The Pharmacogenomics Journal (2008) 8, 152–161 & 2008 Nature Publishing Group All rights reserved 1470-269X/08 $30.00 www.nature.com/tpj ORIGINAL ARTICLE Hepatocyte nuclear factor-1 alpha is associated with UGT1A1, UGT1A9 and UGT2B7 mRNA expression in human liver J Ramı´rez1, S Mirkov1, Experimental evidence suggests HNF1a regulates UGT expression. This study 1 2 2,3 investigates (1) whether the variability in HNF1a expression is associated with W Zhang , P Chen , S Das , the variability in UGT1A1, UGT1A9 and UGT2B7 expression in human livers 1 1,3,4 W Liu , MJ Ratain and (2) the functionality of 12 HNF1a variants using mRNA expression as and F Innocenti1,3,4 phenotype. Controlling for known UGT variation in cis-acting elements known to affect UGT expression, we demonstrate that a combination of 1Department of Medicine, University of Chicago, HNF1a mRNA levels and UGT genotype predicts variance in UGT expression 2 Chicago, IL, USA; Department of Human to a higher extent than UGT genotype alone. None of the HNF1a Genetics, University of Chicago, Chicago, IL, USA; 3Committee on Clinical Pharmacology and polymorphisms studied, however, seem to have an effect on HNF1a, Pharmacogenomics, University of Chicago, UGT1A1, UGT1A9 and UGT2B7 expression, ruling out their functional role. Chicago, IL, USA and 4Cancer Research Center, Our data provide evidence for HNF1a being a determinant of UGT1A1, University of Chicago, Chicago, IL, USA UGT1A9 and UGT2B7 mRNA expression. However, the amount of UGT a Correspondence: intergenotype variability explained by HNF1 expression appears to be Dr F Innocenti, Section of Hematology- modest, and further studies should investigate the role of multiple Oncology, Department of Medicine, University transcription factors. of Chicago, 5841 S, Maryland Avenue, The Pharmacogenomics Journal (2008) 8, 152–161; doi:10.1038/sj.tpj.6500454; MC2115, Chicago, IL 60637, USA; E-mail: [email protected] published online 17 April 2007 Keywords: HNF1a; UGT1A1; UGT1A9; UGT2B7; gene regulation Introduction Glucuronidation by the uridine diphosphate-glucuronosyltransferase (UGT) enzymes is a major metabolic pathway that facilitates the elimination of a large variety of molecules into urine and bile by increasing their water solubility. The UGT1 gene codes for nine functional UGT1A enzymes (UGT1A1, UGT1A3, UGT1A4, UGT1A5, UGT1A6, UGT1A7, UGT1A8, UGT1A9 and UGT1A10) generated through a process of RNA splicing of one of the multiple first exons to the set of common exons 2–5. The UGT2 family is encoded by separate homologous genes and is subdivided into two subfamilies: UGT2A (encoding UGT2A1) and UGT2B (encoding proteins UGT2B4, UGT2B7, UGT2B10, UGT2B11, UGT2B15, UGT2B17 and UGT2B28). Most UGTs are expressed in the liver but they can also be found in extrahepatic tissues. Genetic polymorphisms in UGTs have been associated with interindividual variability in glucuronidation. The genetic variation in UGT1A1 is the most extensively studied. UGT1A1 gene transcriptional activity is affected by the length of the TATA box in the promoter, where a larger number of TA repeats Received 26 November 2006; revised 18 February 2007; accepted 15 March 2007; results in reduced transcriptional activity. Homozygosity for the (TA)7 allele published online 17 April 2007 (UGT1A1*28) is associated with Gilbert’s syndrome (a mild form of conjugated HNF1a regulation of UGT1A1, UGT1A9 and UGT2B7 J Ramı´rez et al 153 hyperbilirubinemia)1,2 and predisposition to severe toxicity might influence the mRNA expression of HNF1a and the of the anticancer drug irinotecan.3,4 Even though the effect UGTs of interest. of the UGT1A1*28 allele on the UGT1A1 glucuronidation rates in humans is well known, the phenotypic variability Results within each genotypic group remains high.4,5 Similar to UGT1A1, the UGT1A9 and UGT2B7 isoforms are highly Effect of HNF1a genotypes on HNF1a expression expressed in the liver and play an important role in The median (not log-transformed) hepatic mRNA level of the hepatic glucuronidation of numerous drugs. Multiple HNF1a was 254 (range 30–944, n ¼ 53). The median studies have been conducted to evaluate the functional mRNA level of 18S was 1.18 (range 0.18–1.84, n ¼ 54). The consequences of common polymorphisms in UGT1A9 and frequencies of the HNF1a variants are listed in Table 1. All UGT2B7, although the results to date have been incon- alleles were in Hardy–Weinberg equilibrium, and their sistent.5–10 It can be hypothesized that the phenotypic frequencies were comparable to the data in the HapMap variability in UGTs that remains unaccounted for by for the CEPH Caucasians. None of the HNF1a genotypes had cis-acting genetic polymorphisms resides in the variability a significant effect (P40.1) on the expression of HNF1a. in transcription factors. Initiation of transcription is an integrated and complex mechanism involving both Effect of UGT genotypes on UGT expression cis-acting elements and trans-acting factors. Transcription The median (not log-transformed) mRNA levels of UGT1A1, factors involved in the regulation of UGT constitutive UGT1A9 and UGT2B7 were 759 (range 71–15,712, n ¼ 54), expression include the hepatocyte nuclear factor 1 (HNF1), 520 (range 6–14,303, n ¼ 44) and 1263 (range 17–17.245, the octamer transcription factor 1 (Oct-1), the pre-B-cell n ¼ 54), respectively. Analysis of the linear relationship homeobox factor 2 (Pbx2), the caudal-related homeo- between the mRNA levels of the UGTs showed that the domain protein 2 (Cdx2, an intestine-specific transcription UGT1A1 mRNA levels were significantly and moderately factor) and Prep1.11–16 UGT regulation by hormones and related to those of UGT1A9 (r2 ¼ 0.49, Po0.0001, n ¼ 44; xenobiotics is mediated through the aryl hydrocarbon Figure 1a) and UGT2B7 (r2 ¼ 0.39, Po0.0001, n ¼ 54; Figure receptor (AhR), the constitutive androgen receptor (CAR), 1b). Similarly, the mRNA content of UGT1A9 and UGT2B7 the pregnane X receptor (PXR), the farnesoid X receptor, were significantly correlated (r2 ¼ 0.54, Po0.0001, n ¼ 44; the peroxisome proliferated-activated receptors and trans- Figure 1c). Similar results were obtained in partial correla- cription factors that respond to stress.17 tions performed to adjust for a potential confounding effect The HNF1 family is comprised of HNF1a and HNF1b, of 18S mRNA levels (data not shown). which increase gene transcription by binding as homo- The genotype frequencies of the UGT variants are shown dimers or heterodimers to DNA sequences in target genes. in Table 1. The alleles were in Hardy–Weinberg equilibrium. HNF1a is well conserved among species and is the pre- The association between UGT1A1*28 and UGT1A1 expres- dominant form expressed in the human liver.18 Experimen- sion was best described by an additive model (r2 ¼ 0.17, tal evidence suggests a prominent role of HNF1a as a P ¼ 0.003, n ¼ 51; 6/6 ¼ 0, 6/7 ¼ 1, 7/7 ¼ 2; Figure 2). A trans-acting factor regulating UGT expression. HNF1a binds recessive model (r2 ¼ 0.08, P ¼ 0.07, n ¼ 43) described the and activates the proximal promoters of several human relationship between UGT1A9*1b genotype and UGT1A9 UGTs, including UGT1A1, UGT1A9 and UGT2B7.11,13,15,16,19 expression (10/10: mean7s.d. ¼ 1.8870.92, n ¼ 6; 9/9 HNF1a is polymorphic. The I27L (79A4C) variant, a and 9/10: mean7s.d. ¼ 2.5870.83, n ¼ 37). The 985A4G common missense polymorphism in HNF1a, is located in the dimerization domain of the protein.20 79A4Cis associated with insulin resistance and glucose intolerance Table 1 Genotype frequencies as well as with differences in high-density lipoprotein cholesterol among individuals.20–22 The molecular function Gene Variant Frequency of this variant has not been established but preliminary studies showed that luciferase UGT2B17 activity was reduced HNF1a 79A4C 0.25 after cells were co-transfected with HNF1a 79A4C compared rs1169286 A/G 0.33 to constructs without this variant.14 Additionally, HNF1a rs11065385 A/G 0.74 rare variants have been associated with maturity onset rs2393791 G/A 0.63 rs1169293 G/A 0.88 diabetes of the young (MODY3).23 The effect of HNF1a rs12427353 G/C 0.22 genetic variation on its mRNA expression in human liver, as rs2071190 T/A 0.26 well as its association with the expression of UGTs known to rs1169302 T/G 0.43 be regulated by HNF1a, has never been studied. rs1169303 A/C 0.54 This study had two aims: (1) to investigate whether HNF1a rs3999413 C/T 0.14 mRNA expression levels predict mRNA expression level rs1169306 C/T 0.37 variability in UGT1A1, UGT2B7 and UGT1A9 while rs1169307 T/C 0.62 taking into account the known effect of cis-acting variants UGT1A1 À53(TA)647 0.38 on UGT expression and (2) to determine whether any of UGT1A9 À118T9410 0.38 the 12 HNF1a polymorphisms typed in normal livers UGT2B7 985A4G 0.13 The Pharmacogenomics Journal HNF1a regulation of UGT1A1, UGT1A9 and UGT2B7 J Ramı´rez et al 154 ab c r2 = 0.49, p<0.0001, n = 44 r2 = 0.39, p<0.0001, n = 54 r2 = 0.54, p<0.0001, n = 44 4 4 4 mRNA) mRNA) mRNA) 18S 18S 18S 3 3 3 mRNA / mRNA mRNA / mRNA mRNA / mRNA 2 2 2 UGT1A1 UGT1A9 UGT1A1 1 log ( log ( log ( 0.5 1.5 2.5 3.5 4.5 12345 12345 log (UGT1A9 mRNA /18S mRNA) log (UGT2B7 mRNA /18S mRNA) log (UGT2B7 mRNA /18S mRNA) Figure 1 Association between the hepatic mRNA levels of (a) UGT1A1 and UGT1A9,(b) UGT1A1 and UGT2B7, and (c) UGT1A9 and UGT2B7. Data points represent means of triplicate determinations from a single experiment. 4.5 associated with UGT1A1 expression in a recessive model r2 = 0.17, p = 0.003, n = 51 (r2 ¼ 0.06, P ¼ 0.09, n ¼ 52; A/A and A/G: mean7s.d.