Splicing Outcomes of 5' Splice Site GT>GC Variants That Generate Wild-Type Transcripts Differ Significantly Between Full-Length and Minigene Splicing Assays.

Jin-Huan Lin,Gerald Le Gac,Wen-Bin Zou,Jian-Min Chen,Claude Férec,David N Cooper,Hao Wu,Emmanuelle Masson,Zhuan Liao,Yann Fichou

doi:10.3389/fgene.2021.701652

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.

Highlights

In principle, both coding and intronic variants within a gene have the potential to affect splicing (Cooper et al, 2009; Scotti and Swanson, 2016; Anna and Monika, 2018; Truty et al, 2021)
Combining data derived from a meta-analysis of human inherited diseaseassociated +2T>C variants and a cell culture-based Full-Length Gene Splicing Assay (FLGSA) of forward engineered +2T>C substitutions, we estimated that ∼15–18% of +2T>C variants can generate up to 84% wild-type transcripts relative to their wild-type counterparts (Lin et al, 2019)
Many +2T>C variants in human disease genes that have been capable of generating some wild-type transcripts are likely to have gone largely unreported; this represents a significant deficiency in terms of our understanding of genotype-phenotype relationships and tailored treatment options given that even the minor retention of wild-type transcripts derived from a variant allele might significantly impact disease expression and severity (Ramalho et al, 2002; Den Uijl et al, 2011; Raraigh et al, 2018; Lin et al, 2019; Scalet et al, 2019; Joynt et al, 2020)

Summary

Introduction

Both coding and intronic variants within a gene have the potential to affect splicing (Cooper et al, 2009; Scotti and Swanson, 2016; Anna and Monika, 2018; Truty et al, 2021). Many +2T>C variants in human disease genes that have been capable of generating some wild-type transcripts are likely to have gone largely unreported; this represents a significant deficiency in terms of our understanding of genotype-phenotype relationships and tailored treatment options given that even the minor retention of wild-type transcripts derived from a variant allele might significantly impact disease expression and severity (Ramalho et al, 2002; Den Uijl et al, 2011; Raraigh et al, 2018; Lin et al, 2019; Scalet et al, 2019; Joynt et al, 2020).

Results

Discussion

Conclusion