Discover and functionally characterize full-penetrance causes of nephrosis / FSGS

摘要 发现并从功能上表征肾病/FSGS的全昏迷原因。 慢性肾脏病（CKD）是人类健康损失最大的疾病之一， 不断上升。类固醇耐药肾病综合征（SRNS）是CKD的第二大最常见原因 局灶节段性肾小球硬化症（FSGS）不可避免地导致CKD，复发率为33% 肾移植的风险SRNS的发病机制尚不清楚，也没有有效的治疗方法。为 SRNS的主要原因（病因学）和疾病机制（发病机制）一直是一个难题， 几十年然而，NS的全突变单基因病因（例如podocin）的鉴定已经暗示了 肾小球足细胞是发病机制的中心。在前两个R 01资助期内，我们： 1)通过全外显子组测序确定了50个目前已知的NS单基因病因中的34个， 功能特征的相关疾病机制; 2）发现，编码的蛋白质， 聚集在蛋白质复合物中从而定义了SRNS的新疾病途径（例如，RhoA/Rac1/Cdc42 3）描述的基因型-表型相关性，其对个性化治疗具有可操作的意义。 疾病管理; 4）在“足细胞迁移试验”中模拟相关疾病机制， 斑马鱼和小鼠模型; 5）揭示了针对特定患者的“个性化治疗”选择（例如辅酶Q10 在COQ 6或ADCK 4突变中）; 6）在世界范围内的队列中证实，约30%的SRNS（<25岁） 由单基因突变引起，从而允许遗传机制研究和个性化 7）发现了激素依赖性NS的第一个遗传原因（6 基因），会聚于RhoA调节。这些遗传学发现使SRNS的研究变得容易， “精确医学”的遗传方法，使遗传诊断成为可能，“个性化”的研究， 疾病机制和治疗方法。因此，我们将追求以下具体目标： SA 1.通过WES在约1,000个SRNS家族中发现SRNS缺失的单基因原因。 SA 2.对新发现的SRNS/SSNS的单基因病因进行功能表征，以描述 发病机制和研究“个性化”基因型-表型和基因型-治疗 相关性 SA 3.在CRISPR k.o.中进行小分子筛选新的SRNS基因模型确定，使用 建立了“足细胞迁移试验”和斑马鱼模型，以发现第一种药物， SRNS。 SA 4.研究我们在类固醇依赖性NS中发现的6个新的单基因原因 对RhoA调节的影响描述了类固醇和其他药物对足细胞的直接作用机制。

ABSTRACT Discover and functionally characterize full-penetrance causes of nephrosis/FSGS. Chronic kidney diseases (CKD) take one of the highest tolls on human health, and their prevalence continuously rises. Steroid-resistant nephrotic syndrome (SRNS) is the 2nd most frequent cause of CKD before 25 yrs. By focal segmental glomerulosclerosis (FSGS) it inevitably leads to CKD with a 33% recurrence risk in a renal transplant. The pathogenesis of SRNS is unknown and no curative treatment is available. For SRNS, the primary causes (etiology) and disease mechanisms (pathogenesis) have been a conundrum for decades. However, identification of full-penetrance single-gene causes of NS (e.g. podocin) has implicated the renal glomerular podocyte at the center of the pathogenesis. Within the 2 previous R01 funding periods we: 1) Identified by whole exome sequencing 34 of the 50 currently known single-gene causes of NS and functionally characterized the related disease mechanisms; 2) Discovered that the encoded proteins cluster in protein complexes thereby defining novel disease pathways for SRNS (e.g., RhoA/Rac1/Cdc42 signaling); 3) Delineated genotype-phenotype correlations with actionable implications for personalized disease management; 4) Modeled the related disease mechanisms in the `podocyte migration assay', zebrafish & mouse models; 5) Revealed `personalized treatment' options for specific patients (e.g. CoQ10 in COQ6 or ADCK4 mutations); 6) Demonstrated in a world-wide cohort that ~30% of SRNS (<25 yrs) is caused by single-gene mutations, thereby permitting genetic mechanistic studies and personalized medicine for patients with SRNS ; 7) Discovered the first genetic causes of steroid-dependent NS (6 genes), converging on RhoA regulation. These genetic discoveries made the study of SRNS accessible to genetic approaches of `precision medicine', enabling genetic diagnostics, the study of `personalized' disease mechanisms, and treatment approaches. We, therefore will pursue the following Specific Aims: SA1. Discover the missing single-gene causes of SRNS by WES in ~1,000 SRNS families. SA2. Functionally characterize newly identified single-gene causes of SRNS/SSNS to delineate the pathogenesis and study `personalized' genotype-phenotype and genotype-treatment correlations. SA3. Perform small molecule screens in CRISPR k.o. models of novel SRNS genes identified, using established `podocyte migration assay' and zebrafish models, to discover the first drugs for SRNS. SA4. Study the 6 novel single-gene causes that we discovered in steroid-dependent NS to converge on RhoA regulation delineate mechanisms of steroid and other direct drug effects on podocytes.