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Institute of Microbiology and Service of Infectious Diseases, University of Lausanne
Swiss HIV Cohort Study Data Center, Lausanne, Division of Infectious Diseases and Hospital Epidemiology, University Hospital, Zurich, Division of Infectious Diseases, University Hospital, Bern, Division of Infectious Diseases, Kantonsspital, St. Gallen, Division of Infectious Diseases, University Hospital, Basel
Division of Infectious Diseases, Ospedale Regionale di Lugano, Lugano, Switzerland
Background.
Unconjugated hyperbilirubinemia results from Gilbert syndrome and from antiretroviral therapy (ART) containing protease inhibitors. An understanding of the interaction between genetic predisposition and ART may help to identify individuals at highest risk for developing jaundice.
Methods.
We quantified the contribution of UGT1A1*28 and ART to hyperbilirubinemia by longitudinally modeling 1386 total bilirubin levels in 96 human immunodeficiency virus (HIV)infected individuals during a median of 6 years.
Results.
The estimated average bilirubin level was 8.8 mol/L (0.51 mg/dL). Atazanavir increased bilirubin levels by 15 mol/L (0.87 mg/dL), and indinavir increased bilirubin levels by 8 mol/L (0.46 mg/dL). Ritonavir, lopinavir, saquinavir, and nelfinavir had no or minimal effect on bilirubin levels. Homozygous UGT1A1*28 increased bilirubin levels by 5.2 mol/L (0.3 mg/dL). As a consequence, 67% of individuals homozygous for UGT1A1*28 and receiving atazanavir or indinavir had 2 episodes of hyperbilirubinemia in the jaundice range (>43 mol/L [>2.5 mg/dL]), versus 7% of those with the common allele and not receiving either of those protease inhibitors (P < .001). Efavirenz resulted in decreased bilirubin levels, which is consistent with the induction of UDP-glucuronosyltransferase 1A1.
Conclusions.
Genotyping for UGT1A1*28 before initiation of ART would identify HIV-infected individuals at risk for hyperbilirubinemia and decrease episodes of jaundice.
Unconjugated bilirubin needs to be conjugated with glucuronic acid to be excreted in the bile. This step is mediated by the microsomal enzyme UDP-glucuronosyltransferase (UGT). Fifteen UGT isoforms with different substrate specificities, including the bilirubin-specific isoform UGT1A1, have been identified [1]. Reduced activity of this enzyme leads to unconjugated hyperbilirubinemia.
Gilbert syndrome is the most common inherited cause of unconjugated hyperbilirubinemia, and it occurs in 3%10% of the general population [2]. The influence of a polymorphism in the promoter TATA element of the gene encoding UGT1A1 has been extensively investigated in the context of Gilbert syndrome. Promoters containing 7 thymine adenine (TA) nucleotide repeats, A(TA)7TAA (the UGT1A1*28 allele), are less active than is the common promoter containing 6 TA repeats. Thus, homozygosis for the allele A(TA)7TAA leads to higher levels of unconjugated bilirubin [24]. The allelic frequency of this polymorphism differs among racial groups. It is more common in African Americans (f = 0.43), whites (f = 0.360.39), and Indians (f = 0.35) and less common in Japanese (f = 0.11), Chinese (f = 0.16), and Malaysians (f = 0.19) [3, 57]. In addition to the UGT1A1*28 allele, other polymorphisms in the UGT1A1 gene have been associated with Gilbert syndrome in the Japanese population [810].
Unconjugated hyperbilirubinemia is also a recognized adverse effect of protease inhibitor (PI) therapy containing indinavir (IDV) or atazanavir (ATV) [1113]. The pathophysiological bases of this adverse effect may be the same as those in Gilbert syndrome. Zucker et al. reported that IDV induces hyperbilirubinemia by competitive inhibition of the UGT1A1 enzyme and found a strong association between the homozygous variant allele A(TA)7TAA and the risk of developing hyperbilirubinemia while receiving IDV [14].
In the present study, we used a longitudinal approach to model the influence that the UGT1A1*28 allele has on antiretroviral therapy (ART)associated hyperbilirubinemia. The aim of the study was to (1) quantify the degree of elevation in bilirubin levels in association with various PIs, (2) assess the interaction between the presence of the UGT1A1*28 allele and the ART regimen, and (3) identify individuals at risk of developing jaundice after the initiation of ART.
PARTICIPANTS, MATERIALS, AND METHODS
Study participants.
A total of 96 participants were recruited from the genetics project of the Swiss HIV Cohort Study (information available at: http://www.shcs.ch). The ethics committees of all participating centers approved the genetics project, and all participants gave written, informed consent for genetic testing. The study population was a convenience sample as determined by a preliminary analysis of the allelic frequency and the size of the genetic effect. The selection criteria included having received ATV, IDV, or lopinavir (LPV) at at least 1 time point during follow-up. The study period was defined as the follow-up period starting with the first study visit at which bilirubin levels were measured (beginning in 1994) until November 2004. Total bilirubin and conjugated bilirubin levels were measured. The unconjugated bilirubin level was calculated by subtracting the conjugated bilirubin level from the total bilirubin level. Hyperbilirubinemia was graded in accordance with the AIDS Clinical Trials Group guidelines for total bilirubin levels: grade 1 (mild), 2332 mol/L (1.31.9 mg/dL); grade 2 (moderate), 3353 mol/L (1.93.1 mg/dL); grade 3 (severe), 54105 mol/L (3.16.1 mg/dL); and grade 4 (serious), >105 mol/L (>6.1 mg/dL) [15]. Jaundice was defined as a total bilirubin level >43 mol/L (>2.5 mg/dL) [16].
Genetic analyses.
Genomic DNA was extracted from frozen peripheral blood mononuclear cells. Genotyping was performed by direct sequencing by use of forward (5-AAGTGAACTCCCTGCTACCTT-3) and reverse (5-CCACTGGGATCAACAGTATCT-3) primers, described elsewhere [5], to generate a product that was 253255 bp.
Statistical analysis.
The data were analyzed longitudinally by modeling the individual effects of the different covariables on log-transformed total bilirubin levels. We used the log transformation instead of the linear scale, to stabilize variances and obtain a more symmetric distribution. The analysis used a multivariate model proposed by Diggle [17, 18] in which the covariance structure arises from the consideration of 3 residual components. This approach allows for disentanglement of the total variation into 3 distinct dimensions: (1) a random effect accounting for the propensity of individuals with intrinsically high or low bilirubin levels, (2) a stationary Gaussian process to allow for some correlation between the repeated measurements from the same individual, and (3) a residual error term. This approach is particularly relevant for cohort data, so that an economical and plausible covariance structure can be obtained. The specific advantages include the ability to accommodate unbalanced repeated measurements made at irregular time intervalsas is usually the case with cohort datato generate a flexible correlation structure with few parameters to be estimated and thereby allowing more precise inferences, a straightforward interpretation of regression coefficients, and a better understanding of the sources of variability [19, 20]. Our model assumes that the covariances are stationary. However, an informal check of the assumed covariance structure as well as of the importance of each component can be gained by nonparametrically estimating the empirical semivariogram [18, 21].
Covariables included in the regression model were ART regimen, age, sex, hepatitis C and B serostatus, comedication (cotrimoxazole, pyrimethamine, or rifampin), smoking status, hemoglobin level, and the genetic factor tested. On the basis of an initial assessment of the impact that each individual antiretroviral drug had on the bilirubin level in an additive multivariate model, the ART agents were grouped as follows: (1) no ART at the time that bilirubin level was measured, (2) ART containing only nucleoside reverse-transcriptase inhibitors (NRTI ART), (3) ART containing NRTI and nonnucleoside reverse-transcriptase inhibitors (NNRTI ART), and ART containing PIs (PI ART), including (4) ATV, (5) IDV, (6) LPV, (7) nelfinavir (NFV), (8) saquinavir (SQV), and (9) full-dose ritonavir (RTV). For adjustment for confounding variables, cross-sectional and longitudinal relationships between the continuous explanatory variables and the response were distinguished [19, 22], and adequate functional form was assessed by use of fractional polynomials [23]. Goodness of fit was evaluated by comparing mean observed and predicted measurements, as well as residuals, on time plots of values in strata defined by polymorphisms and ART regimens. We also compared the estimated variogram from our multivariate model with the nonparametric estimate [17, 18, 21].
The analysis of interactions between genotypes and ART regimens was first tested globally. If the global test was significant, subcategories contributing to the P value were screened, and significant and consistent strata were retained. All statistical analyses were conducted using Stata (version 8.2 for Windows; StataCorp) and SAS (version 8.02; SAS Institute).
RESULTS
Study participants.
The UGT1A1*28 allele was present with an allelic frequency of 0.41 and followed Hardy-Weinberg equilibrium. The characteristics of the study participants and the median total bilirubin levels during the follow-up period are summarized in table 1. A total of 1386 total bilirubin and 226 unconjugated bilirubin levels were recorded during the study period. Analyses showed a highly significant correlation between the UGT1A1 genotype and log-transformed total and unconjugated bilirubin levels (r = 0.95). These results allowed the elaboration of a longitudinal model using the log-transformed total bilirubin level as the phenotype. The key results were thereafter confirmed on the data set of unconjugated bilirubin levels (see below).
The median number of measurements of total bilirubin levels during the follow-up period was comparable in study participants with normal (23 mol/L [1.3 mg/dL]; n = 10 measurements) and elevated (>23 mol/L [1.3 mg/dL]; n = 15.5 measurements) total bilirubin values. Of the covariables tested, hemoglobin levels and chronic hepatitis C or B infection were associated with increased bilirubin levels (table 2).
Effect of ART on bilirubin levels.
A total of 152 total bilirubin levels (11%) were measured in the absence of ART; 57 (4.1%) were measured in participants who received NRTI ART; 49 (3.5%) were measured in participants who received NNRTI ART; and 1128 (81.4%) were measured in participants who received PI ART that contained ATV (n = 185; 13.3%), IDV (n = 534; 38.5%), LPV (n = 152; 11.0%), NFV (n = 149; 10.8%), RTV (n = 32; 2.3%), or SQV (n = 76; 5.5%). The median number of changes in the ART regimen per individual was 3 (range, 114 changes). The influence of ART on total bilirubin levels is shown in table 2. ATV had a stronger association with elevated total bilirubin levels than IDV did. We evaluated the effect of receiving ATV or IDV alone or in combination with RTV. We did not find significant differences (P = .30) in the estimated effect on total bilirubin levels for individuals who received ATV alone (0.38 log10 mol/L), ATV in combination with RTV (0.45 log10 mol/L), IDV alone (0.27 log10 mol/L), or IDV in combination with RTV (0.31 log10 mol/L). The estimated average bilirubin level was 8.8 mol/L (0.51 mg/dL). PI ART containing ATV increased this level by an estimated 15 mol/L (0.87 mg/dL), and PI ART containing IDV increased this level by 8 mol/L (0.46 mg/dL). LPV, NFV RTV, and SQV had modest effects on total bilirubin levels. NNRTI ART (consisting of efavirenz in 98% of the data points) was associated with a decrease of 3.1 mol/L (0.18 mg/dL) in total bilirubin levels.
Effect of the UGT1A1*28 allele on bilirubin levels.
The presence of 1 or 2 copies of the variant allele resulted in a gene dose effect on total bilirubin levels (table 2). The genetic effect was observed in the absence of treatment and across all treatment groups (figure 1). The overall effect of homozygous UGT1A1*28 was to increase bilirubin levels by 5.2 mol/L (0.3 mg/dL), which was less than the increase attributed to the use of ATV or IDV. The simultaneous presence of homozygous UGT1A1*28 and PI ART containing ATV or IDV resulted in nearly half of the bilirubin levels signaling grade 2 toxicity or greater (figure 1).
Because the models were built on total bilirubin levels, we confirmed the main findings in the restricted data set of 226 measurements of unconjugated bilirubin levels. The estimates for the effect of treatment or genotype on unconjugated bilirubin levels were comparable to those obtained by using total bilirubin levels. Compared with that in individuals who did not receive PIs, the estimated effect on unconjugated bilirubin levels in individuals who received PI ART containing IDV or ATV was an increase of 0.41 (SE, 0.07) log10 mol/L (P < .001). For PI ART containing LPV, NFV, RTV, or SQV, the effect was estimated to be a decrease of 0.10 (SE, 0.084) log10 mol/L (P = .22). The estimated effect of homozygous UGT1A1*28 on unconjugated bilirubin levels was an increase of 0.21 (SE, 0.09) log10 mol/L (P = .021). The bilirubin levels reflected the effect of the influences exerted by the antiretroviral drugs and by the genetic background. The estimated total bilirubin level for individuals homozygous for the UGT1A1*28 allele and who received PI ART containing ATV was 37.7 mol/L (2.19 mg/dL). The global test for interactions showed only a trend for significance (P = .062), and, thus, subcategories contributing to the P value were not screened.
Twenty-seven participants (28%) had 2 bilirubin levels that were >43 mol/L (>2.5 mg/dL) (the threshold for jaundice). However, 6 (67%) of 9 participants homozygous for the UGT1A1*28 allele who received ATV or IDV had bilirubin levels in the jaundice range, versus 4 (7%) of 54 participants with the common allele who did not receive ATV or IDV (P < .001) (figure 1).
DISCUSSION
We quantified the relative contribution of the UGT1A1*28 allele and different ART combinations to hyperbilirubinemia in HIV-infected individuals. The most relevant finding of the study is the identification of a gene-ART association that could lead to severe hyperbilirubinemia. Individuals with the UGT1A1*28 allele may develop jaundice when exposed to ATV or IDV. This study was conducted primarily on a white population; however, the allele exists in other populations and is associated with the same physiological effect, which extends the validity of the conclusions to other ethnic groups [24].
We used an objective measurement of bilirubinemia and not a subjective assessment of jaundice to establish the quantitative influence of the various treatments and genotypes. Although hyperbilirubinemia is not a serious adverse effect, clinical jaundice can stigmatize the HIV-infected individual and result in additional consultations and in treatment modification. However, because we wanted to develop a model of drug-gene interaction, our goal was not primarily to assess clinical usefulness but, rather, to present a concept for the use of genetic data in clinics. These results extend the findings of Zucker et al. [16] and O'Mara et al. [25], who documented an association between the UGT1A1*28 allele and hyperbilirubinemia in individuals who receive PI ART containing IDV or ATV. A genetic predisposition to hyperbilirubinemia has also been reported in individuals undergoing irinotecan treatment for cancer [26]. In contrast, the use of efavirenz resulted in decreased bilirubin levels. This observation is consistent with the known activation by efavirenz of pregnane X receptor [27], which leads to the induction of UGT1A isoforms [28].
The present study explored 1386 measurements of total bilirubin levels in 96 participants, during a median follow-up of 6 years. This allowed us to quantitate the precise influence of multiple factors of possible relevance to bilirubin levels (ART regimen, age, sex, hepatitis C and B serostatus, comedication, smoking status, and hemoglobin level) against the genetic background of Gilbert syndrome. The study participants served as their own controls through multiple changes in ART regimens and periods when treatment was not used. We have previously used this longitudinal modeling in the investigation of interactions between apolipoprotein (APO) E and APOC3 and the risk of severe ritonavir-associated hypertriglyceridemia [29].
The frequency of jaundice-range bilirubin levels observed under various treatments and genetic backgrounds allows a preliminary modeling of the potential value of introducing genetic testing before the initiation of ART. We tested the scenario "no genotyping/ATV or IDV as first-line agent" versus "genotype-guided ART" and narrowed the use of ATV or IDV to individuals without the UGT1A1*28 allele. Implementation of such a program would lead to a theoretical 75% reduction in the number of bilirubin levels in the jaundice range. The high frequency of the UGT1A1*28 allele, and the high risk of developing jaundice in the setting of Gilbert syndrome when exposed to specific PIs, is a good example of how genetic testing for this allele, ideally in conjunction with testing for other markers of toxicity, may be used in the future in the clinical setting.
SWISS HIV COHORT STUDY TEAM MEMBERS
The members of the Swiss HIV Cohort Study are M. Battegay, E. Bernasconi, J. Bni, H. Bucher, P. Bürgisser, S. Cattacin, M. Cavassini, R. Dubs, M. Egger, L. Elzi, P. Erb, K. Fantelli, M. Fischer, M. Flepp, A. Fontana, P. Francioli (president), H. Furrer (chairman of the Clinical and Laboratory Committee), M. Gorgievski, H. Günthard, B. Hirschel, L. Kaiser, C. Kind, T. Klimkait, B. Ledergerber, U. Lauper, M. Opravil, F. Paccaud, G. Pantaleo, L. Perrin, J.-C. Piffaretti, M. Rickenbach (head of the Data Center), C. Rudin (chairman of the Mother and Child Substudy), P. Schmid, J. Schüpbach, R. Speck, A. Telenti, A. Trkola, P. Vernazza (chairman of the Scientific Board), R. Weber, and S. Yerly.
Acknowledgment
We thank T. Buclin, for useful comments.
References
1. Tukey RH, Strassburg CP. Human UDP-glucuronosyltransferases: metabolism, expression, and disease. Annu Rev Pharmacol Toxicol 2000; 40:581616. First citation in article
2. Bosma PJ, Chowdhury JR, Bakker C, et al. The genetic basis of the reduced expression of bilirubin UDP-glucuronosyltransferase 1 in Gilbert's syndrome. N Engl J Med 1995; 333:11715. First citation in article
3. Balram C, Sabapathy K, Fei G, Khoo KS, Lee EJ. Genetic polymorphisms of UDP-glucuronosyltransferase in Asians: UGT1A1*28 is a common allele in Indians. Pharmacogenetics 2002; 12:813. First citation in article
4. Monaghan G, Ryan M, Seddon R, Hume R, Burchell B. Genetic variation in bilirubin UPD-glucuronosyltransferase gene promoter and Gilbert's syndrome. Lancet 1996; 347:57881. First citation in article
5. Beutler E, Gelbart T, Demina A. Racial variability in the UDP-glucuronosyltransferase 1 (UGT1A1) promoter: a balanced polymorphism for regulation of bilirubin metabolism Proc Natl Acad Sci USA 1998; 95:81704. First citation in article
6. Biondi ML, Turri O, Dilillo D, Stival G, Guagnellini E. Contribution of the TATA-box genotype (Gilbert syndrome) to serum bilirubin concentrations in the Italian population. Clin Chem 1999; 45:8978. First citation in article
7. Takeuchi K, Kobayashi Y, Tamaki S, et al. Genetic polymorphisms of bilirubin uridine diphosphate-glucuronosyltransferase gene in Japanese patients with Crigler-Najjar syndrome or Gilbert's syndrome as well as in healthy Japanese subjects. J Gastroenterol Hepatol 2004; 19:10238. First citation in article
8. Aono S, Adachi Y, Uyama E, et al. Analysis of genes for bilirubin UDP-glucuronosyltransferase in Gilbert's syndrome. Lancet 1995; 345:9589. First citation in article
9. Maruo Y, Addario D, Mori A, et al. Two linked polymorphic mutations (A(TA)7TAA and T-3279G) of UGT1A1 as the principal cause of Gilbert syndrome. Hum Genet 2004; 115:5256. First citation in article
10. Sato H, Adachi Y, Koiwai O. The genetic basis of Gilbert's syndrome. Lancet 1996; 347:5578. First citation in article
11. Boffito M, Kurowski M, Kruse G, et al. Atazanavir enhances saquinavir hard-gel concentrations in a ritonavir-boosted once-daily regimen. AIDS 2004; 18:12917. First citation in article
12. Hammer SM, Squires KE, Hughes MD, et al. A controlled trial of two nucleoside analogues plus indinavir in persons with human immunodeficiency virus infection and CD4 cell counts of 200 per cubic millimeter or less. AIDS Clinical Trials Group 320 Study Team. N Engl J Med 1997; 337:72533. First citation in article
13. Sulkowski MS. Drug-induced liver injury associated with antiretroviral therapy that includes HIV-1 protease inhibitors. Clin Infect Dis 2004; 38(Suppl 2):S90S7. First citation in article
14. Zucker SD, Qin X, Rouster SD, et al. Mechanism of indinavir-induced hyperbilirubinemia. Proc Natl Acad Sci USA 2001; 98:126716. First citation in article
15. Fellay J, Boubaker K, Ledergerber B, et al. Prevalence of adverse events associated with potent antiretroviral treatment: Swiss HIV Cohort Study. Lancet 2001; 358:13227. First citation in article
16. Kasper DL, Fauci AS, Longo DL, Braunwald E, Hauser SL, Jameson JL. Harrison's principles of internal medicine. New York: McGraw-Hill, 2005. First citation in article
17. Diggle PJ. Analysis of longitudinal data. New York: Oxford University Press, 2002. First citation in article
18. Diggle PJ. An approach to the analysis of repeated measurements. Biometrics 1988; 44:95971. First citation in article
19. Laird NM, Donnelly C, Ware JH. Longitudinal studies with continuous responses. Stat Methods Med Res 1992; 1:22547. First citation in article
20. Jones RH, Boadi-Boateng F. Unequally spaced longitudinal data with AR(1) serial correlation. Biometrics 1991; 47:16175. First citation in article
21. Diggle PJ, Verbyla AP. Nonparametric estimation of covariance structure in longitudinal data. Biometrics 1998; 54:40115. First citation in article
22. Ware JH, Dockery DW, Louis TA, Xu XP, Ferris BG Jr, Speizer FE. Longitudinal and cross-sectional estimates of pulmonary function decline in never-smoking adults. Am J Epidemiol 1990; 132:685700. First citation in article
23. Royston P, Ambler G, Sauerbrei W. The use of fractional polynomials to model continuous risk variables in epidemiology. Int J Epidemiol 1999; 28:96474. First citation in article
24. Ioannidis JP, Ntzani EE, Trikalinos TA. "Racial" differences in genetic effects for complex diseases. Nat Genet 2004; 36:13128. First citation in article
25. O'Mara E, Randall D, Passarell J, et al. Population pharmacodynamic assessment of atazanavir exposure, uridine diphosphatase-glucoronosyl transferase (UGT) 1A1 genotype and safety in healthy human subjects (abstract A-1253, 2002b). Program and abstracts of the 42nd Interscience Conference on Antimicrobial Agents and Chemotherapy (San Diego). Washington, DC: American Society for Microbiology, 2002. First citation in article
26. Sai K, Saeki M, Saito Y, et al. UGT1A1 haplotypes associated with reduced glucuronidation and increased serum bilirubin in irinotecan-administered Japanese patients with cancer. Clin Pharmacol Ther 2004; 75:50115. First citation in article
27. Hariparsad N, Nallani SC, Sane RS, Buckley DJ, Buckley AR, Desai PB. Induction of CYP3A4 by efavirenz in primary human hepatocytes: comparison with rifampin and phenobarbital. J Clin Pharmacol 2004; 44:127381. First citation in article
28. Xie W, Yeuh MF, Radominska-Pandya A, et al. Control of steroid, heme, and carcinogen metabolism by nuclear pregnane X receptor and constitutive androstane receptor. Proc Natl Acad Sci USA 2003; 100:41505. First citation in article
29. Tarr PE, Taffe P, Bleiber G, et al. Modeling the influence of APOC3, APOE, and TNF polymorphisms on the risk of antiretroviral therapyassociated lipid disorders. J Infect Dis 2005; 191:141926 [erratum: J Infect Dis 2005; 191:1997]. First citation in article