Dataset Information

Minigene-Based Splice Assays Reveal the Effect of Non-Canonical Splice Site Variants in USH2A.

ABSTRACT: Non-canonical splice site variants are increasingly recognized as a relevant cause of the USH2A-associated diseases, non-syndromic autosomal recessive retinitis pigmentosa and Usher syndrome type 2. Many non-canonical splice site variants have been reported in public databases, but an effect on pre-mRNA splicing has only been functionally verified for a subset of these variants. In this study, we aimed to extend the knowledge regarding splicing events by assessing a selected set of USH2A non-canonical splice site variants and to study their potential pathogenicity. Eleven non-canonical splice site variants were selected based on four splice prediction tools. Ten different USH2A constructs were generated and minigene splice assays were performed in HEK293T cells. An effect on pre-mRNA splicing was observed for all 11 variants. Various events, such as exon skipping, dual exon skipping and partial exon skipping were observed and eight of the tested variants had a full effect on splicing as no conventionally spliced mRNA was detected. We demonstrated that non-canonical splice site variants in USH2A are an important contributor to the genetic etiology of the associated disorders. This type of variant generally should not be neglected in genetic screening, both in USH2A-associated disease as well as other hereditary disorders. In addition, cases with these specific variants may now receive a conclusive genetic diagnosis.

SUBMITTER: Reurink J

PROVIDER: S-EPMC9654511 | biostudies-literature | 2022 Nov

REPOSITORIES: biostudies-literature

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Publications

Minigene-Based Splice Assays Reveal the Effect of Non-Canonical Splice Site Variants in USH2A.

Reurink Janine J Oostrik Jaap J Aben Marco M Ramos Mariana Guimarães MG van Berkel Emma E Ołdak Monika M van Wijk Erwin E Kremer Hannie H Roosing Susanne S Cremers Frans P M FPM

International journal of molecular sciences 20221101 21

Non-canonical splice site variants are increasingly recognized as a relevant cause of the USH2A-associated diseases, non-syndromic autosomal recessive retinitis pigmentosa and Usher syndrome type 2. Many non-canonical splice site variants have been reported in public databases, but an effect on pre-mRNA splicing has only been functionally verified for a subset of these variants. In this study, we aimed to extend the knowledge regarding splicing events by assessing a selected set of USH ...[more]

PMID: 36362125

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Project description:PALB2 loss-of-function variants confer high risk of developing breast cancer. Here we present a systematic functional analysis of PALB2 splice-site variants detected in approximately 113,000 women in the large-scale sequencing project Breast Cancer After Diagnostic Gene Sequencing (BRIDGES; https://bridges-research.eu/). Eighty-two PALB2 variants at the intron-exon boundaries were analyzed with MaxEntScan. Forty-two variants were selected for the subsequent splicing functional assays. For this purpose, three splicing reporter minigenes comprising exons 1-12 were constructed. The 42 potential spliceogenic variants were introduced into the minigenes by site-directed mutagenesis and assayed in MCF-7/MDA-MB-231 cells. Splicing anomalies were observed in 35 variants, 23 of which showed no traces or minimal amounts of the expected full-length transcripts of each minigene. More than 30 different variant-induced transcripts were characterized, 23 of which were predicted to truncate the PALB2 protein. The pathogenicity of all variants was interpreted according to an in-house adaptation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG-AMP) variant classification scheme. Up to 23 variants were classified as pathogenic/likely pathogenic. Remarkably, three ±1,2 variants (c.49-2A>T, c.108+2T>C, and c.211+1G>A) were classified as variants of unknown significance, as they produced significant amounts of either in-frame transcripts of unknown impact on the PALB2 protein function or the minigene full-length transcripts. In conclusion, we have significantly contributed to the ongoing effort of identifying spliceogenic variants in the clinically relevant PALB2 cancer susceptibility gene. Moreover, we suggest some approaches to classify the findings in accordance with the ACMG-AMP rationale. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Dataset Information

Minigene-Based Splice Assays Reveal the Effect of Non-Canonical Splice Site Variants in USH2A.

Publications

Minigene-Based Splice Assays Reveal the Effect of Non-Canonical Splice Site Variants in <i>USH2A</i>.

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