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.