中国人局灶节段肾小球硬化症致病基因INF2突变及功能研究

Focal segmental glomerulosclerosis (FSGS) is one of the most common primary glomerulonephritis in Chinese patients. The major clinical manifestations is moderate to massive proteinuria. A considerable portion of the patients is insensitive or resistant to steroid and immunosuppressive therapy and progress to end stage renal failure, and eventually require renal replacement therapy,such as hemodialysis or kidney transplantation. Previous study indicated that the genetic background was involved in the incidence of FSGS. And recently INF 2 gene(encoding inverted formin 2) mutations were confirmed to be the major cause of autosomal dominant inherited FSGS and also play an important role in the pathogenesis of the sporadic FSGS. But the exact mechanism remains to be elucidated. We have collected ninety-one late onset FSGS families , and more than nine hundred cases of sporadic FSGS patients during the past fifteen years. We also carried out the screening and functional studies for some of the pathogenic genes cause familial FSGS. Recnetly, we sequenced the INF2 gene on the part of the familial FSGS patients and identified two novel pathogenic mutations. Based on the fundamental work, we will continue the INF2 gene mutation screening work in the remanent familial FSGS patients and also in the steroid-resistant primary FSGS patients. When pathogenic mutations were identified，we will carry out functional analysis to confirm. We plan to study the INF2 gene function both in vitro and in vivo. We will perform plasmid construction,site-directed mutagenesis, podocyte culture and transfection, real-time PCR, western blot, immunohistochemistry, confocal microscopy to study the gene function in molecular, cellular and tissue level.Through the study, we want to clarify the pathgneic role of INF2 mutations in Chinese late-onset familal and steroid-resistant sporadic FSGS patients and to explore the possible mechanism of podocyte injury and incidence of FSGS due to INF2 mutations.

局灶节段性肾小球硬化症（FSGS）是国人最常见的原发性肾小球肾炎之一，临床主要表现为中到大量蛋白尿。其病因不明，患者对现有药物治疗不敏感导致肾功能不断恶化进入终末期肾病。目前认为遗传因素参与FSGS发病，特别是近期发现的编码反转成蛋白-2(inverted formin 2)的基因INF2突变在FSGS发病中起重要作用，但机制有待深入研究。我们前期共收集迟发性FSGS家系91个，散发患者900余例，开展过致病基因ACTN4、TRPC6的筛查和功能研究。最近对部分FSGS家系患者筛查中又发现了INF2新突变位点。本研究将在此工作基础上继续在家系及部分原发FSGS患者中筛查INF2基因突变，并选取发现的突变位点采用构建质粒、定点诱变、细胞转染、免疫组化、共聚焦显微镜等技术于体内外分别在细胞、分子和组织水平进行功能研究。以期明确INF2突变在国人FSGS发病中的作用，并在致病机制研究中取得突破。

局灶节段肾小球硬化症(FSGS)是我国最常见的肾小球肾炎之一,临床治疗效果差,易进展到终末期肾功能衰竭（ESRD），其发病机制尚未完全明确。国外近期研究显示编码反转蛋白-2(inverted formin 2)的基因INF2突变在成人发病型FSGS中起到重要作用。本课题对收集到的FSGS病例进行了从临床病理到致病基因的系统分析，并聚焦于INF2基因突变在国人FSGS发病中的作用及机制研究。首先通过比较家族性FSGS及散发性FSGS患者在临床、病理表现及预后方面的差异，发现家族性FSGS患者病理损伤更严重，预后较差，更值得临床关注。随后在排除其它FSGS致病基因如ACTN4、TRPC6等突变后，在FSGS患者中对INF2基因进行了突变分析：先是结合连锁分析和候选基因测序结果在一个FSGS大家系中发现INF2基因的致病突变S85W，证实INF2突变可导致国人FSGS的发病。随后分批对另外93个FSGS家系进行了INF2的筛查，共发现了5个致病突变（其中3个为新发现基因突变），但在82个激素抵抗的散发性FSGS患者中未能发现致病突变。从而证实INF2突变在我国家族性迟发型FSGS患者发病中起重要作用，可以解释6.4%的家族性FSGS的病因。在进一步的功能研究中，通过全基因组合成和定点诱变技术构建了含有INF2突变位点的质粒。将其和野生型质粒转染到体外培养的足细胞中，结果发现新突变S85W可使足细胞中a-actinin4的细胞定位由纤维丝状改变为弥散性分布，并显著下调a-actinin4蛋白及mRNA的表达水平。同时S85W也可显著下调血清反应因子（SRF）磷酸化水平并使SRF的细胞分布发生改变，并且S85W突变还可以显著抑制INF2与Cdc42的相互作用从而影响和Rho／Rock信号通路的激活。因而可以初步得出结论INF2的致病突变S85W可通过抑制INF2与cdc42之间的相互作用影响INF2活化，进而抑制SRF活化，影响a-actinin4的转录，最终影响正常足细胞骨架的形成。而另一个插入突变S129_Q130ins对该通路的影响不明显，可能存在其它致病途径，需要进一步研究。目前，在第二批家族性FSGS患者筛查中发现的两个致病突变S154F和F248C的功能研究也正在进行中，期望能进一步揭示INF2基因突变造成FSGS的发病机制。