Genomic DNA was extracted from peripheral blood leukocytes or lymphoblastoid cell lines using the NucleoSpin Blood L kit (Macherey-Nagel, Duren, Germany). MLPA was performed using the P064 MR1 (mental retardation 1) and P250 DiGeorge kits (MRC-Holland, Amsterdam, The Netherlands). The P064 MR1 kit detects copy number changes both at the 15q and 22q loci, and includes 5 probes specific for sequences in or near the 15q11.2 Prader-Willi syndrome/Angelman syndrome critical region (one probe in the MKRN3, NDN, and GABRB3, and two in UBE3A) and 6 probes in the 22q11.21 DiGeorge region (in chromosomal order: CLTCL1, CDC45L, CLDN5, ARVCF, KLHL22, SNAP29). Information regarding the probe sequences and ligation sites can be obtained at http://www.mlpa.com. The P064 kit also screens for other mental retardation syndromes: Smith-Magenis syndrome (17p11.2), Williams syndrome (7q11.23), 1p deletion syndrome (1p36), Sotos syndrome (5q35.3), Miller-Dieker syndrome (17p13.3), Alagille syndrome (20p12.2), and Saethre-Chotzen syndrome (7p21). All patients (n = 236) were screened with the P064 MLPA kit; in addition, 126 patients were also screened with the P250 DiGeorge kit, which contains 14 different probes in the 22q11.2 region.
