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Acamprosate is a Food and Drug Administration (FDA) approved medication for the treatment of alcohol use disorder (AUD). However, only a subset of patients achieves optimal treatment outcomes. Currently, no biological measures are utilized to predict response to acamprosate treatment. We applied our established pharmaco-omics informed genomics strategy to identify potential biomarkers associated with acamprosate treatment response. Specifically, our previous open-label acamprosate clinical trial recruited 442 patients with AUD who were treated with acamprosate for three months. We first performed proteomics using baseline plasma samples to identify potential biomarkers associated with acamprosate treatment outcomes. Next, we applied our established "proteomics-informed genome-wide association study (GWAS)" research strategy, and identified 12 proteins, including interleukin-17 receptor B (IL17RB), associated with acamprosate treatment response.​ A GWAS for IL17RB concentrations identified several genome-wide significant signals. Specifically, the top hit single nucleotide polymorphism (SNP) rs6801605 with a minor allele frequency of 38% in the European American population mapped 4 kilobase (Kb) upstream of IL17RB, and intron 1 of the choline dehydrogenase (CHDH) gene on chromosome 3 (p: 4.8E-20). The variant genotype (AA) for the SNP rs6801605 was associated with lower IL17RB protein expression. In addition, we identified a series of genetic variants in IL17RB that were associated with acamprosate treatment outcomes. Furthermore, the variantgenotypes for all of those IL17RB SNPs were protective for alcohol relapse. Finally, we demonstrated that the basal level of mRNA expression of IL17RB was inversely correlated with those of nuclear factor-κB (NF-κB) subunits, and a significantly higher expression of NF-κB subunits was observed in AUD patients who relapsed to alcohol use. In summary, this study illustrates that IL17RB genetic variants might contribute to acamprosate treatment outcomes. This series of studies represents an important step toward generating functional hypotheses that could be tested to gain insight into mechanisms underlying acamprosate treatment response phenotypes. (The ClinicalTrials.gov Identifier: NCT00662571)., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Purpose: The Mayo-Baylor RIGHT 10K Study enabled preemptive, sequence-based pharmacogenomics (PGx)-driven drug prescribing practices in routine clinical care within a large cohort. We also generated the tools and resources necessary for clinical PGx implementation and identified challenges that need to be overcome. Furthermore, we measured the frequency of both common genetic variation for which clinical guidelines already exist and rare variation that could be detected by DNA sequencing, rather than genotyping., Methods: Targeted oligonucleotide-capture sequencing of 77 pharmacogenes was performed using DNA from 10,077 consented Mayo Clinic Biobank volunteers. The resulting predicted drug response-related phenotypes for 13 genes, including CYP2D6 and HLA, affecting 21 drug-gene pairs, were deposited preemptively in the Mayo electronic health record., Results: For the 13 pharmacogenes of interest, the genomes of 79% of participants carried clinically actionable variants in 3 or more genes, and DNA sequencing identified an average of 3.3 additional conservatively predicted deleterious variants that would not have been evident using genotyping., Conclusion: Implementation of preemptive rather than reactive and sequence-based rather than genotype-based PGx prescribing revealed nearly universal patient applicability and required integrated institution-wide resources to fully realize individualized drug therapy and to show more efficient use of health care resources., Competing Interests: Conflict of Interest Liewei Wang, John Logan Black III, and Richard M. Weinshilboum are cofounders of and stockholders in OneOme, LLC, which was used only to return results to the study participants. Additionally, John Logan Black III and Mayo Clinic Ventures have applied for a patent on the CNVAR software cited in this study as well as the methodology upon which the software is based. All other authors declare no conflicts of interest., (Copyright © 2022 American College of Medical Genetics and Genomics. Published by Elsevier Inc. All rights reserved.)

Clinical pharmacogenomic testing typically uses targeted genotyping, which only detects variants included in the test design and may vary among laboratories. To evaluate the potential patient impact of genotyping compared with sequencing, which can detect common and rare variants, an in silico targeted genotyping panel was developed based on the variants most commonly included in clinical tests and applied to a cohort of 10,030 participants who underwent sequencing for CYP1A2, CYP2C19, CYP2C9, CYP2D6, CYP3A4, CYP3A5, DPYD, SLCO1B1, TPMT, UGT1A1, and VKORC1. The results of in silico targeted genotyping were compared with the clinically reported sequencing results. Of the 10,030 participants, 2780 (28%) had at least one potentially clinically relevant variant/allele identified by sequencing that would not have been detected in a standard targeted genotyping panel. The genes with the largest number of participants with variants only detected by sequencing were SLCO1B1, DPYD, and CYP2D6, which affected 13%, 6.3%, and 3.5% of participants, respectively. DPYD (112 variants) and CYP2D6 (103 variants) had the largest number of unique variants detected only by sequencing. Although targeted genotyping detects most clinically significant pharmacogenomic variants, sequencing-based approaches are necessary to detect rare variants that collectively affect many patients. However, efforts to establish pharmacogenomic variant classification systems and nomenclature to accommodate rare variants will be required to adopt sequencing-based pharmacogenomics., (Copyright © 2022 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Pulsatilla Decoction (Bai-Tou-Weng-Tang) has been used medically in China for thousands of years for the treatment of diseases caused by bacteria. In recent decades, Pulsatilla Decoction is becoming a well-known formula prescription used for the treatment of ulcerative colitis in traditional Chinese medicine. Pulsatilla chinensis is the chief herbal source of Pulsatilla Decoction, and it is rich in triterpenoid saponins, such as anemoside B4, anemoside A3, and 23-hydroxybetulinic acid. Anemoside B4 is the most abundant of that group and has been used as a quality control marker for Pulsatilla chinensis. As the major active component of Pulsatilla chinensis, anemoside B4 has also received attention as a pure compound for its therapeutic potential. In this review, we systematically analyze the findings on triterpenoid saponins, especially anemoside B4, anemoside A3 and 23-hydroxybetulinic acid, included in Pulsatilla chinensis and Pulsatilla Decoction. We discuss the pharmacokinetics and tissue distribution of these triterpenoid saponins as well as their biological activities. We also summarize the pharmacological effects of anemoside B4 and its two possible metabolites, anemoside A3 and 23-hydroxybetulinic acid, as pure compounds. In summary, this review sketches a profile of the state of existing knowledge with regard to the pharmacological effects of anemoside B4, especially its anti-inflammatory and immunomodulatory effects. These findings point to the possibility that anemoside B4 has potential to be studied further as a natural compound-originated immunomodulatory agent for the treatment of inflammatory diseases such as ulcerative colitis and thus, may represent one of the most important active components of Pulsatilla Decoction responsible for its anti-ulcerative colitis efficacy., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Cytochrome (CYP) P450 enzymes have a primary role in antidepressant metabolism and variants in these polymorphic genes are targets for pharmacogenetic investigation. This is the first meta-analysis to investigate how CYP2C19 polymorphisms predict citalopram/escitalopram efficacy and side effects. CYP2C19 metabolic phenotypes comprise poor metabolizers (PM), intermediate and intermediate+ metabolizers (IM; IM+), extensive and extensive+ metabolizers (EM [wild type]; EM+) and ultra-rapid metabolizers (UM) defined by the two most common CYP2C19 functional polymorphisms (rs4244285 and rs12248560) in Caucasians. These polymorphisms were genotyped or imputed from genome-wide data in four samples treated with citalopram or escitalopram (GENDEP, STAR*D, GenPod, PGRN-AMPS). Treatment efficacy was assessed by standardized percentage symptom improvement and by remission. Side effect data were available at weeks 2-4, 6 and 9 in three samples. A fixed-effects meta-analysis was performed using EM as the reference group. Analysis of 2558 patients for efficacy and 2037 patients for side effects showed that PMs had higher symptom improvement (SMD = 0.43, CI = 0.19-0.66) and higher remission rates (OR = 1.55, CI = 1.23-1.96) compared to EMs. At weeks 2-4, PMs showed higher risk of gastro-intestinal (OR = 1.26, CI = 1.08-1.47), neurological (OR = 1.28, CI = 1.07-1.53) and sexual side effects (OR = 1.52, CI = 1.23-1.87; week 6 values were similar). No difference was seen at week 9 or in total side effect burden. PMs did not have higher risk of dropout at week 4 compared to EMs. Antidepressant dose was not different among CYP2C19 groups. CYP2C19 polymorphisms may provide helpful information for guiding citalopram/escitalopram treatment, despite PMs being relatively rare among Caucasians (∼2%)., (Copyright © 2018 Elsevier B.V. and ECNP. All rights reserved.)

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/β-catenin pathway were more frequently mutated and negative regulators of Wnt/β-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/β-catenin pathway activation and increased cell cycle progression scores can serve as molecular markers for predicting resistance to AA/P therapy., (© The Author 2017. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Purpose: Despite potential clinical benefits, implementation of pharmacogenomics (PGx) faces many technical and clinical challenges. These challenges can be overcome with a comprehensive and systematic implementation model., Methods: The development and implementation of PGx were organized into eight interdependent components addressing resources, governance, clinical practice, education, testing, knowledge translation, clinical decision support (CDS), and maintenance. Several aspects of implementation were assessed, including adherence to the model, production of PGx-CDS interventions, and access to educational resources., Results: Between August 2012 and June 2015, 21 specific drug-gene interactions were reviewed and 18 of them were implemented in the electronic medical record as PGx-CDS interventions. There was complete adherence to the model with variable production time (98-392 days) and delay time (0-148 days). The implementation impacted approximately 1,247 unique providers and 3,788 unique patients. A total of 11 educational resources complementary to the drug-gene interactions and 5 modules specific for pharmacists were developed and implemented., Conclusion: A comprehensive operational model can support PGx implementation in routine prescribing. Institutions can use this model as a roadmap to support similar efforts. However, we also identified challenges that will require major multidisciplinary and multi-institutional efforts to make PGx a universal reality.Genet Med 19 4, 421-429.

Significant barriers, such as lack of professional guidelines, specialized training for interpretation of pharmacogenomics (PGx) data, and insufficient evidence to support clinical utility, prevent preemptive PGx testing from being widely clinically implemented. The current study, as a pilot project for the Right Drug, Right Dose, Right Time-Using Genomic Data to Individualize Treatment Protocol, was designed to evaluate the impact of preemptive PGx and to optimize the workflow in the clinic setting. We used an 84-gene next-generation sequencing panel that included SLCO1B1, CYP2C19, CYP2C9, and VKORC1 together with a custom-designed CYP2D6 testing cascade to genotype the 1013 subjects in laboratories approved by the Clinical Laboratory Improvement Act. Actionable PGx variants were placed in patient's electronic medical records where integrated clinical decision support rules alert providers when a relevant medication is ordered. The fraction of this cohort carrying actionable PGx variant(s) in individual genes ranged from 30% (SLCO1B1) to 79% (CYP2D6). When considering all five genes together, 99% of the subjects carried an actionable PGx variant(s) in at least one gene. Our study provides evidence in favor of preemptive PGx testing by identifying the risk of a variant being present in the population we studied., (Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

Background: Medication history discrepancies have the potential to cause significant adverse clinical effects for patients. More than 40% of medication errors can be traced to inadequate reconciliation., Objective: The objective of this study was to determine the accuracy of electronic medical record (EMR)-reconciled medication lists obtained in an academic emergency department (ED)., Methods: Comprehensive research medication ingestion histories for the 48 h preceding ED visit were performed and compared to reconciled EMR medication lists in a convenience sample of ED patients. The reconciled EMR list of prescription, nonprescription, vitamins, herbals, and supplement medications were compared against a structured research medication history tool. We measured the accuracy of the reconciled EMR list vs. the research history for all classes of medications as the primary outcome., Results: Five hundred and two subjects were enrolled. The overall accuracy of EMR-recorded ingestion histories in the preceding 48 h was poor. The EMR was accurate in only 21.9% of cases. Neither age ≥ 65 years (odds ratio [OR] = 1.3; 95% confidence interval [CI] 0.6-2.6) nor sex (female vs. male: OR = 1.5; 95% CI 0.9-2.5) were predictors of accurate EMR history. In the inaccurate EMRs, prescription lists were more likely to include medications that the subject did not report using (78.9%), while the EMR was more likely not to capture nonprescriptions (76.1%), vitamins (73.0%), supplements (67.3%), and herbals (89.1%) that the subject reported using., Conclusions: Medication ingestion histories procured through triage EMR reconciliation are often inaccurate, and additional strategies are needed to obtain an accurate list., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Purpose: We determined hormone concentrations (estradiol [E2], estrone [E1], estrone conjugates [E1-C], androstenedione [A], testosterone [T]) before and on anastrozole therapy where we also determined plasma concentrations of anastrozole and its metabolites., Experimental: Postmenopausal women who were to receive adjuvant anastrozole for resected early breast cancer were studied. Pretreatment, blood samples were obtained for the acquisition of DNA and for plasma hormone measurements (E2, E1, E1-C, A, and T). A second blood draw was obtained at least 4 weeks after starting anastrozole for hormone, anastrozole and metabolite measurements. For hormone assays, a validated bioanalytical method using gas chromatography negative ionization tandem mass spectrometry was used. Anastrozole and metabolite assays involved extraction of plasma followed by LC/MS/MS assays., Results: 649 patients were evaluable. Pretreatment and during anastrozole, there was large inter-individual variability in E2, E1, and E1-C as well as anastrozole and anastrozole metabolite concentrations. E2 and E1 concentrations were below the lower limits of quantitation in 79% and 70%, respectively, of patients on anastrozole therapy, but those with reliable concentrations had a broad range (0.627-234.0 pg/mL, 1.562-183.2 pg/mL, respectively). Considering E2, 8.9% had the same or higher concentration relative to baseline while on anastrozole, documented by the presence of drug., Conclusions: We demonstrated large inter-individual variability in anastrozole and anastrozole metabolite concentrations as well as E1, E2, E1-C, A, and T concentrations before and while on anastrozole. These findings suggest that the standard 1mg daily dose of anastrozole is not optimal for a substantial proportion of women with breast cancer., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Purpose: Pemetrexed, an antifolate involved in purine and pyrimidine formation, is a potential alternative to fluoropyrimidines in the treatment of colorectal cancer. A phase I trial was performed to establish the maximum tolerated dose (MTD) of pemetrexed and oxaliplatin when B(12) and folate supplementation is used., Patients and Methods: Patients with metastatic colorectal cancer received folate (> 350 microg) daily and vitamin B(12) (1000 microg) every 9 weeks starting 7 days before chemotherapy. Pemetrexed over 10 minutes and oxaliplatin over 2 hours were given every 3 weeks in escalating dose cohorts., Results: Twenty-two patients were entered on 6 dose levels. The MTD was established at the highest dose level, pemetrexed 900 mg/m(2) and oxaliplatin 130 mg/m(2). Toxicities related to treatment at the MTD included grade 3 neutropenia and thrombocytopenia. For all dose levels combined, grade 3/4 toxicities included hematologic, neurologic, and gastrointestinal. Nine of 21 evaluable patients responded overall (response rate, 43%). The time to tumor progression was 11.9 months., Conclusion: The MTD was determined to be pemetrexed 900 mg/m(2) and oxaliplatin 130 mg/m(2) every 21 days when folate and B (12) supplementation are used. Because of the observed tolerability and activity of this regimen, further evaluation is warranted.

A common cause for pharmacogenetic alteration in drug response is genetic variation in encoded amino acid sequence. We have used the catecholamine and drug-metabolizing enzyme sulfotransferase (SULT)1A3 to create an artificial model system to study mechanisms-especially possible aggresome formation-by which genetic alteration in amino acid sequence might influence function. Specifically, we created a double variant SULT1A3 allozyme that included the naturally occurring Asn234 polymorphism plus an additional Trp172Arg mutation. Analysis of the SULT1A3 X-ray crystal structure had indicated that the Trp172Arg mutation might destabilize the protein's structure. Expression of SULT1A3 Arg172,Asn234 in COS-1 cells resulted in undetectable enzyme activity and a virtual lack of enzyme protein. Rabbit reticulocyte lysate degradation studies showed that the double variant allozyme was degraded much more rapidly than was wild type SULT1A3 by a ubiquitin-proteasome-dependent process. In addition, after expression in COS-1 cells, the double variant allozyme localized to aggresomes, a process not previously described or studied in pharmacogenetics. Therefore, the alteration of only one or two amino acids can lead to decreased levels of protein as a result of both aggresome formation and accelerated degradation. The possible role of aggresome formation in pharmacogenetics should be evaluated in naturally occurring systems with inherited alteration in encoded amino acid sequence.

Sulfotransferase (SULT) 1A3 catalyzes the sulfate conjugation of catecholamines. Inheritance is an important factor responsible for individual variation in SULT1A3 activity, and gene resequencing studies have shown the presence of one functionally significant SULT1A3 nonsynonymous cSNP. However, following completion of the Human Genome Project, it appeared that SULT1A3 might be duplicated. We used specific PCR-based assays and fluorescence in situ hybridization to verify that 2 SULT1A3 genes-SULT1A3 and SULT1A4-were present on chromosome 16 in all human DNA samples studied. Furthermore, reanalysis of previous gene resequencing data confirmed the presence of the SULT1A3 SNPs identified previously, but also revealed 11 novel polymorphisms, including 3 nonsynonymous cSNPs. Functional genomic studies showed that two of those cSNPs, C302T, and C302A, resulted in decreased enzyme activity without striking changes in substrate kinetics but with parallel changes in levels of immunoreactive protein. In addition, RT-PCR revealed that both SULT1A3 and SULT1A4 can be transcriptionally active. The duplication of SULT1A3 will have to be taken into account in future efforts to understand individual variation in SULT1A3 activity or properties.

A growing body of evidence supports the hypothesis that estrogens can be carcinogens as a result of their conversion to genotoxins after biotransformation to form the catecholestrogens (CEs) 2-hydroxyestrone (2-OHE1), 2-hydroxyestradiol (2-OHE2), 4-hydroxyestrone (4-OHE1) and 4-hydroxyestradiol (4-OHE2). CEs can then undergo further metabolism to form quinones that interact with DNA to form either stable or depurinating adducts. These events could potentially be interrupted by the sulfate conjugation of both the parent estrogens and/or the CEs. We set out to determine whether CEs can serve as substrates for sulfate conjugation, and-if so-which of the growing family of human sulfotransferase (SULT) isoforms are capable of catalyzing those reactions. We determined apparent K(m) values for 10 recombinant human SULT isoforms, as well as the three most common allozymes for SULT1A1 and SULT1A2, with 2-OHE1, 2-OHE2, 4-OHE1, and 4-OHE2, and with the endogenous estrogens, estrone (E1) and 17beta-estradiol (E2), as substrates. With the exception of SULT1B1, SULT1C1, and SULT4A1, all of the human SULTs studied catalyzed the sulfate conjugation of CEs. SULT1E1 had the lowest apparent K(m) values, 0.31, 0.18, 0.27, and 0.22 microM for 4-OHE1, 4-OHE2, 2-OHE1, and 2-OHE2, respectively. These results demonstrate that SULTs can catalyze the sulfate conjugation of CEs, and they raise the possibility that individual variation in this pathway for estrogen and CE metabolism as a result of common genetic polymorphisms could represent a risk factor for estrogen-dependent carcinogenesis., ((c)2002 Elsevier Science (USA).)

Histamine N-methyltransferase (HNMT) catalyzes the N-methylation of histamine in mammals. The experimentally determined HNMT three-dimensional (3D) structure is not available. However, there is a common genetic polymorphism for human HNMT (Thr105Ile) that reduces enzymatic activity and is a risk factor for asthma. To obtain insights into mechanisms responsible for the effects of that polymorphism on enzymatic activity and thermal stability, we predicted the 3D structure of HNMT using the threading method and molecular dynamics simulations in water. Herein, we report a theoretical 3D model of human HNMT which reveals that polymorphic residue Thr105Ile is located in the turn between a beta strand and an alpha helix on the protein surface away from the active site of HNMT. Ile105 energetically destabilizes folded HNMT because of its low Chou-Fasman score for forming a turn conformation and the exposure of its hydrophobic side chain to aqueous solution. It thus promotes the formation of misfolded proteins that are prone to the clearance by proteasomes. This information explains, for the first time, how genetic polymorphisms can cause enhanced protein degradation and why the thermal stability of allozyme Ile105 is lower than that of Thr105. It also supports the hypothesis that the experimental observation of a significantly lower level of HNMT enzymatic activity for allozyme Ile105 than that with Thr105 is due to a decreased concentration of allozyme Ile105, but not an alternation of the active-site topology of HNMT caused by the difference at residue 105., (Copyright 2001 Academic Press.)

Sulfae conjugation is an important pathway in the metabolism of a large number of exogenous and endogenous compounds. These reactions are catalyzed by sulfotransferase (SULT) enzymes that utilize 3'-phosphoadenosine 5'-phosphosulfate (PAPS) as a sulfate donor. PAPS is synthesized from ATP and inorganic sulfate by PAPS synthetase (PAPSS). Two separate PAPSS cDNAs, PAPSS1 and PAPSS2, have been identified in human tissues. We have cloned and characterized the genes for human PAPSS1 and PAPSS2 to make it possible to study the pharmacogenomics of these enzymes. Both genes consisted of 12 exons with virtually identical exon-intron splice junction locations. All splice junctions conformed to the "GT-AG" rule. The total length of PAPSS1 was approximately 108 kb, while that of PAPSS2 was greater than 37 kb. The 5'-flanking region of PAPSS1 did not include a TATA box sequence near the site of transcription initiation, but PAPSS2 had a TATA motif located 21 bp upstream from the site of transcription initiation. Northern blot analysis showed that the major PAPSS1 and PAPSS2 transcripts were approximately 2.7 and 4.2 kb in length, respectively. PAPSS1 mapped to human chromosome band 4q24 while PAPSS2 mapped to 10q22-23 by fluorescence in situ hybridization analysis. Cloning and structural characterization of PAPSS1 and PAPSS2 will make it possible to perform molecular genetic and pharmacogenomic studies of these important enzymes in humans., (Copyright 2000 Academic Press.)

Protein L-isoaspartyl methyltransferase (PIMT) is believed to play an important role in the disposition of age-damaged proteins by catalyzing the repair of abnormal isoaspartyl linkages resulting from the spontaneous deamidation of asparaginyl residues or isomerization of aspartyl residues. As a step toward testing the hypothesis that human disease- or age-related pathology might be associated with a deficiency in PIMT, we investigated basal activity and thermal stability of PIMT in erythrocyte lysates from 299 U.S. family members. Thermal stability was measured because it is a sensitive measure of variation in amino acid sequence. Basal activity was normally distributed with a mean+/-SD of 558+/-43 units/ml erythrocytes. Statistical analysis of the data revealed that basal PIMT activity exhibited a high degree of heritability. Enzyme thermal stability showed a skewed bimodal frequency distribution, and segregation analysis of family member pedigrees was consistent with Mendelian inheritance of two major alleles. No DNA was available from the family samples, so we tested two additional population samples for a known Ile/Val polymorphism at codon 119 and for PIMT activity and thermal stability, using blood donated by 25 Norwegians and by 20 Koreans. Single-stranded conformational polymorphism analysis using polymerase chain reaction revealed a 100% correlation between thermal stability grouping and this polymorphism. The high thermal stability samples were all homozygous Ile, the low thermal stability samples were all homozygous Val, and the intermediate thermal stability samples were all heterozygous. Furthermore, this polymorphism was responsible, in part, for the variation observed in basal erythrocyte PIMT activity. These results will help provide a foundation for future studies aimed at correlating levels of PIMT activity, or other properties of this enzyme, with human disease.

The phenol sulfotransferases (PSTs) catalyze the sulfation of both small planar phenols and phenolic monoamines. Three highly homologous PST genes, SULT1A1, SULT1A2, and SULT1A3, are known to exist in humans. The prototypic biochemical phenotype associated with the enzyme encoded by SULT1A1 is the thermal stable (TS) sulfation of 4 microM 4-nitrophenol (TS PST activity). Biochemical pharmacogenetic studies have demonstrated that individual variation in both TS PST activity and thermal stability in humans are inherited. As a step toward understanding molecular mechanisms responsible for the genetic regulation of PSTs in humans, we report here common SULT1A1 nucleotide polymorphisms that are associated with phenotypic variation in both platelet TS PST activity and thermal stability. When 905 human subjects were phenotyped for platelet TS PST activity and thermal stability, activity varied more than 50-fold, and thermal stability varied over 10-fold. DNA was isolated from the blood of 33 of these subjects selected on the basis of "extreme" TS PST phenotypes: high activity and high thermal stability; low activity and low thermal stability; or low activity and high thermal stability. These 33 subjects were genotyped for SULT1A1 by PCR amplification and sequencing of the entire open reading frame (ORF) as well as approximately 1 kb of intron DNA sequence. One common allele, SULT1A1*2, was uniformly associated with both very low TS PST activity and low thermal stability. The allele frequency of SULT1A1*2 in a randomly selected population sample of 150 Caucasian blood donors was 0.31 (31%), indicating that approximately 9% of this population would be homozygous for that allele.

Thermolabile (TL) phenol sulfotransferase (PST) catalyzes the sulfate conjugation of phenolic monoamines such as dopamine. The level of activity of TL PST in at least one human tissue, the blood platelet, is controlled by a genetic polymorphism. We previously cloned and expressed a cDNA for human liver TL PST and localized its gene, STM, to human chromosome 16p11.2, a region of the chromosome to which the Batten disease gene is also localized. A cDNA for human brain TL PST with an identical open reading frame (ORF) has also been cloned. We have now isolated the human TL PST gene, STM, and have characterized its structural organization as an additional step toward understanding molecular mechanisms involved in the regulation of levels of TL PST activity in human tissue. STM consists of ten exons and nine introns, with a total length of approximately 8.4 kb. Exons range from 88-499 bp in length, while introns vary from 89-1855 bp. Many of the exon-intron splice junctions in STM are located at positions identical to those of splice junctions in the human dehydroepiandrosterone (DHEA) ST gene, STD, and the rat phenol or aryl ST gene. The first two STM exons are represented in the 5'-UTR of a longer TL PST cDNA expressed in both brain and liver, while exon III is represented in a shorter cDNA 5'-UTR expressed in both tissues. These observations suggest alternative transcription initiation and/or alternative splicing as explanations for the existence of TL PST mRNA species with two different 5'-UTRs. 5'-Flanking region(s) of STM contained neither canonical TATA nor CCAAT elements, but they did contain pyrimidine-rich stretches. Northern blot analyses showed that an mRNA species approximately 1.4 kb in length was expressed in human liver, kidney, lung, small intestine, spleen and leukocyte. Molecular cloning and structural characterization of STM will make it possible to study molecular genetic mechanisms involved in the regulation of TL PST activity in human tissue.

The sulfate conjugation of phenolic biogenic amines in humans is catalyzed by the thermolabile (TL) form of phenol sulfotransferase (PST). As a first step toward cloning a cDNA for TL PST, the enzyme was purified from jejunal mucosa, the human tissue with the highest known specific activity, and purified TL PST was subjected to limited proteolysis and amino acid sequencing. The PCR was then performed with human liver cDNA as template and primers designed on the basis of an 18 amino acid stretch of TL PST sequence to obtain a probe for screening cDNA libraries. When cDNA library screening proved unsuccessful, PCR primers were designed based on the nucleotide sequence of a functionally uncharacterized human brain cDNA that had been speculated to represent an aryl sulfotransferase. This brain cDNA encoded the 18 amino acid sequence that we had determined in TL PST. With human liver cDNA as template, these primers amplified a PCR product that contained an 885 nucleotide open reading frame that encoded 295 amino acids--including the 18 amino acids of TL PST sequence. In vitro transcription and translation of this human liver cDNA resulted in the synthesis of a 35.5 kDa protein. The human liver cDNA was also expressed in COS-1 cells, and the biochemical and physical characteristics of the encoded enzyme were identical with those of human liver TL PST. The cloning of a cDNA for human liver TL PST represents an important step toward understanding the molecular basis for the regulation of this enzyme in humans.

Analysis of the catechol-O-methyltransferase (COMT) enzyme in human RBC lysates from 15 samples exhibiting inherited variations in level of activity and thermal stability was performed. Electrophoretic blotting and immune fixation was carried out following sodium dodecyl sulfate polyacrylamide gel electrophoresis or isoelectric focusing of lysate protein. These techniques did not reveal a major structural alteration of the protein that could account for the observed variation in enzyme activity or thermal stability. Future studies utilizing molecular genetic techniques should make it possible to determine the basis for inherited variations in human RBC COMT activity and thermal stability.

6-mercaptopurine (6-MP) can be inactivated by S-methylation, which is catalysed by thiopurine methyltransferase (TPMT). An alternative metabolic route leads to the formation of cytotoxic 6-thioguanine nucleotides (6-TGN). To investigate whether these two pathways compete with each other to affect the therapeutic response to 6-MP, 6-TGN concentrations and TPMT enzymatic activity were measured in erythrocytes (RBC) from 95 children on long-term 6-MP therapy for lymphoblastic leukaemia (ALL). RBC TPMT activities were also measured in 130 control children and 104 long-term survivors of ALL no longer on treatment. The 95 children on 6-MP showed wide interindividual differences in RBC 6-TGN concentrations at the full protocol dose of 75 mg/m2, and RBC 6-TGN concentrations correlated negatively with RBC TPMT activity. Children with 6-TGN concentrations below the group median had higher TPMT activities and a higher subsequent relapse rate. 50 of the 104 long-term survivors had been treated with "gentle" low-dose protocols, and this subgroup contained an excess of children with lower TPMT activities compared with normal controls. These results indicate that genetically determined TPMT activity may be a substantial regulator of the cytotoxic effect of 6-MP, an effect which in turn could be important in influencing the outcome of therapy for childhood ALL.

Nicotinamide N-methyltransferase (NNMT) catalyzes the N-methylation of nicotinamide, pyridine, and structurally related compounds. The products of this reaction are positively charged pyridinium ions that cannot be removed from aqueous solution by simple organic solvent extraction. We developed an assay for human liver NNMT based on the extraction of the positively charged reaction product into 60% isoamyl alcohol in toluene in the presence of the ion-pairing reagent 1-heptanesulfonic acid. Nicotinamide was the methyl acceptor for the reaction, and [14C-methyl]-S-adenosyl-L-methionine (Ado-Met) served as the methyl donor. Apparent Km values of human liver NNMT for nicotinamide and Ado-Met were 347 and 1.76 mumol/l, respectively. The product of the reaction was identified as N1-methylnicotinamide (NMN) by high performance liquid chromatography. NNMT activity was inhibited by the reaction products, NMN and S-adenosyl-L-homocysteine. NNMT activity was not affected by inhibitors of other methyltransferases including Ca2+, SKF 525A and 3,4-dimethoxy-5-hydroxybenzoic acid. Individual variation in NNMT activity was studied by measuring hepatic enzyme activities in 163 human liver biopsy samples obtained during clinically-indicated surgery. The average NNMT activity in these samples was 51.5 +/- 32.5 U per mg protein (mean +/- SD), and there was not a significant correlation of enzyme activity with patient age or with the time of storage of the biopsy samples at -80 degrees C. The distribution of activities was bimodal, and approximately 26% of the samples were included in a subgroup with high NNMT activity. It will now be possible to test the hypothesis that individual differences in hepatic NNMT activity might be related to variation in the N-methylation of pyridine compounds and to individual differences in either toxicity or the therapeutic efficacy of such compounds.

A radiochemical assay for the measurement of histamine N-methyltransferase (HNMT) activity in human erythrocytes (RBCs) has been developed. This assay was developed as a first step toward testing the hypothesis that the biochemical properties and regulation of HNMT in an easily obtainable human cell, the RBC, might reflect those of the enzyme in less accessible cells and tissues. The Michaelis (Km) constant in the RBC for histamine, the methyl acceptor substrate for the reaction, was 5.0 X 10(-5) mol/l. The Km constant for S-adenosyl-L-methionine, the methyl donor, was 2.8 X 10(-6) mol/l. The assay was performed at a reaction pH of 7.4 with a potassium phosphate buffer. The product of the reaction was identified as N tau-methylhistamine by high performance liquid chromatography. The Kii for inhibition of the RBC enzyme by amodiaquine, an HNMT inhibitor, was 1.0 X 10(-7) mol/l, while the Kis value was 0.48 X 10(-7) mol/l. Blood samples obtained from 39 randomly selected adult white subjects had a mean activity of 130 +/- 30 U/ml of packed RBCs (mean +/- SD). Enzyme activities varied over a range from 74-213 U. There were no differences between men and women in mean activities, nor was there a significant correlation between RBC HNMT activity and age. The results of experiments in which lysates with 'low' and 'high' activities were mixed gave no indication that individual variations in RBC HNMT activities were due to the effects of endogenous enzyme inhibitors or activators. RBC HNMT activities measured in blood samples from 17 individual subjects four times over 6 wk were quite constant in each subject, with an average coefficient of variation of 6.2%. The availability of this assay will make it possible to test the hypothesis that individual variations in RBC HNMT activity might be used to predict individual differences in HNMT activity in other human cells and tissues.

Procedures for the precise assay of human platelet phenol sulphotransferase activity were determined. The coefficient of variation of the assay was 5.8% when the enzyme activity was expressed per 10(8) platelets, and was 9.4% when it was expressed per mg soluble platelet protein. Mean platelet phenol sulphotransferase (PST) activity in samples from 102 randomly selected adults was 1.2 +/- 0.4 units/10(8) platelets (mean +/- S.D.), with a range from 0.2 to 2.9. The mean activity for umbilical cord blood platelet PST was 0.93 +/- 0.3 units/10(8) platelets (mean +/- S.D., n = 27). The substrate used routinely for the assay was 3-methoxy-4-hydroxyphenylglycol (MHPG). There was a significant correlation between the formation of MHPG sulfate by individual platelet preparations and the formation of sulfated product with each of the following substrates: tyramine (r = 0.92, n = 21); dopamine (r = 0.82, n = 16); 5-hydroxytryptamine (r = 0.94, n = 20); acetaminophen (r = 0.77, n = 17); and alphamethyldopa (r = 0.77, n = 17) (p less than 0.001 for each). Platelet PST activity correlated significantly with human renal cortex PST activity (r = 0.54, n = 20, p less than 0.02). The correlation coefficient between platelet PST activity and jejunal mucosal enzyme activity in eight patients was 0.67. These results raise the possibility that human platelet PST activity measured with MHPG as substrate might reflect the enzyme activity in other tissues and the degree of sulfate conjugation of a variety of substrates.

Human red blood cell (RBC) membranes have been reported to contain both high and low affinity 'forms' of the drug metabolizing enzyme thiol methyltransferase (TMT). The biochemical characteristics of the two 'forms' of human RBC TMT were compared. Apparent Km constants of the high affinity activity for 2-mercaptoethanol and S-adenosyl-L-methionine, cosubstrates for the TMT reaction, were 0.38 mumol/l and 2.6 mumol/l, respectively. These constants may be compared with values of 20 mmol/l and 43 mumol/l, respectively, previously reported for the low affinity form of RBC TMT activity. The properties and regulation of the two forms of TMT were then compared with each other and also with those of two 'control' enzymes, phenol methyltransferase (PMT) and beta-glucuronidase. When high and low affinity TMT, PMT and beta-glucuronidase activities were measured in RBC membranes from 22 individual subjects, there were highly significant correlations among all three methyltransferase activities (all r values greater than 0.95), but beta-glucuronidase activity did not correlate significantly with any of the methyltransferase activities (all r values less than 0.40). The thermal stabilities of the three methyltransferases were very similar. They were all inactivated approximately 50% by incubation at 48 degrees C for 15 min. beta-Glucuronidase activity was approximately 50% inactivated by incubation at 76 degrees C for 15 min. PMT and both TMT activities had similar subcellular distributions and similar responses to ions and to enzyme inhibitors. These results suggested that high and low affinity TMT and PMT activities might be catalyzed by the same enzyme. Alternatively, these three RBC membrane methyltransferase activities might be regulated in a parallel fashion.

PST (E.C. 2.8.2.1) plays an important role in the metabolism of many drugs, catecholamine metabolites, and catecholamines. PST activity was detected in each of 178 human RBC lysates. When MHPG was used as a substrate, the activity ranged from 28 to 1385 U/ml of RBC, with an average value of 217.7 +/- 13.1 (mean +/- S.E.M.). However, there was not a direct relationship between quantity of RBC lysate and enzyme activity, an observation that raised the possibility of endogenous enzyme inhibitors. Therefore human RBC PST was partially purified (415-fold) to use in the study of tissue enzyme inhibitors. The pH optimum of the partially purified enzyme was 7.5, with an apparent Km value for [35S]PAPS of 0.46 microM and an apparent Km value for MHPG of 260 microM. When the partially purified enzyme was added to each of 20 human RBC lysates, its activity was inhibited an average of 91% +/- 0.6 (mean +/- S.E.M.). Endogenous inhibitors were also present in homogenates of human renal cortex and in homogenates of a variety of rat tissues. RBC lysates contained at least two PST inhibitors: a low-molecular-weight inhibitor (less than 2000) that was heat-, acid-, and base-stable, dialyzable, and resistant to digestion by chymotrypsin; and a high-molecular-weight inhibitor (greater than 65,000) that was heat-labile and nondialyzable. Whether the RBC enzyme activity may serve as an indicator of PST activity in less accessible tissues remains to be determined. However, the first step toward testing this hypothesis will require the accurate measurement of PST activity in tissue preparations by a procedure that removes or inactivates enzyme inhibitors.

A radiochemical microassay for the determination of phenol O-methyltransferase (PMT) activity in human red blood cell membranes has been developed. Acetaminophen was used as the substrate. The apparent Michaelis-Menten (KM) value for acetaminophen was 21.2 X 10(-3) M. The apparent KM value for S-adenosyl-L-methionine, a co-substrate for the reaction, was 4.8 X 10(-6) M, and the pH optimum of the reaction was approximately 9.0 with four different buffer systems. Phenol was also tested as a substrate and had an apparent KM value of 2.0 X 10(-3) M. Human erythrocyte (RBC) membrane PMT activity did not have the biochemical characteristics of catechol O-methyltransferase, another RBC membrane methyltransferase enzyme activity. Blood samples obtained from 212 randomly selected adult white subjects had a mean activity of 134.5 +/- 41.5 pmol of p-acetanisidide formed per mg protein per hour (mean +/- S.D.). Activities varied from 44 to 282 units. There were no differences in the mean activities of samples from men and women. Experiments in which mixtures of "low" and "high" activity RBC membrane preparations were assayed for PMT provided no evidence that the variations in enzyme activity were due to the presence of endogenous PMT activators or inhibitors. RBC membrane PMT activity in blood from 9 patients with renal failure, a pathological state in which there are elevated circulating levels of phenols, was found to be significantly decreased with average activity of 76.2 +/- 9.7 (mean +/- S.E.M., P less than 0.001).

A radiochemical microassay for the measurement of thiol methyltransferase (TMT) activity in human red blood cell (RBC) membranes has been developed. Both 2-mercaptoethanol and dithiothreitol were used as substrates for the enzyme. The pH optimum of the reaction was approximately 9.0 when glycine NaOH was used as a buffer. The apparent Michaelis-Menten (KM) value for the methyl donor for the reaction, S-adenosyl-L-methionine, was 43 mumol/l. Human RBC TMT activity was neither activated nor inhibited by Ca2+, Mg2+, or tropolone, but the enzyme was inhibited by SKF 525A and by reagents that react with sulfhydryl groups. The mean TMT activity in blood from 289 randomly selected adult white subjects was 10.93 +/- 3.22 units per mg protein (mean +/- S.D.). The activity was the same in samples from men and women. The results of experiments in which TMT activity was measured in mextures of RBC membranes with relatively "low" and relatively "high" activities provided no evidence that individual variations in the enzyme activity were due to variations in endogenous TMT activators or inhibitors.

A radiochemical micromethod for the determination of thiopurine methyltransferase (TPMT) activity in human red blood cells (RBC) is described. Both 6-mercaptopurine and 6-thioguanine were substrates for the TPMT activity in the human RBC. Apparent Michaelis-Menten (KM) values for 6-mercaptopurine and 6-thioguanine were 3.2 X 10(-4) M and 2.0 X 10(-4) M, respectively. The apparent KM value for S-adenosyl-L-methionine, a co-substrate for the reaction, was 1.7 X 10(-6) M. The pH optimum for the reaction was approximately 7.5. Blood samples from 73 randomly selected adult subjects had a mean activity of 10.2 +/- 2.4 (mean +/- S.D.) units/ml packed red blood cells. The range of activities was from 4.6 to 14.2 units/ml. The results of experiments in which partially purified human kidney TPMT was added to RBC lysates and of experiments in which "low" and "high" activity lysates were mixed gave no indication that individual variations in RBC TPMT activity were due to endogenous inhibitors or activators of the enzyme.

Phenosulphotransferase (EC 2.8.2.1) (PST) is an important catecholamine and drug metabolizing enzyme. Optimal conditions have been determined for the accurate measurement of PST activity in the human platelet, human renal cortex, and human jejunum with a radiochemical microassay. 3-Methoxy-4-hydroxyphenylglycol (MHPG) and 35S-3'-phosphoadenosine-5'-phosphosulfate (35S-PAPS) were the substrates for the reaction. The apparent Michaelis-Menten (Km) values for MHPG with platelet, renal cortex, and jejunum were 1.09, 0.46 and 1.16 mmol/l, respectively. Apparent Km values for PAPS in the same tissues were 0.14, 0.13 and 0.21 mumol/l. The pH optimum of the reaction in all three tissues was approximately 6.2--6.8 with three different buffer systems. The coefficients of variation for the assay of platelet, renal cortex, and jejunal activities were 6.2%, 3.4% and 4.4%, respectively. Mean platelet PST activity in blood samples from 75 randomly selected adult subjects was 5.0 +/- 1.72 nmol of MHPG sulfate formed per hour per mg of platelet protein (8.3 x 10(-5) +/- 2.9 x 10(-5) mumol . min-1 . mg-1, mean +/- S.D.). There was a 5-fold intersubject variation in platelet PST activity within two standard deviations of the mean value. Experiments in which partially purified human erythrocyte PST was added to platelet, kidney and gut homogenates under these assay conditions provided evidence that endogenous PST inhibitors did not affect the observed enzyme activity.

A radiochemical micromethod for the determination of catechol-O-methyltransferase (COMT) activity in human red blood cells (RBC) is described. The method avoids artifacts that occur with other assay procedures. Erythrocyte lysates are exposed to a particulate chelating agent (Chelex-100) to remove endogenous divalent cations that inhibit COMT. 3,4-Dihydroxybenzoic acid rather than norepinephrine is used as a substrate to increase both the sensitivity and the reproducibility of the procedure. An internal standard of purified rat liver COMT is added to lysates to detect possible variations in endogenous activators or inhibitors of the enzyme. Blood samples from 373 unselected subjects age 16-18 were assayed for RBC COMT activity with this assay procedure. The COMT activity in these blood samples ranged from 3-25 units with a mean activity of 11.3 plus or minus 4.2 (mean plus or minus S.D.).