Molecular Inversion Probe-Based Sequencing of USH2A Exons and Splice Sites as a Cost-Effective Screening Tool in USH2 and arRP Cases.

Janine Reurink,Jacoline B Ten Brink,Helger G Yntema,Dominika Oziębło,G Jane Farrar,L Ingeborgh Van Den Born,Erwin Van Wijk,Hanka Venselaar,Susanne Roosing,Astrid S Plomp,Marco Aben,Hannie Kremer,Monika Ołdak,Jaap Oostrik,Tuula Rinne,Adrian Dockery,Ronald J.e Pennings ,Frans P.m Cremers ,Mubeen Khan ,Arthur A B Bergen

doi:10.3390/ijms22126419

Abstract

A substantial proportion of subjects with autosomal recessive retinitis pigmentosa (arRP) or Usher syndrome type II (USH2) lacks a genetic diagnosis due to incomplete USH2A screening in the early days of genetic testing. These cases lack eligibility for optimal genetic counseling and future therapy. USH2A defects are the most frequent cause of USH2 and are also causative in individuals with arRP. Therefore, USH2A is an important target for genetic screening. The aim of this study was to assess unscreened or incompletely screened and unexplained USH2 and arRP cases for (likely) pathogenic USH2A variants. Molecular inversion probe (MIP)-based sequencing was performed for the USH2A exons and their flanking regions, as well as published deep-intronic variants. This was done to identify single nucleotide variants (SNVs) and copy number variants (CNVs) in 29 unscreened or partially pre-screened USH2 and 11 partially pre-screened arRP subjects. In 29 out of these 40 cases, two (likely) pathogenic variants were successfully identified. Four of the identified SNVs and one CNV were novel. One previously identified synonymous variant was demonstrated to affect pre-mRNA splicing. In conclusion, genetic diagnoses were obtained for a majority of cases, which confirms that MIP-based sequencing is an effective screening tool for USH2A. Seven unexplained cases were selected for future analysis with whole genome sequencing.

Highlights

A substantial proportion of individuals with autosomal recessive retinitis pigmentosa or Usher syndrome (USH) lacks a genetic diagnosis and has reduced possibilities for future therapy and optimal genetic counseling
Eleven autosomal recessive retinitis pigmentosa (arRP) cases with monoallelic USH2A variants that met our criteria were included in the study
The deletion is predicted to result in the loss of 12 fibronectin type-III domains of usherin, and this copy number variants (CNVs) was classified as pathogenic. We identified this deletion in USH type 2 (USH2) cases from five families in our cohort, and variable-number tandem repeat (VNTR) marker analysis in four out of five families revealed a shared haplotype (Figure S1)

Summary

Introduction

A substantial proportion of individuals with autosomal recessive retinitis pigmentosa (arRP) or Usher syndrome (USH) lacks a genetic diagnosis and has reduced possibilities for future therapy and optimal genetic counseling. According to RetNet, variants in 63 and 16 genes have been associated with arRP and (atypical forms of) USH, respectively (RetNet consulted on 9 March 2021) [1]. USH2A defects are estimated to be causative of 8–19% of non-syndromic arRP cases [2,3], 29–55% of all USH cases and 57–90%. The prevalence of arRP and USH is estimated to range from 22–67 per 100,000 individuals [7,8] and from 4–6 per 100,000 individuals [9,10], respectively. This highlights the importance of screening the USH2A gene in medical genetic testing. Screening of USH2A for defects can be challenging as the gene spans 800,503 nucleotides (nt) harboring 73 exons (NM_206933.2), including a cochlea-specific exon, exon

Objectives

Methods

Conclusion