Novel detection assay by PCR-RFLP and frequency of the CYP3A5 SNPs,CYP3A5*3 and *6, in a Japanese population

In this study, we established useful and reliable methods for the direct detection of the variants of CYP3A5 gene by polymerase chain reaction (PCR) and DdeI restriction analysis. The frequency of CYP3A5 related SNPs in 200 healthy Japanese male subjects was determined. The homozygous wild-type (*1/*1) frequency was 7.0% (14/200), the heterozygous (*1/*3) frequency was 32.5% (65/200) and the homozygous mutant-type (*3/*3) frequency was 60.5% (121/200). The *6 allele was not detected in any of the Japanese individuals. This result suggests that an estimated 40% of the Japanese express relatively high levels of metabolically active CYP3A5 protein. The proposed detection assays are useful for screening the CYP3A5 related SNPs in pharmacogenetic research.     

CYP3A5 genetic polymorphisms in different ethnic populations.

Cyp3A5 activity varies within any given ethnic population, suggesting that genetic variation within the Cyp3A5 gene may be the most important contributor to interindividual and interracial differences in Cyp3A-dependent drug clearance and response. The full extent of Cyp3A5 polymorphism in a white and an indigenous African population was analyzed using DNA direct sequencing procedures. The presence of 10 and 12 single nucleotide polymorphisms was detected in the white and African samples, respectively. Thirteen novel mutations occurring at low frequencies were identified in these populations. Significant differences were observed in the distribution of Cyp3A5*3, Cyp3A5*6, and Cyp3A5*7 alleles among white and African populations. The frequency of Cyp3A5*3 allele in white Canadians (approximately 93%) is higher than in Zimbabweans (77.6%) (p < 0.001). In contrast, Cyp3A5*6 and Cyp3A5*7 alleles are relatively frequent in African subjects (10-22%) but absent in white subjects (p < 0.001). These differences may reflect evolutionary pressures generated by environmental factors in geographically distinct regions. However, the genetic polymorphism of Cyp3A5 alone does not explain the interindividual differences in Cyp3A-mediated metabolism.     

Ethnic-related differences in the frequency distribution of genetic polymorphisms in the CYP1A1 and CYP1B1 genes in Japanese and Caucasian populations

1. Race-related differences in the frequency distribution of genetic polymorphisms in the CYP1A1 and CYP1B1 genes were studied in 39 Japanese and 45 Caucasians. 2. Four types of CYP1A1 polymorphism, namely m1 (a nucleotide change at T6235C in the 3'-flanking region), m2 (A4889G at exon 7), m3 (T5639C in the 3'-flanking region) and m4 (C4887A at exon 7), and three types of CYP1B1 genetic polymorphism, namely m1 (C488G and G701T leading to Arg48Gly and Ala119Ser exchanges respectively), m2 (C1294G leading to a Leu432Val exchange) and m3 (A1358G leading to an Asn453Ser exchange) were studied. 3. The distribution of the m1-, m2-, m3-, and m4-types of CYP1A1 polymorphism in the Japanese population was 30.8, 17.9, 0 and 0% respectively; those in Caucasians were 3.3, 6.7, 0 and 2.2% respectively. Two types (m1, and m2) of CYP1B1 polymorphism were expressed at 14.1 and 21.8% respectively in the Japanese, and by 28.9 and 37.5% respectively in the Caucasian. Ethnic differences were also noted in the m3-type CYP1B1 polymorphism in which the incidence in Caucasians was 23.9%, whereas no cases in the 39 Japanese subjects were observed. 4. No apparent association was found in the incidence in each of the genetic polymorphisms of CYP1A1 and CYP1B1 genes, nor in methylenetetrahydrofolate reductase gene, except that the occurrence of the m2-type of CYP1A1 genetic polymorphism was related to that of the m1-type CYP1A1 polymorphism in the Japanese population. 5. These results suggest that there are race-related differences in the occurrence of genetic polymorphisms in both CYP1A1 and CYP1B1 genes in Japanese and Caucasian populations and that these differences in P450 genetic polymorphisms may, in part, cause differences in the occurrence of lung and breast cancers in these ethnic groups.     

Ethnic-related differences in the frequency distribution of genetic polymorphisms in the CYP1A1 and CYP1B1 genes in Japanese and Caucasian populations

1. Race-related differences in the frequency distribution of genetic polymorphisms in the CYP1A1 and CYP1B1 genes were studied in 39 Japanese and 45 Caucasians. 2. Four types of CYP1A1 polymorphism, namely m1 (a nucleotide change at T6235C in the 3'-flanking region), m2 (A4889G at exon 7), m3 (T5639C in the 3'-flanking region) and m4 (C4887A at exon 7), and three types of CYP1B1 genetic polymorphism, namely m1 (C488G and G701T leading to Arg48Gly and Ala119Ser exchanges respectively), m2 (C1294G leading to a Leu432Val exchange) and m3 (A1358G leading to an Asn453Ser exchange) were studied. 3. The distribution of the m1-, m2-, m3-, and m4-types of CYP1A1 polymorphism in the Japanese population was 30.8, 17.9, 0 and 0% respectively; those in Caucasians were 3.3, 6.7, 0 and 2.2% respectively. Two types (m1, and m2) of CYP1B1 polymorphism were expressed at 14.1 and 21.8% respectively in the Japanese, and by 28.9 and 37.5% respectively in the Caucasian. Ethnic differences were also noted in the m3-type CYP1B1 polymorphism in which the incidence in Caucasians was 23.9%, whereas no cases in the 39 Japanese subjects were observed. 4. No apparent association was found in the incidence in each of the genetic polymorphisms of CYP1A1 and CYP1B1 genes, nor in methylenetetrahydrofolate reductase gene, except that the occurrence of the m2-type of CYP1A1 genetic polymorphism was related to that of the m1-type CYP1A1 polymorphism in the Japanese population. 5. These results suggest that there are race-related differences in the occurrence of genetic polymorphisms in both CYP1A1 and CYP1B1 genes in Japanese and Caucasian populations and that these differences in P450 genetic polymorphisms may, in part, cause differences in the occurrence of lung and breast cancers in these ethnic groups.     

Comparative metabolic capabilities of CYP3A4, CYP3A5, and CYP3A7.

The human cytochromes P450 (P450) CYP3A contribute to the biotransformation of 50% of oxidatively metabolized drugs. The predominant hepatic form is CYP3A4, but recent evidence indicates that CYP3A5 contributes more significantly to the total liver CYP3A than was originally thought. CYP3A7 is the major fetal form and is rarely expressed in adults. To compare the metabolic capabilities of CYP3A forms for 10 substrates, incubations were performed using a consistent molar ratio (1:7:9) of recombinant CYP3A, P450 reductase, and cytochrome b5. A wide range of substrate concentrations was examined to determine the best fit to kinetic models for metabolite formation. In general, K(m) or S(50) values for the substrates were 3 to 4 times lower for CYP3A4 than for CYP3A5 or CYP3A7. For a more direct comparison of these P450 forms, clearance to the metabolites was determined as a linear relationship of rate of metabolite formation for the lowest substrate concentrations examined. The clearance for 1'-hydroxy midazolam formation at low substrate concentrations was similar for CYP3A4 and CYP3A5. For CYP3A5 versus CYP3A4, clearance values at low substrate concentrations were 2 to 20 times lower for the other biotransformations. The clearance values for CYP3A7-catalyzed metabolite formation at low substrate concentrations were substantially lower than for CYP3A4 or CYP3A5, except for clarithromycin, 4-OH triazolam, and N-desmethyl diltiazem (CYP3A5 - CYP3A7). The CYP3A forms demonstrated regioselective differences in some of the biotransformations. These results demonstrate an equal or reduced metabolic capability for CYP3A5 compared with CYP3A4 and a significantly lower capability for CYP3A7.     

Genetic polymorphisms of the CYP3A4, CYP3A5, CYP3A7 and CYP1A2 among the Jordanian population

Cytochromes P450 (CYP450) plays an extremely vital role in oxidation, reduction, and peroxidation of numerous endogenous and exogenous compounds, like drugs and procarcinogens. Mainly, expression occurs in the liver, in varying polymorphic forms. Therefore, proposed as biomarkers of susceptibility to carcinogenicity and toxicity. The objective of this study was to find the allelic frequencies of CYP3A5*2,*3,*4,*5,*6,*7, CYP3A4*1B, CYP3A7*1C and CYP1A2*1C, *1D, *1E, *1F enzymes in Jordanians, and to compare them with other ethnic groups. We used polymerase chain reaction-restriction fragment length (PCR-RFLP) to genotype alleles, and we calculated frequencies using Hardy Weinberg's equation (HWE). Allelic frequencies results were: CYP3A5*2 (0.2%), CYP3A5*3 (86.6%), CYP3A5*6 (1.7%), CYP*3A5*4,*5*7 not detected, CYP3A4*1B (11.7%), CYP3A7*1C (1.7%). Finally 6.5%, 18.2%, 6.0%, 67.3% were the results of CYP1A2*1C, 1D, 1E and 1F, respectively. In conclusion, genotyping method and results of this study can be adopted or used in pharmacotherapy, toxicity and carcinogenic studies in Jordan.     

Differences in CYP3A5*3 genotype distribution and combinations with other polymorphisms between Spaniards and Other Caucasian populations

The goal of this study was to detect genotypic differences between Spaniards and other related populations regarding CYP3A4*1B, CYP3A5*3, and ABCB1 (MDR1) C3435T polymorphisms. DNA from 177 Spanish patients were analyzed for the presence of these mutations using PCR-restriction fragment length polymorphism or direct sequencing. The observed frequencies for CYP3A4*1B, CYP3A5*3, and C3435T alleles were within normal values in Caucasians (0.04, 0.91, and 0.5, respectively). However, 2.8% of the patients were homozygous for the wild-type CYP3A5*1 allele, an extremely uncommon genotype in other Caucasians. In addition, analysis of CYP3A4-3A5 haplotypes revealed the existence of 2 unusual subgroups: patients who were homozygous wild-type for both polymorphisms, and patients showing a CYP3A4*1A/*1B-CYP3A5*3/*3 genotype combination. The incidence of CYP3A5*1/*1 carriers and the occurrence of subjects combining the 2 above-mentioned unusual genotype combinations were more frequent in Spanish-Caucasians compared with American- or European-Caucasians. ABCB1 C3435T genotype frequencies were equally distributed between both single and combined CYP3A4 and 3A5 genotypes. These findings suggest that dose requirements for drugs metabolized by CYP3A and certain allele-disease association studies in white populations could show discrepancies in Spaniards.     

CYP3A5 polymorphism and the ethnic differences in cyclosporine pharmacokinetics in healthy subjects

To determine the relationship between CYP3A5 polymorphism and cyclosporine pharmacokinetic parameters among healthy volunteers, an oral cyclosporine (CsA) pharmacokinetic study was performed in 16 healthy subjects. Blood CsA concentrations were measured by high-performance liquid chromatography. Concentration-versus-time data were analyzed by a noncompartmental method using WinNonLin, and the blood samples were genotyped for the CYP3A5 using the polymerase chain reaction and pyrosequencing. CsA pharmacokinetic parameters were dichotomized and compared using the 1-way ANOVA test according to the CYP3A5*3C genotype. There were 6 homozygous A/A (wild type), 6 homozygous G/G (variant), and 4 heterozygous A/G genotypes for CYP3A5*3 C in these 16 healthy volunteers. All whites were G/G group, and all African Americans except 1 were either A/A or A/G group. The mean AUC (ng x h/mL) of CsA for the 3 genotype groups were 4962 +/- 1074 (A/A), 6677 +/- 1153 (G/G), and 5416 +/- 1817 (A/G), (A/A versus G/G, P = 0.03), and the mean CL/F (mL/min/kg) were 15.6 +/- 3.1 (A/A), 12.0 +/- 2.3 (G/G), and 14.7 +/- 5.9 (A/G), (A/A versus G/G, P = 0.04). None of the other parameters were significantly different among the 3 genotypes. In conclusion, the CYP3A5*3C polymorphism appears to affect AUC and CL/F of oral CsA significantly in healthy subjects, which may partly explain some of the differences of pharmacokinetics in CsA between African Americans and whites.     

Growth hormone regulation and developmental expression of rat hepatic CYP3A18, CYP3A9, and CYP3A2

The present study investigated the role of growth hormone (GH) in hepatic CYP3A18 and CYP3A9 expression in prepubertal and adult male rats. For comparison, the effects of GH on CYP3A2 expression were also measured. Initial experiments demonstrated that CYP3A18 mRNA levels were greater during puberty and adulthood than during the prepubertal period, CYP3A9 mRNA was not expressed until puberty and its expression increased in adulthood, and CYP3A2 mRNA levels were relatively constant from prepuberty to adult life. Hypophysectomy, which results in the loss of multiple pituitary factors including GH, increased CYP3A2 and CYP3A18 mRNA expression 3- to 4-fold, but it did not affect CYP3A9 mRNA levels or CYP3A-mediated testosterone 2beta- or 6beta-hydroxylase activity in adult rats. GH administered as twice daily s.c. injections (0.12 microg/g body weight) to hypophysectomized or intact adult rats did not affect CYP3A18 or CYP3A9 mRNA expression. The same treatment decreased CYP3A2 mRNA and protein and testosterone 2beta- and 6beta-hydroxylase activity levels in intact but not hypophysectomized rats. However, in intact prepubertal rats, intermittent GH administration decreased CYP3A18 and CYP3A2 mRNA levels, but a higher dosage (3.6 microg/g) was required to suppress CYP3A2. Overall, the present study demonstrated that: (a) the constitutive expression of CYP3A18, CYP3A9, and CYP3A2 does not require the presence of GH, (b) CYP3A18 is more sensitive than CYP3A9 to GH modulation in adult rats; and (c) CYP3A2 is less sensitive to the suppressive influence of GH during the prepubertal period than during adult life.     

Co-regulation of CYP3A4 and CYP3A5 and contribution to hepatic and intestinal midazolam metabolism

We recently demonstrated that a variant allele of CYP3A5 (CYP3A5*3) confers low CYP3A5 expression as a result of improper mRNA splicing. In this study, we further evaluated the regulation of CYP3A5 in liver and jejunal mucosa from white donors. For all tissues, high levels of CYP3A5 protein were strongly concordant with the presence of a wild-type allele of the CYP3A5 gene (CYP3A5*1). CYP3A5 represented greater than 50% of total CYP3A content in nearly all of the livers and jejuna that carried the CYP3A5*1 wild-type allele. Overall, CYP3A5 protein content accounted for 31% of the variability in hepatic midazolam hydroxylation activity. Improperly spliced mRNA (SV1-CYP3A5) was found only in tissues containing a CYP3A5*3 allele. Properly spliced CYP3A5 mRNA (wt-CYP3A5) was detected in all tissues, but the median wt-CYP3A5 mRNA was 4-fold higher in CYP3A5*1/*3 livers compared with CYP3A5*3/*3 livers. Differences in wt-CYP3A5 and CYP3A4 mRNA content explained 53 and 51% of the interliver variability in CYP3A5 and CYP3A4 content, respectively. Hepatic CYP3A4 and CYP3A5 contents were not correlated when all livers were compared. However, for CYP3A5*1/*3 livers, levels of the two proteins were strongly correlated (r = 0.93) as were wt-CYP3A5 and CYP3A4 mRNA (r = 0.76). These findings suggest that CYP3A4 and CYP3A5 genes share a common regulatory pathway for constitutive expression, possibly involving conserved elements in the 5'-flanking region.     

Allele and genotype frequencies of CYP2B6 and CYP3A5 in the Japanese population

OBJECTIVE: The goal of this study was to determine the frequencies of allelic variants of CYP2B6and CYP3A5 in the Japanese population. METHODS: Genotyping of CYP2B6 (*2, *3, *4, *5, *6, and *7) and CYP3A5 ( *2, *3, *4, *5, and *6) was carried out in 265 unrelated Japanese subjects by polymerase chain reaction (PCR), restriction fragment length polymorphism and allele-specific, real-time PCR assays. RESULTS: Allele frequencies for CYP2B6*2, *3, *4, *5, *6, and *7 in 256 Japanese subjects were 0.047, 0, 0.093, 0.011, 0.164, and 0, respectively. Ethnic variation in allele frequencies relative to that in Caucasian subjects was observed for CYP2B6*4 (0.093 vs 0.040), *5 (0.011 vs 0.109), *6 (0.164 vs 0.256), and *7 (0 vs 0.030). Allele frequencies for CYP3A5*2, *3, *4, *5, and *6 in 265 Japanese subjects were 0, 0.740, 0, 0.004, and 0, respectively. The frequency of the CYP3A5*1 allele is 2.8 times higher in Japanese than in Caucasians. CONCLUSIONS: Our results contribute to a better understanding of the molecular basis of ethnic differences in drug response, which may help to improve individualization of drug therapy and offer a preliminary basis for more rational use of drugs that are substrates for CYP2B6 and CYP3A5 in the Japanese population.     

Linkage between the distribution of mutations in the CYP2C18 and CYP2C19 genes in the Japanese and Caucasian

1. Two different types of genetic polymorphisms in each of CYP2C18 and CYP2C19 genes were examined and compared with respect to their frequencies in distribution in liver DNA of 39 Japanese and 45 Caucasians. 2. Individuals who were classified into CYP2C19m1 (as detected with SmaI digestion) in exon 5 of CYP2C19 gene were found to display a CYP2C18m1 polymorphism (as detected with DdeI digestion) in the 5-flanking region of CYP2C18 gene in Japanese and Caucasian populations. The Japanese subjects who were classified into CYP2C19m2 (as detected with BamHI digestion) in exon 4 of CYP2C19 gene were found to have a CYP2C18m2 genetic polymorphism (as detected with Tsp509I digestion) in exon 2 of CYP2C18 gene. None of the Caucasians had the CYP2C18m2 nor CYP2C19m2 alleles. 3. Frequencies in two types (C416T in exon 3, A1061C in exon 7) of CYP2C9 genetic polymorphism were found to be independent to those of CYP2C18 and CYP2C19 genetic polymorphisms in these samples. 4. Thus, the results suggest that the CYP2C18 gene is localized very closely to the CYP2C19 gene on the same human chromosome.     

No major difference in inhibitory susceptibility between CYP2C9.1 and CYP2C9.3

OBJECTIVE: CYP2C9 is a polymorphic enzyme, and CYP2C9*3 is associated with decreased metabolic activity. In addition to the impaired metabolism, we investigated whether the CYP2C9*3 exhibited altered inhibitory susceptibility compared with CYP2C9*1. METHOD: In the present study, CYP2C9.1 and CYP2C9.3 were expressed in yeast. Using typical CYP2C9 substrates (diclofenac, tolbutamide and S-warfarin) and a potent CYP2C9 inhibitor (nicardipine), the Ki values for nicardipine on the three metabolisms in CYP2C9*1 and CYP2C9*3 were determined. RESULT: The ratios of Ki(CYP2C9*3)/Ki(CYP2C9*1) on tolbutamide, diclofenac and S-warfarin metabolisms were 1.2, 3.1 and 0.8, respectively. CONCLUSION: In conclusion, there are no significant differences in the inhibitory susceptibility between the two CYP2C9 enzymes.