The Pharmacogenomics Journal (2010) 10, 524–536 & 2010 Macmillan Publishers Limited. All rights reserved 1470-269X/10 www.nature.com/tpj ORIGINAL ARTICLE Genetic variation in carboxylesterase genes and susceptibility to isoniazid-induced hepatotoxicity S Yamada1,2, K Richardson3, Treatment of latent tuberculosis infection (LTBI) generally includes isoniazid 4 1 (INH), a drug that can cause serious hepatotoxicity. Carboxylesterases (CES) M Tang , J Halaschek-Wiener , are important in the metabolism of a variety of substrates, including 4,5 4 VJ Cook , JM FitzGerald , xenobiotics. We hypothesized that genetic variation in CES genes expressed K Elwood4, F Marra4,6,8 in the liver could affect INH-induced hepatotoxicity. Three CES genes are and A Brooks-Wilson1,7,8 known to be expressed in human liver: CES1, CES2 and CES4. Our aim was to systematically characterize genetic variation in these novel candidate genes 1Cancer Genetics, Canada’s Michael Smith and test whether it is associated with this adverse drug reaction. As part of a Genome Sciences Centre, British Columbia pilot study, 170 subjects with LTBI who received only INH were recruited, Cancer Agency, Vancouver, Canada; including 23 cases with hepatotoxicity and 147 controls. All exons and the 2Department of Life Science, Ritsumeikan University, Kusatsu Shiga, Japan; 3Centre for promoters of CES1, CES2 and CES4 were bidirectionally sequenced. A large Clinical Epidemiology and Evaluation, Vancouver polymorphic deletion was found to encompass exons 2 to 6 of CES4.No Coastal Health Research Institute, Vancouver, significant association was found. Eleven single-nucleotide polymorphisms 4 Canada; British Columbia Centre for Disease (SNPs) in CES1 were in high linkage disequilibrium with each other. One of Control, Vancouver, Canada; 5Faculty of Medicine, University of British Columbia, these SNPs, C(À2)G, alters the translation initiation sequence of CES1 and Vancouver, Canada; 6Department of Family represents a candidate functional polymorphism. Replication of this possible Practice, University of British Columbia, association in a larger sample set and functional studies will be necessary to 7 Vancouver, Canada and Department of determine if this CES1 variant has a role in INH-induced hepatotoxicity. Biomedical Physiology and Kinesiology, Simon Fraser University, Burnaby, British Columbia, The Pharmacogenomics Journal (2010) 10, 524–536; doi:10.1038/tpj.2010.5; Canada published online 2 March 2010 Correspondence: Keywords: pharmacogenetics; tuberculosis; genetic association; single-nucleotide polymorph- Dr A Brooks-Wilson, Cancer Genetics, Canada’s ism; haplotype; case–control study Michael Smith Genome Sciences Centre, British Columbia Cancer Agency, 675 W 10th Ave., Vancouver BC V5Z 4E6, Canada. E-mail: [email protected] Introduction Tuberculosis (TB) is a major global health problem. In Canada, groups at increased risk include Aboriginal persons, the foreign-born and inner city populations.1–3 Isoniazid (INH) is recommended as the drug of choice to treat latent tuberculosis infection (LTBI). Several adverse drug reactions (ADRs) are associated with INH, including hepatitis. A better understanding of the basis of this potentially life-threatening ADR is needed to inform preventive measures.4–6 The occurrence of ADRs related to INH, especially hepatotoxicity, has been well characterized.7 The incidence of INH-induced hepatotoxicity ranges from 1 to 36%, depending on different regimens, the population being treated and the definition of hepatic injury used.8,9 Alcohol consumption, presence of HIV, advanced age and chronic liver disease have been reported to increase the risk of INH-induced hepatotoxicity.10–14 8These authors co-led the study. N-acetyltransferase 2 (NAT2) is directly involved in INH metabolism, and genetic variation in the NAT2 gene has been reported to be a risk factor for INH- Received 23 August 2009; revised 22 November 2009; accepted 29 December 2009; published induced hepatotoxicity. There is wide variability in reported associations of NAT2 online 2 March 2010 variants with INH-induced hepatotoxicity in different populations;15–18 however, Genetic variation in CES genes and INH hepatotoxicity S Yamada et al 525 controversy remains regarding the importance of NAT2 ing exons of the three genes in all study subjects. This not variation for this ADR in different ethnic groups. Factors only addresses the effect of genetic variation in these genes that may contribute to heterogeneity of NAT2/hepatotoxi- on INH-induced hepatotoxicity in our population, but also city associations include genetic differences between popu- provides an extensive catalog of genetic variation to support lations17 and contributions of variation in other genes to other pharmacogenetic analyses of these genes. this ADR. It is likely that NAT2 variation accounts for only a portion of INH-induced hepatotoxicity.15–18 An amidase enzyme(s) catalyzes two steps in the metabo- Patients and methods lism of INH.19,20 There is strong evidence from an animal model that amidase activity levels influence hepatotoxicity; Study subjects rabbits treated with an amidase inhibitor, bis-p-nitrophenyl All individuals in British Columbia who are identified to phosphate, at the same time they are dosed with INH fail to have LTBI are eligible to receive preventative treatment develop the severe hepatotoxicity developed by animals through a publicly funded program. We enrolled subjects treated with INH alone.21 Modulation of hepatic amidase receiving treatment with INH (300 mg daily) for LTBI at the activity therefore affects the development of hepatotoxicity, Vancouver or Victoria TB Clinics from 2004 to 2006. likely by altering INH metabolism. By extension, naturally Inclusion criteria were as follows: subjects were included if occurring genetic variation in amidase genes may account they were 19 years of age or older, not receiving other anti- for at least some of the variation in susceptibility to INH- TB drugs concurrently with INH, nonreactive to hepatitis B induced hepatotoxicity. Given that INH metabolism occurs surface antigen and negative for antibody to hepatitis C by in the liver and that toxic metabolites released there are the serology, not having any liver or metabolic diseases, without cause of hepatotoxicity, amidase genes expressed in the liver an HIV þ test result, not consuming seven or more alcoholic are logical candidate genes for INH hepatotoxicity. Candi- beverages per day and had sufficient aspartate aminotrans- date genes for INH-induced hepatotoxicity were chosen ferase (AST) monitoring to detect an INH-induced hepato- based on three criteria: their ability to cleave amide bonds, toxicity event. their expression in the liver and their inhibition by bis-p- We selected serum AST at baseline and follow-up as a nitrophenyl phosphate. Cleavage of amide bonds is carried marker of hepatotoxicity. Although alanine transaminase is out by esterases, which can also cleave ester bonds. There are more specific for liver dysfunction, our local BC Centre for many esterases, but only a subset of them is inhibited by bis- Disease Control TB Clinic uses AST alone and considers a rise p-nitrophenyl phosphate, a selective inhibitor of ‘type B’ in AST after drug initiation without other confounders esterases, or cholinesterases and carboxylesterases (CES). (heart disease, muscle disease and so on) to be attributable to Cholinesterases (acetylcholinesterase and pseudocholines- medication. In addition, our research was conducted to terase) break down the neurotransmitter acetylcholine and, impact policy within our local TB Clinic and, as such, we as such, would not be expected to be involved in INH have conducted this study using measures that are consis- metabolism. tent with the Clinic’s policies and procedures. Three CES genes are well characterized in the human Information was collected regarding subject age, sex, genome: CES1, CES2 and CES3. All three are expressed in the ethnicity of each grandparent, concurrent medical illnesses liver. CES3 is more highly expressed in brain endothelial and alcohol and cigarette consumption. Confirmation of cells than in liver and has been suggested to function at the medication use, duration of treatment, all AST test dates and blood–brain barrier.22 CES1 (OMIM 114835) and CES2 results and hepatitis serology were obtained from the TB (OMIM 605278) are both highly expressed in the liver and Control database at the BC Centre for Disease Control. represent good candidates for genes involved in INH Baseline AST was measured before the initiation of INH metabolism. Both are located on human chromosome 16; treatment or as the first value entered into the subject’s CES1 has 14 exons and CES2 has 12.23 CES423 is a transcribed medical record within the first 2 weeks of treatment pseudogene located adjacent to and 28 kb upstream from initiation. Values were measured monthly thereafter until CES1. It has six transcribed exons and spans approximately treatment discontinuation or whenever subjects had symp- 14 kb.24 CES1 and CES4 have very high sequence similarity toms of suspected hepatitis (anorexia, nausea, vomiting, to each other; CES4 appears to be an inverted duplication of malaise and tea-colored urine). Serum hepatitis B virus CES1.25,26 Because of the proximity of CES1 and CES4 to surface antigen, immunoglobulin M antibody to hepatitis A each other and their similarity, we also characterized CES4 as virus and antibody to hepatitis C virus were tested at part