Materials and Methods
Data collectionThis study was approved by the Research Ethics Committee of the Third Affiliated Hospital of Guangzhou Medical University. When fetal structural anomalies are detected by ultrasound or results of noninvasive prenatal testing (NIPT) indicates high risk for a chromosomal anomaly, pregnant women were suggested to undergo amniocentesis and prenatal genetic test (CMA, MES or WES) with informed consents. Since 2014, CMA was adopted as the first-tier test for patients with above indications in our center. Subsequently, at the beginning of 2017, MES and WES became available in our clinical setting which allowed further definite diagnoses in patients with complex phenotypes. In China, termination of pregnancies after 24 gestational weeks is legal in cases where the continuation of the pregnancy constitutes a danger to the mother’s physical or mental health or life, or major fetal abnormalities (based on ultrasound findings, genetic testing, or both). The fetal medicine system (Astraia software gmbh, Munich, Germany) was used to analyze the appropriateness of fetal growth. From January 2014 to June 2019, except for 75 women who were lost to follow-up, we record the indications, genetic test results, complications and pregnancy outcomes for all target women who underwent late amniocentesis (≥24 gestational weeks) in the Third Affiliated Hospital of Guangzhou Medical University. Pregnancy outcomes (deliveries or terminations) were obtained from medical records or by phone contact if the participant did not give birth in our hospital.CMAGenomic DNA was obtained from amniotic fluid (10 ml) collected by amniocentesis using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. DNA (50 ng) was labeled using Affymetrix Cytogenetics Reagent Kit, and the labeled DNA was applied to an Affymetrix Cytoscan 750K array (Affymetrix Inc., Santa Clara, CA). The platform contains 550,000 non-polymorphic Copy Number Variation (CNV) probes and more than 200,000 Single Nucleotide polymorphism (SNP) probes with an average resolution of 100 kb. Practical procedures were carried out according to the manufacturer’s instructions. The data files generated for each sample were analyzed using Chromosome Analysis Suite (ChAS) Software. The characteristics and spectrum of CNV including the type of aberrations (gains/duplications or losses/deletions), genomic loci, sizes, and the mode of inheritance (familial or de-novo) were studied. The data were interpreted by using information available in the scientific literature and public databases (CLIVAR, Database of Genomic Variants, etc.). These information were used to classify detected CNVs based on their expected clinical significance as benign, likely benign, variants of uncertain significance (VOUS), likely pathogenic or pathogenic [11], in accordance with the recommended guidelines from the International Standard Cytogenomic Array and the American College of Medical Genetics (ACMG). Quantitative Fluorescence Polymerase Chain Reaction and multiplex probe ligation assay (MLPA) for common aneuploidies (chromosomes 21, 18, 13, X, and Y) were performed when a rapid result was required. In some cases, with pathognostic ultrasound findings or known family history, targeted fetal molecular diagnosis for specific single gene mutations was also made.ES (MES/WES)
Parental blood samples were collected for DNA extraction using the SolPure Blood DNA kit (Magen, Guangzhou, China) according to the manufacturer’s instructions. Genomic DNA of the fetuses was obtained from amniotic fluid as described above. The genomic DNA was fragmented by a Q800R Sonicator (Qsonica, Newtown, USA) to generate 300‐500 bp DNA fragments. The paired-end libraries were prepared using the library preparation protocol (Illumina, San Diego, CA). Custom designed NimbleGen SeqCap probes (Roche NimbleGen, Madison, WI) were used for in-solution hybridization to enrich target sequences. Genes with the phenotype-causing mutation were identified from Online Mendelian Inheritance in Man (OMIM). Subsequent sequencing of the enriched DNA was performed on a NextSeq500 sequencer (Illumina, San Diego, CA).
Sequencing reads from the fetal DNA were mapped to the reference human genome version hg19 (http://genome.ucsc.edu/). Variants were called and reviewed by NextGENe software (SoftGenetics, State College, PA) and in-house annotation pipeline. Literature, mutation and population databases were used for variant annotation, including 1000 Genomes, dbSNP, GnomAD, Clinvar, HGMD, and OMIM. The synonymous and common SNPs (MAF>0.1%) were filtered out, and rare variants with high confidence were considered as a disease-causing candidate for further genetic evaluation. Multiple computational algorithms were applied to assist the genetic evaluation of pathogenicity, including SIFT (https://sift.bii.a-star.edu.sg/, Craig Venter Institute), Polyphen-2 (https://genetics.bwh.harvard.edu/pph2/, Harvard University), and Mutation Taster (https://www.mutationtaster.org, NeuroCure Cluster of Excellence). The interpretation of variants was performed according to the ACMG guidelines.