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PURPOSE: The interpretation of genetic information has always been challenging, but next-generation sequencing produces data on such a vast scale that many more variants of uncertain pathogenicity will be found. We exemplify this issue with reference to human rhodopsin, in which pathogenic mutations can lead to autosomal dominant retinitis pigmentosa. METHODS: Rhodopsin variants, with unknown pathogenicity, were found in patients by next-generation and Sanger sequencing and a multidisciplinary approach was used to determine their functional significance. RESULTS: Four variants in rhodopsin were identified: F45L, P53R, R69H, and M39R, with the latter two substitutions being novel. We investigated the cellular transport and photopigment function of all four human substitutions and found that the F45L and R69H variants behave like wild-type and are highly unlikely to be pathogenic. By contrast, P53R (a de novo change) and M39R were retained in the endoplasmic reticulum with significantly reduced functionality and are clearly pathogenic. CONCLUSION: Potential pathogenicity of variants requires careful assessment using clinical, genetic, and functional data. We suggest that a multidisciplinary pathway of assessment, using several functional assays, will be required if next-generation sequencing is to be used effectively, reliably, and safely in the clinical environment.

Original publication




Journal article


Genet Med

Publication Date





891 - 899


Adult, Alleles, Amino Acid Sequence, Amino Acid Substitution, Biological Transport, Child, Preschool, Computational Biology, DNA Copy Number Variations, Endoplasmic Reticulum, Female, Genetic Predisposition to Disease, Genetic Testing, Genome, Human, Humans, Male, Middle Aged, Molecular Sequence Data, Mutation, Reproducibility of Results, Retinitis Pigmentosa, Rhodopsin, Sensitivity and Specificity, Sequence Analysis, DNA