The Identification And Pathogenic Mechanism Of The Novel Mitochondrial16S RRNA2336T>C Mutation Associated With Hypertrophic Cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is a primary disorder characterized by asymmetric thickening of the septum and left ventricular wall, with a prevalence of0.2%in the general population. And it is the main cause of sudden cardiac death (SCD) in adolescents and athletes.Here we reported a family with characteristic of maternally transmitted hypertrophic cardiomyopathy. All the maternal members (4/4) of this family were affected with hypertrophic cardiomyopathy. Echocardiography (ECHO) analysis showed that their maximum left ventricular wall thickness (MLVWT) ranged from26.8mm to46.6mm, and the ratio of interventricular septum thickness to left posterior wall thickness ranged from2.86to3.99, which suggested typical characteristic of hypertrophic cardiomyopathy. In this family, hypertrophic cardiomyopathy was accompanied by complete atrioventricular block (AVB). Furthermore, left ventricular remodeling was also observed in the proband, and finally resulted in the progression of hypertrophic cardiomyopathy to dilated cardiomyopathy (DCM).PCR amplification and sequencing of the mitochondrial DNA derived from maternal members of this family identified a novel16S rRNA2336T>C mutation. This mutation, with high conservation index of94.1%, was absent in2704controls (http://www.genpat.uu.se/mtDB) and350Han Chinese controls. The2336T>C mutation disturbs the2336U-A2438base pair in the stem-loop structure of16S rRNA domain III, which might affect the stability of16S rRNA secondary structure. Through the construction of patient-specific lymphoblastoid cell lines, we found that the mitochondrial DNA copy number was increased about50%in mutant cell lines carrying the2336T>C mutation compared with wild-type cell lines, while the steady-state level of16S rRNA showed no significant difference between this two groups. Meanwhile, the transcription level of binding protein of mitochondrial large ribosomal subunit, including MRPL19and MRPL23, was decreased about22%and28%respectively, and the translation level of MRPL19was decreased about20%. Those data indicated that the2336T>C mutation resulted in decreased expression level of genes associated with mitochondrial ribosomal RNA, and might affect mitochondrial ribosomal assembly. Western blot analysis showed that, in relative to wild-type cell lines, the translation level of subunits of oxidative phosphorylation complex encoded by mitochondrial DNA, including CⅠ-ND1, CⅢ-cytb, CⅣ-MTCO2and C V-ATP6was significantly reduced about39%,41%,21%and49%respectively (P<0.05). Meanwhile, the translation level of subunits of oxidative phosphorylation complex encoded by nuclear DNA, including CⅠ-NDUFA9, CⅡ-SDHA, CIII-UQCRC2, CⅣ-COXⅣ and CⅣ-ATP5A, was reduced about30%,37%,28%,38%and18%respectively (P<0.05). Those data suggested that abnormal assembly of mitochondrial ribosome induced by the2336T>C mutation might further affect mitochondrial DNA translation.Electron microscopic analysis suggested mitochondria with circular appearance, abundant and well-defined cristae in wild-type cell lines, while extremely elongated mitochondria and mitochondria with disorganized and fragmented cristae in mutant cell lines. In addition, the number of mitochondria appeared greater for mutant cell lines than those in wild-type cell lines, which support the hypothesis of energy compromise as a critical factor in the pathogenesis of hypertrophic cardiomyopathy. Functional analysis indicated that the oxygen consumption rate (OCR) in mutant cell lines was decreased about37%, and the mitochondrial ATP synthesis was decreased about29%. The radical oxidative species (ROS) production resulted by mitochondrial dysfunction was increased about52%. Those data suggested that the2336T>C mutation resulted in mitochondrial dysfunction.In addition, mutation screening was also performed in four common pathogenic genes of hypertrophic cardiomyopathy (including MYH7, MYBPC3, TnT2and TnI3) through PCR amplification and sequencing of exons. However, we failed to identify the potential pathogenic mutations. Considering the heteroplasmic clinical presentations of the affected individuals in this family, other factors (including nuclear pathogenic genes, nuclear modifier genes, environmental factors and so on) could not be completely excluded from the involvement in hypertrophic cardiomyopathy of this family.Therefore, the16S rRNA2336T>C mutation resulted in decreased expression level of genes associated with mitochondrial ribosomal RNA, and might affect mitochondrial assembly, and resulted in mitochondrial translation deficiency and abnormal mitochondrial function. Insufficient energy supply (ATP) and oxidative damage (ROS) of myocardial cells, induced by mitochondrial dysfunction, might be the main mechanism that the16S rRNA2336T>C mutation participated in the pathogenesis of hypertrophic cardiomyopathy in this family. In conclusion, for the first time, our study identified the16S rRNA2336T>C mutation associated with maternally inherited hypertrophic cardiomyopathy which was accompanied by complete atrioventricular block, and systematically explored the pathogenic mechanism of this mutation in hypertrophic cardiomyopathy. Our study will provide a new insight in the diagnosis and treatment of hypertrophic cardiomyopathy.