Plasma consists of DNA released from multiple tissues within the body. Using genome-wide bisulfite sequencing of plasma DNA and deconvolution of the sequencing data with reference to methylation ...profiles of different tissues, we developed a general approach for studying the major tissue contributors to the circulating DNA pool. We tested this method in pregnant women, patients with hepatocellular carcinoma, and subjects following bone marrow and liver transplantation. In most subjects, white blood cells were the predominant contributors to the circulating DNA pool. The placental contributions in the plasma of pregnant women correlated with the proportional contributions as revealed by fetal-specific genetic markers. The graft-derived contributions to the plasma in the transplant recipients correlated with those determined using donor-specific genetic markers. Patients with hepatocellular carcinoma showed elevated plasma DNA contributions from the liver, which correlated with measurements made using tumor-associated copy number aberrations. In hepatocellular carcinoma patients and in pregnant women exhibiting copy number aberrations in plasma, comparison of methylation deconvolution results using genomic regions with different copy number status pinpointed the tissue type responsible for the aberrations. In a pregnant woman diagnosed as having follicular lymphoma during pregnancy, methylation deconvolution indicated a grossly elevated contribution from B cells into the plasma DNA pool and localized B cells as the origin of the copy number aberrations observed in plasma. This method may serve as a powerful tool for assessing a wide range of physiological and pathological conditions based on the identification of perturbed proportional contributions of different tissues into plasma.
Cell-free DNA in human plasma is nonrandomly fragmented and reflects genomewide nucleosomal organization. Previous studies had demonstrated tissue-specific preferred end sites in plasma DNA of ...pregnant women. In this study, we performed integrative analysis of preferred end sites with the size characteristics of plasma DNA fragments. We mined the preferred end sites in short and long plasma DNA molecules separately and found that these “size-tagged” ends showed improved accuracy in fetal DNA fraction estimation and enhanced noninvasive fetal trisomy 21 testing. Further analysis revealed that the fetal and maternal preferred ends were generated from different locations within the nucleosomal structure. Hence, fetal DNA was frequently cut within the nucleosome core while maternal DNA was mostly cut within the linker region. We further demonstrated that the nucleosome accessibility in placental cells was higher than that for white blood cells, which might explain the difference in the cutting positions and the shortness of fetal DNA in maternal plasma. Interestingly, short and long size-tagged ends were also observable in the plasma of nonpregnant healthy subjects and demonstrated size differences similar to those in the pregnant samples. Because the nonpregnant samples did not contain fetal DNA, the data suggested that the interrelationship of preferred DNA ends, chromatin accessibility, and plasma DNA size profile is likely a general one, extending beyond the context of pregnancy. Plasma DNA fragment end patterns have thus shed light on production mechanisms and show utility in future developments in plasma DNA-based noninvasive molecular diagnostics.
The human placenta is a dynamic and heterogeneous organ critical in the establishment of the fetomaternal interface and the maintenance of gestational well-being. It is also the major source of ...cell-free fetal nucleic acids in the maternal circulation. Placental dysfunction contributes to significant complications, such as preeclampsia, a potentially lethal hypertensive disorder during pregnancy. Previous studies have identified significant changes in the expression profiles of preeclamptic placentas using whole-tissue analysis. Moreover, studies have shown increased levels of targeted RNA transcripts, overall and placental contributions in maternal cell-free nucleic acids during pregnancy progression and gestational complications, but it remains infeasible to noninvasively delineate placental cellular dynamics and dysfunction at the cellular level using maternal cell-free nucleic acid analysis. In this study, we addressed this issue by first dissecting the cellular heterogeneity of the human placenta and defined individual cell-type–specific gene signatures by analyzing more than 24,000 nonmarker selected cells from full-term and early preeclamptic placentas using large-scale microfluidic single-cell transcriptomic technology. Our dataset identified diverse cellular subtypes in the human placenta and enabled reconstruction of the trophoblast differentiation trajectory. Through integrative analysis with maternal plasma cell-free RNA, we resolved the longitudinal cellular dynamics of hematopoietic and placental cells in pregnancy progression. Furthermore, we were able to noninvasively uncover the cellular dysfunction of extravillous trophoblasts in early preeclamptic placentas. Our work showed the potential of integrating transcriptomic information derived from single cells into the interpretation of cell-free plasma RNA, enabling the noninvasive elucidation of cellular dynamics in complex pathological conditions.
Researchers have developed approaches for the noninvasive prenatal testing of single gene diseases. One approach that allows for the noninvasive assessment of both maternally and paternally inherited ...mutations involves the analysis of single nucleotide polymorphisms (SNPs) in maternal plasma DNA with reference to parental haplotype information. In the past, parental haplotypes were resolved by complex experimental methods or inferential approaches, such as through the analysis of DNA from other affected family members. Recently, microfluidics-based linked-read sequencing technology has become available and allows the direct haplotype phasing of the whole genome rapidly. We explored the feasibility of applying this direct haplotyping technology in noninvasive prenatal testing.
We first resolved the haplotypes of parental genomes with the use of linked-read sequencing technology. Then, we identified SNPs within and flanking the genes of interest in maternal plasma DNA by targeted sequencing. Finally, we applied relative haplotype dosage analysis to deduce the mutation inheritance status of the fetus.
Haplotype phasing and relative haplotype dosage analysis of 12 out of 13 families were successfully achieved. The mutational status of these 12 fetuses was correctly classified.
High-throughput linked-read sequencing followed by maternal plasma-based relative haplotype dosage analysis represents a streamlined approach for noninvasive prenatal testing of inherited single gene diseases. The approach bypasses the need for mutation-specific assays and is not dependent on the availability of DNA from other affected family members. Thus, the approach is universally applicable to pregnancies at risk for the inheritance of a single gene disease.
Plasma DNA obtained from a pregnant woman was sequenced to a depth of 270× haploid genome coverage. Comparing the maternal plasma DNA sequencing data with the parental genomic DNA data and using a ...series of bioinformatics filters, fetal de novo mutations were detected at a sensitivity of 85% and a positive predictive value of 74%. These results represent a 169-fold improvement in the positive predictive value over previous attempts. Improvements in the interpretation of the sequence information of every base position in the genome allowed us to interrogate the maternal inheritance of the fetus for 618,271 of 656,676 (94.2%) heterozygous SNPs within the maternal genome. The fetal genotype at each of these sites was deduced individually, unlike previously, where the inheritance was determined for a collection of sites within a haplotype. These results represent a 90-fold enhancement in the resolution in determining the fetus’s maternal inheritance. Selected genomic locations were more likely to be found at the ends of plasma DNA molecules. We found that a subset of such preferred ends exhibited selectivity for fetal- or maternal-derived DNA in maternal plasma. The ratio of the number of maternal plasma DNA molecules with fetal preferred ends to those with maternal preferred ends showed a correlation with the fetal DNA fraction. Finally, this second generation approach for noninvasive fetal whole-genome analysis was validated in a pregnancy diagnosed with cardiofaciocutaneous syndrome with maternal plasma DNA sequenced to 195× coverage. The causative de novo BRAF mutation was successfully detected through the maternal plasma DNA analysis.
The current methods for distinguishing the zygosities of twins include ultrasound scanning, which is nondefinitive, and amniocentesis, which is invasive. We explored the use of massively parallel ...sequencing of maternal plasma DNA for the noninvasive prenatal assessment of the zygosities of twin pregnancies.
Plasma DNA was extracted from blood collected from 8 women pregnant with twins. Target enrichment and massively parallel sequencing were performed for each plasma DNA library. Apparent fractional fetal DNA concentrations were calculated for multiple genomic regions by determining the ratio of minor to major alleles among single-nucleotide polymorphism sites. Variations in the apparent fractional fetal DNA concentrations between genomic regions were used to infer whether individual fetuses in a twin pair were genotypically different and hence dizygotic.
The extent of the variation in the apparent fractional fetal DNA concentration across chromosomes was 0.82-1.35 SDs for monozygotic twin pregnancies and 2.42-4.80 SDs for dizygotic twin pregnancies. The proportions of apparent fractional fetal DNA concentration values that deviated beyond the range expected for stochastic variation were 0.00%-1.93% for monozygotic twin pregnancies and 36.2%-78.1% for dizygotic twin pregnancies. After identifying a pair of twins as likely dizygotic, the method also allowed determination of the fractional fetal DNA concentrations contributed by the individual fetuses of a dizygotic twin pair.
Noninvasive prenatal determination of twin zygosity by maternal plasma DNA sequencing is feasible. It is also possible to determine the relative fractional fetal DNA concentrations for each fetus for dizygotic twin pregnancies.
The objectives of this study were to compare the concentrations, size profiles and major tissue contributors of cell-free DNA (cfDNA) in plasma and in serum.
Thirteen pregnant women in the third ...trimester were recruited for this study. We collected EDTA-plasma and serum samples using various collection tubes. We determined their cfDNA concentrations and fetal cfDNA fractions using a zinc-finger X (ZFX)/zinc-finger Y (ZFY) droplet digital polymerase chain reaction (ZFX/ZFY ddPCR) assay. We used paired-end massively parallel sequencing (MPS) to measure plasma and serum cfDNA sizes at single-base resolution. We deconvoluted the genome-wide bisulfite sequencing data with reference to the methylation profiles of different tissues.
The concentrations of cfDNA collected in Sarstedt Serum Z tubes were found to be significantly higher than those in Greiner Bio-One Vacuette® Z Serum Separator Clot Activator tubes or Vacuette® Z Serum Clot Activator tubes. The concentrations of fetal cfDNA were significantly reduced in samples collected in the Vacuette® serum collection tubes. Fetal cfDNA fractions were significantly reduced in all sera compared to plasma. MPS of serum cfDNA revealed a right shift of the size distributions compared to plasma. Methylation-based tissue mapping of serum cfDNA revealed an increase of cfDNA from neutrophils and B cells but not T cells.
The use of different serum collection tubes has a significant impact on serum cfDNA concentrations. This effect is likely mediated through the combined effect of genomic DNA release from white blood cells and DNA degradation or removal.
•The choice of collection tubes affects the yield of serum cell-free DNA (cfDNA).•Serum cfDNA has longer fragment sizes than plasma cfDNA.•White cell contamination of serum cfDNA may be revealed by epigenomic analysis.•Plasma is superior to serum as a source of cfDNA analysis.
To investigate the age-specific prevalence of hepatitis B virus (HBV) infection in young pregnant women in Hong Kong Special Administrative Region (SAR), China, and to determine whether an increase ...in prevalence occurs during adolescence.
HBV prevalence was quantified using data from routine antenatal screening for hepatitis B surface antigen (HBsAg) in 10 808 women aged 25 years or younger born in Hong Kong SAR and managed at a single hospital between 1998 and 2011. The effect on prevalence of maternal age, parity and birth before or after HBV vaccine availability in 1984 was assessed, using Spearman's correlation and multiple logistic regression analysis.
Overall, 7.5% of women were HBsAg-positive. The prevalence ranged from 2.3% to 8.4% in those aged ≤ 16 and 23 years, respectively. Women born in or after 1984 and those younger than 18 years of age were less likely to be HBsAg-positive (odds ratio, OR: 0.679; 95% confidence interval, CI: 0.578-0.797) and (OR: 0.311; 95% CI: 0.160-0.604), respectively. For women born before 1984, there was no association between HBsAg carriage and being younger than 18 years of age (OR: 0.60; 95% CI: 0.262-1.370) Logistic regression analysis showed that the prevalence of HBsAg carriage was influenced more by the woman being 18 years old or older (adjusted OR, aOR: 2.80; 95% CI: 1.46-5.47) than being born before 1984 (aOR: 1.42; 95% CI: 1.21-1.67).
Immunity to HBV in young pregnant women who had been vaccinated as neonates decreased in late adolescence.
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