Identification of a novel CYP2D6 allele associated with poor metabolism of sparteine in a Japanese population.

The relationship between genotypes associated with restriction fragment length polymorphisms at the P450 CYP2D locus and the metabolic phenotypes for sparteine in healthy Japanese subjects was investigated. Four distinct restriction fragments of 44 kb, 29 kb, 11.5 kb or 16 + 9 kb after digestion with Xba I were observed. The genotype of Xba I 11.5 kb, which is predictive of the poor metabolizer phenotype in Caucasians is also associated with Japanese poor metabolizers. Most Xba I 44 kb are associated with the extensive metabolizer phenotype. However, one Xba I 44 kb haplotype in linkage disequilibrium with a Bam HI restriction fragment length polymorphism allele, designated Bam HI 2.3 kb-, appears to be a novel mutant CYP2D6 allele in Japanese subjects having the poor metabolizer phenotype.     

Characterization of the novel defective CYP2C9*24 allele.

CYP2C9 is one of the major drug-metabolizing enzymes, and it is involved in the oxidative metabolism of approximately 10% of clinically important drugs, among which some, such as the anticoagulant warfarin, have a narrow therapeutic index. The human CYP2C9 gene is highly polymorphic. We found a new sequence variation in exon 7 of the CYP2C9 gene (1060G>A) resulting in a substitution of acidic amino acid glutamate to basic lysine (E354K) when translated. The allele, designated CYP2C9(*)24, was present in heterozygous state in one warfarin-treated patient. To characterize the CYP2C9(*)24 allele, we expressed the wild-type and CYP2C9.24 protein in a recombinant yeast expression system and a human embryonic kidney (HEK)-293 cell system. Carbon monoxide difference spectra were recorded on dithionite-reduced microsomes, and protein was determined by Western blotting. Transfection with CYP2C9.1 cDNA resulted in detectable CYP2C9 protein in yeast or HEK-293 cells, whereas only small amounts of the protein were detected in yeast transfected with CYP2C9.24 cDNA. A strong differential absorption peak at 450 nm was observed with microsomes of yeast transfected with CYP2C9.1 cDNA, whereas no peak was detected with microsomes of yeast transfected with CYP2C9.24 cDNA or empty pYeDP60 plasmid. These results suggest that CYP2C9.24 may be improperly folded, both in yeast and mammalian cells, resulting in improper heme incorporation and rapid intracellular degradation. The data obtained in the expression systems are consistent with our findings in vivo. In conclusion, we have identified a novel defective CYP2C9 variant allele of potential importance for drug metabolism in vivo.     

Functional analysis of three CYP1A2 variants found in a Japanese population.

Human cytochrome P450 1A2 (CYP1A2) catalyzes the metabolism of many important drugs and environmental chemicals. We previously reported three naturally occurring genetic polymorphisms (125C>G, Pro42Arg, CYP1A2*15; 1130G>A, Arg377Gln, *16; and 1367G>A, Arg456His, *8) found in a Japanese population. In this study, these variant enzymes were expressed in Chinese hamster V79 cells, and their mRNA and protein expression levels as well as catalytic activities were determined. All three variant enzymes showed reduced protein expression levels (66% for Pro42Arg and approximately 30% for Arg377Gln and Arg456His) compared with that of the wild type (WT) without any change in mRNA expression levels. Kinetic analysis for 7-ethoxyresorufin O-deethylation revealed that V(max) and V(max)/K(m) of all three variants were less than 3 and 1% of the WT, respectively, although the K(m) value was significantly increased only in the Arg377Gln variant (approximately a 9-fold increase). Markedly reduced activities of the three variants were also observed for phenacetin O-deethylation. In the reduced CO difference spectral analysis using recombinant proteins produced in the Sf21/baculovirus system, the peak at 450 nm seen in the WT protein was hardly observed in the three variants, suggesting marked reductions in their hemoprotein formation. These results suggest that Pro42, Arg377, and Arg456 are critical amino acids for the production of catalytically active CYP1A2 holoenzyme.     

A novel variant allele of OATP-C (SLCO1B1) found in a Japanese patient with pravastatin-induced myopathy

We have recently found that the frequency of OATP-C*15 is significantly higher in patients who experienced myopathy after receiving pravastatin or atorvastatin than in patients without myopathy. However, there were two patients who experienced pravastatin-induced myopathy despite the fact that they did not possess OATP-C*15 or other known mutations of OATP-C that have been reported to decrease the function of OATP-C. In this study, we sequenced all of the exons and exon-intron junctions of OATP-C of the two patients and found a novel mutation in exon 12 of OATP-C in one of the patients. In this mutation (1628T>G), there is a substitution of Leu to Trp at position 543 in transmembrane-spanning domain 10 of OATP-C. However, the frequency of this mutation in the Japanese population appears to be very low (<1%).     

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.     

Four novel defective alleles and comprehensive haplotype analysis of CYP2C9 in Japanese

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.     

Limited frequency of the CYP2C19*17 allele and its minor role in a Japanese population

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT
• A novel CYP2C19 gene variant, CYP2C19 * 17 , is associated with increased metabolic activity.
• Ethnic differences in the frequency of the variant allele have been reported. However, the frequency of the CYP2C19 * 17 allele has not been studied in the Japanese population.
WHAT THIS STUDY ADDS
• In a population of 265 healthy Japanese subjects, a low frequency (1.3%) of the CYP2C19 * 17 allele was observed.
• The limited frequency of the * 17 allele and the absence of a subject homozygous for * 17 indicated that CYP2C19 * 17 would play a minor role in a Japanese population.  

AIMS

We investigated the CYP2C19 * 17 allelic frequency in Japanese subjects, and evaluated whether CYP2C19 * 17 is an important determinant of interindividual variability of CYP2C19 activity.  

METHODS

We enrolled 265 subjects to determine their CYP2C19 genotype and plasma metabolic ratio following a single dose of 40 mg omeprazole.  

RESULTS

Seven subjects heterozygous for CYP2C19 * 17 and no * 17/ * 17 subjects resulted in the CYP2C19 * 17 frequency being 1.3%. These heterozygotes had moderate metabolic activities when compared with the metabolic ratio of the other subjects.  

CONCLUSIONS

The low frequency of CYP2C19 * 17 and the absence of * 17/ * 17 indicates that CYP2C19 * 17 plays a minor role in the Japanese population.     

Functional characterization of two novel CYP2C19 variants (CYP2C19*18 and CYP2C19*19) found in a Japanese population

Cytochrome P450 2C19 (CYP2C19) plays an important role in the metabolism of a wide range of therapeutic drugs and exhibits genetic polymorphism with interindividual differences in metabolic activity. We have previously described two CYP2C19 allelic variants, namely CYP2C19*18 and CYP2C19*19 with Arg329His/Ile331Val and Ser51Gly/Ile331Val substitutions, respectively. In order to investigate precisely the effect of amino acid substitutions on CYP2C19 function, CYP2C19 proteins of the wild-type (CYP2C19.1B having Ile331Val) and variants (CYP2C19.18 and CYP2C19.19) were heterologously expressed in yeast cells, and their S-mephenytoin 4'-hydroxylation activities were determined. The K(m) value of CYP2C19.19 for S-mephenytoin 4'-hydroxylation was significantly higher (3.0-fold) than that of CYP2C19.1B. Although no significant differences in V(max) values on the basis of microsomal and functional CYP protein levels were observed between CYP2C19.1B and CYP2C19.19, the V(max)/K(m) values of CYP2C19.19 were significantly reduced to 29-47% of CYP2C19.1B. By contrast, the K(m), V(max) or V(max)/K(m) values of CYP2C19.18 were similar to those of CYP2C19.1B. These results suggest that Ser51Gly substitution in CYP2C19.19 decreases the affinity toward S-mephenytoin of CYP2C19 enzyme, and imply that the genetic polymorphism of CYP2C19*19 also causes variations in the clinical response to drugs metabolized by CYP2C19.     

Functional characterization of two novel CYP2C19 variants (CYP2C19*18 and CYP2C19*19) found in a Japanese population

Cytochrome P450 2C19 (CYP2C19) plays an important role in the metabolism of a wide range of therapeutic drugs and exhibits genetic polymorphism with interindividual differences in metabolic activity. We have previously described two CYP2C19 allelic variants, namely CYP2C19*18 and CYP2C19*19 with Arg329His/Ile331Val and Ser51Gly/Ile331Val substitutions, respectively. In order to investigate precisely the effect of amino acid substitutions on CYP2C19 function, CYP2C19 proteins of the wild-type (CYP2C19.1B having Ile331Val) and variants (CYP2C19.18 and CYP2C19.19) were heterologously expressed in yeast cells, and their S-mephenytoin 4′-hydroxylation activities were determined. The K_m value of CYP2C19.19 for S-mephenytoin 4′-hydroxylation was significantly higher (3.0-fold) than that of CYP2C19.1B. Although no significant differences in V_max values on the basis of microsomal and functional CYP protein levels were observed between CYP2C19.1B and CYP2C19.19, the V_max/K_m values of CYP2C19.19 were significantly reduced to 29–47% of CYP2C19.1B. By contrast, the K_m, V_max or V_max/K_m values of CYP2C19.18 were similar to those of CYP2C19.1B. These results suggest that Ser51Gly substitution in CYP2C19.19 decreases the affinity toward S-mephenytoin of CYP2C19 enzyme, and imply that the genetic polymorphism of CYP2C19*19 also causes variations in the clinical response to drugs metabolized by CYP2C19.     

Haplotype structure and allele frequencies of CYP2B6 in a Korean population.

Cytochrome P450 2B6 (CYP2B6) metabolizes a number of therapeutic drugs and its metabolic activity varies markedly in human liver. Although genetic polymorphisms of CYP2B6 have been reported in noncoding and coding regions, little information is available regarding single nucleotide polymorphisms (SNPs) and their haplotypes in noncoding regions in Asians. Fourteen previously reported SNPs were determined by polymerase chain reaction-restriction fragment length polymorphism or SNaPshot analysis in a Korean population and their haplotypes were inferred from genotype data using an expectation-maximization algorithm. The most common haplotypes were haplotype I, the reference sequence (frequency 0.35), haplotype II (0.19), haplotype III (0.19), and haplotype V (0.12), which together accounted for 85% of all haplotypes. The frequency of haplotype III, which contains -2320C, -1778G, -1186G, -750C, and 15582T, was found to be 2.4-fold higher than that of the *1J allele in Caucasians, and the frequency of haplotype V, which contains -8207C, -1456C, -750C, 516T, and 785G, was 55% of that of the *6B allele in Caucasians. Moreover, haplotype V, the *6B allele, appeared to be completely linked to -8207 within a putative nuclear receptor binding motif, suggesting that lower expressions of the *6B allele may be associated with the presence of noncoding SNPs such as -8207G>C linked to nonsynonymous SNPs. In conclusion, we found 11 previously described polymorphisms and identified four major haplotypes of CYP2B6 in Koreans. The frequencies of the *1J or *6B alleles, which may reduce CYP2B6 enzyme expression, were found to be significantly different between Koreans and Caucasians.     

Identification of a novel variant CYP2C9 allele in Chinese

OBJECTIVES: Cytochrome P450 (CYP) 2C9 metabolizes about 16% of drugs in current clinical use, including lornoxicam and tolbutamide. SNPs in the CYP2C9 gene have increasingly been recognized as determinants of the metabolic phenotype that underlies interindividual and ethnic differences. METHODS: The present study focused on a Chinese poor metabolizer (PM) whose apparent genotype (CYP2C9*1/CYP2C9*3) did not agree with his PM phenotype for both lornoxicam and tolbutamide. By sequencing his CYP2C9 gene, we identified a new variant CYP2C9 allele involving a T269C transversion in exon 2 that leads to a Leu90Pro substitution in the encoded protein. RESULTS: The CYP2C9 genotype analysis in the family of the poor metabolizer showed the new exon 2 change and CYP2C9*3 occurred on different alleles. Thus, the PM status of this subject could be attributed to his being heterozygous for the CYP2C9 T269C allele together with the CYP2C9*3. Frequency analysis in 147 unrelated Chinese males indicated approximately 2% of the Chinese population carry the allele. CONCLUSION: This study suggests that this novel CYP2C9 allele was correlated with reduced plasma clearance of drugs that are substrates for CYP2C9.     

N-acetylation polymorphism of dapsone in a Japanese population.

1. The N-acetylation of dapsone (DDS) was studied in 182 unrelated healthy Japanese subjects. The frequency of slow acetylators determined using the plasma monoacetyldapsone (MADDS) to DDS ratio (MADDS/DDS, slow acetylators less than 0.30 and rapid acetylators greater than 0.35) at 3 h after an oral dose of DDS (100 mg) was 6.6% (12 of the 182 subjects) with a 95% confidence interval of 3.8 to 11.2%. 2. The frequency distribution histogram of the plasma MADDS/DDS ratio showed an apparent trimodal pattern. However, the numbers of heterozygous (n = 105) and homozygous rapid acetylators (n = 65) derived from the observed data did not agree with those predicted for the respective rapid acetylators (n = 70, and n = 100) by applying the Hardy-Weinberg Law, when the suggested antimode of 0.85 discriminating these two rapid acetylators was employed. 3. The incidence of slow acetylators was unexpectedly lower in the males (1.4%, 1 of the 69 subjects, with a 95% confidence interval of 0.2 to 7.7%) compared with the incidence in the females (9.7%, 11 of the 113 subjects, with a 95% confidence interval of 5.5 to 16.6%). The difference reached a marginally significant level (Fisher's exact probability test, P = 0.02). 4. The mean plasma concentration of MADDS was significantly (P less than 0.001) lower in the slow compared to the rapid acetylators and there was a highly significant correlation (rs = 0.757, P less than 0.001) between plasma MADDS levels and MADDS/DDS ratios. 5. Slow acetylators showed a significantly (P less than 0.001) lower urinary MADDS/DDS ratio and excreted less (P less than 0.001) MADDS than rapid acetylators.(ABSTRACT TRUNCATED AT 250 WORDS)     

Genetic polymorphisms of CYP2C8 in Japanese population.

CYP2C8 plays important roles in metabolizing therapeutic drugs and endogenous compounds. Although genetic polymorphisms of CYP2C8 were reported, there is little information on CYP2C8 polymorphisms in the Japanese population. In the present study, we screened for previously described polymorphisms in the coding region of this gene using polymerase chain reaction (PCR)-restriction fragment length polymorphism or allele specific-PCR analyses. Eleven polymorphisms of CYP2C8*2 (I269F), CYP2C8*3 (R139K, K399R), CYP2C8*4 (I264M), CYP2C8*5 (frameshift), T130N, E154D, N193K, K249R, L390S, P404A, and H411L have been comprehensively investigated in at least 200 Japanese individuals. A single subject was heterozygous for CYP2C8*5, and the allele frequency was calculated as 0.0025. The other single nucleotide polymorphisms (SNPs) were not found in the Japanese subjects in the present study. Thus, it appears that the frequencies of these alleles in Japanese are extremely low. In addition, concerning the SNPs of T130N, E154D, N193K, K249R, and H411L, it remains clear that these alleles exist as polymorphisms or represent sequence errors or cloning artifacts. Although several SNPs such as CYP2C8*2, CYP2C8*3, CYP2C8*4, and P404A have been reported to reduce the enzymatic activity, pharmacokinetic abnormalities of drugs metabolized by polymorphic CYP2C8 might be rare in Japanese.     

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.     

Genotype and allele frequency of CYP2D6 in Tamilian population

Objective To assess the frequency of CYP2D6 *3, *4, *5 and *10 allelic variants in a South Indian population and compare the frequencies with other major populations.Methods Polymerase chain reaction (PCR) or PCR-restriction fragment length polymorphism (RFLP)-based methods were used to identify the CYP2D6 genotypes of 106 healthy unrelated male and female volunteers of Tamilian origin. The allele and genotype frequencies observed were compared with other major populations.Results The *10 allele was the most frequent mutant allele in Tamilians (20.3%). The *5 allele occurred at 0.9% and the *3 allele was not detected. The most frequent allele causing enzyme inactivation was *4 allele in Tamilians (6.6%), which is significantly higher than that reported in Japanese (0%).Conclusions The *10 allele is the most common mutant allele in Tamilians. The CYP2D6*4 and CYP2D6*5 alleles are distributed in a significantly different way in the Tamil population relative to Oriental populations.     

Three novel single nucleotide polymorphisms of the FMO3 gene in a Japanese population

We sequenced all exons and exon-intron junctions of the flavin-containing monooxygenase 3 (FMO3) gene from 2 Japanese individuals and their family members, who were case subjects that showed low FMO3 metabolic capacity among a population of self-reported trimethylaminuria Japanese volunteers. We found two novel single nucleotide polymorphisms (SNPs) (21,254 C>A and 24,006 A>G) causing amino acid substitutions, Thr(201)Lys in exon 5 and Met(260)Val in exon 6, respectively. The Thr(201)Lys and Met(260)Val also presented together with known SNPs (Glu(158)Lys-Glu(308)Gly and Val(257)Met, respectively) in the same alleles of the FMO3 gene to form novel haplotypes. A SNP (30,398 C>T) in the FMO3 gene causing a stop codon at Arg(500) in exon 9 was also discovered. These sequences are as follows: 1) SNP, 060116Shimizu001; GENE NAME, FMO3; ACCESSION NUMBER, AL021026; LENGTH, 25 base; 5'-GTGATATTGCCAC/AAGAACTCAGCCG-3'. 2) SNP, 060116Shimizu002; GENE NAME, FMO3; ACCESSION NUMBER, AL021026; LENGTH, 25 base; 5'-TAC(G/A)TGAAGCAGA/GTGAATGCAAGAT-3'. 3) SNP, 060116Shimizu003; GENE NAME, FMO3; ACCESSION NUMBER, AL021026; LENGTH, 25 base; 5'-CCCATGCAGACAC/TGAGTGGTCGGGA-3'.     

Limited effects of frequent CYP2D6*36-*10 tandem duplication allele on in vivo dextromethorphan metabolism in a Japanese population

Purpose  

Although CYP2D6*36 was thought to be one of the alleles causing the poor metabolizer phenotype, several in vitro studies clarified that the enzyme produced by CYP2D6*36 showed enzymatic activities. However, the effects of CYP2D6*36 in tandem with CYP2D6*10 on the in vivo CYP2D6 activity have been unclear. In this study, we investigated in vivo metabolic capacities of CYP2D6 among the subjects carrying different numbers of CYP2D6*36 in tandem with CYP2D6*10.