variations, dont sept mutations (8,0 %) étaient exclusivement détectées chez les patients indiens atteints de CMD pour la première fois. Parmi ces mutations, nous avons observé quatre mutations faux-sens­Arg723His, Phe510Leu, His358Leu et Ser384Tyr (2,9 %), une mutation par déphasage­Asn676_T-del (1,5 %) et deux mutations des sites d'épissage (IVS17+2T) T>G et (IVS19-1G) G>A (3,6 %). Étonnamment, les quatre mutations faux-sens changeaient les acides aminés évolutivement conservés. Selon deux outils bioinformatiques­PolyPhen-2 (de l'anglais, polymorphism phenotyping v2) et SIFT (de l'anglais, sorting intolerant from tolerant), les quatre mutations faux-sens devaient être pathogènes. De plus, les quatre modélisations de ß-MYH7 par homologie­p.Leu358, p.Tyr384, p.Leu510 et p.His723­affichaient de façon respective des écarts quadratiques moyens de ∼2,55 Å, ∼1,24 Å, ∼3,36 Å et ∼3,86 Å. CONCLUSIONS: Dans la présente étude, nous avons détecté de nombreuses nouvelles mutations, uniques et rares, dans le gène ß-MYH7, exclusivement chez les patients indiens atteints de CMD (8,0 %). Ici, nous avons démontré comment chaque mutant (faux-sens) perturbe de manière unique un réseau essentiel d'interactions non liantes au site de mutation (moléculaire) et peut contribuer à la survenue de la CMD. Par conséquent, les conclusions de notre étude peuvent donner un aperçu des bases moléculaires de la maladie et du diagnostic tout en favorisant la promotion de nouvelles stratégies thérapeutiques (par la médecine personnalisée).

Ribosomal protein S6 kinase beta-1 gene variants cause hypertrophic cardiomyopathy.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is a genetic heart muscle disease with preserved or increased ejection fraction in the absence of secondary causes. Mutations in the sarcomeric protein-encoding genes predominantly cause HCM. However, relatively little is known about the genetic impact of signalling proteins on HCM. METHODS AND RESULTS: Here, using exome and targeted sequencing methods, we analysed two independent cohorts comprising 401 Indian patients with HCM and 3521 Indian controls. We identified novel variants in ribosomal protein S6 kinase beta-1 (RPS6KB1 or S6K1) gene in two unrelated Indian families as a potential candidate gene for HCM. The two unrelated HCM families had the same heterozygous missense S6K1 variant (p.G47W). In a replication association study, we identified two S6K1 heterozygotes variants (p.Q49K and p.Y62H) in the UK Biobank cardiomyopathy cohort (n=190) compared with matched controls (n=16 479). These variants are neither detected in region-specific controls nor in the human population genome data. Additionally, we observed an S6K1 variant (p.P445S) in an Arab patient with HCM. Functional consequences were evaluated using representative S6K1 mutated proteins compared with wild type in cellular models. The mutated proteins activated the S6K1 and hyperphosphorylated the rpS6 and ERK1/2 signalling cascades, suggesting a gain-of-function effect. CONCLUSIONS: Our study demonstrates for the first time that the variants in the S6K1 gene are associated with HCM, and early detection of the S6K1 variant carriers can help to identify family members at risk and subsequent preventive measures. Further screening in patients with HCM with different ethnic populations will establish the specificity and frequency of S6K1 gene variants.

RAF1 mutations in childhood-onset dilated cardiomyopathy.

Dilated cardiomyopathy (DCM) is a highly heterogeneous trait with sarcomeric gene mutations predominating. The cause of a substantial percentage of DCMs remains unknown, and no gene-specific therapy is available. On the basis of resequencing of 513 DCM cases and 1,150 matched controls from various cohorts of distinct ancestry, we discovered rare, functional RAF1 mutations in 3 of the cohorts (South Indian, North Indian and Japanese). The prevalence of RAF1 mutations was ~9% in childhood-onset DCM cases in these three cohorts. Biochemical studies showed that DCM-associated RAF1 mutants had altered kinase activity, resulting in largely unaltered ERK activation but in AKT that was hyperactivated in a BRAF-dependent manner. Constitutive expression of these mutants in zebrafish embryos resulted in a heart failure phenotype with AKT hyperactivation that was rescued by treatment with rapamycin. These findings provide new mechanistic insights and potential therapeutic targets for RAF1-associated DCM and further expand the clinical spectrum of RAF1-related human disorders.

Promoter hypermethylation analysis in myelodysplastic syndromes: diagnostic & prognostic implication.

BACKGROUND & OBJECTIVE: Myelodysplastic syndromes (MDS) are a heterogenous group of haematopoietic stem cell disorders that are multifactorial in their aetiology. Unique genetic alterations in combinations or in isolation account for a small fraction of MDS suggesting the epigenetic hypermethylation as a possible leading cause for MDS and its transformation to acute myelocytic leukaemia (AML). Therefore, in this study, promoter hypermethylation status of key cell cycle regulators was assessed as markers in MDS patients and association of hypermethylation with clinical progression of disease was also studied. METHODS: Promoter hypermethylation analysis of five tumour associated genes namely p16, p15, MGMT, hMLH1 and E-cadherin were done for 41 MDS patient samples with its various subtype. The hypermethylation analysis was done by using semi-nested multiplex PCR. RESULTS: Eighty per cent of (33/41) of the MDS samples were found to be methylated in any one of the four genes (p16, p15, MGMT and E-cadherin). The p15 methylation was found to be the most frequent 61 per cent (25/41), E-cadherin was methylated in 39 per cent (16/41) and p16 in 37 per cent (15/41) of the cases. MGMT gene showed a low 5 per cent (2/41) methylation whereas hMLH1 gene was not methylated in any one of the samples analysed. INTERPRETATION & CONCLUSION: Differential rate of methylation of the four genes (p16, p15, MGMT and E-cadherin) was observed in MDS samples. All the samples analysed showed the absence of a methylator phenotype in MDS. The methylation frequency of all these genes increased with the clinical severity of the MDS subtypes. Therefore, hypermethylation may be used as a diagnostic and prognostic tool in ascertaining the clinical severity of MDS.

ACE I/D polymorphism in Indian patients with hypertrophic cardiomyopathy and dilated cardiomyopathy.

AIM: The study was carried to determine the association of angiotensin converting enzyme (ACE) insertion/deletion (I/D) polymorphism with the risk of hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), and restrictive cardiomyopathy (RCM). METHODS AND RESULTS: A total of 174 patients diagnosed with cardiomyopathy (118 with HCM, 51 with DCM, and 5 with RCM) and 164 ethnically, age- and gender-matched controls were included in the study. ACE I/D genotyping was performed by PCR. In total, 25.86% of the patients were in New York Heart Association (NYHA) class III and IV at presentation. A total of 67.24% patients had dyspnea, 56.89% had angina pectoris, and 25.28% of the patients had at least one event of syncope. Frequency of occurrence of the disease was more in male patients compared to female patients (P < 0.05). After adjustment for age, sex, body mass index (BMI), and smoking habit, the prevalence of ACE DD genotype, and ACE 'D' allele was significantly higher in patients as compared to controls and was associated with increased risk (DD: OR 2.11, 95% CI 1.27-3.52, P < 0.05; 'D': OR 1.91, 95% CI 1.08-3.35, P < 0.05). The mean septal thickness was higher for DD and ID genotypes (20.40 +/- 3.73 mm and 21.82 +/- 5.35 mm, respectively) when compared with II genotype (18.63 +/- 6.69 mm) in HCM patients, however, the differences were not significant statistically (P > 0.05). The DCM patients with ID genotype showed significantly decreased left ventricular ejection fraction (LVEF) at enrolment (26.50 +/- 8.04%) (P = 0.04). CONCLUSION: Our results suggest that D allele of ACE I/D polymorphism significantly influences the HCM and DCM phenotypes.