Significance We used massively parallel sequencing to study the size profiles of plasma DNA samples at single-base resolution and in a genome-wide manner. We used chromosome arm-level z -score ...analysis (CAZA) to identify tumor-derived plasma DNA for studying their specific size profiles. We showed that populations of aberrantly short and long DNA molecules existed in the plasma of patients with hepatocellular carcinoma. The short ones preferentially carried the tumor-associated copy number aberrations. We further showed that there were elevated amounts of mitochondrial DNA in the plasma of hepatocellular carcinoma patients. Such molecules were much shorter than the nuclear DNA in plasma. These findings have shed light on fundamental biological characteristics of plasma DNA and related diagnostic applications for cancer.
The analysis of tumor-derived circulating cell-free DNA opens up new possibilities for performing liquid biopsies for the assessment of solid tumors. Although its clinical potential has been increasingly recognized, many aspects of the biological characteristics of tumor-derived cell-free DNA remain unclear. With respect to the size profile of such plasma DNA molecules, a number of studies reported the finding of increased integrity of tumor-derived plasma DNA, whereas others found evidence to suggest that plasma DNA molecules released by tumors might be shorter. Here, we performed a detailed analysis of the size profiles of plasma DNA in 90 patients with hepatocellular carcinoma, 67 with chronic hepatitis B, 36 with hepatitis B-associated cirrhosis, and 32 healthy controls. We used massively parallel sequencing to achieve plasma DNA size measurement at single-base resolution and in a genome-wide manner. Tumor-derived plasma DNA molecules were further identified with the use of chromosome arm-level z -score analysis (CAZA), which facilitated the studying of their specific size profiles. We showed that populations of aberrantly short and long DNA molecules existed in the plasma of patients with hepatocellular carcinoma. The short ones preferentially carried the tumor-associated copy number aberrations. We further showed that there were elevated amounts of plasma mitochondrial DNA in the plasma of hepatocellular carcinoma patients. Such molecules were much shorter than the nuclear DNA in plasma. These results have improved our understanding of the size profile of tumor-derived circulating cell-free DNA and might further enhance our ability to use plasma DNA as a molecular diagnostic tool.
Cell-free DNA in plasma has been used for noninvasive prenatal testing and cancer liquid biopsy. The physical properties of cell-free DNA fragments in plasma, such as fragment sizes and ends, have ...attracted much recent interest, leading to the emerging field of cell-free DNA fragmentomics. However, one aspect of plasma DNA fragmentomics as to whether double-stranded plasma molecules might carry single-stranded ends, termed a jagged end in this study, remains underexplored. We have developed two approaches for investigating the presence of jagged ends in a plasma DNA pool. These approaches utilized DNA end repair to introduce differential methylation signals between the original sequence and the jagged ends, depending on whether unmethylated or methylated cytosines were used in the DNA end-repair procedure. The majority of plasma DNA molecules (87.8%) were found to bear jagged ends. The jaggedness varied according to plasma DNA fragment sizes and appeared to be in association with nucleosomal patterns. In the plasma of pregnant women, the jaggedness of fetal DNA molecules was higher than that of the maternal counterparts. The jaggedness of plasma DNA correlated with the fetal DNA fraction. Similarly, in the plasma of cancer patients, tumor-derived DNA molecules in patients with hepatocellular carcinoma showed an elevated jaggedness compared with nontumoral DNA. In mouse models, knocking out of the
gene reduced jaggedness, whereas knocking out of the
gene enhanced jaggedness. Hence, plasma DNA jagged ends represent an intrinsic property of plasma DNA and provide a link between nuclease activities and the fragmentation of plasma DNA.
The precise measurement of cell-free fetal DNA in maternal plasma facilitates noninvasive prenatal diagnosis of fetal chromosomal aneuploidies and other applications. We tested the hypothesis that ...microfluidics digital PCR, in which individual fetal-DNA molecules are counted, could enhance the precision of measuring circulating fetal DNA.
We first determined whether microfluidics digital PCR, real-time PCR, and mass spectrometry produced different estimates of male-DNA concentrations in artificial mixtures of male and female DNA. We then focused on comparing the imprecision of microfluidics digital PCR with that of a well-established nondigital PCR assay for measuring male fetal DNA in maternal plasma.
Of the tested platforms, microfluidics digital PCR demonstrated the least quantitative bias for measuring the fractional concentration of male DNA. This assay had a lower imprecision and higher clinical sensitivity compared with nondigital real-time PCR. With the ZFY/ZFX assay on the microfluidics digital PCR platform, the median fractional concentration of fetal DNA in maternal plasma was > or =2 times higher for all 3 trimesters of pregnancy than previously reported.
Microfluidics digital PCR represents an improvement over previous methods for quantifying fetal DNA in maternal plasma, enabling diagnostic and research applications requiring precise quantification. This approach may also impact other diagnostic applications of plasma nucleic acids, e.g., in oncology and transplantation.
Epigenetic mechanisms play an important role in prenatal development, but fetal tissues are not readily accessible. Fetal DNA molecules are present in maternal plasma and can be analyzed ...noninvasively.
We applied genomewide bisulfite sequencing via 2 approaches to analyze the methylation profile of maternal plasma DNA at single-nucleotide resolution. The first approach used maternal blood samples and polymorphic differences between the mother and fetus to analyze the fetal methylome across the genome. The second approach used the methylation profile of maternal blood cells and the fractional fetal DNA concentration in maternal plasma to deduce the placental methylomic profile from maternal plasma DNA-sequencing data.
Because of the noninvasive nature of these approaches, we were able to serially assess the methylation profiles of fetal, placental, and maternal plasma with maternal blood samples collected in the first and third trimesters and after delivery. Gestation-related changes were observed. The fetal methylation profile deduced from maternal plasma data resembled that of the placental methylome, both on a genomewide level and per CpG site. Imprinted genes and differentially methylated regions were identified from the maternal plasma data. We demonstrated one potential clinical application of maternal plasma bisulfite sequencing with the successful detection of fetal trisomy 21.
We successfully analyzed fetal and placental methylomes on a genomewide scale, noninvasively and serially. This development offers a powerful method for research, biomarker discovery, and clinical testing for pregnancy-related disorders.
5-Methylcytosine (5mC) is an important type of epigenetic modification. Bisulfite sequencing (BS-seq) has limitations, such as severe DNA degradation. Using single molecule real-time sequencing, we ...developed a methodology to directly examine 5mC. This approach holistically examined kinetic signals of a DNA polymerase (including interpulse duration and pulse width) and sequence context for every nucleotide within a measurement window, termed the holistic kinetic (HK) model. The measurement window of each analyzed double-stranded DNA molecule comprised 21 nucleotides with a cytosine in a CpG site in the center. We used amplified DNA (unmethylated) and M.SssI-treated DNA (methylated) (M.SssI being a CpG methyltransferase) to train a convolutional neural network. The area under the curve for differentiating methylation states using such samples was up to 0.97. The sensitivity and specificity for genome-wide 5mC detection at single-base resolution reached 90% and 94%, respectively. The HK model was then tested on human-mouse hybrid fragments in which each member of the hybrid had a different methylation status. The model was also tested on human genomic DNA molecules extracted from various biological samples, such as buffy coat, placental, and tumoral tissues. The overall methylation levels deduced by the HK model were well correlated with those by BS-seq (
= 0.99;
< 0.0001) and allowed the measurement of allele-specific methylation patterns in imprinted genes. Taken together, this methodology has provided a system for simultaneous genome-wide genetic and epigenetic analyses.
Noninvasive prenatal testing (NIPT) is accomplished by analysis of circulating cell-free fetal nucleic acids in maternal plasma. The advent of massively parallel sequencing (MPS) has enabled NIPT of ...chromosomal aneuploidies with unprecedented robustness, and these tests are now widely available for clinical use. Moreover, MPS-based NIPT of subchromosomal deletions duplications and single-gene disorders has also been achieved, and the number of applications is growing. In addition to specific fetal genetic disorders, the whole fetal genome, transcriptome, and methylome have been revealed by deep sequencing of maternal plasma. The analysis of the fetal transcriptome and methylome may yield valuable information on fetal and maternal health. With continued improvement in sequencing technology and reduction in sequencing costs, the analysis of cell-free nucleic acids would play an increasingly important role in prenatal screening, diagnosis, monitoring, and risk stratification of fetal as well as maternal conditions.
Cell-free fetal DNA is present in the plasma of pregnant women. It consists of short DNA fragments among primarily maternally derived DNA fragments. We sequenced a maternal plasma DNA sample at up to ...65-fold genomic coverage. We showed that the entire fetal and maternal genomes were represented in maternal plasma at a constant relative proportion. Plasma DNA molecules showed a predictable fragmentation pattern reminiscent of nuclease-cleaved nucleosomes, with the fetal DNA showing a reduction in a 166-base pair (bp) peak relative to a 143-bp peak, when compared with maternal DNA. We constructed a genome-wide genetic map and determined the mutational status of the fetus from the maternal plasma DNA sequences and from information about the paternal genotype and maternal haplotype. Our study suggests the feasibility of using genome-wide scanning to diagnose fetal genetic disorders prenatally in a noninvasive way.
Context:
Congenital adrenal hyperplasia (CAH) is an autosomal recessive condition that arises from mutations in CYP21A2 gene, which encodes for the steroidogenic enzyme 21-hydroxylase. To prevent ...genital ambiguity in affected female fetuses, prenatal treatment with dexamethasone must begin on or before gestational week 9. Currently used chorionic villus sampling and amniocentesis provide genetic results at approximately 14 weeks of gestation at the earliest. This means that mothers who want to undergo prenatal dexamethasone treatment will be unnecessarily treating seven of eight fetuses (males and three of four unaffected females), emphasizing the desirability of earlier genetic diagnosis in utero.
Objective:
The objective of the study was to develop a noninvasive method for early prenatal diagnosis of fetuses at risk for CAH.
Patients:
Fourteen families, each with a proband affected by phenotypically classical CAH, were recruited.
Design:
Cell-free fetal DNA was obtained from 3.6 mL of maternal plasma. Using hybridization probes designed to capture a 6-Mb region flanking CYP21A2, targeted massively parallel sequencing (MPS) was performed to analyze genomic DNA samples from parents and proband to determine parental haplotypes. Plasma DNA from pregnant mothers also underwent targeted MPS to deduce fetal inheritance of parental haplotypes.
Results:
In all 14 families, the fetal CAH status was correctly deduced by targeted MPS of DNA in maternal plasma, as early as 5 weeks 6 days of gestation.
Conclusions:
MPS on 3.6 mL plasma from pregnant mothers could potentially provide the diagnosis of CAH, noninvasively, before the ninth week of gestation. Only affected female fetuses will thus be treated. Our strategy represents a generic approach for noninvasive prenatal testing for an array of autosomal recessive disorders.
PDF
