A prospective genome-wide study of prostate cancer metastases reveals association of wnt pathway activation and increased cell cycle proliferation with primary resistance to abiraterone acetate-prednisone.

Background: Genomic aberrations have been identified in metastatic castration-resistant prostate cancer (mCRPC), but molecular predictors of resistance to abiraterone acetate/prednisone (AA/P) treatment are not known. Patients and methods: In a prospective clinical trial, mCRPC patients underwent whole-exome sequencing (n = 82) and RNA sequencing (n = 75) of metastatic biopsies before initiating AA/P with the objective of identifying genomic alterations associated with resistance to AA/P. Primary resistance was determined at 12 weeks of treatment using criteria for progression that included serum prostate-specific antigen measurement, bone and computerized tomography imaging and symptom assessments. Acquired resistance was determined using the end point of time to treatment change (TTTC), defined as time from enrollment until change in treatment from progressive disease. Associations of genomic and transcriptomic alterations with primary resistance were determined using logistic regression, Fisher's exact test, single and multivariate analyses. Cox regression models were utilized for determining association of genomic and transcriptomic alterations with TTTC. Results: At 12 weeks, 32 patients in the cohort had progressed (nonresponders). Median study follow-up was 32.1 months by which time 58 patients had switched treatments due to progression. Median TTTC was 10.1 months (interquartile range: 4.4-24.1). Genes in the Wnt/ß-catenin pathway were more frequently mutated and negative regulators of Wnt/ß-catenin signaling were more frequently deleted or displayed reduced mRNA expression in nonresponders. Additionally, mRNA expression of cell cycle regulatory genes was increased in nonresponders. In multivariate models, increased cell cycle proliferation scores (≥ 50) were associated with shorter TTTC (hazard ratio = 2.11, 95% confidence interval: 1.17-3.80; P = 0.01). Conclusions: Wnt/ß-catenin pathway activation and increased cell cycle progression scores can serve as molecular markers for predicting resistance to AA/P therapy.

Genetic Breast Cancer Susceptibility Variants and Prognosis in the Prospectively Randomized SUCCESS A Study.

Large-scale genotyping studies have identified over 70 single nucleotide polymorphisms (SNPs) associated with breast cancer (BC) risk. However, knowledge regarding genetic risk factors associated with the prognosis is limited. The aim of this study was therefore to investigate the prognostic effect of nine known breast cancer risk SNPs. BC patients (n = 1687) randomly sampled in an adjuvant, randomized phase III trial (SUCCESS A study) were genotyped for nine BC risk SNPs: rs17468277 (CASP8) , rs2981582 (FGFR2) , rs13281615(8q24), rs3817198 (LSP1) , rs889312 (MAP3K1) , rs3803662 (TOX3) , rs13387042(2q35), rs4973768 (SLC4A7) , rs6504950 (COX11) . Cox proportional hazards models were used to test the SNPs' association with overall survival (OS) and progression-free survival (PFS). Additional analyses were carried out for molecular subgroups. rs3817198 in LSP1 (lymphocyte-specific protein 1) was the only SNP that significantly influenced OS (p = 0.01) and PFS (p < 0.01) in the likelihood ratio test comparing the genetic survival model with the clinical survival model. In the molecular subgroups, triple-negative patients with two minor alleles in rs3817198 had a much better prognosis relative to OS (adjusted HR 0.03; 95% CI 0.002 - 0.279) and PFS (HR 0.09; 95% CI 0.02 - 0.36) than patients with the common alleles. The same effect on PFS was shown for patients with luminal A tumors (HR 0.19; 95% CI 0.05 - 0.84), whereas patients with luminal B tumors had a poorer PFS with two minor alleles (HR 2.13; 95% CI 1.02 - 4.40). The variant in rs3817198 has a prognostic effect particularly in the subgroup of patients with triple-negative BC, suggesting a possible link with immunomodulation and BC.

TSPAN5, ERICH3 and selective serotonin reuptake inhibitors in major depressive disorder: pharmacometabolomics-informed pharmacogenomics.

Millions of patients suffer from major depressive disorder (MDD), but many do not respond to selective serotonin reuptake inhibitor (SSRI) therapy. We used a pharmacometabolomics-informed pharmacogenomics research strategy to identify genes associated with metabolites that were related to SSRI response. Specifically, 306 MDD patients were treated with citalopram or escitalopram and blood was drawn at baseline, 4 and 8 weeks for blood drug levels, genome-wide single nucleotide polymorphism (SNP) genotyping and metabolomic analyses. SSRI treatment decreased plasma serotonin concentrations (P<0.0001). Baseline and plasma serotonin concentration changes were associated with clinical outcomes (P<0.05). Therefore, baseline and serotonin concentration changes were used as phenotypes for genome-wide association studies (GWAS). GWAS for baseline plasma serotonin concentrations revealed a genome-wide significant (P=7.84E-09) SNP cluster on chromosome four 5' of TSPAN5 and a cluster across ERICH3 on chromosome one (P=9.28E-08) that were also observed during GWAS for change in serotonin at 4 (P=5.6E-08 and P=7.54E-07, respectively) and 8 weeks (P=1.25E-06 and P=3.99E-07, respectively). The SNPs on chromosome four were expression quantitative trait loci for TSPAN5. Knockdown (KD) and overexpression (OE) of TSPAN5 in a neuroblastoma cell line significantly altered the expression of serotonin pathway genes (TPH1, TPH2, DDC and MAOA). Chromosome one SNPs included two ERICH3 nonsynonymous SNPs that resulted in accelerated proteasome-mediated degradation. In addition, ERICH3 and TSPAN5 KD and OE altered media serotonin concentrations. Application of a pharmacometabolomics-informed pharmacogenomic research strategy, followed by functional validation, indicated that TSPAN5 and ERICH3 are associated with plasma serotonin concentrations and may have a role in SSRI treatment outcomes.

Inflammation-related genetic variants predict toxicity following definitive radiotherapy for lung cancer.

Definitive radiotherapy improves locoregional control and survival in inoperable non-small cell lung cancer patients. However, radiation-induced toxicities (pneumonitis/esophagitis) are common dose-limiting inflammatory conditions. We therefore conducted a pathway-based analysis to identify inflammation-related single-nucleotide polymorphisms associated with radiation-induced pneumonitis or esophagitis. A total of 11,930 single-nucleotide polymorphisms were genotyped in 201 stage I-III non-small cell lung cancer patients treated with definitive radiotherapy. Validation was performed in an additional 220 non-small cell lung cancer cases. After validation, 19 single-nucleotide polymorphisms remained significant. A polygenic risk score was generated to summarize the effect from validated single-nucleotide polymorphisms. Significant improvements in discriminative ability were observed when the polygenic risk score was added into the clinical/epidemiological variable-based model. We then used 277 lymphoblastoid cell lines to assess radiation sensitivity and expression quantitative trait loci (eQTL) relationships of the identified single-nucleotide polymorphisms. Three genes (PRKCE, DDX58, and TNFSF7) were associated with radiation sensitivity. We concluded that inflammation-related genetic variants could contribute to the development of radiation-induced toxicities.

Catechol O-methyltransferase pharmacogenomics and selective serotonin reuptake inhibitor response.

We applied a systematic pharmacogenetic approach to investigate the role of genetic variation in the gene encoding catechol O-methyltransferase (COMT) in individual variation in selective serotonin reuptake inhibitor (SSRI) response among depressed patients. In all, 23 single-nucleotide polymorphisms (SNPs) in COMT were genotyped using DNA from the Sequenced Treatment Alternatives to Relieve Depression (STAR(*)D) study (N=1914). One SNP, rs13306278, located in the distal promoter region of COMT, showed significant association with remission in White non-Hispanic (WNH) subjects (P=0.038). Electromobility shift assay for rs13306278 showed alternation in the ability of the variant sequence to bind nuclear proteins. A replication study was performed using samples from the Mayo Clinic Pharmacogenetics Research Network Citalopram/Escitalopram Pharmacogenomic study (N=422) that demonstrated a similar trend for association. Our findings suggest that novel genetic markers in the COMT distal promoter may influence SSRI response phenotypes.

The pharmacogenetics research network: from SNP discovery to clinical drug response.

The NIH Pharmacogenetics Research Network (PGRN) is a collaborative group of investigators with a wide range of research interests, but all attempting to correlate drug response with genetic variation. Several research groups concentrate on drugs used to treat specific medical disorders (asthma, depression, cardiovascular disease, addiction of nicotine, and cancer), whereas others are focused on specific groups of proteins that interact with drugs (membrane transporters and phase II drug-metabolizing enzymes). The diverse scientific information is stored and annotated in a publicly accessible knowledge base, the Pharmacogenetics and Pharmacogenomics Knowledge base (PharmGKB). This report highlights selected achievements and scientific approaches as well as hypotheses about future directions of each of the groups within the PGRN. Seven major topics are included: informatics (PharmGKB), cardiovascular, pulmonary, addiction, cancer, transport, and metabolism.

Human catechol O-methyltransferase genetic variation: gene resequencing and functional characterization of variant allozymes.

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.

L-DOPA biotransformation: correlations of dosage, erythrocyte catechol O-methyltransferase and platelet SULT1A3 activities with metabolic pathways in Parkinsonian patients.

The objectives of this study were to determine (1) the effects of dose and drug absorption on pathways of biotransformation of L-DOPA in Parkinsonian patients treated with Sinemet, and (2) the extent to which genetically-determined variations in the activities of erythrocyte catechol O-methyltransferase and/or platelet phenol sulfotransferase might be reflected in individual differences in L-DOPA metabolism. In the 19 patients studied, there were negative correlations between dosage or absorption and extent of O-methylation and of sulfation of L-DOPA or its metabolites. Levels of activity for erythrocyte COMT were also reflected in individual variation in the metabolism of L-DOPA. In contrast, differences in platelet phenol sulfotransferase were not reflected in differences in sulfation of L-DOPA or of its metabolites. If such a relationship did exist, it might have been obscured by the effects of high dosage of L-DOPA, effects which might have resulted from a deficiency of the sulfation cosubstrate 3'-phosphoadenosine 5'-phosphosulfate in patients taking higher doses of drug.

Mouse histamine N-methyltransferase: cDNA cloning, expression, gene cloning and chromosomal localization.

OBJECTIVE: Histamine N-methyltransferase (HNMT) catalyzes the Ntau-methylation of histamine. We set out to clone a mouse liver HNMT cDNA and the mouse HNMT gene as steps toward characterizing molecular genetic mechanisms involved in the regulation of this important histamine-metabolizing enzyme. DESIGN: A PCR-based strategy was used to clone both the mouse HNMT cDNA and the gene encoding that cDNA, Hnmt. The cDNA was used both to express recombinant mouse HNMT and to determine the chromosomal localization of Hnmt. RESULTS: The mouse liver HNMT cDNA was 1657 bp in length with an 888 bp open reading frame (ORF) that encoded a 296 amino acid protein with a predicted Mr value of approximately 32.5 kDa. The amino acid sequence of the encoded protein was 84% identical to that of human kidney HNMT. Mouse HNMT was expressed in COS-1 cells, and its apparent Km values for histamine and S-adenosyl-L-methionine (Ado-Met), the two cosubstrates for the reaction, were 5.3 and 5.8 microM, respectively. The mouse HNMT gene, Hnmt, spanned approximately 25 kb and had 7 exons. Its structure differed from that of the human gene primarily by the presence of an additional exon at the 5'-terminus. Hnmt mapped to mouse chromosome 2 in an area of conserved synteny to human chromosome 2q, the location of the human gene (2q22) on the basis of fluorescence in situ hybridization. CONCLUSIONS: Cloning and functional characterization of the mouse HNMT cDNA and gene will now make it possible to study in the mouse molecular genetic mechanisms involved the regulation of this important histamine-metabolizing enzyme.

Human 3'-phosphoadenosine 5'-phosphosulfate synthetase: radiochemical enzymatic assay, biochemical properties, and hepatic variation.

Sulfation is a major pathway in the biotransformation of many drugs and other xenobiotic compounds. The sulfotransferase (SULT) enzymes that catalyze these reactions use 3'-phosphoadenosine 5'-phosphosulfate (PAPS) as a sulfate donor cosubstrate. The synthesis of PAPS from inorganic sulfate and ATP is catalyzed by PAPS synthetase (PAPSS). We previously cloned the genes for human PAPSS1 and PAPSS2 as a step toward pharmacogenetic studies of these enzymes. We have now developed a sensitive PAPSS radiochemical enzymatic assay for use in genotype-phenotype correlation analyses. This coupled assay uses the sulfation of 17beta-[(3)H]estradiol catalyzed by recombinant human SULT1E1 to measure PAPS, which has been generated by PAPSS during the initial step of the assay. SULT1E1 proved to be ideal for this application both because of its relative resistance to inhibition by ATP, a substrate for the PAPSS-catalyzed step, and because of its low K(m) values for both PAPS (58 nM) and estradiol (29 nM). After optimal PAPSS assay conditions had been established, substrate kinetic studies were performed with cytosol preparations from human liver and cerebral cortex, two tissues with very different expression patterns for PAPSS1 and PAPSS2 mRNA. Brain and liver cytosol PAPSS activities had apparent K(m) values for ATP of 0.26 and 0.62 mM, respectively, and for SO(4)(2-) of 0.08 and 0.31 mM, respectively. PAPSS activity was then measured in 83 human liver biopsy samples to determine the nature and extent of individual variation in this enzyme activity. An 18-fold variation was observed. This sensitive new radiochemical assay can now be used in pharmacogenetic studies of PAPSS in humans.

Severe 6-thioguanine-induced marrow aplasia in a child with acute lymphoblastic leukemia and inherited thiopurine methyltransferase deficiency.

6-thioguanine (6TG) is undergoing investigation for use in the maintenance phase of acute lymphoblastic leukemia (ALL). Just as with 6-mercaptopurine (6MP), it is be expected that 6TG would cause pancytopenia in individuals with inherited thiopurine methyltransferase (TPMT) deficiency. We report the first case of severe and prolonged pancytopenia caused by 6-thioguanine in an 8-year-old boy with ALL and inherited TPMT deficiency. Neutropenia lasted 67 days, whereas anemia and thrombocytopenia did not recover for 96 days. To obviate this life-threatening complication, clinicians should consider assaying TPMT activity before initiating therapy with 6MP and, particularly, 6TG in children with ALL.

Human nicotinamide N-methyltransferase pharmacogenetics: gene sequence analysis and promoter characterization.

Nicotinamide N-methyltransferase (NNMT) catalyses the N-methylation of nicotinamide and structurally related pyridines. NNMT enzymatic activity in human liver varies over a five-fold range with a bimodal frequency distribution - raising the possibility of regulation by a genetic polymorphism. We set out to characterize molecular genetic mechanisms that might be involved in the regulation of individual variation in human liver NNMT activity. After Northern blot analysis confirmed that NNMT is highly expressed in the liver, eight human hepatic biopsy samples, four each with 'low' or 'high' levels of activity, were used to perform quantitative Western blot analysis. There was a highly significant correlation (r(s) = 0.96, P < 0.0001) between NNMT activity and immunoreactive protein in these samples. We next determined that a potent promoter was located within the initial 700 bp of the 5'-flanking region of the human NNMT gene. That gene consists of 3 exons, with an initial 1240 bp intron and a second intron that is approximately 14 kb in length. We subsequently isolated DNA from 27 human liver biopsy samples with low, intermediate or high levels of NNMT activity. The three exons, all 1240 bp of intron 1 and approximately 700 bp of the 5'-flanking region of the NNMT gene were amplified from each of these samples with the polymerase chain reaction, followed by DNA sequencing to identify genetic polymorphisms that might correlate with 'NNMT phenotype'. No single nucleotide polymorphisms (SNPs) or insertion/deletion events were detected within either the exons or 5'-flanking regions of NNMT for these 27 samples. Although there were eight SNPs within intron 1, none were systematically related to level of NNMT activity. These results indicate that the exons and 5'-flanking region of the NNMT gene display little or no sequence variation. Therefore, polymorphisms within these areas of the gene are unlikely to be related to wide individual variations in the level of this enzyme activity in the human liver.

Human phenol sulfotransferases SULT1A2 and SULT1A1: genetic polymorphisms, allozyme properties, and human liver genotype-phenotype correlations.

Phenol sulfotransferases (PSTs or phenol SULTs) catalyze the sulfate conjugation of phenolic drugs, xenobiotics, and monoamines. Two human PST isoforms have been defined biochemically, a thermostable (TS), or phenol-preferring, and a thermolabile (TL), or monoamine-preferring form. Pharmacogenetic studies showed that levels of both TS PST activity and TS PST thermal stability (an indirect measure of variation in amino acid sequence) in the platelet were regulated by genetic polymorphisms. Subsequent molecular genetic experiments revealed the existence of three human PST genes, two of which, SULT1A1 and SULT1A2, encode proteins with "TS PST-like" activity. We recently reported common nucleotide polymorphisms for SULT1A1 that are associated with variations in platelet TS PST activity and thermal stability. In the present experiments, we set out to determine whether functionally significant DNA polymorphisms also might exist for SULT1A2, to compare the biochemical properties of all common allozymes encoded by SULT1A2 and SULT1A1, and to study phenol SULT genotype-phenotype correlations in the human liver. We phenotyped 61 human liver biopsy samples for TS PST thermal stability and activity. The open reading frames of SULT1A2 and SULT1A1 then were amplified with the polymerase chain reaction and sequenced for each of these hepatic tissue samples. We observed 13 SULT1A2 alleles that encoded 6 allozymes. These alleles were in linkage disequilibrium with alleles for SULT1A1. Biochemical characterization of common allozymes encoded by both genes suggested that SULT1A1 was primarily responsible for "TS PST phenotype" in the human liver. In summary, both SULT1A2 and SULT1A1 have a series of common alleles encoding enzymes that differ functionally and are associated with individual differences in phenol SULT properties in the liver.

Rabbit lung indolethylamine N-methyltransferase. cDNA and gene cloning and characterization.

Indolethylamine N-methyltransferase (INMT) catalyzes the N-methylation of tryptamine and structurally related compounds. This reaction has been studied because of its possible role in the in vivo synthesis of psychoactive compounds or neurotoxins and has been characterized biochemically in preparations of rabbit lung. Therefore, we set out to purify rabbit lung INMT, to clone and express its cDNA, and to clone and structurally characterize its gene as steps toward understanding the function and regulation of this enzyme. Rabbit lung INMT was purified and partial amino acid sequence was obtained. A polymerase chain reaction-based approach was then used to clone a rabbit lung INMT cDNA with a 792-base pair open reading frame that encoded a 263-amino acid protein with a predicted molecular mass of 29 kDa. When the cDNA was expressed in COS-1 cells, the encoded protein catalyzed the methylation of tryptamine and structurally related compounds, and was inhibited by two products of the reaction, S-adenosyl-L-homocysteine (AdoHcy) and N,N-dimethyltryptamine, as well as antimigraine drugs that are structurally related to N,N-dimethyltryptamine. Northern blot analysis demonstrated the presence of 2.0-kilobase mRNA species in rabbit lung, liver and, at lower levels, in brain. The cDNA was then used to clone the rabbit INMT gene. That gene had three exons and was structurally similar to the genes for nicotinamide N-methyltransferase and phenylethanolamine N-methyltransferase in several species. Cloning and expression of a rabbit lung INMT cDNA and cloning of the rabbit INMT gene represent important steps toward determination of the function and regulation of this mammalian methyltransferase enzyme.

Mouse nicotinamide N-methyltransferase gene: molecular cloning, structural characterization, and chromosomal localization.

Nicotinamide N-methyltransferase (NNMT) catalyzes the N-methylation of nicotinamide and structurally related compounds. There are large strain-dependent variations in the expression of NNMT activity in mouse liver during growth and development, raising the possibility of developmental regulation of the gene. Therefore, we set out to clone and structurally characterize the mouse NNMT gene, Nnmt. The gene spanned approximately 16 kb and consisted of three exons, 348 bp, 208 bp, and 487 bp in length, with an initial 1228-bp intron and a second intron that was approximately 14 kb in length. The locations of the splice junctions within the gene were highly conserved compared with those in genes for structurally related methyltransferase enzymes. The Nnmt gene contained no canonical TATA box sequences, but an "initiator" (Inr) sequence was located at the site of transcription initiation as determined by 5' rapid amplification of cDNAs ends. A promoter was located within the initial 750 bp of the 5' flanking region of the gene according to studies of the expression of a reporter gene in HepG2 cells. 5'-Flanking region sequences for mouse strains with high and low hepatic NNMT activity differed with regard to a series of nucleotide substitutions, insertions, and deletions, with the most striking difference being a 12-bp insertion/deletion. The Nnmt gene mapped to mouse chromosome 9 in an area of conserved synteny to human chromosome 11q, consistent with the localization of the human NNMT gene to 11q23. Cloning and structural characterization of the mouse Nnmt gene will make it possible to study molecular genetic mechanisms involved in the expression of this important methyltransferase.

Human histamine N-methyltransferase pharmacogenetics: common genetic polymorphisms that alter activity.

Histamine N-methyltransferase (HNMT) catalyzes a major pathway in histamine metabolism. Levels of HNMT activity in humans are regulated by inheritance. We set out to study the molecular basis for this genetic regulation. Northern blot analysis showed that HNMT is highly expressed in the kidney, so we determined levels of enzyme activity and thermal stability in 127 human renal biopsy samples. DNA was isolated from 12 kidney samples with widely different HNMT phenotypes, and exons of the HNMT gene were amplified with the polymerase chain reaction. In these 12 samples, we observed a C314T transition that resulted in a Thr105Ile change in encoded amino acid, as well as an A939G transition within the 3'-untranslated region. All remaining renal biopsy samples then were genotyped for these two variant sequences. Frequencies of the alleles encoding Thr105 and Ile105 in the 114 samples studied were 0.90 and 0.10, respectively, whereas frequencies for the nucleotide A939 and G alleles were 0.79 and 0.21, respectively. Kidney samples with the allele encoding Ile105 had significantly lower levels of HNMT activity and thermal stability than did those with the allele that encoded Thr105. These observations were confirmed by transient expression in COS-1 cells of constructs that contained all four alleles for these two polymorphisms. COS-1 cells transfected with the Ile105 allele had significantly lower HNMT activity and immunoreactive HNMT protein than did those transfected with the Thr105 allele. These observations will make it possible to test the hypothesis that genetic polymorphisms for HNMT may play a role in the pathophysiology of human disease.

Pharmacokinetics, dose adjustments, and 6-mercaptopurine/methotrexate drug interactions in two patients with thiopurine methyltransferase deficiency.

Two children with acute lymphoblastic leukaemia (ALL) were found to be thiopurine methyltransferase (TPMT)-deficient by both genotype and phenotype. They were monitored with haematological parameters and red blood cell concentrations of 6-thioguanine nucleotides (E-6TGN) and methotrexate (E-MTX, including MTX polyglutamates), in relation to the doses of 6-mercaptopurine (6MP) and methotrexate (MTX), during their maintenance chemotherapy. Both patients developed severe pancytopenia at the standard protocol dose of 6MP. Even at 25% and 5%, respectively, of the protocol dose of 6MP, they achieved E-6TGN values several-fold above the population median, but without unacceptable bone-marrow toxicity. Their high E-6TGN values had only a minor influence on their E-MTX values and their tolerance to oral MTX, but severe pancytopenia followed high-dose MTX infusions. Due to the risk of fatal myelosuppression we recommend up-front determination of TPMT activity in patients treated with 6MP or azathioprine.

Olsalazine and 6-mercaptopurine-related bone marrow suppression: a possible drug-drug interaction.

A patient with refractory Crohn's disease had two separate episodes of bone marrow suppression while receiving 50 to 75 mg 6-mercaptopurine a day and 1000 to 1750 mg olsalazine a day. This adverse reaction necessitated dose reduction of 6-mercaptopurine on the first occasion and withdrawal of 6-mercaptopurine and olsalazine on the second occasion. The patient's red blood cell thiopurine methyltransferase (TPMT) activity was 1.2 U per milliliter red blood cells (low normal range) and her TPMT genotype was wild-type sequence for all known alleles of TPMT that result in low TPMT enzyme activity. In vitro enzyme kinetic studies confirmed the hypothesis that olsalazine and olsalazine-O-sulfate are potent noncompetitive inhibitors of recombinant human TPMT. We suggest that the patient's relatively low baseline level of TPMT activity was inhibited by olsalazine and olsalazine-O-sulfate, leading to decreased clearance of 6-mercaptopurine and its accumulation. This ultimately increased intracellular 6-thiopurine nucleotide levels to toxic concentrations, which caused bone marrow suppression.

Mouse liver nicotinamide N-methyltransferase: cDNA cloning, expression, and nucleotide sequence polymorphisms.

Nicotinamide N-methyltransferase (NNMT) catalyzes the N-methylation of nicotinamide and structurally related compounds. We cloned mouse liver NNMT cDNA to make it possible to test the hypothesis that large differences among strains in levels of hepatic NNMT activity might be associated with strain-dependent variation in NNMT amino acid sequence. Mouse liver NNMT cDNA was 1015 nucleotides in length with a 792 nucleotide open reading frame (ORF) that was 83% identical to the nucleotide sequence of the human liver NNMT cDNA ORF. The mouse liver cDNA encoded a 264 amino acid protein with a calculated Mr value of 29.6 kDa. NNMT cDNA ORF sequences were then determined in five inbred strains of mice with very different levels of hepatic NNMT enzymatic activity. Although multiple differences among strains in nucleotide sequence were observed, none altered encoded amino acids. cDNA sequences for C57BL/6J and C3H/HeJ mice, prototypic strains with "high" and "low" levels of hepatic NNMT activity, respectively, were then expressed in COS-1 cells. Both expression constructs yielded comparable levels of enzyme activity, and biochemical properties of the expressed enzyme, including apparent Km values for substrates and IC50 values for inhibition by N1-methylnicotinamide, were very similar to those of mouse liver NNMT. Growth and development experiments were then conducted, which demonstrated that, although at 8 weeks of age average hepatic NNMT activity in C57BL/6J mice was 5-fold higher than that in C3H/HeJ mice, activities in the two strains were comparable by 30 weeks of age--indicating strain-dependent variation in the developmental expression of NNMT in mouse liver. These observations will serve to focus future studies of strain-dependent differences in murine hepatic NNMT on the regulation of the enzyme activity during growth and development.

Human dehydroepiandrosterone sulfotransferase pharmacogenetics: quantitative Western analysis and gene sequence polymorphisms.

Dehydroepiandrosterone sulfotransferase (DHEA ST) catalyzes the sulfation of DHEA and other hydroxysteroids. DHEA ST enzymatic activity in individual human liver biopsy samples has been shown to vary over a five-fold range, and frequency distribution histograms are bimodal, with approximately 25% of subjects included in a high activity subgroup. We set out to characterize the molecular basis for variation in human liver DHEA ST activity. The first step involved performing quantitative Western analysis of cytosol preparations from 92 human liver samples that had been phenotyped with regard to level of DHEA ST enzymatic activity. There was a highly significant correlation (r(s) = 0.635, P < 0.0001) between levels of DHEA ST activity and immunoreactive protein. We next attempted to determine whether the expression of DHEA ST might be controlled, in part, by a genetic polymorphism. DNA was isolated from three "low" and three "high" DHEA ST activity liver samples. Exons and the 5'-flanking region of the DHEA ST gene (STD) were amplified for each of these samples with the polymerase chain reaction (PCR). When compared with "wild type" STD sequence, some of the samples contained a T --> C transition at DHEA ST cDNA nucleotide 170, located within exon 2, resulting in a Met 57 --> Thr change in amino acid. Other samples contained an A --> T transversion at nucleotide 557 within STD exon 4 that resulted in a Glu 186 --> Val change. STD exons 2 and 4 were then sequenced for DNA isolated from an additional 87 liver samples that had been phenotyped with regard to level of DHEA ST enzymatic activity. The allele frequency for the exon 2 polymorphism in these samples was 0.027, whereas that for the exon 4 polymorphism was 0.038, but neither polymorphism was systematically related to the level of enzyme activity in these samples. Transient expression in COS-1 cells of cDNA that contained the nucleotide 170 and 557 polymorphisms, either separately or together, resulted in decreased expression of both DHEA ST enzymatic activity and level of immunoreactive protein, but only when the nucleotide 557 variant was present. Identification of common genetic polymorphisms within STD will now make it possible to test the hypothesis that those polymorphisms might alter in vivo expression and/or function of this important human steroid-metabolizing enzyme.