RT Journal Article
SR Electronic
T1 Interpreting TMEM67 missense variants of uncertain significance (VUS) in an animal model
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2021.06.17.448799
DO 10.1101/2021.06.17.448799
A1 Karen I. Lange
A1 Sunayna Best
A1 Sofia Tsiropoulou
A1 Ian Berry
A1 Colin A. Johnson
A1 Oliver E. Blacque
YR 2021
UL http://biorxiv.org/content/early/2021/06/17/2021.06.17.448799.abstract
AB Purpose A molecular genetic diagnosis is essential for accurate counselling and management of patients with ciliopathies. Uncharacterized missense alleles are often classified as variants of uncertain significance (VUS) and are not clinically useful. In this study, we explore the use of a tractable animal model (C. elegans) for in vivo interpretation of missense VUS alleles of TMEM67, a gene frequently mutated as a cause of ciliopathies.Methods CRISPR/Cas9 gene editing was used to generate homozygous worm strains carrying TMEM67 patient variants. Quantitative phenotypic assays (dye filling, roaming, chemotaxis) assessed cilia structure and function. Results were validated by genetic complementation assays in a human TMEM67 knock-out hTERT-RPE1 cell line.Results Quantitative assays in C. elegans distinguished between known benign (Asp359Glu, Thr360Ala) and pathogenic (Glu361Ter, Gln376Pro) variants. Analysis of seven missense VUS alleles predicted two benign (Cys173Arg, Thr176Ile) and four pathogenic variants (Cys170Tyr, His782Arg, Gly786Glu, His790Arg). Results from one VUS (Gly979Arg) were inconclusive in worms, but additional in vitro validation suggested it was likely benign.Conclusion Efficient genome editing and quantitative functional assays in C. elegans make it a tractable in vivo animal model that allows stratification and rapid, cost-effective interpretation of ciliopathy-associated missense VUS alleles.Competing Interest StatementThe authors have declared no competing interest.