Fifty nanograms of DNA were used in the MLPA protocol. Experiments were performed according to the manufacturer's instructions but the volume of all kit reagents was decreased by 20%. Reactions were performed on a GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA, USA). PCR products were analyzed by capillary electrophoresis on an ABI Prism 3730 Genetic Analyzer (Applied Biosystems). The resultant traces were analyzed using the software GeneMarker 1.70 (SoftGenetics, State College, PA, USA). After population normalization, the peak height from each sample was compared to a synthetic control, which represents the median of all normal samples in each experiment. Peak heights below 0.75 were considered as deletions and values above 1.3 as duplications. Cases with apparent deletions or duplications were confirmed with quantitative PCR. Apparent deletions of a single probe were sequenced to rule out single-base changes within the probe-binding region. Analysis of positive controls (with confirmed 15q11-13 and 22q11.2 deletions and duplications) under the same experimental conditions ensured the reliable detection of copy number gains and loses.
