Validation of ambiguous MLPA results by targeted next-generation sequencing discloses a nonsense mutation in the DMD gene.

Background: Duchenne muscular dystrophy (DMD) is the most common inherited muscular disease and caused by mutations in the DMD gene on the X-chromosome. Multiplex ligation-dependent probe amplification (MLPA) is recognized as a convenient and reliable technique to detect exon deletion/duplication mutations in the DMD gene. Here, we applied targeted semi-conductor next-generation sequencing to clarify the cause of ambiguous MLPA results.

Methods: Targeted semi-conductor next-generation sequencing was carried out using the Inherited Disease Panel (IDP) on the Ion Torrent Personal Genome Machine (PGM).

Results: MLPA analysis disclosed unclassifiable relative peak ratio of exon 18 in a DMD boy. His female cousin was indicated to have exon 18 deletion in one allele. To validate these incompatible results, targeted next-generation sequencing was conducted. A nucleotide change, C.2227 C>T creating a premature stop codon, was in exon 18. Concomitantly, both C and T nucleotides were identified in his cousin's genome. Ambiguous values of the relative peak ratio in MLPA were considered due to the one nucleotide mismatch between the genomic sequence and the probe used in MLPA.

Conclusion: Analysis using IDP on PGM disclosed a nonsense mutation in the DMD gene as a cause of ambiguous results of MLPA.