Small mutations detected by multiplex ligation-dependent probe amplification of the dystrophin gene

Currently, multiplex ligation-dependent probe amplification (MLPA) has been recognized as the most powerful and convenient method to identify exon deletions or duplications in the dystrophin gene, the mutation of which causes Duchenne and Becker muscular dystrophies (DMD/BMD). The mutation diagnosis is easily done by assessing the amounts amplified by MLPA (loss, single, or double). However, an ambiguous amount of amplified product has never been reported. When 77 Japanese DMD/BMD patients were examined by MLPA from MRC-Holland (Amsterdam, The Netherlands), deletions/duplications in the dystrophin gene were identified in 64.8%. Ten male patients showed loss of a single exon by MLPA, but one of them was found to have not an exon deletion, but a four-nucleotide deletion (c.3347-3350delAGAA) within the exon. Remarkably, two patients showed ambiguous amounts of product with less than half of that of a single copy, making the genetic diagnosis impossible. In one patient, a novel single-nucleotide change (c.4303G>T) leading to a nonsense mutation was identified. In another patient, a novel five-nucleotide deletion (c.4536-4540delGAGTG) was identified. It was considered that these two mutations partially disturbed MLPA amplification, resulting in ambiguous amplification. These results show that MLPA can serve as a tool for screening small mutations, as well as for detecting exon deletions or duplications.