Circulatory Fetal DNA Research Articles

Multiplex fluorescent PCR for noninvasive prenatal detection of fetal-derived paternally inherited diseases using circulatory fetal DNA in maternal plasma

Objective To develop a fluorescent polymerase chain reaction (PCR) assay for the detection of circulating fetal DNA in maternal plasma and use the established multiplex in noninvasive prenatal genetic diagnosis and its further applications in forensic casework. Study design The DNA template was extracted from 47 pregnant women and the whole blood samples from the stated biological fathers were used to detect genotype. Using multiplex fluorescent PCR at 16 different polymorphic short tandem repeat (STR) loci, maternal DNA extracted from plasma samples at early pregnancy, medium pregnancy and late pregnancy were used to detect genotype. Their husbands’ DNA was also used for fetal genotype ascertainment. Results Multiplex fluorescent PCR with 16 polymorphic short tandem repeats revealed the presence of fetal DNA in all cases. Every pregnant women/husband pair was informative in at least 3 of 16 loci. The chances of detecting paternally inherited fetal alleles ranged from 66.67 to 94.12%. They are 66.67% in early pregnancy, 85.71% in medium pregnancy and 94.12% in late pregnancy. The accuracy of Multiplex PCR assay to detect fetal DNA was 100%. Conclusions Circulating fetal DNA analysis can be used as a possible alternative tool in routine laboratory prenatal diagnosis in the near future; this highly polymorphic STR multiplex has greatly improved the chances of detecting paternally inherited fetal alleles compared with other fetal DNA detection systems that use fetus-derived Y sequences to detect only male fetal DNA in maternal plasma. Our proposed technique can be applied to both female and male fetuses, which provides a sensitive, accurate and efficient method for noninvasive prenatal genetic diagnosis and forensic casework.

Quantification of Fetal and Total Circulatory DNA in Maternal Plasma Samples Before and After Size Fractionation by Agarose Gel Electrophoresis

Fetal extracellular DNA is mainly derived from apoptotic bodies of trophoblast. Recent studies have shown size differences between fetal and maternal extracellular DNA. We have examined the quantification of fetal (SRY gene) and total (GLO gene) extracellular DNA in maternal plasma in different fractions (100-300, 300-500, 500-700, 700-900, and >900 bp) after size fractionation by agarose gel electrophoresis. DNA was extracted from maternal plasma samples from 11 pregnant women carrying male foetuses at the 16th week of gestation. Fetal circulatory DNA was mainly detected in the 100-300 bp fraction with the median concentration being 14.4 GE/ml. A lower median amount of 4.9 GE/ml was also found in the 300-500 bp fraction. Circulatory DNA extracted from the 100-300 bp fraction contained 4.2 times enriched fetal DNA when compared with unseparated DNA sample. Fetal DNA within the 300-500 bp fraction was 2.5 times enriched. Circulatory fetal DNA is predominantly present in a fraction with molecular size <500 bp, which can be used for the detection of paternally inherited alleles. However, the usage of size-separated DNA is not suitable for routine clinical applications because of risk of contamination.

Optimized Real‐Time Quantitative PCR Measurement of Male Fetal DNA in Maternal Plasma

DNA of fetal origin is present in the plasma of pregnant women. The quantitative measurement of circulatory fetal DNA (cfDNA) by real-time quantitative PCR (qPCR) has been applied to investigate a possible correlation between increased levels and pregnancy-related disorders. However, as the levels of cfDNA are close to the detection limit (LOD) of the method used, the measurements may not be reliable. This is also problematic for the evaluation of preanalytical steps, such as DNA extraction and cfDNA enrichment by size separation. We optimized a protocol for the qPCR analysis of the multi-copy sequence DYS14 on the Y chromosome. This was compared with an established assay for the single-copy SRY gene. Probit regression analysis showed that the limit of detection (LOD) of the DYS14 assay, (0.4 genome equivalents (GE)) and limit of quantification (LOQ) were 10-fold lower in comparison to SRY (4 GE). The levels of cfDNA obtained from the first trimester of pregnancy could be quantified with high precision by the DYS14 assay (CV below 25%) as opposed to the SRY measurements (26-140%). Additionally, fetal sex was correctly determined in all instances. The low copy numbers of fetal DNA in plasma of women in the first trimester of pregnancy can be measured reliably, targeting the DYS14 that is present in multiple copies per Y chromosome.

Minimal Alteration in the Ratio of Circulatory Fetal DNA to Fetal Corticotropin-Releasing Hormone mRNA Level in Preeclampsia

Objectives: We have recently observed that fetal DNA and fetal corticotropin-releasing hormone (CRH) mRNA are associated with in vitro generated syncytiotrophoblast-derived microparticles, and that the ratio of fetal DNA to mRNA (CRH) varied according to whether the particles were derived by predominantly apoptotic, apo-necrotic or necrotic pathways. Hence, we examined whether these ratios varied in maternal plasma samples taken from normotensive and preeclamptic pregnancies in vivo. Methods: Maternal plasma samples were collected from 18 cases with preeclampsia and 29 normotensive term controls. Circulatory fetal CRH mRNA and DNA levels were quantified by real-time PCR and RT-PCR. Results: Circulatory fetal mRNA and fetal DNA levels were significantly elevated in the preeclampsia study group when compared to normotensive controls. Alterations in the fetal mRNA to DNA ratio between the study and control groups were minimal, even when stratified into early (<34 weeks of gestation) and late (>34 weeks of gestation) onset preeclampsia. Conclusions: Our data suggest that although circulatory fetal DNA and mRNA levels are significantly elevated in preeclampsia, the ratios in maternal plasma are not dramatically altered.

Noninvasive genotyping of 9 Y‐chromosome specific STR loci using circulatory fetal DNA in maternal plasma by multiplex PCR

Human Y-Chromosome specific STR (Y-STR) has now become a useful loci in casework. However, noninvasive genotyping of multiple Y-STR loci and its application in prenatal genetic diagnosis haven't been reported. The purpose of this study is to develop a Y-STR multiplex PCR amplification system that is suitable for the amplification of short-sized templates of circulatory male fetal DNA and use the established multiplex in noninvasive prenatal genetic diagnosis and its further applications in forensic casework. On the basis of the characteristic of circulatory fetal DNA in maternal plasma, we selected 9 Y-STR loci in which the allele size was less than 180 bp in length and developed two multiplexes that allowed fluorescent genotyping of 9 Y-STR loci simultaneously. These Y-STR loci include two trinucleotide repeats (DYS426 and DYS388) and seven tetranucleotide repeats (DYS393, DYS460, H4; DYS391, DYS389 I, DYS456 and DYS458). Sixty-four pairs of plasma DNA samples from pregnant women and genomic DNA samples from their husbands were detected by our method. As a result, an average of 7.3 Y-STR specific alleles was detected in each of the 30 plasma DNA samples from pregnancies with male fetuses. However, none of these 9 Y-STR specific alleles was detected in 34 plasma samples from pregnant women carrying female babies. The chances of detecting Y-STR alleles ranged from 66.7 to 93.3%. Fifty-eight haplotypes were detected in 64 unrelated Chinese male individuals; haplotype diversity was 0.9966. This highly polymorphic Y-STR multiplex has greatly improved the chances of detecting the Y-STR allele. This assay provides a sensitive, accurate and efficient method for noninvasive prenatal genetic diagnosis and forensic casework.

Parallel Assessment of Circulatory Fetal DNA and Corticotropin-Releasing Hormone mRNA in Early- and Late-Onset Preeclampsia

Preeclampsia, a severe disorder of human pregnancy of unknown etiology, remains a major cause of fetal and maternal mortality (1)(2). Because of the heterogeneous nature of this disorder, it has been suggested that preeclampsia could be subclassified into two distinct forms to better understand the underlying causes (3). These two forms, termed early and late onset, are defined as the development of symptoms before or after 34 weeks of pregnancy, respectively (3). Results of several studies have indicated that the early-onset form is more severe, frequently leading to the delivery of growth-retarded premature babies, whereas the late-onset form is more evanescent and clinically of lesser importance (4). Previous studies have indicated that cell-free fetal DNA and mRNA concentrations are increased in preeclampsia (5)(6)(7)(8). Because these studies focused on either cell-free DNA or RNA alone, we examined the concentrations of these nucleic acids simultaneously in cases with early- and late-onset preeclampsia. Approval for this study was granted by the respective Institutional Review Boards (Basel and Stellenbosch), and export of biological material was approved by the South African Department of Health. Written informed consent was obtained in all instances. In our study, maternal blood samples were collected from 37 pregnant women with manifest preeclampsia. Preeclampsia was defined by new-onset blood pressure of at least 140/90 mmHg in 2 determinations 4 h apart or by a diastolic blood pressure >110 mmHg, as well an associated proteinuria of at least 300 mg/24 h after 20 weeks of gestation (6). Early-onset preeclampsia was defined as having symptoms before 34 weeks of pregnancy, whereas late-onset was defined as having symptoms after 34 weeks of …

Detection of Paternally Inherited Fetal Point Mutations for β-Thalassemia Using Size-Fractionated Cell-Free DNA in Maternal Plasma

Currently, fetal point mutations cannot be reliably analyzed from circulatory fetal DNA in maternal plasma, due to the predominance of maternal DNA sequences. However, analysis of circulatory fetal DNA sequences in maternal plasma have been shown to selectively enrich for fetal DNA molecules on the basis of a smaller molecular size than maternal DNA. To examine the prenatal analysis of 4 common beta-thalassemia point mutations: IVSI-1, IVSI-6, IVSI-110, and codon 39. A total of 32 maternal blood samples were collected at 10 to 12 weeks of gestation (mean, 10.7 weeks) between February 15, 2003, and February 25, 2004, in Bari, Italy, from women with risk for beta-thalassemia in their newborns immediately prior to chorionic villous sampling. Samples in which the father and mother did not carry the same mutation were examined. Circulatory DNA was size-fractionated by gel electrophoresis and polymerase chain reaction (PCR) amplified with a peptide-nucleic-acid clamp, which suppresses amplification of the normal maternal allele. Presence of the paternal mutant allele was detected by allele-specific real-time PCR. Detection of paternally inherited beta-globin gene point mutations. Presence or absence of the paternal mutant allele was correctly determined in 6 (86%) of 7 cases with the IVSI-1 mutation, 4 (100%) of 4 with the IVSI-6 mutation, 5 (100%) of 5 with the IVSI-110 mutation, and 13 (81%) of 16 with the codon 39 mutation. One false-positive test result was scored for the IVSI-1 mutation. Two cases with the codon 39 mutation were classified as uncertain and 1 case was excluded due to lack of a diagnostic test result at the time of analysis. These results yielded an overall sensitivity of 100% and specificity of 93.8%, with classified cases removed. Our recently described technique of the size-fractionation of circulatory DNA in maternal plasma may be potentially useful for the noninvasive prenatal determination of fetal point mutations.

Improved prenatal detection of a fetal point mutation for achondroplasia by the use of size‐fractionated circulatory DNA in maternal plasma—case report

The efficacious analysis of fetal loci involving point mutations from circulatory fetal DNA in maternal plasma is hindered by the preponderance of maternal DNA. It has recently been shown that the size difference between fetal and maternal DNA species can be used for the selective enrichment of circulatory fetal DNA in maternal plasma. We have now tested this approach for the detection of a fetal point mutation in the fibroblast growth factor receptor 3 (FGFR3) gene that causes achondroplasia. Circulatory DNA was extracted from maternal plasma and size-fractionated by agarose gel electrophoresis. The fraction with a size less than 300 bp was examined by a touchdown PCR assay specific for the FGFR3 gene, and the mutation was identified by SfcI restriction analysis. Our analysis indicated that although the fetal mutation was discernible in the analysis of total plasma DNA, the result using size-fractionated DNA was much more evident. The enrichment of circulatory fetal DNA in maternal plasma by size-fractionation facilitates the detection of subtle feto-maternal genetic differences, such as those involving point mutations. This approach can easily be extended for the non-invasive prenatal determination of other fetal loci.

Size separation of circulatory DNA in maternal plasma permits ready detection of fetal DNA polymorphisms.

Analysis of fetal DNA in maternal plasma has recently been introduced as a new method for noninvasive prenatal diagnosis, particularly for the analysis of fetal genetic traits, which are absent from the maternal genome, e.g., RHD or Y-chromosome-specific sequences. To date, the analysis of other fetal genetic traits has been more problematic because of the overwhelming presence of maternal DNA sequences in the circulation. We examined whether different biochemical properties can be discerned between fetal and maternal circulatory DNA. Plasma DNA was examined by agarose gel electrophoresis. The fractions of fetal and maternal DNA in size-fractionated fragments were assayed by real-time PCR. The determination of paternally and maternally inherited fetal genetic traits was examined by use of highly polymorphic chromosome-21-specific microsatellite markers. Size fractionation of circulatory DNA indicated that the major portion of cell-free fetal DNA had an approximate molecular size of <0.3 kb, whereas maternally derived sequences were, on average, considerably larger than 1 kb. Analysis of size-fractionated DNA (</=0.3 kb) from maternal plasma samples facilitated the ready detection of paternally and maternally inherited microsatellite markers. Circulatory fetal DNA can be enriched by size selection of fragment sizes less than approximately 0.3 kb. Such selection permits easier analysis of both paternally and maternally inherited DNA polymorphisms.

Antiphospholipid and Anti‐DNA Antibodies Are Not Associated with the Elevated Release of Circulatory Fetal DNA in Pregnancies Affected by Preeclampsia

Objectives: We have previously shown that the levels of circulatory fetal DNA are elevated in preeclampsia and that these increases correspond to disease severity. Several reports have indicated that increased levels of antiphospholipid (anti‐PL) and anti‐DNA antibodies may be associated with preeclampsia, in particular with the severe forms of the disorder. Since the release of cell‐free DNA by the placenta is attributed to some form of cell death or damage and as anti‐PL and anti‐double‐stranded DNA (dsDNA) antibodies have been proposed to lead to placental damage, we have studied the relationship between these parameters in preeclampsia. Methods: Circulating fetal DNA levels in samples taken from pregnant women with mild (n = 12) or severe (n = 12) preeclampsia and from normal pregnant controls (n = 35) were quantified using a Taqman real‐time Polymerase Chain Reaction (PCR) assay. The Anti‐PL antibodies (IgG and IgM) were assayed by anticardiolipin ELISA and by commercial anti‐β2‐Glycoprotein I (GPI) ELISA kits. Anti‐dsDNA antibodies (IgG and IgM) were analyzed by a commercially available anti‐dsDNA ELISA kit. Results: No correlation could be drawn with the quantity of circulatory fetal DNA in the samples analyzed and corresponding anti‐PL or anti‐dsDNA antibody levels. Furthermore, no significant difference existed between the levels of these antibodies in the two study groups and the control cohort. Conclusion: Our data suggest that the mechanism leading to the increased release of cell‐free circulatory DNA from the placenta does not involve trophoblast damage mediated by these agents. Our analysis also questions the reported involvement of anti‐PL and anti‐DNA antibodies in preeclampsia.

Circulatory fetal and maternal DNA in pregnancies at risk and those affected by preeclampsia.

Elevations in fetal cell traffic as well as increased release of cell-free fetal DNA into the maternal periphery have previously been shown to occur in pregnancies affected by preeclampsia. Our own investigations have shown that manifestation of preeclampsia is associated with an increased accumulation of circulatory fetal DNA as well as cell-free maternal DNA in maternal plasma. We further established that the increments in these two molecular genetic analytes corresponded to the severity of the disease and to each other. This latter phenomenon was evident in preeclamptic pregnancies, but not in normal ones. As we had recently performed a prospective study to investigate fetal cell traffic prior to onset of preeclamptic symptoms, we examined the levels of cell-free fetal and maternal DNA in these samples. This analysis indicated that circulatory fetal DNA concentrations were significantly elevated prior to onset of the disease symptoms. No similar feature was observed for cell-free maternal DNA levels.

Both maternal and fetal cell-free DNA in plasma fluctuate.

Elevations in the concentration of cell-free fetal DNA in maternal plasma have recently been determined in various pregnancy-related disorders, including preeclampsia, preterm labor, and polyhydramnios. In addition, almost 2-fold increments in cell-free fetal DNA levels have been recorded in pregnancies with certain aneuploid fetuses, in particular trisomy 21. These findings have led to the speculation that quantitative assessment of circulatory fetal DNA may be useful in the noninvasive prenatal diagnosis of certain fetal genetic constellations or pregnancy-related disorders. A premise for any quantitative analysis is that the quantity of the analyte being assayed does not vary greatly over time. As this aspect has not been examined for circulatory DNA levels, we examined these in normal healthy individuals as well as in pregnant women. Our data indicate that severalfold alterations in circulatory DNA amounts do occur over short periods of time. Of particular note is that we observed almost 2-fold variations in free fetal DNA levels over a period of 3 days, which are in a similar range to the elevations noted in aneuploid pregnancies. Our results, therefore, imply that caution should be used when using small increments in circulatory fetal DNA concentrations for potential diagnostic applications.