A pipeline for identifying disease-causing variants in transmembrane proteins

项目总结/摘要 候选人背景：Glazer博士获得学士学位。麻省理工学院的生物学博士从 加州大学伯克利分校的进化遗传学。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 还开始对通过生物库遗传学发现的候选疾病变体进行体外研究。 研究策略：孟德尔遗传中存在数千种意义不明的罕见变异体（VUS）。 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 用于鉴定与疾病相关的变异并检测这些变异的有效、可扩展的方法 体外该建议集中在细胞表面跨膜蛋白，其占细胞膜蛋白的约20%。 蛋白质组，并与许多孟德尔疾病。Aim 1将利用基因发现， 大型生物库，以确定新的疾病变异的关联，使用一种方法来聚合多器官 表型风险评分（PheRS）。将对PheRS评分进行细化和验证， ~50个候选跨膜基因-疾病对。这些分数将在BioVU中部署， 范德比尔特的生物库（最近扩大到超过10万个基因型个体）和其他队列。 将检测变体与PheRS评分的相关性，以在统计学上发现高优先级变体 与疾病有关。Aim 2将使用一种新的方法测试高优先级跨膜变体的体外功能。 基因特异性和一般测定的混合物。这些研究开始时将有4种变体， 根据PheRS评分与孟德尔疾病相关，但仍被归类为VUS。这种方法 将扩展到测试目标1中发现的其他变体的体外功能。Aim 3将开发 并验证了Surface-seq，一种用于全面测量细胞表面的通用方法 跨膜蛋白中几乎所有变体的运输。此方法将首先在 小基因KCNE 1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BMPR 2，一个与肺动脉高压相关的基因。总的来说， 这项工作有可能鉴定出许多新的致病/可能致病的变异体， 跨膜基因将根据目标1-3的数据对变体进行重新分类， 将发表并存入ClinVar数据库。 职业发展和培训：该提案利用申请人在以下方面的专业知识： 遗传学、基因组学和高通量测定。它包括许多新的培训机会， 罗登和丹尼小组和其他合作者，特别是涉及生物库遗传学和新的 研究跨膜基因的方法。额外的培训将有助于导致候选人的 目标是建立一个研究基因组医学和跨膜蛋白的独立实验室。

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.