MYH6尾部突变扰乱心肌粗肌丝组装导致房间隔缺损中的作用

Atrial septal defect (ASD) is the most common congenital heart disease (CHD), its etiology is complex. We performed the whole-genome sequencing for a large ASD family, and identified and confirmed that a new MYH6 tail insertion mutation E1823_L1824insRE was the etiology of this family. Previous studies have shown that: MYH6 tail mutation can reduce the content of alpha helix, and affect the assembly of thick filaments, however, the proliferation ability of the myocardial cells with abnormal thick filament are unknown. We propose a new mechanisms that the MYH6 tail mutation cause ASD: MYH6 tail mutation not only disrupt the thick filament assembly, and the MYH7 can not compensation normally, which leads to reduce proliferation of cardiomyocytes in atrial septum. This study further revealed the key problem that the MYH6 mutation affects the tail of the thick filament assembly, and lead to myocardial cells proliferation in atrial septum by immunohistochemistry, protein denaturation and cell proliferation analysis in the Myh6 mutant mouse model, on the basis of previous work. This project will clarify the mechanism of MYH6 tail mutation in ASD, and provide a scientific basis for molecular diagnosis and prenatal screening of CHD.

房间隔缺损是最常见的先心病，病因复杂。本课题组前期对一个大型的房间隔缺损家系进行全基因组测序，发现并验证了一个新的MYH6尾部插入突变E1823_L1824insRE是此家系的病因。研究表明：MYH6尾部突变能降低α-螺旋含量，影响粗肌丝组装；然而这种异常粗肌丝心肌的增殖能力尚且未知。由此我们提出MYH6尾部突变导致房间隔缺损的新机制：MYH6尾部突变不仅扰乱粗肌丝组装，而且使MYH7不能正常补偿，同时导致房间隔区域心肌细胞增殖障碍。本研究拟在前期工作的基础上，在Myh6突变小鼠模型中，进一步采用免疫组化、蛋白质变性和细胞增殖分析等技术，揭示这个MYH6尾部插入突变如何影响粗肌丝组装，并导致房间隔心肌细胞增殖降低这一关键科学假设。本项研究不仅将阐明MYH6尾部插入突变在房间隔缺损中的致病机制，还为先心病分子诊断和产前筛查提供科学依据。

继发孔型房间隔缺损（Atrial septal defect，secundum ，ASDⅡ，OMIM:603642）是我国第二常见的先天性心脏缺陷（congenital heart defect，CHD）。然而，家族性ASDⅡ的遗传病因学仍不清楚。. 本研究采用全外显子组测序（whole-exome sequencing，WES）和Sanger测序，在一个患有ASDⅡ的中国汉族四代大家系中鉴定了一个新的肌球蛋白重链6（MYH6）基因插入变异NM_.3:c.5465 dup（R1822_E1823dup）。MYH6 R1822_E1823dup变异在该家系中呈常染色体显性遗传，并且与疾病共分离。插入变异位于MYH6蛋白的卷曲螺旋结构域，这个结构域上的氨基酸在同源肌球蛋白和不同物种之间高度保守。在转染成肌细胞C2C12细胞系中，MYH6 R1822_E1823dup变异显著地损害了肌原纤维的组装，并且显著地使细胞凋亡增加，但是并未显著降低细胞活性。此外，分子模拟显示R1822_E1823dup变异损伤了肌球蛋白α-螺旋，增加了肌球蛋白通过卷曲螺旋形成二聚体时的稳定性，表明该变异对卷曲螺旋结构域的自聚集存在显著影响。这些结果表明R1822_E1823dup变异在ASDⅡ的发病机制中起了重要作用。. 本研究结果扩大了家族性ASDⅡ相关MYH6基因变异谱，为该家系的遗传咨询和产前诊断提供了分子基础和数据支持。