Application of long-read sequencing in the diagnosis of Duchenne/Becker muscular dystrophy: unveiling complex structural variations and deep intronic mutations.

BACKGROUND: Despite the widespread use of Multiplex Ligation-dependent Probe Amplification (MLPA) and Next-Generation Sequencing (NGS) in Duchenne/Becker Muscular Dystrophy (DMD/BMD), these methods have limitations when dealing with complex genetic backgrounds. Long-Read Sequencing (LRS), an emerging technology that provides longer read lengths, is advantageous for uncovering complex gene rearrangements and deep intronic mutations. METHODS: This study conducted LRS on 5 children with suspected DMD/BMD. One patient had not underwent genetic testing before, while the others had previously underwent MLPA and WES and were unable to find pathogenic variants. Chromosome analysis and X inactivation analysis were performed on female patient. RESULTS: Among the four patients who had not received a diagnosis through MLPA and NGS, LRS successfully identified translocations and inversions in two patients and deep intronic mutations in the other two. The fifth patient, who underwent LRS, initially showed no apparent mutations. However, muscle biopsy confirmed the disease diagnosis, and RNA sequencing revealed a partial deletion of exon 19 in the mRNA, ultimately pinpointing the causative mutation. CONCLUSIONS: The results of this study highlight the advantages of LRS in revealing complex genetic variations, particularly those challenging to detect with conventional methods, such as structural variations and deep intronic regions. Furthermore, combining muscle biopsy and RNA sequencing provides more comprehensive diagnostic information for patients not diagnosed through standard genetic tests. In the future, this technology is expected to complement routine genetic testing, aiding clinicians in achieving precise diagnoses across a broader patient population.