Epigenetic DNA diagnostics

We used GlaI/FatI-PCR assay for determination of 5mC/T polymorphism frequencies at position xp1: 245618129 (according to the GRCh38.p13 genomic assembly) in DNA preparations isolated from blood cells of 51 breast cancer (BC) patients and 63 gastric cancer patients (GC). The data obtained were compared with those previously obtained for 92 healthy blood donors. It was shown that the cytosine in this position is predominantly is methylated in all DNA samples. The incidence of a diploid C/C set in this position in patients with breast cancer and gastric cancer is 9.8% and 7.94%, respectively. This is significantly different from the incidence of C homozygotes in healthy women (26.09%) and healthy donors of both sexes (22.83%). Thus, C/C homozygotes at position xp1: 245618129 is an favorable sign indicating a lower risk (2.7-2.9 times) of two types of oncological diseases. At the same time, the incidence of C/T heterozygotes and T/T homozygotes in patients with gastric cancer and breast cancer exceeds similar indicators for control groups of healthy people by 5.5-10.7%.

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