Cytoscan hd array

Genomic DNA was isolated using the Chemagen automated DNA isolation work station (Janus Chemagic 360, Perkin Elmer). Single nucleotide variation and copy number variation (CNV) analysis of the IGF1 gene (NM_000618.5) was performed using Sanger sequencing and multiplex ligation-dependent probe amplification (SALSA MLPA Probemix P262 GHI, MRC Holland) according to standard procedures (details available upon request). Single nucleotide variation and CNV classification was performed using ACMG/AMP criteria [34, 35, 44] . Genome-wide CNV analysis was performed using Affymetrix CytoScan HD array, Illumina Infinium CytoSNP-850K BeadChip or Illumina Infinium GSA+MD-24 v1.0 Beadchip according to standard procedures (details available upon request).

Deletions and duplications were detected by microarray analysis of DNA extracted from peripheral blood. Most subjects were tested with a version of the EmArray oligonucleotide array [Baldwin et al. 2008 Gene Technology (OGT) array based on the GRCh37/hg19 genome assembly. Subjects 4-7 were tested using the Affymetrix CytoScan HD array and Subjects 11 and 14 were tested using the Illumina CytoSNP-850K BeadChip, both based on the GRCh37/hg19 genome assembly.
Subjects 2 and 3 were tested using the 180k CMA-HR, based on the NCBI36/hg18 genome assembly. Coordinates from arrays using the NCBI36/hg18 build were converted to GRCh37/hg19 using the LiftOver tool (http://genome.ucsc.edu) [Kent et al. 2002] .
DNA digestion, labeling, purification, hybridization, array scanning, and analysis were performed following manufacturers' instructions. To determine the inheritance of CNVs, fluorescence in situ hybridization (FISH) analysis was performed on peripheral blood samples from parents using standard cytogenetic procedures with probes corresponding to the proband's CNV.