Detecting copy number changes in genomic DNA: MAPH and MLPA.

Using Multiplex Amplifiable Probe Hybridization (MAPH) and Multiplex Ligation-dependent ProbeAmplification (MLPA) we have screened different cohorts of Duchenne/Becker Muscular Dystrophy (DMD/BMD) patients for duplications. In an unselected series the duplication frequency was 8%; in agroup of patients already screened for deletions and point mutations we found a duplication in 64% ofcases. The majority were simple, contiguous duplications, however we detected 4 non-contiguousduplications, with two also including a triplication. In two instances the 3’ end of the gene was affected, aregion not usually screened by multiplex PCR. These mutations would therefore go undetected, whilst potentially disturbing the reading frame of the mRNA. This emphasizes the importance of screening theentire gene for rearrangements.More than 50% of the duplications found were at the 5` end of the gene, whereas most deletions are foundin the middle of the gene. A more detailed comparison of the regions affected showed that a duplication of exon 2 only was the single most common duplication found. Analysis of the breakpoints of 11 such casesrevealed two recombination hotspots within intron 2, whereas the breakpoints within intron 1 werescattered. We propose that unequal crossing over between sister chromatids is not responsible for exon 2duplications. Instead, a mechanism such as synthesis-dependent non-homologous end joining may beresponsible. Assuming this also applies to other duplications within the gene, this may explain the different distributions seen for deletions and duplications.67