Identification and function of new genes causing childhood nephrotic syndrome

儿童局灶节段性肾小球硬化（FSGS）是一种越来越普遍的疾病，表现为类固醇抵抗性肾病综合征（SRNS），并导致终末期肾脏疾病。该病严重干扰受影响儿童的心理社会发育。由于这种疾病的发病机制在很大程度上是未知的，治疗方案是有限的和有争议的。使用多种免疫抑制剂和细胞毒性药物治疗这些患者会带来相当高的发病率和死亡率。儿童FSGS发病机制的最新突破来自于定位克隆在儿童FSGS和先天性肾病综合征中发生突变的新基因（NPHS1/nephrin, NPHS2/podocin， LAMB2等）。这些发现表明，单个基因的隐性突变足以引起FSGS。这为在了解这些疾病的分子发病机制的基础上开发新的治疗和预防方法提供了机会。为了实现这一目标，我们取得了以下进展：首先，我们发现NPHS2突变是FSGS的常见原因，占所有儿童病例的28%，并且在生命第一年的所有SRNS病例中，66%可以通过仅四个基因的突变来解释。我们对来自全球队列的bb101300名个体进行了突变分析，并检测了基因型-表型相关性，即所有具有两个NPHS2突变的儿童都具有类固醇抗性，但在肾移植中FSGS复发的风险降低。其次，自首次提交以来，我们通过定位克隆和新的候选基因选择策略确定了我们定位到SRN3位点的基因，通过显示PLCE1基因（磷脂酶C epsilon）突变是隐性早发性肾病综合征的新原因。根据突变类型的不同，儿童的组织学表现为弥漫性系膜硬化（DMS）或FSGS。值得注意的是，一些患者对类固醇或环孢素A治疗有反应。因此，我们确定了第一个在类固醇敏感性肾病综合征中描述的基因。我们通过证明PLCel在肾小球发育中对nephrin和podocin表达的作用，并检测到其与肾小球蛋白IQGAP1的相互作用，来表征PLCel的致病作用。我们为这种疾病建立了一个斑马鱼模型，这将对治疗研究有用。第三，我们分别在14q24.3和13q染色体上定位了两个新的隐性FSGS基因位点（SRN2和SRN4）。在这些初步数据的基础上，我们打算：1)鉴定一个导致FSGS/SRNS的新基因（SRN2）并对其进行功能表征。2)拓展PLCE1突变作为SRNS/SSNS新发现病因的发病机制功能研究。3)通过定位克隆鉴定SRN4基因，并对其功能进行鉴定和定位。正如PLCE1/SRN3突变所显示的那样，这些研究将为儿童FSGS的分子基础提供新的见解，将为遗传学发现转化为临床应用提供有用的生物标志物，并将有助于开发用于肾病综合征药物测试的动物模型。

DESCRIPTION (provided by applicant): Focal segmental glomerulosclerosis (FSGS) of childhood is an increasingly prevalent disease that manifests as steroid resistant nephrotic syndrome (SRNS) and leads to end stage kidney disease. The disease severely interferes with psychosocial development of affected children. Since the pathogenesis of this disease group is largely unknown, treatment options are limited and controversial. Treatment of these patients with multiple immunosuppressive and cytotoxic agents brings about considerable morbidity and mortality. Recent breakthroughs in understanding the pathogenesis of childhood FSGS have come from positional cloning of novel genes (NPHS1/nephrin, NPHS2/podocin, LAMB2, and others) that are mutated in children with FSGS and congenital nephrotic syndrome. These findings demonstrated that recessive mutations in a single gene are sufficient to cause FSGS. This offers the opportunity to develop new approaches towards treatment and prevention based on understanding the molecular pathogenesis of these conditions. Towards this goal we have made the following progress: First, we showed that mutations in NPHS2 are a frequent cause of FSGS, accounting for 28% of all childhood cases, and that 66% of all SRNS cases in the first year of life can be explained by mutations in four genes only. We performed mutational analysis in >1,300 individuals from a worldwide cohort and detected the genotype-phenotype correlation that all children with two NPHS2 mutations are steroid resistant, but have a reduced risk for FSGS recurrence in a kidney transplant. Second, since first submission, we identified the gene that we mapped to the SRN3 locus using positional cloning and a new candidate gene selection strategy, by showing that mutations in the PLCE1 gene (phospholipase C epsilon) are a new cause of recessive early-onset nephrotic syndrome. Depending on the type of mutation, children have either the histologic picture of diffuse mesangial sclerosis (DMS) or of FSGS. Remarkably, some patients responded to steroid or cyclosporine A treatment. We thereby identified the first gene ever described in steroid sensitive nephrotic syndrome. We characterized the pathogenic role of PLCel by demonstrating its role for nephrin and podocin expression in glomerular development, and detected an interaction with the glomerular protein IQGAP1. We generated a zebrafish model for this disease, which will be useful for therapeutic studies. Third, we have mapped two new gene loci (SRN2 and SRN4) for recessive FSGS on chromosomes 14q24.3 and 13q, respectively. On the basis of these preliminary data we intend to: 1) Identify and functionally characterize a new gene (SRN2) that causes FSGS/SRNS. 2) Expand functional studies on the disease mechanisms of PLCE1 mutations as a newly discovered cause of SRNS/SSNS. 3) Identify by positional cloning and functionally characterize the SRN4 gene and map additional loci. As shown for PLCE1/SRN3 mutations, these studies will generate new insights into the molecular basis of childhood FSGS, will provide useful biomarkers for translation of genetic findings into clinical applications, and will help develop animal models for drug testing in nephrotic syndrome.