Correlation of mitochondrial tRNA variants with coronary heart disease in a Chinese pedigree
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目的 探讨线粒体DNA变异和冠心病的相关性，研究1个母系遗传冠状动脉粥样硬化性心脏病(CHD)的可能机制。 方法 选取2022年5月10日在杭州市第一人民医院就诊的1例女性CHD先证者及其母系遗传CHD家系成员作为研究对象，收集该家系的先证者和母系CHD成员的临床资料。通过对先证者及家系成员进行线粒体DNA测序，对照正常线粒体基因，经过数据比对筛选变异位点。针对变异位点，进行物种间的保守性分析并使用生物信息学软件预测变异位点对tRNA二级结构的影响。Real-time PCR检测线粒体拷贝数，并建立转线粒体细胞系进行线粒体功能分析，包括膜电位和ATP水平测定。 结果 该家系共4代32人，母系成员10人，患CHD有4人，外显率为40%。测序结果发现先证者和母系成员携带m.4420A>T及m.10463T>C变异，2个变异位点在物种间均具有高度保守性。m.4420A>T位于tRNAMet D环第22位碱基上，m.4420A>T变异破坏了原有13T-22A碱基配对，可能会引起tRNA代谢障碍。m.10463T>C位于tRNAArg接收臂的第67位碱基，该位点碱基与tRNA结构稳定性相关。实验发现携带m.4420A>T和m.10463T>C变异的家系成员的线粒体拷贝数、膜电位和ATP水平均低于正常对照(P<0.05)，分别平均下降约50.47%、39.6%及47.4%。 结论 线粒体tRNAMet 4420A>T和tRNAArg 10463T>C变异可能是这个母系CHD家系的重要分子基础。该家系表现出mtDNA同质性变异、发病年龄以及临床表型等差异，提示核基因、环境因素和线粒体遗传背景对家系成员的冠心病发病进程有一定的影响。 Objective To explore the correlation of mitochondrial DNA (mtDNA) variants and coronary heart disease (CHD) in a Chinese pedigree and the possible molecular mechanisms. Methods A Chinese pedigree featuring matrilineal inheritance of CHD who visited Hangzhou First People′s Hospital in May 2022 was selected as the study subject. Clinical data of the proband and her affected relatives was collected. By sequencing the mtDNA of the proband and her pedigree members, candidate variants were identified through comparison with wild type mitochondrial genes. Conservative analysis among various species was conducted, and bioinformatics software was used to predict the impact of variants on the secondary structure of tRNA. Real-time PCR was carried out to determine the copy number of mtDNA, and a transmitochondrial cell line was established for analyzing the mitochondrial functions, including membrane potential and ATP level. Results This pedigree had contained thirty-two members from four generations. Among ten maternal members, four had CHD, which yielded a penetrance rate of 40%. Sequence analysis of proband and her matrilineal relatives revealed the presence of a novel m. 4420A>T variant and a m. 10463T>C variant, both of which were highly conserved among various species. Structurally, the m. 4420A>T variant had occurred at position 22 in the D-arm of tRNAMet, which disrupted the 13T-22A base-pairing, while the m. 10463T>C variant was located at position 67 in the acceptor arm of tRNAArg, a position critical for steady-state level of the tRNA. Functional analysis revealed that patients with the m. 4420A>T and m. 10463T>C variants exhibited much fewer copy number of mtDNA and lower mitochondrial membrane potential (MMP) and ATP contents (P< 0.05), which were decreased by approximately 50.47%, 39.6% and 47.4%, respectively. Conclusion Mitochondrial tRNAMet 4420A>T and tRNAArg 10463T>C variants may underlay the maternally transmitted CHD in this pedigree, which had shown variation in mtDNA homogeneity, age of onset, clinical phenotype and other differences, suggesting that nuclear genes, environmental factors and mitochondrial genetic background have certain influence on the pathogenesis of CHD.
Objective To explore the correlation of mitochondrial DNA (mtDNA) variants and coronary heart disease (CHD) in a Chinese pedigree and the possible molecular mechanisms. Methods A Chinese pedigree featuring matrilineal inheritance of CHD who visited Hangzhou First People′s Hospital in May 2022 was selected as the study subject. Clinical data of the proband and her affected relatives was collected. By sequencing the mtDNA of the proband and her pedigree members, candidate variants were identified through comparison with wild type mitochondrial genes. Conservative analysis among various species was conducted, and bioinformatics software was used to predict the impact of variants on the secondary structure of tRNA. Real-time PCR was carried out to determine the copy number of mtDNA, and a transmitochondrial cell line was established for analyzing the mitochondrial functions, including membrane potential and ATP level. Results This pedigree had contained thirty-two members from four generations. Among ten maternal members, four had CHD, which yielded a penetrance rate of 40%. Sequence analysis of proband and her matrilineal relatives revealed the presence of a novel m. 4420A>T variant and a m. 10463T>C variant, both of which were highly conserved among various species. Structurally, the m. 4420A>T variant had occurred at position 22 in the D-arm of tRNAMet, which disrupted the 13T-22A base-pairing, while the m. 10463T>C variant was located at position 67 in the acceptor arm of tRNAArg, a position critical for steady-state level of the tRNA. Functional analysis revealed that patients with the m. 4420A>T and m. 10463T>C variants exhibited much fewer copy number of mtDNA and lower mitochondrial membrane potential (MMP) and ATP contents (P< 0.05), which were decreased by approximately 50.47%, 39.6% and 47.4%, respectively. Conclusion Mitochondrial tRNAMet 4420A>T and tRNAArg 10463T>C variants may underlay the maternally transmitted CHD in this pedigree, which had shown variation in mtDNA homogeneity, age of onset, clinical phenotype and other differences, suggesting that nuclear genes, environmental factors and mitochondrial genetic background have certain influence on the pathogenesis of CHD.
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