Genetic polymorphisms and haplotype structures of the human CYP2W1 gene in a Japanese population.

A novel human cytochrome P450, designated CYP2W1, has recently been identified and is found to be present mainly in tumor cells, particularly in colon cancer cells. In the present study, we report the first systematic investigation of polymorphisms in the human CYP2W1 gene. Based on denaturing high performance liquid chromatography analyses of polymerase chain reaction products, we analyzed nine exons and exon-intron junctions of the gene in DNA samples from 200 Japanese subjects and identified six single nucleotide polymorphisms (SNP). Three of the novel nonsynonymous SNPs were as follows: 173A>C (Glu58Ala) in exon 1 and 5432G>A (Val432Ile) and 5584G>C (Gln482His) in exon 9. Two previously known nonsynonymous SNPs, that is, 2008G>A (Ala181Thr) in exon 4 and 5601C>T (Pro488Leu) in exon 9, were also found. On haplotype analyses, in addition to the wild-type CYP2W1*1A (frequency, 0.295) allele, other alleles, namely, CYP2W1*1B (0.318), CYP2W1*2 (0.005), CYP2W1*3 (0.005), CYP2W1*4 (0.008), CYP2W1*5 (0.003), and CYP2W1*6 (0.368), were also characterized. The most common allele, CYP2W1*6, exhibited the amino acid substitution Pro488Leu. These results were in good agreement with the expected genotype distributions that were calculated using the Hardy-Weinberg equation. The data on variant alleles and comprehensive haplotype structures would be useful for predicting the metabolic phenotypes of CYP2W1 substrates in the Japanese population.

Functional characterization of 17 CYP2D6 allelic variants (CYP2D6.2, 10, 14A-B, 18, 27, 36, 39, 47-51, 53-55, and 57).

Cytochrome P450 2D6 (CYP2D6) is an enzyme of potential importance for the metabolism of drugs used clinically, and it exhibits genetic polymorphism with interindividual differences in metabolic activity. To date, 21 CYP2D6 allelic variants have been identified in the Japanese population. The aim of this study was to investigate the functional characterization of CYP2D6 variants identified in Japanese subjects. Wild-type CYP2D6 and its variants, namely, CYP2D6.2, CYP2D6.10, CYP2D6.14A, CYP2D6.14B, CYP2D6.18, CYP2D6.27, CYP2D6.36, CYP2D6.39, CYP2D6.47, CYP2D6.48, CYP2D6.49, CYP2D6.50, CYP2D6.51, CYP2D6.53, CYP2D6.54, CYP2D6.55, and CYP2D6.57 were transiently expressed in COS-7 cells, and enzymatic activities of the CYP2D6 variant proteins were characterized using bufuralol and dextromethorphan. Functional characterization of 17 CYP2D6 variants revealed an absence of enzyme activity in four (CYP2D6.14A, CYP2D6.36, CYP2D6.47, and CYP2D6.57), low activity in eight (CYP2D6.10, CYP2D6.14B, CYP2D6.18, CYP2D6.49, CYP2D6.50, CYP2D6.51, CYP2D6.54, and CYP2D6.55), and high activity in one (CYP2D6.53) compared with the wild type. Analysis of CYP2D6 variant proteins can be useful for predicting CYP2D6 phenotypes and could be applied to personalized drug therapy.

Possible relationship between the risk of Japanese bladder cancer cases and the CYP4B1 genotype.

Cytochrome P450 4B1 (CYP4B1) is involved in the metabolism of several xenobiotics, such as 2-aminofluorene, 2-naphthylamine and benzidine. CYP4B1 allelic variants CYP4B1*1-*7 were recently identified. We thus hypothesized that CYP4B1 genotypes may modify bladder cancer risk. We examined the CYP4B1 genotypes in 169 bladder cancer cases and 190 hospital controls using a hybridization probe assay. Among the CYP4B1 genotypes observed, the most frequent genotypes in both the groups were CYP4B1*1/*1, *1/*2, *1/*3 and *2/*2. Logistic regression analysis revealed that the subjects carrying the CYP4B1*1/*2 or *2/*2 genotypes had a 1.75-fold increased risk of bladder cancer (95% CI=1.03-2.95, P = 0.038) compared with the subjects carrying the CYP4B1*1/*1 genotype. We demonstrated the first genetic study regarding the association of CYP4B1 with bladder cancer. Our results suggest that CYP4B1 genotypes might have an effect on the risk of bladder cancer.

Three novel single nucleotide polymorphisms (SNPs) of CYP2S1 gene in Japanese individuals.

We analyzed all nine exons and exon-intron junctions of the CYP2S1 gene in 200 Japanese individuals and identified the following three novel single nucleotide polymorphisms (SNPs): 4612G>A (Glu147Glu) in exon 3, 5478C>T (Leu230Leu) and 5479T>G (Leu230Arg, CYP2S1*5A) in exon 5. The allele frequencies were 0.013 for 4612G>A, 0.058 for 5478C>T, and 0.003 for 5479T>G. In addition, a known SNP 1324C>G (Pro74Pro) was detected at a frequency of 0.300.

Genetic testing for pharmacogenetics and its clinical application in drug therapy.

There is wide individual variation in drug responses and adverse effects. As the main causes of the variation in drug responses, attention has focused on the genetic polymorphisms that encode metabolic enzymes regulating pharmacodynamics and receptors modulating the affinity with the responsive sites. Tailor-made drug therapy analyzes genetic polymorphisms involved in drug responses before drug administration and selects drugs and doses suitable for the individual genetic background. Establishment of tailor-made drug therapy is expected to contribute to medical economy by avoiding wasteful drug administration. To promote such medical practice, it is necessary to use simple genetic testing that is clinically convenient. Currently, genetic testing using real-time PCR has been frequently employed at laboratories with its clinical application anticipated. As to the many genes involved in drug responses, to date, the application of patient genetic information to tailor-made drug therapy has been achieved at the practical level. Information on pharmacogenetics will be a critical factor in medical practice in the near future.

Three novel single nucleotide polymorphisms of the human thiopurine S-methyltransferase gene in Japanese individuals.

In this study, the entire coding sequence and the exon-intron junctions of the thiopurine S-methyltransferase (TPMT) gene from 200 Japanese individuals were screened for mutation. Three novel single nucleotide polymorphisms (SNPs) were identified-106G>A in exon 3 (Gly36Ser, *20 allele), 967A>G in 3'-untranslated region, and -87C>T in intron 8. The allele frequencies were 0.003 for 106G>A, 0.003 for 967A>G, and 0.010 for IVS8 -87C>T. In addition, the three known SNPs, 474T>C (Ile158Ile), 719A>G (Tyr240Cys, *3C allele), and IVS4 +35C>T were detected at frequencies of 0.299, 0.010, and 0.421, respectively.

Gender difference in association between polymorphism of serotonin transporter gene regulatory region and anxiety.

OBJECTIVE: The objective of this study was to verify the hypothesis that variation of the serotonin transporter gene promoter region (5-HTTLPR) is associated with sensitivity to stress. METHODS: Genotyping of 5-HTTLPR and evaluation of emotional states were performed on 194 participants. Participants' emotional states were evaluated using the Perceived-Stress Scale (PSS), the State-Trait Anxiety Inventory (STAI), and the Self-rating Depression Scale (SDS). RESULTS: There was significant GenderxGenotype interaction in STAI (state, P<.05; trait, P<.05). Females with the l/s genotype showed higher anxiety than those with the s/s genotype in both state and trait anxiety. Oppositely, males with the s/s genotype showed higher anxiety than those with the l/s genotype. CONCLUSION: On all emotional scales, females with the l/s genotype showed high scores, contrary to males with the same genotype. Therefore, our results suggest that 5-HTTLPR l allele may be one pathway that activates negative emotion in females but acts contrary in males.

Genetic polymorphisms and haplotype structures of the CYP4A22 gene in a Japanese population.

The CYP4A fatty acid monooxygenases oxidize endogenous arachidonic acid to 20-hydroxyeicosatetraenoic acid that acts as a regulator of blood pressure. Among the isoforms of the CYP4A subfamily, the human CYP4A22 was recently identified. In this study, we report the comprehensive investigation of polymorphisms in the CYP4A22 gene. To investigate genetic variation in CYP4A22 in 191 Japanese subjects, we used denaturing HPLC (DHPLC) and direct sequencing. Our investigation has enabled the identification of 13 sequence variations in the CYP4A22 coding region, thereby demonstrating for the first time that this gene is subject to polymorphism. Two of these sequence variations correspond to silent mutations located in exons 8 (His323His) and 9 (Gly390Gly). Nine of these sequence variations correspond to missense mutations located in exons 1 (Arg11Cys), 3 (Arg126Trp), 4 (Gly130Ser and Asn152Tyr), 5 (Val185Phe), 6 (Cys231Arg), 7 (Lys276Thr), 10 (Leu428Pro), and 12 (Leu509Phe). One of these sequence variations corresponds to nonsense mutations located in exon 9 (Gln368stop). The 13th mutation corresponds to a nucleotide deletion (G7067del) that causes a frameshift and consequently results in a stop codon 80 nucleotides downstream. In addition to the wild-type CYP4A22*1 allele, 20 variants, namely CYP4A22*2-15, were characterized by haplotype analysis. Based on these data, we concluded that allelic variants of the human CYP4A22 gene exist and speculated that some of these variants may be functionally relevant.

Competitive allele-specific short oligonucleotide hybridization (CASSOH) with enzyme-linked immunosorbent assay (ELISA) for the detection of pharmacogenetic single nucleotide polymorphisms (SNPs).

Individualization of drug therapy through genetic testing would maximize the effectiveness of medication and minimize its risks. Recent progress in genetic testing technologies has been remarkable, and they have been applied for the analysis of genetic polymorphisms that regulate drug responses. Clinical application of genetic information to individual health care requires simple and rapid identification of nucleotide changes in clinical settings. We previously reported a novel DNA diagnostic method for detecting single nucleotide polymorphisms (SNPs) using competitive allele-specific short oligonucleotide hybridization (CASSOH) with an immunochromatographic strip. We have developed the method further in order to incorporate an enzyme-linked immunosorbent assay (ELISA) into the final detection step; this enables multiple SNP detection. Special ELISA chips have been fabricated so that disposal of buffer waste is not required and handling procedures are minimized. This method (CASSOH-ELISA) has been successfully applied for the detection of clinically important SNPs in drug metabolism, such as N-acetyltransferase 2, NAT2*6 (590G>A) and NAT*7 (857G>A), and mitochondrial DNA (1555A>G). It would also facilitate point-of-care genetic testing for potentially diverse clinical applications.

Involvement of reactive oxygen species and stress-activated MAPKs in satratoxin H-induced apoptosis.

Satratoxins, members of the trichothecene mycotoxin family, have been known to be harmful to health. However, the mechanisms underlying the toxicity still remain unclear. The present study is undertaken to elucidate the mechanisms of the satratoxin H-induced cytotoxicity in PC12 cells. Satratoxin H caused cytotoxicity, which was reflected from apoptosis determined by chromatin staining and flow cytometry. Satratoxin H stimulated the phosphorylation of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK). Pre-incubation with SB203580, a p38 MAPK inhibitor, or SP600125, a JNK inhibitor, but not PD98059, an ERK inhibitor, reduced satratoxin-induced cytotoxicity. Co-incubation of cells with glutathione, N-acetyl-L-cysteine or glutathione reductase inhibited cytotoxicity and the phosphorylation of p38 MAPK induced by satratoxin H. Our data suggest that satratoxin H-induced apoptosis in PC12 cells is dependent on the activation of p38 MAPK/JNK and the increase in reactive oxygen species.

Two novel single nucleotide polymorphisms (SNPs) of the CYP2D6 gene in Japanese individuals.

We analyzed all the exons and exon-intron junctions of the CYP2D6 gene from 286 Japanese individuals. We detected two novel single nucleotide polymorphisms (SNPs) 2556C>T in exon 5 (Thr261Ile) and 3835A>C in exon 8 (Lys404Gln). Both these SNPs showed a frequency of 0.002.

A novel single nucleotide polymorphism of the human methylenetetrahydrofolate reductase gene in Japanese individuals.

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%).

Altered water barrier function in epidermal-type fatty acid binding protein-deficient mice.

We have generated mutant mice for epidermal-type fatty acid binding protein by the gene targeting technique and examined the phenotype in detail. Despite a lack in the expression of epidermal-type fatty acid binding protein mRNA and its protein in the skin and other tissues of the mutant mice, the animals appeared normal in gross and histologic examination. Northern blot analysis of other fatty acid binding proteins revealed a distinct elevated gene expression of heart-type fatty acid binding protein in the skin of the homozygous mice. In analyses of the skin, no differences were observed in contents of major fatty acids, electron microscopic appearance as well as inflammatory responses in ear skin between the mutant and wild-type mice. Basal transepidermal water loss of homozygous mice was lower than that of the wild mice. When acetone was applied to the skin for disruption of the water permeability barrier, recovery in transepidermal water loss was delayed, although maximum transepidermal water loss upon acetone treatment was similar between homozygous and wild-type mice in terms of size and time course. The molecular mechanism by which epidermal-type fatty acid binding protein contributes to the water barrier function of the skin remains to be elucidated.

Phage-display selection of antibodies to the left end of CTX3C using synthetic fragments.

Ciguatoxins (CTXs) are a family of toxins that contaminate a variety of fish and cause ciguatera seafood poisoning. The limited availability of CTXs from natural sources has hampered preparation of antibodies and, thus, the development of immunoassays for these toxins. In the current studies, we utilized synthetic fragments as haptens to prepare antibodies against CTX3C, a congener of CTXs, thereby avoiding the problem of its scarcity. Synthetic ABC-ring fragment (ABC) of CTX3C was conjugated with keyhole limpet hemocyanin (KLH) and immunized on mice. Phage-displayed antibodies were first screened based on affinity to a soluble biotin-linked ABC-ring fragment that was captured on streptavidin-linked magnetic beads. The beads were then eluted with the ABCD-ring fragment (ABCD), and recovered phages were amplified. This elution with a synthetic fragment allowed the preparation of antibodies to ABCD as well as to ABC. Three antibodies with affinities of K(d) approximately 10(-5) M for ABCD were selected and prepared as soluble recombinant Fabs (rFabs). One rFab, 1C49, bound to CTX3C itself, although the binding affinity for CTX3C was weaker than for ABCD.

Elevated expression level of 60-kDa subunit of tRNA-guanine transglycosylase in colon cancer.

tRNA-guanine transglycosylase (TGT) is an enzyme which synthesizes a modified nucleoside, queuosine, by exchanging the base moiety of guanosine for queuine in tRNA. We have reported that the expression level of the 60-kDa subunit of TGT (TGT60kD) is elevated in leukemic cells, however, there is no other report on the expression of TGT60kD in cancer cells. The expression levels of the TGT60kD protein are elevated in four of the five colon cancer cell lines and 83% of colon cancer tissues compared with normal tissues. The expression levels of the TGT60kD protein decreased in two colon cancer cell lines, after cell differentiation was induced. A marked positive staining of cancer cells in colon tissues was observed, and the subcellular staining pattern was mainly cytosolic. These data suggest that the role of TGT60kD in colon carcinogenesis.

Design, production, and characterization of recombinant neocarzinostatin apoprotein in Escherichia coli.

Neocarzinostatin (NCS) is the first discovered anti-tumor antibiotic having an enediyne-containing chromophore and an apoprotein with a 1:1 complex. An artificial gene library for NCS apoprotein (apo-NCS) production in Escherichia coli was designed and constructed on a phage-display vector, pJuFo. The recombinant phages expressing pre-apo-NCS protein were enriched with a mouse anti-apo-NCS monoclonal antibody, 1C7D4. The apo-NCS gene (encsA) for E. coli was successfully cloned, and then re-cloned into the pRSET A vector. After the his-tagged apo-NCS protein had been purified and cleaved with enterokinase, the binding properties of the recombinant protein as to ethidium bromide (EtBr) were studied by monitoring of total fluorescence intensity and fluorescence polarization with a BEACON 2000 system and GraphPad Prism software. A dissociation constant of 4.4 +/- 0.3 microM was obtained for recombinant apo-NCS in the fluorescence polarization study. This suggests that fluorescence polarization monitoring with EtBr as a chromophore mimic may be a simplified method for the characterization of recombinant apo-NCS binding to the NCS chromophore. When Phe78 on apo-NCS was substituted with Trp78 by site-directed mutagenesis using a two stage megaprimer polymerase chain reaction, the association of the apo-NCS mutant and EtBr observed on fluorescence polarization analysis was of the same degree as in the case of the wild type, although the calculated maximum change (DeltaIT(max)) in total fluorescence intensity decreased from 113.9 to 31.3. It was suggested that an environmental change of the bound EtBr molecule on F78W might have dramatically occurred as compared with in the case of wild type apo-NCS. This combination of monitoring of fluorescence polarization and total fluorescence intensity will be applicable for determination and prediction of the ligand state bound or associated with the target protein. The histone-specific proteolytic activity was also re-investigated using this recombinant apo-NCS preparation, and calf thymus histone H1, H2A, H2B, H3, and H4. The recombinant apo-NCS does not act as a histone protease because a noticeable difference was not observed between the incubation mixtures with and without apo-NCS under our experimental conditions.

Characterization of 11-dehydro-thromboxane B2 recombinant antibody obtained by phage display technology.

Recombinant monoclonal antibodies specific for 11-dehydro-thromboxane B(2) (11D-TX) were isolated from the combinatorial libraries on a pComb3 phage-display vector using a magnetic cell sorting (MACS) system. The libraries were constructed from repertories of light and heavy-chains derived from the total RNA of 11D-TX conjugated keyhole limpet haemocyanin-immunized mice. Biotinylation of 11D-TX conjugated bovine serum albumin (BSA) was performed through free thiol groups on BSA using 1-biotinamido-4-[4'-(maleimidomethyl) cyclohexanecarboxamido] butane (Biotin-BMCC). Affinity bio-panning was performed to enrich the phage display libraries against biotinylated 11D-TX conjugated BSA with the MACS system. Results indicated that the selected anti-11D-TX Fab fragments expressed by E. coli exhibited a five-fold higher affinity for BSA conjugated 11D-TX compared to BSA alone and little specificity to other related compounds as determined by the binding assay and inhibition enzyme-linked immunosorbent assay (ELISA). This is the first report of an antibody against prostaglandin produced by phage display technology and also determination of the DNA sequence of this antibody. The MACS system was shown to be a simpler and more efficient method of panning than the conventional ELISA procedure. According to our results, we concluded that the phage display technique combined with the MACS system allowed the selection of the antibody with high affinity and some specificity.

A simultaneous LightCycler detection assay for five genetic polymorphisms influencing drug sensitivity.

OBJECTIVES: The routine detection of polymorphisms affecting drug sensitivity in patients before treatment is important in the identification of drug responders or nonresponders, and patients at increased risk of drug toxicity. Here, we present an assay for the simultaneous and rapid genotyping of five polymorphisms influencing drug sensitivity. DESIGN AND METHODS: We used a hybridization probe assay on the LightCycler to detect five single nucleotide polymorphisms (SNPs): INPP1 (973C>A), ADRB2 (R16G and Q27E), HTR2A (102T>C), and mtDNA (1555A>G). Two fluorescent labeled hybridization probes were designed for the simultaneous detection of the five SNPs and detection of the variant alleles was performed by melting curve analysis. RESULTS: All five SNPs were detected with a single thermocycle protocol within 40 min. The genotypes determined in this assay were identical to those obtained with conventional PCR and restriction fragment length polymorphism analysis. CONCLUSIONS: To our knowledge, we report here for the first time a method for simultaneous detection of five SNPs, on a single thermocycle protocol by the LightCycler. This method is rapid, highly sensitive, and high-throughput, and is thus suitable for routine clinical use and large-scale epidemiologic studies.

Thiazolidinediones increase arachidonic acid release and subsequent prostanoid production in a peroxisome proliferator-activated receptor gamma-independent manner.

Thiazolidinedione, peroxisome proliferator-activated receptor gamma (PPARgamma) agonist, has been used as an anti-diabetic drug and as an useful tool to elucidate multiple PPARgamma functions by in vitro and in vivo studies. We investigated the effects of thiazolidinediones on prostanoid production in lipopolysaccharide-stimulated cells. The high concentrations (>10 microM) of rosiglitazone and pioglitazone significantly increased lipopolysaccharide-stimulated prostanoid production such as thromboxane A2 and prostaglandin E2. However, PPARgamma antagonist could not inhibit them. In PPARgamma-deficient cells, thiazolidinediones increased prostaglandin E2 production. Thiazolidinediones increased arachidonic acid (AA) release from the cell membrane by not stimulating AA releasing process involving several phospholipase A2s but inhibiting AA reuptaking process. The expression of cyclooxygenase-1 and cyclooxygenase-2 were not affected by thiazolidinediones. In this study, we demonstrated that high concentrations of TZDs increased AA release by the inhibition of AA reuptaking process, leading to subsequent increase in the prostanoid production in a PPARgamma-independent manner. This mechanism provides useful information for the elucidation of multiple PPARgamma functions and diabetic drug therapy.

Quantitative liquid chromatography-tandem mass spectrometric analysis of 11-dehydro TXB2 in urine.

A simple and selective determination method of 11-dehydrothromboxane B2 (11-dehydroTXB2), which is urinary metabolite of TXA2, has been developed employing liquid chromatography-tandem mass spectrometry (LC-MS-MS). 11-DehydroTXB2 and its deuterium-labeled analogue as an internal standard were extracted from urine by simple solid-phase extraction (SPE). These compounds were analyzed using LC-MS-MS in the selected reaction monitoring (SRM) mode, by monitoring the transitions from m/z 367 to m/z 161 for 11-dehydroTXB2 and from m/z 371 to m/z 165 for its internal standard. A good linear response over the range 50 pg-10 ng per tube was demonstrated. The values determined by LC-MS-MS were well validated and closely corresponded to the values determined by gas chromatography-mass spectrometry (GC-MS). The mean concentration of 11-dehydroTXB2 in urine of healthy adults was 635 +/- 427 pg/mg creatinine (mean +/- S.D., n = 13). This simple, accurate and selective determination method described in this study should greatly aid in evaluating the role of TXA2 in vivo.