Mario Harvey

Analysis of single nucleotide polymorphisms in genes in the chromosome 12Q24.31 region points to P2RX7 as a susceptibility gene to bipolar affective disorder

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.

Glucuronidation of the Antiretroviral Drug Efavirenz by UGT2B7 and an in Vitro Investigation of Drug-Drug Interaction with Zidovudine

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 Km value of UGT2B7 (21 μM) is similar to the value observed for human liver microsomes (24 μM), whereas the variant allozyme UGT2B7*2 (Tyr268) 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 concentration-dependent manner. At Km values (25 and 1000 μM for EFV and AZT, respectively), EFV inhibited AZT glucuronidation by 47%, whereas AZT inhibited EFV glucuronidation by 23%. With a Ki value of 17 μM 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 (Cmax = 12.9 μM) are in a range similar to its Ki 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.

UGT genomic diversity: beyond gene duplication

The human uridine diphospho (UDP)-glucuronosyltransferase (UGT) superfamily comprises enzymes responsible for a major biotransformation phase II pathway: the glucuronidation process. The UGT enzymes are located in the endoplasmic reticulum of almost all tissues, where they catalyze the inactivation of several endogenous and exogenous molecules, including bilirubin, sex steroids, numerous prescribed drugs, and environmental toxins. This metabolic pathway is particularly variable. The influence of inheritable polymorphisms in human UGT-encoding genes has been extensively documented and was shown to be responsible for a fraction of the observed phenotypic variability. Other key genomic processes are likely underlying this diversity; these include copy-number variations, epigenetic factors, and newly discovered splicing mechanisms. This review will discuss novel molecular aspects that may be determinant to UGT phenotypes.

Copy-number variations (CNVs) of the human sex steroid metabolizing genes UGT2B17 and UGT2B28 and their associations with a UGT2B15 functional polymorphism.

UGT2B17 and UGT2B28 are among the most commonly deleted genes in humans and encode members of the uridine diphosphate (UDP)‐glucuronosyltransferase 2B (UGT2B) subfamily. They are involved, along with UGT2B15, in the catabolism of sex‐steroid hormones. Despite the recent biomedical interest in UGT2B17 and UGT2B28 copy‐number variations (CNVs) within human populations, the impact of their gene dosage has been hampered by the lack of precise molecular identification of the common deletion breakpoints within high homology sequence regions on chromosome 4. We have characterized these common deletions and report their coexistence in Caucasians, along with the p.D85Y (rs1902023:G>T) functional polymorphism of UGT2B15. Segmental duplications of 4.9 kb for UGT2B17 and 6.8 kb for UGT2B28 comprise purine‐rich recombination sites located 117 kb and 108 kb apart on both ends of the deletions. CNVs of UGT2B17 and UGT2B28 occur in Caucasians at 27% and 13.5%, respectively. While only 43% have two copies of both genes, 57% harbor at least one deletion. Their co‐occurrence on 5% of chromosomes creates a 225‐kb genomic gap. CNVs of both UGT2B17 and UGT2B28, with the co‐occurrence of UGT2B15:p.D85Y, generate seven distinct haplotypes. Restricting the analyses to CNV of the UGT2B17 gene without evaluating UGT2B28 CNV, along with the genotype of UGT2B15, may over‐ or underestimate the impact of each gene under physiological conditions or disease states. Hum Mutat 30:1–10, 2009.

Analysis of microsatellite markers and single nucleotide polymorphisms in candidate genes for susceptibility to bipolar affective disorder in the chromosome 12Q24.31 region.

Previous results from our genetic analyses using pedigrees from a French Canadian population suggested that the interval delimited by markers D12S86 and D12S378 on chromosome 12 was the most probable genomic region to contain a susceptibility gene for affective disorders. Here we present a more detailed genetic analysis of a 7.7 Mb genomic region located on 12q24.31. This region was saturated with 20 microsatellite markers to refine the candidate region and linkage analysis performed in 41 families from the Saguenay‐Lac‐St‐Jean (SLSJ) region of Quebec. The results of two point parametric analysis using MFLINK supported the presence of a susceptibility locus on chromosome 12q24.31. Association studies with microsatellite markers using a case/control sample from the same population (n = 401) and analyzed with CLUMP revealed significant allelic associations between the bipolar phenotype and markers NBG6 (P = 0.008) and NBG12 (P < 10−3). According to these results, we investigated candidate genes in the NBG12 area. We analyzed 32 genes for the presence of polymorphisms in coding sequences and intron/exon junctions and genotyped 22 non‐synonymous SNPs in the SLSJ case/control sample. Two uncommon polymorphisms (minor allele frequency ≤ 0.03) found in KIAA1595 and FLJ22471 genes, gave P‐values below 0.05 with the T1 statistic. Moreover, using haplotype analysis, a nearly significant haplotypic association was observed at the HM74 gene. These results do not give strong support for a role in the susceptibility to bipolar disorder of any of these genes analyzed. However, the significance of rare polymorphisms should be explored by further analyses.

Modulation of the Human Glucuronosyltransferase UGT1A Pathway by Splice Isoform Polypeptides Is Mediated through Protein-Protein Interactions

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, co-expression of i1 and i2 in HEK293 cells leads to a significant reduction of the velocity of the glucuronidation reaction without affecting the affinity (Km app) for all substrates tested and the Km 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.

Analysis of inherited genetic variations at the UGT1 locus in the French-Canadian population.

The UDP‐glucuronosyltransferase UGT1 locus is composed of nine exon 1s, each flanked by a unique promoter region, and common exons (2, 3, 4, and the alternatively spliced exons 5a and 5b). Here, we characterized the genetic architecture of the UGT1 gene in a Caucasian sample. Overall, 98 variations in regulatory domains, exons and exon–intron boundaries were genotyped in 254 unrelated subjects, including 12 unreported UGT1 polymorphisms. We determined allele frequencies, computed pairwise linkage disequilibrium (LD), and inferred haplotypes; this thorough analysis yielding a limited number of common UGT1 haplotypes. Moreover, only 17 haplotype tagging single nucleotide polymorphisms (htSNPs) are required to capture most of the allelic diversity of the locus. Four haplotype blocks were inferred: Block 9/6 (UGT1A9, UGT1A7 and UGT1A6), Block 4 (UGT1A4), Block 3/1 (UGT1A3 and UGT1A1), and Block C (3′UTR). A high level of linkage exists between Blocks 9/6 and 3/1, while the 3′UTR SNPs are genetically isolated. The most common haplotype (16.5%) presents multiple deleterious alleles, mainly 1A1*28, 1A3*2, 1A6*2, and 1A7*4. More interestingly, we reveal the co‐occurrences of multiple deleterious variations, some of which could be associated with interindividual differences in glucuronidation. Comparison with the HapMap data set demonstrated differences in haplotypic diversity between ethnic samples, but similarity between Caucasian cohorts, as observed previously. This report provides relevant data for further pharmacogenomic studies. Hum Mutat 0, 1–12, 2009.

Refining the UGT1A Haplotype Associated with Irinotecan-Induced Hematological Toxicity in Metastatic Colorectal Cancer Patients Treated with 5-Fluorouracil/Irinotecan-Based Regimens

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 = 21) and toxicity were prospectively evaluated in 167 patients. We included variants in the 3′untranscribed region (3′UTR) 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) = 2.43; P = 0.004]. The inclusion of a 3′UTR single-nucleotide polymorphism (SNP) permitted refinement of the previously defined HI, in which HIa was associated with the absence of severe neutropenia in combined cohorts (OR = 0.55; P = 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 3′UTR SNP allowed the identification of a protective HI (OR = 0.50; P = 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.

Immunohistochemical expression of conjugating UGT1A-derived isoforms in normal and tumoral drug-metabolizing tissues in humans

Glucuronidation by UDP‐glucuronyltransferase (UGT) enzymes is the prevailing conjugative pathway for the metabolism of both xenobiotics and endogenous compounds. Alterations in this pathway, such as those generated by common genetic polymorphisms, have been shown to significantly impact on the health of individuals, influencing cancer susceptibility, responsiveness to drugs and drug‐induced toxicity. Alternative usage of terminal exons leads to UGT1A‐derived splice variants, namely the classical and enzymatically active isoforms 1 (i1) and the novel enzymatically inactive isoforms 2 (i2). In vitro functional data from heterologous expression and RNA interference experiments indicate that these i2 isoforms act as negative modulators of glucuronidation, likely by forming inactive complexes with active isoform 1. We used specific antibodies against either active i1 or inactive i2 proteins to examine their distribution in major drug‐metabolizing tissues. Data revealed that UGT1A_i1 and inactive UGT1A_i2 are co‐produced in the same tissue structures, including liver, kidney, stomach, intestine and colon. Examination of the cellular distribution and semi‐quantitative level of expression of UGT1As revealed heterogeneous expression of i1 and i2 proteins, with increased expression of i2 in liver tumours and decreased levels of i1 and i2 in colon cancer specimens, compared to normal tissues. These differences in expression may be relevant to human colon and liver cancer tumorigenesis. Our data clearly demonstrate the similar immunolocalization of active and inactive UGT1A isoforms in most UGT1A‐expressing cell types of major tissues involved in drug metabolism. These expression patterns are consistent with a dominant‐negative function for the i2 encoded by the UGT1A gene. Copyright

Alternatively Spliced Products of the UGT1A Gene Interact with the Enzymatically Active Proteins to Inhibit Glucuronosyltransferase Activity In Vitro

UDP-glucuronosyltransferases (UGTs) are major mediators in conjugative metabolism. Current data suggest that UGTs, which are anchored in the endoplasmic reticulum membrane, can oligomerize with each other and/or with other metabolic enzymes, a process that may influence their enzymatic activities. We demonstrated previously that the UGT1A locus encodes previously unknown isoforms (denoted “i2”), by alternative usage of the terminal exon 5. Although i2 proteins lack transferase activity, we showed that knockdown of endogenous i2 levels enhanced cellular UGT1A-i1 activity. In this study, we explored the potential of multiple active UGT1A_i1 proteins (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, and UGT1A10) to interact with all spliced i2s by coimmunoprecipitation. We further studied the functional consequences of coexpressing various combinations of spliced i1s and i2s from highly similar UGTs, namely UGT1A7, UGT1A8, and UGT1A9, based on expression profiles observed in human tissues. The i1 isoform of each UGT1A coimmunoprecipitated its respective i2 homolog as well as all other i2s, indicating that they can form heteromeric complexes. Functional data further support the fact that i2 splice species alter glucuronidation activity of i1s independently of the identity of the i2, although the degree of inhibition varied, suggesting that this phenomenon may occur in tissues expressing such combinations of splice forms. These results provide biochemical evidence to support the inhibitory effect of i2s on multiple active UGT1As, probably through formation of inactive heteromeric assemblies of i1s and inactive i2s. The relative abundance of active/inactive oligomeric complexes may thus determine transferase activity.