Whole Genome Sequencing for Nephrotic Syndrome Discovery

摘要 原发性肾病综合征（NS）是一种罕见的肾小球滤过屏障功能衰竭的疾病， 大量的蛋白质从尿中排出。NS的发病率和死亡率与疾病和非疾病相关。 NS目前根据组织学分类和治疗， 外观（如局灶节段性肾小球硬化[FSGS]、微小病变疾病[MCD]）或对 类固醇治疗这种描述性分类是非特异性的，并没有阐明NS的潜在病理生物学。 为了实现对NS患者越来越精确和有效的护理，更好地了解其潜在的 分子机制是必要的。NS中的人类遗传学研究已证明是一种有前途的策略， 通过分子亚型对患者进行分类，并确定将刺激生物标志物和药物发现的靶点。 发现新的，并确定已知的，遗传决定因素的NS，其临床相关性，和 我们正在生成和分析深层的全基因组测序， 和RNA-seq从北美基于人群的队列中的受影响患者获得转录组数据。 利用NS患者肾脏的基因表达，我们的目标是发现与NS相关的遗传变异。 NS通过表达数量性状基因座（eQTL）研究。利用eQTL作为中间分子表型 提高了检测重要关联的能力，也将直接阐明生物学途径， 没有被发现，否则。我们还将确定已知的单基因型NS在本研究中的流行率。 使用强大的生物信息学过滤与功能测试配对的队列。我们将通过以下方式描述临床相关性： 与基线和纵向表型的整合。在多基因模型之外，我们同样决心 目的：探讨孟德尔遗传性肾病综合征患者免疫抑制剂耐药表型的发生率。 到目前为止，我们的实验室已经对肾病患者进行了初步的eQTL和孟德尔研究。 综合征研究网络，取得了可喜的成果。在这里，我们将扩展这些研究，从目标1开始， 使用新的WGS和RNA-seq数据集来发现肾小球和肾小管间质eQTL，并鉴定这些eQTL。 对这些肾脏组织和其他组织的特异性。在目标2中，我们将这些eQTL定位到特定的细胞谱系， 通过整合单细胞RNA-Seq数据，鉴定那些重叠的肾源性调控区， 发现它们与生物过程和临床表型的关联。我们将对顶级候选人进行功能测试 果蝇和肾类器官系统中的eQTL。在目标3中，我们将使用WGS数据来发现 由于已知的孟德尔原因导致的NS。我们将在美国患者中研究孟德尔NS的临床相关性， 确定与孟德尔疾病完全缓解相关的因素。最后，我们将使用 果蝇和类器官模型来功能性地筛选我们发现的假定致病变体。总之， 我们希望这些发现能扩大我们对NS相关遗传变异谱的了解， 这些窝藏他们的临床意义，其致病性的机制。

Abstract Primary nephrotic syndrome (NS) is a rare disease of glomerular filtration barrier failure that causes massive urinary excretion of protein. Morbidity and mortality from NS is related both to the disease and the non- specific medications used to try to treat it. NS is currently classified and treated according to histologic appearance (e.g. focal segmental glomerulosclerosis [FSGS], minimal change disease[MCD]) or response to steroid therapy. This descriptive taxonomy is nonspecific and does not illuminate NS's underlying pathobiology. To achieve increasingly precise and effective care for NS patients, a better understanding of its underlying molecular mechanisms is necessary. Human genetics studies in NS have proven a promising strategy to permit patient classification by molecular subtype and identify targets that will spur biomarker and drug discovery. To discover novel, and identify known, genetic determinants of NS, their clinical correlates, and the mechanisms by which they confer their harm, we are generating and analyzing deep whole genome sequencing and RNA-seq derived transcriptomic data from affected patients in a North American, population-based cohort. Using gene expression from the kidneys of patients with NS, we aim to discover genetic variants associated with NS via expression quantitative trait loci (eQTL) studies. Using eQTLs as an intermediate molecular phenotype improves power to detect significant associations and will also directly illuminate biologic pathways that would not have been detected otherwise. We will also determine the prevalence of known monogenic forms NS in this cohort using robust bioinformatics filtering paired with functional testing. We will describe clinical correlates via integration with baseline and longitudinal phenotyping. Beyond the polygenic model, we are equally determined to discover the penetrance of the immunosuppressant resistant phenotype among patients with Mendelian NS. Our lab has thus far performed preliminary eQTL and Mendelian studies on enrollees in the Nephrotic Syndrome Study Network, with promising results. Here, we will expand these studies, beginning in Aim 1 by using new WGS and RNA-seq datasets to discover glomerular and tubulointerstitial eQTLs and identify those specific to these kidney tissues vs other tissues. In Aim 2, we will localize these eQTLs to specific cell lineages via integration with single cell RNA-Seq data, identify those overlapping kidney-derived regulatory regions, and discover their association with biologic processes and clinical phenotypes. We will functionally test top candidate eQTLs in drosophila and kidney organoid systems. In Aim 3, we will use WGS data to discover the prevalence of NS due to known Mendelian causes. We will study clinical correlates of Mendelian NS in US patients and identify factors associated with complete remission among those with Mendelian disease. Finally, we will use the drosophila and organoids models to functionally screen the putative pathogenic variants we discover. Altogether, we expect these discoveries to expand our knowledge of the spectrum of NS-associated genetic variation, the clinical implications for those harboring them, and the mechanisms underlying their pathogenicity.