Multiplex functional assay of variant effect in the retinal transcription factor CRX

视网膜转录因子 CRX 变异效应的多重功能测定

• 批准号: 10555204
• 负责人: James Lewis Shepherdson
• 金额: $ 3.36万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-02-01 至 2026-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10555204
• 关键词: Address; Age of Onset; Benign; Biological Assay; Blindness; Catalogs; Cells; Classification; ClinVar; Clinical; Clinical Management; Clinical Research; Databases; Decision Making; Defect; Diagnosis; Disease; Genes; Genetic; Genetic Transcription; Genetic Variation; Genotype; Homeobox; Human; Human Genetics; Inherited; Leber' s amaurosis; Libraries; Location; Measures; Molecular; Mutation; Pathogenesis; Pathogenicity; Patients; Phenotype; Photoreceptors; Proteins; Reporter; Reporter Genes; Reporting; Retina; Retinal Diseases; Retinitis Pigmentosa; Severities; System; Techniques; Technology; Testing; Time; Translating; United States National Institutes of Health; Variant; Vertebrate Photoreceptors; Vision; Work; clinical sequencing; cone-rod dystrophy; disease-causing mutation; experimental study; gene therapy; genetic variant; human disease; interest; loss of function; multiplex assay; mutation screening; patient variability; recessive genetic trait; transcription factor; variant of unknown significance

PROJECT SUMMARY The transcription factor Cone-Rod Homeobox (CRX ) is a master regulator of photoreceptor cell fate. Sequence variants in CRX can cause Retinitis Pigmentosa, Cone-Rod Dystrophy, and Leber Congenital Amaurosis, all inherited causes of vision loss and blindness. CRX is the only gene implicated in the pathogenesis of all three of these diseases, which present with both rod- and cone-centric phenotypes of varying age of onset and severity. Several CRX variants have been reported to cause severe dominant disease through antimorphic genetic interac- tions with wild-type CRX, and yet these mutations are adjacent to variants which are benign or only cause mild, recessive disease. Determining which mutations in CRX are pathogenic and quantifying their effect on functional activity is prerequisite to interpreting patient variation and predicting patient phenotypes. However, most variants in CRX are “Variants of Uncertain Significance” (VUS), meaning that insufficient clinical or functional evidence exists to determine their pathogenicity. Without a robust catalog of human genetic variation, advances in patient sequencing cannot be translated into clinical guidance or therapies for patients with uncharacterized variants. One potential solution to this challenge is Deep Mutational Scanning (DMS), which uses massive libraries of variant sequences in multiplexed assays to simultaneously measure the functional consequence of thousands of variants in a gene of interest in a single experiment. In DMS, each gene variant is assigned a quantitative functional pathogenicity score based on its activity in a molecular assay. This proposal will use DMS to simultaneously assay the transcriptional activity and abundance of all point substitutions, truncations, and frameshifts in CRX. The direct product of this work will be a “look-up table” listing the functional consequence of every CRX variant on protein activity and stability, which will be directly applicable to clinical variant classification and decision making. Given the retina's privileged location as a facile target for gene therapy, a catalog of human variation in CRX could inform the clinical management of patients afflicted with inherited retinopathies in the near term. Furthermore, this work will establish an extensible platform for additional DMS studies of other retinal transcription factors, broadly expanding our understanding of inherited retinal disease.

项目摘要 转录因子锥体型同型（CRX）是光感受器细胞命运的主要调节剂。顺序 CRX中的变体会导致色素性视网膜炎，锥体杆营养不良和Leber先天性肌动蛋白，所有这些都 继承视力丧失和失明的原因。 CRX是与所有三个的发病机理有关的唯一基因 这些疾病以不同年龄和严重程度的不同年龄为中心的杆和锥体表型存在。 据报道，几种CRX变体通过抗鲜艳的遗传间间疾病引起严重的显性疾病 具有野生型CRX的特征，但是这些突变与良性或仅引起轻度的变体相邻 隐性疾病。确定CRX中的哪些突变是致病性的，并量化其对功能的影响 活动是解释患者变异和预测患者表型的先决条件。 但是，CRX中的大多数变体都是“不确定的变体”（VUS），这意味着不足的临床 或功能证据存在以确定其致病性。没有强大的人类遗传目录 变异，患者测序的进展不能转化为患者的临床指导或疗法 未表征的变体。解决这一挑战的一种潜在解决方案是深突变扫描（DMS）， 在多路复用测定中使用大量变体序列的库同时测量功能 单个实验中有成千上万种变体的结果。在DMS中，每个基因变体是 根据分子测定中的活性，分配了定量功能致病性评分。 该建议将使用DMS简单地主张所有点的转录活动和抽象 CRX中的取代，截断和移架。这项工作的直接产品将是“查找表”列表 每个CRX变体对蛋白质活性和稳定性的功能后果，这将是直接适用的 进行临床变体分类和决策。鉴于视网膜特权位置作为轻松的目标 基因疗法，CRX中人类变异的目录可以告知与患者的临床管理 在短期内遗传性视网膜病。此外，这项工作将建立一个可扩展的平台 DMS对其他视网膜转录因子的研究，广泛地扩展了我们对遗传残留疾病的理解。