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Splicing outcomes of 5′ splice site GT>GC variants that generate wild-type transcripts differ significantly between full-length and minigene splicing assays

Lin, Jin-Huan, Wu, Hao, Zou, Wen-Bin, Masson, Emmanuelle, Fichou, Yann, Le Gac, Gerald, Cooper, David N., Férec, Claude, Liao, Zhuan and Chen, Jian-Min 2021. Splicing outcomes of 5′ splice site GT>GC variants that generate wild-type transcripts differ significantly between full-length and minigene splicing assays. Frontiers in Genetics 12 , 701652. 10.3389/fgene.2021.701652

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Abstract

Combining data derived from a meta-analysis of human disease-associated 5′ splice site GT>GC (i.e., +2T>C) variants and a cell culture-based full-length gene splicing assay (FLGSA) of forward engineered +2T>C substitutions, we recently estimated that ∼15–18% of +2T>C variants can generate up to 84% wild-type transcripts relative to their wild-type counterparts. Herein, we analyzed the splicing outcomes of 20 +2T>C variants that generate some wild-type transcripts in two minigene assays. We found a high discordance rate in terms of the generation of wild-type transcripts, not only between FLGSA and the minigene assays but also between the different minigene assays. In the pET01 context, all 20 wild-type minigene constructs generated the expected wild-type transcripts; of the 20 corresponding variant minigene constructs, 14 (70%) generated wild-type transcripts. In the pSPL3 context, only 18 of the 20 wild-type minigene constructs generated the expected wild-type transcripts whereas 8 of the 18 (44%) corresponding variant minigene constructs generated wild-type transcripts. Thus, in the context of a particular type of variant, we raise awareness of the limitations of minigene splicing assays and emphasize the importance of sequence context in regulating splicing. Whether or not our findings apply to other types of splice-altering variant remains to be investigated.

Item Type: Article
Date Type: Publication
Status: Published
Schools: Medicine
Additional Information: This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY)
Publisher: Frontiers Media
ISSN: 1664-8021
Date of First Compliant Deposit: 14 September 2021
Date of Acceptance: 13 July 2021
Last Modified: 15 Sep 2021 09:30
URI: http://orca.cardiff.ac.uk/id/eprint/144099

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