Genetic polymorphisms of UGT1A6 in a Japanese population

Thirteen single nucleotide polymorphisms (SNPs), including 6 novel ones, were found in exon 1 and its flanking region of UDP-glucuronosyltransferase (UGT) 1A6 from 195 Japanese subjects. Several novel SNPs were identified, including 269G>A (R90H), 279A>G (S93S), and 308C>A (S103X) in exon 1, and IVS1+109C>T, IVS1+120A>G, and IVS1+142C>T in the intron downstream of exon 1. Among these SNPs, 308C>A confers termination of translation at codon 103, resulting in the production of an immature protein that probably lacks enzymatic activity. The allele frequencies were 0.003 for all the 6 SNPs. In addition, the 3 known nonsynonymous SNPs were detected: 19T>G (S7A), 541A>G (T181A), and 552A>C (R184S) with frequencies of 0.226, 0.218, and 0.226, respectively. High linkage disequilibrium was observed among 19T>G (S7A), 315A>G (L105L), 541A>G (T181A), 552A>C (R184S), and IVS1+130G>T, as reported in Caucasian and African-American populations. Then, 11 haplotypes in UGT1A6 were estimated. The novel nonsynonymous variant, 269A or 308A, was shown to be located on the same DNA strand together with 19G, 315G, 541G, 552C, and IVS1+130T. Our results provide fundamental and useful information for genotyping UGT1A6 in the Japanese, and probably Asian populations.     

Genetic variations and haplotypes of CYP2C19 in a Japanese population

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.     

Genetic variations and haplotypes of UDP-glucuronosyltransferase 1A locus in a Korean population

Glucuronidation catalyzed by UDP-glucuronosyltransferases (UGTs) is one of the most important mechanisms for host defense against xenobiotics and endobiotics. Although genetic polymorphisms of several UGT1A isoforms have been reported separately, the haplotypes in all functional exons have not been identified, and little information is available regarding single nucleotide polymorphisms in Koreans. We analyzed genetic polymorphisms in all functional exons of the UGT1A locus by direct sequencing of genomic DNA from 50 healthy Korean subjects, and their haplotypes were inferred from genotype data using an expectation-maximization algorithm. We identified 67 polymorphisms, including three novel single nucleotide polymorphisms, 233C>T in UGT1A1 (T78M), 292C>T in 1A4 (Q98Stop), and 701T>C in 1A7 (I234T). Two amino acid substitutions, 1A4 Q98Stop and 1A7 I234T, were each associated with a decrease of enzymatic activity, whereas UGT1A1 T78M had no significant influence on catalytic function. The frequencies of the known variants in Koreans differed significantly from those reported in other ethnic groups. Haplotype analysis was performed within the polymorphisms in each UGT1A isoform as well as across the isoforms. Based on strong linkage disequilibrium within UGT1A7, between 1A5 and 1A4, and within 1A3, the complex was divided into three blocks, Block 7, Block 5/4, and Block 3. The haplotypes for each block were subsequently determined, revealing a profile that differed from those of other ethnic groups. These results suggest that genetic polymorphisms of the UGT1A locus differ between Koreans and other ethnic populations. Such differences should be considered in pharmacogenetic studies.     

Genetic variations and haplotypes of ABCC2 encoding MRP2 in a Japanese population

The multidrug resistance-associated protein 2 (MRP2) encoded by the ABCC2 gene is expressed in the liver, intestine and kidneys and preferentially exports organic anions or conjugates with glucuronide or glutathione. In this study, all 32 exons and the 5'-flanking region of ABCC2 in 236 Japanese were resequenced, and 61 genetic variations including 5 novel nonsynonymous ones were detected. A total of 64 haplotypes were determined/inferred and classified into five *1 haplotype groups (*1A, *1B, *1C, *1G, and *1H) without nonsynonymous substitutions and *2 to *9 groups with nonsynonymous variations. Frequencies of the major 4 haplotype groups *1A (-1774delG), *1B (no common SNP), *1C (-24C>T and 3972C>T), and *2 [1249G>A (Val417Ile)] were 0.331, 0.292, 0.172, and 0.093, respectively. This study revealed that haplotype *1A, which has lowered activity, is quite common in Japanese, and that the frequency of *1C, another functional haplotype, was comparable to frequencies in Asians and Caucasians. In contrast, the haplotypes harboring 3972C>T but not -24C>T (*1G group), which are reportedly common in Caucasians, were minor in Japanese. Moreover, the allele 1446C>T (Thr482Thr), which has increased activity, was not detected in our Japanese population. These findings imply possible differences in MRP2-mediated drug responses between Asians and Caucasians.     

Genetic variations and haplotype structures of the ABC transporter gene ABCC1 in a Japanese population

Multidrug resistance-related protein 1 (MRP1), an ATP-binding cassette transporter encoded by the ABCC1 gene, is expressed in many tissues, and functions as an efflux transporter for glutathione-, glucuronate- and sulfate-conjugates as well as unconjugated substrates. In this study, the 31 exons and their flanking introns of ABCC1 were comprehensively screened for genetic variations in 153 Japanese subjects to elucidate the linkage disequilibrium (LD) profiles and haplotype structures of ABCC1 that is necessary for pharmacogenetic studies of the substrate drugs. Eighty-six genetic variations including 31 novel ones were found: 1 in the 5'-flanking region, 1 in the 5'-untranslated region (UTR), 20 in the coding exons (9 synonymous and 11 nonsynonymous variations), 4 in the 3'-UTR, and 60 in the introns. Of these, eight novel nonsynonymous variations, 726G>T (Trp242Cys), 1199T>C (Ile400Thr), 1967G>C (Ser656Thr), 2530G>A (Gly844Ser), 3490G>A (Val1164Ile), 3550G>A (Glu1184Lys), 3901C>T (Arg1301Cys), and 4502A>G (Asp1501Gly), were detected with an allele frequency of 0.003. Based on the LD profiles, the analyzed regions of the gene were divided into five LD blocks (Blocks -1 and 1 to 4). The multiallelic repeat polymorphism in the 5'-UTR was defined as Block -1. For Blocks 1, 2, 3 and 4, 32, 23, 23 and 13 haplotypes were inferred, and 9, 7, 7 and 6 haplotypes commonly found on > or = 10 chromosomes accounted for > or = 91% of the inferred haplotypes in each block. Haplotype-tagging single nucleotide polymorphisms for each block were identified to capture the common haplotypes. This study would provide fundamental and useful information for the pharmacogenetic studies of MRP1-dependently effluxed drugs in Japanese.     

Single nucleotide polymorphisms and haplotype frequencies of UGT2B4 and UGT2B7 in a Japanese population.

Both UDP-glucuronosyltransferase 2B4 (UGT2B4) and UGT2B7 are expressed mainly in the human liver and have several overlapping substrates; e.g., catechol estrogens, bile acids, codeine, and carvedilol. To identify novel single nucleotide polymorphisms (SNPs) and haplotypes in a Japanese population, the enhancer/promoter regions, all the exons, and the surrounding intronic regions of UGT2B4 and UGT2B7 were sequenced from 136 Japanese individuals. We found 16 and 21 polymorphisms, including 10 and 4 novel ones in UGT2B4 and UGT2B7, respectively. The novel nonsynonymous SNPs were 1364A>G (K455R) and 1531T>C (C511R) in UGT2B4 and 1192G> A (D398N) in UGT2B7. From linkage disequilibrium analysis, several SNPs in UGT2B7 were found to be highly linked with each other. No close linkage between the SNPs in UGT2B4 and UGT2B7 was observed, indicating that each gene is located within an independent haplotype block. Thus, haplotype analysis was separately performed for the two genes. In UGT2B4, we unambiguously determined 8 haplotypes and inferred an additional 12 haplotypes using an expectation-maximization-based program. In UGT2B7, five haplotypes were unambiguously assigned and an additional eight haplotypes were inferred. The haplotype structure of UGT2B7 was more diverse than that of UGT2B4 in terms of the number of frequent SNPs. In addition, ethnic differences in the UGT2B4(*)2 and UGT2B7(*)2 haplotypes between the Japanese and the Caucasian and/or African populations were found. Our findings provide fundamental and useful information for genotyping UGT2B4 and UGT2B7 in the Japanese, and probably other populations.     

Genetic variations of the ABC transporter gene ABCC3 in a Japanese population

An ATP-binding cassette transporter, multidrug resistance-related protein 3 (MRP3), is encoded by the ABCC3 gene. The MRP3 protein is expressed in several tissues, and functions as an efflux transporter for conjugated as well as unconjugated substrates. In this study, the 31 ABCC3 exons and their flanking introns were comprehensively screened for genetic variations in 89 Japanese subjects. Forty-six genetic variations, including 21 novel ones, were found: 8 were located in the 5'-flanking region, 14 in the coding exons (8 synonymous and 6 nonsynonymous variations), and 24 in the introns. Of these 46 variations, five novel nonsynonymous variations, 2221C>T (Gln741Stop), 2395G>A (Val799Met), 2798_2799delAG (Gln933ArgfsX64), 3657C>A (Ser1219Arg), and 4217C>T (Thr1406Met), were found as heterozygous variations. The allele frequencies were 0.011 for Ser1219Arg and 0.006 for the other four variations. Gln741Stop induces a stop codon at codon 741. Gln933ArgfsX64 causes a frame-shift at codon 933, resulting in early termination at codon 997. Both variations result in loss of 6 transmembrane helices (from the 12th to 17th helices) in the C-terminus and all regions of nucleotide binding domain 2. Thus, both variant proteins are assumed to be inactive. These data provide fundamental and useful information for pharmacogenetic studies on MRP3-transported drugs in Japanese.     

Genetic polymorphisms and haplotypes of por, encoding cytochrome p450 oxidoreductase, in a Japanese population

Cytochrome P450 oxidoreductase (POR) transfers electrons from NADPH to all microsomal cytochrome P450 (CYP) enzymes and is necessary for microsomal CYP activities. In this study, to find genetic variations and to elucidate the haplotype structures of POR, we comprehensively screened the genetic variations in the 5'-flanking region, all the exons and their flanking introns of POR for 235 Japanese subjects. Seventy-five genetic variations including 26 novel ones were found: 7 were in the 5'-flanking region, 2 in the 5'-untranslated region (5'-UTR, non-coding exon 1), 16 in the coding exons (10 nonsynonymous and 6 synonymous), 45 in the introns, 4 in the 3'-UTR and 1 in the 3'-flanking region. Of these, 4 novel nonsynonymous variations, 86C>T (T29M), 1648C>T (R550W), 1708C>T (R570C) and 1975G>A (A659T), were detected with allele frequencies of 0.002. We also detected known nonsynonymous SNPs 683C>T (P228L), 1237G>A (G413S), 1453G>A (A485T), 1508C>T (A503V), 1510G>A (G504R) and 1738G>C (E580Q) with frequencies of 0.002, 0.009, 0.002, 0.434, 0.002 and 0.002, respectively. Based on the linkage disequilibrium (LD) profiles, the analyzed region could be divided into two LD blocks. For Blocks 1 and 2, 14 and 46 haplotypes were inferred, respectively, and 2 and 6 common haplotypes found in more than 0.03 frequencies accounted for more than 81% of the inferred haplotypes. This study provides fundamental and useful information for the pharmacogenetic studies of drugs metabolized by CYPs in the Japanese population.     

Genetic polymorphisms in CYP1A1, CYP2D6, UGT1A6, UGT1A7, and SULT1A1 genes and correlation with benzene exposure in a Chinese occupational population

Metabolic enzymes involved in benzene activation or detoxification, including cytochrome P-450 1A1 (CYP1A1), cytochrome P-450 2D6 (CYP2D6), UDP-glucuronosyltransferase 1A6 (UGT1A6), UDP-glucuronosyltransferase1A7 (UGT1A7), and sulfotransferase 1A1 (SULT1A1), were studied for their roles in human susceptibility to benzene poisoning. All 304 subjects were investigated with a unitary questionnaire and their DNA was isolated from blood samples by a routine phenol-chloroform extraction. The study included 152 benzene poisoning patients, and 152 control workers occupationally exposed to benzene in South China. The genotypes were determined by polymerase chain reaction-restricted fragment length polymorphism (PCR-RFLP) technique with genomic DNA. No individuals had the CYP 2D6 c.212 G>A variant alleles in this study. There is no association between the UGT1A6 c.181 T>A, UGT1A7 c.208 Trp>Arg, and SULT1A1 c.638 G>A genotypes and increased risk of benzene-induced carcinogenesis. Although most of the CYP2D6 haplotypes did not show any significant difference, the CYP2D6 haplotype CYP2D6 c.188 C/C, C/T, and c.4268 C/C was significantly overrepresented in the case group (OR 4.02, 95% CI: 2.53-6.39) compared with in controls. Overall, our data suggested that individuals with CYP1A1 c.5639 T/T, CYP2D6 c.188 C/C, C/T, and CYP2D6 c.4268 C/C genotypes tend to be more susceptible to benzene toxicity.     

Interactions between human UGT1A1, UGT1A4, and UGT1A6 affect their enzymatic activities.

Protein-protein interactions between human UDP-glucuronosyltransferase (UGT) 1A1, UGT1A4, and UGT1A6 were investigated using double expression systems in HEK293 cells (UGT1A1/UGT1A4, UGT1A1/UGT1A6, and UGT1A4/UGT1A6). The substrates specific for UGT1A1 (estradiol and bilirubin), UGT1A4 (imipramine and trifluoperazine), and UGT1A6 (serotonin and diclofenac) were used to determine the effects of the coexpression of the other UGT1A isoforms on the enzymatic activity. The coexpression of UGT1A4 and UGT1A6 decreased the S(50) and V(max) values of UGT1A1-catalyzed estradiol 3-O-glucuronide formation and increased the V(max) value of UGT1A1-catalyzed bilirubin O-glucuronide formation. The coexpression of UGT1A1 decreased the V(max) value of UGT1A4-catalyzed imipramine N-glucuronide formation but had no effect on UGT1A4-catalyzed trifluoperazine N-glucuronide formation. The coexpression of UGT1A6 had no effect on UGT1A4-catalyzed imipramine N-glucuronide formation but increased the K(m) and V(max) of UGT1A4-catalyzed trifluoperazine N-glucuronide formation. The coexpression of both UGT1A1 and UGT1A4 increased the V(max) values of UGT1A6-catalyzed serotonin and diclofenac O-glucuronide formation. Thus, the effects of the coexpression of other UGT1A isoforms on the kinetics of specific activities were different depending on the UGT1A isoforms and substrates. Native polyacrylamide gel electrophoresis analysis of the double expression systems showed multiple bands at approximately 110 kDa, indicating the existence of heterodimers as well as homodimers of UGTs. In conclusion, we found that human UGT1A1, UGT1A4, and UGT1A6 interact with each other, possibly by heterodimerization, and that their effects on the enzymatic activities are complex depending on the isoforms and substrates.     

Genetic variants of human UGT1A3: functional characterization and frequency distribution in a Chinese Han population.

UDP-glucuronosyltransferase 1A3 (UGT1A3) contributes to glucuronidation of many important endogenous compounds and xenobiotics, including some flavonoids. Recently, a total of six single nucleotide polymorphisms (SNPs) have been identified in the human UGT1A3 gene. Among them, four SNPs (A17G, Q6R; T31C, W11R; C133T, R45W; and T140C, V47A) cause amino acid substitutions. Variants caused by these SNPs showed an activity change in estrone metabolism, whereas their activities toward other substrates were not examined. In the present study, three common flavonoids, quercetin, luteolin, and kaempferol, were used as substrates for glucuronidation by wild-type and variant UGT1A3s. Our results demonstrated that the activities of three variants, UGT1A3.2, UGT1A3.3, and UGT1A3.5, were remarkably lower than that of UGT1A3.1. In contrast, UGT1A3.4 exhibited an increase in glucuronidation efficiency of approximately 4 times and a clear preference to quercetin 7- and 3-hydroxyl groups. The frequency distributions of UGT1A3 alleles and SNPs in UGT1A3 in a Chinese Han population were statistically different from the reported value in German-Caucasians (p < 0.05). UGT1A3 variants have an altered glucuronidation activity toward quercetin, luteolin, and kaempferol and may alter human susceptibility to flavonoid exposure.     

Novel genetic variations and haplotypes of hepatocyte nuclear factor 4alpha (HNF4A) found in Japanese type II diabetic patients

Thirty-nine single nucleotide variations, including 16 novel ones, were found in the 5' promoter region, all of the exons and their surrounding introns of HNF4A in 74 Japanese type II diabetic patients. The following novel variations were identified (based on the amino acid numbering of splicing variant 2): -208G>C in the 5' promoter region; 1154C>T (A385V) and 1193T>C (M398T) in the coding exons; 1580G>A, 1852G>T, 2180C>T, 2190G>A, and 2362_2380delAAGAATGGTGTGGGAGAGG in the 3'-untranslated region, and IVS1+231G>A, IVS2-83C>T, IVS3+50C>T, IVS3-54delC, IVS5+173_176delTTAG, IVS5-181_-180delAT, IVS8-106A>G, and IVS9-151A>C in the introns. The allele frequencies were 0.311 for 2362_2380delAAGAATGGTGTGGGAGAGG, 0.054 for 1580G>A, 0.047 for 1852G>T, 0.020 for IVS1+231G>A, 0.014 for IVS9-151A>C, and 0.007 for the other 11 variations. In addition, one known nonsynonymous single nucleotide polymorphism, 416C>T (T139I), was detected at a 0.007 frequency. Based on the linkage disequilibrium profiles, the region analyzed was divided into three blocks. Haplotype analysis determined/inferred 10, 16, and 12 haplotypes for block 1, 2, and 3, respectively. Our results on HNF4A variations and haplotypes would be useful for pharmacogenetic studies in Japanese.     

UDP-glucuronosyltransferase 1A4 polymorphisms in a Japanese population and kinetics of clozapine glucuronidation.

The UDP-glucuronosyltransferase (UGT) family plays a major role in the excretion of endobiotics and xenobiotics and their metabolites. Human UGT1A4 catalyzes the glucuronidation of primary, secondary, and tertiary amines, sapogenins, androgens, and progestins. We directly sequenced polymerase chain reaction-amplified fragments of the UGT1A4 gene from 100 healthy adult Japanese volunteers and calculated their mutation frequency. We identified four single nucleotide polymorphisms (SNPs): three in exon 1 (142T > G: L48V, 448T > C: L150L, 804G > A: P268P), and one in intron 1 (867 + 43C > T). We found three types of alleles with distinct SNP combinations that coded for different amino acid sequences: L48V-L150L-P268P-867 + 43C > T (frequency, 0.155), L48V (0.01), and P268P (0.01) (wild-type frequency was 0.825). The L48V mutant gave twice the efficiency (V(max)/K(m)) for the antipsychotic drug clozapine as the wild-type. Efficiencies of L48V for trans-androsterone, imipramine, and cyproheptadine were increased, but the efficiency for tigogenin was reduced. L48V therefore increased or decreased the glucuronidation activity, depending upon the substrates. This study shows the importance of identifying patients with the L48V polymorphism when calculating dosage, and when considering the potential adverse effects of drugs that are substrates of UGT1A4.     

Two novel haplotypes of CYP2D6 gene in a Japanese population

Two novel haplotypes of CYP2D6 were found in Japanese subjects. One haplotype of the human CYP2D6 gene, newly designated as CYP2D6(*)44 allele, had both a novel single nucleotide polymorphism (SNP) of 2950G>C in intron 6 donor splice junction and a known SNP (82CG, -1235A>G, -740C>T, -678G>A, and a gene conversion with CYP2D7 gene in intron 1 associated with CYP2D6(*)21. Both CYP2D6(*)44 and CYP2D6(*)21B alleles would cause a splicing error or a frameshift with impaired drug metabolizing function mediated by CYP2D6.     

CYP1A1 and CYP3A4 polymorphic variations in Delhi population of Northern India

Cytochrome P450 (CYP) 1A1 and CYP3A4 are important phase I xenobiotic metabolizing enzymes involved in the metabolism of numbers of toxins, endogenous hormones and pharmaceutical drugs. Polymorphisms in these phase I genes can alter enzyme activity and are known to be associated with cancer susceptibility related to environmental toxins and hormone exposure. Their genotypes may also display ethnicity dependent population frequencies. The present study was aimed to determine the frequencies of commonly known functional polymorphisms of CYP1A1 and CYP3A4 in North Indian population. Allelic frequency of CYP1A1 polymorphisms, m1, m2 and m4 were observed to be 40.3, 31.2 and 0% respectively. Frequency of CYP3A4*1B polymorphism was 0%. We observed inter as well as intra ethnic variation in the distribution of frequency of these polymorphisms. Analysis of polymorphisms in these genes might help in predicting the risk of cancer. Our results emphasize the need for more such studies in "high risk populations".