新发现候选致病基因NDUFA7在心脏发育中的功能及在肥厚型心肌病中致病机制的研究

Hypertrophic cardiomyopathy (HCM), the most common familial form of cardiomyopathy, is a primary disease of the cardiac muscle affecting one in every 500 people in general population. Although over twenty genes mutations have been associated with the cause of HCM, around 30% of HCM cases have unknown genetic cause. Our group recently performed exome sequencing date of 74 HCM samples and.identified that NDUFA7 gene acts as a potential HCM associated gene. Our previous study showed that zebrafish homologous gene ndufa7 is expressed in somites and heart. In addition, we found that the injection of ndufa7 morpholino leads to low circulation, heart defects and increased expression level of anf and bnf. Further study showed that ndufa7 is involved in cardiac hypertrophy by regulating calcium signaling pathway and ROS level. In this proposal, we will perform pedigree analysis of NDUFA7 gene mutation, and finish the functional analysis of ndufa7 in zebrafish heart development. Furthermore, by establishing NDUFA7 knockout mice and mutation knockin mice, as well as inducing cardiac hypertrophy by transverse aortic constriction, we will study the role of NDUFA7 in hypertrophic cardiomyopathy under physiological and pathological condition. In summary, the bioinformatics and functional analysis of NDUFA7 will improve the understanding of the pathogenesis of hypertrophic cardiomyopathy, and provide novel insights into the clinical diagnosis.

肥厚型心肌病是一种原发于心肌的遗传疾病，目前仍有30%患者的致病基因及突变未知。申请人前期对74例肥厚型心肌病病人的样品进行全基因组外显子组测序，筛选到新的致病基因NDUFA7。前期研究发现，人NDUFA7的同源基因ndufa7在斑马鱼胚胎的体节和心脏中表达；干扰ndufa7的表达导致斑马鱼胚胎的心脏异常、心脏肥大标志物anf、bnf表达量升高。进一步研究发现，ndufa7通过调节钙信号通路和ROS的产生在心肌肥大中发挥作用。在本项目中，我们将在前期工作的基础上，完成肥厚型心肌病病例的测序分析工作，并对NDUFA7基因突变进行家系分析；完成ndufa7在斑马鱼心脏发育中的功能分析；建立小鼠NDUFA7基因敲除和突变敲入模型，并通过主动脉弓缩窄术诱导小鼠心肌肥厚，研究在生理及病理条件下NDUFA7在心肌肥大中的功能及作用机制。本项目将为肥厚型心肌病的临床诊断提供新的思路。

肥厚型心肌病是一种原发于心肌的遗传疾病，目前仍有30%患者的致病基因及突变未知。申请人前期对74例肥厚型心肌病病人的样品进行全基因组外显子组测序，筛选到新的致病基因NDUFA7。首先采用斑马鱼为模式生物进行研究，发现人NDUFA7的同源基因ndufa7在斑马鱼胚胎的体节和心脏中表达；干扰ndufa7的表达导致斑马鱼胚胎的心脏异常、心脏肥大标志物anf、bnf表达量升高。进一步研究发现，ndufa7通过调节钙信号通路和ROS的产生在心肌肥大中发挥作用。进一步构建小鼠心肌肥厚模型，发现NDUFA7在心肌肥厚的小鼠心脏中表达量显著下降。而且，NDUFA7在肥大的心肌细胞中的表达水平也显著下降。与野生型小鼠对比，Ndufa7-/-小鼠的心脏体积、心脏胫骨比以及心脏体重比显著增加。心功能分析发现，Ndufa7-/-小鼠心脏的射血分数和左室短轴缩短率显著下降。而且，Ndufa7-/-小鼠心脏中心肌肥厚标志基因ANP和β-MHC，以及纤维化基因Collagen3的表达水平显著升高。分离小鼠心脏，进行免疫组化病理分析，发现与对照小鼠相比，Ndufa7-/-小鼠的心肌细胞排列紊乱，心肌细胞发生肥大，同时伴有一定程度的心肌纤维化。随着月龄的增加，Ndufa7-/-小鼠的心肌肥大和纤维化程度加剧。进一步的机制研究发现，Ndufa7-/-小鼠的心脏中自噬水平受到显著抑制，而且NDUFA7与自噬重要蛋白LC3相互作用，表明NDUFA7可能通过自噬调节心肌肥厚的发生。本项目研究了NDUFA7在心肌肥厚发生中的功能及作用机制，有望为肥厚型心肌病的临床诊断提供新的思路和分子靶点。