Stepwise genetic testing strategy identified pathogenic variants in 10 Chinese duchenne muscular dystrophy patients

Duchenne muscular dystrophy (DMD) results from pathogenic variants in the DMD gene. Despite routine screening using Multiplex Ligation-dependent Probe Amplification (MLPA) and Whole-Exome Sequencing (WES), a subset of cases remains molecularly unexplained. This diagnostic gap is often attributed to the inherent limitations of these methods in detecting deep intronic variants or complex structural rearrangements.

We implemented a tiered molecular diagnostic workflow for 10 male patients with suspected DMD. Primary screening for copy number variations (CNVs) was performed via MLPA. Negative cases were sequentially analyzed using WES and Whole-Genome Sequencing (WGS). Pathogenicity was validated through in silico splicing predictions, mRNA transcript analysis, and chromosomal breakpoint mapping.

MLPA identified CNVs in six cases. WES resolved two additional patients, identifying a frameshift variant (c.7392delC) and a nonsense variant (c.4729C>T). In the remaining two cases, WGS identified a deep intronic variant (c.9225-287C>A) and a 9.4 Mb chromosomal inversion. Functional analysis of the c.9225-287C>A variant revealed the activation of a 58-nucleotide pseudoexon, resulting in a frameshift (p.H3076Vfs*15) and truncated dystrophin expression. For the 9.4 Mb inversion, WGS successfully mapped the breakpoints to DMD intron 7, enabling definitive carrier identification within the family.

Our findings demonstrate that WGS is a robust tool for detecting pathogenic variations that evade standard MLPA and WES protocols, including deep intronic variants and large-scale inversions. This study emphasizes the clinical necessity of an integrated, tiered genomic approach to ensure accurate genetic counseling and facilitate access to precision therapies.