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 non-synonymous 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 Ile(306) was linked to Thr(287), so this double variant allozyme was also studied functionally. After expression in COS-1 cells and correction for transfection efficiency, the Trp(173) allozyme displayed 31%, Thr(287) 350%, Ile(306) 4.8%, and Thr(287)/Ile(306) 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 mum for WT, Trp(173), and Thr(287) allozymes, with K(m) values for sodium arsenite with the same allozymes of 11.8, 8.9, and 4.5mum. The Ile(306) and Thr(287)/Ile(306) 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.
Wood
Thomas C
TC
Division of Clinical Pharmacology, Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic College of Medicine, 200 First Street SW, Rochester, MN 55905, USA.
Salavagionne
Oreste E
OE
Mukherjee
Baidehi
B
Wang
Liewei
L
Klumpp
Annette F
AF
Thomae
Bianca A
BA
Eckloff
Bruce W
BW
Schaid
Daniel J
DJ
Wieben
Eric D
ED
Weinshilboum
Richard M
RM
D020121:5' Untranslated Regions; D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001151:Arsenic / Q000737:chemistry; D018053:Arsenites / Q000494:pharmacology; D001483:Base Sequence; D015153:Blotting, Western; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D005091:Exons; D015870:Gene Expression; D017930:Genes, Reporter; D014644:Genetic Variation; D023281:Genomics; D006239:Haplotypes; D006801:Humans; D007532:Isoleucine / Q000737:chemistry; D007700:Kinetics; D015810:Linkage Disequilibrium; D008780:Methyltransferases / Q000235:genetics* / Q000502:physiology*; D008969:Molecular Sequence Data; D009154:Mutation; D016366:Open Reading Frames; D010597:Pharmacogenetics; D011110:Polymorphism, Genetic; D020641:Polymorphism, Single Nucleotide; D020033:Protein Isoforms; D017422:Sequence Analysis, DNA; D017670:Sodium Compounds / Q000494:pharmacology; D013912:Threonine / Q000737:chemistry; D014158:Transcription, Genetic; D014162:Transfection
D020121:5' Untranslated Regions; D018053:Arsenites; D017931:DNA Primers; D018076:DNA, Complementary; D020033:Protein Isoforms; D017670:Sodium Compounds; D007532:Isoleucine; D013912:Threonine; C017947:sodium arsenite; D008780:Methyltransferases; C505756:AS3MT protein, human; D001151:Arsenic