Next-generation sequencing in health-care delivery: lessons from the functional analysis of rhodopsin

Abstract

original research articlePurpose: 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. 2012:14(11):891-899 Genet Med

Next-generation sequencing (NGS) as a diagnostic tool for retinal degeneration reveals a much higher detection rate in early-onset disease

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Targeted Next-Generation Sequencing Reveals Novel USH2A Mutations Associated with Diverse Disease Phenotypes: Implications for Clinical and Molecular Diagnosis

“…Among all, interpretation of genetic datasets is most important while also most problematic [4]. Bioinformatics predictions are useful, but the results can sometimes be confusing or even incorrect [37]. Therefore, more functional analyses are needed to confirm the pathogenesis of distinct mutations, and also to gain a clearer insight into the mechanism underlying the selectively clinical phenotypes caused by different mutations.…”

Genomic evidence for rod monochromacy in sloths and armadillos suggests early subterranean history for Xenarthra

“…Dasypus novemcinctus and C. hoffmanni also share a unique missense mutation in SWS1, possessing a leucine at residue 23 rather than a proline (bovine RH1 numbering; figure 1; electronic supplementary material, table S3). A proline is present in all opsins across vertebrates [49], with missense mutations in rod opsin (RH1) resulting in a non-functional pigment in vitro (P23H [50]), progressive photoreceptor degeneration in vivo (P23H [51,52]), reduction in chromophore yield owing to a decrease in cell-surface transportation (P23H, P23L [53]) and high amounts of misfolding, with P23L having the highest degree of misfolding among seven RH1 mutants [54]. Consistent with these data, Kogia breviceps ( pygmy sperm whale [6]) and Megaderma lyra (greater false vampire bat (AWHB01305061-AWHB01305064)) both have SWS1 pseudogenes with L23.…”