Catechol O-methyltransferase (COMT) plays an important role in the metabolism of catecholamines, catecholestrogens and catechol drugs. A common COMT G472A genetic polymorphism (Val108/158Met) that was identified previously is associated with decreased levels of enzyme activity and has been implicated as a possible risk factor for neuropsychiatric disease. We set out to 'resequence' the human COMT gene using DNA samples from 60 African-American and 60 Caucasian-American subjects. A total of 23 single nucleotide polymorphisms (SNPs), including a novel nonsynonymous cSNP present only in DNA from African-American subjects, and one insertion/deletion were observed. The wild type (WT) and two variant allozymes, Thr52 and Met108, were transiently expressed in COS-1 and HEK293 cells. There was no significant change in level of COMT activity for the Thr52 variant allozyme, but there was a 40% decrease in the level of activity in cells transfected with the Met108 construct. Apparent K m values of the WT and variant allozymes for the two reaction cosubstrates differed slightly, but significantly, for 3,4-dihydroxybenzoic acid but not for S-adenosyl-L-methionine. The Met108 allozyme displayed a 70-90% decrease in immunoreactive protein when compared with WT, but there was no significant change in the level of immunoreactive protein for Thr52. A significant decrease in the level of immunoreactive protein was also observed in hepatic biopsy samples from patients homozygous for the allele encoding Met108. These observations represent steps toward an understanding of molecular genetic mechanisms responsible for variation in COMT level and/or properties, variation that may contribute to the pathophysiology of neuropsychiatric disease.

1 Estrogens are used as drugs and estrogen exposure is a risk factor for hormone-dependent diseases such as breast cancer. Sulfate conjugation is an important pathway for estrogen metabolism. The sulfotransferase (SULT) enzyme SULT1E1 has the lowest K m values for estrogens and catecholestrogens of the 10 known human SULT isoforms. 2 We previously cloned and characterized the human SULT1E1 cDNA and gene as steps toward pharmacogenetic studies. In the present experiments, we set out to determine whether common, functionally significant genetic polymorphisms might exist for SULT1E1. As a first step, we 'resequenced' the eight SULT1E1 exons and exon -intron splice junctions as well as portions of the 5 0 -flanking region using DNA from 60 African-American and 60 Caucasian-American subjects. 3 In all, 23 polymorphisms, 22 single nucleotide polymorphisms (SNPs) and one insertion deletion were observed. There were three nonsynonymous coding SNPs (cSNPs) that altered the following encoded amino acids: Asp22Tyr, Ala32Val and Pro253His. Among these, 12 pairs of SNPs were tightly linked. In addition, 12 unambiguous SULT1E1 haplotypes were identified, including six that were common to both populations studied. 4 Transient expression in COS-1 cells of constructs containing the three nonsynonymous cSNPs showed significant decreases in SULT1E1 activity for the Tyr22 and Val32 allozymes, with corresponding decreases in levels of immunoreactive protein. There were no changes in levels of either activity or immunoreactive protein for the His253 allozyme. Apparent K m values of the Val32 allozyme for the two cosubstrates for the reaction, 17b-estradiol and 3 0 -phosphoadenosine 5 0 -phosphosulfate, were not significantly different from those of the wild-type enzyme, but there was a two-to three-fold increase in K m values for the His253 allozyme and a greater than five-fold increase for the Tyr22 allozyme. 5 These observations raise the possibility that genetically determined variation in SULT1E1-catalyzed estrogen sulfation might contribute to the pathophysiology of estrogen-dependent diseases as well as variation in the biotransformation of exogenously administered estrogens.

SULT2A1 catalyzes the sulfate conjugation of dehydroepiandrosterone (DHEA) as well as other steroids. As a step toward pharmacogenetic studies, we have 'resequenced' SULT2A1 using 60 DNA samples from African-American and 60 samples from Caucasian-American subjects. All exons, splice junctions and approximately 370 bp located 5' of the site of transcription initiation were sequenced. We observed 15 single nucleotide polymorphisms (SNPs), including three non-synonymous coding SNPs (cSNPs) that were present only in DNA from African-American subjects. Linkage analysis revealed that two of the nonsynonymous cSNPs were tightly linked. Expression constructs were created for all nonsynonymous cSNPs observed, including a 'double variant' construct that included the two linked cSNPs, and those constructs were expressed in COS-1 cells. SULT2A1 activity was significantly decreased for three of the four variant allozymes. Western blot analysis demonstrated that decreased levels of immunoreactive protein appeared to be the major mechanism responsible for decreases in activity, although apparent Km values also varied among the recombinant allozymes. In addition, the most common of the nonsynonymous cSNPs disrupted the portion of SULT2A1 involved with dimerization, and this variant allozyme behaved as a monomer rather than a dimer during gel filtration chromatography. These observations indicate that common genetic polymorphisms for SULT2A1 can result in reductions in levels of both activity and enzyme protein. They also raise the possibility of ethnic-specific pharmacogenetic variation in SULT2A1-catalyzed sulfation of both endogenous and exogenous substrates for this phase II drug-metabolizing enzyme.

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

Sulfotransferase (SULT) 1A3 catalyzes the sulfate conjugation of catecholamines and structurally related drugs. As a step toward studies of the possible contribution of inherited variation in SULT1A3 to the pathophysiology of human disease and/or variation in response to drugs related to catecholamines, we have resequenced all seven coding exons, three upstream non-coding exons, exon-intron splice junctions and the 5¢-flanking region of SULT1A3 using DNA samples from 60 African-American (AA) and 60 CaucasianAmerican (CA) subjects. Eight single nucleotide polymorphisms (SNPs) were observed in AA and five in CA subjects, including one non-synonymous cSNP (Lys234Asn) that was observed only in AA subjects with an allele frequency of 4.2%. This change in amino acid sequence resulted in only 28 ± 4.5% (mean ± SEM) of the enzyme activity of the wildtype (WT) sequence after transient expression in COS-1 cells, with a parallel decrease (54 ± 2.2% of WT) in level of SULT1A3 immunoreactive protein. Substrate kinetic studies failed to show significant differences in apparent K m values of the two allozymes for either dopamine (10.5 versus 10.2 lM for WT and variant, respectively) or the cosubstrate 3¢-phosphoadenosine 5¢-phosphosulfate (0.114 versus 0.122 lM, respectively). The decrease in level of immunoreactive protein in response to this single change in amino acid sequence was due, at least in part, to accelerated SULT1A3 degradation through a proteasome-mediated process. These observations raise the possibility of ethnic-specific inherited alterations in catecholamine sulfation in humans.