外显子组测序技术检测家族性激素耐药型肾病综合征基因突变的研究

Steroid-resistant nephrotic syndrome (SRNS) in childhood may be a risk factor for the development of the end-stage renal disease. SRNS can be expected to be a monogenic disorder. Over the past decade, mutations in nearly 21 different genes that affect multiple podocyte functions have been linked with focal segmental glomerulosclerosis (FSGS). Many patients with this disease do not respond to treatment with glucocorticoids, and immunosuppressive therapy often fails as well. The pathogenic mechanisms in the vast majority (>80%) of childhood-onset SRNS is still uncertain. Since 2006, we have apply traditional Sanger sequencing in patients with SRNS to explore the mutations in genes encoding protein of the slit-diaphragm complex (NPHS1, which encodes nephrin, NPHS2, which encodes podocin, and CD2AP, which encodes CD2AP-associated protein) and in genes encoding proteins associated with the podocyte cell membrane (PLCE1, which encodes phospholipase C, epsilon 1, a membrane phospholipid catalyst), as well as mutations in WT1 (the Wilms' tumor gene, mainly in connection with syndromes in which FSGS may be seen). As a result, the genetic cause of 3 families and some sporadic patients with SRNS remains undiscovered. In this further study, the mutations in the other 16 genes reported on FSGS will firstly be excluded by Sanger sequencing in this 3 families, which are strongly indicative of a simple autosomal recessive inheritance. Secondly, linkage analysis followed by exome sequencing will be carried out to narrow the candidate loci and define the candidate variants within these loci. Thirdly, the causative variants will be further determined by direct sequencing. Finally, routine histologic and immunohistochemical investigation will be performed on human kidney-biopsy specimens and cultured podocytes. Expression studies in knockout mice are also performed to characterize the functional consequences of the mutations identified. The objective of this study is to unravel the genetic basis of SRNS and provide new starting points for the treatment of SRNS.

儿童激素耐药型肾病综合征（SRNS）是导致终末期肾脏病的常见病因。目前已证实原发性SRNS是一种单基因遗传病。尽管至今研究发现21个单基因突变可致病，但是仍有80%的儿童SRNS的病因尚不清楚。自2006年以来本课题组采用传统测序方法相继对所收集家族性和散发性SRNS患者进行NPHS1、NPHS2、CD2AP、PLCE1、WT1基因检测，至今仍有3个家系及部分散发病例病因不明。此次研究计划首先在排除其他16个已知致病基因突变后，对这3个家系采用全基因组连锁分析进行致病基因的染色体定位，然后进行外显子组测序以筛选特定区域内的候选基因，再用Sanger法直接测序进行验证，以确定致病基因。最后应用免疫组化技术、真核细胞表达技术和基因敲除技术，在患者肾病理组织、体外培养的足细胞以及动物模型上进行致病基因表达分析和功能鉴定，以进一步揭示SRNS发病的遗传机制，给临床治疗用药提供新的思路和理论依据。

原发性肾病综合征（nephrotic syndrome， NS）是儿童期常见的肾小球疾病，主要表现为大量蛋白尿、低白蛋白血症、不同程度的水肿和高脂血症。大部分原发性NS患儿对激素敏感，即激素敏感型NS，预后好。但是，10%～20%的患儿对激素抵抗，即激素耐药型NS（steroid-resistant nephrotic syndrome，SRNS），预后差，30%～40%的SRNS患儿在10年内逐渐进展至终末期肾脏疾病。研究表明，部分SRNS的发病与肾小球滤过屏障的原发缺陷有关。迄今已报道20多个单基因突变可导致SRNS，然而，仍有80%的SRNS患儿病因不明。本课题组对一个隐性遗传型SRNS家系的先证者及其父母和姐姐进行全外显子组测序。对照人群为520例尿检正常健康志愿者。提取所有研究对象外周血基因组DNA。全外显子组测序采用人外显子组序列捕获系统（Agilent SureSelect V4，51M）和Illumina HiSeq 2000测序技术。对所发现的基因变异通过PCR和DNA直接测序法进行验证。先证者及其父母和姐姐全外显子组测序总共得到56387个单个核苷酸变异和6383个小片段插入缺失变异。经生物信息学分析排除了大部分变异，最终筛选出19个基因。在这19个基因中，1个新候选基因的复合杂合变异(E803K和R1173X)不存在于SNP数据库。先证者携带复合杂合变异E803K和 R1173X，其父亲、母亲和姐姐分别携带E803K 、R1173X和 E803K杂合变异。E803K 和R1173X在520例对照人群中未检出，表明E803K和R1173X为基因突变。生物信息学分析预测E803K和R1173X为致病突变。研究结果提示新候选基因突变可能导致家族性SRNS，尚需进一步开展新候选基因突变致足细胞损伤及其机制的研究。