一个新的限制性心肌病致病基因鉴定及致病机制研究

Restrictive cardiomyopathy (RCM) is characterized by increased stiffness of the myocardium with diastolic dysfunction without significant hypertrophy. Genetic factor has showed to be important in RCM. In preliminary work, one pedigree was collected, and target resequencing of known genes related to RCM was done in order to identify pathogenic mutation, but no mutation was found. Then whole exome sequencing was performed. After data analysis, one SLC4A3 nonsense mutation (p.S1210X) was found, and the mutation is co-separation with the phenotype in this pedigree. Based on these, this research will focus on the study the pathogenic possibility and the molecular mechanism of SLC4A3 in RCM..In this study, firstly, mutation screening of SLC4A3 will be performed in more sporadic cases or familial individuals, to provide genetic evidence that SCL4A3 is a novel causative gene for RCM. Secondly, in vitro protein expression studies, Cl-/HCO3- exchange assays were performed, and subcellular localization and interaction with other proteins of mutant SLC4A3 proteins will be detected by confocal microscopy and co-immunoprecipitation. Finaly, Slc4a3S1210X knock-in mouse model was made by TALEN technology. In transgenic mouse model, M-mode echocardiography was used to monitor cardiac function, immunohistochemical staining, whole genome microarray and co-immunoprecipitation methods were performed to investigate the influence of Slc4a3S1210X on cardiac structure and function; to explain the molecular mechanism of its roles in RCM; to provide the molecular basis of SLC4A3 regulation roles in myocardial cell; and finally maybe provide a new therapy target for RCM.

限制性心肌病（RCM）是由于心室壁僵硬使得心腔充盈压升高而导致舒张功能异常的一种心肌病。遗传因素在RCM的发病中起重要作用。本项目前期收集到一个RCM家系，通过靶基因再测序后排除了已报道基因致病的可能，采用全外显子组测序发现一个SLC4A3基因无义突变（p.S1210X），该突变在家系中与患者表型共分离。本项目以此为切入点，进行SLC4A3基因致病的可能性及致病机制研究。首先继续收集RCM病例进行SLC4A3基因突变筛查，寻求SLC4A3基因突变致病的遗传学证据；其次从细胞水平上检测突变体对蛋白功能的影响，蛋白亚细胞定位和蛋白间相互作用的变化；最后构建Slc4a3S1210X knock-in小鼠模型，通过心脏超声、免疫组化、表达芯片及免疫沉淀等方法，探讨Slc4a3S1210X对心脏结构和功能的影响及其分子机制，为SLC4A3在心肌细胞中的重要作用提供理论基础，为RCM的治疗提供新靶点。

限制性心肌病（restrictive cardiomyopathy，RCM）是由于心室壁僵硬使得心腔充盈压升高而导致舒张功能异常的一种心肌病。项目组收集到一个RCM家系，通过靶基因再测序排除了已报道基因致病的可能，采用全外显子组测序发现一个SLC4A3基因无义突变（p.S1210X）。该突变在家系中与患者表型共分离。随后，用CRISPR/Cas9方法构建Slc4a3S1210X knock-in小鼠模型，对获得的小鼠模型通过心脏超声、免疫组化、表达芯片及信号通路蛋白表达实验等方法，探讨Slc4a3S1210X对心脏结构和功能的影响及其分子机制。结果显示：1、超声心动结果显示：突变小鼠组室间隔厚度（IVS）略有增厚，左心室大小（LVID）有所减小，左室后壁厚度（LVPW）也略有增加，但统计学差异不显著处理。但在8月龄小鼠组，突变小鼠MV E/e'比值有所增加，统计学差异显著（P＜0.05），说明模型小鼠心脏舒张功能受损。2、免疫组化：选取性别、月龄相匹配的转基因鼠和野生型小鼠，将包埋的心脏切成4μm厚的切片，用苏木精-伊红（hematoxylin and eosin，HE）染色观察各腔室大小及各房室间壁厚度，用天狼猩红染色（Sirius red stain）观察胶原在心肌中分布的情况。免疫组化显示AE3蛋白在心肌细胞中的分布情况。结果显示：HE染色未见不同基因型小鼠心脏各腔室大小及各室间隔壁厚度有明显差异；AE3蛋白在不同基因型小鼠心肌细胞分布无明显差异；天狼猩红染色结果显示：未见明显差异。3、表达芯片：选取性别、月龄相匹配的转基因鼠和野生型小鼠，提取心室组织总RNA，进行RNAseq。结果显示：经KEGG富集分析，突变鼠与野生鼠之间RNA表达水平在钙离子信号通路及MAPK信号通路上有差异。4、信号通路蛋白表达实验：选取MAPK信号通路相关蛋白（p-Raf，p-Mek，p-Erk）进行Western-blot实验，结果显示，这些蛋白在突变鼠组与野生鼠组之间表达差异不显著。结论：通过构建knock-in小鼠模型并对模型进行体内及体外实验，发现大龄鼠（8月龄以上）的心脏舒张功能有受损，与人类表型相一致。但经一系列体内生化分析，目前还没有找到Slc4a3S1210X致病的直接证据。