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.

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.

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.

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.

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%).