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Technical Briefs |
1 Department of Laboratory Medicine, Sungkyunkwan University School of Medicine, Samsung Medical Center, Ilwon-Dong, Kangnam-Gu, Seoul 135-710, Korea;2 Department of Laboratory Medicine, College of Medicine, Korea University, Anam-Dong, Seongbuk-Gu, Seoul, Korea;3 Clinical Research Center, Samsung Biomedical Research Institute, Ilwon-Dong, Kangnam-Gu, Seoul, Korea
aauthor for correspondence: fax 82-2-3410-2719; e-mail jwonk{at}smc.samsung.co.kr
UDP-glucuronosyltransferase 1A1 (UGT1A1) is the key enzyme for bilirubin conjugation. Defects in this enzyme can cause a nonhemolytic unconjugated hyperbilirubinemia, such as Crigler–Najjar syndrome type 1 (CN1) and 2 (CN2) and Gilbert syndrome (GS). In 1991, the cDNA of the human UGT1A1 gene was cloned, and this led to the identification of genetic defects in patients with CN1, CN2, and GS (1)(2)(3). It was shown that homozygous or compound heterozygous mutations of the UGT1A1 gene can lead to these inheritable unconjugated hyperbilirubinemias, and >30 variants have been identified (4)(5).
In GS, a TATAA box variant [A(TA)6TAA>A(TA)7TAA] in the promoter region of the UGT1A1 gene has been reported in Caucasian populations, and several polymorphisms in the coding region, including 211G>A (G71R), have been reported to have similar associations with GS in Japanese populations (6)(7)(8). Recently, Sugatani et al. (9) identified a T-to-G substitution in the phenobarbital-responsive enhancer module 3279 bp upstream from the UGT1A1 gene. They suggested that the -3279T>G polymorphism could be another risk factor for the development of mild hyperbilirubinemia
Presumably, different combinations of the polymorphisms (haplotypes) in the UGT1A1 gene associated with GS or mild hyperbilirubinemia might produce a variety of serum total bilirubin (T-Bil) concentrations. Because these polymorphisms in the UGT1A1 gene lie in a relatively small region (Fig. 1A
), a certain extent of linkage disequilibrium (LD) among these polymorphisms is expected. Therefore, haplotype analysis is more reasonable than association analysis using any single polymorphism to reveal the genetic background of an increased serum T-Bil concentration. We analyzed the haplotype structure of the UGT1A1 gene and investigated its relationship to the serum T-Bil concentration in healthy Korean males.
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The study participants were 324 healthy Korean males [mean (SD) age, 49.8 (5.4) years] randomly selected from the registry for routine health checks at Samsung Medical Center in Seoul, Korea. We restricted the study participants to males because the prevalence of unconjugated hyperbilirubinemia is higher in men than in women because of the lower rate of daily bilirubin production or other unidentified factors in females (10)(11) and thus the genetic influence on the bilirubin concentration might be more evident in men. None had a history of hepatic or hematologic disorders such as anemia, excessive alcohol intake, or chronic use of medications or narcotics. Informed consent was obtained from all participants, and the Institutional Review Board of the Samsung Medical Center approved the study protocol. Serum samples were collected from each participant in the morning after overnight fasting. The serum T-Bil concentration was measured by the diazo method (Daiichi Pure Chemicals) with the Hitachi 747 system (Hitachi) at least twice with a >1-month interval to provide a mean value for each participant. The mean values were used in all statistical analyses.
Two promoter regions containing the -3279T>G polymorphism and the TATAA box, and a part of exon 1 containing the 211G>A polymorphism of the UGT1A1 gene were amplified and sequenced with use of appropriate primers, as given in the Data Supplement that accompanies the online version of this Technical Brief (available at http://www.clinchem.org/content/vol49/issue12/). The differences in the serum T-Bil concentration among the subgroups according to the UGT1A1 alleles or haplotypes were tested by ANOVA. The influence of smoking status, polymorphisms, and haplotypes of the UGT1A1 gene on serum T-Bil was also estimated by the GLM procedure incorporated in the SAS System for Windows (SAS Institute Inc.). The Hardy–Weinberg equilibrium was tested with Arlequin software (12). Multisite haplotypes and their frequencies were estimated with use of SAS Genetics software (SAS Institute Inc.), and the assignment of haplotypes to each individual was performed with the PHASE program (13). Pairwise LD was estimated as D, D', and r2 (14). These LD coefficients were calculated with use of the SAS Genetics software.
The mean (SD) serum T-Bil concentration in the total group of participants was 16.3 (5.9) µmol/L [0.95 (0.34) mg/dL]. When we investigated the association of smoking with serum T-Bil concentrations, we observed a significant difference among groups by smoking status (P = 0.0058, ANOVA). Current smokers had lower serum T-Bil concentrations [14.3 (5.3) µmol/L; n = 71] compared with never smokers [17.9 (6.6) µmol/L; n = 45], and former smokers had serum T-Bil concentrations [16.8 (5.8) µmol/L; n = 155] between those of current smokers and never smokers. Smoking status was estimated to explain 
3.8% of the variation in serum T-Bil concentrations.
The genotype frequencies of all three polymorphisms were in Hardy–Weinberg equilibrium, and the frequencies of the -3279G, (TA)7, and 211A alleles were calculated as 0.267, 0.127, and 0.213, respectively. As shown in the table in the online Data Supplement, the mean serum T-Bil concentrations in homozygous carriers of the -3279G, (TA)7, or 211A allele were significantly higher than those in heterozygous carriers or homozygous carriers of wild-type alleles (P <0.0001). Among the three polymorphisms, homozygous carriers of (TA)7 showed the highest mean serum T-Bil concentration [33.2 (4.1) µmol/L], followed by homozygous carriers of 211A [24.2 (8.3) µmol/L] and -3279G [23.7 (6.2) µmol/L]. The variabilities in serum T-Bil explained by the -3279T>G, (TA)6/7, and 211G>A polymorphisms were 15.1% (P <0.0001), 28.1% (P <0.0001), and 12.9% (P <0.0001), respectively.
A likelihood ratio test using Arlequin software (12) detected significant pairwise LD with D' values of 1.0 between all pairs of the three polymorphisms (P <0.0001; Fig. 1B
). The highest correlation was observed between -3279T>G and (TA)6/7 polymorphisms (r2 = 0.3999; Fig. 1B
). Multisite haplotype inference revealed that the participants had four of eight possible haplotypes: 3279T-(TA)6-211G (ht1), 3279T-(TA)6-211A (ht2), 3279G-(TA)6-211G (ht3), and 3279G-(TA)7-211G (ht4). ht1 was the most common with an estimated frequency of 0.5201 [95% confidence interval (CI), 0.4816–0.5586], followed by ht2 (0.2129; 95% CI, 0.1814–0.2445), ht3 (0.1404; 95% CI, 0.1137–0.1672), and ht4 (0.1265; 95% CI, 0.1009–0.1521).
When the participants were stratified into 10 groups according to their UGT1A1 haplotypes, we observed significant differences in the mean serum T-Bil concentration among the groups (P = 0.0001; Table 1
). The lowest concentration was observed in group A [ht1/ht1; 13.1 (3.6) µmol/L], whereas the highest concentration was observed in group J [ht4/ht4; 33.2 (4.1) µmol/L]. Homozygous as well as compound heterozygous carriers of the variant haplotypes (ht2, ht3, or ht4) had increased serum T-Bil concentrations compared with homozygous or heterozygous carriers of the wild haplotype (ht1). Approximately 61.4% and 53.5% of the variability in serum T-Bil concentrations could be explained by the UGT1A1 haplotype with or without consideration of smoking status, respectively.
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Multisite haplotype inference revealed that our study population had only four of eight possible haplotypes. The four missing haplotypes, 3279T-(TA)7-211G, 3279T-(TA)7-211A, 3279G-(TA)6-211A, and 3279G-(TA)7-211A, suggest that -3279T plus (TA)7 and (TA)7 plus 211A alleles never exist on the same chromosome, at least in a Korean population. From the above results, we also suggest that a transition of G to A at nucleotide 211 in exon 1 of the UGT1A1 gene would form the ht2 haplotype, whereas a transversion of T to G at nucleotide -3279 in the promoter region of the UGT1A1 gene would form the ht3 haplotype. In addition, ht4 could be the result of an introduction of an extra (TA) repeat to the ht3.
Disclosure of the molecular genetic basis of reduced expression of UGT1A1 is important not only for understanding the molecular pathophysiology of increased serum T-Bil concentrations in GS but also for predicting severe toxicity by irinotecan in cancer patients (15)(16). In addition, increased serum T-Bil concentrations have been associated with a low risk of coronary artery disease (17)(18). To reveal the genetic background of an increased serum T-Bil concentration, haplotype analysis is more reasonable than association analysis using any single polymorphism because of tight LD among polymorphisms in the UGT1A1 gene.
In summary, we demonstrated that there is complete LD among three common polymorphisms of the UGT1A1 gene in a male Korean population. In addition, we could unequivocally construct haplotypes for the UGT1A1 gene, which were revealed to be significantly associated with the serum T-Bil concentration. However, there are two major limitations in the present study. One is that the study population comprised only Korean males and thus the results cannot be freely applied to other populations, and the other is that only three common polymorphisms were included in the analysis. Nevertheless, to the best of our knowledge, this is one of the first studies to analyze the haplotype structure of the UGT1A1 gene including both promoter and coding regions and to assess the haplotype–phenotype correlation between the UGT1A1 gene and the serum T-Bil concentration.
Acknowledgments
This work was supported by National Research Laboratory Grants from the Korea Institute of Science & Technology Evaluation and Planning, Korea.
References
The following articles in journals at HighWire Press have cited this article:
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  A. L. Hong, D. Huo, H.-J. Kim, Q. Niu, D. L. Fackenthal, S. A. Cummings, E. M. John, D. W. West, A. S. Whittemore, S. Das, et al. UDP-Glucuronosyltransferase 1A1 Gene Polymorphisms and Total Bilirubin Levels in an Ethnically Diverse Cohort of Women Drug Metab. Dispos., August 1, 2007; 35(8): 1254 - 1261. [Abstract] [Full Text] [PDF]
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 F. Innocenti, E. E. Vokes, and M. J. Ratain Irinogenetics: What Is the Right Star? J. Clin. Oncol., May 20, 2006; 24(15): 2221 - 2224. [Full Text] [PDF]
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N. Kaniwa, K. Kurose, H. Jinno, T. Tanaka-Kagawa, Y. Saito, M. Saeki, J.-i. Sawada, M. Tohkin, and R. Hasegawa RACIAL VARIABILITY IN HAPLOTYPE FREQUENCIES OF UGT1A1 AND GLUCURONIDATION ACTIVITY OF A NOVEL SINGLE NUCLEOTIDE POLYMORPHISM 686C> T (P229L) FOUND IN AN AFRICAN-AMERICAN Drug Metab. Dispos., March 1, 2005; 33(3): 458 - 465. [Abstract] [Full Text] [PDF]
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