Cytoscan 750k array

The DNA from MDS was prepared for hybridization to the Affymetrix CytoScan 750 K array (750,000 probes) according to the manufacturer's protocol. A total of 250 ng of isolated DNA per sample was digested with NspI, and the sample was subsequently ligated, PCR-amplified and purified, fragmented, biotin-labelled and hybridized for use in a CytoScan 750 K array (Affymetrix). The data were analysed using the Nexus Copy Number (version 7.5; Biodiscovery Inc.) software programme, and they were normalized using the SNP-FASST2 segmentation algorithm. The normalized probe intensity and allele ratio data were visualized in Nexus v7.5. In addition to the microarray analysis, the TaqMan Copy Number Assay was also used to quantitatively analyse the CN of ROBO1 and ROBO2. The primers and probes were purchased from Applied Biosystems Inc, and the assay was performed according to the manufacturer's instructions. Each replicate was normalized to RPP14 (a reference gene) to obtain a ΔCt (FAM dye Ct- VIC dye Ct), and an average ΔCt for each sample was calculated. All of the samples were normalized to a calibrator sample to determine the ΔΔCt. The relative quantity was 2-ΔΔCt, and the copy number was 2 × relative quantity.

In total, 119 amniotic fluid samples, 53 chorionic villi, 13 cord blood samples, and one skin tissue were collected by the nurse in accordance with the Declaration of Helsinki. Fetal DNA was extracted using a QIAGEN DNA Mini Kit, according to the manufacturer’s instructions. SNP array experiments were carried out according to Affymetrix CytoScan 750K array standard protocols. The Affymetrix CytoScan 750K array includes 550,000 CNV probes and 200,000 SNP probes for the CNV analysis and is wildly used for prenatal diagnosis. The microarray was scanned using a GeneChip Scanner 3000 system and annotated using Chromosome Analysis Suite (ChAS) software based on the hg19 human reference sequence. The chromosomal abnormalities in each sample were assessed by the American College of Medical Genetics and Genomics (ACMG) guidelines by ChAS software.

Genomic DNA was extracted from peripheral blood leucocytes of the proband and his parents using the QIAamp Blood Mini Kit (QIAGEN, Hilden, Germany) following manufacturer protocol. CMA-SNP array analysis of the prband’s DNA was performed using the Affymetrix^® CytoScan^TM 750K Array (Affymetrix, Santa Clara, CA, United States) following the manufacturer’s recommended protocols. When all quality control tests were passed, we analyzed deletions of ≥ 50 kb (marker ≥ 20 kb), repeats of ≥ 100 kb (marker ≥ 20 kb), and homozygous chromosomal fragments of > 5 Mb.