Research method:
- 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.
- 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] .
- 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: