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

Abstract

Inherited retinal degeneration (IRD) is a common cause of visual impairment (prevalence B1/3500). There is considerable phenotype and genotype heterogeneity, making a specific diagnosis very difficult without molecular testing. We investigated targeted capture combined with next-generation sequencing using Nimblegen 12plex arrays and the Roche 454 sequencing platform to explore its potential for clinical diagnostics in two common types of IRD, retinitis pigmentosa and cone-rod dystrophy. 50 patients (36 unknowns and 14 positive controls) were screened, and pathogenic mutations were identified in 25% of patients in the unknown, with 53% in the early-onset cases. All patients with new mutations detected had an age of onset o21 years and 44% had a family history. Thirty-one percent of mutations detected were novel. A de novo mutation in rhodopsin was identified in one early-onset case without a family history. Bioinformatic pipelines were developed to identify likely pathogenic mutations and stringent criteria were used for assignment of pathogenicity. Analysis of sequencing metrics revealed significant variability in capture efficiency and depth of coverage. We conclude that targeted capture and next-generation sequencing are likely to be very useful in a diagnostic setting, but patients with earlier onset of disease are more likely to benefit from using this strategy. The mutation-detection rate suggests that many patients are likely to have mutations in novel genes.

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

“…6 Patients 2 and 4 were screened in a pilot study of NGS in patients negative for mutations in the ATP-binding cassette, sub-family A, member 4 gene (ABCA4). Patient 3 had early-onset RP and no family history and was screened as part of a wider study on NGS in clinical diagnostics, whereas Patient 1 had a family history of ADRP and was screened in a UK National Health Service diagnostics laboratory.…”

“…DNA was sequenced on the 454 GS FLX platform (Roche, Branford, CT). 6 The pathogenicity of all sequence variants was assessed using standard bioinformatic analyses (Supplementary Materials and Methods online). Multiplex ligation-dependent probe amplification (MLPA) was used to exclude the possibility of recessive disease caused by an undetected copy-number variant in the second allele and was performed using the SALSA MLPA RP kit (MRC-Holland, Amsterdam, Netherlands), according to the manufacturer's instructions (Supplementary Materials and Methods online).…”

“…6 Variants were filtered to include nonsynonymous single-nucleotide polymorphisms (SNPs), variants within 25 bp of the splice sites, and indels. All data were then analyzed using standard bioinformatic programs including the annotation software Alamut, and pathogenicity prediction programs PolyPhen, SIFT, and MutPred, plus literature searches and comparison with frequency data in dbSNP and the 1000 Genomes project.…”

Streamlined ion torrent PGM-based diagnostics: BRCA1 and BRCA2 genes as a model

“…As a result, many diagnostic laboratories are shifting from Sanger sequencing platforms to higher throughput NGS platforms, [1][2][3][4][5] already used in research. However, the performance and quality criteria expected from NGS in diagnostic and research settings are strikingly different.…”

Understanding the impact of genetic testing for inherited retinal dystrophy

“…Until recently, conventional molecular diagnostics were limited to conditions such as X-linked retinitis pigmentosa (xlRP), choroideraemia and retinoschisis 5 where family history or clinical examination can steer molecular testing, while array-based detection methods were implemented to identify known mutations for certain heterogeneous conditions such as Leber congenital amaurosis. 4 The advent of next generation sequencing (NGS) strategies has made molecular diagnosis and genetic testing available to significantly greater numbers of families with RD 3,6,7 and as new NGS tests become available, it will be important to understand where there is significant patient need for testing and to define how clinical services should meet those needs.…”