Unveiling non-coding DMD variants: synergising RNA sequencing and DNA sequencing for enhanced molecular diagnosis

Pathogenic variants in the DMD gene are associated with dystrophinopathy including Duchenne and Becker muscular dystrophy (DMD/BMD). Targeted DMD gene, gene panels, exomes and genome sequencing have advanced genetic diagnostics, yet some cases remain elusive.

We performed total RNA sequencing (RNAseq) on muscle biopsy from 13 male patients with a clinical diagnosis of DMD/BMD. Splice aberration events are evaluated using the Integrative Genomics Viewers. Targeted DNA sequencing result was used/re-analysed to confirm complex rearrangement events identified.

RNAseq identified aberration splicing or expression events in the DMD gene of 12 cases. Splice-altering intronic single nucleotide variant events including c.7309+5G>T, c.7309+5G>A, c.3276+1G>A and c.3603+820G>T were identified in four cases. Splice-altering events were also detected in one case with small indel c.94-38_94del and two cases with intragenic deletions (exons 51-52 and 45-47 deletions). Furthermore, complex DNA rearrangements inducing aberration splicing/expression events were identified in five cases involving exons 44-45, 55-56, 2-79, 45-79 or 68-79, which were concordant with their DNA sequencing reanalysis results. Some cases with exon deletions have presented non-canonical transcripts expression. The RNAseq result showing aberrant splicing/expression in different exon regions in most of these cases corresponded with their immunohistochemical dystrophin staining results and/or clinical symptoms.

Our data demonstrated RNAseq is a powerful tool to provide functional data for DMD splice aberration events, especially when integrating with immunohistochemical testing and DNA sequencing, for elucidating the pathogenicity of DMD variants and achieving a precise genetic diagnosis in patients with DMD/BMD clinical presentation but without definitive diagnoses after routine genetic testing.