DNA In Maternal Blood Research Articles

Non-invasive prenatal diagnosis of fetal trisomy 21 using cell-free fetal DNA in maternal blood

Since the existence of cell-free fetal DNA (cff-DNA) in maternal circulation was discovered, it has been identified as a promising source of fetal genetic material in the development of reliable methods for non-invasive prenatal diagnosis (NIPD) of fetal trisomy 21 (T21). Currently, a prenatal diagnosis of fetal T21 is achieved through invasive techniques, such as chorionic villus sampling or amniocentesis. However, such invasive diagnostic tests are expensive, require expert technicians, and have a miscarriage risk approximately 1%. Therefore, NIPD using cff-DNA in the detection of fetal T21 is significant in prenatal care. Recently, the application of new techniques using single-molecular counting methods and the development of fetal-specific epigenetic markers has opened up new possibilities in the NIPD of fetal T21 using cff-DNA. These new technologies will facilitate safer, more sensitive and accurate prenatal tests in the near future. In this review, we investigate the recent methods for the NIPD of fetal T21 and discuss their implications in future clinical practice.

Prenatal diagnosis of 21 trisomy by quantification of methylated fetal DNA in maternal blood: study on 10 pregnancies

Prenatal diagnosis of 21 trisomy by quantification of methylated fetal DNA in maternal blood: study on 10 pregnancies

Prenatal diagnosis of single gene disorders

Genetic disease can be due to imbalance of whole chromosomes, smaller microdeletions/duplications or at the single gene level where even a single base change can cause significant disease. This review focuses on those disorders caused by single gene defects and their diagnosis during pregnancy. In many cases the genomic location of a specific gene is known and the disease-causing mutation in a particular family may have been identified. In others, diagnosis may rely on other methodologies such as ultrasound or biochemical testing. Until recently this testing has relied upon invasive testing of a pregnancy. However non-invasive prenatal diagnosis to detect fetal DNA in maternal blood has now been introduced and its current and future utility is discussed.

Evaluation of the association between maternal HCMV viremia and the course of pregnancy and neonatal outcome

Congenital cytomegaly is caused by intrauterine mother-to-fetus HCMV transmission and constitutes the most common vertical infection. The aim of the study was to analyze the viremia level in maternal blood and its influence on the course and duration of pregnancy as well as newborn condition. The material included blood samples collected from 117 pregnant women with serological features of HCMV infection and from 29 neonates hospitalized at DFMMG in Lodz between 1999 and 2009. The presence of HCMV DNA in the maternal and fetal blood was tested using real-time PCR. Prevalence of maternal viremia was observed to increase the risk of viremia in neonates, as compared to children born to mothers with no viremia. However; lack of HCMV DNA in maternal blood does not exclude fetal infection in utero. Newborn condition assessed by Apgar scores was significantly lower in the group of infants born to mothers with serological features of acute cytomegaly (p < 0.05). The assessment of viremia level in maternal blood can be helpful in assessing the risk of intrauterine infection in the fetus as well as in predicting the neonatal outcome of newborn.

596: First trimester screening markers in women with pregestational diabetes mellitus: is a correction factor needed?

595 NIPDT registry–a critical utilization of non-invasive prenatal diagnosis test recently introduced to clinical practice Ossie Geifman-Holtzman, Janet Berman, Amen L Ness, Amy Cohen, Richard Fischer, Amanda Carre, Stuart Weiner Drexel College of Medicine, Obstetrics & Gynecology, Philadelphia, PA, Temple School of Medicine, Obstetrics & Gynecology, Philadelphia, PA, Saint Peters University Hospital, Obstetrics & Gynecology, New Brunswick, NJ, Einstein Medical Center, Genetics & Obstetrics/ Gynecology, Philadelphia, PA, Cooper University Hospital, Obstetrics & Gynecology, Camden, NJ, Thomas Jefferson University Hospital, Obstetrics & Gynecology, Philadelphia, PA OBJECTIVE: To evaluate the accuracy and utilization of noninvasive prenatal diagnosis test (NIPDT) using fetal DNA in maternal blood and to establish a multicenter registry to accomplish this aim. STUDY DESIGN: NIPDT by using fetal DNA in maternal blood to diagnose chromosomal aneuploidy is currently available and its utilization is rapidly expanding. We collected data from 6 centers and tabulated it by patient age, gestational age (GA), indication, maternal serum screen, ultrasound finding. IRB approval was obtained. We have also established the NIPDT Registry to accomplish the above goal. RESULTS: A total of 175 patients underwent testing over the past 3 months. Tests were performed at 11-13 weeks (12%), at 14-17 weeks (36%), at 18-22 (40%) weeks, and at 23-25 (12%) weeks’ gestation. The following indications were used to perform the test: Maternal age (above 35) 54% , Abnormal maternal serum screening 41%, Abnormal ultrasound finding 27% Positive family or personal history 4%. At maternal age of less than 35 about 77%, 15% and 8% were tested because of abnormal ultrasound finding, abnormal serum screen or both abnormal and others, respectively. Inconclusive results were reported to be between 3.5%-5% possible causes: testing at early GA or fetuses with hydrops. Repeat testing at a later GA yielded conclusive results except for one patient. At this time, as most tested patients are undelivered accuracy is determined based on CVS/amniocentesis results with 2% incorrect diagnosis by NIPDT, for example trisomy 18 was reported by NIPDT and was normal by FISH and Karyotype. CONCLUSION: The clinical utilization of NIPDT is rapidly expanding with significant clinical and economical implications. Larger sample size and independent evaluation of the accuracy of all results in clinical setting are clearly required and will be studied by the NIPDT Registry. Current standard of prenatal diagnosis care should continue along with critical assessment of the NIPDT results. 596 First trimester screening markers in women with pregestational diabetes mellitus: is a correction factor needed? Padmalatha Gurram, Peter Benn, Ann-Marie Prabulos, Kisti Fuller, Crawford Christine, Campbell Winston University of Connecticut, Maternal-Fetal Medicine, Farmington, CT, University of Connecticut, Clinical Genetics and developmental biology, Farmington, CT OBJECTIVE: To investigate if maternal serum pregnancy associated plasma protein-A (PAPP-A), total beta hCG and nuchal translucency (NT) measurements differ in women with pregestational diabetes (PGDM) vs. non-diabetic controls (NDC) and if so is a correction factor needed for diabetic women. STUDY DESIGN: We performed a retrospective cohort analysis of all women having a first trimester aneuploidy screen (11 to 13 6 wks) between 1/2005-12/2011 at our facility. The primary outcome was difference in PAPP-A, hCG & NT MoM (multiples of the median, corrected for maternal weight, race, gestational age [GA]) between PGDM and NDC. Women using insulin (IDDM) or oral hypoglycemics (OHG) prior to pregnancy or at the time of screening were classified as PGDM. Known aneuploidy & multifetal gestations were excluded. We also examined if any difference in PAPP-A, hCG and NT between PGDM & NDC is affected by GA or HbA1C.We compared screen positive (1:270)Down syndrome rates (DSR) between PGDM & NDC. Chi square & Mann-Whitney tests were used (significant at p 0.05). RESULTS: Of 6741 eligible patients, 103(1.5%) had IDDM & 4 (0.06%) were on OHG (excluded due to small number). Outcomes were available for 1512 patients, of which 67 (4%) developed gestational diabetes and were excluded. HbA1C was available for 43 IDDM patients. Mean maternal weight in IDDM patients was 202 lbs vs. NDC 161 lbs, (p 0.05). There was a 12% reduction of median PAPP-A & 18 % reduction of median total hCG in women with IDDM (Table). The effect of IDDM on PAPP-A & hCG did not differ with GA (p 0.05).The mean HgbA1C% was 7.3 1.5. There was no significant correlation between HbA1C and PAPP-A (Figure), hCG or NT (p 0.05). Screen positive DSR was 8.74% in IDDM vs. NDC 7.19%, (p 0.03). CONCLUSION: In women with IDDM, PAPP-A and hCG were significantly lower compared to NDC and correction factors should be considered in the aneuploidy calculation of risks for IDDM patients. This effect is not GA dependent or associated with glycemic control. Poster Session IV Academic Issues, Antepartum Fetal, Clinical Ob, Fetus, Genetics, Hypertension, Med-Surg-Diseases, Operative Ob, U/S www.AJOG.org

591: Using targeted sequencing of SNPs to achieve a highly accurate non-invasive detection of fetal aneuploidy of 13, 18, 21 and sex chromosomes

accurate non-invasive detection of fetal aneuploidy of 13, 18, 21 and sex chromosomes Matthew Rabinowitz, Matthew Hill, Zachary Demko, Sallie McAdoo, Bernhard Zimmermann, Brynn Levy Natera, Inc, Research and Development, San Carlos, CA, Columbia Univeristy, Department of Pathology and Cell Biology, New York, NY OBJECTIVE: To non-invasively detect all abnormalities of chromosomes 13, 18, 21, X and Y, in the fetus through analysis of cell-free fetal DNA in maternal blood. STUDY DESIGN: 500 maternal plasma samples (including 415 euploid and 85 aneuploid samples) were collected from patients at greater than 9 weeks of gestation under an institutional review board (IRB) approved protocol. Aneuploid samples included Trisomy 21, Trisomy 18, Trisomy 13, Turner syndrome (45,X), Klinefelter (47,XXY), and Triploidy. Cell free DNA was isolated, and a targeted multiplex PCR amplification of 19,000 SNP loci on 13, 18, 21, X, and Y was performed. Sequencing data was analyzed using novel Parental Support (PS) technology that uses Bayesian statistics to analyze multiple copy number hypotheses and determine the Maximum likelihood estimation (MLE) given the sequencing data. Unlike other reported methods, the PS method determines a confidence, or calculated accuracy, without requiring a reference chromosome. RESULTS: In those samples that passed quality control thresholds, all calls across all 5 chromosomes tested were correct. The mean calculated accuracy of calls in samples that passed the quality threshold was 99.8%. Samples that failed to meet this threshold (10%) were typically of poor DNA quality and/or low fetal fraction ( 4%). Identification of these samples enables a quick redraw and retesting of the patient rather than increasing the risk of false negatives/positives. CONCLUSION: The PS informatics approach is able to calculate a sample-specific accuracy and detect chromosomally abnormal fetuses from a maternal blood sample with high accuracy at chromosomes 13, 18, 21, X, and Y. Use of the calculated accuracy enables equivalent detection across the chromosomes, and permits maternal blood draws early in pregnancy without increasing the probability of false negatives or positives.

Cell-free fetal DNA in maternal blood: new possibilities in prenatal diagnostics1)

Abstract Cell free fetal DNA (cff-DNA) in maternal blood has given rise to the possibility of new non-invasive approaches in prenatal genetic diagnoses. In contrast to the established invasive techniques chorionic villi sampling and amniocentesis, both associated with specific risk (0.5%–1%) for procedure-related abortions, cff-DNA is simply gained by maternal venous blood sampling, without any risk for the embryo or fetus. Therefore, cff-DNA offers the possibility for riskless genetic diagnoses of ongoing pregnancies. Molecular genetic techniques are already used for the qualitative detection of specific fetal sequences, such as paternal inherited or spontaneous originated (de novo) mutations. Until recently, the introduction of digital PCR and next generation sequencing technologies has shown that a reliable quantitative detection of mutant alleles as well as of clinical relevant aneuploidies (Trisomy 13, 18 and 21) from fetal DNA in maternal blood is also possible.

Assessment of Foetal DNA in Maternal Blood – A Useful Tool in the Hands of Prenatal Specialists

Over the last few years, first trimester screening between 11+ and 13+ weeks of gestation has become one of the most important ultrasound examinations in pregnancy, as it allows physicians to predict several pregnancy complications including pre-eclampsia or pre-term birth. Screening for trisomies 21/18 and 13 using maternal and gestational age, foetal nuchal translucency, and maternal serum biochemistry was formerly the main reason for first trimester screening. However, today this is only one part of the overall examination. In the near future, the analysis of foetal DNA obtained from maternal blood will be used to supplement first trimester screening for aneuploidy or even replace current screening methods. In this review we show how prenatal medicine specialists can use foetal DNA analysis.

Clinical evaluations of cell‐free fetal DNA quantities in pre‐eclamptic pregnancies

Quantitative changes of cell-free fetal DNA in maternal plasma as an indicator for impending pre-eclampsia was reported in different studies. Cell-free fetal nucleic acids can be detected in maternal circulation during pregnancy. Our aim was to determine the higher rate of fetal DNA levels in maternal blood in pre-eclampsia compared to normal pregnancies and the clinical use of real-time polymerase chain reaction (PCR) in the Turkish population as a marker. According to their gestational ages, the plasma levels of 30 pre-eclamptic women at 26-40 weeks of pregnancy were matched with 18 healthy pregnant women. Cell-free fetal DNA levels in maternal plasma were compared using real-time PCR technology. For the quantitative measurement of fetal DNA from maternal blood, the relative quantification PCR process was applied to all samples, using SRY and GAPDH genes. These patients were classified as pre-eclamptic and control groups and were matched according to weeks of pregnancy. Free fetal DNA levels of 30 pre-eclamptic patients were compared to healthy pregnant women and an average 3.06-fold increase was observed. During the second trimester, free fetal DNA levels were 1.5 times higher in pre-eclamptic patients. This increase was 3.5-fold during the third trimester. The DNA increase of pre-eclamptic patients was 4.1-fold and 3.4-fold during 29th-33rd and 34th-40th weeks, respectively. Cell-free fetal DNA in maternal blood could be used as a marker for identifying subjects at increased risk of developing pre-eclampsia.

Noninvasive prenatal aneuploidy testing of chromosomes 13, 18, 21, X, and Y

To develop a non-invasive prenatal aneuploidy test based on analysis of cell-free DNA in maternal blood that is capable of detecting whole chromosomal aneuploidy of chromosomes 13, 18, 21, X and Y with high accuracy in the first trimester.

Noninvasive fetal trisomy 21 detection using chromosome-selective sequencing: a variation of the molecular counting theme

Evaluation of: Sparks A, Wang ET, Struble CA et al. Selective analysis of cell-free DNA in maternal blood for evaluation of fetal trisomy 21. Prenat. Diagn. 32, 3–9 (2012).Fetal DNA is present in the plasma of pregnant women. A fetus with trisomy of a chromosome will release an increased amount of DNA from that chromosome into maternal plasma. Such an increase has previously been measured using methods that allow individual DNA molecules to be counted. One such method involves the use of random massively parallel sequencing of maternal plasma DNA. As the sequencing process is random, sequence tags from a potentially aneuploid chromosome only represent a fraction of the sequencing data. The performance of selective amplification and sequencing of specific genomic regions is a recently reported approach for focusing the sequencing power onto genomic regions of diagnostic interest. This article provides a critical analysis of this approach and puts this method in the perspective of other recent works in the field.

The Potential Usefulness of Free Fetal DNA in Maternal Blood for Prenatal Fetal Gender Determination in Multiple Pregnancies

We applied a noninvasive prenatal test for the determination of fetal gender in multiple pregnancies by using free fetal DNA circulating in maternal blood in order to evaluate whether the quantification of male DNA could distinguish the fetal gender and the number of male and female fetuses in multiple pregnancies. We enrolled consecutively 44 women with twin pregnancies between 11-14 weeks of gestation. Peripheral maternal blood was collected, and genomic DNA was extracted from maternal plasma and analyzed for the multicopy DYS 14 sequence by using real-time PCR to quantify male DNA. Results showed that male DNA concentration was significantly higher in twin pregnancies with at least one male fetus, compared to twin pregnancies with only female fetuses. Comparing male DNA concentration in pregnancies with two male fetuses versus pregnancies with one female fetus and one male fetus, we did not obtain a significant difference between the two groups due to a slight overlapping of the range values. Therefore, our test correctly predicted fetal gender, distinguishing twin pregnancies with at least one male fetus from twin pregnancies with only female fetuses, with a diagnostic accuracy of 100%. For distinguishing pregnancies with two male fetuses from pregnancies with both female and male fetuses, a diagnostic accuracy of 76.1% was achieved.

DNA Sequencing of Maternal Plasma to Detect Down Syndrome

The use of current screening for Down syndrome is relatively effective and can detect up to 90% of cases with a false-positive rate of about 5%. However, follow-up invasive procedures are associated fetal loss in as many as 1 in 200 tests. Alternative noninvasive diagnostic tests have been investigated that can detect fetal cell-free DNA in maternal blood. In 2008, 2 reports described the use of such a test, massively parallel shotgun sequencing (MPSS), which is an internally validated test based on next-generation sequencing. Subsequent studies with the MPSS test were promising and suggested a detection rate of at least 98% at a false-positive rate of 2% or lower. However, sample sizes were small (13–86 Down syndrome cases), and the test was not performed in Clinical Laboratory Improvement Amendments (CLIA)-certified laboratories. This blinded, nested case-control study analyzed the use of MPSS within a cohort of 4664 pregnancies. Women at high risk for Down syndrome were enrolled at 27 prenatal diagnostic centers worldwide. The test was evaluated in 212 Down syndrome and 1484 matched euploid pregnancies. Similar numbers of Down syndrome cases were diagnosed in the first and second trimester. None of the samples had been previously tested. A CLIA-certified commercial laboratory performed the primary testing. A subset of samples was sent to a CLIA-certified university laboratory for confirmatory testing. MPSS testing correctly identified 209 of 212 Down syndrome fetuses; the detection rate was 98.6%, with a 95% confidence interval of 95.9–99.7. The false-positive rate was 0.2% (3/1471), with a 95% confidence interval of <0.1–0.6. Testing failed in 13 euploid pregnancies (0.8%). After testing was concluded but before unblinding, the primary testing laboratory adjusted the chromosome 21 data for guanine cytosine base content to assess alternative methods of interpreting the MPSS results. Adjusting chromosome 21 counts for guanine cytosine base content had the largest impact on improving test performance (reduced false-negative and false-positive results). These findings show that measuring maternal plasma DNA in a large cohort of high-risk pregnancies can identify nearly all cases of Down syndrome at a very low false-positive rate. Although confirmation by invasive testing is needed, data from this and previous studies suggest the benefit of this test in women at high risk of Down syndrome.

Selective analysis of cell‐free DNA in maternal blood for evaluation of fetal trisomy

ObjectiveTo develop a novel prenatal assay based on selective analysis of cell-free DNA in maternal blood for evaluation of fetal Trisomy 21 (T21) and Trisomy 18 (T18).MethodsTwo hundred ninety-eight pregnancies, including 39 T21 and seven T18 confirmed fetal aneuploidies, were analyzed using a novel, highly multiplexed assay, termed digital analysis of selected regions (DANSR™). Cell-free DNA from maternal blood samples was analyzed using DANSR assays for loci on chromosomes 21 and 18. Products from 96 separate patients were pooled and sequenced together. A standard Z-test of chromosomal proportions was used to distinguish aneuploid samples from average-risk pregnancy samples. DANSR aneuploidy discrimination was evaluated at various sequence depths.ResultsAt the lowest sequencing depth, corresponding to 204 000 sequencing counts per sample, average-risk cases where distinguished from T21 and T18 cases, with Z statistics for all cases exceeding 3.6. Increasing the sequencing depth to 410 000 counts per sample substantially improved separation of aneuploid and average-risk cases. A further increase to 620 000 counts per sample resulted in only marginal improvement. This depth of sequencing represents less than 5% of that required by massively parallel shotgun sequencing approaches.ConclusionDigital analysis of selected regions enables highly accurate, cost efficient, and scalable noninvasive fetal aneuploidy assessment. © 2012 John Wiley & Sons, Ltd.

Noninvasive Detection of Fetal Trisomy 21 by Sequencing of DNA in Maternal Blood: A Study in a Clinical Setting

Objective We sought to evaluate a multiplexed massively parallel shotgun sequencing assay for noninvasive trisomy 21 detection using circulating cell-free fetal DNA. Study design Sample multiplexing and cost-optimized reagents were evaluated as improvements to a noninvasive fetal trisomy 21 detection assay. A total of 480 plasma samples from high-risk pregnant women were employed. Results In all, 480 prospectively collected samples were obtained from our third-party storage site; 13 of these were removed due to insufficient quantity or quality. Eighteen samples failed prespecified assay quality control parameters. In all, 449 samples remained: 39 trisomy 21 samples were correctly classified; 1 sample was misclassified as trisomy 21. The overall classification showed 100% sensitivity (95% confidence interval, 89–100%) and 99.7% specificity (95% confidence interval, 98.5–99.9%). Conclusion Extending the scope of previous reports, this study demonstrates that plasma DNA sequencing is a viable method for noninvasive detection of fetal trisomy 21 and warrants clinical validation in a larger multicenter study.

Noninvasive prenatal tests for Down syndrome are near

Noninvasive prenatal tests for Down syndrome are near

Determination of fetal chromosome aberrations from fetal DNA in maternal blood: has the challenge finally been met?

The analysis of cell-free fetal nucleic acids in maternal blood for prenatal diagnosis has been transformed by several recent profound technology developments. The most noteworthy of these are ‘digital PCR’ and ‘next-generation sequencing’ (NGS), which might finally deliver the long-sought goal of noninvasive detection of fetal aneuploidy. Recent data, however, indicate that NGS might even be able to offer a much more detailed appraisal of the fetal genome, including paternal and maternal inheritance of point mutations for mendelian disorders such as β-thalassaemia. Although these developments are very exciting, in their current form they are still too complex and costly, and will need to be simplified considerably for their optimal translation to the clinic. In this regard, targeted NGS does appear to be a step in the right direction, although this should be seen in the context of ongoing progress with the isolation of fetal cells and with proteomic screening markers.

Non-invasive tool for foetal sex determination in early gestational age

Free foetal DNA in maternal blood during early pregnancy is an ideal source of foetal genetic material for non-invasive prenatal diagnosis. The aim of this study was to evaluate the use of free foetal DNA analysis at early gestational age as pretest for the detection of specific Y-chromosome sequences in maternal plasma of women who are carriers of X-linked disorders, such as haemophilia. Real-time quantitative PCR analysis of maternal plasma was performed for the detection of the SRY or DYS14 sequence. A group of 208 pregnant women, at different gestational periods from 4 to 12 weeks, were tested to identify the optimal period to obtain an adequate amount of foetal DNA for prenatal diagnosis. Foetal gender was determined in 181 pregnant women sampled throughout pregnancy. Pregnancy outcome and foetal gender were confirmed using karyotyping, ultrasonography or after birth. The sensitivity, which was low between 4th and 7th week (mean 73%), increased significantly after 7+1th weeks of gestation (mean 94%). The latter sensitivity after 7+1th week of gestation is associated to a high specificity (100%), with an overall accuracy of 96% for foetal gender determination. This analysis demonstrates that foetal gender determination in maternal plasma is reliable after the 9th week of gestation and it can be used, in association with ultrasonography, for screening to determine the need for chorionic villus sampling for prenatal diagnosis of X-linked disorders, such as haemophilia.

Fetal DNA: A Needle in the Haystack

A fetal epigenetic footprint in mothers’ blood paves the way for noninvasively diagnosing Down’s syndrome.

Telegony, the Sire Effect and non‐Mendelian Inheritance Mediated by Spermatozoa: A Historical Overview and Modern Mechanistic Speculations

Telegony is the belief that the sire first mated to a female will have an influence upon some of that female's later offspring by another male. Although the reality of telegony was acknowledged by such authorities as Darwin, Spencer, Romanes and many experienced breeders, it has been met with scepticism because of Weismann's unfavourable comments and negative results obtained in several test experiments. In this article, alleged cases of telegony are provided. A search of the literature of cell biology and biochemistry reveals several plausible mechanisms that may form the basis for telegony. These involve the penetration of spermatozoa into the somatic tissues of the female genital tract, the incorporation of the DNA released by spermatozoa into maternal somatic cells, the presence of foetal DNA in maternal blood, as well as sperm RNA-mediated non-Mendelian inheritance of epigenetic changes.