Chromosomal Microarray Analysis Protocol

Chromosomal microarray analysis was performed using Human OmniExpress-24 v1.0 BeadChip (Illumina Inc., San Diego, CA, USA), which contains 716,503 genome-wide markers at an average spacing of 4 kb. It targets a minor allele frequency of 5%, as reported in the HapMap data. It includes SNPs within 10 kb of RefSeq genes, nonsynonymous SNPs (NCBI annotated), MHC/ADME SNPs, and sex chromosomes. DNA amplification, tagging, and hybridization were performed according to the manufacturer’s protocol, aided by the Tecan Freedom Evo (Tecan, Mannedorf, Switzerland). The array slides were scanned on an iScan Reader (Illumina, Inc., San Diego, CA, USA). All data collected were evaluated using Illumina Genome Studio v2011.1 software (Illumina, Inc., San Diego, CA, USA) and genome build GRCh37/hg19. Data were analyzed using Nexus Copy Number 7.5 (BioDiscovery, El Segundo, CA, USA). CNV classification was based on the American College of Medical Genetics and Genomics (ACMG) and the Clinical Genome Resource (ClinGen) 2020 guidelines [21 (link)].