Gene Discovery in Autosomal Dominant Focal Segmental Glomerulosclerosis

描述（由申请人提供）：局灶节段性肾小球硬化（FSGS）是终末期肾脏疾病的常见原因。FSGS的发病机制尚未明确，也没有一致有效的治疗方法。最近的研究发现了罕见遗传性FSGS的致病基因，包括我们自己的研究，至少有六个基因的突变与家族性FSGS相关，每一个发现都阐明了肾小球损伤的分子机制。为了建立这一富有成效的调查线，我们已经确定并仔细表征了118个家庭的家庭FSGS。我们筛选了其余家族中已知导致FSGS的基因突变，并在另外6种中确定了致病突变；其余111个家庭疾病的遗传基础尚不清楚。本建议的目的是利用这一宝贵而独特的家庭资源系统地识别家族性FSGS的致病基因。目前传统的连锁和定位克隆方法的局限性包括它们对大的、多家族的要求。此外，在传统的连锁分析中，由于缺乏重组事件的大区域，缩小候选区域可能是困难的，因此这些区域需要繁琐和漫长的致病突变筛选。强大的新遗传工具可以促进这一筛选过程，并改善在较小家庭中的变异发现。特别是，高效的全外显子组测序，靶向捕获蛋白质编码基因序列，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靶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利用我们的家族数据，并加速鉴定FSGS的新突变。在初步研究中，我们使用这种组合在一个FSGS家族中发现了WT1 （Wilms' Tumor-1）基因的一个新变体，我们有证据表明它是致病突变。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FSGS的原因将是先前与家族性FSGS无关的基因编码区域的序列变异。我们的目标是：1)使用GWLS和全外显子组测序来鉴定家族性FSGS相关的遗传变异。2)表征候选致病突变的功能后果，3)确定目标2中发现的这些新致病突变在Duke FSGS数据集中的患病率。任何发现有致病突变的基因都将在剩余的家族中进行测序，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功能后果的表征，我们将显著提高对正常肾小球生物学和FSGS及相关肾小球疾病发病机制的理解。此外，我们的发现很可能为改善目前几乎没有有效治疗方法的疾病提供新的机会。

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.