Maternal DNA Sequencing Research Articles

Noninvasive Prenatal Testing for Fetal XXY Aneuploidies Among Pregnancies in Beijing of China

Abstract Objective: To evaluate the screening performance of noninvasive prenatal testing (NIPT) based on high-throughput massively parallel sequencing technology for the fetal XXY aneuploidies among pregnancies in Beijing of China. Methods: The study enrolled 26 913 consecutive pregnancies, 20–50 years old, who attended the Peking Union Medical College Hospital, Beijing, China, for prenatal screening from January 1, 2016 to December 31, 2019. Cell-free DNA was extracted from maternal peripheral blood to have a high-throughput massively parallel sequencing procedure. Cases with high-risk of fetal XXY were suggested to take invasive prenatal diagnosis (IPD) for confirmation. Maternal DNA sequencing was performed, if necessary, to find other potential factors that may lead to high-risk results of XXY by NIPT. Results: Among a cohort of 26 913 pregnant women, 34 were high-risk for fetal XXY, among which 30 accepted IPD while 4 declined. In those who accepted IPD, 19 cases were confirmed fetal XXY by chromosome karyotyping analysis while 11 were verified as false positive. Among the 19 confirmed fetal XXY cases, 14 elected pregnancy termination. For all the 34 high-risk cases, two were verified maternal sex chromosome aneuploidy. The calculated detection rate, positive predictive value, and false-positive rate of NIPT for fetal XXY in this cohort was 100.00% (19/19), 63.33% (19/30), and 0.04% (11/26 890), respectively. And the percentage of pregnancy termination was 73.68% (14/19). Conclusion: NIPT could be used as a potential method for fetal XXY screening, although the accuracy needs to be improved. As NIPT is not diagnostic, IPD is strongly recommended for those with high-risk results. For cases with discordance between NIPT and fetal karyotyping, maternal DNA sequencing would help to identify the cause of false-positive/false-negative results.

Contribution of maternal copy number variations to false-positive fetal trisomies detected by noninvasive prenatal testing.

The aim of the study was to determine the contribution and significance of maternal copy number variations (CNVs) to false-positive noninvasive prenatal testing (NIPT) trisomy results. A total of 112 021 patients were referred for NIPT. Fetal aneuploidy testing was performed using low coverage massively parallel sequencing, and results reported as chromosome Z-scores. Copy number variation sequencing (CNV-Seq) was used to detect maternal DNA CNVs. Confirmatory amniocentesis and karyotyping of 563 of 781 patients (72%) receiving a positive trisomy result revealed 489 true and 74 false positives. In 6 of these 74 patients (8.1%), CNV-Seq revealed non-pathogenic maternal duplications (1.76-10.90 megabases) on the chromosome associated with the fetal trisomy. There was a strong correlation of higher Z-scores with increasing size of the maternal CNVs (R2 = 0.94). When the contribution of the maternal CNV-Seq reads to chromosome Z-scores were removed, all original Z-scores shifted to the normal range. Maternal CNVs can potentially contribute to a small but significant number of false-positive fetal trisomies detected by NIPT. To avoid unnecessary invasive procedures and better manage patients, we recommend that confirmatory maternal DNA sequencing is performed when the NIPT methodology used indicates a high risk of a maternal CNV. © 2017 John Wiley & Sons, Ltd.

The role of transplacental microtransfusions of maternal lymphocytes in HIV transmission to newborns

Perinatal HIV transmission could occur via microtransfused maternal blood during delivery. If so, detecting maternal cells in umbilical cord blood should correlate with infection risk. To develop sensitive assays for maternal DNA in infant's blood stored as dried blood spots (DBS) and examine the correlation between microtransfusion and perinatal HIV infection risk. Blood-in-blood serial dilutions were prepared as DBS. Extracted DNA was amplified for unique minor-population sequences using 24 allele-specific polymerase chain reaction assays. Using newborns born to HIV+ mothers, paired mother-infant samples were similarly examined to identify unique maternal sequences targeted by allele-specific polymerase chain reaction of DNA extracted from cord blood DBS. Cord-blood PCR-negative infants were categorized as uninfected or perinatally infected by HIV PCR on samples collected 4-8 weeks after birth. Sequences from added cells were detected at less than 1: 1000 dilutions in 19 of 20 aliquots, and less than 1: 10 000 dilutions in seven of 20 aliquots; the median limit of detection (probit analysis) was one added genomic sequence in 9500 background sequences of amplifiable DNA. Maternal sequences were detected in cord-blood DBS of 50% of infected infants (N = 18) and 44% of uninfected infants (N = 43). Infection did not correlate with more frequent detection of maternal sequences. This semiquantitative assay reliably detected maternal DNA sequences in DBS at levels of less than 1: 1000 cells. Maternal sequences were frequently detected but did not correlate infection risk with detection or level of maternal DNA in umbilical cord blood. Therefore, we could not demonstrate that microtransfusions at parturition were responsible for perinatal HIV transmission.

Recent Developments in the Detection of Fetal Single Gene Differences in Maternal Plasma and the Role of Size Fractionation

The presence of cell-free fetal DNA in maternal plasma allowed noninvasive prenatal diagnosis of fetal loci completely absent from the maternal genome, such as SRY gene and RhD gene. However, the detection of fetal point mutations is hindered by the predominance of maternal DNA sequences. Recent studies have shown that cell-free fetal DNA exists in maternal plasma in small fragments. Thus, cell-free fetal DNA can be enriched by size fractionation, which improves detection of fetal gene mutations. Furthermore, it has been shown that Matrix Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF) mass spectrometry also permits the detection of fetal SNPs from maternal plasma. These two new developments are discussed.

Detection of Paternally Inherited Fetal Point Mutations for β-Thalassemia Using Size-Fractionated Cell-Free DNA in Maternal Plasma

Currently, fetal point mutations cannot be reliably analyzed from circulatory fetal DNA in maternal plasma, due to the predominance of maternal DNA sequences. However, analysis of circulatory fetal DNA sequences in maternal plasma have been shown to selectively enrich for fetal DNA molecules on the basis of a smaller molecular size than maternal DNA. To examine the prenatal analysis of 4 common beta-thalassemia point mutations: IVSI-1, IVSI-6, IVSI-110, and codon 39. A total of 32 maternal blood samples were collected at 10 to 12 weeks of gestation (mean, 10.7 weeks) between February 15, 2003, and February 25, 2004, in Bari, Italy, from women with risk for beta-thalassemia in their newborns immediately prior to chorionic villous sampling. Samples in which the father and mother did not carry the same mutation were examined. Circulatory DNA was size-fractionated by gel electrophoresis and polymerase chain reaction (PCR) amplified with a peptide-nucleic-acid clamp, which suppresses amplification of the normal maternal allele. Presence of the paternal mutant allele was detected by allele-specific real-time PCR. Detection of paternally inherited beta-globin gene point mutations. Presence or absence of the paternal mutant allele was correctly determined in 6 (86%) of 7 cases with the IVSI-1 mutation, 4 (100%) of 4 with the IVSI-6 mutation, 5 (100%) of 5 with the IVSI-110 mutation, and 13 (81%) of 16 with the codon 39 mutation. One false-positive test result was scored for the IVSI-1 mutation. Two cases with the codon 39 mutation were classified as uncertain and 1 case was excluded due to lack of a diagnostic test result at the time of analysis. These results yielded an overall sensitivity of 100% and specificity of 93.8%, with classified cases removed. Our recently described technique of the size-fractionation of circulatory DNA in maternal plasma may be potentially useful for the noninvasive prenatal determination of fetal point mutations.

Size separation of circulatory DNA in maternal plasma permits ready detection of fetal DNA polymorphisms.

Analysis of fetal DNA in maternal plasma has recently been introduced as a new method for noninvasive prenatal diagnosis, particularly for the analysis of fetal genetic traits, which are absent from the maternal genome, e.g., RHD or Y-chromosome-specific sequences. To date, the analysis of other fetal genetic traits has been more problematic because of the overwhelming presence of maternal DNA sequences in the circulation. We examined whether different biochemical properties can be discerned between fetal and maternal circulatory DNA. Plasma DNA was examined by agarose gel electrophoresis. The fractions of fetal and maternal DNA in size-fractionated fragments were assayed by real-time PCR. The determination of paternally and maternally inherited fetal genetic traits was examined by use of highly polymorphic chromosome-21-specific microsatellite markers. Size fractionation of circulatory DNA indicated that the major portion of cell-free fetal DNA had an approximate molecular size of <0.3 kb, whereas maternally derived sequences were, on average, considerably larger than 1 kb. Analysis of size-fractionated DNA (</=0.3 kb) from maternal plasma samples facilitated the ready detection of paternally and maternally inherited microsatellite markers. Circulatory fetal DNA can be enriched by size selection of fragment sizes less than approximately 0.3 kb. Such selection permits easier analysis of both paternally and maternally inherited DNA polymorphisms.

Cell‐Free DNA in Urine

Intrigued by the rapid clearance of free fetal DNA from the maternal circulation, we have investigated whether this fetal genetic material could be cleared via the kidney. For this purpose, we examined for the presence of Y chromosome-specific DNA sequences in urine samples obtained from 8 women pregnant with male fetuses. No male-specific sequences could be detected, despite the use of a very sensitive nested PCR assay nor a highly reproducible real-time PCR assay. We did, however, detect maternal DNA sequences. To determine if this cell-free DNA was derived from the kidney or another source, we next examined urine from female kidney transplant patients who had received male kidneys. Y chromosome-specific sequences were indeed detectable by both nested and real-time PCR in these samples, thereby confirming a recent report describing urinary DNA microchimerism. Quantitative analysis of serially obtained samples furthermore suggests that transplant-derived sequences are elevated during periods of graft rejection. These results imply that the measurement of graft-derived urinary DNA may serve as a new marker for kidney graft tolerance.

Reliability of prenatal diagnosis of genetic diseases by analysis of amplified trophoblast DNA.

Dot blot analysis on enzymatically amplified trophoblast DNA with allele specific oligonucleotide probes is currently used for the prenatal diagnosis of single gene disorders characterised at the molecular level, such as the beta thalassaemias, phenylketonuria, sickle cell anaemia, and alpha 1-anti-trypsin deficiency. A potential problem with the use of this procedure is the co-amplification of maternal sequences, which may obscure the diagnosis in the fetus. To address this question, we carried out prenatal diagnosis of beta thalassaemia in 300 couples at risk by dot blot analysis on enzymatically amplified DNA with 32P or horseradish peroxidase labelled allele specific oligonucleotide probes. We verified the diagnosis obtained by this procedure with oligonucleotide hybridisation on electrophoretically separated non-amplified trophoblast DNA fragments. We detected no co-amplified maternal sequences, even with a faint signal, in the dot blot of trophoblast DNA from those fetuses diagnosed as normal or homozygotes, nor in those diagnosed as heterozygotes, who were born to parents carrying different mutations and had inherited the paternal mutation. These results indicate that, when careful dissection of trophoblast tissue from maternal decidua is carried out, amplification of chorionic villi DNA is not associated with amplification of maternal DNA sequences. We may thus conclude that dot blot analysis of trophoblast DNA is a very reliable procedure for prenatal diagnosis.