Chromosomal aneuploidy is the major reason why couples opt for prenatal diagnosis. Current methods for definitive diagnosis rely on invasive procedures, such as chorionic villus sampling and ...amniocentesis, and are associated with a risk of fetal miscarriage. Fetal DNA has been found in maternal plasma but exists as a minor fraction among a high background of maternal DNA. Hence, quantitative perturbations caused by an aneuploid chromosome in the fetal genome to the overall representation of sequences from that chromosome in maternal plasma would be small. Even with highly precise single molecule counting methods such as digital PCR, a large number of DNA molecules and hence maternal plasma volume would need to be analyzed to achieve the necessary analytical precision. Here we reasoned that instead of using approaches that target specific gene loci, the use of a locus-independent method would greatly increase the number of target molecules from the aneuploid chromosome that could be analyzed within the same fixed volume of plasma. Hence, we used massively parallel genomic sequencing to quantify maternal plasma DNA sequences for the noninvasive prenatal detection of fetal trisomy 21. Twenty-eight first and second trimester maternal plasma samples were tested. All 14 trisomy 21 fetuses and 14 euploid fetuses were correctly identified. Massively parallel plasma DNA sequencing represents a new approach that is potentially applicable to all pregnancies for the noninvasive prenatal diagnosis of fetal chromosomal aneuploidies.
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.
Plasma DNA is predominantly hematopoietic in origin. The size difference between maternal- and fetal-derived DNA in maternal plasma prompted us to investigate whether there was any discrepancy in ...molecular size between hematopoietically and nonhematopoietically derived DNA in plasma.
Plasma DNA samples from 6 hematopoietic stem cell transplant recipients and 1 liver transplant recipient were analyzed by massively parallel paired-end sequencing. The size of each fragment was deduced from the alignment positions of the paired reads. In sex-mismatched transplant recipients, the reads from chromosome Y were used as markers for the male donor/recipient. For other transplant recipients, the reads of the donor- and recipient-specific alleles were identified from the single-nucleotide polymorphism genotypes.
In male patients receiving female hematopoietic stem cells, more chromosome Y-derived DNA molecules (nonhematopoietically derived) were ≤150 bp than the autosome-derived ones (mainly hematopoietically derived) (median difference, 9.9%). In other hematopoietic stem cell transplant recipients, more recipient-specific DNA molecules (nonhematopoietically derived) were ≤150 bp than the donor-specific ones (hematopoietically derived) (median difference, 14.8%). In the liver transplant recipient, more donor-derived DNA molecules (liver derived) were ≤150 bp than the recipient-derived ones (mainly hematopoietically derived) (difference, 13.4%). The nonhematopoietically derived DNA exhibited a reduction in a 166-bp peak compared with the hematopoietically derived DNA. A 10-bp periodicity in size distribution below approximately 143 bp was observed in both DNA populations.
Massively parallel sequencing is a powerful tool for studying posttransplantation chimerism. Plasma DNA molecules exhibit a distinct fragmentation pattern, with the nonhematopoietically derived molecules being shorter than the hematopoietically derived ones.
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.
DNA methylation, one of the most important epigenetic modifications, plays a crucial role in various biological processes. The level of DNA methylation can be measured using whole-genome bisulfite ...sequencing at single base resolution. However, until now, there is a paucity of publicly available software for carrying out integrated methylation data analysis. In this study, we implemented Methy-Pipe, which not only fulfills the core data analysis requirements (e.g. sequence alignment, differential methylation analysis, etc.) but also provides useful tools for methylation data annotation and visualization. Specifically, it uses Burrow-Wheeler Transform (BWT) algorithm to directly align bisulfite sequencing reads to a reference genome and implements a novel sliding window based approach with statistical methods for the identification of differentially methylated regions (DMRs). The capability of processing data parallelly allows it to outperform a number of other bisulfite alignment software packages. To demonstrate its utility and performance, we applied it to both real and simulated bisulfite sequencing datasets. The results indicate that Methy-Pipe can accurately estimate methylation densities, identify DMRs and provide a variety of utility programs for downstream methylation data analysis. In summary, Methy-Pipe is a useful pipeline that can process whole genome bisulfite sequencing data in an efficient, accurate, and user-friendly manner. Software and test dataset are available at http://sunlab.lihs.cuhk.edu.hk/methy-pipe/.
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.
Trisomy 21 is the most common reason that women opt for prenatal diagnosis. Conventional prenatal diagnostic methods involve the sampling of fetal materials by invasive procedures such as ...amniocentesis. Screening by ultrasonography and biochemical markers have been used to risk-stratify pregnant women before definitive invasive diagnostic procedures. However, these screening methods generally target epiphenomena, such as nuchal translucency, associated with trisomy 21. It would be ideal if noninvasive genetic methods were available for the direct detection of the core pathology of trisomy 21. Here we outline an approach using digital PCR for the noninvasive detection of fetal trisomy 21 by analysis of fetal nucleic acids in maternal plasma. First, we demonstrate the use of digital PCR to determine the allelic imbalance of a SNP on PLAC4 mRNA, a placenta-expressed transcript on chromosome 21, in the maternal plasma of women bearing trisomy 21 fetuses. We named this the digital RNA SNP strategy. Second, we developed a nonpolymorphism-based method for the noninvasive prenatal detection of trisomy 21. We named this the digital relative chromosome dosage (RCD) method. Digital RCD involves the direct assessment of whether the total copy number of chromosome 21 in a sample containing fetal DNA is overrepresented with respect to a reference chromosome. Even without elaborate instrumentation, digital RCD allows the detection of trisomy 21 in samples containing 25% fetal DNA. We applied the sequential probability ratio test to interpret the digital PCR data. Computer simulation and empirical validation confirmed the high accuracy of the disease classification algorithm.
Massively parallel sequencing has recently been used in noninvasive prenatal diagnosis. The current costs of this technology are still relatively expensive, however, and sample throughput is still ...relatively low when it is used as a molecular diagnostic tool. Rather than nonselectively sequencing the genome, target enrichment provides a logical approach for more efficient and cost-effective massively parallel sequencing because it increases the proportion of informative data from the targeted region(s). Existing applications of targeted sequencing have mainly been qualitative analyses of genomic DNA. In this study, we investigated its applicability in enriching selected genomic regions from plasma DNA and the quantitative performance of this approach.
DNA was extracted from plasma samples collected from 12 pregnant women carrying female fetuses. The SureSelect Target Enrichment System (Agilent Technologies) was used to enrich for exons on chromosome X. Plasma DNA libraries with and without target enrichment were analyzed by massively parallel sequencing. Genomic DNA samples of the mother and fetus for each case were genotyped by microarray.
For the regions targeted by the enrichment kit, the mean sequence coverage of the enriched samples was 213-fold higher than that of the nonenriched samples. Maternal and fetal DNA molecules were enriched evenly. After target enrichment, the coverage of fetus-specific alleles within the targeted region increased from 3.5% to 95.9%.
Targeted sequencing of maternal plasma DNA permits efficient and unbiased detection of fetal alleles at genomic regions of interest and is a powerful method for measuring the proportion of fetal DNA in a maternal plasma sample.
Prenatal diagnosis of monogenic diseases, such as cystic fibrosis and β-thalassemia, is currently offered as part of public health programs. However, current methods based on chorionic villus ...sampling and amniocentesis for obtaining fetal genetic material pose a risk to the fetus. Since the discovery of cell-free fetal DNA in maternal plasma, the noninvasive prenatal assessment of paternally inherited traits or mutations has been achieved. Due to the presence of background maternal DNA, which interferes with the analysis of fetal DNA in maternal plasma, noninvasive prenatal diagnosis of maternally inherited mutations has not been possible. Here we describe a digital relative mutation dosage (RMD) approach that determines if the dosages of the mutant and wild-type alleles of a disease-causing gene are balanced or unbalanced in maternal plasma. When applied to the testing of women heterozygous for the CD41/42 (-CTTT) and hemoglobin E mutations on HBB, digital RMD allows the fetal genotype to be deduced. The diagnostic performance of digital RMD is dependent on interplay between the fractional fetal DNA concentration and number of DNA molecules in maternal plasma. To achieve fetal genotype diagnosis at lower volumes of maternal plasma, fetal DNA enrichment is desired. We thus developed a digital nucleic acid size selection (NASS) strategy that effectively enriches the fetal DNA without additional plasma sampling or experimental time. We show that digital NASS can work in concert with digital RMD to increase the proportion of cases with classifiable fetal genotypes and to bring noninvasive prenatal diagnosis of monogenic diseases closer to reality.
The presence of fetal DNA in maternal plasma represents a source of fetal genetic material for noninvasive prenatal diagnosis; however, the coexisting background maternal DNA complicates the analysis ...of aneuploidy in such fetal DNA. Recently, the SERPINB5 gene on chromosome 18 was shown to exhibit different DNA-methylation patterns in the placenta and maternal blood cells, and the allelic ratio for placenta-derived hypomethylated SERPINB5 in maternal plasma was further shown to be useful for noninvasive detection of fetal trisomy 18.
To develop a similar method for the noninvasive detection of trisomy 21, we used methylation-sensitive single nucleotide primer extension and/or bisulfite sequencing to systematically search 114 CpG islands (CGIs)-76% of the 149 CGIs on chromosome 21 identified by bioinformatic criteria-for differentially methylated DNA patterns. The methylation index (MI) of a CpG site was estimated as the proportion of molecules methylated at that site.
We identified 22 CGIs which were shown to contain CpG sites that were either completely unmethylated (MI = 0.00) in maternal blood cells and methylated in the placenta (MI range, 0.22-0.65), or completely methylated (MI = 1.00) in maternal blood cells and hypomethylated in the placenta (MI range, 0.00-0.75). We detected, for the first time, placental DNA-methylation patterns on chromosome 21 in maternal plasma during pregnancy and observed their postpartum clearance.
Twenty-two (19%) of the 114 studied CGIs on chromosome 21 showed epigenetic differences between samples of placenta and maternal blood cells; these CGIs may provide a rich source of markers for noninvasive prenatal diagnosis.
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