Evaluation of Human Variants in Disease Models for End Stage Renal Disease

描述(申请人提供)：在过去的资助周期中，使用各种大鼠模型，我们已经证明Rab38负责名为RF-2的数量性状基因座(QTL)，鉴定和验证了Sorcs1负责名为RF-1的QTL，并发现Shroom3导致肾小球通透性的变化。通过定位另外两个QTL，RF-3和RF-4，我们在克隆方面取得了相当大的进展。在与慢性肾脏疾病遗传学协会(CKDGen)的合作中，我们发现SORCS1和SHROOM3与人类肾脏疾病显著相关，表明这项工作具有临床意义。我们的长期目标是确定终末期肾病(ESRD)的遗传结构。我们的数据表明，蛋白尿需要肾小球通透性和近端小管蛋白转运的缺陷，这现在是我们的中心假说。拟议的具体目标是：1)测试SHROOM3的人类序列变异体，预测为功能障碍；2)确定参与肾小球通透性的QTL RF-3(区间大小1.81 Mb)和QTL RF-4(区间大小0.9 Mb)的基因/基因组元件，这被认为是引发蛋白尿的；以及3)测试关于肾小球通透性障碍和近端小管蛋白质运输功能障碍共同参与蛋白尿发生的中心假设。测试此中心 假设需要使用整个动物，而不能在人类身上实现。我们对这一建议的遗传/基因组基础设施包括：亲本菌株的完整基因组序列，包括生物信息学分析的NextGen测序工具，“狭窄的”同源菌株，三个已识别的致病基因，以及详细的生理特征，为发现重要的新基因和检验这一新的中心假设创造了一个理想的平台。我们将在HEK293细胞、斑马鱼和人源化致敏的CKD FHH大鼠模型中研究SHROOM3的人类变异。具体地说，AIM 1测试了一组人类肾脏疾病的变种，并展示了如何使用模型系统来跟踪许多全基因组关联研究(GWAS)提名的基因。我们与CKDGen的合作使我们能够测试已确定的RF-3和RF-4基因是否有助于人类疾病；从而提供关于人类疾病过程的知识。这项拟议研究的基本原理是测试SHROOM3的人类变异，识别新的基因，以及一个新的假说，这将为这一疾病过程提供新的见解，并可能为未来发病率上升和有效治疗有限的肾脏疾病的治疗确定新的靶点。特定的目标1-3有望揭示突变的SHROOM3是如何发挥作用的知识，并识别两个新的基因，它们要么与已知的途径相连，要么揭开新的途径的面纱。我们还将确定是否涉及两次撞击(一次在肾小球，一次在近端小管)。该提案的每个目标都将为确定蛋白尿的遗传结构提供基本的进展，蛋白尿通常是终末期肾病的先兆。

DESCRIPTION (provided by applicant): During the past grant cycle, using a variety of rat models, we have proven that Rab38 is responsible for the quantitative trait locus (QTL) named Rf-2, identified and validated that Sorcs1 is responsible for the QTL named Rf-1, and have identified that Shroom3 causes changes in glomerular permeability. We have made considerable strides towards cloning by position two other QTL, Rf-3 and Rf-4. In collaboration with the Chronic Kidney Disease Genetics (CKDGen) Consortium, we found that SORCS1 and SHROOM3 are significantly associated with renal disease in humans, demonstrating that this work has clinical relevance. Our long-term goal is to define the genetic architecture of end stage renal disease (ESRD). Our data suggests that a defect in both the glomerular permeability and protein trafficking in the proximal tubules is required for proteinuria, which is now our centrl hypothesis. The proposed Specific Aims are to 1) test a collection of human sequence variants of SHROOM3 predicted to be dysfunctional; 2) identify the genes/genomic elements underlying the QTL Rf-3 (interval size 1.81 Mb) and QTL Rf-4 (interval size 0.9 Mb) participating in glomerular permeability, which is thought to initiate proteinuria; and 3) test the central hypothesis that dysfunction in both glomerular permeability and protein trafficking in the proximal tubules are participating together in the development of proteinuria. Testing this central hypothesis requires the use of whole animals and cannot be done in human. Our genetic/genomic infrastructure for this proposal includes: complete genomic sequence of parental strains, NextGen sequencing tools including bioinformatic analysis, "narrow" congenic strains, three identified causative genes, as well as detailed physiological characterization creating an ideal platform for discovering important new genes and testing this new central hypothesis. We will study human variants of SHROOM3 in HEK293 cells, zebrafish and humanized sensitized FHH rat model for CKD. Specifically, Aim 1 tests a collection of human variants for renal disease and demonstrates how model systems can be used to follow-up genes nominated by many genome-wide association studies (GWAS). Our collaboration with the CKDGen enables us to test if the genes identified for Rf-3 and Rf-4 contribute to human disease; thereby, providing knowledge about the disease process in humans. The rationale for the proposed research is the test of human variation in SHROOM3, identification of new genes, and a new hypothesis which will provide new insights into this disease process and could identify new targets for future treatment of renal disease with a rising incidence and limited effective treatments. Specific aims 1-3 are expected to reveal the knowledge about how mutated SHROOM3 functions and identify two new genes, which will either link to known pathways or unmask new ones. We will also determine if two hits (one in the glomeruli and one in proximal tubules) are involved. Each aim of this proposal will provide fundamental advances toward defining the genetic architecture of proteinuria which is often the precursor of ESRD.