Plasma placental RNA allelic ratio permits noninvasive prenatal chromosomal aneuploidy detection.

Current methods for prenatal diagnosis of chromosomal aneuploidies involve the invasive sampling of fetal materials using procedures such as amniocentesis or chorionic villus sampling and constitute a finite risk to the fetus. Here, we outline a strategy for fetal chromosome dosage assessment that can be performed noninvasively through analysis of placental expressed mRNA in maternal plasma. We achieved noninvasive prenatal diagnosis of fetal trisomy 21 by determining the ratio between alleles of a single-nucleotide polymorphism (SNP) in PLAC4 mRNA, which is transcribed from chromosome 21 and expressed by the placenta, in maternal plasma. PLAC4 mRNA in maternal plasma was fetal derived and cleared after delivery. The allelic ratios in maternal plasma correlated with those in the placenta. Fetal trisomy 21 was detected noninvasively in 90% of cases and excluded in 96.5% of controls.

Noninvasive prenatal diagnosis of hemophilia by microfluidics digital PCR analysis of maternal plasma DNA.

Hemophilia is a bleeding disorder with X-linked inheritance. Current prenatal diagnostic methods for hemophilia are invasive and pose a risk to the fetus. Cell-free fetal DNA analysis in maternal plasma provides a noninvasive mean of assessing fetal sex in such pregnancies. However, the disease status of male fetuses remains unknown if mutation-specific confirmatory analysis is not performed. Here we have developed a noninvasive test to diagnose whether the fetus has inherited a causative mutation for hemophilia from its mother. The strategy is based on a relative mutation dosage approach, which we have previously established for determining the mutational status of fetuses for autosomal disease mutations. In this study, the relative mutation dosage method is used to deduce whether a fetus has inherited a hemophilia mutation on chromosome X by detecting whether the concentration of the mutant or wild-type allele is overrepresented in the plasma of heterozygous women carrying male fetuses. We correctly detected fetal genotypes for hemophilia mutations in all of the 12 studied maternal plasma samples obtained from at-risk pregnancies from as early as the 11th week of gestation. This development would make the decision to undertake prenatal testing less traumatic and safer for at-risk families.

Targeted massively parallel sequencing of maternal plasma DNA permits efficient and unbiased detection of fetal alleles.

BACKGROUND: 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. METHODS: 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. RESULTS: 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%. CONCLUSIONS: 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.

Noninvasive prenatal diagnosis of monogenic diseases by digital size selection and relative mutation dosage on DNA in maternal plasma.

Prenatal diagnosis of monogenic diseases, such as cystic fibrosis and beta-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.

Noninvasive prenatal diagnosis of fetal chromosomal aneuploidy by massively parallel genomic sequencing of DNA in maternal plasma.

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.

Noninvasive prenatal detection of trisomy 21 by an epigenetic-genetic chromosome-dosage approach.

BACKGROUND: The use of fetal DNA in maternal plasma for noninvasive prenatal diagnosis of trisomy 21 (T21) is an actively researched area. We propose a novel method of T21 detection that combines fetal-specific epigenetic and genetic markers. METHODS: We used combined bisulfite restriction analysis to search for fetal DNA markers on chromosome 21 that were differentially methylated in the placenta and maternal blood cells and confirmed any target locus with bisulfite sequencing. We then used methylation-sensitive restriction endonuclease digestion followed by microfluidics digital PCR analysis to investigate the identified marker. Chromosome-dosage analysis was performed by comparing the dosage of this epigenetic marker with that of the ZFY (zinc finger protein, Y-linked) gene on chromosome Y. RESULTS: The putative promoter of the HLCS (holocarboxylase synthetase) gene was hypermethylated in the placenta and hypomethylated in maternal blood cells. A chromosome-dosage comparison of the hypermethylated HLCS and ZFY loci could distinguish samples of T21 and euploid placental DNA. Twenty-four maternal plasma samples from euploid pregnancies and 5 maternal plasma samples from T21 pregnancies were analyzed. All but 1 of the euploid samples were correctly classified. CONCLUSIONS: The epigenetic-genetic chromosome-dosage approach is a new method for noninvasive prenatal detection of T21. The epigenetic part of the analysis can be applied to all pregnancies. Because the genetic part of the analysis uses paternally inherited, fetal-specific genetic markers that are abundant in the genome, broad population coverage should be readily achievable. This approach has the potential to become a generally usable technique for noninvasive prenatal diagnosis.

Synergy of total PLAC4 RNA concentration and measurement of the RNA single-nucleotide polymorphism allelic ratio for the noninvasive prenatal detection of trisomy 21.

BACKGROUND: Maternal plasma mRNA encoded by the PLAC4 gene (placenta-specific 4), which is transcribed from chromosome 21 in placental cells, is a potential marker for the noninvasive assessment of chromosome 21 dosage in the fetus. We evaluated the diagnostic sensitivities and specificities of 2 trisomy 21-screening approaches that use maternal plasma PLAC4 mRNA. METHODS: We studied maternal plasma samples from 153 pregnant women carrying euploid and trisomy 21 fetuses. For the samples in which the fetuses were heterozygous for the studied PLAC4 single-nucleotide polymorphism (SNP), we measured the ratio between 2 alleles of the SNP in maternal plasma PLAC4 mRNA (RNA-SNP) by mass spectrometric (MS) and digital PCR methods. For pregnancies involving fetuses homozygous for the SNP, we quantified the total PLAC4 mRNA concentration in maternal plasma by real-time PCR and digital PCR. RESULTS: For the RNA-SNP approach, we achieved a diagnostic sensitivity and specificity of 100% (95% CI, 40.2%-100%) and 89.7% (95% CI, 78.8%-96.1%), respectively, for both the MS and the digital PCR methods. For the mRNA-quantification approach, the areas under the ROC curves were 0.859 (95% CI, 0.741-0.903) and 0.833 (95% CI, 0.770-0.923) for plasma PLAC4 mRNA concentrations measured by the real-time PCR and the digital PCR methods, respectively. CONCLUSIONS: For prenatal screening of trisomy 21, the quantification of the total PLAC4 mRNA concentration can be used in a synergistic manner with the RNA-SNP allelic ratio approach to increase the population coverage of cases in which diagnostic information can be obtained.

Non-invasive prenatal detection of fetal trisomy 18 by RNA-SNP allelic ratio analysis using maternal plasma SERPINB2 mRNA: a feasibility study.

OBJECTIVE: Non-invasive prenatal diagnosis of chromosome aneuploidies has been achieved by measuring the ratio of two alleles of a single nucleotide polymorphism (SNP) in circulating placental mRNA (the RNA-SNP allelic ratio approach) in maternal plasma. We investigated the feasibility of applying this approach for the non-invasive prenatal detection of fetal trisomy 18. METHOD: We targeted serpin peptidase inhibitor, clade B (ovalbumin), membrane 2 (SERPINB2) mRNA, which is transcribed from chromosome 18 and is preferentially expressed by the placenta. We developed a mass-spectrometric assay to measure the SERPINB2 RNA-SNP allelic ratios in the placental samples and maternal plasma obtained from pregnancies involving euploid and trisomy 18 fetuses. RESULTS: We were able to separate all the euploid and trisomy 18 placentas by their SERPINB2 RNA-SNP allelic ratios. The allelic ratios of the trisomy 18 placentas deviated from the reference interval established from the euploid placentas. Due to the relatively low concentrations of SERPINB2 mRNA in maternal plasma, we used pooled maternal plasma samples for analysis. We were able to identify three of the four pooled trisomy 18 plasma samples by their deviated allelic ratios when compared with the reference interval obtained from pooled euploid plasma samples. CONCLUSION: It is feasible to detect fetal trisomy 18 non-invasively by maternal plasma SERPINB2 RNA-SNP analysis provided that sufficient quantities of plasma samples are used.

A strategy for identifying circulating placental RNA markers for fetal growth assessment.

OBJECTIVE: To evaluate whether circulating placental mRNAs in maternal plasma could serve as markers for the assessment of fetal growth or intrauterine growth restriction (IUGR). METHODS: From a panel of placental transcripts detectable in maternal plasma identified by microarray previously, we chose growth-related transcripts, namely CSH1, GH2, KISS1, and ADAM12, as potential growth markers. Relationships between the maternal plasma mRNA concentrations with several fetal growth indicators were studied. Maternal plasma mRNA concentrations from IUGR pregnancies with or without pre-eclampsia (PET) were compared with gestational age matched controls cross-sectionally and longitudinally. The four transcripts were quantified by one-step real-time RT-PCR. RESULTS: Maternal plasma GH2 mRNA significantly correlated with birth weight and fetal biometric measurements. Maternal plasma ADAM12 mRNA concentration was significantly higher in IUGR with PET than normal pregnancies in the cross-sectional comparison. No significant difference was observed for all markers between IUGR without PET and controls in both the cross-sectional and longitudinal comparisons. CONCLUSION: This study presents a potential strategy in identifying surrogate markers for the study of fetal growth. Circulating GH2 mRNA in maternal plasma appeared to be associated with fetal growth. The utility of this strategy and the currently assessed markers could be explored in further studies.

Embryotoxicity study of ginsenoside Rc and Re in in vitro rat whole embryo culture.

BACKGROUND: Pregnant women commonly consume ginseng. However, there is little data concerning the effects of ginseng on early pregnancy. METHODS: Rat embryos were exposed in vitro to different concentrations of Rc and Re from day 9.5 to day 11.5 after conception. Embryos were scored for growth and differentiation at the end of the culture period. RESULTS: Embryos exposed to 50.0 microg/ml Re had significantly lower median morphological score (29.0 versus 48.0), fewer number of somites (15.0 versus 21.0), and smaller yolk sac diameter (3.5 versus 4.1 mm) and crown-rump length (CRL) (2.9 versus 3.4 mm) compare to control embryos. There was no significant difference between embryos exposed to 5.0 microg/ml Re and control embryos. There was also no difference in the biometric and morphologic parameters among control and embryos exposed to 5.0 and 50.0 microg/ml Rc. CONCLUSION: There is a significant variability in embryotoxic effects of different ginsenosides. Further studies to evaluate the synergistic embryotoxic effects of ginsenosides are warranted.

Corticotropin-releasing hormone peptide and human first-trimester placental growth.

The aim of this study was to determine whether corticotropin-releasing hormone (CRH) regulates human trophoblast cell growth. The results showed that exogenous CRH significantly stimulated human trophoblast proliferation in first-trimester primary cultures. In vivo, CRH was strongly immunolocalised to cytotrophoblastic cells in proliferative cell columns and in chorionic villi. We postulate that CRH may have an important role in early placental development and successful pregnancy.

Treatment of intractable hyperemesis gravidarum by ondansetron.

Hyperemesis gravidarum is a disabling condition. It is not uncommon that patients request termination of pregnancy because of intolerable symptoms and psychological stress. We report a case in which termination of pregnancy was avoided by the use of ondansetron to treat the hyperemesis gravidarum.

The use of three-dimensional ultrasound does not improve training in fetal biometric measurements.

OBJECTIVE: To investigate whether three-dimensional (3D) technology offers any advantage over two-dimensional (2D) ultrasound in fetal biometric measurement training. METHODS: Ten midwives with no hands-on experience in ultrasound were randomized to receive training on 2D or 3D ultrasound fetal biometry assessment. Midwives were taught how to obtain fetal biometric measurements (biparietal diameter (BPD), head circumference (HC), abdominal circumference (AC), and femur length (FL)) by a trainer. Subsequently, each midwife measured the parameters on another 10 fetuses. The same set of measurements was repeated by the trainer. The percentage deviation between the midwives' and the trainer's measurements was determined and compared between training groups. Time required for completion was recorded. Frozen images were reviewed by another sonographer to assess the image quality using a standardized scoring system. RESULTS: The median time for the complete set of measurements was significantly shorter in the 2D than in 3D group (13.4 min versus 17.8 min, P = 0.03). The mean percentage deviations did not reach statistical significance between the two groups except for FL (3.83% in 2D group versus 2.23% in 3D group (P = 0.046)). There were no significant differences in the quality scores. CONCLUSIONS: This study showed that the only demonstrable advantage of 3D ultrasound was a slightly more accurate measurement of FL, at the expense of a significantly longer time required.

Non-invasive prenatal assessment of trisomy 21 by multiplexed maternal plasma DNA sequencing: large scale validity study.

OBJECTIVES: To validate the clinical efficacy and practical feasibility of massively parallel maternal plasma DNA sequencing to screen for fetal trisomy 21 among high risk pregnancies clinically indicated for amniocentesis or chorionic villus sampling. DESIGN: Diagnostic accuracy validated against full karyotyping, using prospectively collected or archived maternal plasma samples. SETTING: Prenatal diagnostic units in Hong Kong, United Kingdom, and the Netherlands. PARTICIPANTS: 753 pregnant women at high risk for fetal trisomy 21 who underwent definitive diagnosis by full karyotyping, of whom 86 had a fetus with trisomy 21. Intervention Multiplexed massively parallel sequencing of DNA molecules in maternal plasma according to two protocols with different levels of sample throughput: 2-plex and 8-plex sequencing. MAIN OUTCOME MEASURES: Proportion of DNA molecules that originated from chromosome 21. A trisomy 21 fetus was diagnosed when the z score for the proportion of chromosome 21 DNA molecules was >3. Diagnostic sensitivity, specificity, positive predictive value, and negative predictive value were calculated for trisomy 21 detection. RESULTS: Results were available from 753 pregnancies with the 8-plex sequencing protocol and from 314 pregnancies with the 2-plex protocol. The performance of the 2-plex protocol was superior to that of the 8-plex protocol. With the 2-plex protocol, trisomy 21 fetuses were detected at 100% sensitivity and 97.9% specificity, which resulted in a positive predictive value of 96.6% and negative predictive value of 100%. The 8-plex protocol detected 79.1% of the trisomy 21 fetuses and 98.9% specificity, giving a positive predictive value of 91.9% and negative predictive value of 96.9%. CONCLUSION: Multiplexed maternal plasma DNA sequencing analysis could be used to rule out fetal trisomy 21 among high risk pregnancies. If referrals for amniocentesis or chorionic villus sampling were based on the sequencing test results, about 98% of the invasive diagnostic procedures could be avoided.

Noninvasive prenatal diagnosis of fetal trisomy 18 and trisomy 13 by maternal plasma DNA sequencing.

Massively parallel sequencing of DNA molecules in the plasma of pregnant women has been shown to allow accurate and noninvasive prenatal detection of fetal trisomy 21. However, whether the sequencing approach is as accurate for the noninvasive prenatal diagnosis of trisomy 13 and 18 is unclear due to the lack of data from a large sample set. We studied 392 pregnancies, among which 25 involved a trisomy 13 fetus and 37 involved a trisomy 18 fetus, by massively parallel sequencing. By using our previously reported standard z-score approach, we demonstrated that this approach could identify 36.0% and 73.0% of trisomy 13 and 18 at specificities of 92.4% and 97.2%, respectively. We aimed to improve the detection of trisomy 13 and 18 by using a non-repeat-masked reference human genome instead of a repeat-masked one to increase the number of aligned sequence reads for each sample. We then applied a bioinformatics approach to correct GC content bias in the sequencing data. With these measures, we detected all (25 out of 25) trisomy 13 fetuses at a specificity of 98.9% (261 out of 264 non-trisomy 13 cases), and 91.9% (34 out of 37) of the trisomy 18 fetuses at 98.0% specificity (247 out of 252 non-trisomy 18 cases). These data indicate that with appropriate bioinformatics analysis, noninvasive prenatal diagnosis of trisomy 13 and trisomy 18 by maternal plasma DNA sequencing is achievable.

Epigenetic-genetic chromosome dosage approach for fetal trisomy 21 detection using an autosomal genetic reference marker.

BACKGROUND: The putative promoter of the holocarboxylase synthetase (HLCS) gene on chromosome 21 is hypermethylated in placental tissues and could be detected as a fetal-specific DNA marker in maternal plasma. Detection of fetal trisomy 21 (T21) has been demonstrated by an epigenetic-genetic chromosome dosage approach where the amount of hypermethylated HLCS in maternal plasma is normalized using a fetal genetic marker on the Y chromosome as a chromosome dosage reference marker. We explore if this method can be applied on both male and female fetuses with the use of a paternally-inherited fetal single nucleotide polymorphism (SNP) allele on a reference chromosome for chromosome dosage normalization. METHODOLOGY: We quantified hypermethylated HLCS molecules using methylation-sensitive restriction endonuclease digestion followed by real-time or digital PCR analyses. For chromosome dosage analysis, we compared the amount of digestion-resistant HLCS to that of a SNP allele (rs6636, a C/G SNP) that the fetus has inherited from the father but absent in the pregnant mother. PRINCIPAL FINDINGS: Using a fetal-specific SNP allele on a reference chromosome, we analyzed 20 euploid and nine T21 placental tissue samples. All samples with the fetal-specific C allele were correctly classified. One sample from each of the euploid and T21 groups were misclassified when the fetal-specific G allele was used as the reference marker. We then analyzed 33 euploid and 14 T21 maternal plasma samples. All but one sample from each of the euploid and T21 groups were correctly classified using the fetal-specific C allele, while correct classification was achieved for all samples using the fetal-specific G allele as the reference marker. CONCLUSIONS: As a proof-of-concept study, we have demonstrated that the epigenetic-genetic chromosome dosage approach can be applied to the prenatal diagnosis of trisomy 21 for both male and female fetuses.

Maternal plasma DNA sequencing reveals the genome-wide genetic and mutational profile of the fetus.

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.

Systematic identification of placental epigenetic signatures for the noninvasive prenatal detection of Edwards syndrome.

BACKGROUND: Noninvasive prenatal diagnosis of fetal aneuploidy by maternal plasma analysis is challenging owing to the low fractional and absolute concentrations of fetal DNA in maternal plasma. Previously, we demonstrated for the first time that fetal DNA in maternal plasma could be specifically targeted by epigenetic (DNA methylation) signatures in the placenta. By comparing one such methylated fetal epigenetic marker located on chromosome 21 with another fetal genetic marker located on a reference chromosome in maternal plasma, we could infer the relative dosage of fetal chromosome 21 and noninvasively detect fetal trisomy 21. Here we apply this epigenetic-genetic (EGG) chromosome dosage approach to detect Edwards syndrome (trisomy 18) in the fetus noninvasively. PRINCIPAL FINDINGS: We have systematically identified methylated fetal epigenetic markers on chromosome 18 by methylated DNA immunoprecipitation (MeDIP) and tiling array analysis with confirmation using quantitative DNA methylation assays. Methylated DNA sequences from an intergenic region between the VAPA and APCDD1 genes (the VAPA-APCDD1 DNA) were detected in pre-delivery, but not post-delivery, maternal plasma samples. The concentrations correlated positively with those of an established fetal genetic marker, ZFY, in pre-delivery maternal plasma. The ratios of methylated VAPA-APCDD1(chr18) to ZFY(chrY) were higher in maternal plasma samples of 9 male trisomy 18 fetuses than those of 27 male euploid fetuses (Mann-Whitney test, P=0.029). We defined the cutoff value for detecting trisomy 18 fetuses as mean+1.96 SD of the EGG ratios of the euploid cases. Eight of 9 trisomy 18 and 1 of 27 euploid cases showed EGG ratios higher than the cutoff value, giving a sensitivity of 88.9% and a specificity of 96.3%. CONCLUSIONS: Our data have shown that the methylated VAPA-APCDD1 DNA in maternal plasma is predominantly derived from the fetus. We have demonstrated that this novel fetal epigenetic marker in maternal plasma is useful for the noninvasive detection of fetal trisomy 18.