High prevalence of the UGT1A1*28 variant in HIV-infected individuals in Greece

Abstract

Hyperbilirubinaemia with or without jaundice is one of the side effects of atazanavir boosted with low-dose ritonavir (ATV/rit) related to the drug plasma levels, as a result of its metabolism by UGT1A1 – uridine diphosphate-glucuronosyl transferase. Genotyping for UGT1A1*28 before initiation of antiretroviral therapy containing atazanavir may aid in identifying individuals at risk of hyperbilirubinaemia. Our objective was to estimate the prevalence of the UGTA1A1*28 polymorphism in HIV-infected individuals in Greece and to determine its potential association with hyperbilirubinaemia in patients receiving ATV/rit. The prevalence of the UGTA1A1*28 variant was estimated in 79 HIV-infected patients prior to the administration of the first-line treatment. The UGTA1A1*28 variant was detected in 46 out of 79 individuals (58.2%). Antiretroviral therapy was administered to 64/79 patients (81%). Among them, 26/64 (40.6%) received ATV/rit. Of the ATV/rit-treated patients, 14 were found to be carriers of the UGT1A1*28 variant (54%), and maximum serum bilirubin levels were significantly higher in the carrier population (4.71 vs. 2.69 mg/dL, p = 0.026). In 50% of the population, maximum levels were recorded in the first month of follow-up. Although carriage of UGT1A1 is linked with the development of hyperbilirubinaemia, the implementation of a pharmacogenomic approach in clinical practice cannot yet be recommended as a standard of care.

Keywords

HIV AIDS treatment antiretroviral therapy atazanavir hyperbilirubinaemia UGT1A1 pharmacogenomics

Introduction

Over the past few years, there has been a remarkable increase in our knowledge of the variation in the human genome. In parallel, genotyping technologies have advanced significantly and allow sufficient throughput to accommodate genome-wide approaches. This knowledge has allowed the implementation of pharmacogenomics in the clinical management of HIV infection.¹ The rationale is based upon optimising or modifying an antiretroviral regimen according to genomic analysis of the patient's susceptibility to an adverse event related to a component of the regimen.² Atazanavir (ATV) boosted with low-dose ritonavir (rit) is used as part of a preferred/recommended regimen in almost all national and regional guidelines.³ Hyperbilirubinaemia is the most common adverse event in patients treated with ATV. Over three-fourths of all patients experienced an elevation of bilirubin while on treatment and approximately one-third of all cases reached grade 3–4 increases.⁴ Jaundice and hyperbilirubinaemia are more common with boosted ATV.⁵ This protease inhibitor (PI) acts as an inhibitor of uridine diphosphate – glucuronosyltransferase (UGT), the enzyme responsible for hepatic conjugation of bilirubin. Variation in the promoter region of the UGT1A1 gene (UGT1A1*28) can influence bilirubin plasma levels, modulating the susceptibility to hyperbilirubinaemia. Previous linkage studies identified a major locus at the chromosome 2q telomere that encodes UGT1A1. In addition, polymorphism in the UGTA1A enzyme and specifically the UGT1A1*28 variant may influence the risk of hyperbilirubinaemia in patients treated with ATV/rit.^6,7

The aim of this study was to estimate the prevalence of the UGT1A1*28 polymorphism in HIV-infected individuals in an HIV Unit of a tertiary University Hospital in Greece and to determine its potential association with hyperbilirubinaemia in patients receiving boosted ATV (ATV/rit).

Patients and methods

Design of the study

This is an observational cross-sectional study.

Patients

HIV-1-infected individuals presenting to the department and attending the HIV outpatient clinic of ATTIKON University Hospital in Athens for the one-year period, 1 April 2009–1 May 2010. All participating patients followed the regular visit schedule and work-up as implemented by the department and recommended by national guidelines. Data retrieved from the medical records included age, gender, antiretroviral therapy, baseline CD4 count (cells/µL) at study entry, stage as per Centers for Disease Control (CDC) definition, maximum value of serum total bilirubin (mg/dL) recorded during follow-up of 48 weeks and for those receiving an ATV/rit-containing regimen total bilirubin/indirect bilirubin serum levels at week 4 visit after treatment initiation and time-point of maximum total serum bilirubin levels. Concomitant medication history was noted, as well as stopping/switching events for patients on antiretrovirals. Patients were also classified into four groups according to the grade of total serum bilirubin level as per WHO criteria, from grade 1 + (mild) [1.1–1.5 × ULN], grade 2 + (moderate) [1.6–2.0 × ULN], grade 3 + (severe) [2.0–3.0 × ULN] up to grade 4 + (potentially life threatening) [>3.0 × ULN].⁸

Laboratory methodology

Genomic DNA was extracted from peripheral blood mononuclear cells. Genotyping was performed by direct sequencing of the region amplified using primers (5′-AAGTGAACTCCCTGCTACCTT-3′) and (5′-CCACTGGGATCAACAGTATCT-3′) in an Applied Biosystems 3100-Avant Genetic Analyzer as described previously.⁹

Ethics

Participation in the study was not associated with management and treatment decisions. Genetic analysis of UGT1A1 polymorphism was not associated with treatment choice, as it was performed retrospectively. All the study participants were informed of test results and use of their anonymised personal data. The study protocol was approved by the University of Athens Medical School Ethics Committee and the Scientific Committee of ATTIKON University General Hospital. This study complied with the Helsinki Declaration.

Statistical analysis

Anonymised data were entered into an electronic database. Proportions and means/medians were calculated for categorical and continuous variables, respectively. Chi squared, Fisher’s exact test and Mann–Whitney U test were performed as appropriate to calculate a 95% level of significance. All statistical analyses were performed using IBM SPSS version 20. Comparisons were made between carriers and non-carriers of the UGT1A1*28 polymorphism. The distribution of time to maximum bilirubin level in ATV/rit-treated individuals was estimated using Kaplan–Meier methodology.

Results

The analysis was performed on 79 Caucasian patients (three women). At baseline the median CD4 count was 320 cells/µL (interquartile range [IQR_1–3 120–590]). Patients were classified as Stage B or C as per CDC classification in 12.6% (10/79) and 25.3% (20/79) of cases, respectively (in order to classify the progression of HIV infection in most clinical settings, the 1993 CDC classification is still being used that takes the clinical presentation and CD4 T cell count into account).¹⁰ Mean age (SD) of the study population was 36.3 years (9.5) (min–max 19.8–65.1 years). The UGT1A1*28 variant was detected in 46 out of 79 individuals (58.2%). In the total population, median values (IQR_1–3) for maximum serum bilirubin were 0.9 mg/dL (0.25–2.2) (min–max 0.25–8.81). No differences were detected between carriers and non-carriers of the UGT1A1 polymorphism (Table 1). Antiretroviral treatment was administered to 64/79 patients (81%). Among them, 26/64 (40.6%) received ATV/rit. Of the ATV/rit-treated patients, 14 were found to be carriers of the UGT1A1*28 variant (54%). For patients not receiving ATV/rit, polymorphism carriers had higher maximum serum bilirubin levels (mean/SD in mg/dL) when compared to non-carriers (0.97/0.53 vs. 0.67/0.25, respectively, p = 0.03).

Table 1.

Study population characteristics with respect to polymorphism status.

Men	76 (96.2)
Caucasian	79 (100)
	UGT			p
	Yes (46)	No (33)	Total (79)	p
Age (years), mean (SD)	37.1 (7.4)	35.3 (10.8)	36.3 (9.5)	0.45
Min–max age (years)	19.8–65.1	20–49.1	19.8–65.1	–
cART (%)	35 (76.1%)	29 (87.9%)	64 (81%)	0.25
ATV/rit	14 (30.4%)	12 (36.4%)	26 (32.9%)	0.63
Median max bilirubin (IQR 1–3) in mg/dL	0.97 (0.7–3.49)	0.77 (0.54–1.47)	0.9 (0.6–2.2)	0.41
Min–max bilirubin (mg/dL)	0.33–8.81	0.25–5.58	0.25–8.81	–

ATV: atazanavir.

In the ATV/rit-treated population, maximum serum bilirubin levels were significantly higher in the carrier population (4.71 vs. 2.69 mg/dL, respectively, p = 0.02) (Table 2). In 50% of the population, maximum values were recorded in the first month of follow-up. Three patients had to switch to another PI due to icterus-related aesthetic reasons (all had total bilirubin serum levels >8 mg/dL and carried the UGT1A1 polymorphism). No pruritus or abnormal transaminases were recorded for these patients. In the ATV/rit-treated group, grade 3/4 bilirubin elevation was significantly associated with carriage of the polymorphism (92.9% vs. 54.5%, p = 0.026). For the 52 patients not on ATV/rit, only one developed grade 3/4 levels of bilirubin (UGT1A1 polymorphism carrier).

Table 2.

Bilirubin levels for individuals receiving an atazanavir/ritonavir-containing antiretroviral regimen with respect to their UGT1A1 polymorphism status.

	Carriers (n = 14)	Non-carriers (n = 12)	Total (n = 26)	p
Age (years), mean (SD)	41.4 (12.8)	38.8 (6.1)	40.2 (10.2)	0.52
Bilirubin (mg/dL) at four weeks, mean (SD)	5.35 (2.59)	2.64 (0.84)	3.99 (2.24)	0.02
Maximum bilirubin (mg/dL), mean (SD)	4.71 (2.45)	2.69 (1.66)	4.12 (2.35)	0.02
Indirect bilirubin (mg/dL)at four weeks, mean (SD)	4.41 (2.46)	2.25 (0.84)	3.65 (2.27)	0.039
When max bilirubin detected (months)
Median (IQR 1–3)	3.5 (1–32)	1 (1–12)	2 (1–18)	0.428
Mode	1	1	1	–
Min–max	1–50	1–50	1–50	–

IQR: interquartile range.

In Figure 1, the time-point of maximum bilirubin levels measured in individuals receiving ATV/rit is presented with respect to their polymorphism status.

Figure 1.

Time-point at follow-up of maximum serum bilirubin levels for people receiving atazanavir/ritonavir-containing regimens with respect to their UGT1A1 polymorphism status.

Discussion

This is the first study performed in Greece with a view to estimate the prevalence of UGT1A1 polymorphism in HIV-infected individuals. A prevalence of approximately 60% was found in our cohort, similar when compared to available national data for cancer patients’ cohorts and healthy controls (58.2% vs. 51.3% and 62.5%, respectively).^11,12

Our study findings confirmed the association of hyperbilirubinaemia with the use of ATV/rit-containing regimen in individuals carrying the UGT1A1 polymorphism, highlighting also the fact that carriers of the polymorphism develop total bilirubin levels >5 mg/dL in the majority of cases when on ATV/rit. On the other hand, no other liver dysfunction was recorded, whereas three individuals switched to another boosted PI due to aesthetic reasons. Hyperbilirubinaemia is a common complication of ATV-containing antiretroviral therapy. Hyperbilirubinaemia severity is related to UGT1A1 polymorphism.⁷ A recent study (ACTG 5202) documented that homozygosity for *28/*28 was present in 8% of whites, 24% of blacks and 18% of Hispanics. An association between this polymorphism and ATV/rit discontinuation was found only among Hispanics.¹³ Discontinuation rates of ATV/rit in our study population were approximately 9% (3/26 in ATV/rit group). In the CASTLE study, <1% of patients discontinued treatment due to hyperbilirubinaemia, and there were no differences in quality of life or adherence between patients with or without hyperbilirubinaemia. Of note, rates of grade 3/4 hyperbilirubinaemia recorded at 4, 24, 48 and 96 weeks were 13%, 15%, 18% and 22%, respectively and 11% had scleral icterus.¹⁴

In view of this finding and the established need for treatment adherence, a question for a pharmacogenomic approach is raised, i.e. if detection of the polymorphism at baseline can either modify a boosted ATV regimen to a non-boosted one or completely refocus regimen choice to an alternative PI. Pharmacogenomics is well established in HIV medicine, particularly with the case of class 1 HLA-B*5701 allele and abacavir hypersensitivity.¹⁵ Other studies have tried to explore possible association between central nervous system adverse events due to efavirenz and CYP2B6 polymorphism or nevirapine side effects and HLA DRB1*0101.¹⁶

In an Italian study, investigators switched patients carrying the UGT1A1 polymorphism from ATV/rit to 400 mg unboosted ATV daily. Bilirubin levels and triglycerides level were reduced and there was no compromise in immunological and virological efficacy of the regimen.¹⁷ This approach has not been attempted on a larger scale, although looks promising. In the Greek setting, use of unboosted ATV is not the general rule. It must also be noted that a favourable association between UGT1A1*28 and cardiovascular disease events has been described, attributed possibly to a protective action of higher bilirubin levels.¹⁸

The cost-effectiveness of a priori pharmacogenomic testing for UGT1A1 polymorphism was assessed by Schackman et al.¹⁹ They concluded that such an approach could be cost-effective only if the assay cost was low and if it contributed to better in-treatment retention rates, provided other comparator PI choices have the same drug cost and efficacy.

In terms of the real-life clinical setting, our study findings suggest that the time-point prediction of highest bilirubin levels is possible and may provide useful data to share with the individual querying about treatment options and the ATV starter candidate. For the majority of the patients, the highest bilirubin levels will be recorded within four weeks after treatment initiation. This time-point generally coincides with the first visit post-treatment initiation, allowing for discussion and handling further adherence issues related to icterus. With this fact in mind in addition to the high prevalence rate of the polymorphism in this Greek cohort, a pharmacogenomic analysis could be of limited value if a watchful approach with appropriate counselling is adopted, especially in times of austerity. A potential limitation of the present study, apart from the number of patients enrolled, is the current lack of further genotype analysis for those homozygous for seven repeats (7/7) and heterozygotes (6/7) versus the wild type of six repeats (6/6).^20,21 Nevertheless, a recent meta-analysis based upon all available evidence on the issue revealed that homozygosity of the UGT1A1*28 allele does not strongly predict the incidence of severe hyperbilirubinaemia. The authors conclude that UGT1A1*28 testing does not appear to provide additional information to aid clinical decision making when initiating ATV treatment in HIV-infected patients, until relevant prospective large-scale studies provide results.²²

In conclusion, although carriage of UGT1A1 is linked with the development of hyperbilirubinaemia, the implementation of a pharmacogenomic approach in clinical practice and treatment decision algorithms cannot yet be recommended as standard of care. Reasons for not following such an approach could be the cost of testing and the lack of data from large-scale trials designed to address the issue of baseline testing and treatment options. Our study also stressed the fact that for most patients the level of expected hyperbilirubinaemia associated with ATV treatment could be anticipated after month 1 of treatment.

Footnotes

Conflict of interest

The authors declare that there is no conflict of interest.

Funding

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

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