From shadows to clarity: A new paradigm for comprehensive variant detection in undiagnosed dystrophinopathy using combined long-read and RNA sequencing

Approximately 2-5 % of dystrophinopathy cases remain undiagnosed at the molecular level following standard multiplex ligation-dependent probe amplification (MLPA) and exome sequencing (ES), precluding these patients from variant-specific genetic counseling and therapy eligibility assessment. We developed an integrated diagnostic pipeline combining targeted long-read sequencing (LRS) with muscle RNA-seq and applied it to 50 Chinese patients (46 males, 4 females) with biopsy-confirmed dystrophinopathy in whom MLPA and ES had yielded negative results. A molecular diagnosis was achieved for all 50 probands: 30 (60 %) harbored intronic single nucleotide variants (SNVs) leading to pseudoexon inclusion or exon skipping, while the remaining 20 (40 %) carried complex structural variants (SVs), including inversions (n = 13), translocations (n = 5), and insertions/deletions (n = 2). Of these, 23 (46 %) were novel variants not previously recorded in disease databases, and RNA-seq confirmed their aberrant splicing effects. A notable discovery was a 1.9 kb intronic inversion that uniquely activated cryptic splice sites on the antisense strand, representing a previously unreported pathogenic mechanism in dystrophinopathies. Cascade screening identified 45 carriers across 28 families, revealing a maternal carrier rate of 84.8 % (28/33). This integrated LRS and RNA-seq approach demonstrates significant value in resolving molecularly undiagnosed dystrophinopathy cases and enabling comprehensive genetic counseling. Furthermore, it establishes a robust diagnostic paradigm for molecularly unresolved neuromuscular disorders.