Human arsenic methyltransferase (AS3MT) pharmacogenetics: Gene resequencing and functional genomics studies

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Abstract

Arsenic contaminates ground water worldwide. Methylation is an important reaction in the biotransformation of arsenic. We set out to study the pharmacogenetics of human arsenic methyltransferase (AS3MT, previously CYT19). After cloning the human AS3MT cDNA, we annotated the human gene and resequenced its 5′-flanking region, exons, and splice junctions using 60 DNA samples from African-American (AA) and 60 samples from Caucasian-American (CA) subjects. We observed 26 single nucleotide polymorphisms (SNPs), including 3 nonsynonymous cSNPs, as well as a variable number of tandem repeats in exon 1 within an area encoding the cDNA 5′-untranslated region. The nonsynonymous cSNPs included T860C (M287T) with frequencies of 10.8 and 10% in AA and CA subjects, respectively, as well as C517T (A173W) in one AA and C917T (T306I) in one CA sample. Haplotype analysis showed that Ile306 was linked to Thr287, so this double variant allozyme was also studied functionally. After expression in COS-1 cells and correction for transfection efficiency, the Trp173 allozyme displayed 31%, Thr287 350%, Ile306 4.8%, and Thr287/Ile306 6.2% of the activity of the wild type (WT) allozyme, with 20, 190, 4.4, and 7.9% of the level of WT immunoreactive protein, respectively. Apparent Km values for S-adenosyl-L-methionine were 4.6, 3.1, and 11 μM for WT, Trp 173, and Thr287 allozymes, with Km values for sodium arsenite with the same allozymes of 11.8, 8.9, and 4.5 μM. The Ile306 and Thr287/Ile306 allozymes expressed too little activity for inclusion in the substrate kinetic studies. Expression of reporter gene constructs for the 5′-flanking region and the variable number of tandem repeats in the 5′-untranslated region demonstrated cell line-dependent variation in reporter gene expression, with shorter repeats associated with increased transcription in HepG2 cells. These results raise the possibility that inherited variation in AS3MT may contribute to variation in arsenic metabolism and, perhaps, arsenic-dependent carcinogenesis in humans.

Original languageEnglish (US)
Pages (from-to)7364-7373
Number of pages10
JournalJournal of Biological Chemistry
Volume281
Issue number11
DOIs
StatePublished - Mar 17 2006

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