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Genetic variations in cytochrome P450 2C9 (CYP2C9) are known to contribute to interindividual and interethnic variability in response to clinical drugs such as warfarin. In the present study, CYP2C9 from 263 Japanese subjects was resequenced, resulting in the discovery of 62 variations including 32 novel ones. In addition to the two known non-synonymous single nucleotide polymorphisms (SNPs), Ile359Leu (*3; allele frequency=0.030) and Leu90Pro (*13; 0.002), seven novel non-synonymous SNPs, Leu17Ile (0.002), Lys118ArgfsX9 (*25; 0.002), Thr130Arg (*26; 0.002), Arg150Leu (*27; 0.004), Gln214Leu (*28; 0.002), Pro279Thr (*29; 0.002) and Ala477Thr (*30; 0.002), were found. Functional characterization of novel alleles using a mammalian cell expression system in vitro revealed that *25 was a null allele and that *26, *28 and *30 were defective alleles. The *26 product showed a 90% decrease in the Vmax value but little change in the Km value towards diclofenac. Both *28 and *30 products showed two-fold higher Km values and three-fold lower Vmax values than the *1 allele, suggesting the importance of Gln214 and Ala477 for substrate recognition. Linkage disequilibrium and haplotype analyses were performed using the detected variations. Only five haplotypes (frequency >0.02) accounted for most (>87%) of the inferred haplotypes, and they were closely associated with the haplotypes of CYP2C19 in Japanese. Although the haplotype structure of CYP2C9 was rather simple in Japanese, the haplotype distribution was quite different from those previously reported in Caucasians and Africans. Taken together, novel defective alleles and detailed haplotype structures would be useful for determining metabolic phenotypes of CYP2C9 substrate drugs in Japanese and probably Asians.

No abstract

Forty-eight single nucleotide variations, including 27 novel ones, were found in the 5'- regulatory region, all of the exons and their surrounding introns of CYP2C19 in 253 Japanese subjects (134 diabetic patients and 119 healthy volunteers). Identified novel variations were as follows: -2772G>A, 2767_-2760delGGTGAACA, -2720T>C, -2547delG, -2545G>T, -2545_-2544 delGC, and -2040C>T in the enhancer region; -778C>T, -777G>A, -529G>C, -189C>A, and -185A>G in the promoter region; 151A>G (S51G), 481G>C (A161P), 986G>A (R329H), 1078G>A (D360N), and 1119C>T (D373D) in the exons, and IVS1+128T>A, IVS3+163G>A, IVS4+271A>G, IVS5-49A>G, IVS6-210C>T, IVS6-196T>A, IVS6-32T>A, IVS7+84G>A, IVS7-174C>T, and IVS8+64C>T in the introns. Since we found no significant differences in the variation frequencies between healthy volunteers and diabetic patients, the data for all subjects were treated as one group in further analysis. The allele frequencies were 0.265 for IVS6-196T>A, 0.045 for -2772G>A and -2720T>C, 0.024 for -2040C>T, 0.014 for IVS7-174C>T, 0.010 for -529G>C, 0.006 for IVS1+128T>A and 481G>C (A161P), 0.004 for -2767_-2760delGGTGAACA and IVS6-210C>T, and 0.002 for the other 17 variations. In addition, the two known nonsynonymous single nucleotide polymorphisms, 681G>A (splicing defect, (*)2 allele) and 636G>A (W212X; (*)3 allele) were detected at 0.267 and 0.128 frequencies, respectively. No variation was detected in the known binding sites for constitutive androstane receptor and glucocorticoid receptor. Linkage disequilibrium analysis showed several close linkages of variations throughout the gene. By using the variations, thirty-one haplotypes of CYP2C19 and their frequencies were estimated. Our results would provide fundamental and useful information for genotyping CYP2C19 in the Japanese and probably other Asian populations.

Abstract.Diabetes mellitus in Long-Evans Tokushima Lean (LETL) rats closely resembles type 1 diabetes in human beings, e.g., no gender differences in the incidence of diabetes and no T lymphopenia. Although the LETL rats have been established as an inbred strain, the incidence of diabetes is only '2O%. In the present study, we established two substrains, one a diabetes-prone (KDP) and the other a nondiabetic (KND) from the original inbred LETL rats. The features of KDP rats are a high incidence of diabetes (over all -7O%) without lymphopenia and 100% development of mild to severe insulitis at 120-220 days of age. In contrast, the KND substrain is characterized by the complete absence of diabetes incidence. Among 165 SSLP marker loci throughout all rat chromosomes, no loci showed variation among KDP and KND substrains and their parental LETL rats. In this regard, the genetic background of these two substrains, KDP and KND, appears to be uniform except for the major gene(s) that is responsible for the diabetes. In this context, these two substrains of LETL rats should serve as useful tools for research on the pathogenesis and for the genetic analysis of type 1 diabetes. In this report, we have not only established, but also characterized these two substrains, and provided their fundamental data.