Francesco Crea

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.

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 Downs 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.