Dataset Information

Restriction enzyme-mediated enhanced detection of circulating cell-free fetal DNA in maternal plasma.

ABSTRACT: A universal method confirming the presence of circulating cell-free fetal (ccff) DNA in maternal plasma is important in the field of noninvasive prenatal diagnostics. Restriction endonuclease digestion of one allele of a single-nucleotide polymorphism (SNP) was used to allow detection of paternal alleles in maternal plasma DNA. Multiplexed genotyping of 92 panethnic high-frequency SNPs predicted >0.99 probability of detecting at least four informative loci per sample. Child-maternal paired DNA samples were used to confirm detection of 2% child's heterozygous DNA in a background of maternal DNA homozygous for the digestible allele. By restriction endonuclease digestion of DNA in a PCR cocktail before thermal cycling, 10 genomic copies of a paternal SNP allele were detectable in a background of 990 maternal SNP alleles. A comparison of 154 pregnant and nonpregnant female plasma DNA samples demonstrated enhanced detection of nondigestible SNP alleles in maternal plasma. Receiver operating characteristic curve analysis showed an optimal detection threshold of four nondigestible SNP alleles in plasma for the confirmation of ccff DNA and 98% sensitivity and 96% specificity at a 95% confidence level. Our study demonstrates the ability of this technique to confirm the presence of paternal alleles from ccff DNA in maternal plasma.

SUBMITTER: Tynan JA

PROVIDER: S-EPMC3123783 | biostudies-literature | 2011 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Restriction enzyme-mediated enhanced detection of circulating cell-free fetal DNA in maternal plasma.

Tynan John A JA Mahboubi Payam P Cagasan Lesley L LL van den Boom Dirk D Ehrich Mathias M Oeth Paul P

The Journal of molecular diagnostics : JMD 20110506 4

A universal method confirming the presence of circulating cell-free fetal (ccff) DNA in maternal plasma is important in the field of noninvasive prenatal diagnostics. Restriction endonuclease digestion of one allele of a single-nucleotide polymorphism (SNP) was used to allow detection of paternal alleles in maternal plasma DNA. Multiplexed genotyping of 92 panethnic high-frequency SNPs predicted >0.99 probability of detecting at least four informative loci per sample. Child-maternal paired DNA s ...[more]

PMID: 21704271

Similar Datasets

Project description:BackgroundCurrent methods fail to accurately predict women at greatest risk of developing fetal growth restriction (FGR) or related adverse outcomes, including stillbirth. Sexual dimorphism in these adverse pregnancy outcomes is well documented as are sex-specific differences in gene and protein expression in the placenta. Circulating maternal serum microRNAs (miRNAs) offer potential as biomarkers that may also be informative of underlying pathology. We hypothesised that FGR would be associated with an altered miRNA profile and would differ depending on fetal sex.MethodsmiRNA expression profiles were assessed in maternal serum (> 36 weeks' gestation) from women delivering a severely FGR infant (defined as an individualised birthweight centile (IBC) < 3rd) and matched control participants (AGA; IBC = 20-80th), using miRNA arrays. qPCR was performed using specific miRNA primers in an expanded cohort of patients with IBC < 5th (n = 15 males, n = 16 females/group). Maternal serum human placental lactogen (hPL) was used as a proxy to determine if serum miRNAs were related to placental dysfunction. In silico analyses were performed to predict the potential functions of altered miRNAs.ResultsInitial analyses revealed 11 miRNAs were altered in maternal serum from FGR pregnancies. In silico analyses revealed all 11 altered miRNAs were located in a network of genes that regulate placental function. Subsequent analysis demonstrated four miRNAs showed sexually dimorphic patterns. miR-28-5p was reduced in FGR pregnancies (p < 0.01) only when there was a female offspring and miR-301a-3p was only reduced in FGR pregnancies with a male fetus (p < 0.05). miR-454-3p was decreased in FGR pregnancies (p < 0.05) regardless of fetal sex but was only positively correlated to hPL when the fetus was female. Conversely, miR-29c-3p was correlated to maternal hPL only when the fetus was male. Target genes for sexually dimorphic miRNAs reveal potential functional roles in the placenta including angiogenesis, placental growth, nutrient transport and apoptosis.ConclusionsThese studies have identified sexually dimorphic patterns for miRNAs in maternal serum in FGR. These miRNAs may have potential as non-invasive biomarkers for FGR and associated placental dysfunction. Further studies to determine if these miRNAs have potential functional roles in the placenta may provide greater understanding of the pathogenesis of placental dysfunction and the differing susceptibility of male and female fetuses to adverse in utero conditions.

Project description:BackgroundCirculating cell-free (ccf) fetal DNA comprises 3-20% of all the cell-free DNA present in maternal plasma. Numerous research and clinical studies have described the analysis of ccf DNA using next generation sequencing for the detection of fetal aneuploidies with high sensitivity and specificity. We sought to extend the utility of this approach by assessing semi-automated library preparation, higher sample multiplexing during sequencing, and improved bioinformatic tools to enable a higher throughput, more efficient assay while maintaining or improving clinical performance.MethodsWhole blood (10mL) was collected from pregnant female donors and plasma separated using centrifugation. Ccf DNA was extracted using column-based methods. Libraries were prepared using an optimized semi-automated library preparation method and sequenced on an Illumina HiSeq2000 sequencer in a 12-plex format. Z-scores were calculated for affected chromosomes using a robust method after normalization and genomic segment filtering. Classification was based upon a standard normal transformed cutoff value of z = 3 for chromosome 21 and z = 3.95 for chromosomes 18 and 13.ResultsTwo parallel assay development studies using a total of more than 1900 ccf DNA samples were performed to evaluate the technical feasibility of automating library preparation and increasing the sample multiplexing level. These processes were subsequently combined and a study of 1587 samples was completed to verify the stability of the process-optimized assay. Finally, an unblinded clinical evaluation of 1269 euploid and aneuploid samples utilizing this high-throughput assay coupled to improved bioinformatic procedures was performed. We were able to correctly detect all aneuploid cases with extremely low false positive rates of 0.09%, <0.01%, and 0.08% for trisomies 21, 18, and 13, respectively.ConclusionsThese data suggest that the developed laboratory methods in concert with improved bioinformatic approaches enable higher sample throughput while maintaining high classification accuracy.

Project description:ObjectivesPreeclampsia and fetal growth restriction are obstetrical syndromes associated with abnormal placental implantation and changes in the activation status of maternal leukocytes. This study is aimed to determine by a simple, rapid fluorescent assay the changes in maternal serum total cell-free DNA (t-cfDNA) concentrations in women with preeclampsia and those with fetal growth restriction (FGR).Study designA cross-sectional study was conducted measuring maternal serum t-cfDNA concentrations. Women were classified into the following groups: 1) patients with preeclampsia (n = 21); 2) FGR-estimated fetal weight below the 10thpercentile (n = 28); and 3) normal pregnancy (n = 39). Serum samples were directly assayed for t-cfDNA using a rapid fluorescent SYBR Gold assay. Elevated maternal serum t-cfDNA concentrations were defined as a cutoff>850ng/ml. Nonparametric statistics were used for analysis.ResultsWomen with preeclampsia had a higher median maternal serum concentration (802 ng/ml, 400-2272 ng/ml) than women with a normal pregnancy (499 ng/ml, 0-1892 ng/ml, p = 0.004) and those with FGR (484 ng/ml, 72-2187 ng/ml, p = 0.012). Moreover, even patients with FGR <5th percentile and abnormal Doppler had a lower median maternal serum t-cfDNA than those with preeclampsia (median 487 ng/ml, 144-1971 ng/ml, p = 0.022). The median concentration of t-cfDNA did not differ between women with a normal pregnancy and those with FGR (p = 0.54), as well as those with fetuses <5th percentile and abnormal Doppler (p = 0.7). Women with preeclampsia had a higher proportion of elevated t-cfDNA than those with a normal pregnancy (p = 0.015) and patients with FGR (p = 0.025).ConclusionsPreeclampsia is associated with higher maternal serum t-cfDNA concentration than normal pregnancy or FGR. This observation may reflect an increased systemic activation of the maternal inflammation, rather than placental; this assumption is supported by the fact that we did not observe a significant change in the maternal serum t-cfDNA in patients with placental-mediated FGR.

Dataset Information

Restriction enzyme-mediated enhanced detection of circulating cell-free fetal DNA in maternal plasma.

Publications

Restriction enzyme-mediated enhanced detection of circulating cell-free fetal DNA in maternal plasma.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets