纤毛病是与能动或原发性非能动纤毛的结构或功能缺陷相关的广泛的遗传发育和退行性疾病。大约有200个已知的纤毛病疾病基因,而基因检测可以提供准确的诊断,24-60%接受基因检测的纤毛病患者没有接受基因诊断。这部分是因为遵循美国医学遗传学学院和分子病理学协会的现行指南,很难对由错义或非编码变异引起的疾病提供可靠的临床诊断,占疾病病例的三分之一以上。PRPF31中的突变是退行性视网膜纤毛病常染色体显性视网膜色素变性的第二最常见原因。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中错义变异的效果,该检测方法符合最近公布的临床变异判读体外基线标准标准.该测定利用使用CRISPR基因编辑产生的新的PRPF31+/-人视网膜细胞系,以提供具有显著更少的纤毛的稳定细胞系,其中表达和表征新的错义变体。我们表明,在无效背景下表达纤毛病基因错义变异的细胞的高含量成像可以根据纤毛表型表征变异。我们希望这将是一个有用的工具,用于不确定意义的PRPF31变体的临床表征,并可以扩展到其他纤毛病的变异分类。
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.