Interpreting ciliopathy-associated missense variants of uncertain significance (VUS) in an animal model

Purpose Better methods are required to interpret the pathogenicity of disease-associated variants of uncertain significance (VUS), which cannot be actioned clinically. In this study, we explore the use of a tractable animal model ( Caenorhabditis elegans ) for in vivo interpretation of missense VUS alleles of TMEM67 , a cilia gene associated with ciliopathies. Methods CRISPR/Cas9 gene editing was used to generate homozygous knock-in C. elegans worm strains carrying TMEM67 patient variants engineered into the orthologous gene ( mks-3 ). Quantitative phenotypic assays of sensory cilia structure and function measured if the variants affect mks-3 gene function. Results from worms were validated by a genetic complementation assay in a human TMEM67 knock-out hTERT-RPE1 cell line that tests a TMEM67 signaling function. Results Assays in C. elegans accurately distinguished between known benign (Asp359Glu, Thr360Ala) and known pathogenic (Glu361Ter, Gln376Pro) variants. Analysis of eight missense VUS generated evidence that three are benign (Cys173Arg, Thr176Ile, Gly979Arg) and five are pathogenic (Cys170Tyr, His782Arg, Gly786Glu, His790Arg, Ser961Tyr). Conclusion Efficient genome editing and quantitative functional assays in C. elegans make it a tractable in vivo animal model that allows rapid, cost-effective interpretation of ciliopathy-associated missense VUS alleles. ### Competing Interest Statement The authors have declared no competing interest.