GlaI

M.A. Abdurashitov, A.G. Akishev, S.Kh. Degtyarev SibEnzyme Ltd, Novosibirsk Corresponding author: M.A. Abdurashitov, SibEnzyme Ltd., 2/12 Ak. Timakova Street,Novosibirsk 630117, Russia; Tel.: +7 (383) 333-4991; Fax: +7 (383) 333-6853; E-mail: abd@sibenzyme.ru Abstract The 5-methylcytosine-dependent DNA endonuclease GlaI recognizes and cleaves DNA sites with the sequence 5′-R(5mC)GY-3′ / 3′-YG(5mC)R-5′, which are formed de novo in the human genome by the DNA methyltransferase DNMT3. Owing to this specificity, GlaI is efficiently used to determine the methylation status of RCGY sites in the human genome. Genome-wide mapping of R(5mC)GY sites can be performed by next-generation sequencing (NGS) of DNA hydrolysates generated by GlaI digestion, whereas analysis of individual DNA fragments can be carried out using GlaI-PCR analysis. In this study, we compared the results of NGS analysis of GlaI DNA hydrolysates with data obtained by GlaI-PCR analysis of regulatory regions of 11 tumor suppressor genes in DNA from human cell lines L68 (normal lung fibroblasts), Raji (Burkitt lymphoma), and U937 (histiocytic lymphoma). It was shown that in L68 cells most of the analyzed regulatory regions are unmethylated, whereas methylation of these regions is observed in the malignant cell lines Raji and U937. With the exception of a single region, for all other analyzed fragments the methylation status determined by GlaI-PCR analysis fully correlates with the number of R(5mC)GY sites detected by NGS. The only discrepancy between the two methods was identified for the regulatory region of the RASSF1A gene in L68 cells and was attributed to the presence of allele-specific methylation, which was confirmed by additional real-time GlaI-PCR analysis. Taking allele-specific methylation of the RASSF1A regulatory region into account, complete concordance was observed between the data obtained by NGS analysis of GlaI DNA hydrolysates and GlaI-PCR analysis of regulatory regions of the studied tumor suppressor genes in the human cell lines L68, Raji, and U937. Thus, NGS analysis of GlaI DNA hydrolysates and GlaI-PCR analysis are complementary approaches that enable reliable identification of aberrantly methylated genomic regions in normal and malignant cells, making them promising tools for epigenetic research and DNA-based diagnostics. Keywords: 5-methylcytosine; DNA methylation; methyl-dependent restriction endonucleases;GlaI; NGS analysis; GlaI-PCR analysis; epigenetic DNA diagnostics; allele-specific methylation. DOI: 10.26213/3034-4298.2025.6.1.001 Citation: M.A. Abdurashitov, A.G. Akishev, S.Kh. Degtyarev (2025) Determination of the Methylation Status of RCGY Sites in the Human Genome: A Comparison of NGS Analysis of GlaI DNA Hydrolysates and GlaI-PCR Data, Epigenetic DNA diagnostics, vol 2025(1), DOI: 10.26213/3034-4298.2025.6.1.001 This work is available at Creative Commons…

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.

DNA methylation in human genome is important for the cells specialization and functioning. An abnormal methylation of the regulation regions of some genes may cause the genes silencing and this phenomenon is often detected in cancer cells. Determination of differences of the genome-wide methylation in normal and tumor cells is useful for understanding the carcinogenesis process and for development of new methods of epigenetic diagnostics. The positions of methylated RCGY sites in the genomes of Raji, U-937 and L68 human cell lines have been determined using the previously developed method of massive parallel sequencing of Glal fragments. A comparison of the obtained data has revealed significant differences in methylation of CpG islands, putative regulatory regions and some repetitive DNA families between genomes of malignant and non-malignant cells. GO enrichment analysis of genes with highly methylated regulatory regions has shown the possible metabolic processes, which may be affected epigenetically in carcinogenesis. The new method allows to determine positions of many modified cytosine bases in the genomes and may be a simple alternative to the existing methods of genome-wide methylation analysis.