Pitfalls of Multiple Ligation-Dependent Probe Amplifications in Detecting DMD Exon Deletions or Duplications

Multiple ligation-dependent probe amplifications (MLPAs) are a key technology for the molecular diagnosis of Duchenne/Becker muscular dystrophy, which is mainly caused by large gene arrangements. However, little is known about the false-positive rates of MLPA for this disease. Here, we review MLPA analysis results from 398 patients suspected to have Duchenne/Becker muscular dystrophy. MLPA assay was used for screening the entire coding region. If these amplifications produced normal results, direct sequencing was performed to search for sequence variations and to determine single-exon deletions, duplications, or indeterminate results. Using MLPA, 290 cases (72.9%) showed exon deletion or duplication results. Among those, 75 cases (25.9%) resulted in a deletion or duplication of a single exon. Direct sequencing revealed that 11 single-exon deletion cases resulted in false-positives due to sequence variations within the patient population interfering with probe binding at the probe-hybridization sites. Abnormal MLPA results were closely related to the type of sequence change and the position within the probe-hybridization locus. The most common type was C-T transition (n = 19, 55.9%). Abnormal MLPA results correlated with CA mismatch and low melting temperature (≤75°C). False-positive events for large gene rearrangements involving a single exon in DMD accounted for approximately 15% (11/75). Therefore, careful design of MLPA probes is required to avoid false-positive results.