High-throughput PRPF31 variant characterisation pipeline consistent with ACMG/AMP clinical variant interpretation guidelines

Mutations in PRPF31 are the second most common cause of the degenerative retinal condition autosomal dominant retinitis pigmentosa. Difficulty in characterising missense variants in this gene presents a significant challenge in providing accurate diagnosis for patients to enable targeted testing of other family members, aid family planning, allow pre-implantation diagnosis and inform eligibility for gene therapy trials. With PRPF31 gene therapy in development, there is an urgent need for tools for accurate molecular diagnosis. 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 and well-characterized PRPF31 +/- human retinal cell line generated using CRISPR gene editing, which allows testing of PRPF31 variants which may be causing disease through either haploinsufficiency or dominant negative effects, or a combination of both. The mutant cells have significantly fewer cilia than wild-type cells, allowing rescue of ciliogenesis with benign or mild variants, but do not totally lack cilia, so dominant negative effects can be observed. The results of the assay provide BS3_supporting evidence to the benign classification of two novel uncharacterized PRPF31 variants and suggest that one novel uncharacterized PRPF31 variant may be pathogenic. We hope that this will be a useful tool for clinical characterisation of PRPF31 variants of unknown significance, and can be extended to variant classification in other ciliopathies.