Epigenetics of cell-free plasma DNA for non-invasive prenatal diagnosis of fetal aneuploidies

Since the discovery of cell-free fetal DNA in the circulation of pregnant women fetal-specific DNA biomarkers for non-invasive prenatal diagnosis of fetal aneuploidy have been sought. A model system assessing the DNA methylation of placental DNA and adult peripheral leukocyte DNA has been developed previously to represent fetal and maternal plasma DNA. To use DNA methylation to detect specific DNA molecules it is desirable that cellfree plasma DNA maintains the methylation profile of its tissue source. Using the imprinted gene GNAS1, a test has been developed to assess, for the first time the relative abundance of methylated and unmethylated DNA circulating in plasma. Methylated and unmethylated DNA sequences were found in equal abundance. If this finding is applicable to all plasma DNA sequences, we conclude that the steadystate concentration of DNA in methylated and unmethylated form is a reliable indicator of its input into the circulation. We have also investigated whether the abundances of different single copy gene sequences in cell-free plasma DNA are equal. Using real-time PCR, the relative abundances of six unique genomic DNA sequences in plasma were assessed. We find that plasma DNA from different sequences is not present in equal abundance in normal healthy individuals. The relative abundance of sequences tested differed by as much as 12.3 fold. We present a panel of novel candidate epigenetic biomarkers which have been identified using the model system. Of 366 DNA regions tested 3% were found to be differential. Further characterisation of these candidate epigenetic biomarkers has revealed limitations of the model system. In view of these results, future epigenetic biomarker development should be achieved by a direct assessment of first trimester plasma DNA

Epigenetics of cell-free plasma DNA for non-invasive prenatal diagnosis of fetal aneuploidies

Since the discovery of cell-free fetal DNA in the circulation of pregnant women fetal-specific DNA biomarkers for non-invasive prenatal diagnosis of fetal aneuploidy have been sought. A model system assessing the DNA methylation of placental DNA and adult peripheral leukocyte DNA has been developed previously to represent fetal and maternal plasma DNA. To use DNA methylation to detect specific DNA molecules it is desirable that cellfree plasma DNA maintains the methylation profile of its tissue source. Using the imprinted gene GNAS1, a test has been developed to assess, for the first time the relative abundance of methylated and unmethylated DNA circulating in plasma. Methylated and unmethylated DNA sequences were found in equal abundance. If this finding is applicable to all plasma DNA sequences, we conclude that the steadystate concentration of DNA in methylated and unmethylated form is a reliable indicator of its input into the circulation. We have also investigated whether the abundances of different single copy gene sequences in cell-free plasma DNA are equal. Using real-time PCR, the relative abundances of six unique genomic DNA sequences in plasma were assessed. We find that plasma DNA from different sequences is not present in equal abundance in normal healthy individuals. The relative abundance of sequences tested differed by as much as 12.3 fold. We present a panel of novel candidate epigenetic biomarkers which have been identified using the model system. Of 366 DNA regions tested 3% were found to be differential. Further characterisation of these candidate epigenetic biomarkers has revealed limitations of the model system. In view of these results, future epigenetic biomarker development should be achieved by a direct assessment of first trimester plasma DNA.EThOS - Electronic Theses Online ServiceSAFE Network of Excellence (LSHB-CT-2004–503243)GBUnited Kingdo

Epigenetics of cell-free plasma DNA for non-invasive prenatal diagnosis of fetal aneuploidies

Since the discovery of cell-free fetal DNA in the circulation of pregnant women fetal-specific DNA biomarkers for non-invasive prenatal diagnosis of fetal aneuploidy have been sought. A model system assessing the DNA methylation of placental DNA and adult peripheral leukocyte DNA has been developed previously to represent fetal and maternal plasma DNA. To use DNA methylation to detect specific DNA molecules it is desirable that cellfree plasma DNA maintains the methylation profile of its tissue source. Using the imprinted gene GNAS1, a test has been developed to assess, for the first time the relative abundance of methylated and unmethylated DNA circulating in plasma. Methylated and unmethylated DNA sequences were found in equal abundance. If this finding is applicable to all plasma DNA sequences, we conclude that the steadystate concentration of DNA in methylated and unmethylated form is a reliable indicator of its input into the circulation. We have also investigated whether the abundances of different single copy gene sequences in cell-free plasma DNA are equal. Using real-time PCR, the relative abundances of six unique genomic DNA sequences in plasma were assessed. We find that plasma DNA from different sequences is not present in equal abundance in normal healthy individuals. The relative abundance of sequences tested differed by as much as 12.3 fold. We present a panel of novel candidate epigenetic biomarkers which have been identified using the model system. Of 366 DNA regions tested 3% were found to be differential. Further characterisation of these candidate epigenetic biomarkers has revealed limitations of the model system. In view of these results, future epigenetic biomarker development should be achieved by a direct assessment of first trimester plasma DNA.EThOS - Electronic Theses Online ServiceSAFE Network of Excellence (LSHB-CT-2004–503243)GBUnited Kingdo

Methylation of the imprinted GNAS1 gene in cell-free plasma DNA : equal steady-state quantities of methylated and unmethylated DNA in plasma

Background Genomic DNA sequences in cell-free plasma are biomarkers of cancer prognosis, where characteristic changes in methylation of tumour suppressor or oncogene DNA regions are indicative of changes in gene activity. Also, cell-free fetal DNA can be distinguished, by its methylation status, from the maternal DNA in the plasma of pregnant women, hence providing DNA biomarkers for the proposed minimally-invasive diagnosis of fetal aneuploidies, including Down's syndrome. However, the production and clearance of cell-free DNA from plasma in relation to its methylation status, are poorly understood processes. Methods We studied the methylation status of DNA derived from the imprinted GNAS1 locus, in cell-free plasma DNA of healthy adults. Heterozygotes were identified that carried the SNP rs1800905 in the imprinted region. The parent-of-origin-dependent DNA methylation was analysed by bisulfite conversion, followed by cloning and sequencing. Results Genomic DNA molecules derived from both the methylated, maternal, allele and the unmethylated, paternal, allele were found in plasma. Methylated and unmethylated DNA molecules were present in equal numbers. Conclusions Our data indicate that the methylation status of a DNA sequence has no effect on its steady state concentration in the cell-free DNA component of plasma, in healthy adults

Imprinted Gene Expression and Function of the Dopa Decarboxylase Gene in the Developing Heart

Dopa decarboxylase (DDC) synthesizes serotonin in the developing mouse heart where it is encoded by Ddc_exon1a, a tissue-specific paternally expressed imprinted gene. Ddc_exon1a shares an imprinting control region (ICR) with the imprinted, maternally expressed (outside of the central nervous system) Grb10 gene on mouse chromosome 11, but little else is known about the tissue-specific imprinted expression of Ddc_exon1a. Fluorescent immunostaining localizes DDC to the developing myocardium in the pre-natal mouse heart, in a region susceptible to abnormal development and implicated in congenital heart defects in human. Ddc_exon1a and Grb10 are not co-expressed in heart nor in brain where Grb10 is also paternally expressed, despite sharing an ICR, indicating they are mechanistically linked by their shared ICR but not by Grb10 gene expression. Evidence from a Ddc_exon1a gene knockout mouse model suggests that it mediates the growth of the developing myocardium and a thinning of the myocardium is observed in a small number of mutant mice examined, with changes in gene expression detected by microarray analysis. Comparative studies in the human developing heart reveal a paternal expression bias with polymorphic imprinting patterns between individual human hearts at DDC_EXON1a, a finding consistent with other imprinted genes in human

Unequal representation of different unique genomic DNA sequences in the cell-free plasma DNA of individual donors

Objectives: To assess whether different genomic cell-free DNAs are equally abundant in the plasma of individual donors, and any relationship between DNA methylation and representation in plasma. Design and methods: The concentrations of DNA in plasma were determined by real-time PCR. Results: Different DNA sequences were not equally represented. The relative abundances were similar in different donors. Conclusions: Different DNA sequences are not equally abundant in plasma, with no relationship between DNA methylation and abundance. (C) 2008 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved

Candidate epigenetic biomarkers for non-invasive prenatal diagnosis of Down syndrome

This report describes the first identification and characterization of three chromosome-21-specific DNA sequences (and reference sequences from other chromosomes) that are differentially methylated between peripheral blood and placental tissue, with the aim of providing epigenetic biomarkers for quantifying cell-free fetal DNA in maternal plasma. To select sequences to be screened for differential methylation, three strategies were adopted: (i) investigating promoters of highly differentially expressed genes; (ii) choosing 'random' promoter regions; and (iii) choosing 'random' non-promoter regions. Over 200 pre-selected DNA sequences were screened using a methylation-specific restriction enzyme assay. Differentially methylated sequences located at 21 q22.3 (AIRE, SIM2 and ERG genes), 1q32.1 (CD48 gene and FAIM3 gene), 2p14 (ARHGAP25 gene) and 12q24 (SELPLG gene) were identified. Bisulphite conversion confirmed that CpG sites within the AIRE promoter region are highly differentially methylated, and optimized methylation-specific primers for this region that are highly specific for placental DNA were devised. Next, it was shown that the methylation status of chorionic villus sample DNA from first trimester pregnancies matched the hypermethylated state of term placenta. Thus there is no indication of a difference in methylation status between early and term pregnancy for the sequences tested. The identified sequences constitute candidate biomarkers for non-invasive prenatal diagnosis of Down syndrome

Epigenetic Upregulation of HGF and c-Met Drives Metastasis in Hepatocellular Carcinoma

<div><p>Hepatocyte growth factor (HGF) and its receptor, c-Met, are important regulators of growth and differentiation of healthy hepatocytes. However, upregulation of HGF and c-Met have been associated with tumor progression and metastasis in hepatocellular carcinoma (HCC). Hematogenous dissemination is the most common route for cancer metastasis, but the role of HGF and c-Met in circulating tumor cells (CTCs) is unknown. We have isolated and established a circulating tumor cell line from the peripheral blood of a mouse HCC model. Our studies show that these CTCs have increased expression of HGF and c-Met in comparison to the primary tumor cells. The CTCs display phenotypic evidence of epithelial-mesenchymal transition (EMT) and the EMT appears to be inducible by HGF. Epigenetic analysis of the c-Met promoter identified significant loss of DNA methylation in CTCs which correlated with overexpression of c-Met and increased expression of HGF. Six specific CpG sites of c-Met promoter demethylation were identified. CTCs show significantly increased tumorigenicity and metastatic potential in a novel orthotopic syngeneic model of metastatic HCC. We conclude that during hematogenous dissemination in HCC, CTCs undergo EMT under the influence of increased HGF. This process also involves up regulation of c-Met via promoter demethylation at 6 CpG sites. Consequently, targeting HGF and c-Met expression by CTCs may be a novel non-invasive approach with potential clinical applications in HCC management.</p></div

The epigenetic regulator PLZF represses L1 retrotransposition in germ and progenitor cells

Germ cells and adult stem cells maintain tissue homeostasis through a finely tuned program of responses to both physiological and stress-related signals. PLZF (Promyelocytic Leukemia Zinc Finger protein), a member of the POK family of transcription factors, acts as an epigenetic regulator of stem cell maintenance in germ cells and haematopoietic stem cells. We identified L1 retrotransposons as the primary targets of PLZF. PLZF-mediated DNA methylation induces silencing of the full-length L1 gene and inhibits L1 retrotransposition. Furthermore, PLZF causes the formation of barrier-type boundaries by acting on inserted truncated L1 sequences in protein coding genes. Cell stress releases PLZF-mediated repression, resulting in L1 activation/retrotransposition and impaired spermatogenesis and myelopoiesis. These results reveal a novel mechanism of action by which, PLZF represses retrotransposons, safeguarding normal progenitor homeostasis