Ciliopathies are a broad range of inherited developmental and degenerative diseases associated with structural or functional defects in motile or primary non-motile cilia. There are around 200 known ciliopathy disease genes and whilst genetic testing can provide an accurate diagnosis, 24–60% of ciliopathy patients who undergo genetic testing do not receive a genetic diagnosis. This is partly because following current guidelines from the American College of Medical Genetics and the Association for Molecular Pathology, it is difficult to provide a confident clinical diagnosis of disease caused by missense or non-coding variants, which account for more than one-third of cases of disease. Mutations in PRPF31 are the second most common cause of the degenerative retinal ciliopathy autosomal dominant retinitis pigmentosa. Here, we present a high-throughput high-content imaging assay providing quantitative measure of effect of missense variants in PRPF31 which meets the recently published criteria for a baseline standard in vitro test for clinical variant interpretation. This assay utilizes a new PRPF31^+/– human retinal cell line generated using CRISPR gene editing to provide a stable cell line with significantly fewer cilia in which novel missense variants are expressed and characterised. We show that high-content imaging of cells expressing missense variants in a ciliopathy gene on a null background can allow characterisation of variants according to the cilia phenotype. We hope that this will be a useful tool for clinical characterisation of PRPF31 variants of uncertain significance, and can be extended to variant classification in other ciliopathies.

小儿麻痹症是与活动性或原发性非活动性纤毛的结构或功能缺陷有关的广泛的遗传性发育和退化性疾病。大约有200种已知的纤毛病基因，虽然基因检测可以提供准确的诊断，但是接受基因检测的24-60％的纤毛病患者没有接受遗传诊断。部分原因是，按照美国医学遗传学学会和分子病理学协会的现行指南，很难对由错义或非编码变体引起的疾病提供可靠的临床诊断，而这种错误占三分之一以上。病例。PRPF31中的突变是变性性睫状肌病常染色体显性遗传性视网膜色素变性的第二大最常见原因。在这里，我们提出了一种高通量高含量成像检测方法，可定量测量PRPF31中错义变异体的作用，符合最近公布的用于临床变异解释的基线标准体外测试标准。该测定法使用了新的PRPF31 ^+/-使用CRISPR基因编辑生成的人类视网膜细胞系可提供稳定的细胞系，纤毛明显更少，其中可以表达和表征新型错义变体。我们表明，在空背景下在纤毛虫基因中表达错义变异的细胞的高内涵成像可以根据纤毛表型表征变异。我们希望这将是对不确定意义的PRPF31变体进行临床表征的有用工具，并可以扩展到其他ciliopathies中的变体分类。