Department of Ophthalmology, Ocular Genomics Institute, Berman-Gund Laboratory for the Study of Retinal Degenerations, Massachusetts Eye and Ear, Harvard Medical School, Boston, Massachusetts.
Hum Mutat. 2019 Aug;40(8):1127-1144. doi: 10.1002/humu.23762. Epub 2019 Jun 22.
Characterizing the pathogenicity of DNA sequence variants of unknown significance (VUS) is a major bottleneck in human genetics, and is increasingly important in determining which patients with inherited retinal diseases could benefit from gene therapy. A library of 210 rhodopsin (RHO) variants from literature and in-house genetic diagnostic testing were created to efficiently detect pathogenic RHO variants that fail to express on the cell surface. This study, while focused on RHO, demonstrates a streamlined, generalizable method for detecting pathogenic VUS. A relatively simple next-generation sequencing-based readout was developed so that a flow cytometry-based assay could be performed simultaneously on all variants in a pooled format, without the need for barcodes or viral transduction. The resulting dataset characterized the surface expression of every RHO library variant with a high degree of reproducibility (r = 0.92-0.95), recategorizing 37 variants. For example, three retinitis pigmentosa pedigrees were solved by identifying VUS which showed low expression levels (p.G18D, p.G101V, and p.P180T). Results were validated across multiple assays and correlated with clinical disease severity. This study presents a parallelized, higher-throughput cell-based assay for the functional characterization of VUS in RHO, and can be applied more broadly to other inherited retinal disease genes and other disorders.
鉴定具有未知意义的 DNA 序列变异（VUS）的致病性是人类遗传学的一个主要瓶颈，对于确定哪些遗传性视网膜疾病患者可能受益于基因治疗越来越重要。为了有效地检测未能在细胞表面表达的致病性视紫红质（RHO）变异体，我们创建了一个包含 210 种来自文献和内部遗传诊断测试的 RHO 变体的文库。虽然本研究专注于 RHO，但它展示了一种简化、可推广的方法，用于检测致病性 VUS。我们开发了一种相对简单的基于下一代测序的读取方法，以便可以同时在所有变体的池格式中进行基于流式细胞术的测定，而无需条形码或病毒转导。该数据集以高度的可重复性（r = 0.92-0.95）对每个 RHO 文库变体的表面表达进行了特征描述，重新分类了 37 个变体。例如，通过鉴定表达水平较低的 VUS（p.G18D、p.G101V 和 p.P180T），解决了三个色素性视网膜炎家系。结果在多个检测中得到了验证，并与临床疾病严重程度相关。本研究提出了一种平行、高通量的基于细胞的 RHO 中 VUS 功能特征分析方法，可更广泛地应用于其他遗传性视网膜疾病基因和其他疾病。
