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

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.

The Study of a 231 French Patient Cohort Significantly Extends the Mutational Spectrum of the Two Major Usher Genes MYO7A and USH2A

“…The age of onset of the RP though, remains unknown. However, we show here that USH2A genotypes with a missense/missense combination also lead to USH in 10% of cases and not to isolated RP, as some studies based on fewer patients would suggest [23]. As expected, the c.2276G>T -p.(Cys759Phe) is present in only 2.2% of our cohort.…”

Whole genome sequencing for USH2A-associated disease reveals several pathogenic deep-intronic variants that are amenable to splice correction

“…In previous studies, WES and targeted sequencing panels solved 36% to 63% of persons with non-syndromic arRP and 84% to 90% of Usher syndrome (USH) cases. 4 , 9 We hypothesized that a substantial portion of the remaining unsolved cases has an SV or a deleterious deep-intronic variant that potentially results in inclusion of a pseudoexon (PE). To date, 80 (likely) pathogenic SVs (larger than 50 nucleotides) have already been submitted to ClinVar (March 29, 2022) and several deep-intronic variants resulting in PE inclusion have been reported for USH2A .…”

Genetic Characteristics and Variation Spectrum of USH2A-Related Retinitis Pigmentosa and Usher Syndrome

“…A considerable number of autosomal recessive retinitis pigmentosa (arRP; MIM#268000) or Usher syndrome type II (USH2, MIM#276901) patients are due to USH2A gene defects, and there is an incomplete understanding of the USH2A-related inherited retinal disease (USH2A-IRD) genotype spectrum, which reduces the use of future gene therapy and optimal genetic counseling. According to RetNet ( https://sph.uth.edu/retnet/sum-dis.htm ), variants in 66 and 16 genes have been associated with arRP and atypical forms of USH, respectively (Date of Visit: December 2021) (Stephen, Daiger, Lori, Sullivan, Sara, Bowne; Reurink et al, 2021 ). Based on severity and progression, three clinical forms of USH can be distinguished: USH type I (USH1), type II (USH2), and type III (USH3).…”

“…To date, a large number of studies have focused on the clinical and genetic characteristics of patients with USH2A mutations. However, most studies were conducted in non-East Asian cohorts ( Blanco-Kelly et al, 2015 ; Lenassi et al, 2015 ; Reurink et al, 2021 ), with limited data on Chinese patients, and a lack of knowledge of mutation spectrum differences between other populations ( Nakanishi et al, 2011 ; Ng et al, 2019 ). In this study, we obtained USH2A variants data of all 8,710 participants.…”