Functional analysis of variants in DMD exon/intron 10 predicted to affect splicing

Abstract

Duchenne muscular dystrophy (DMD, MIM #310200) and Becker muscular dystrophy (BMD, MIM #300376) are X-linked recessive hereditary diseases caused by pathogenic variants in the DMD gene. Genetic testing of DMD identifies a certain number of variants of uncertain clinical significance (VUS) whose functional interpretations pose a challenge for gene-based diagnosis. To improve the accuracy of variant interpretation in public mutation repositories, we used computational tools to prioritize VUS and developed a cell-based minigene assay to confirm aberrant splicing. Using this procedure, we evaluated rare variants in exon and intron 10 of the DMD gene. We demonstrated that 16 variants, including both canonical and non-canonical splice sites, altered RNA splicing in variable patterns. Using the example of exon and intron 10 of the DMD gene, we demonstrated the utility of the in vitro minigene assay in the effective assessment of the spliceogenic effect for VUS identified in clinical practice and underlined the necessity of precise variant classification. This is the first systematic characterization of DMD splicing variants, besides, through our study, some undetermined variants are demonstrated to be pathogenic by altering RNA splicing of DMD.