Basic evaluation of diagnosis of nucleic acid metabolizing enzyme deficiency by flow cytometry

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Methylthioadeosine phosphorylase (MTAP) is frequently deficient in a variety of malignancies, although the enzyme is active in normal cells or tissues. This difference in enzyme activities can be exploited to design the tumor-specific chemotherapy.We tried to develop the diagnostic method of MTAP enzyme deficiency which can be easily applied to clinical samples compared to genetic diagnosis. Anti-MTAP monoclonal antibody which we generated worked well with certain cell lines, whereas it did not with other cell lines. This might be caused by the difference in the amount of MTAP protein expressed.Since MTAP deficiency is known to be caused by the gene deletion or promoter hypermethylation, we developed the rapid high-pressure liquid chromatography (HPLC) to detect methylated DNA. PCR products were amplified from the methyl sulfite-modified DNA and PCR products with single nucleotide substituions were separated by HPLC.