60k cytosure constitutional v3 microarray

Chromosome preparation of iPSC lines was performed as described previously [16 (link)]. Twenty metaphases were analyzed for each cell line. To exclude clinically relevant copy number variants array, comparative genomic hybridization (aCGH) was performed using the 60K CytoSure Constitutional v3 microarray (Oxford Gene Technology, Oxford, UK) according to the manufacturer’s instructions.

For invasive prenatal studies, DNA was extracted from amniocytes using the Sherlock AX DNA isolation kit (A&A Biotechnology, Gdansk, Poland), according to manufacturer’s instructions. Array comparative genomic hybridization (aCGH) in the fetus was performed using the 60K CytoSure Constitutional v3 microarray (Oxford Gene Technology, Oxford, UK).
For further genetic testing, DNA was extracted from the peripheral blood of the newborn and his parents using Gentra Purgene Blood Kit (Qiagen, Germantown, MD, USA). Parental and proband DNA samples were tested for the presence of the CNV deletion using junction-specific PCR with DreamTaq DNA Polymerase (Thermo Scientific, Waltham, MA, USA), followed by Sanger sequencing to map the deletion breakpoints. To determine the parental origin of the detected CNV deletion, trio-based genome sequencing (GS) was performed using NEBNext^® Ultra™ II FS DNA Library Prep Kit for Illumina (New England BioLabs, Inc. Ipswich, MA, USA) and paired-end sequenced (2 × 150 bp) on NovaSeq 6000 (Illumina, San Diego, CA, USA). The parental origin of the observed chromosomal abnormality was determined by analyzing the informative single-nucleotide polymorphisms (SNPs) within the deletion region.

We performed array comparative genomic hybridization (arrayCGH), with commercially available arrays such as the 60K CytoSure Constitutional v3 microarray (Oxford Gene Technology, Oxford, UK) according to manufacturer’s instructions. CNVs that showed partial or complete overlap with known segmental duplications were excluded from further analysis, due to the high variability of copy number variations in those regions. CNVs were classified as pathogenic, likely pathogenic, likely benign, benign and of uncertain significance (VUS) based on clinical data in known CNV databases: Database of Genomic Variants (http://dgv.tcag.ca/dgv/app/home accessed on 18 November 2022), ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/ accessed on 18 November 2022), DECIPHER (https://www.deciphergenomics.org/ accessed on 18 November 2022), ClinGen (https://www.clinicalgenome.org/ accessed on 18 November 2022). Recurrent CNVs or CNVs associated with known microdeletion or microduplication syndromes were classified as pathogenic or likely pathogenic depending on the penetrance and clinical features present in probands. In 159 patients (82 prenatal and 77 postnatal), array CGH was performed as a first diagnostic test. Additionally, we conducted aCGH where exome sequencing revealed CNV, which could be confirmed (due to size and array coverage).