Functional and Phenotypic Characterization of a New FSGS Gene

描述(由申请人提供)：局灶性节段性肾小球硬化(FSGS)是肾功能衰竭的常见原因，是改变肾小球滤过屏障(GFB)功能完整性的致病改变的结果。对家族性FSGS病例的研究指出足细胞在其发病机制中的中心作用。我们的长期目标是通过识别对维持GFB功能完整性至关重要的通路来了解FSGS的分子发病机制，并确定FSGS的新治疗靶点。这项应用的总体目标是研究F-肌动蛋白结合细胞周期基因ANLN突变导致FSGS的机制。我们的方法是可行的，因为我们最近发现ANLN的F-肌动蛋白结合域R431C突变是家族性FSGS的原因之一。我们发现，与正常肾组织相比，FSGS塌陷型患者的肾活检组织中苯胺素表达上调。阿尼林与关键的足细胞蛋白共定位，这些蛋白对维持肌动蛋白细胞骨架的完整性很重要。我们还发现，敲除斑马鱼胚胎中的苯胺素会破坏GFB。我们的主要假设是，苯丙胺的F-肌动蛋白结合域的突变影响F-肌动蛋白细胞骨架聚合，导致足细胞异常增殖和迁移；足细胞动态平衡的破坏，进而扰乱正常的GFB功能，导致FSGS的发病。我们将通过以下具体目标来探讨这一假说：1)确定ANLN R431C导致足细胞动态平衡中断的机制。我们将研究R431C变异体对足细胞运动和增殖的影响，以及对苯青素及其受体亚细胞定位的影响。 与已知和新鉴定的足细胞蛋白的相互作用。2)研究R431C突变和其他ANLN变异对斑马鱼胚胎GFB的功能影响。我们将使用体内互补试验来确定R431C ANLN突变和其他新变异的等位基因致病性，使用肾小球滤过作为生理相关读数。3)分析一组FSGS患者ANLN基因突变情况。我们将对我们的FSGS患者队列中ANLN的外显子进行测序，并比较有无突变的受试者的疾病表型。所有新变种的致病性将在斑马鱼中进行测量。创新：这项提案代表了第一项旨在定义苯青素变异导致FSGS的机制的研究。意义：解开ANLN突变导致FSGS的机制可能会确定对维持足细胞细胞骨架功能完整性至关重要的途径。此外，通过探讨苯青素在细胞增殖和运动中的作用，我们将深入了解足细胞更新的机制。 在健康和疾病方面。我们的遗传和机制方法将促进我们对FSGS足细胞表型变化的分子发病机制的理解，并导致识别新的治疗靶点和毒性较低的药理学方法。

DESCRIPTION (provided by applicant): Focal segmental glomerulosclerosis (FSGS), a common cause of kidney failure, is the result of pathogenic changes that alter the functional integrity of the glomerular filtration barrier (GFB). The study of familial FSGS cases points to a central role of the podocyte in its pathogenesis. Our long term goals are to understand the molecular pathogenesis of FSGS by identifying pathways that are critical for the maintenance of the functional integrity of the GFB and identify novel therapeutic targets for FSGS. The overall objective of this application is to study the mechanisms by which mutations in an F-actin binding cell cycle gene, ANLN, causes FSGS. Our approach is feasible because we recently identified a mutation in F-actin binding domain of ANLN, R431C, as a cause of familial FSGS. We showed that anillin is upregulated in kidney biopsies of subjects with collapsing FSGS compared with normal kidney. Anillin co-localizes with key podocyte proteins that are important in maintaining the integrity of the actin cytoskeleton. We also showed that knockdown of anillin in zebrafish embryos disrupts the GFB. Our overarching hypothesis is that mutations in the F-actin binding domain of anillin affect F-actin cytoskeleton polymerization and lead to aberrant podocyte proliferation and migration; disruption of podocyte homeostasis then disrupts normal GFB function and leads to the pathogenesis of FSGS. We will explore this hypothesis through the following specific aims: 1) To determine the mechanisms by which ANLN R431C causes disruption of podocyte homeostasis. We will characterize the effect of R431C variant on a) podocyte motility and proliferation in vitro and b) the subcellular localization of anillin and its interactions with known and newly identified podocyte proteins. 2) To determine the functional effect of the R431C mutation and other ANLN variants on the GFB of zebrafish embryos. We will use an in vivo complementation assay to determine allele pathogenicity of R431C ANLN mutations and other new variants using glomerular filtration as a physiologically relevant readout. 3) To analyze mutations of the ANLN gene in a cohort of patients with FSGS. We will sequence the exons of ANLN in our cohort of FSGS patients and compare the disease phenotype in subjects with and without mutations. The pathogenicity of all new variants will be measure in zebrafish. Innovation: This proposal represents the first study designed to define the mechanisms by which anillin variants cause FSGS. Significance: Unraveling the mechanisms by which mutations in ANLN cause FSGS may identify pathways that are important for maintaining the functional integrity of the podocyte cytoskeleton. Furthermore, by probing the role of anillin in cell proliferation and motility, we will provide insight into the mechanisms of podocyte renewal in health and disease. Our genetic and mechanistic approaches will advance our understanding of the molecular pathogenesis of podocyte phenotype changes in FSGS and lead to identification of novel therapeutic targets and less toxic pharmacologic approaches.