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

摘要 发现肾病/FSGS的完全外显性原因并从功能上确定其特征。 慢性肾脏疾病(CKD)是危害人类健康的最高疾病之一，其发病率 不断上升。激素抵抗型肾病综合征(SRNS)是CKD的第二大常见原因 在25年前。局灶性节段性肾小球硬化(FSGS)不可避免地导致CKD，复发33% 肾移植的风险。SRNS的发病机制尚不清楚，目前尚无根治性治疗方法。为 自发性肾病综合征的主要原因(病因学)和发病机制(发病机制)一直是一个难题 几十年。然而，识别NS的全外显单基因原因(例如，podocin)已经牵涉到 肾小球足细胞是发病机制的中心。在之前的两个R01资助期内，我们： 1)通过外显子组全序列测序，确定了目前已知的50个NS和 从功能上描述了相关的疾病机制；2)发现编码的蛋白质 蛋白质复合体中的簇从而定义了SRN的新的疾病途径(例如，RhoA/rac1/CDc42 信号)；3)描述的基因型-表型相关性与个人化的可操作含义 疾病管理；4)在足细胞迁移试验中模拟相关的疾病机制， 斑马鱼和老鼠模型；5)展示了针对特定患者的个性化治疗选项(例如CoQ10 在COQ6或ADCK4突变中)；6)在世界范围的队列中显示，约30%的SRN(25年)是 由单基因突变引起，从而允许进行遗传机制研究和个性化 SRNS患者的药物；7)发现了类固醇依赖型NS的第一个遗传原因(6 基因)，集中在RhoA调控上。这些基因发现使SRN的研究变得容易 “精确医学”的遗传学方法，使基因诊断成为可能，“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： SA1.通过WES在约1,000个SRNS家系中发现SRNS缺失的单基因原因。 SA2.对新发现的SRNS/SSNS的单基因病因进行功能表征，以描绘 发病机制和研究“个体化”的基因--表型和基因治疗 关联性。 SA3.在CRISPR k.o中进行小分子筛选。发现了新的SRNS基因模型，使用 建立了足细胞迁移试验和斑马鱼模型，以发现第一批治疗 SRNS。 SA4.研究我们在类固醇依赖型NS中发现的6个新的单基因收敛原因 关于RhoA的调节，阐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.