Mrassf1a-Pap, a Novel Methylation-Based Assay for the Detection of Cell-Free Fetal DNA in Maternal Plasma
نویسندگان
چکیده
OBJECTIVES RASSF1A has been described to be differentially methylated between fetal and maternal DNA and can therefore be used as a universal sex-independent marker to confirm the presence of fetal sequences in maternal plasma. However, this requires highly sensitive methods. We have previously shown that Pyrophosphorolysis-activated Polymerization (PAP) is a highly sensitive technique that can be used in noninvasive prenatal diagnosis. In this study, we have used PAP in combination with bisulfite conversion to develop a new universal methylation-based assay for the detection of fetal methylated RASSF1A sequences in maternal plasma. METHODS Bisulfite sequencing was performed on maternal genomic (g)DNA and fetal gDNA from chorionic villi to determine differentially methylated regions in the RASSF1A gene using bisulfite specific PCR primers. Methylation specific primers for PAP were designed for the detection of fetal methylated RASSF1A sequences after bisulfite conversion and validated. RESULTS Serial dilutions of fetal gDNA in a background of maternal gDNA show a relative percentage of ~3% can be detected using this assay. Furthermore, fetal methylated RASSF1A sequences were detected both retrospectively as well as prospectively in all maternal plasma samples tested (n = 71). No methylated RASSF1A specific bands were observed in corresponding maternal gDNA. Specificity was further determined by testing anonymized plasma from non-pregnant females (n = 24) and males (n = 21). Also, no methylated RASSF1A sequences were detected here, showing this assay is very specific for methylated fetal DNA. Combining all samples and controls, we obtain an overall sensitivity and specificity of 100% (95% CI 98.4%-100%). CONCLUSIONS Our data demonstrate that using a combination of bisulfite conversion and PAP fetal methylated RASSF1A sequences can be detected with extreme sensitivity in a universal and sex-independent manner. Therefore, this assay could be of great value as an addition to current techniques used in noninvasive prenatal diagnostics.
منابع مشابه
O-45: Quantification of Cell-Free-Fetal-DNAfrom Maternal Plasma for the First Time in Pakistan:Implications for Non-Invasive PrenatalDiagnosis of Genetic Disorders
Background: Current prenatal diagnosis requires invasive testing which carries a 1-4% procedure-related-risk of miscarriage; hence, non-invasive techniques are desired. The recent demonstration of cell-free-fetal-DNA enriched from maternal plasma has opened new possibilities for non-invasive-prenatal-diagnosis of not only genetic-disorders such as β-thalassaemia and haemophilia but also chromos...
متن کاملAn Effective Method for Detecting Y-chromosome Specific Sequences of Circulating Fetal DNA in Maternal Plasma During the First-trimester
Background and Aims: New advances in the use of cell-free fetal DNA (cffDNA) in maternal plasma of pregnant women has provided the possibility of applying cffDNA in prenatal diagnosis as a non-invasive method. One of the applications of prenatal diagnosis is fetal gender determination. Early prenatal determination of fetal sex is required for pregnant women at risk of X-linked and some endocrin...
متن کاملP-211: Quantitative Changes of Fetal DNA in Maternal Circulation during Pregnancy Based on Detection of SRY Gene in Ovine Species
Background: It is well documented that fetal DNA can cross the placenta and is present in peripheral maternal blood during pregnancy in human. This fetal DNA also named circulating cell free fetal DNA, has emerged as a valuable source for genetic evaluation. Compared with humans, ovine species have a different structure of placental (synepitheliochorial) with no direct contact between the troph...
متن کاملO-45: Quantification of Cell-Free-Fetal-DNAfrom Maternal Plasma for the First Time in Pakistan:Diagnosis of Genetic Disorders
Background: Current prenatal diagnosis requires invasive testing which carries a 1-4% procedure-related-risk of miscarriage; hence, non-invasive techniques are desired. The recent demonstration of cell-free-fetal-DNA enriched from maternal plasma has opened new possibilities for non-invasive-prenatal-diagnosis of not only genetic-disorders such as β-thalassaemia and haemophilia but also chromos...
متن کاملO-37: Pseudomalignant Nature of Placenta during Normal and Pathological Gestation Is Regulated by Epigenetic Mechanisms which Can be Exploited To Design Non-Invasive Fetal Dna Markers
Background Placentation shares many analogues with the development of tumors such as rapid proliferation, invasiveness, gene expression profiles especially the expression of tumor suppressor genes, oncogenes and matrixmetallo proteinases (MMPs). Thus, a placenta has been described as a pseudomalignant tissue. However, placentation is tightly regulated and any deregulation of this pseudomalignan...
متن کامل