Inhibition of 17ß-estradiol activation by CYP1A1: genotype- and regioselective inhibition by St. John's Wort and several natural polyphenols.

Several epidemiological studies associate certain CYP1A1 genotypes, alone or in combination, with an increased risk of estrogen-related cancers. Previously we demonstrated that metabolic activation of estrogens by CYP1A1 is a genotype-dependent reaction with the CYP1A1.2 (Ile462Val) variant being the most efficient catalyst (Kisselev et al.). To answer the question whether genotype-dependent inhibition of activation of estrogens by CYP1A1 could also contribute, we studied the inhibition of hydroxylation activity of the most common allelic variants of human CYP1A1 towards 17ß-estradiol. We expressed and purified CYP1A1.1 (wild-type), CYP1A1.2 (Ile462Val), and CYP1A1.4 (Thr461Asn) and performed inhibition assays by natural polyphenols of our diet and drugs of NADPH-dependent estradiol hydroxylation in reconstituted CYP1A1 systems. From the polyphenols studied, a St. John's Wort (Hypericum perforatum) extract, some of its main single constituents hypericin, pseudohypericin, and quercetin, as well as the flavonols kaempferol, myricetin and the phytoestrogens resveratrol and tetramethyl-stilbene exhibited strong inhibition. For the St. John's Wort extract and its single constituents hypericin, pseudohypericin, and quercetin, inhibition exhibited a remarkable dependency on the CYP1A1 genotype. Whereas (wild-type) CYP1A1.1 was most inhibited by the whole crude extract, the variant CYP1A1.2 (Ile462Val) was significantly stronger inhibited by the constituents in its pure form: IC50 values for 2-hydroxylation was more than two times lower compared with the wild-type enzyme and the variant CYP1A1.4 (Thr461Asn). Besides this, the inhibition exhibited a remarkable regioselectivity. The data suggest that risk of estrogen-mediated diseases might be not only influenced by CYP1A1 genotype-dependent activation but also its inhibition by natural polyphenols of our diet and drugs.

Moxifloxacin does not alter ciclosporin pharmacokinetics in transplant patients: a multiple-dose, uncontrolled, single-centre study.

BACKGROUND: Moxifloxacin has a broad antibacterial spectrum and rapid bactericidal activity, and is thus a good option for the treatment of bacterial infections in patients who have undergone organ or bone marrow transplantation. Transplant patients also receive immunosuppressant therapy such as ciclosporin. OBJECTIVE: The primary objective of this study was to assess the steady-state pharmacokinetics of ciclosporin with and without concomitant treatment with moxifloxacin in transplant recipients. A secondary objective was to determine the safety and tolerability of the combined treatment. METHODS: Patients (n = 9) with stable graft function after bone marrow or renal transplantation and who were already receiving ciclosporin therapy were enrolled into the study. The patients were given ciclosporin (Sandimmun Optoral) capsules twice daily (total daily dosage 150-380 mg/day) throughout the study period. Moxifloxacin (Avolox) tablets 400 mg once daily were given on days 2-8 inclusive. The primary outcome measure was the change in ciclosporin pharmacokinetics on coadministration with moxifloxacin. Secondary outcomes were the steady-state pharmacokinetics of moxifloxacin and ciclosporin plus its metabolites in patients receiving moxifloxacin and ciclosporin concomitantly. Moxifloxacin pharmacokinetic parameters in the presence of ciclosporin were compared with previously published pharmacokinetic data for moxifloxacin in healthy individuals. RESULTS: No significant changes occurred in the concentration-time curves of ciclosporin and its metabolites following combination therapy with moxifloxacin. The geometric means of whole blood concentrations of ciclosporin and ciclosporin plus its metabolites on day 1 were similar to those on day 8 following combined administration of ciclosporin and moxifloxacin for 7 days. The ratio of combination treatment to monotherapy for ciclosporin was 1.01 (90% CI 0.91, 1.11) for the area under the blood concentration-time curve from time zero to 12 hours at steady state (AUC(12,ss)) and 0.96 (90% CI 0.88, 1.04) for the maximum steady-state blood drug concentration (C(max,ss)). For ciclosporin plus its metabolites the ratio was 1.07 (90% CI 0.99, 1.17) for AUC(12,ss) and 1.03 (90% CI 0.98, 1.09) for C(max,ss). The pharmacokinetic parameters for moxifloxacin were unaffected by the presence of ciclosporin. CONCLUSIONS: Concomitant administration of moxifloxacin does not alter the pharmacokinetic parameters of ciclosporin or ciclosporin plus its metabolites in immunosuppressed patients. Therefore, no dose adjustments or additional drug monitoring are required when ciclosporin is coadministered with moxifloxacin.

Genetic polymorphisms in endothelin-receptor-subtype-a-gene as susceptibility factor for obstructive sleep apnea syndrome.

INTRODUCTION: Traits of obstructive sleep apnea syndrome (OSAS) such as impaired ventilatory control, craniofacial abnormalities, and concomitant cardiovascular diseases are associated with modified endothelin-1 gene (EDN-1) or endothelin-receptor-subtype-a (EDNRA) gene. The endothelin system regulates the cardiovascular homeostasis. EDN-1 interacts mainly with EDNRA for signal transduction. In our study we investigate associations of EDNRA-polymorphisms (four frequent polymorphisms with an allele frequency >5%) and OSAS severity. METHODS: Three hundred ninety-three patients older than 18years, of Caucasian origin and with OSAS (AHI>5/h and daytime sleepiness) were investigated by cardiorespiratory polysomnography. In addition 58 control subjects with healthy sleep were recruited from nearly 300 volunteers. We analysed the EDNRA-polymorphisms E335E, H323H, G-231A and G+70C by polymerase-chain-reaction, restriction-fragment-length-polymorphism and real-time-PCR. RESULTS: Carrier of the mutant G-231A allele had a significantly lower AHI (p=0.03, OR 0.53, 95% CI 0.3-0.94) when comparing patients and controls. When comparing OSAS severity groups without controls we could not detect significant correlations for the four investigated EDNRA-polymorphisms. Our data confirm that BMI (p<0.001) and male gender (p=0.02) are significantly associated with AHI. The allele frequencies were similar. DISCUSSION: The genetic investigation of OSA remains important. Our control group was relatively small and we investigated 4 reasonable candidates out of more than 100 EDNRA-polymorphisms. The detected protective effect of the mutant G-231A allele needs further confirmation. Gene based research in OSAS should use genome wide scan and should still consider the endothelin system.

St John's wort extract (Ze 117) does not alter the pharmacokinetics of a low-dose oral contraceptive.

PURPOSE: St John's wort (Hypericum perforatum) is an herbal remedy that is widely used in the treatment of depression. Recent clinical data have demonstrated that St John's wort extracts interfere with the action of various drugs and possibly also with combined oral contraceptives. Therefore, we investigated the effects of a St John's wort extract (Ze 117) with low hyperforin content on the pharmacokinetics of ethinylestradiol and 3-ketodesogestrel. METHOD: Sixteen healthy female volunteers, who had taken a low-dose oral contraceptive (Lovelle contains 0.02 mg ethinylestradiol + 0.15 mg desogestrel) for at least 3 months, participated in the study. Pharmacokinetic data (AUC, C(max), t(max)) were determined the day before (reference) and after (test) a 14-day period of Ze 117 intake (250 mg twice daily). RESULTS: Before the co-administration of Ze 117 on day 7, the geometric mean (geometric coefficient of variation) for the AUC(0-24) of ethinylestradiol was 152.53 pg.h/ml (87.39%) and after co-administration on day 21 it was 196.57 pg.h/ml (78.14%). The respective values for ketodesogestrel were 36.37 pg.h/ml (34.18%) and 41.12 pg.h/ml (34.36%). The mean of individual ratios (reference-to-test) of log-transformed AUC values (90% confidence interval) were 0.951 (0.915-0.986) for ethinylestradiol and 0.968 (0.944-0.992) for ketodesogestrel indicating a small gain [corrected] in bioavilability, but bioequivalence nevertheless. CONCLUSION: These results indicate that the recommended dose of the hypericum extract Ze117, which has a low hyperforin content, does not interact with the pharmacokinetics of the hormonal components of the low-dose oral contraceptive.

Endothelin-1 gene variant Lys198Asn and plasma endothelin level in obstructive sleep apnea.

Obstructive sleep apnea (OSA) is a recognized risk factor for cardiovascular disorders. Thus, an association between endothelin-1 (EDN1) and OSA can be assumed. We investigated a cohort of 364 consecutive patients (age 57 +/- 10 years) with mild to severe OSA for the EDN1 variant Lys198Asn (G/T) and endothelin plasma levels and compared them with 57 controls. The Lys198Asn genotype was significantly associated with the apnea/hypopnea index (AHI) with a median of 30/h of sleep for GG, 27/h for GT and 59/h for TT genotype (p < 0.05). Further stratification of patients into 2 groups by body mass index (BMI) revealed a strong association between AHI and Lys198Asn polymorphism in 191 obese patients (p = 0.005), whereas in 173 nonobese patients, we observed no association. A substantial effect by BMI on OSA severity was seen with multiple linear regression (p < 0.001). However, this effect was modified by the Lys198Asn polymorphism and by gender: the AHI increase per unit of BMI was more pronounced in males than in females, and about 1.3 times greater in homozygous carriers of the mutant allele than in other carrier groups. EDN1 plasma levels of untreated OSA patients and of patients treated with nasal continuous positive airway pressure were not elevated compared with controls. Our results indicate that the Lys198Asn polymorphism is associated with the severity of OSA in obese subjects. The EDN1 plasma level cannot be used as a marker for OSA or its severity.

Vitamin D receptor poly(A) microsatellite and colorectal cancer risk in Caucasians.

BACKGROUND: Colorectal cancer incidence and prognosis are influenced by vitamin D intake and expression of the vitamin D receptor (VDR). Polymorphisms of the VDR are linked to several diseases. This study was aimed to investigate whether variants of the VDR poly(A) microsatellite are associated with colorectal cancer incidence. MATERIALS AND METHODS: The poly(A) polymorphism was analyzed in a series of 255 colorectal cancer patients and 255 controls of Caucasian origin (case-control study) by a combination of GeneScan and sequencing. RESULTS: There was a distinct separation between long and short alleles. We found 19, 20, 21, and 22 A-repeats for the long variant and 14 and 15 A-repeats for the short variant. Frequencies of long and short alleles did not differ between cases and controls, nor did frequencies of any single variant. CONCLUSION: Our findings do not support an association between VDR poly(A) variants and the incidence of colorectal cancer.

Genetic variants in FKBP5 affecting response to antidepressant drug treatment.

INTRODUCTION: Hyperactivity of the hypothalamic-pituitary-adrenal (HPA) axis is a pathogenic mechanism of depression, and genetic polymorphisms in HPA axis genes have been described to influence response to antidepressant drugs. In particular, two polymorphisms in FKBP5, a co-chaperone of the glucocorticoid receptor, were strongly associated with response to therapy. We aimed to analyze whether these findings could be reproduced in a different sample of otherwise comparable inpatients with major depression. METHODS: Genotyping for the two variants within the FKBP5 gene was performed using PCR-restriction fragment length polymorphism and Taqman real-time PCR in a cohort of 179 inpatients who were monitored for the first 3 weeks of antidepressant drug treatment. The early response to antidepressant drugs was assessed as percentage of decline in Hamilton depression score after 3 weeks, responders versus nonresponders were distinguished by a 50% decrease. RESULTS: The FKBP5 variants rs3800373 and rs1360780 were highly linked, and carriers of the FKBP5 variants had a trend towards a higher chance to respond (p = 0.04; odds ratio: 1.8; 95% CI: 0.98-3.3). When analyzing drug-specific subgroups, the effect was seen mainly in the subgroups of patients treated with antidepressant drug combinations or with venlafaxine. CONCLUSION: In this study, an effect of FKBP5 variants on antidepressant drug response was confirmed in an independent cohort of depressed patients; however, with an odds ratio of 1.8 the effect size was smaller than that described earlier.

Below median birth weight in appropriate-for-gestational-age preterm infants as a risk factor for bronchopulmonary dysplasia.

AIMS: To assess the presence of chorioamnionitis and intrauterine growth as prenatal risk factors for broncho pulmonary dysplasia (BPD) in appropriate-for-gestational-age (AGA) infants of <28 weeks' gestation. METHODS: Gender, race, birth weight, gestational age, histology of the placenta, diagnosis of BPD at 36 weeks' gestation, postnatal dexamethasone treatment, and death were recorded in 150 preterm infants born at <28 weeks' gestation, and admitted between 1996 and 2001. RESULTS: In 122 AGA infants (mean gestational age: 26.18 weeks, mean birth weight: 837 g), BPD was associated with gestational age-related birth weights below the 50(th) centile. Intrauterine growth deceleration started between 25 and 26 weeks' gestation. Chorioamnionitis was not related to BPD. CONCLUSIONS: AGA infants of 26-28 weeks' gestation with birth weights below the median showed an increased risk of developing BPD.

Common genetic variations in human brain-specific tryptophan hydroxylase-2 and response to antidepressant treatment.

OBJECTIVE: Genetic variability within the serotoninergic system may predict the response to antidepressant drugs. Several polymorphisms in the gene coding for the brain-specific tryptophan hydroxylase (TPH2) have been associated with susceptibility to psychiatric diseases. In this study, we analyzed the correlation between TPH2 polymorphisms and response to antidepressant drugs. METHODS: The study included 182 patients who received drug treatment for major depression. To assess the variability in the TPH2 gene, four single nucleotide polymorphisms (SNPs) tagging the common TPH2 haplotypes and six SNPs medically relevant according to data from other studies were analyzed in a multiplex single base primer extension reaction. RESULTS: Two SNPs, rs10897346 and rs1487278, were significantly associated with response to therapy (P=0.003 and 0.007). The rs10897346 variant showed the highest predictive values with carriers of null C alleles showing a 2.6-fold increased risk (95% confidence interval 1.4-4.8) for nonresponse compared with the others. The effect was found in all major types of antidepressant medications administered in this study and was statistically significant in the subgroup on selective serotonin reuptake inhibitors. Multiple logistic regression analyses confirmed the rs10879346 polymorphism as an independent predictor of the antidepressant response (odds ratio: 3.86; 1.75-8.55, P=0.0008). The therapeutically relevant variant rs10897346 is completely linked with the functional Pro312Pro polymorphism, which is known to affect TPH2 expression and may influence serotonin synthesis in the brain. CONCLUSION: The polymorphisms rs10897346 and Pro312Pro in the TPH2 gene might play an important role for TPH2 expression and antidepressant drug response.

An anomalous haplotype distribution of the arrestin domain-containing 4 gene (ARRDC4) haplotypes in Caucasians.

Little was known about the sequence variability of the human Arrestin domain-containing 4 gene (ARRDC4). We sequenced its DNA from exon 2 to exon 8 in a sample of 92 Russians. Seven variants were identified; one of them has not been described yet. It causes an amino acid change from Thr to Met. Identified variants were genotyped in the complete sample of 253 unrelated men and women to analyze haplotype distribution. Fifteen haplotypes were inferred. Nine haplotypes had estimated frequencies > 1%. Ninety-five percent of all haplotypes were determined by five haplotype-tagging single nucleotide polymorphisms. Haplotypes form two clades. The two most common haplotypes cover 76% of all haplotypes. The certainty of the haplotype reconstruction does not depend on the haplotype-inferring algorithms, but is a result of the anomalous haplotype distribution of ARRDC4, which makes this gene a suitable candidate gene for haplotype association studies. Interestingly, there is a great evolutionary distance between the two most common haplotypes, which could suggest a more complicated coalescent process with either past gene flow, selections, or bottlenecks.

Comparison of SLCO1B1 sequence variability among German, Turkish, and African populations.

BACKGROUND: OATP1B1 is one of the key hepatocellular uptake transporters providing extraction of diverse compounds, including bile acids, xenobiotics, and a variety of drugs, from portal venous blood into the liver. Polymorphisms of the SLCO1B1 gene have been demonstrated to influence in vitro transport function and the pharmacokinetic profile of compounds. OBJECTIVE: The goal of our study was the comparison of SLCO1B1 gene sequence variability in three ethnic groups as a basis for future genetic association studies. METHODS: Eighteen exonic SLCO1B1 single nucleotide polymorphisms (SNPs) were genotyped by PCR and RFLP analysis in 300 German, 94 Turkish, and 115 African subjects. Calculation of pairwise linkage disequilibrium and estimation of population haplotype frequencies were carried out, and haplotype block structure was determined. RESULTS: Only eight genotyped SNPs (c.388A>G, c.411G>A, c.463C>A, c.521T>C, c.571C>T, c.597C>T, c.1463G>>C, c.1929A>C) were found in at least one of our German, Turkish, or African samples. A total of 12 haplotypes with a frequency >or=1% in at least one of the three populations could be inferred. Between the Caucasian and African samples, significant differences in sequence variability were observed leading to a different haplotype profile in these populations. CONCLUSION: Our results demonstrate a high sequence variability of OATP1B1 within different popuations. In the future, distinct haplotypes should be taken into account when studying the effect of OATP1B1 on drugs in different populations.

Effects of the CYP2D6 gene duplication on the pharmacokinetics and pharmacodynamics of tramadol.

The analgesic drug tramadol is bioactivated by CYP2D6 to the opioid receptor agonist O-desmethyltramadol. Case reports indicated that carriers of the CYP2D6 gene duplication may be at high risk for opioid adverse events. However, the effects of the CYP2D6 duplication on kinetics and dynamics of tramadol have not been systematically studied. Pharmacokinetics and effects were monitored after a single dose of 100 mg racemic tramadol in 11 carriers of a CYP2D6 gene duplication allele (ultrarapid metabolizer [UM]) and compared with 11 carriers of 2 active CYP2D6 genes (extensive metabolizer [EM]). Pharmacodynamics was measured by cold pressure test, pupillometry, and standardized adverse event recording. The maximum plasma concentrations of the active metabolite (+)R,R-O-desmethyltramadol were significantly higher in the UM group compared with the EM group (P = 0.005; t test) with a mean difference of 14 ng/mL (95% confidence limit of difference, 2-26 ng/mL). Median (+)R,R-tramadol area under the curve was 786 and 587 mug.h.L in EMs and UMs, and the corresponding median (+)R,R-O-desmethyltramadol area under the curve was 416 and 448 mug.h.L (P = 0.005, t test). There was an increased pain threshold and pain tolerance and a stronger miosis after tramadol in UMs compared with EMs. Almost 50% of the UM group experienced nausea compared with only 9% of the EM group. In conclusion, pharmacokinetic differences between EMs and UMs were smaller than expected; nevertheless, UMs were more sensitive to tramadol than EMs. Therefore, tramadol may frequently cause adverse effects in southern European and Northern African populations with a high proportion of UMs.

Effect of tolterodine on sleep structure modulated by CYP2D6 genotype.

OBJECTIVE: Tolterodine, a drug for the treatment of overactive bladder symptoms, has a limited entry into the brain, which makes cognitive side effects seldom. However, some case reports have described central-nervous side effects such as sleepiness. The aim of this retrospective analysis was to investigate whether tolterodine-related effects on sleep stage parameters could be explained by different CYP2D6 metabolizer characteristics of subjects. METHODS: Data were taken from two randomized, double-blind, placebo-controlled studies conducted in a cross-over design. Forty-eight volunteers underwent 4 two-night attended polysomnographic studies. Subjective quality of sleep and cognitive function were assessed. A single dose of 4 mg tolterodine or placebo was administered before sleep. Forty-four volunteers gave informed consent for genotyping. We found 19 extensive metabolizers (EM), 20 intermediate metabolizers (IM), 4 poor metabolizers (PM) and 1 ultrarapid metabolizer. There were no significant differences between the groups regarding demographic data. RESULTS: Rapid eye movement (REM) sleep duration as a percentage of total sleep time showed significant reduction (p=0.019) in the group carrying one or more deficient alleles (IM+PM). No significant difference was found with two active alleles of CYP2D6 in the EM group. REM latencies under tolterodine displayed a tendency towards prolongation, which was irrespective of the metabolizer status. Subjective sleep parameters did not show statistically significant changes after tolterodine. Cognitive skills were not affected. CONCLUSION: Our retrospective analysis reveals that a decrease of REM sleep under tolterodine is found only in individuals carrying one or two deficient CYP2D6 alleles.

Vitamin D receptor haplotypes protect against development of colorectal cancer.

OBJECTIVE: We investigated the association of vitamin D receptor polymorphisms and colorectal cancer incidence in a Caucasian population. METHODS: Frequencies of the vitamin D receptor gene polymorphisms 23005G>A (CDX-2), 27823C>T (FokI), 60890G>A (BsmI), 61050G>A (Tru9I), 61888G>T (ApaI), and 61968T>C (TaqI) were determined in a series of 256 colorectal cancer patients and 256 patients without malignant disease (case-control study) using polymerase chain reaction and restriction fragment length polymorphism genotyping assays (PCR-RFLP). Haplotype analysis based on the six genetic loci was applied to the received genotypes. RESULTS: Pairwise linkage disequilibrium between BsmI, ApaI, TaqI, and Tru9I was confirmed (P < 0.001). Allele frequencies did not differ between the groups. There was no association between any single variant and colorectal cancer. However, haplotypes BsmI(G)#TaqI(C) and BsmI(A)#TaqI(T) were inversely associated with colorectal cancer incidence (P < 0.001), the odds being 15.0 times smaller [odds ratio (OR) 0.067; 95% confidence interval (CI), 0.016-0.284] and 5.3 times smaller (OR 0.188; 95% CI 0.077-0.461), respectively, compared with noncarriers. CONCLUSION: Our findings suggest that vitamin D receptor haplotypes BsmI(G)#TaqI(C) and BsmI(A)#TaqI(T) have a protective effect against colorectal cancer in Caucasians.

Recalling P446. P4501A1 (CYP1A1) opting for clinical application.

This review goes back to spectral studies [see Hildebrandt et al., 1968]. The findings of apparent absolute spectra of two interconvertible forms of microsomal mixed function oxidases are looked back on to recall whether their impact sustained scrutiny or are rather remembered as of sentimental value only. The second part summarizes studies on the clinical relevance of CYP1A1 with special reference to our investigations. The impact of genetic variability of CYP1A1 on cancer susceptibility, differential effects of polyphenols and hyperforin on toxification and detoxification pathways of benzo[a]pyrene, and differential metabolite patterns of 17 beta-estradiol, estrone, and eicosapentaenoic acid are presented.

Risk of coronary artery disease as influenced by variants of the human endothelin and endothelin-converting enzyme genes.

OBJECTIVE: The potent vasoconstrictor endothelin (ET) is generated by enzymatic cleavage catalyzed by the endothelin-converting enzyme 1 (ECE-1) and plays a crucial role in the regulation of vascular tone and endothelial function. Polymorphisms of the ET and ECE genes may contribute to the development and progression of coronary artery disease. Recently, we have shown the functional relevance of the +138 adenine ins/del polymorphism on ET-1 expression in vitro. The aim of our case-control study was to investigate the impact of known and novel variants of the ET and ECE genes on the risk of coronary artery disease in vivo. METHODS: In a prestudy, 36 single nucleotide polymorphisms in the ET-1, ET-2, ET-3 and ECE-1 genes were identified in 55 participants by sequencing analysis. Subsequently, 1000 matched pairs of angiographically confirmed coronary artery disease patients and hospital controls were genotyped for the eight most common or functionally relevant variants of the ET-1 (138 A ins/del, 2176T>G, 3660G>A, 5665G>T (Lys198Asn)) and ECE-1 gene (-854C>T, -839T>G, -377G>A, and exon 9 +2C>T). RESULTS: Carriers of at least one copy of the dysfunctional ET-1 5665 T allele were at increased risk of coronary artery disease (odds ratio 1.25; 95% confidence interval 1.03-1.52, P=0.025), particularly among men (odds ratio 1.32; 95% confidence interval 1.06-1.65, P=0.014). Homozygous carriers of the ECE-1 -839G variant allele exhibited a decreased risk of coronary artery disease (odds ratio 0.41; 95% confidence interval 0.18-0.90, P=0.024). The other six screened variants showed no association with coronary artery disease, the overall haplotype distribution differed slightly but significantly. CONCLUSIONS: This large case-control study argues for an only minor-if any-role of the ET-1 and ECE-1 genotype for the risk of coronary artery disease development.

Association of MDR1 genotypes with susceptibility to colorectal cancer in older non-smokers.

OBJECTIVE: The multidrug resistance gene 1 (MDR1) seems to play a role in the carcinogenesis of colorectal tumors. The importance of MDR1 SNPs 2677G > T/A in exon 21 and 3435C > T in exon 26 for cancer susceptibility, however, has not yet been clearly defined. METHODS: Two hundred and eighty-five colorectal cancer patients and 275 controls from five hospitals in the European part of Russia were genotyped for the polymorphisms -129T > C (rs3213619) in exon 1b, 2677G > T/A (rs2032582), and 3435C > T (rs1045642) in this population-based case-control study. Genotype-phenotype analysis was performed with simultaneous consideration of lifestyle risk factors. RESULTS: Our analysis confirmed the preponderate impact of smoking on colorectal cancer development. The risk of heavy smokers (>/=60 pack years) to develop colorectal cancer by far exceeded that of lifelong non-smokers (OR = 3.9, 95% CI: 1.4 to 10.6). Smoking is a more potent risk factor than is the genetic influence of MDR1 in our study. However, a smoking and age-stratified analysis, revealed a statistically significant association between MDR1 genotypes and colorectal cancer in life-long non-smokers with an age > or =63 years (the median age in our sample). The association was stronger for rectal cancer than for colon cancer. Patients who carried the genotypes (-129TT; 2677GG; 3435CC) or (-129TT; 2677TT; 3435TT) developed more frequently colorectal cancer than others (OR = 3.9; 95% CI: 2.0 to 7.7). CONCLUSIONS: Our results show that the interaction of genetic and lifestyle risk factors should be taken into account to elucidate the genetic influence of MDR1 variability on cancer susceptibility.

In-vitro transport characteristics discriminate wild-type ABCB1 (MDR1) from ALA893SER and ALA893THR polymorphisms.

BACKGROUND: Genetic variation of the human ABCB1 (P-glycoprotein; MDR1 gene product) efflux transporter is strongly suggested as a determinant factor governing the pharmacokinetics of diverse drugs and xenobiotics. Despite various efforts to associate polymorphisms in ABCB1 to actual clinical effect and transport function, information is still inconsistent or even controversial. METHODS AND RESULTS: Using membrane vesicle preparation from ABCB1-expressing HighFive insect cells, we report here that saturation kinectic parameters of the frequently occurring ABCB1 triallelic variants 893Ser (exon 21, 2677T) and 893Thr (2677A) were considerably different from wild-type 893Ala (2677G), despite similar protein expression levels. Of importance were significant differences in transport capacities between the tested 893Ala/Ser/Thr variants. In comparison with 893Ala, maximal transport rates for vincristine of 893Ser and 893Thr increased 50% and three-fold, respectively. Cis-inhibition by digoxin, didanosine or fexofenadine was least pronounced in 893Ser, whereas no genotype differences could be observed using verapamil. CONCLUSION: These results suggest an influence of ABCB1-893 triallelic variants on transport function and drug-drug interaction, which might be most pronounced in 893Thr. Furthermore, some of the mechanisms of 2677G/T/A-based haplotype-associated alterations in ABCB1 activity may have been unveiled.

Systematic analysis of sequence variability of the endothelin-1 gene: a prerequisite for association studies.

We analyzed allele frequencies and pairwise linkage disequilibria of 13 variants in the EDN1 gene of 298 young males, the majority of German ancestry. Our analysis comprises all common variants in the five exons and flanking intronic regions, as well as known polymorphisms in the promoter sequence. In addition to previously analyzed polymorphisms, our haplotype reconstruction included five recently described variants and was done by using three different algorithms to allow inference of result stability. More than 30 haplotypes were predicted. All haplotypes with frequencies > or = 1% were inferred by all three methods and can be described by seven haplotype tagging single-nucleotide polymorphisms (htSNPs), reducing the genotyping load to 65%. Three of these haplotypes with frequencies of about 11%, 9%, and 4% had been mistaken for one haplotype in the previous analysis, which included only six polymorphisms, some of them not being htSNPs. Systematic analysis of sequence variability and comprehensive haplotype analysis of the EDN1 gene determined a substantial part of its genetic variability for further association studies and helped to reduce the genotyping load for common phenotypes.

Fifteen polymorphisms in endothelin-1, endothelin-2 and endothelin-receptor-A genotyped by four duplex assays and seven simple assays on a LightCycler using hybridization probes.

Endothelins (EDNs) are peptides, produced by various tissues, with potent vasoactive and mitogenic properties. Endothelin actions are mediated via specific G protein-coupled receptors of two subtypes, endothelin-receptor-A (EDNRA) and endothelin-receptor-B (EDNRB). Some polymorphisms of the EDN1, EDN2 and EDNRA genes may influence susceptibility to vascular diseases. Thus, genotyping for polymorphisms of these genes may represent a tool for predicting individual susceptibility to vascular diseases. Here, we report 11 fluorescence resonance energy transfer (FRET) assays for the detection of 15 polymorphisms, because the assays used in previous studies (allele-specific PCR and restriction fragment-length polymorphism assays) for some of these polymorphisms are laborious and time-consuming. The newly developed assays are fast and work without expensive ready-to-use mixtures.