NPPA点突变R123Q在先天性室间隔缺损发病中的作用及机制研究

Ventricular septal defect (VSD) is the most common congenital heart disease (CHD), but its etiology and pathogenesis have not been elucidated. Recent studies have shown that gene mutation may play an important role in the pathogenesis of VSD. In the preliminary work, our research group screened an unreported NPPA point mutation p.R123Q by whole exome sequencing of 6 VSD pedigrees, which may be a pathogenic mutation for VSD. It was reported that myocardial enzyme Corin activates mature ANP by cleaving proANP. This mutation is located at the cleavage site of Corin cleaving proANP. In addition, closure of VSD delayed in Corin knockout embryonic heart. Based on this, we hypothesize that the NPPA R123Q mutation affects the activation of ANP by blocking the cleavage of proANP by Corin, consequently resulting in VSD. To verify the hypothesis, we constructed lentiviral vector, mutant cell line, point mutation knock-in mouse, and performed co-immunoprecipitate, and cell function experiments to investigate the effect of NPPA R123Q mutation on the pathogenesis of VSD, so as to provide a new target for VSD diagnosis and treatment in clinic.

室间隔缺损（VSD）是最常见的先天性心脏病（CHD），其病因及发病机制尚未阐明，研究表明基因突变在VSD的发病过程中发挥重要作用。课题组前期通过6个VSD家系的全外显子组测序筛选出一个未被报道的NPPA点突变R123Q，可能为VSD发生的致病突变。目前已知，心肌酶Corin通过裂解proANP而活化具有心肌保护作用的成熟ANP，该突变位于Corin裂解proANP的切点位置，而Corin基因敲除小鼠胚胎的室间隔闭合延迟。基于此，我们提出假说：NPPA R123Q突变通过阻碍Corin对proANP的裂解作用而影响ANP的活化，从而导致VSD的发生。为验证该假说，我们通过慢病毒载体构建、突变细胞株构建、点突变敲入小鼠构建、免疫共沉淀、细胞功能学实验等，从分子、细胞及动物等水平探讨NPPA R123Q突变在VSD发病中的作用及分子机制，从而为临床上VSD的诊疗提供新的靶点。

室间隔缺损（VSD）是最常见的先天性心脏病（CHD），其病因及发病机制尚未阐明，研究表明基因突变在VSD的发病过程中发挥重要作用。课题组前期通过6个VSD家系的全外显子组测序筛选出一个未被报道的NPPA点突变R123Q，可能为VSD发生的致病突变。本课题主要研究内容包括：(1)NPPA R123Q对Corin裂解proANP作用的影响及分子机制；(2)NPPA R123Q对心肌细胞和心脏成纤维细胞功能的影响；(3)NPPA R123Q点突变小鼠胚胎心脏表型及细胞功能。我们通过慢病毒载体构建、突变细胞株构建、点突变敲入小鼠构建、免疫共沉淀、细胞功能学实验等，从分子、细胞及动物等水平探讨NPPA R123Q突变在VSD发病中的作用及分子机制。结果显示：NPPA R123Q可以阻碍Corin对proANP的裂解作用，其作用机制是该突变通过减弱Corin与proANP的结合作用，从而影响ANP的活化，进而对心肌细胞和成纤维细胞的功能产生影响，导致VSD的产生。基于此，我们得出结论：NPPA R123Q突变通过阻碍Corin对proANP的裂解作用而影响ANP的活化，从而导致VSD的发生，可为临床上VSD的诊疗提供新的靶点。