Variants in the Sequence of the Probe Hybridization Site May Affect MLPA Performance in Patients with Duchenne/Becker Muscular Dystrophy

Background The MLPA (multiplex ligation dependent probe amplification) technique is currently the test most widely used to detect mutations in the Duchenne/Becker muscular dystrophy (DMD) gene in the initial assessment. However, several studies have suggested that MLPA results require implementing other detection methods due to false duplication. Our aim was to evaluate variables that could alter the peak ratio in MLPA in individuals with Duchenne/Becker muscular dystrophy (DMD/BMD) who present sequence variants at the probe hybridization site, such as the location of sequence variants (SVs), melting temperature of the probe, and the type of variant. Methods We analyzed patients with clinical suspicion of DMD/BMD through the MLPA technique. The DMD gene was sequenced in patients with normal results in MLPA. Results Of 111 patients, 72 had an abnormal MLPA result, of which 10 had a single exon abnormal peak, and 39 had a normal peak ratio. Out of 10 patients, 4 (40%) with a single exon abnormal peak ratio had SV at the hybridization site of the probe. In the other 6, the deletion was confirmed. Out of 39 patients with a normal peak ratio, 11 presented SVs at the hybridization site of the probe, and DMD/BMD was confirmed. Conclusions In cases of abnormal peak ratio results of MLPA in a single exon, it would be valuable to sequence the DMD gene to assess whether variants in the probe hybridization site might result in a false positive that could be interpreted as an exon deletion.