Bertrand Caillier*, Johanie Lépine*, Jelena Tojcic, Vincent Ménard, Louis Pérusse, Alain Bélanger, Olivier Barbier et Chantai Guillemette
*Co-permier auteurs
Pharmacogenetics and Genomics (facteur d'impact: 5.391) 2007 Jul; 17(7):481-95
Résumé
Nous avons identifié les mécanismes génétiques expliquant les variations interindividuelles en expression et en activité de UGT1A3, conjuguant efficacement l'estrone (Ei). UGT1A3 a été séquence chez 249 Caucasiens et nous avons identifié 7 polymorphismes en 5' et 10 dans l'exon 1, dont 6 affectent la séquence protéique. Une activité transcriptionnelle réduite a été associée avec les 6 haplotypes du promoteur suite à des essais d'un gène rapporteur à la luciférase (2-fois, p<0.0001). Les analyses de retard sur gel indiquent que les variations -
148 T>C et -581 C>T, liant respectivement les facteurs HNF-la et HNF-4a, pourraient être impliqués. Parallèlement, trois phénotypes ont été observés pour les formes variantes de protéines UGT1A3 envers Ei : 1) Clairance élevée ; UGT1A3*1, *2 (WnR-V*7A) et *3 (WnR), 2) Intermédiaire ; *5 (Q6R-WnR), *7 (F110I), *9 (WnR-M208L), *10 (Y41 A) et *11 (WnR-V47A-M,14I) et 3) Faible ; *4 (R45W), *6 (WnR-V47A-M270V) et *8 (A158V). Basé sur ces études in vitro, 20.1% des Caucasiens ont deux alleles affectant potentiellement l'expression et/ou l'activité de UGT1A3. Des polymorphismes de UGT1A3 pourraient contribuer aux variations dans l'exposition d'une femme à Ei au cours de sa vie et modifier le risque de cancer.
A PHARMACOGENOMICS STUDY OF THE HUMAN ESTROGEN GLUCURONOSYLTRANSFERASE
UGT1A3
Bertrand Cailliert1'2, Johanie Lépinet1,2, Jelena Tojcic1'2, Vincent Ménard1'2, Louis Perusse3, Alain Bélanger4, Olivier Barbier2 and Chantai Guillemette1'2*
'Canada Research Chair in Pharmacogenomics, Laboratory of Pharmacogenomics, 2Oncology and Molecular Endocrinology Research Center, CHUQ Research Center and Faculty of Pharmacy, Laval University, Québec, Canada. 3Division of Kinesiology, Department of Social and Preventive Medicine, Faculty of Medicine, Laval University, Québec, Canada. 4Oncology and Molecular Endocrinology Research Center, CHUQ Research Center and Faculty of Medicine, Laval University, Québec, Canada.
tThese authors have contributed equally to this work.
* Author to whom correspondence should be sent: Chantai Guillemette, Ph.D.
Canada Research Chair in Pharmacogenomics Laboratory of Pharmacogenomics
CHUL Research Center, T3-48 2705 Boulevard Laurier
Québec, Canada, Gl V 4G2
Tel. (418) 654-2296, Fax. (418) 654-2761 E-mail: chantai.guillemette@crchul.ulaval.ca
These results were partially presented at the Annual Meeting of The American Society for Clinical Pharmacology and Therapeutics (ASCPT) held at Baltimore in Maryland, USA, March 8-11, 2006, number OV-A-3 and also at the 97th Annual Meeting of the American Association for Cancer Research (AACR) held at Washington in the District of Columbia, USA, April 1-5, 2006, number #5389.
Short title: Pharmacogenomics study of human UGT 1 A3
Footnotes: This work was supported by the Canada Research Chair program, the Canadian Institutes of Health Research (CIHR MOP-68964) and Canada research chair program (C.G.). J.L. is a recipient of a studentship from CIHR. B.C. and J.T. are recipients of a studentship from the Fonds de la recherche en santé du Québec (FRSQ). O.B. is granted by the Health Research Foundation Rx&D/CIHR. C.G. is holder of the Canada Research Chair in Pharmacogenomics. Genbank accession numbers: UGT1A3*1 (AY724451), *2 (AY724454), *3(AY724452), *6(AY724455), *7(AY724450), *8(DQ408604) and *9(AY724453).
Number of: Text pages: 47 Tables: 3 Figures: 7 References: 42
Number of words: Abstract: 250 words Introduction: 689 words Discussion: 1736 words
The abbreviations used are : UGT, UDP-glucuronosyltransferase; SNP, Single Nucleotide Polymorphism; Ei, estrone; E2, estradiol; -G, glucuronide; -S, sulphate; EMSA, Electrophoresis Mobility Shift Assay; HNF, Hepatocyte Nuclear Factor; NE, nuclear extracts; UDPGA, UDP-glucuronic acid; HPLC, high-performance liquid chromatography; MS/MS, tandem mass spectrometry; H, Haplotype.
Abstract
UGT 1 A3 is one of the most efficient at conjugating estrone (Ei), a precursor for biosynthesis of estradiol (E2) in peripheral tissues. We established the genetic mechanisms that might contribute to individual variation in UGT 1 A3 expression and activity. UGT 1 A3 first exon and 5'-flanking regions were sequenced in 249 Caucasians. We identified 17 polymorphisms, among them 7 regulatory and 10 exonic polymorphisms with 6 leading to amino acid changes. Luciferase reporter assays, site directed mutagenesis and electrophoretic mobility shift assays using hepatoma HepG2 cells were carried out to show functionality of variant promoters. Reduce transcriptional activity was associated with all 6 variant promoters (2-fold; p<0.001). One of the potential mechanisms would involved the -
148 T>C and -581 C>T variations that modulate gene function by affecting HNF-la and HNF-4oc binding, respectively. Then, Ei-conjugating activity was assessed with eleven heterologously expressed allozymes. Three phenotypes were observed; UGT1A3*1, *2 (WnR, V47A) and *3 (WUR) with high intrinsic clearance values; UGT1A3*5 (Q6R, WUR), *7 (FnoI), *9 (WnR, M208L), *10 (V47A) and *11 (WnR, V47A and M114I) had intermediate CLjnt (2X to 10X lower vs *\), whereas UGT1A3*4 (R45W), *6 (WUR, V47A, M270V) and *8 (A158V) had low CLint (>10X lower vs *1). Diplotype analyses indicate that 20.1% of individuals carry two alleles affecting UGT 1 A3 expression and/or activity. This study did not investigate genotype-phenotype association, but raise the possibility that genetically determined variation might contribute to variability in the inactivation of Ei by UGT 1 A3 and subsequence changes in lifetime exposure to estrogens potentially modifying risk of cancer.
INTRODUCTION
UDP-glucuronosyltransferase (UGT) enzymes are a superfamily of proteins that catalyzes the glucuronidation of a wide range of xenobiotics and endogenous compounds. This metabolic process plays an important role in drug metabolism, in the detoxification and excretion of environmental carcinogens and in maintaining the homeostasis of several biochemical processes, including the metabolism of bilirubin, steroid hormones and bile acids. Eighteen functional proteins have been described in humans and have been categorized in two subfamilies UGT1 and UGT2. Among those, UGT 1 A3 is one of the enzymes responsible for the addition of glucuronic acid on amino functions (N- glucuronidation) [1], UGT 1 A3 is also active towards steroid hormones and biliary acids [2, 3] and conjugates several benzo[a]pyrene metabolites, some phenolic compounds, opioids (morphine, buprenorphine) and other drugs [4, 5]. UGT 1 A3 is mostly expressed in the liver, but is also found in bile ducts and the gastric and the intestine tissues [4, 5].
There is currently a growing interest regarding the role of UGT in the metabolism of steroid hormones in peripheral tissues, such as the breast, ovary and uterus, and their role in protecting against excessive exposure to estrogens that represents a known risk factor for cancer development in these organs [6-11]. Our recent work revealed six UGT proteins that are particularly active for the conjugation of parent estrogens estrone (Ei) and 17(3-estradiol (E2), as well as for their hydroxylated and methoxylated metabolites [2]. Among the active enzymes are UGT2B7 and the bilirubin-conjugating enzyme UGT1A1 in addition to other UGT1 family members, namely UGT1A3, UGT1A8, UGT1A9 and UGT1A10. Their expression in tissues such as mammary epithelium cells and uterine cells have been demonstrated [2, 8, 12, 13]. UGT 1 A3 is of particular interest since its is one of the most efficient enzymes in catalyzing the conjugation of Ei to produce the inactive Ei-3- glucuronide (Ei-3G) [2]. In postmenopausal women, Ei conjugated with sulfate (Ei-S) constitutes the most abundant circulating precursor of estrogen [6] and represents a major immediate source of E2 for peripheral tissues that express sulfatase (EpS—>E\) and 17-beta- hydroxysteroid dehydrogenase enzymes types 1, 7 and 12 (Ei—>E2) [14-16] (Figure la). These enzymes are expressed in endometrial and mammary tissues supporting that Ei-S is a significant supply of active E2 that can be locally produced in target cells. Because
UGT 1 A3 is one of the most reactive enzymes predicted to be involved in the inactivation of Ei to Ei-3G in vivo, it would be important to determine the possible contribution of inheritance to variation in the expression and/or activity of UGT 1 A3.
UGT1A3 is encoded by the singular UGT1 gene on chromosome 2q37. Four of the five exons that constitute the UGT 1 A3 mRNA encode the C-terminal half of the molecule and are shared by all eight other functional UGT isoforms expressed from the UGT1A locus. Several polymorphisms at the UGT1A locus have been linked to altered conjugation of estrogens in vitro and were shown to contribute significantly to the genetic susceptibility of breast, ovarian and endometrial cancers [7, 8, 10-12, 17, 18]. These studies reinforce the concept that the inactivation by UGT conjugating enzymes modifies, in peripheral tissues, local hormonal exposure. Most studies were focused on coding region polymorphisms while nucleotide changes affecting the 5'-untranslated regions of the UGT genes are only a recent concern. Single nucleotide polymorphisms (SNPs) in regulatory DNA sequences, or other structural modifications in the genomic DNA such as nucleotide repeats, may alter gene expression by influencing the binding affinity of transcription factors. An important example is the UGT1A1*2S variant, located in the TATA box region of the gene, which dramatically impairs transcriptional activity and, consequently, UGT1A1 protein expression [19]. A limited number of polymorphisms have been reported for the UGT 1 A3 gene [20- 23]. In addition there is currently no report examining promoter SNPs and haplotype of the UGT1A3 gene while the regulation of the UGT1A3 gene has yet to be studied.
This study was designed to explore molecular genetic mechanism(s) that might contribute to individual variation in UGT 1 A3 activity and expression. We scanned for polymorphisms in the coding region of UGT 1 A3 and its proximal promoter, characterized the effect of SNP haplotypes on UGT1A3 promoter activity and enzyme function and assessed the co- occurrence of coding region and regulatory SNPs.
M A T E R I A L S A N D M E T H O D S
Chemicals. Ei and Ei-3-glucuronides (Ep3G) were purchased from Steraloids (Newport, RI). All chemicals were of the highest grade available. Cell culture reagents were from Invitrogen (Carlsbad, CA). Restriction enzymes and other molecular biology reagents were from Stratagene (La Jolla, CA), Promega Corp. (Madison, WI) and Roche (Mannheim, Germany). Protein assay reagents were obtained from Bio-Rad Laboratories Inc. (Hercules, CA). [<x-32P]dCTP was purchased from NEN-Life Sciences (Boston, MA). ExGen 500 was from Invitrogen (Burlington, Canada). Anti-HNF-la, anti-HNFl|3 and anti-HNF-4a were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
Human DNA samples and sequencing. DNA samples from 249 healthy and unrelated Caucasian subjects were obtained from the Québec Family Study (QFS) for UGT1A3 single-nucleotide polymorphism (SNP) genotyping [24]. These samples were anonymized prior to their reception in our laboratory. All subjects have provided written consent for use of their DNA for experimental purposes, and the present study was reviewed and approved by Institutional Review Boards (CHUL Research Center and Laval University).
The first exon of UGT 1 A3 (-80/+1122) and the promoter region (-885/+171) were amplified using PCR with specific primers (Table 1). The reaction volume for the PCR was 50 [tl containing 2 mM MgC^, 0.2 mM of each dNTP, 0.4 pM of each primer. Two units of TaqDNA polymerase were added and the reaction was initially incubated at 95°C for 3 min followed by 35 and 40 cycles for the coding region and promoter region, respectively, at 95°C 30 sec, 53°C 30 sec and 72°C 40 sec (coding region, 1st strategy), or 95°C 30 sec, 63°C 30 sec and 72°C 40 sec (coding region, 2nd strategy), or 95°C 30 sec, 65°C 30 sec and 72°C 30 sec (coding region, 3rd strategy), or 95°C 30 sec, 58°C 40 sec and 72°C 1 min (promoter region, both strategies). The PCR reaction was completed with an incubation of 7 min at 72°C. Amplicons were sequenced with an ABI 3700 automated sequencer using Big Dye™ primer chemistry (Perkin Elmer, Wellesley, MA, USA). Sequences were analyzed with Staden preGap4 and Gap4 programs (staden.sourceforge.net). Polymorphisms in DNA are described according to the UGT allele's nomenclature guidelines available at http://galien.pha.ulaval.ca/alleles/alleles.html. The indicated positions are given with
respect to the UGT 1 Al translation start site with the A of ATG depicted as +1 and the immediately following 5' base as -1.
UGT1A3 stable cell lines expressing variant allozymes. Directed mutagenesis was used to generate variant UGT 1 A3 cDNA plasmids (reference sequence: Genbank AF297093) (Table 1). Individual UGT 1 A3 variants were stably expressed in HEK-293 cells and microsomal fraction was prepared by differential centrifugation, as previously described [2]. For a more accurate assessment of the quantitative differences in glucuronidation activity between variant allozymes, HEK-293-derived cell lines were characterized for UGT 1 A3 protein expression level by Western blot analysis (data not shown). Briefly, 10 pg of microsomal variant UGT 1 A3 proteins were separated on SDS-PAGE and transferred onto nitrocellulose. The quantification of UGT 1 A3 protein was assessed using the anti- human antibody RC-71 that recognizes the carboxyl-terminus region common to all UGT1A [2]. In parallel, the expression of calnexin, that is constitutively expressed in the reticulum endoplasmic membrane, was determined in order to normalize sample loading (Stressgen Biotechnologies, Victoria, Canada). Bands were visualized using enhanced chemiluninescence (ECL, Amersham, Piscataway, NJ) and quantified by Bioimage Visage
110s from Genomic Solutions Inc (Ann Harbor, MI). The expression level of each UGT 1 A3 variant was determined by the ratio UGT/calnexin.
Enzyme assays, mass spectrometry analyses and substrate kinetic. To establish the functional impact of nonsynonymous polymorphisms of UGT 1 A3 on Ei glucuronidation, enzymatic assays were conducted with microsomal proteins obtained from HEK-293 cells stably overexpressing each variant protein. Enzymatic assays were performed with 40 to 60 pg of total microsomal proteins in 100 pi reaction volume containing; 50 mMTris-HCl (pH 7.5), 10 mM MgCh, 5 ng/ml pepstatine, 0.5 ng/ml leupeptine, 10 pg/ml phosphatidylcholine and 1 mM UDPGA. Reactions were started by adding varying concentrations of Ei ranging from 1 to 200 [iM and incubating at 37°C. After three hours, the assays were stopped by adding 100 pi of ice-cold methanol and then centrifuged at
14,000 x g for 10 min in order to remove proteins. Glucuronide formation was assessed by mass spectrometry analyses, as previously described [2]. Briefly, the HPLC and tandem
mass spectrometry (MS/MS) system consisted of a mass spectrometer (model API 3000, Perkin-Elmer Sciex, Thornhill, Canada) equipped with an electrospray ionization source in the negative ion mode and a HPLC pump plus autosampler (model 2690, Waters, Milford, MA). Relative glucuronidation activities (Rel. Vmax) are expressed as pmol/h/mg/UGT level and are obtained after dividing the absolute activity (Abs. Vmax; pmol/h/mg) by UGT expression level determined by Western blot. Three independent experiments were done in duplicates for each UGT 1 A3 variants except for UGT1A3*1 which is from five experiments done in duplicates.
Determination of the kinetic parameters was performed using Sigmaplot 8.02a with Enzyme Kinetic 1.1 (Systat Software, Point Richmond, CA). Best kinetic enzyme model was chosen after visual inspection of the fitted curve, the Eadie-Hofstee plot and the Akaike Information Criterion values (AIC). All UGT 1 A3 variants displayed a sigmoid profile toward Ei conjugation. The equation used for sigmoid kinetic corresponds to the Hill equation: V = (Vmax x Sn) / (Km" + Sn), where Vmax is the maximal velocity, Km is the substrate concentration at half-maximal velocity, and 'n' is the degree of curve sigmoidicity. Based on this model, the intrinsic clearance estimation values were calculated using the following equation: CLjnt = (Vmax/Km) x ((n - 1) / (n x (n - l)1/n)) [25].
UGT1A3 promoter functional assays. A 1146 pb fragment of the human UGT1A3 promoter (-1144/+2) was amplified by PCR from human genomic DNA samples with known haplotypes using Pfu polymerase (Stratagene) and 100 pmol of sense (5- CTAGCTGCTCGAGGCATCAGCAATCTTGTGAGCACAGGAC-3') and antisense (5'- CTAGCTGAAGCTTATCTCAGCAGAAGACACGGACAGC-3 ' ) primers based on the UGT1A gene sequence AF297093 [26]. Xhol and Hindlll restriction sites were inserted in the sense and antisense primers, respectively (underlined sequence). The PCR products were digested and inserted in the pGL3 luciferase vector to generate the UGT 1 A3 reporter constructs. The reporter constructs for the UGT 1 A3 reference promoter and UGT 1 A3 promoter haplotype variants were generated from individuals of known genotypes or by site directed mutagenesis. Human HepG2 hepatoma cells were from the American Type Culture Collection (Rockville, MD) and cultured in Minimum Essential Medium Eagle (MEM) (Wisent, Ont., Canada) supplemented by 10% Fetal Bovine Serum (FBS), 1000U/L of
Streptomycin Penicillin, ImM of Sodium Pyruvate and O.lmM of non essential amino acids. HepG2 cells were plated at a density of 60 x 103 cells/well of 24-well plates and transfected with 100 ng of the indicated luciferase reporter plasmids and 30 ng of the pRL- NULL expression vector with 2 \iL ExGen™500 reagent (Invitrogen, Burlington, Canada). for 6 hours at 37°C. All samples were complemented with pBS-SK+ plasmid (Stratagene) to an identical amount of 270 ng/well. pJB5-HNF-la is a gift of Dr Crabtree (Howard Hughes Medical Institute, Standford, CA) and psG5-HNF-4 is a gift from Dr Staels (Institut Pasteur de Lille, Lille, France). Then, 30 hours after the transfection, cells were analyzed using the Promega Dual Luciferase reporter assay system Kit (Promega) in a Micro plate LB 96 V Micro plate reader from EG&G Berthold. To normalize the level of firefly luciferase, the Renilla luciferase was used. Results in relative light units (RLU) were analyzed in Microsoft Excel software.
Electrophoretic Mobility Shift Assays. HepG2 nuclear extracts were prepared using a previously described method [27]. Sense and anti-sense oligonucleotides of 25 nucleotides (5.0 pg each) encompassing the different mutations sites were annealed at a final concentration of 100 ng/uX. One microliter of double stranded oligonucleotides was end- labeled with y-32P ATP using T4-polynucleotide kinase to produce radiolabeled probes. 10 pg nuclear extracts (NE) were incubated 10 min at 4°C in a total volume of 20 pL containing HEPES 10 mM pH 7.8 buffer, KC1 60 mM, 0.2% IGEPAL, 6% Glycerol, 2 mM DTT, 200 ng poly dldC and 100 ng salmon sperm DNA. The radiolabeled probes were added, and the binding reaction was incubated 15 more minutes at 4°C. The proteins complexes were resolved in a 6% nondenaturating polyacrylamide gel electrophoresis in 0.5x Tris-Borate-EDTA at 4°C. For supershift experiments, 0.2 pg of antibodies were incubated 10 min before the addition of the radiolabeled probes at 4°C. For competition experiments, the indicated excess quantities of unlabeled oligonucleotides were added to the binding reaction just before the labeled probes.
Statistical analysis. The haplotype frequencies were estimated using the PHASE 2.1 software (http://www.stat.washington.edu/stephens/software.html). The linkage was
assessed with the LDplotter tool program found at (https://innateimmunity.net/). Student's t test was used for comparisons between groups using SIGMASTAT 3.1 (Systat software). Differences were considered significant whenP < 0.05.
RESULTS
Identification of polymorphisms in the UGT1A3 gene. The resequencing of the UGT1A3 first exon (-80 to +1122) led to the discovery of 10 polymorphisms in the Caucasian population (n=249) at nucleotides 31 (T>C), 81 (G>A), 140 (T>C), 234 (A>G), 328 (T>A), 473 (C>T), 477 (A>G), 537 (T>C), 622 (A>C) and 808 (A>G) relative to the start codon. Six nonsynonymous coding SNPs that alter the encoded amino acids were observed: WUR, V*7A, FU0I, A158V, M208L and M270V (Figure lb). Overall, four novel polymorphisms were discovered into UGT1A3 exon-1 including three non-synonymous polymorphisms, F110I, A158V and M208L, and one silent variant D179D. The variants WnR and V47A are the most prevalent in Caucasians with allelic frequencies of 44.1% and 38.5%, respectively. The variant M270V is present at 2% in the population tested while the F110I, A158V, and M208L) were less common (0.2%, 0.6% and 0.2%, respectively).
Haplotype analyses of the coding sequences were inferred computationally using PHASE v2.1 program and UGT1A3 alleles are listed in Table 2. The reference sequence of UGT1A3 (allele *1) and the UGT1A3*2 (WUR and V47A) were found in 54.4% and 35.9% of the population, respectively. The UGT1A3*3 (WnR) and UGT1A3*6 (WUR, V47A and M270V) were less common with frequencies of 6.0% and 2.0%, respectively. The remaining haplotypes, namely UGT1A3*! (F,10T), UGT1A3*8 (A,58V), UGT1A3*9 (WnR and M208L) and UGT1A3*10 (V47A) were present in less than 1% of the population. The UGT1A3H (R45W), UGT1A3*5 (Q6R and WnR) and UGT1A3*11 (WnR, V^A, and M114I) were not found in the French-Canadian population tested but were reported previously [20, 22, 23] (Table 2).
We also sequenced 1146 bp of the UGT 1 A3 proximal promoter and identified seven novel common single nucleotide changes at positions -758 A>G, -751 T>C, -581 C>T, -553 G>A, -204 A>G, -148 T>C, -66 T>C from the ATG start site (Figure 1). These variations are observed at an allelic frequency of 43.3%, 37.3%, 44.9%, 4.0%, 43.3%, 4.6% and 43.3%, respectively. Haplotype analyses with the promoter sequences only, revealed the existence of 6 variant promoters (HI to H6) (Table 2). The reference promoter HI (Genbank AF297093) [26] is the most frequent (55.0%). Variants at positions -758G, -58IT, -204G and -66C are tightly linked (r2=0.934) and present in variant haplotypes 2 (28.7%), 3 (4.6%)
4 (6.0%) and 5 (4.0%). Haplotype 2 also contains the SNP at -75 IC. Haplotype 3 is characterised by the presence of an additional variant at position -148C while a variant at position -553A is also present in the haplotype 5, both of them also possess the -75 IC SNP. Haplotype 6 includes only the -58 IT variation (1.6%).
Altered estrone glucuronidation by recombinant UGT 1 A3 variant allozymes. The functional significance of nonsynonymous cSNPs was studied by stably expressing variant allozymes in HEK-293 cells. In vitro enzymatic assays were conducted using microsomal fraction of individual UGT 1 A3 enzymes with Ei as a substrate (Table 3). For a more accurate assessment of the quantitative differences in glucuronidation activity, immunoreactive UGT protein levels were then determined by Western blot.
Kinetic characteristics of UGT 1 A3 allozymes show that apparent Km values were not significantly different when compared to the UGT1A3*1 protein (Km= 50 ± 7 pM, P>0.05). After normalisation of enzyme activities (Absolute Vmax) by levels of UGT 1 A3 expression (Relative Vmax), all variants except UGT 1 A3 *2 and *10 enzymes, demonstrated significantly altered velocity to inactivate Ej. Velocities were reduced by 3- to 21- fold for allozymes *3 (R11), *4 (W45), *5 (R6RU), *7 (I110), *9 (RnL208) and *11 (RnA47I114) (P<0.05). A more dramatic decrease was observed for UGT1A3*8 (V158) and UGT1A3*6 (RnA47V270), reducing by 100- to 1280- fold the velocity compared to the UGT1A3*1 protein, respectively (Table 3). In order to gain insight into the nucleic acid positions potentially responsible for those critical changes for UGT 1 A3 kinetics, two hypothetic mutant enzymes were generated and included UGT 1 A3 RnV270 and UGT 1 A3 I114 alone. Results indicate that the substitution of a methionine by an isoleucine at codon 114 (I114) alone do not affect significantly the enzyme activity of UGT1A3 (P>0.05). In contrast, the combined occurrence of an arginine at codon 11 and a valine at codon 270 abolishes the glucuronidation activity of UGT 1 A3 (P<0.005) (Figure 2 and Table 3).
Based on the observed kinetic parameters, UGT 1 A3 allozymes were divided in three groups of predicted phenotypes based on CLjnt values; namely the UGT 1 A3 enzymes with