A pipeline for identifying disease-causing variants in transmembrane proteins

项目摘要/摘要 候选人背景：格雷泽博士获得麻省理工学院生物学学士学位和麻省理工学院生物学博士学位 加州大学伯克利分校进化遗传学。作为丹·罗登实验室的博士后研究员 在范德比尔特，他开发了研究心脏离子通道的高通量方法。他有 还开始了通过生物库遗传学发现的候选疾病变异的体外研究。 研究策略：孟德尔人中存在数千种罕见的未知意义(VU)变体 疾病基因。为了提高基因组医学的准确性和影响力，该领域必须发展 高效、可扩展的方法，用于识别变异与疾病的关联并测试这些变异 在试管中。这项建议侧重于细胞表面跨膜蛋白，它占细胞表面跨膜蛋白的20%左右。 蛋白质组，与许多孟德尔疾病有关。目标1将利用基因发现在一个 大型生物库，使用聚合多器官的方法识别新的疾病变异关联 表型称为表型风险评分(PHERS)。PHERS分数将经过细化和验证，以 大约50对候选跨膜基因-疾病对。这些分数将部署在BioVU， 范德比尔特的生物库(最近扩大到10万个基因分型的个体)和其他队列。 变异将被测试与PHERS分数的关联，以从统计上发现高优先级的变异 与疾病有关。目标2将测试高优先级跨膜变异体的体外功能 基因特异性分析和一般分析的混合。这些研究将从统计上的4个变种开始 根据PHERS评分与孟德尔疾病有关，但仍被归类为VUS。这种方法 将被扩展以测试在Aim 1中发现的其他变体的体外功能。Aim 3将开发 并验证了一种综合测量细胞表面的通用方法Surface-seq 几乎每一种跨膜蛋白的变种都会被贩卖。此方法将首先在 小基因KCNE1，与心律失常和耳聋有关。然后，它将扩展到 更大的跨膜基因，包括与肺动脉高压有关的BMPR2基因。总的来说， 这项工作有可能发现许多新的致病/可能致病变异体 跨膜基因。变体将使用AIMS 1-3中的数据进行重新分类，并且分类 将发布并存入ClinVar数据库。 职业发展和培训：该建议利用申请人在以下方面的专长 遗传学、基因组学和高通量分析。它包括许多新的培训机会， Roden和Denny团队以及其他合作者，特别是涉及生物库遗传学和新 跨膜基因的研究方法。额外的培训将有助于应聘者 目标是建立一个独立的实验室，研究基因组药物和跨膜蛋白。

PROJECT SUMMARY/ABSTRACT Candidate background: Dr. Glazer received a B.S. from MIT in biology and a Ph.D. from the University of California-Berkeley in evolutionary genetics. As a postdoctoral fellow in Dan Roden's lab at Vanderbilt, he has developed high-throughput methods for studying cardiac ion channels. He has also begun in vitro studies of candidate disease variants discovered through biobank genetics. Research strategy: Thousands of rare Variants of Unknown Significance (VUS) exist in Mendelian disease genes. To improve the accuracy and impact of genomic medicine, the field must develop efficient, scalable methods for identifying variant associations with disease and testing these variants in vitro. This proposal focuses on cell surface transmembrane proteins, which comprise ~20% of the proteome and are associated with many Mendelian diseases. Aim 1 will use genetic discovery in a large biobank to identify new disease-variant associations, using a method to aggregate multi-organ phenotypes called the Phenotype Risk Score (PheRS). PheRS scores will be refined and validated for ~50 candidate transmembrane gene-disease pairs. These scores will be deployed in BioVU, Vanderbilt's biobank (recently expanded to >100,000 genotyped individuals), and other cohorts. Variants will be tested for association with PheRS scores to discover high-priority variants statistically linked with disease. Aim 2 will test the in vitro function of high-priority transmembrane variants using a mixture of gene-specific and general assays. These studies will begin with 4 variants statistically associated with Mendelian diseases by PheRS scores, but still classified as a VUS. This approach will be expanded to test in vitro function of additional variants discovered in Aim 1. Aim 3 will develop and validate Surface-seq, a general method for comprehensively measuring the cell surface trafficking of nearly every variant in a transmembrane protein. This method will first be optimized on a small gene, KCNE1, which is associated with arrhythmias and deafness. It will then be extended to larger transmembrane genes, including BMPR2, a gene linked to pulmonary hypertension. Overall, this work has the potential to identify many new pathogenic/likely pathogenic variants in transmembrane genes. Variants will be reclassified with data from Aims 1-3, and the classifications will be published and deposited into the ClinVar database. Career development and training: This proposal takes advantage of the applicant's expertise in genetics, genomics, and high-throughput assays. It includes many new training opportunities with the Roden and Denny groups and other collaborators, especially involving biobank genetics and new methods for studying transmembrane genes. The additional training will help lead to the candidate's goal of establishing an independent lab studying genomic medicine and transmembrane proteins.