Methyl conjugation is an important pathway in the metabolism of many drugs, xenobiotic compounds, and neurotransmitters. Over the past two decades, the applicant's laboratory has systematically explored the pharmacogenetics of enzymes that catalyze methyl conjugation. We have described and characterized, both biochemically and clinically, a series of genetic polymorphisms for methyltransferase enzymes in humans. Several of those polymorphisms result in significant individual variation in drug metabolism, toxicity and effect. The present proposal is focussed on studies of the molecular basis in humans for the genetic regulation of two of these enzymes, thiopurine methyltransferase (TPMT) and histamine N- methyltransferase (HNMT). The TPMT genetic polymorphism represents a striking example of the clinical importance of pharmacogenetics, since it is a major factor responsible for individual differences in the toxicity and therapeutic efficacy of drugs such as 6-mercaptopurine and azathioprine. We now propose to extend our studies of the pharmacogenetics of TPMT beyond our recent cloning of a cDNA and a processed pseudogene for this enzyme to include cloning of the gene or genes for TPMT in humans. We will then use a series of techniques in an attempt to determine the molecular basis for this clinically important genetic polymorphism. Another goal of these experiments is the development of DNA-based diagnostic tests that might ultimately be used to predict thiopurine toxicity. We have also demonstrated that the level of activity of the neurotransmitter and autacoid metabolizing enzyme HNMT in humans is controlled by a genetic polymorphism, and we have recently cloned a cDNA for human kidney HNMT. Our studies of HNMT pharmacogenetics will now be expanded to include cloning of the gene or genes for HNMT in humans as a step toward determination of the molecular basis for this genetic polymorphism. It should then be possible to develop DNA-based tests for use in non-invasive studies of a possible role of inherited variation in HNMT activity in the pathophysiology of human disease. The results of the proposed experiments will increase our understanding of molecular mechanisms responsible for pharmacogenetic variation in the S- and N- methylation of drugs, xenobiotic compounds and neurotransmitters. They will also help make it possible to predict individual differences in the biotransformation and effects of drugs and endogenous compounds metabolized by methylation catalyzed by TPMT and HNMT.
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