Gene Discovery in Autosomal Dominant Focal Segmental Glomerulosclerosis

描述(申请人提供)：局灶性节段性肾小球硬化(FSGS)是终末期肾脏疾病的常见原因。FSGS的发病机制尚未明确，也没有始终如一有效的治疗方法。最近在罕见的遗传性FSGS中发现的致病基因，包括我们自己的研究，至少有6个基因突变与家族性FSGS相关，每个发现都阐明了肾小球损伤的分子机制。在这一富有成效的调查基础上，我们确定了118个患有家族性FSGS的家庭并仔细描述了这些家庭的特征。我们已经对我们的其余家庭进行了筛查，以寻找已知的导致FSGS的基因突变，并在另外6个家族中确定了原因突变；其余111个家庭的疾病遗传基础尚不清楚。这项建议的目的是利用这一宝贵而独特的家庭资源来系统地鉴定家族性FSGS的致病基因。目前传统的连锁和位置克隆方法的局限性包括它们对大型、多基因家族的要求。此外，在传统的连锁分析中，缩小候选区域可能很困难，因为大区域缺乏重组事件，因此这些区域需要繁琐而漫长的致病突变筛查。强大的新基因工具可以促进这一筛查过程，并在较小的家庭中改进变异发现。特别是，有效的完整外显子组测序，即有针对性地捕获编码蛋白质的基因序列，在我们的研究中应该特别有用，因为大多数孟德尔疾病是由影响目标基因外显子组的突变引起的。因此，通过结合全基因组连锁分析(GWLS)和全外显子组测序，我们可以最大限度地发挥我们的家族数据的影响，并加速识别FSGS的新突变。在初步研究中，我们已经使用这种组合在一个FSGS家族中鉴定了WT1(Wilms‘Tumor-1)基因的一个新的变异，我们有证据表明它是因果突变。这一成功提供了概念验证，并为如何在拟议的研究中识别和评估基因提供了路线图。我们的假设是，在我们的家族队列中，遗传性FSGS的原因将是以前与家族性FSGS无关的基因编码区的序列变异。我们的目标是：1)使用GWLS和全外显子组测序来识别与家族性FSGS相关的遗传变异。2)确定候选致病突变的功能后果，3)确定这些在AIM 2中发现的新致病突变在Duke FSGS数据库中的流行程度。任何被发现有致病突变的基因都将在剩余的家系中进行测序，并充分利用我们的家族资源。通过结合全基因组连锁分析(GWLS)、全外显子组测序和变异的功能后果的表征，我们将显著提高对正常肾小球生物学以及FSGS和相关肾小球疾病的发病机制的理解。此外，我们的发现可能会揭示改进这种目前几乎没有有效治疗方法的疾病的治疗的新机会。

DESCRIPTION (provided by applicant): Focal segmental glomerulosclerosis (FSGS) is a frequent cause of end-stage renal disease. The pathogenesis of FSGS has not been precisely defined and there are no consistently effective treatments. Recent studies identifying causal genes in rare, inherited FSGS, including our own study, have associated mutations in at least six genes with familial FSGS, and each discovery has clarified molecular mechanisms of glomerular injury. To build on this productive line of inquiry, we have ascertained and carefully characterized 118 families with familial FSGS. We have screened the remainder of our families for mutations in genes known to cause FSGS and identified the causal mutations in an additional 6 kindreds; the genetic basis of disease in the remaining 111 families is unknown. The objective of this proposal is to use this valuable and unique family resource to systematically identify causal genes for familial FSGS. Limitations of current conventional linkage and positional cloning approaches include their requirement for large, multiplex families. In addition, narrowing candidate areas in traditional linkage analysis can be difficult due to larg regions that lack recombination events and hence these regions have required cumbersome and lengthy screening for causative mutations. Powerful new genetic tools can facilitate this screening process and improve variant discovery in smaller families. In particular, efficient whole-exome sequencing, the targeted capture of protein-coding gene sequences, should be particularly useful in our studies since most Mendelian disorders are caused by mutations affecting exomes of the target gene. Thus, by combining genome-wide linkage analysis (GWLS) and whole-exome sequencing, we can maximize impact of our family data and accelerate identification of novel mutations in FSGS. In preliminary studies, we have used this combination to identify a novel variant in the WT1 (Wilms' Tumor-1) gene in one FSGS family, and we have evidence suggesting it is the causal mutation. This success provides proof-of-concept and provides a roadmap of how genes will be identified and evaluated in the proposed studies. Our hypothesis is that causes of inherited FSGS in our cohort of families will be sequence variants in the coding region of genes not previously associated with familial FSGS. We aim to: 1) Use GWLS and whole-exome sequencing to identify genetic variants associated with familial FSGS. 2) Characterize functional consequences of candidate causative mutations and 3) Determine the prevalence in the Duke FSGS dataset of these new causative mutations identified in Aim 2. Any genes found to have causative mutations will be sequenced in the remaining families and take full advantage of our family resource. By combining genome-wide linkage analysis (GWLS), whole-exome sequencing, and characterization of variants' functional consequences, we will significantly improve understanding of normal glomerular biology and of the pathogenesis of FSGS and related glomerular diseases. Moreover, our discoveries are likely to reveal new opportunities to improve therapy for a disease that currently has few effective treatments.