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

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 K_m values for S-adenosyl-L-methionine were 4.6, 3.1, and 11 μM for WT, Trp ¹⁷³, and Thr²⁸⁷ allozymes, with K_m 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.