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GLAD-PCR Assay of R(5mC)GY Sites in the Regulatory Region of Tumor-Suppressor Genes Associated with Gastric Cancer
B S Malyshev1, N A Netesova1, N A Smetannikova1
1State Research Center of Virology and Biotechnology «Vector», Novosibirsk region, Koltsovo, 630559 Russia.
Abstract:
At early stages of carcinogenesis, the regulatory regions of some tumor suppressor genes become aberrantly methylated at RCGY sites, which are substrates of DNA methyltransferase Dnmt3. Identification of aberrantly methylated sites in tumor DNA is considered to be the first step in the development of epigenetic PCR test systems for early diagnosis of cancer. Recently, we have developed a GLAD-PCR assay, a method for detecting the R(5mC)GY site in the genome position of interest even at significant excess of DNA molecules with a non-methylated RCGY site in this location. The aim of the present work is to use the GLAD-PCR assay to detect the aberrantly methylated R(5mC)GY sites in the regulatory regions of tumor suppressor genes (brinp1, bves, cacna2d3, cdh11, cpeb1, epha7, fgf2, galr1, gata4, hopx, hs3st2, irx1, lrrc3b, pcdh10, rprm, runx3, sfrp2, sox17, tcf21, tfpi2, wnt5a, zfp82, and znf331) in DNA samples obtained from gastric cancer (GC) tissues. The study of the DNA samples derived from 29 tumor and 25 normal gastric tissue samples demonstrated a high diagnostic potential of the selected RCGY sites in the regulatory regions of the irx1, cacna2d3, and epha7 genes; the total indices of sensitivity and specificity for GC detection being 96.6% and 100%, respectively.
Insights
Aberrantly methylated RCGY sites in tumor suppressor genes can indicate early cancer. A new GLAD-PCR assay effectively detects these epigenetic markers in gastric cancer tissues, showing high diagnostic accuracy.
Area of Science:
- Molecular Biology
- Epigenetics
- Oncology
Background:
- Aberrant methylation of RCGY sites in tumor suppressor genes occurs during early carcinogenesis.
- DNA methyltransferase Dnmt3 targets these RCGY sites.
- Identifying methylated sites is crucial for developing epigenetic PCR tests for early cancer diagnosis.
Purpose of the Study:
- To utilize the developed GLAD-PCR assay for detecting aberrant methylation at RCGY sites.
- To investigate these methylated sites within the regulatory regions of specific tumor suppressor genes in gastric cancer tissues.
Main Methods:
- Development of the GLAD-PCR assay for detecting methylated R(5mC)GY sites.
- Application of GLAD-PCR to analyze DNA from 29 gastric cancer and 25 normal gastric tissue samples.
- Focus on RCGY sites in 23 tumor suppressor genes including irx1, cacna2d3, and epha7.
Main Results:
- The GLAD-PCR assay demonstrated high diagnostic potential for specific RCGY sites.
- The irx1, cacna2d3, and epha7 genes showed significant methylation patterns in gastric cancer.
- Achieved a total sensitivity of 96.6% and specificity of 100% for gastric cancer detection.
Conclusions:
- The GLAD-PCR assay is effective in detecting aberrantly methylated RCGY sites in gastric cancer.
- Selected RCGY sites in irx1, cacna2d3, and epha7 are promising biomarkers for early gastric cancer diagnosis.
- This approach holds significant potential for developing novel epigenetic PCR-based diagnostic tools.
