Title: Human Arsenic Methyltransferase (AS3MT) Pharmacogenetics
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 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 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, Trp173, and Thr287 allozymes, with Km values for sodium arsenite with the same allozymes of 11.8, 8.9, and 4.5μm. 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. 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 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, Trp173, and Thr287 allozymes, with Km values for sodium arsenite with the same allozymes of 11.8, 8.9, and 4.5μm. 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. Acute arsenic exposure can result in cardiac failure, peripheral neuropathy, leukopenia, and death, whereas chronic exposure can lead to neurotoxicity, hepatic injury, and carcinogenesis (1Simeonova P.P. Luster M.I. Toxicol. Appl. Pharmacol. 2004; 198: 444-449Crossref PubMed Scopus (118) Google Scholar, 2Yoshida T. Yamauchi H. Fan Sun G. Toxicol. Appl. Pharmacol. 2004; 198: 243-252Crossref PubMed Scopus (465) Google Scholar, 3Chen C.J. Hsu L.I. Wang C.H. Shih W.L. Hsu Y.H. Tseng M.P. Lin Y.C. Chou W.L. Chen C.Y. Lee C.Y. 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Finally, As2O3 is used as a therapeutic agent, particularly for the treatment of promyelocytic leukemia (8Sanz M.A. Fenaux P. Lo Coco F. European APL Group of Experts Haematologica. 2005; 90: 1231-1235PubMed Google Scholar, 9Sirulnik L.A. Stone R.M. Clin. Adv. Hematol. Oncol. 2005; 3 (429): 391-397PubMed Google Scholar). Methylated and dimethylated arsenic are the major urinary arsenic metabolites in humans (10Smith T.J. Crecelius E.A. Reading J.C. Environ. Health Perspect. 1977; 19: 89-93Crossref PubMed Scopus (105) Google Scholar, 11Crecelius E.A. Environ. Health Perspect. 1977; 19: 147-150Crossref PubMed Scopus (291) Google Scholar). Although methylation has been regarded as a detoxification reaction, methylated AsIII is more cytotoxic and genotoxic than are arsenate (the most stable form of arsenic) and arsenite (12Petrick J.S. Ayala-Fierro F. Cullen W.R. Carter D.E. Vasken Aposhian H. Toxicol. Appl. Pharmacol. 2000; 163: 203-207Crossref PubMed Scopus (595) Google Scholar, 13Styblo M. Del Razo L.M. Vega L. Germolec D.R. LeCluyse E.L. Hamilton G.A. Reed W. Wang C. Cullen W.R. Thomas D.J. Arch. Toxicol. 2000; 74: 289-299Crossref PubMed Scopus (843) Google Scholar, 14Mass M.J. Tennant A. Roop B.C. Cullen W.R. Styblo M. Thomas D.J. Kligerman A.D. Chem. Res. Toxicol. 2001; 14: 355-361Crossref PubMed Scopus (459) Google Scholar). Methylated derivatives are also more potent inhibitors of glutathione reductase (15Styblo M. Serves S.V. Cullen W.R. Thomas D.J. Chem. Res. Toxicol. 1997; 10: 27-33Crossref PubMed Scopus (251) Google Scholar, 16Chouchane S. Snow E.T. Chem. Res. Toxicol. 2001; 14: 517-522Crossref PubMed Scopus (103) Google Scholar), thioredoxin reductase (17Lin S. Cullen W.R. Thomas D.J. Chem. Res. Toxicol. 1999; 12: 924-930Crossref PubMed Scopus (201) Google Scholar, 18Lin S. Del Razo L.M. Styblo M. Wang C. Cullen W.R. Thomas D.J. Chem. Res. Toxicol. 2001; 14: 305-311Crossref PubMed Scopus (144) Google Scholar) and pyruvate dehydrogenase (19Petrick J.S. Jagadish B. Mash E.A. Aposhian H.V. Chem. Res. Toxicol. 2001; 14: 651-656Crossref PubMed Scopus (298) Google Scholar) than is arsenite. Therefore, knowledge of individual variation in the function of the enzyme that catalyzes the formation of methylated arsenicals might be an important step toward increasing our understanding of the biological and pathological consequences of chronic exposure to inorganic arsenic. An arsenic methyltransferase activity was recently described in the rat that catalyzes the methylation of arsenite, with S-adenosyl-l-methionine (AdoMet) 2The abbreviations used are: AdoMet, S-adenosyl-l-methionine; SNP, single nucleotide polymorphisms; VNTR, variable number of tandem repeats; UTR, untranslated region; CA, Caucasian-American; AA, African-American; WT, wild type; ORF, open reading frame; RRL, rabbit reticulocyte lysate; TPMT*3A, thiopurine S-methyltransferase *3A. as the methyl donor (20Lin S. Shi Q. Nix F.B. Styblo M. Beck M.A. Herbin-Davis K.M. Hall L.L. Simeonsson J.B. Thomas D.J. J. Biol. Chem. 2002; 277: 10795-10803Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar). In rats, that activity is expressed in a variety of organs, and a similar activity is expressed in the human liver, kidney, and brain (21Zakharyan R.A. Ayala-Fierro F. Cullen W.R. Carter D.M. Aposhian H.V. Toxicol. Appl. Pharmacol. 1999; 158: 9-15Crossref PubMed Scopus (102) Google Scholar). The cloning of a rat cDNA, encoded by a gene that was originally annotated as "Cyt19," and the subsequent demonstration that the encoded protein was an "arsenic methyltransferase" (20Lin S. Shi Q. Nix F.B. Styblo M. Beck M.A. Herbin-Davis K.M. Hall L.L. Simeonsson J.B. Thomas D.J. J. Biol. Chem. 2002; 277: 10795-10803Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar), opened the way for studies of the possible contribution of variation in this gene (subsequently annotated as As3mt) to individual differences in arsenic methylation. In the present study, we set out to characterize the human ortholog for the rat Cyt19-As3mt gene to make it possible to resequence the human gene using DNA from subjects of two different ethnic groups. During gene resequencing, we observed a series of genetic polymorphisms and haplotypes in the human AS3MT gene. Functional genomic studies of all variant allozymes encoded by AS3MT alleles that contained nonsynonymous cSNPs, as well as a VNTR located in the 5′-UTR of the cDNA, were then performed. These experiments represent a step toward studies of individual variation in arsenic methylation, arsenic pharmacogenetics, and arsenic toxicogenetics in humans. DNA Samples—DNA samples from 60 CA and 60 AA subjects, as well as a panel of 10 primate DNA samples (sample sets HD100CAU, HD100AA, and PRP00001), were obtained from the Coriell Cell Repository (Camden, NJ). The human DNA samples had been collected and anonymized by the NIGMS, National Institutes of Health. All subjects had provided written informed consent for the use of their DNA for research purposes, and the present studies were reviewed and approved by the Mayo Clinic Institutional Review Board. cDNA Cloning—The human AS3MT cDNA was cloned by PCR amplification based on an assumption of homology between the sequences of the rat As3mt cDNA and its human ortholog, using human liver cDNA as template. 5′- and 3′-rapid amplification of cDNA ends were performed with Marathon-Ready human liver and prostate cDNA (Clontech, Palo Alto, CA) using nested PCR amplifications with AS3MT-specific primers paired with the AP1 and AP2 primers supplied by the manufacturer. The sequences of primers used to perform these and subsequent amplifications are listed in supplementary materials Table 1. Gene Resequencing—Annotation of the human AS3MT gene (originally designated CYT19, GenBank® accession number NT_008804.8) was performed using the human cDNA sequence obtained by performing cDNA cloning. The PCR was then used to amplify each of the 11 AS3MT exons. We also attempted to amplify one exon using the primate DNA samples obtained from the Coriell Cell Repository as template. Primers were designed to amplify each of the human gene exons, plus portions of the introns flanking that exon. The primers used for the gene resequencing studies included M13 tags at their 5′-ends to make it possible to use dye primer sequencing chemistry (see supplementary materials Table 1). Dye primer sequencing was used to enhance our ability to identify heterozygous bases (22Chadwick R.B. Conrad M.P. McGinnis M.D. Johnston-Dow L. Spurgeon S.L. Kronick M.N. BioTechniques. 1996; 20: 676-683PubMed Google Scholar). Amplicons from these reactions were sequenced in the Mayo Molecular Biology Sequencing Core Facility with an ABI 3700 DNA sequencer (Applied Biosystems, Foster City, CA) using Big Dye® (PerkinElmer Life Sciences) dye primer chemistry. All samples were sequenced on both strands, and those with ambiguous chromatograms, as well as samples that contained SNPs that were observed in only a single sample, were subjected to a second, independent round of amplification, followed by DNA sequencing. Samples resulting from amplifications of the initial exon that contained the VNTR were also subjected to agarose gel electrophoresis to determine VNTR length. Samples homozygous for the VNTR were sequenced directly, and selected heterozygous samples were subcloned into pCR2.1, followed by sequencing of each of the alleles present in that sample. Transient Expression—The "wild type" (WT) open reading frame (ORF) of the human AS3MT cDNA was amplified, and site-directed mutagenesis was performed to create a synonymous cSNP that removed an EcoRI site. That ORF sequence was then cloned into pCR2.1 (Promega). The insert was excised with EcoRI and was cloned into the eukaryotic expression vector p91023(B), and "circular PCR" was used to perform site-directed mutagenesis to create constructs encoding variant allozymes (see supplementary materials Table 1 for sequences of these primers). Insert sequences after circular PCR were verified by sequencing the entire ORF. These expression constructs were then used to transfect COS-1 cells in serum-free Dulbeccos's modified Eagle's medium (BioWhittaker, Walkersville, MD) using the TransFast reagent (Promega) at a charge ratio of 1:1, with a 1-h transfection time. "Empty" p91023(B), vector that did not contain insert, was used as a control to make it possible to correct for possible endogenous AS3MT in COS-1 cells. However, endogenous activity was found to be negligible, averaging less than 1% of the activity present after transfection with the WT construct. During transfection, 15 μg of AS3MT construct DNA was cotransfected with 1 μg of pSV-β-galactosidase DNA (Promega) to make it possible to correct for transfection efficiency. Six separate plates were transfected with each construct studied. The transfected COS-1 cells were incubated at 37 °C for 48 h, washed with phosphate-buffered saline, and resuspended in 2 ml of homogenization buffer. They were then lysed with a Polytron homogenizer (Brinkmann Instruments), and the homogenates were centrifuged at 100,000 × g for 1 h at 4 °C. The resulting supernatant cytosol preparations were stored at -80 °C prior to assay. AS3MT Enzyme Assay—Recombinant allozymes were assayed for AS3MT activity using a modification of the method described by Zakharyan et al. (23Zakharyan R. Wu Y. Bogdan G.M. Aposhian H.V. Chem. Res. Toxicol. 1995; 8: 1029-1038Crossref PubMed Scopus (135) Google Scholar). Briefly, 0.10 m Tris-HCl buffer, pH 8.0, 4 mm glutathione, 1 mm MgCl2, 12.5 mm sodium arsenite (the methyl acceptor substrate), [methyl-3H]AdoMet, the methyl donor, (11.8 Ci/mmol, 0.55 mCi/ml, 10 μm final concentration), and recombinant enzyme were combined in a final volume of 250 μl. Blanks were samples that contained no methyl acceptor substrate. Reaction mixtures were incubated at 37 °C for 60 min, and the reaction was terminated by the addition of 750 μlof12 m HCl. Methylated arsenic compounds were isolated using the organic solvent extraction procedure described by Zakharyan et al. (23Zakharyan R. Wu Y. Bogdan G.M. Aposhian H.V. Chem. Res. Toxicol. 1995; 8: 1029-1038Crossref PubMed Scopus (135) Google Scholar). Enzyme activity was corrected for transfection efficiency by measuring β-galactosidase activity spectrophotometrically with the β-Galactosidase Enzyme Assay system (Promega). The same AS3MT activity assay was used to determine apparent Km values for the two cosubstrates for the reaction. Specifically, five concentrations of sodium arsenite that varied from 2.5 to 100 μm were tested in the presence of 10 μm [methyl-3H]AdoMet. Activity was also measured in the presence of 12.5 μm sodium arsenite with five concentrations of AdoMet that varied from 1.25 to 20 μm. Quantitative Western Blot Analyses—Levels of immunoreactive AS3MT protein were determined for each recombinant allozyme by performing quantitative Western blot analysis. A rabbit polyclonal anti-body directed against AS3MT amino acids 341-360 was used to perform these studies. This peptide, linked to keyhole limpit hemocyanin, was used to generate a rabbit polyclonal antibody (Cocalico, Reamstown, PA). The antibody had been tested for specificity by performing Western blot analyses with recombinant AS3MT as well as human liver, kidney, and prostate cytosol preparations. During the Western blot analyses, COS-1 cell cytosol was loaded on 12% SDS mini-gels (Bio-Rad) in quantities that resulted in equal β-galactosidase activity to correct for possible variation in transfection efficiency. Electrophoresis was then performed for 1 h at 150V,andthe proteins were transferred to nitro-cellulose membranes. The membranes were blocked overnight with 5% milk in Tris-buffered saline with Tween 20 (TBST). The following day, the membranes were incubated for 0.5 h with primary antibody diluted 1:2000 with 5% milk in TBST, followed by three washes. The secondary antibody was a 1:20,000 dilution of goat anti-rabbit horseradish peroxidase (Bio-Rad) that was applied for 1 h in 5% milk in TBST, followed by three washes. Bound antibody was detected by enhanced chemiluminescence performed with the ECL Western blotting system (Amersham Biosciences). The Western blot data were analyzed with the AMBIS radioanalytic Imaging System, Quant Probe version 4.31 (AMBIS, San Diego, CA). Multiple blots were performed for each allozyme, and immunoreactive protein levels were expressed as a percentage of the intensity of the band for the WT construct on the same gel. Rabbit Reticulocyte Lysate (RRL) Protein Synthesis and Degradation—Transcription and translation of WT AS3MT and allozymes encoded by the variant alleles (Trp173, Thr287, and Ile306) were performed using the TnT® coupled RRL System (Promega) with constructs that had been cloned into pCR3.1 (Promega). Specifically, 25 μl of "treated" RRL, plus 2 μl of T7 buffer, 1 μl of T7 polymerase, 1 μl of RNasin, and 2 μl of [35S]methionine (1000 Ci/mmol, 10 mCi/ml, 0.4 μm final concentration) were used to perform these experiments. With the exception of the RNasin (Promega) and the [35S]methionine (Amersham Biosciences), all reagents were included in the Promega kit. One μg of the pCR3.1 expression construct DNA was added to the mixture, the reaction volume was increased to 50 μl with nuclease-free water (Promega), and the mixture was incubated at 30 °C for 90 min. A 5-μl aliquot was then used to perform electrophoresis with a 12% SDS-PAGE gel that was dried and exposed to x-ray film (Kodak). To perform the protein degradation experiments, 60 μl of an ATP generating system, 60 μl of "untreated" RRL and 12 μl of radioactively labeled protein were combined. The ATP generating system consisted of 100 μl each of 1 m Tris-HCl, pH 7.8, 160 mm MgCl2, 120 mm KCl, 100 mm dithiothreitol, 100 mm ATP, 200 mm creatine phosphate, and 2 mg/ml ATP kinase (all from Sigma), plus 300 μl of nuclease-free water. This mixture was incubated at 37 or 40 °C. Aliquots were removed at various times and were subjected to electrophoresis on a 12% SDS-PAGE gel. A variant allozyme for a genetically polymorphic drug metabolizing enzyme that has been shown to be degraded rapidly by a proteasome-dependent process, thiopurine S-methyltransferase (TPMT)*3A (24Wang L. Sullivan W. Toft D. Weinshilboum R. Pharmacogenetics. 2003; 13: 555-564Crossref PubMed Scopus (80) Google Scholar), was used as a positive control for these experiments. The gels were dried and exposed to x-ray film, and levels of [35S]methionine radioactively labeled proteins were determined by using the AMBIS system. Reporter Gene Constructs—Firefly luciferase reporter gene constructs in pGL3-Basic were created for the four different length AS3MT 5′-flanking region and 5′-UTR VNTR sequences. Inserts in these constructs were then sequenced, and the constructs were used to transfect HepG2 and HEK293 cells using the TransFast reagent at a charge ratio of 1:1, with a 1-h transfection time. During transfection, 2 μg of construct DNA was cotransfected with 0.2 μg of pRL-TK DNA to make it possible to correct for transfection efficiency. Transfected cells were incubated at 37 °C for 48 h, washed with phosphate-buffered saline, and lysed in 1 ml of lysis buffer. Cell lysates were then used to measure firefly luciferase activity with the Dual Luciferase Reporter Assay system (Promega). Data Analysis—The DNA sequence from the resequencing studies was analyzed using the PolyPhred 4.0 (25Nickerson D.A. Tobe V.O. Taylor S.L. Nucleic Acids Res. 1997; 25: 2745-2751Crossref PubMed Scopus (823) Google Scholar) and Consed 8.0 (26Gordon D. Abajian C. Green P. Genome Res. 1998; 8: 195-202Crossref PubMed Scopus (2849) Google Scholar) programs. The Wisconsin Genetics Computer Group (GCG) package, version 10, was also used to analyze nucleotide sequence. Human SNP data bases, including dbSNP and the HapMap, build 18, were searched to determine whether the polymorphisms that we observed had been reported previously. The RepeatMasker (University of Washington) program was used to screen for repeat sequences. Values for π, θ, and D values were calculated as described by Tajima (27Tajima F. Genetics. 1989; 123: 585-595Crossref PubMed Google Scholar). Linkage disequilibrium was analyzed by calculating D′ values for all polymorphism pairs as described by Hartl and Clark (28Hartl D.L. Clark A.G. Principles of Population Genetics. Third. Sinauer Associates, Inc., Sunderland, MA2000: 95-107Google Scholar) and Hendrick (29Hendrick P.W. Genetics of Populations. 2nd. Jones and Bartlett, Sudbury, MA2000: 396-405Google Scholar), and those data were displayed graphically. Haplotype analysis was performed as described by Schaid et al. (30Schaid D.J. Rowland C.M. Tines D.E. Jacobson R.M. Poland G.A. Am. J. Hum. Genet. 2002; 70: 425-434Abstract Full Text Full Text PDF PubMed Scopus (1566) Google Scholar). Apparent Km values were calculated with the method of Wilkinson (31Wilkinson G.N. Biochem. J. 1961; 80: 324-332Crossref PubMed Scopus (2724) Google Scholar) using a computer program developed by Cleland (32Cleland W.W. Nature. 1963; 198: 463-465Crossref PubMed Scopus (392) Google Scholar). Differences between mean values were evaluated by use of the Student's t test calculated with the Statview 4.5 program (Abacus Concepts, Berkeley, CA). Human AS3MT Resequencing—The human AS3MT cDNA sequence, as well as that of the encoded protein, are shown in Fig. 1. The cDNA had a 1125-bp ORF that encoded a 375-amino acid protein. The ORF nucleotide and encoded amino acid sequences were 81 and 76% identical with those of rat As3mt, respectively. The human AS3MT gene mapped to chromosome 10q24, contained 11 exons, and was ∼32 kb in length (Fig. 2). The location of the human gene was syntenic with the rat ortholog that maps to rat chromosome 1q54, and with the location of the mouse ortholog on chromosome 19D1. It should be pointed out that the sequences of the cDNA and, as a result, the gene that we have shown in Figs. 1 and 2 differ from the current NCBI annotation for the cDNA (NM_020682.2), an annotation that is based on the application of exon prediction software to an early version of the human chromosome 10 sequence. Specifically, ORF nucleotide 1057 in our sequence is missing from the NCBI cDNA annotation, resulting in a frameshift and truncation of the protein within exon 10, with exon 11 converted entirely to 3′-UTR. Furthermore, there are 4 variant nucleotides in exon 5 of the present NCBI cDNA annotation, one synonymous and three resulting in the following alterations in encoded amino acids: I132F, Y135N, and G140A (33Li J. Waters S.B. Drobna Z. Devesa V. Styblo M. Thomas D.J. Toxicol. Appl. Pharmacol. 2005; 204: 164-169Crossref PubMed Scopus (52) Google Scholar). Those nucleotides were not polymorphic in any of our resequencing studies of 240 alleles, and the deletion at nucleotide 1057 was also not present in any of our 120 DNA samples. As a result, we concluded that the current NCBI annotation, which is labeled "provisional," is almost certainly in error at those locations. Of note is the fact that the current NCBI genomic sequence for AS3MT matches our WT sequence and that the encoded protein in SwissProt is that which we have shown in Fig. 1.FIGURE 2Human AS3MT polymorphisms. Shaded rectangles represent coding exons, and open rectangles represent portions of exons that encode the UTR sequence. Exon and introns lengths are also indicated. Arrows show the locations of polymorphisms, with polymorphism frequencies indicated by the colors of the arrows. Amino acids altered as a result of nonsynonymous cSNPs are also indicated. "VNTR" refers to a variable number of tandem repeats located in the 5′-UTR (see Fig. 3).View Large Image Figure ViewerDownload Hi-res image Download (PPT) AS3MT was resequenced using DNA samples from 60 AA and 60 CA subjects. Eleven PCR amplifications were performed for each sample and, as a result, a total of over 1,300,000 bp of DNA sequence was analyzed on both strands. Those experiments resulted in the identification of 26 SNPs, 22 in DNA samples from AA and 21 in DNA from CA subjects. The locations of the polymorphisms that we observed are listed in Table 1 and are depicted graphically in Fig. 2. All polymorphisms were in Hardy-Weinberg equilibrium. Three of the SNPs were nonsynonymous and altered encoded amino acids in the following codons: R173W, M287T, and T306I. The frequency of the polymorphism resulting in the M287T alteration in sequence was 10% or greater in both populations studied. This polymorphism was located in exon 9 of human AS3MT. Therefore, exon 9 was also amplified in the 10 primate DNA samples that we had obtained (rhesus monkey, pigtailed macaque, bonobo, gorilla, chimpanzee, Sumatian orangutan, red-chested mustached tamarin, black-handed spider monkey, common woolly monkey, and ring-tailed lemur). This codon encoded Thr in all 10 of these primate DNA samples, Ile in the rat, mouse, and dog orthologs, and Glu in the chicken gene. Twenty-one of the 26 human AS3MT SNPs had frequencies of greater than 1% in at least one of the two ethnic groups and, as a result, would be considered "common" in these populations. An additional polymorphism involved a VNTR in exon 1 within an area encoding the 5′-UTR. This VNTR consisted of repeat elements that were either 35 or 36 bp in length, differing only in the loss of the 3′-terminal "a," with 2 to 4 repeats in each allele (Fig. 3). The repeat consisted of 1 to 3 copies of the 36-bp "A" sequence, plus one copy of the 35-bp "B" sequence, with the B repeat at the 3′-terminus of the VNTR (Fig. 3). Four repeat elements (three A and one B sequence) were observed only in AA subjects. Allele and genotype frequencies for the VNTR in both populations are listed in Table 2. When public data bases, including dbSNP, were searched, 7 of the AS3MT polymorphisms that we observed were already in data bases. The HapMap, build 18, included only 3 of the polymorphisms that we observed. Our AS3MT resequencing data have been deposited in the NIH data base PharmGKB, with accession number PA128747780.TABLE 1Human AS3MT polymorphisms Locations and frequencies of the 26 SNPs and one VNTR observed in the human AS3MT gene are listed. Nucleotide locations, except those in introns, have been numbered on the basis of the A in the translation initiation codon, with that nucleotide designated (+1), and with positive numbers 3′ and negative numbers 5′ to that location. Intron (IVS) nucleotides have been numbered relative to splice junctions, with the initial 5′ nucleotide in an intron designated (+1) and the final 3′ nucleotide designated (-1). Polymorphisms in exons have been "boxed." The numbers at the right are those used to designate polymorphisms for the linkage disequilibrium analysis. Asterisk, polymorphism present in dbSNP.FIGURE 3Human AS3MT VNTR. The area of AS3MT amplified for the VNTR luciferase reporter gene constructs is indicated. The bold sequence is that of AS3MT exon 1. The bracketed sequences are VNTR repeat sequences A and B.View Large Image Figure ViewerDownload Hi-res image Download (PPT)TABLE 2Human AS3MT VNTR *V2 has the AB structure, *V3 the A2B structure, and *V4 the A3B VNTR structure (see Fig. 3). Observed allele and genotype frequencies for the 2 ethnic groups studied are listed. CA, Caucasian-American. AA, African-American.TABLE 2Human AS3MT VNTR *V2 has the AB structure, *V3 the A2B structure, and *V4 the A3B VNTR structure (see Fig. 3). Observed allele and genotype frequencies for the 2 ethnic groups studied are listed. CA, Caucasian-American. AA, African-American. We calculated "nucleotide diversity," a quantitative measure of genetic variation, adjusted for the number of alleles studied, for these data. Two commonly used measures of nucleotide diversity are π, average heterozygosity per site, and θ, a population mutation measure that is theoretically equal to the neutral mutation parameter (34Fullerton S.M. Clark A.G. Weiss K.M. Nickerson D.A. Taylor S.L. Stengard J.H. Sa