ABSTRACT
Xanthine oxidase (XO) catalyzes the oxidation of endogenous and exogenous purines and pyrimidines. In the present study, we investigated polymorphisms in the promoter region of the XO gene. Sequence variations in the 5'-flanking region were screened using denaturing high-performance liquid chromatography (DHPLC) on DNA samples from 196 unrelated Japanese individuals. Thirteen polymorphisms were identified and 13 haplotypes were classified by haplotype analysis. The promoter activities of these polymorphisms were measured by luciferase assay in the human hepatoma cell lines HepG2 and Huh-7. Transcriptional activity was significantly lower in cell lines transfected with the reporter construct containing 5-kb upstream fragments with -1756T than in those with wild-type -1756C. Our results indicate that genetic variation in the promoter region of XO may determine interindividual differences in XO gene expression.
Subject(s)
Promoter Regions, Genetic/genetics , Xanthine Oxidase/genetics , Asian People/genetics , Cell Line, Tumor , Gene Frequency , Hep G2 Cells , Humans , Japan , Polymorphism, Genetic , TransfectionABSTRACT
Our previous studies show that 10 xanthine oxidase (XO) variants (Arg149Cys, Pro555Ser, Arg607Gln, Thr623Ile, Ile703Val Asn909Lys, Thr910Lys, Pro1150Arg, His1221Arg, and Cys1318Tyr) exhibit altered activity toward the endogenous substrate xanthine. This study investigates whether these variants also exhibit altered kinetics for the exogenous substrate 6-thioxanthine (6-TX). To investigate the kinetics of wild-type XO and these variants, expression constructs were transfected into mammalian COS-7 cells. S-9 fractions containing the expressed proteins were used to determine their kinetic parameters, i.e., K(m), V(max), and intrinsic clearance (CL(int)), for the substrate 6-TX. Functional characterization of the 10 XO variants revealed that 4 of the variants (Arg149Cys, Asn909Lys, Thr910Lys, and Pro1150Arg) were inactive, 2 (Arg607Gln, and Cys1318Tyr) had reduced activity (CL(int), 55.5% and 64.7% less than that of wild-type XO, respectively). This study provides comprehensive data regarding how genetic variation in XO affects its activity toward 6-TX. We found that the in vitro activity of 8 of the XO variants toward 6-TX was functionally affected. These results suggeste that polymorphism in the gene encoding XO may increase the toxicity of thiopurine drugs such as 6-mercaputopurine.
Subject(s)
Polymorphism, Genetic , Xanthine Oxidase/genetics , Xanthines/metabolism , Alleles , Animals , COS Cells , Chlorocebus aethiops , Kinetics , Xanthine Oxidase/metabolismABSTRACT
Thiopurines (such as azathioprine and 6-mercaptopurine) are widely used for the treatment of patients suffering from malignancies, rheumatic disease, inflammatory bowel disease and solid organ transplant rejection. These drugs are activated and eliminated by a number of enzymes in the human body. This analyzes all the exons and exon-intron junctions of 5 enzyme genes (hypoxanthine-guanine phosphoribosyltransferase, HGPRT; inosine triphosphate pyrophosphatase, ITPA; inosine monophosphate dehydrogenases 1 and 2, IMPDH1 and IMPDH2 and guanosine monophosphate synthetase, GMPS) involved in the metabolism of thiopurine drugs. Twelve novel single nucleotide polymorphisms (SNPs) (HGPRT: IVS6-12C>A (frequency:0.003); ITPA: 569T>C (Phe189Phe, 0.003); IMPDH1: IVS8-15C>A (0.003), IVS9+227A>G (0.003), IVS17+115C>T (0.003), and 930C>T (Thr310Thr, 0.005); IMPDH2: IVS1+50G>T (0.003), IVS2+15G>A (0.010), IVS3-20G>A (0.003), 609C>T (Arg203Arg, 0.003), and 1534C>T (Arg512Trp, 0.003); and GMPS: 1563T>C (Gly521Gly, 0.003)) and 7 known SNPs (ITPA: 94C>A (Pro32Thr, 0.005), 138G>A (Gln46Gln, 0.586), and 563G>A (Glu187Glu, 0.433); IMPDH1: 987G>C (Leu329Leu, 0.113) and 1575A>G (Ala525Ala, 0.620) and GMPS: IVS5-7T>C (0.153), 993A>G (Thr331Thr, 0.153)) were identified in 200 Japanese subjects. These data should provide useful information for thiopurine therapy in the Japanese and as well as other Asian populations.
Subject(s)
Asian People/genetics , Gene Frequency , Genetic Variation , Hypoxanthine Phosphoribosyltransferase/genetics , Inflammatory Bowel Diseases/genetics , Myeloid-Lymphoid Leukemia Protein/genetics , Oncogene Proteins, Fusion/genetics , Azathioprine/pharmacology , Carbon-Nitrogen Ligases , Exons , Genes, Recessive , Genotype , Guanosine Monophosphate/metabolism , Humans , Introns , Mercaptopurine/pharmacology , Methyltransferases/genetics , Molecular Sequence Data , Mutation , Pharmacogenetics , Phenotype , Polymorphism, Single Nucleotide , Pyrophosphatases/genetics , Thionucleotides/metabolism , Inosine TriphosphataseABSTRACT
OBJECTIVE: Xanthine oxidase (XO) catalyzes the oxidation of endogenous and exogenous purines and pyrimidines. In this study, we speculated that individual variations in XO activity are caused by genetic variations in the XO gene. METHODS: To investigate the genetic variations in XO in 96 Japanese participants, denaturing high-performance liquid chromatography was used. To assess the effects of these variations on enzymatic activity, wild-type XO and 21 types of variant XO--including those in the database and those just discovered--were transiently expressed in COS-7 cells. RESULTS: Three nonsynonymous single nucleotide polymorphisms, including 514G>A (Gly172Arg), 3326A>C (Asp1109Thr), and 3662A>G (His1221Arg) were identified in Japanese participants. Functional characterization of 21 XO variants showed a deficiency in enzyme activity in two variants (Arg149Cys and Thr910Lys); low activity (intrinsic clearance, CLint: 22-69% compared with the wild-type) in six variants (Pro555Ser, Arg607Gln, Thr623Ile, Asn909Lys, Pro1150Arg, and Cys1318Tyr); and high activity (CLint: approximately two-fold higher than that in the wild-type) in two variants (Ile703Val and His1221Arg). CONCLUSION: These results suggest that several single nucleotide polymorphisms in the XO gene are involved in individual variations in XO activity. In addition, such findings will be useful to identify xanthinuria patients.
Subject(s)
Genetic Variation , Xanthine Oxidase/genetics , Xanthine Oxidase/metabolism , Alleles , Animals , Base Sequence , COS Cells , Chlorocebus aethiops , Chromatography, High Pressure Liquid , Cloning, Molecular , DNA Primers/genetics , Humans , Japan , Kinetics , Mutagenesis, Site-Directed , Pharmacogenetics , Polymorphism, Single Nucleotide , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Xanthine/urine , Xanthine Oxidase/deficiencyABSTRACT
The genetic polymorphisms of methylenetetrahydrofolate reductase (MTHFR) have been associated with increased toxicity of methotrexate (MTX), a folic acid antagonist that is widely used to treat cancer and immunosuppressive disorders such as rheumatoid arthritis. In this study, we analyzed all the exons and exon/intron junctions of the MTHFR gene from 200 Japanese individuals. We detected a novel single nucleotide polymorphism (SNP) 148C>T (Arg46Trp) in exon 1. The allele frequency of this polymorphism in the Japanese population appears to be extremely low (0.25%).
