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GLAD-PCR Assay of DNA Methylation Markers Associated with Colorectal Cancer

Alexey A. Evdokimov , Nina A. Netesova , Natalia A. Smetannikova , Murat A. Abdurashitov, Alexandr G. Akishev , Boris S. Malyshev , Evgeniya S. Davidovich , Vladimir V. Fedotov , Vitaliy V. Kuznetsov , Yuriy D. Ermolaev , Andrey B. Karpov , Alexey E. Sazonov , Ravil M. Tahauov , Sergey Kh. Degtyarev
Biol Med (Aligarh) , 8:7(2016)

Hypermethylation of the gene regulatory regions is documented for many cancer diseases. Such an aberrant DNA methylation in cancer cells is catalyzed by DNA methyltransferases Dnmt3a and Dnmt3b, which predominantly recognize and methylate RCGY sequences with formation of R(5mC)GY sites. Recently, based on a new methyl-directed DNA endonuclease GlaI, we developed a GLAD-PCR assay, which allows determining R(5mC)GY site in a defi ned position of the genomic DNA. In this work we applied GLAD-PCR assay for identifi cation of the methylated RCGY sites in the regulatory regions of some downregulated genes associated with colorectal cancer (CRC). This list includes ADHFE1, ALX4, CNRIP1, EID3, ELMO1, ESR1, FBN1, HLTF, LAMA1, NEUROG1, NGFR, RARB, RXRG, RYR2, SDC2, SEPT9, SFRP2, SOCS3, SOX17, THBD, TMEFF2, UCHL1, and VIM genes. GLAD-PCR analysis of selected RCGY sites within the regulatory regions of some of these genes demonstrates a good prognostic potential with relatively high sensitivity and specifi city of CRC detection in tumor DNA

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MteI-PCR assay – a new method of methylation status determination for GC-rich regulatory regions of human tumor suppressor genes

M.A. Abdurashitov, A.N. Kuksova, A.G. Akishev, E.V. Zemlyanskaya, S.Kh. Degtyarev 
Bulletin of biotechnology and physico-chemical biology named by Yu.A.Ovchinnikov (Moscow), V.9, No.3, p.15-23 (2013)

A novel method of MteI-PCR analysis of GC-rich DNA regions has been proposed based on the unique methyl-dependent site-specific DNA endonuclease MteI, which recognizes a long highly methylated site. The method includes DNA hydrolysis with MteI followed by real-time PCR or PCR with electrophoretic determination of the obtained products. Methylation status of regulatory regions of a number of tumor supressor genes has been determined by this method in comparison with the similar data obtained by previously proposed method of BlsI- and GlaI-PCR analysis. An applicability of MteI-PCR analysis has been shown by analysis of methylation status of CEBPD, HS3ST2, RASSF1A, SEPT9b and TWIST1 genes regulatory regions. In case of RASSF1A regulatory region, real-time MteI-PCR analysis in contrast to BlsI- and GlaI-PCR analysis, does not show methylation of this DNA region in a control healthy lung fibroblast cell line. Thus, MteI-PCR analysis allows to perform more distinct determination and discrimination of RASSF1A regulatory region methylation and, probably, some other GC-rich regions of human DNA as well.

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Real time GlaI-PCR assay of regulation regions of human genes HDAC4, RARB and URB1

This email address is being protected from spambots. You need JavaScript enabled to view it. , AG Akishev, MA Abdurashitov, SB Oleynikova, VL Sitko, and S Kh Degtyarev 

RJPBCS, vol 7(2), 2016, p.p. 667-676

Real-time GlaI-PCR assay is developed to determine DNA methylation status of the regulation regions of HDAC4, URB1 and RARB genes in DNA preparations from human leukocytes. Real-time GlaI-PCR assay is DNA hydrolysis with methyl-directed site-specific DNA endonuclese GlaI followed by real-time PCR from the primers located upstream and downstream of the studied DNA region. The obtained data confirm a full methylation of the studied ACGT and GCGC sites in the regulatory regions of the HDAC4 and URB1 genes and a complete hydrolysis of these sites with GlaI. A first exon of RARB gene is slightly methylated in the leukocytes DNA preparations and according to the results of GlaI-PCR assay we don’t observe GlaI hydrolysis of ACGCG site in RARB gene. The data obtained correspond to the literature data. The proposed method of real-time GlaI-PCR assay may be used to determine the methylation status of any unique parts in human and mammalian genomes.

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Application of GLAD-PCR assay for determination of the methylation sites in the regulatory regions of tumor-supressors gene ELMO1 and ESR1 in colorectal cancer

A.A. Evdokimov, This email address is being protected from spambots. You need JavaScript enabled to view it. , N.A. Smetannikova, M.A. Abdurashitov, A.G. Akishev,  E.S. Davidovich, Yu.D. Ermolaev, A.B. Karpov, A.E. Sazonov, R.M. Tahauov, S.Kh. Degtyarev
Translated from Problems in oncology, #1, 2016 p.116-120

Aberrant methylation of regulation regions of tumor-suppressor genes (TSGs) is shown for many cancer diseases. In course of this modification the enzyme DNMT3 methylates RCGY sites in CpG-islands of regulation regions producing R(5mC)GY sites. Earlier we developed GLAD-PCR assay to determine R(5mC)GY site in a definite position of human genome [3]. In this work we have applied GLAD-PCR assay to determine R(5mC)GY sites in regulation regions of ESR1 and ELMO1 TSGs. We have studied methylation of DNA fragment in first exon of ELMO1 gene and in ESR1 promoter region in DNA preparations from malignant cell line SW837 and colorectal tumor samples. We have determined methylation of four sites in each region and found two highly methylated sites: GCGC in first exon of ELMO1 gene and GCGT in promoter region of ESR1 gene. Site GCGT is weakly methylated in healthy tissues and more methylated in the most of colorectal samples. Site GCGC is not methylated in healthy tissues and significantly methylated in 60% of colorectal samples. A possibility to use GLAD-PCR assay for cancer diagnostics is discussed.

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Mapping of R(5mC)GY Sites in the Genome of Human Malignant Cell Line Raji

Abdurashitov M.A., Tomilov V.N., Gonchar D.A., Kuznetsov V.V., Degtyarev S.Kh. 
Biology and Medicine, Volume 7, Issue 4, BM-135-15 (2015)

The purpose of this study was to develop a method for the wide-scale determination of methylated RCGY sites in a genome using the next-generation sequencing (NGS) approach. We hydrolyzed genomic DNA from human, malignant cell line Raji with methyl-directed site-specific endonuclease GlaI and isolated DNA fragments 140-400 bp in length. DNA structure of 75 bp was determined for both ends of each DNA fragment based on NGS technology with Illumina MiSeq. The mapping of the obtained sequences on the human reference genome allowed to define the positions of more than one million R(5mC)GY sites represented in Raji genome with relatively high frequency. These data were compared with the results of the methylation study of regulatory regions of tumor suppressor genes studied earlier with a new version of PCR analysis. We have concluded that a suggested method of R(5mC)GY sites determination in a whole genome may be used for epigenetic studies, particularly for determination of methylation of regulatory regions of genes, which may affect the gene expression.

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