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衰竭的疾病，可导致 大量的尿液蛋白质排泄。NS的发病率和死亡率既与疾病有关，也与非 试图治疗它的特定药物。目前，NS是根据组织学分类和治疗的 表现(例如局灶性节段性肾小球硬化[FSGS]、微小病变病[MCD])或对 类固醇疗法。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患者日益精确和有效的护理，更好地了解其潜在的 分子机制是必要的。在NS中进行的人类遗传学研究已经证明是一种有希望的策略 根据分子亚型对患者进行分类，并确定将刺激生物标记物和药物发现的靶点。 发现新的和识别已知的NS的遗传决定因素，它们的临床相关性，以及 它们带来危害的机制，我们正在产生和分析深度全基因组测序 RNA-seq从北美人群队列中受影响的患者那里获得转录数据。 利用NS患者肾脏的基因表达，我们的目标是发现与 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 提高检测重大关联的能力，并将直接照亮生物途径，从而 否则就没有被检测到。我们还将确定已知的单基因形式NS在本 使用稳健的生物信息学过滤和功能测试相结合的队列。我们将通过以下方式描述临床相关性 与基线和纵向表型相结合。在多基因模型之外，我们同样有决心 目的：了解孟德尔肾病患者免疫抑制耐药表型的外显性。 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 症状研究网络，结果令人振奋。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数据集发现肾小球和肾小管间质eQTL并识别这些eQTL 与其他组织相比，这些肾组织具有特异性。在目标2中，我们将这些eQTL定位到特定的细胞系 通过与单细胞RNA-Seq数据的整合，识别那些重叠的肾脏来源的调节区，以及 发现它们与生物学过程和临床表型的关系。我们将从功能上测试顶尖候选人 果蝇和肾脏器官系统中的eQTL。在目标3中，我们将使用WGS数据来发现流行率 由于已知的孟德尔原因导致的NS。我们将研究美国患者孟德尔肾病的临床相关性和 在孟德尔病患者中确定与完全缓解相关的因素。最后，我们将使用 果蝇和有机类化合物模型，从功能上筛选我们发现的可能的致病变异。总而言之， 我们希望这些发现将扩大我们对NS相关遗传变异频谱的了解 对携带这些病毒的人的临床意义，以及它们致病的机制。

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.