Mutation Screening of Multiple Genes in Spanish Patients with Autosomal Recessive Retinitis Pigmentosa by Targeted Resequencing

Abstract

Retinitis Pigmentosa (RP) is a heterogeneous group of inherited retinal dystrophies characterised ultimately by the loss of photoreceptor cells. RP is the leading cause of visual loss in individuals younger than 60 years, with a prevalence of about 1 in 4000. The molecular genetic diagnosis of autosomal recessive RP (arRP) is challenging due to the large genetic and clinical heterogeneity. Traditional methods for sequencing arRP genes are often laborious and not easily available and a screening technique that enables the rapid detection of the genetic cause would be very helpful in the clinical practice. The goal of this study was to develop and apply microarray-based resequencing technology capable of detecting both known and novel mutations on a single high-throughput platform. Hence, the coding regions and exon/intron boundaries of 16 arRP genes were resequenced using microarrays in 102 Spanish patients with clinical diagnosis of arRP. All the detected variations were confirmed by direct sequencing and potential pathogenicity was assessed by functional predictions and frequency in controls. For validation purposes 4 positive controls for variants consisting of previously identified changes were hybridized on the array. As a result of the screening, we detected 44 variants, of which 15 are very likely pathogenic detected in 14 arRP families (14%). Finally, the design of this array can easily be transformed in an equivalent diagnostic system based on targeted enrichment followed by next generation sequencing.

Targeted Sequencing of 179 Genes Associated with Hereditary Retinal Dystrophies and 10 Candidate Genes Identifies Novel and Known Mutations in Patients with Various Retinal Diseases

“…Compared with these methods, next-generation massively parallel sequencing (NGS) with various applications provides high sensitivity and speed of variation detection and is currently considered the most efficient approach for mutation screening. [11][12][13] While several chip-based approaches for the NGS platform have been previously developed to screen various panels of HRDs genes (e.g., for autosomal dominant and recessive RP genes [adRP genes and arRP genes] 14,15 ) exome sequencing is also considered a first tier screening method for genetically heterogeneous diseases. 16 Targeted gene approaches save time, cost less, and achieve higher enrichment of the targeted sequences, but at the expense of missing mutations in genes not previously associated with HRDs.…”

“…Several microarrays have been developed recently to capture various panels of HRD genes. 14,15,[31][32][33] Most of these arrays were generated on the Affymetrix resequencing chip platform (Affymetrix, Inc., Cleveland, OH). 15,31,32 The nucleotide call rates for the previously reported resequencing chips ranged from 90% to 99%.…”

“…14,15,[31][32][33] Most of these arrays were generated on the Affymetrix resequencing chip platform (Affymetrix, Inc., Cleveland, OH). 15,31,32 The nucleotide call rates for the previously reported resequencing chips ranged from 90% to 99%. 15 The RDs189-array yielded superior call rates (>99.9%), presumably due to the removal of repetitive elements (see Supplementary Material and Supplementary Table S3, http://www.iovs.org/lookup/suppl/doi:10.…”

“…15,31,32 The nucleotide call rates for the previously reported resequencing chips ranged from 90% to 99%. 15 The RDs189-array yielded superior call rates (>99.9%), presumably due to the removal of repetitive elements (see Supplementary Material and Supplementary Table S3, http://www.iovs.org/lookup/suppl/doi:10. 1167/iovs.12-10967/-/DCSupplemental).…”

Panel-based next generation sequencing as a reliable and efficient technique to detect mutations in unselected patients with retinal dystrophies

“…5,6 In contrast to the APEX technology, Sanger and microarray-based sequencing approaches identify also novel base changes. 7,8 Approaches to apply Sanger sequencing to all known disease genes implicated in a subtype of RD, namely RP, result in a detection rate of 50-60% among the autosomal dominant RP cases, however, only at the expense of an enormous effort in time and costs for the analysis of hundreds of exons. 2,9 Recently, studies applying next generation sequencing (NGS) techniques to analyze genetically precharacterized patient collections showed similar mutation detection rates for RP cases.…”