The P2RX7 gene is located within a region on chromosome 12q24.31 that has been identified as a susceptibility locus for affective disorders by linkage and association studies. P2RX7 is a purinergic ATP-binding calcium channel expressed in neurons as well as in microglial cells in various brain regions. We investigated 29 single nucleotide polymorphisms (SNPs) within the P2RX7 gene and adjacent genes in a sample of 1000 German Caucasian patients suffering from recurrent major depressive disorder (MDD). These were contrasted with diagnosed healthy Caucasian controls from the same population (n=1029). A non-synonymous coding SNP in the P2RX7 gene (rs2230912), previously found to be associated with bipolar disorder, was significantly associated (P=0.0019) with MDD. This polymorphism results in an amino acid exchange in the C-terminal cytosolic domain of the P2RX7 channel protein, suggesting that the observed P2RX7 polymorphism might play a causal role in the development of depression.
Previous results from our genetic analyses using pedigrees from a French Canadian population suggested that the interval delimited by markers on chromosome 12, D12S86 and D12S378, was the most probable genomic region to contain a susceptibility gene for affective disorders. Association studies with microsatellite markers using a case/control sample from the same population (n = 427) revealed significant allelic associations between the bipolar phenotype and marker NBG6. Since this marker is located in intron 9 of the P2RX7 gene, we analyzed the surrounding genomic region for the presence of polymorphisms in regulatory, coding and intron/exon junction sequences. Twenty four (24) SNPs were genotyped in a case/control sample and 12 SNPs in all pedigrees used for linkage analysis. Allelic, genotypic or family-based association studies suggest the presence of two susceptibility loci, the P2RX7 and CaMKK2 genes. The strongest association was observed in bipolar families at the non-synonymous SNP P2RX7-E13A (rs2230912, P-value = 0.000708), which results from an over-transmission of the mutant G-allele to affected offspring. This Gln460Arg polymorphism occurs at an amino acid that is conserved between humans and rodents and is located in the C-terminal domain of the P2X7 receptor, known to be essential for normal P2RX7 function.
ABSTRACT:The non-nucleoside reverse transcriptase inhibitor efavirenz (EFV) is directly conjugated by the UDP-glucuronosyltransferase (UGT) pathway to form EFV-N-glucuronide (EFV-G), but the enzyme(s) involved has not yet been identified. The glucuronidation of EFV was screened with UGT1A and UGT2B enzymes expressed in a heterologous system, and UGT2B7 was shown to be the only reactive enzyme. The apparent K m value of UGT2B7 (21 M) is similar to the value observed for human liver microsomes (24 M), whereas the variant allozyme UGT2B7*2 (Tyr 268 ) displayed similar kinetic parameters. Because 3-azido-3-deoxythymidine (AZT), one of the most current nucleotide reverse transcriptase inhibitors prescribed in combination with EFV, is also conjugated by UGT2B7, the potential metabolic interaction between EFV and AZT has been studied using human liver microsomes. Glucuronidation of both drugs was inhibited by one another, in a concentrationdependent manner. At K m values (25 and 1000 M for EFV and AZT, respectively), EFV inhibited AZT glucuronidation by 47%, whereas AZT inhibited EFV glucuronidation by 23%. With a K i value of 17 M for AZT-glucuronide formation, EFV appears to be one of the most selective and potent competitive inhibitor of AZT glucuronidation in vitro. Moreover, assuming that concentrations of EFV achieved in plasma (C max ؍ 12.9 M) are in a range similar to its K i value, it was estimated that EFV could produce a theoretical 43% inhibition of AZT glucuronidation in vivo. We conclude that UGT2B7 has a major role in EFV glucuronidation and that EFV could potentially interfere with the hepatic glucuronidation of AZT.Efavirenz [(EFV) (Sustiva); Bristol-Myers Squibb Co., Princeton, NJ] is the preferred non-nucleoside reverse transcriptase inhibitor (NNRTI) agent for initial therapy for HIV infection. A typical antiretroviral treatment regimen consists of two nucleoside reverse transcriptase inhibitors (NRTIs) plus a protease inhibitor or an NNRTI. The 2008 IAS-USA guidelines recommend either of two basic threedrug regimens for treatment of antiretroviral-naive patients: 1) EFV plus two NRTIs; or 2) a ritonavir-boosted protease inhibitor (lopinavir, atazanavir, fosamprenavir, darunavir, or saquinavir) plus two NRTIs [tenofovir and emtricitabine; abacavir and lamivudine; or 3Ј-azido-3Ј-deoxythymidine (zidovudine; AZT) and lamivudine] (Hammer et al., 2008). A recent study on HIV-infected adults of Southern Africa further revealed that among patients who began nevirapine-based or EFV-based antiretroviral therapy between January 1998 and September 2004, 1321 of 1822 EFV-treated patients (72.5%) received it in combination with the NRTI pair AZT plus lamivudine (Nachega et al., 2008). Thus, a significant proportion of HIV-infected, highly active antiretroviral therapy-naive adults receive EFV and AZT in combination.A large interpatient variability was found to affect EFV bioavailability (coefficient of variation up to 118%) (Marzolini et al., 2001), which might be explained in part by its extensive metabolism....
BackgroundUDP-glucuronosyltransferase 1A1 (UGT1A1) is a pivotal enzyme involved in metabolism of SN-38, the active metabolite of irinotecan commonly used to treat metastatic colorectal cancer. We previously demonstrated aberrant methylation of specific CpG dinucleotides in UGT1A1-negative cells, and revealed that methylation state of the UGT1A1 5'-flanking sequence is negatively correlated with gene transcription. Interestingly, one of these CpG dinucleotides (CpG -4) is found close to a HNF1 response element (HRE), known to be involved in activation of UGT1A1 gene expression, and within an upstream stimulating factor (USF) binding site.ResultsGel retardation assays revealed that methylation of CpG-4 directly affect the interaction of USF1/2 with its cognate sequence without altering the binding for HNF1-alpha. Luciferase assays sustained a role for USF1/2 and HNF1-alpha in UGT1A1 regulation in colon cancer cells. Based on the differential expression profiles of HNF1A gene in colon cell lines, we also assessed whether methylation affects its expression. In agreement with the presence of CpG islands in the HNF1A promoter, treatments of UGT1A1-negative HCT116 colon cancer cells with a DNA methyltransferase inhibitor restore HNF1A gene expression, as observed for UGT1A1.ConclusionsThis study reveals that basal UGT1A1 expression in colon cells is positively regulated by HNF1-alpha and USF, and negatively regulated by DNA methylation. Besides, DNA methylation of HNF1A could also play an important role in regulating additional cellular drug metabolism and transporter pathways. This process may contribute to determine local inactivation of drugs such as the anticancer agent SN-38 by glucuronidation and define tumoral response.
This study investigated the molecular mechanisms underlying the regulatory effect of the newly discovered 45-kDa enzymatically inactive UGT1A spliced polypeptides, named isoform i2, upon UGT1A-mediated glucuronidation. Initially, using an inducible system that mimics the relative abundance of isoforms 1 and 2 of UGT1A1 in human tissues, the rates of formation of glucuronides were significantly reduced. We then used a heterologous system constitutively expressing both isoforms i1 and i2 for an in-depth investigation of the presence of spliced i2 on glucuronidation kinetics. UGT1A1, UGT1A7, and UGT1A8 were selected as candidates for these studies. In all cases, coexpression of i1 and i2 in HEK293 cells leads to a significant reduction of the velocity of the glucuronidation reaction without affecting the affinity (K m app ) for all substrates tested and the K m for the co-substrate, UDP-glucuronic acid. The data are consistent with a dominant-negative model of inhibition but do not sustain with an UGT1A_i2-mediated inhibition by competitive binding for substrate or the co-substrate. In contrast, the data from the co-immunoprecipitation experiments are indicative of the existence of a mixture homo-oligomeric (i1-i1 or i2-i2) and hetero-oligomeric (i1-i2) complexes in which the i2-i2 and i1-i2 subunits would be inactive. Thus, protein-protein interactions are likely responsible for the inhibition of active UGT1A_i1 by i2 spliced polypeptides. This new regulatory mechanism may alternatively modulate cellular response to endo/xeno stimulus.
Summary. Factor VIII activity (factor VIII:C) and factor VIII antigen (factor VIII:Ag) levels above150 IU/dl are associated with a five-to sixfold increased risk of venous thrombosis compared with levels , 100 IU/dl. These high levels are present in 25% of patients with a first episode of deep-vein thrombosis and in 11% of healthy controls. von Willebrand factor (VWF) and blood group are important determinants of the factor VIII level in plasma and therefore contribute to thrombotic risk, while factor VIII appears to be the final effector. Previously, we found familial clustering of factor VIII:C levels in women, which remained after adjustment for VWF and blood group. In the present study, we analysed the familial influence on factor VIII:Ag levels exceeding 150 IU/dl in 12 large families with thrombophilia in which high factor VIII:Ag levels contribute to thrombotic risk. As expected, blood group was a main determinant of the plasma factor VIII level: 58 relatives (32%) had factor VIII levels above 150 IU/dl and 50 (86%) of these had blood group non-O. After adjustment for blood group and age, we found an association between factor VIII:Ag levels in sister pairs (0´35, P 0´003), brother pairs (0´35, P 0´003), brother±sister pairs (0´35, P , 0´001) and in mother±son pairs (0´45, P 0´02), but not in father±daughter or father±son pairs. The familial aggregation test was strongly positive for factor VIII:Ag levels (P , 0´001) and remained so after adjustment for the influence of blood group. We conclude that high factor VIII:Ag levels are a highly prevalent risk factor for venous thrombosis and contribute to risk in families with thrombophilia, and that these high levels are likely to be genetically determined by factors other than just blood group.
Despite the importance of UDP-glucuronosyltransferase (UGT) 1A1*28 in irinotecan pharmacogenetics, our capability to predict drug-induced severe toxicity remains limited. We aimed at identifying novel genetic markers that would improve prediction of irinotecan toxicity and response in advanced colorectal cancer patients treated with folic acid (leucovorin), fluorouracil (5-FU), and irinotecan (camptosar)-based regimens. The relationships between UGT1A candidate markers across the gene (n 5 21) and toxicity were prospectively evaluated in 167 patients. We included variants in the 39untranscribed region (39UTR) of the UGT1A locus, not studied in this context yet. These genetic markers were further investigated in 250 Italian FOLFIRI-treated patients. Several functional UGT1A variants, including UGT1A1*28, significantly influenced risk of severe hematologic toxicity. As previously reported in the Italian cohort, a 5-marker risk haplotype [haplotype II (HII); UGTs 1A9/1A7/1A1] was associated with severe neutropenia in our cohort [odds ratio (OR) 5 2.43; P 5 0.004]. The inclusion of a 39UTR singlenucleotide polymorphism (SNP) permitted refinement of the previously defined HI, in which HIa was associated with the absence of severe neutropenia in combined cohorts (OR 5 0.55; P 5 0.038). Among all tested UGT1A variations and upon multivariate analyses, no UGT1A1 SNPs remained significant, whereas three SNPs located in the central region of UGT1A were linked to neutropenia grade 3-4. Haplotype analyses of these markers with the 39UTR SNP allowed the identification of a protective HI (OR 5 0.50; P 5 0.048) and two risk haplotypes, HII and HIII, characterized by 2 and 3 unfavorable alleles, respectively, revealing a dosage effect (ORs of 2.15 and 5.28; P # 0.030). Our results suggest that specific SNPs in UGT1A, other than UGT1A1*28, may influence irinotecan toxicity and should be considered to refine pharmacogenetic testing.
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