Epigenetic DNA diagnostics

The method of GLAD-PCR analysis was used to determine the methylation of RCGY sites in the regulatory region of the FAM19A4 gene (Xp3: 68932451 - 68932800) in the DNA preparations of the malignant human cell lines Raji, Jurkat, HeLa, U-937 and the control DNA of the lung fibroblast cell line L68. It is shown that the octanucleotide GCGCGCGC (Xp3: 68932500), located in the first exon of the FAM19A4 gene, is cleaved approximately equally in the DNA of all 4 malignant human cell lines, whereas in the DNA of L68 this site is hydrolyzed 10-15 times weaker. These results are consistent with data of epigenomic sequencing of L68, Raji, and U-937 DNA. Methylation of CG-dinucleotides at positions 68932727 and/or 68932740 in the promoter of the FAM19A4 gene occurs only in tumor DNA and is not observed in L68 DNA, whereas methylation of CG-dinucleotide at position 68932704 is observed only in HeLa DNA.

SNP 5mC/T (Chr1: 245618129) in DNA samples from the human blood of 92donors was studied by GlaI and FatI-PCR assay. The work includes a) isolation of DNA from the blood cells, b) GlaI and FatI-PCR assay of DNA fragment Chr1: 245617889 - 245618464, c) determination of 5mC (Chr1: 245618129) and thymine in DNA preparations and d) comparative analysis of the obtained results. It was shown that in position (Chr1: 245618129) 43 donors (46,74%) have a heterozygote (5mC/T), 28 donors (30,43%) have homozygote (T/T) and 21 donors (22,83%) have homozygote (5mC/5mC). Thus, taking into account a diploid set of chromosomes in the blood cells, SNP 5mC/T is observed in 99 positions from 184 (53,8%). The results obtained have shown that cytosine in position Chr1: 245618129 in the most of the blood DNA is methylated.

SNP 5mC/T in AluSx repeat (Chr16: 75033884) in DNA samples from the human blood of 92donors was studied by GlaI and FatI-PCR assay. The work includes a) isolation of DNA from the blood cells, b) GlaI and FatI-PCR assay of DNA fragment Chr16: 75033860 – 75033957, c) determination of 5mC (Chr16: 75033884) and thymine (Chr16: 75033884) в in DNA preparations and d) comparative analysis of the obtained results. It was shown that in position (Chr16: 75033884) 53 donors (57,61%) have a heterozygote (5mC-T), 19 donors (20,65%) have homozygote (T-T) and 20 donors (21,74%) have homozygote (5mC-5mC). Thus, taking into account a diploid set of chromosomes in the blood cells, SNP 5mC/T is observed in 70 positions from 156 (49,46%). The results obtained have shown that cytosine in position Chr16: 75033884 in the most of the blood DNA is methylated in accordance with a literature data about a significant methylation of CG-dinucleotides in Alu-repeats

Methylation of ACGC site in position Chr11: 65647266 (according to GRCh38 PrimaryAssembly) in DNA preparations from blood cells of healthy donors and early stage breast cancers was studied with GlaI-PCR assay. The work includes a) preparation of light fraction of the blood cells, b) isolation of genomic DNA, c) determination of genomic DNA concentration by real-time PCR, d) determination of concentration of unmethylated ACGC site in position Chr11: 65647266 with GlaI-PCR assay and e) calculation of percent of DNA molecules with unmethylated ACGC site in position Chr11: 65647266. GLAD-PCR analysis showed that in more than 82% of donors, the level of ACGC methylation in the SIPA1 gene is 3% or less, while in approximately 70% of patients with breast cancer, the level of ACGC site methylation in SIPA1 gene is in the range of 3.2 to 6.4%. These data suggest that at early stages of breast cancer, demethylation of A(5mC)GC site in the SIPA1 gene at position 65647266 takes place in 2/3 of patients. The data of this work may be used for a development of PCR test-system for exclusion of diagnosis “early stage breast cancer”. GlaI-PCR assay doesn’t require special and expensive equipment. The study may be carried out in a standard PCR laboratory when a blood analysis is taken place.