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

项目摘要 转录因子锥-杆同源异型盒（CRX）是感光细胞命运的主要调节因子。序列 CRX的变异可导致视网膜色素变性、视锥-视杆细胞营养不良和莱伯先天性黑蒙， 视力丧失和失明的遗传原因。CRX是唯一与这三种疾病的发病机制有关的基因。 这些疾病，其中提出了杆和锥中心的表型不同的发病年龄和严重程度。 据报道，几种CRX变体通过反形遗传相互作用引起严重的显性疾病， 与野生型CRX的突变，但这些突变是相邻的变异是良性的或只引起轻度， 隐性疾病确定CRX中的哪些突变是致病性的，并量化它们对功能的影响。 活性是解释患者变异和预测患者表型的先决条件。 然而，CRX中的大多数变体都是“不确定意义的变体”（VUS），这意味着不充分的临床 或功能性证据来确定其致病性。如果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患者， 不确定的变种针对这一挑战的一个潜在解决方案是深度突变扫描（DMS）， 在多重测定中使用大量的变异序列文库， 在一个实验中，一个感兴趣的基因中的数千个变体的结果。在DMS中，每个基因变体都是 基于其在分子测定中的活性分配定量功能致病性评分。 该方案将使用DMS同时检测所有点的转录活性和丰度， CRX中的取代、截短和移码。这项工作的直接产品将是一个“查找表”清单 每个CRX变体对蛋白质活性和稳定性的功能性后果，这将直接适用于 临床变异分类和决策。鉴于视网膜的优越位置， 基因治疗，人类CRX变异的目录可以为患有CRX的患者的临床管理提供信息。 遗传性视网膜病变此外，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，广泛扩展了我们对遗传性视网膜疾病的理解。

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.