Yvonne Kwun Yue Cheng

Plasma DNA tissue mapping by genome-wide methylation sequencing for noninvasive prenatal, cancer, and transplantation assessments

Significance Plasma consists of DNA released from multiple tissues within the body. Using genome-wide bisulfite sequencing of plasma DNA, we obtained a bird’s eye view of the identities and contributions of these tissues to the circulating DNA pool. The tissue contributors and their relative proportions are identified by a bioinformatics deconvolution process that draws reference from DNA methylation signatures representative of each tissue type. We validated this approach in pregnant women, cancer patients, and transplant recipients. This method also allows one to identify the tissue of origin of genomic aberrations observed in plasma DNA. This approach has numerous research and diagnostic applications in prenatal testing, oncology, transplantation monitoring, and other fields. 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.

Noninvasive twin zygosity assessment and aneuploidy detection by maternal plasma DNA sequencing

This study aimed to provide an individualized assessment of fetal trisomy 21 and trisomy 18 status for twin pregnancies by maternal plasma DNA sequencing.

Noninvasive Prenatal Determination of Twin Zygosity by Maternal Plasma DNA Analysis

BACKGROUND 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. METHODS 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. RESULTS 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. CONCLUSIONS 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.

Second generation noninvasive fetal genome analysis reveals de novo mutations, single-base parental inheritance, and preferred DNA ends

Significance We explored the limit of noninvasive prenatal testing by performing genome-wide sequencing of maternal plasma DNA at 195× and 270× haploid genome coverages. Combined with the use of a series of bioinformatics filters, fetal de novo mutations could be detected with a positive predictive value that was two orders of magnitude higher than previously reported. A de novo BRAF mutation was noninvasively detected in a case with cardiofaciocutaneous syndrome. The maternal inheritance of the fetus could be ascertained on a genome-wide level without the use of maternal haplotypes, hence greatly increasing the resolution of such analysis. Finally, we showed that certain genomic locations were overrepresented at the ends of plasma DNA fragments with fetal or maternal selectivity. 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.

Use of birth weight threshold for macrosomia to identify fetuses at risk of shoulder dystocia among Chinese populations

To assess the incidence of macrosomia and the influence of birth weight on shoulder dystocia risk among a cohort of Chinese women.

Women's uptake of non‐invasive DNA testing following a high‐risk screening test for trisomy 21 within a publicly funded healthcare system: findings from a retrospective review

The objective of the study was to evaluate the uptake of non‐invasive cell‐free fetal DNA screening test (NIDT) after a high‐risk screening result for trisomy 21

Noninvasive Prenatal Testing by Nanopore Sequencing of Maternal Plasma DNA: Feasibility Assessment

To the Editor: Noninvasive prenatal testing (NIPT) by maternal plasma DNA sequencing is now clinically available for screening fetal chromosomal aneuploidies; these tests have close to 99% sensitivity and 99% specificity (1). Unlike amniocentesis, maternal peripheral blood sampling does not pose any risk of miscarriage. Consequently, the clinical demand for NIPT has increased substantially since it first became commercially available in 2011. Massively parallel sequencing is a core component of most of the currently used laboratory protocols for NIPT of chromosomal aneuploidies (2). Because of the high instrumentation cost, those tests are currently performed at reference laboratories. Oxford Nanopore Technologies has developed a nanopore-based DNA sequencing platform (3). Nanopore sequencers have a comparatively low equipment cost and a small footprint. Each flow cell costs US

Cell-free DNA in maternal plasma and serum: A comparison of quantity, quality and tissue origin using genomic and epigenomic approaches.

500–900 and can be used multiple times for up to 48 h. The sequencing speed is also relatively fast, reaching 30 bases per second from each nanopore. Such features would be advantageous for use in clinical laboratories. In this study, we assessed whether nanopore sequencing could be applied to plasma DNA analysis for NIPT. We obtained plasma samples from 4 groups of individuals recruited with informed consent and institutional approval: women pregnant with male fetuses (third trimester), women pregnant with female fetuses (third trimester), nonpregnant women, and men. …

Integrative single-cell and cell-free plasma RNA transcriptomics elucidates placental cellular dynamics

OBJECTIVES 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. DESIGN AND METHODS 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. RESULTS 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. CONCLUSIONS 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.

Advanced maternal age and postpartum hemorrhage – risk factor or red herring?

Significance The human placenta is a dynamic and cellular heterogeneous organ, which is critical in fetomaternal homeostasis and the development of preeclampsia. Previous work has shown that placenta-derived cell-free RNA increases during pregnancy. We applied large-scale microfluidic single-cell transcriptomic technology to comprehensively characterize cellular heterogeneity of the human placentas and identified multiple placental cell-type–specific gene signatures. Analysis of the cellular signature expression in maternal plasma enabled noninvasive delineation of the cellular dynamics of the placenta during pregnancy and the elucidation of extravillous trophoblastic dysfunction in early preeclampsia. 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.