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.

Keywords: USH2A; Usher syndrome type IIa; molecular inversion probes (MIPs); retinitis pigmentosa.

Figure 1

Overview of combinations of variant types identified in autosomal recessive retinitis pigmentosa (arRP) (n = 11) and Usher syndrome type 2 (USH2) (n = 29) cases. Splice variant: Canonical splice site variants and variants with any effect on splicing (both in frame and out-of-frame). CNV: Copy number variant.

Figure 2

Results of minigene splice assays for variants c.8709C>T and c.14583−20C>G. (A) Variant c.8709C>T (indicated in red) is predicted by splice prediction tool SpliceAI to result in a 39-nt deletion of exon 44 in the USH2A transcript. This effect was indeed observed in a splice assay for the construct containing the variant (M), but not for the wildtype construct (WT). (B) SpliceAI predicts an increased strength of a non-canonical splice donor site due to variant c.14583−20C>G. A splice assay did not demonstrate the use of this splice site as no elongation of exon 67 was observed. Exonic sequences are boxed (blue). Green triangles indicate SpliceAI predictions.