Christine Bellanné-Chantelot

Variants in GCK, HNF1A, and HNF4A genes are the three main causes of monogenic diabetes. Determining the molecular etiology is essential for patients with monogenic diabetes to benefit from the most appropriate treatment. The increasing number of variants of unknown significance (VUS) is a major issue in genetic diagnosis, and assessing the impact of variants on RNA splicing is challenging, particularly for genes expressed in tissues not easily accessible as in monogenic diabetes. The in vitro functional splicing assay based on a minigene construct is an appropriate approach. Here, we performed in silico analysis using SpliceAI and SPiP and prioritized 36 spliceogenic variants in GCK, HNF1A, and HNF4A. Predictions were secondarily compared with Pangolin and AbSplice-DNA bioinformatics tools which include tissue-specific annotations. We assessed the effect of selected variants on RNA splicing using minigene assays. These assays validated splicing defects for 33 out of 36 spliceogenic variants consisting of exon skipping (15%), exonic deletions (18%), intronic retentions (24%), and complex splicing patterns (42%). This provided additional evidence to reclassify 23 out of 31 (74%) VUS including missense, synonymous, and intronic noncanonical splice site variants as likely pathogenic variants. Comparison of in silico analysis with minigene results showed the robustness of bioinformatics tools to prioritize spliceogenic variants, but revealed inconsistencies in the location of cryptic splice sites underlying the importance of confirming predicted splicing alterations with functional splicing assays. Our study underlines the feasibility and the benefits of implementing minigene-splicing assays in the genetic testing of monogenic diabetes after a prior in-depth in silico analysis.