Research method:
  1. Amniocentesis and karyotyping: Amniocentesis were conducted under ultrasonic guidance under aseptic conditions. 20 mL amniotic fluid was obtained. 15ml of it was used for G banding karyotyping, and 5 mL of it was used for CNV-seq detection. Genomic DNA of amniotic fluid cells was extracted by purified column method (QIAamp DNA Blood Mini Kit, QIAGEN company, Germany). Quantitative fluorescence polymerase chain reaction (QF-PCR) was used to exclude maternal cell contamination and polyploidy. When the maternal signal was more than 10%, it indicated maternal cell contamination.
  2. G-banding Karyotyping: amniotic fluid cells were cultured and harvested after being stimulated with phytohemagglutinin for 72 h. Metaphase chromosomes were prepared according to the standard cytogenetic protocols[4] .
  3. CNV-seq detection: The commercial CNV detection library construction kit (Berry Genomics corporation, Beijing) was used. The CNVs were detected by Illumina NextSeq 500. Their sequencing type is SE45 (single ended sequencing, read length: 45bp) and average sequencing depth is 0.1x. Human genome reference sequence version GRCh37 (UCSC database, http://genome.ucsc.edu/cgibin/hgGateway) was selected. Tattini et al[5] analyzed sequencing data by the CNV detection algorithm. The resolution of CNVs was more than 100kb. The main reference for pathogenicity analysis of CNVs is the Database of Genomic Variants (DGV, http://dgv.tcag.ca/dgv/app/home), DECIPHER (https://decipher.sanger.ac.uk), OMIM (https://omim.org), PubMed (https://www.ncbi.nlm.nih.gov/pubmed/) and ClinGen (https://www.ncbi.nlm.nih.gov/projects/dbvar/clingen). CNVs is divided into pCNVs, benign CNVs(bCNVs) and variants of uncertain significance(VOUS). CNVs classification was performed based on the guidelines of the American College of Medical Genetics[6] .
Results: