Next‐generation genetic testing for retinitis pigmentosa

Kornelia Neveling,Anneke I Den Hollander,L Ingeborgh Van Den Born,Joep De Ligt,Christian Gilissen,Marijke N Zonneveld,Michael F Buckley,Hans Scheffer,B Jeroen Klevering,Nienke Wieskamp,Rob W.J Collin,Lies H Hoefsloot,Frans P.M Cremers,Joris A Veltman,Alexander Hoischen,Linda Visser,Ulrich Kellner,Anna Siemiątkowska ,Ramon A C Van Huet ,Kari Branham ,Sabine Gijsen ,Michael Kwint ,Carel B Hoyng

doi:10.1002/humu.22045

Abstract

Molecular diagnostics for patients with retinitis pigmentosa (RP) has been hampered by extreme genetic and clinical heterogeneity, with 52 causative genes known to date. Here, we developed a comprehensive next-generation sequencing (NGS) approach for the clinical molecular diagnostics of RP. All known inherited retinal disease genes (n = 111) were captured and simultaneously analyzed using NGS in 100 RP patients without a molecular diagnosis. A systematic data analysis pipeline was developed and validated to prioritize and predict the pathogenicity of all genetic variants identified in each patient, which enabled us to reduce the number of potential pathogenic variants from approximately 1,200 to zero to nine per patient. Subsequent segregation analysis and in silico predictions of pathogenicity resulted in a molecular diagnosis in 36 RP patients, comprising 27 recessive, six dominant, and three X-linked cases. Intriguingly, De novo mutations were present in at least three out of 28 isolated cases with causative mutations. This study demonstrates the enormous potential and clinical utility of NGS in molecular diagnosis of genetically heterogeneous diseases such as RP. De novo dominant mutations appear to play a significant role in patients with isolated RP, having major implications for genetic counselling.

Highlights

Retinitis pigmentosa (RP; MIM# 268,000) is the most frequent subtype of inherited retinal disease and is clinically and genetically a highly heterogeneous disorder [den Hollander at al., 2010]
The coding regions of retinal dystrophy (RD) genes were enriched by target capture and screened for mutations by nextgeneration sequencing (NGS) in subjects—12 RD patients carrying two known heterozygous mutations in one of the retinal disease genes and 100 RP patients without a molecular diagnosis
We developed a comprehensive diagnostic tool for RP, consisting of a massive parallel sequencing approach for all known retinal disease genes, with systematic analysis and interpretation of all detected genetic variants

Summary

Introduction

Retinitis pigmentosa (RP; MIM# 268,000) is the most frequent subtype of inherited retinal disease and is clinically and genetically a highly heterogeneous disorder [den Hollander at al., 2010]. Fifty-two genes are known to be associated with nonsyndromic RP, involving all modes of inheritance [Berger et al, 2010]. The most widely applied diagnostic test for allelic and genetic heterogeneous diseases, arrayed primer extension (APEX) chip technology, is only able to detect known mutations [Avila-Fernandez et al, 2010]. These chips are designed to separately test for the presence of mutations in autosomal dominant or recessive RP genes, resulting in a diagnostic yield for autosomal recessive RP of only ∼10% [Avila-Fernandez et al, 2010]. The yield of diagnostic testing has remained disappointingly low for RP patients, despite many important disease gene discoveries in the last two decades [Berger et al, 2010]

Methods

Results

Conclusion