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

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

Methods: 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.

Results: RNAseq identified aberration splicing or expression events in the 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.

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