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: 10389419
• 负责人: James Lewis Shepherdson
• 金额: $ 3.27万
• 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/10389419
• 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; Lead; 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; base; 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.

项目总结 转录因子Cone-Rod Homeobox(CRX)是光感受器细胞命运的主要调节者。数列 CRX基因的变异可导致视网膜色素变性、视锥-杆状营养不良和Leber先天性黑便 导致视力丧失和失明的遗传性原因。CRX是与这三种疾病的发病机制有关的唯一基因 这些疾病都表现为杆状和锥状中心表型，发病年龄和严重程度各不相同。 据报道，一些CRX变异体通过反形态遗传交互作用引起严重的显性疾病。 带有野生型CRX的突变，但这些突变与良性或仅引起轻微的变异相邻， 隐性疾病。确定CRX的哪些突变是致病的，并量化它们对功能的影响 活动性是解释患者变异和预测患者表型的先决条件。 然而，CRX中的大多数变种都是不确定信号的变种(VUS)，这意味着临床上不确定的fifi 或者有功能证据来确定它们的致病性。没有一个强大的人类基因目录 变异，患者测序的进展不能转化为临床指导或治疗 没有特征的变种。解决这一挑战的一个潜在解决方案是深度突变扫描(DMS)，它 在多重分析中使用大量变异序列文库来同时测量功能 在一次实验中，一个感兴趣的基因的数千个变异的结果。在DMS中，每个基因变体都是 根据其在分子检测中的活性，给出了一个定量的功能致病性分数。 这项建议将使用DMS来同时检测所有点的转录活性和丰度 CRX中的替换、截断和移帧。这项工作的直接成果将是一个“查找表”列表 每个CRX变体对蛋白质活性和稳定性的功能影响，这将直接适用 对临床变异fi的判定和决策。鉴于视网膜的特殊位置，它很容易成为 基因治疗，人类CRX变异的目录可以为fl患者的临床治疗提供信息 遗传性视网膜病变在短期内。此外，这项工作还将为其他工作建立一个可扩展的平台 DMS对其他视网膜转录因子的研究，广泛地扩大了我们对遗传性视网膜疾病的理解。