In the original arrays (e.g., 100K), Epacadostat mouse copy number was assessed based on intensity of signal from each SNP. For the Affymetrix 6.0 arrays, copy number probes are included in addition to the full array of SNPs, and both are used for quantitation. The Gaussian-smoothed signal log2-ratio of all probe intensities normalized to a reference of 270 normal HapMap samples was calculated by Affymetrix Genotyping Console with standard settings. Additional DNA from HMG-1 and two other samples was assessed by using the Affymetrix 6.0 SNP array. The

software dChipSNP was used for analysis. For HMG-1 and HMG-2, we performed qPCR in cases in which copy number change was detected. Primers were designed to 1q44 and 1p21.1. DNA from two control individuals (Promega) was used for comparison.

We repeated qPCR in an additional specimen from HMG-1 for confirmation by using primers targeting 1p (1p13.3, 1p32.3, and 1p36.2) and 1q (1q21.3, 1q31.1, and 1q42.2). Leukocytes were obtained from six of the cases; DNA was extracted by using standard methods and was used for SNP analysis as above. For HMG-1, we performed SNP analysis and clinical karyotype to assess for the presence of the trisomy 1q in peripheral blood leukocytes (evaluating 50 cells to detect even a low level of mosaicism). Based on the hypothesis that our cases harbor somatic mutations in genes that result in dysregulated growth, we screened the DNA from the brain

samples for a panel of known point mutations in cancer-associated genes (OncoMap Project, Dana Farber Cancer Institute) (MacConaill et al., 2009). This panel EPZ5676 concentration did not include AKT3; genes included in the 1q region were ABL2, DDR2, and NTRK1. We designed primers by using Primer 3 software (http://primer3.sourceforge.net) for the second exon of AKT1, AKT2, and AKT3 in order to evaluate nucleotide position 49. In cases without trisomy 1q, we sequenced DNA from brain tissue (six HMG cases) and leukocytes from the same cases (five cases). To determine the degree of mosaicism in the brain tissue specimen of HMG-3, Cetuximab molecular weight we performed TOPO TA cloning by using standard methods (Invitrogen), successfully analyzing 46 clones for the AKT3 c.49G→A mutation. Published sequencing data indicate that each individual has approximately one to two de novo nonsynonymous variants per diploid genome generation (Awadalla et al., 2010). The likelihood that this would affect this one base pair in all of the 6 × 107 base pairs of the diploid genome is therefore 2× 10−8–3 × 10−8. Correcting by a factor of 10 to reflect the increased somatic versus germline mutation rate (Lynch, 2010a) and accounting for three potential mutations at a given nucleotide position, the estimated likelihood that our mutation would occur by chance is at most 1 × 10−7. We labeled embryonic mouse cortex at embryonic day 10.5 (E10.5), E12.5, E14.5, E16.5, and E18.