Human hydroxysteroid sulfotransferase SULT2B1 pharmacogenomics: gene sequence variation and functional genomics.

The human hydroxysteroid sulfotransferase (SULT) 2B1 gene is a member of the cytosolic SULT gene superfamily. The two SULT2B1 isoforms, SULT2B1a and SULT2B1b, are encoded by a single gene as a result of alternative transcription initiation and alternative splicing. SULT2B1b catalyzes the sulfonation of 3beta-hydroxysteroid hormones and cholesterol, whereas SULT2B1a preferentially catalyzes pregnenolone sulfonation. We used a genotype-to-phenotype approach to identify and characterize common sequence variation in SULT2B1. Specifically, we resequenced all exons, splice junctions, and approximately 2.5 kb of the 5'-flanking regions (FRs) for each isoform using 60 DNA samples each from African-American and Caucasian-American subjects. We observed 100 polymorphisms, including four nonsynonymous coding single nucleotide polymorphisms and one 6-base pair deletion-all within the "shared" region of the open reading frame. Functional genomic studies of the wild type (WT) and five variant allozymes for each isoform performed with a mammalian expression system showed that variant allozyme activities ranged from 64 to 88% of WT for SULT2B1a and from 76 to 98% for SULT2B1b. Relative levels of immunoreactive protein were similar to those for enzyme activity. Luciferase reporter gene constructs for 2.5 kb of the SULT2B1b 5'-FR displayed a cell line-dependent pattern of variation in activity. Finally, deletion of the proline-rich SULT2B1 carboxyl terminus resulted in intracellular protein aggregate formation and accelerated degradation of the truncated protein. These studies resulted in the identification of common SULT2B1 gene sequence variation, as well as insight into the effects of that variation on the function of this important steroid-metabolizing enzyme.

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.

Cat red blood cell thiopurine S-methyltransferase: companion animal pharmacogenetics.

A common genetic polymorphism for thiopurine S-methyltransferase (TPMT) is a major factor responsible for individual variation in the toxicity and therapeutic efficacy of thiopurine drugs in humans. We set out to determine whether inheritance might also influence the level of TPMT activity in the domestic cat, Felis domesticus. As a first step, red blood cell (RBC) TPMT activity was measured in blood samples from 104 cats. The average level of cat RBC TPMT activity was lower than that observed in humans and was not related to either age or sex of the animal. We then cloned and characterized the F. domesticus TPMT cDNA and gene. Genotype-phenotype correlation analysis was performed by resequencing the cat TPMT gene using DNA samples from 12 animals with high and 12 with low levels of RBC TPMT activity. Thirty-one single nucleotide polymorphisms (SNPs) were observed in these 24 DNA samples, including five that altered the encoded amino acid, resulting in nine allozymes (six observed and three inferred). Twelve of the 31 feline TPMT SNPs were associated, collectively, with 56% of the variation in level of RBC TPMT activity in these 24 animals. When those 12 SNPs were assayed in all 89 cats for which DNA was available, 30% of the variation in level of RBC TPMT activity was associated with these 12 polymorphisms. After expression in COS-1 cells, five of the eight variant cat allozymes displayed decreased levels of both TPMT activity and immunoreactive protein compared with the wild-type allozyme. These observations are compatible with the conclusion that inheritance is an important factor responsible for variation in levels of RBC TPMT activity in the cat. They also represent a step toward the application of pharmacogenetic principles to companion animal thiopurine drug therapy.

Human catecholamine sulfotransferase (SULT1A3) pharmacogenetics: functional genetic polymorphism.

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 Caucasian-American (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 wild-type (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 Km values of the two allozymes for either dopamine (10.5 versus 10.2 micro m for WT and variant, respectively) or the cosubstrate 3'-phosphoadenosine 5'-phosphosulfate (0.114 versus 0.122 micro m, 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.

Human estrogen sulfotransferase (SULT1E1) pharmacogenomics: gene resequencing and functional genomics.

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'-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, 17beta-estradiol and 3'-phosphoadenosine 5'-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.

Pharmacogenetics of human 3'-phosphoadenosine 5'-phosphosulfate synthetase 1 (PAPSS1): gene resequencing, sequence variation, and functional genomics.

3'-Phosphoadenosine 5'-phosphosulfate (PAPS) is the high-energy "sulfate donor" for reactions catalyzed by sulfotransferase (SULT) enzymes. The strict requirement of SULTs for PAPS suggests that PAPS synthesis might influence the rate of sulfate conjugation. In humans, PAPS is synthesized from ATP and SO(4)(2-) by two isoforms of PAPS synthetase (PAPSS): PAPSS1 and PAPSS2. As a step toward pharmacogenetic studies, we have resequenced the entire coding sequence of the human PAPSS1 gene, including exon-intron splice junctions, using DNA samples from 60 Caucasian-American and 58 African-American subjects. Twenty-one genetic polymorphisms were observed-1 insertion-deletion event and 20 single nucleotide polymorphisms (SNPs)-including two non-synonymous coding SNPs (cSNPs) that altered the following amino acids: Arg333Cys and Glu531Gln. Twelve pairs of these polymorphisms were tightly linked, and a total of twelve unequivocal haplotypes could be identified-two that were common to both ethnic groups and ten that were ethnic-specific. The Arg333Cys polymorphism, with an allele frequency of 2.5%, was observed only in DNA samples from Caucasian subjects. The Glu531Gln polymorphism was rare, with only a single copy of that allele in a DNA sample from an African-American subject. Transient expression in mammalian cells showed that neither of the non-synonymous cSNPs resulted in a change in the basal level of enzyme activity measured under optimal assay conditions. However, the Glu531Gln polymorphism altered the substrate kinetic properties of the enzyme. The Gln531 variant allozyme had a 5-fold higher K(m) value for SO(4)(2-) than did the wild-type allozyme and displayed monophasic kinetics for Na(2)SO(4). The wild-type allozyme (Glu531) showed biphasic kinetics for that substrate. These observations represent a step toward testing the hypothesis that genetic variation in PAPS synthesis catalyzed by PAPSS1 might alter in vivo sulfate conjugation.