Original Article

The Pharmacogenomics Journal (2010) 10, 524–536; doi:10.1038/tpj.2010.5; published online 2 March 2010

Genetic variation in carboxylesterase genes and susceptibility to isoniazid-induced hepatotoxicity

S Yamada1,2, K Richardson3, M Tang4, J Halaschek-Wiener1, V J Cook4,5, J M FitzGerald4, K Elwood4, F Marra4,6,8 and A Brooks-Wilson1,7,8

  1. 1Cancer Genetics, Canada's Michael Smith Genome Sciences Centre, British Columbia Cancer Agency, Vancouver, Canada
  2. 2Department of Life Science, Ritsumeikan University, Kusatsu Shiga, Japan
  3. 3Centre for Clinical Epidemiology and Evaluation, Vancouver Coastal Health Research Institute, Vancouver, Canada
  4. 4British Columbia Centre for Disease Control, Vancouver, Canada
  5. 5Faculty of Medicine, University of British Columbia, Vancouver, Canada
  6. 6Department of Family Practice, University of British Columbia, Vancouver, Canada
  7. 7Department of Biomedical Physiology and Kinesiology, Simon Fraser University, Burnaby, British Columbia, Canada

Correspondence: Dr A Brooks-Wilson, Cancer Genetics, Canada's Michael Smith Genome Sciences Centre, British Columbia Cancer Agency, 675 W 10th Ave., Vancouver BC V5Z 4E6, Canada. E-mail: abrooks-wilson@bcgsc.ca

8These authors co-led the study.

Received 23 August 2009; Revised 22 November 2009; Accepted 29 December 2009; Published online 2 March 2010.

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Abstract

Treatment of latent tuberculosis infection (LTBI) generally includes isoniazid (INH), a drug that can cause serious hepatotoxicity. Carboxylesterases (CES) are important in the metabolism of a variety of substrates, including xenobiotics. We hypothesized that genetic variation in CES genes expressed in the liver could affect INH-induced hepatotoxicity. Three CES genes are known to be expressed in human liver: CES1, CES2 and CES4. Our aim was to systematically characterize genetic variation in these novel candidate genes and test whether it is associated with this adverse drug reaction. As part of a pilot study, 170 subjects with LTBI who received only INH were recruited, including 23 cases with hepatotoxicity and 147 controls. All exons and the promoters of CES1, CES2 and CES4 were bidirectionally sequenced. A large polymorphic deletion was found to encompass exons 2 to 6 of CES4. No significant association was found. Eleven single-nucleotide polymorphisms (SNPs) in CES1 were in high linkage disequilibrium with each other. One of these SNPs, C(−2)G, alters the translation initiation sequence of CES1 and represents a candidate functional polymorphism. Replication of this possible association in a larger sample set and functional studies will be necessary to determine if this CES1 variant has a role in INH-induced hepatotoxicity.

Keywords:

pharmacogenetics; tuberculosis; genetic association; single-nucleotide polymorphism; haplotype; case–control study

Abbreviations:

INH, isoniazid; ADRs, adverse drug reactions; CES, carboxylesterases; AST, aspartate aminotransferase; OR, odds ratio; UTR, untranslated region; LD, linkage disequilibrium.

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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, 2, 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, 5, 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, 11, 12, 13, 14

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-induced hepatotoxicity. There is wide variability in reported associations of NAT2 variants with INH-induced hepatotoxicity in different populations;15, 16, 17, 18 however, controversy remains regarding the importance of NAT2 variation for this ADR in different ethnic groups. Factors that may contribute to heterogeneity of NAT2/hepatotoxicity associations include genetic differences between populations17 and contributions of variation in other genes to this ADR. It is likely that NAT2 variation accounts for only a portion of INH-induced hepatotoxicity.15, 16, 17, 18

An amidase enzyme(s) catalyzes two steps in the metabolism of INH.19, 20 There is strong evidence from an animal model that amidase activity levels influence hepatotoxicity; rabbits treated with an amidase inhibitor, bis-p-nitrophenyl phosphate, at the same time they are dosed with INH fail to develop the severe hepatotoxicity developed by animals treated with INH alone.21 Modulation of hepatic amidase activity therefore affects the development of hepatotoxicity, likely by altering INH metabolism. By extension, naturally occurring genetic variation in amidase genes may account for at least some of the variation in susceptibility to INH-induced hepatotoxicity. Given that INH metabolism occurs in the liver and that toxic metabolites released there are the cause of hepatotoxicity, amidase genes expressed in the liver are logical candidate genes for INH hepatotoxicity. Candidate genes for INH-induced hepatotoxicity were chosen based on three criteria: their ability to cleave amide bonds, their expression in the liver and their inhibition by bis-p-nitrophenyl phosphate. Cleavage of amide bonds is carried out by esterases, which can also cleave ester bonds. There are many esterases, but only a subset of them is inhibited by bis-p-nitrophenyl phosphate, a selective inhibitor of ‘type B’ esterases, or cholinesterases and carboxylesterases (CES). Cholinesterases (acetylcholinesterase and pseudocholinesterase) break down the neurotransmitter acetylcholine and, as such, would not be expected to be involved in INH metabolism.

Three CES genes are well characterized in the human genome: CES1, CES2 and CES3. All three are expressed in the liver. CES3 is more highly expressed in brain endothelial cells than in liver and has been suggested to function at the blood–brain barrier.22 CES1 (OMIM 114835) and CES2 (OMIM 605278) are both highly expressed in the liver and represent good candidates for genes involved in INH metabolism. Both are located on human chromosome 16; CES1 has 14 exons and CES2 has 12.23 CES423 is a transcribed pseudogene located adjacent to and 28kb upstream from CES1. It has six transcribed exons and spans approximately 14kb.24 CES1 and CES4 have very high sequence similarity to each other; CES4 appears to be an inverted duplication of CES1.25, 26 Because of the proximity of CES1 and CES4 to each other and their similarity, we also characterized CES4 as part of this study.

To assess the effect of genetic variation in these three CES genes on INH-induced hepatotoxicity, we have performed a case-control-based association study of 170 subjects in British Columbia (BC) using single-nucleotide polymorphisms (SNPs) and haplotypes of these genes. Because these genes are novel candidate genes for this ADR, we have carried out genotyping for this study by means of complete re-sequencing of the promoter and all coding and noncoding exons of the three genes in all study subjects. This not only addresses the effect of genetic variation in these genes on INH-induced hepatotoxicity in our population, but also provides an extensive catalog of genetic variation to support other pharmacogenetic analyses of these genes.

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Patients and methods

Study subjects

All individuals in British Columbia who are identified to have LTBI are eligible to receive preventative treatment through a publicly funded program. We enrolled subjects receiving treatment with INH (300mg daily) for LTBI at the Vancouver or Victoria TB Clinics from 2004 to 2006. Inclusion criteria were as follows: subjects were included if they were 19 years of age or older, not receiving other anti-TB drugs concurrently with INH, nonreactive to hepatitis B surface antigen and negative for antibody to hepatitis C by serology, not having any liver or metabolic diseases, without an HIV+ test result, not consuming seven or more alcoholic beverages per day and had sufficient aspartate aminotransferase (AST) monitoring to detect an INH-induced hepatotoxicity event.

We selected serum AST at baseline and follow-up as a marker of hepatotoxicity. Although alanine transaminase is more specific for liver dysfunction, our local BC Centre for Disease Control TB Clinic uses AST alone and considers a rise in AST after drug initiation without other confounders (heart disease, muscle disease and so on) to be attributable to medication. In addition, our research was conducted to impact policy within our local TB Clinic and, as such, we have conducted this study using measures that are consistent with the Clinic's policies and procedures.

Information was collected regarding subject age, sex, ethnicity of each grandparent, concurrent medical illnesses and alcohol and cigarette consumption. Confirmation of medication use, duration of treatment, all AST test dates and results and hepatitis serology were obtained from the TB Control database at the BC Centre for Disease Control. Baseline AST was measured before the initiation of INH treatment or as the first value entered into the subject's medical record within the first 2 weeks of treatment initiation. Values were measured monthly thereafter until treatment discontinuation or whenever subjects had symptoms of suspected hepatitis (anorexia, nausea, vomiting, malaise and tea-colored urine). Serum hepatitis B virus surface antigen, immunoglobulin M antibody to hepatitis A virus and antibody to hepatitis C virus were tested at baseline. Drug-induced hepatitis was defined according to the criteria of the International Consensus Meeting in Paris27 as an increase in serum AST level more than two times the upper limit of normal value during the 9-month treatment with INH, normalization of serum AST level after discontinuation of INH and a causality assessment score greater than 8.

Signed informed consent was obtained from each subject. This study was approved by the joint research ethics board of the University of British Columbia and the BC Cancer Agency.

Genotyping of CES1, CES2 and CES4 by re-sequencing

A 30ml blood sample was collected from each study subject in EDTA tubes, 6ml was immediately used for hepB and hepC testing and the remainder was frozen for later DNA extraction. DNA was extracted from blood of 170 hepB- and hepC-negative subjects using the PureGene DNA isolation kit following the manufacture's instructions (Gentra Systems, Minneapolis, MN, USA). DNA samples were quantified by fluorometry using PicoGreen (Invitrogen, Carlsbad, CA, USA) and a Victor2 fluorescence plate reader (Perkin-Elmer, Waltham, MA, USA).

For each of CES1, CES2 and CES4, all exons and intron/exon boundaries including the 5′ and 3′ untranslated regions (UTRs), 5′ upstream region including the promoter and conserved noncoding sequences were sequenced in each subject. Supplementary Online Table A lists the amplicons, primer pairs and PCR conditions used. For CES1, a total of 5916bp were PCR amplified in each sample using 13 primer pairs and 3 nested primer pairs. For CES2, a total of 8825bp were PCR amplified using 25 primer pairs and for CES4, a total of 5738bp were sequenced using 14 primer pairs. The VISTA browser28 was used to identify conserved noncoding sequences with 70% or greater sequence similarity over at least 100bp between human, mouse and rat orthologous gene sequences.28, 29 Exons were amplified using primers designed in the intronic sequences near the exon boundaries to allow re-sequencing across all splice sites. The 5′ and 3′ UTRs were amplified in overlapping segments. Primer design was completes using CES2, CES1 and CES4 genomic sequences (NM_001266, NM_003869 and NM_016280, respectively) retrieved from the UCSC genome browser23 and Primer3.30 In silico PCR31 was used to check primer specificity. All PCR primers incorporated M13 forward or reverse sequencing tags to allow efficient sequencing of the PCR products. PCR, sequencing and sequence analysis were carried out as described previously,32 with the exception that we used Big Dye Terminator Mix v3.1 (Applied Biosystems, Foster City, CA, USA) at 0.33μl of mix per reaction in a total volume of 4μl with 50 cycles amplification. Haploview33 was used to calculate and display inter-SNP linkage disequilibrium (LD) information derived from sequence data.

CES1 and CES4 are located adjacent to each other and in opposite orientation (Figure 1a), and show high sequence similarity to each other (Figure 1b). Exons 12, 13 and 14, and adjacent intron sequences of CES1 are nearly 100% identical to predicted but nontranscribed exons of CES4. To specifically amplify exons 12, 13 and 14 of CES1, we used nested PCR34 with a first primer set positioned such that the forward primer had one mismatch with the CES4 sequence and the reverse primer was located outside the nearly identical region. Unique localization of this specific PCR primer pair was verified using NCBI Blast.26 This primer set amplified a single 7959-bp-long PCR amplicon containing these three exons of CES1. Long PCR was carried out using Expand Long Range dNTPack PCR master mix (Roche, Basel, Switzerland). Subsequently, primers specific to exons 12, 13 and 14 were used to amplify nested PCR products for sequencing.

Figure 1.
Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

The CES1CES4 locus. (a) Genomic organization of the CES1 and CES4 genes. (b) Sequence similarity between the CES1 and CES4 genes. Red boxes indicate >99%, orange boxes 95–99% and yellow boxes indicate <95% sequence similarity, as estimated by NCBI blast.26 The diagrams are not to scale.

Full figure and legend (124K)

Genotypes of the CES2, CES1 and CES4 gene variants were determined for each of the 170 subjects from bidirectional sequence reads. Detection of polymorphisms was carried out using PolyPhred35 and Consed36 or Mutation Surveyor (SoftGenetics, State College, PA, USA). Each genotype was confirmed by independent examination of sequence traces by two individuals.

Statistical methods

SNPs with less than 5% minor allele frequency (MAF) were excluded from statistical analyses. For each common SNP, logistic regression was used to assess whether the genotypic and allelic frequencies were independently associated with hepatotoxicity after adjustment for age and sex. When the number of rare homozygotes was less than five, the rare homozygotes and heterozygotes were combined for analysis. Tests for trend were performed when five or more rare homozygous alleles were present. Hardy–Weinberg equilibrium was tested in the control groups by Fisher's exact test. Adjustment was made for multiple testing by computing the false discovery rate.37

The Haplo.stats package38 was used to test associations between statistically inferred haplotypes and hepatotoxicity, and to calculate adjusted odds ratios (ORs) and 95% confidence intervals for each haplotype. Haplotypes with frequencies >5% were tested. Rare haplotypes (with individual frequencies of <5%) were combined in the association test. When no haplotypes showed >5% frequency, the threshold for grouping rare haplotypes was lowered to 3%. All analyses were performed with SAS version 9 (SAS, Cary, NC, USA) and R version 2.8 (The R Project for Statistical Computing; www.r-project.org).

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Results

A total of 170 subjects were enrolled (Table 1). The mean duration of treatment was 239.6 days (s.d. 96.8) and the mean age was 40.8 years (s.d. 12.1). More than half of the participants were women (61.6%). Asian was the most common ethnic group (40.7%), followed by Caucasian (30.5%). The mean AST at baseline was 23.7 (s.d. 6.4). There were no statistically significant differences between cases and controls with respect to demographic variables and baseline AST. Twenty-three subjects (13.5%) met our criteria for INH-induced hepatitis. The maximum AST for these subjects was a mean of 125 (s.d. 131) compared to 31 (s.d. 9) for the controls. The case group had significantly higher maximum AST and during-treatment AST than controls.


Bidirectional sequencing was used to detect and genotype genetic variants in each gene in each of the 170 subjects. Of the 57 amplicons used for CES1, CES2 and CES4, four had low-quality sequence reads in both directions. Three amplicons had mononucleotide stretches that prevented good quality sequence reads in one direction but not in the other. Six amplicons contained a small insertion or deletion in more than 5% of individuals, causing those sequence reads to appear superimposed, even though they were of good quality. Good quality sequence reads were obtained in both forward and reverse direction for 77.2% of sample/amplicon combinations. On average 93.0% of sample/amplicon combinations had good quality sequence reads in at least one direction.

We observed 48 variants, including 38 novel variants, in the CES2 gene and 194 variants in the CES1CES4 region (40 and 42 novel, respectively). Genetic variants are listed in Table 2. Of these 48 variants, 31 (64.6%) were transitions, 13 (27.1%) were transversions and 4 (8.3%) were small insertions or deletions. Of the coding region changes, seven resulted in amino-acid changes. There was a large deletion, approximately 10kb in the CES4 gene in some samples, as revealed by 14 samples failing to amplify PCR products from exon 2 to the 3′ end of the gene (Figure 1). Sequencing of the samples that did not have a large deletion revealed 85 variants within this region, including 64 (59.3%) transitions, 40 (37.0%) transversions and 4 (3.7%) small insertions or deletions.


Of the 48 observed variants in CES2, 31 (64.6%) were singletons (observed only once in this data set). Six variants (12.5%) had MAF of 5% or greater. In contrast, only 37 of the 109 variants observed in the CES1 and nondeletion region of CES4 (33.9%) were singletons. Forty-four of these (40.7%) had an MAF of 5% or greater.

The large polymorphic deletion in CES4 spanning exons 2 to 6 was characterized by both PCR and sequencing. Homozygous deletion was deduced for 14 DNA samples in which exons from 2 to 6 did not produce PCR products. PCR alone, however, could not distinguish homozygous nondeleted samples from heterozygous samples. For that, sequence data from PCR products within the deleted region were used to categorize samples. Ninety-one samples that did not appear to be heterozygous at any of 85 polymorphic sites within the deleted region were interpreted to be hemizygous, that is, heterozygous for the large deletion. Fifty-seven samples that showed three or more heterozygous sites within the deletion region were interpreted to be nondeleted on both alleles. Sixteen samples with only one or two heterozygotes were conservatively considered ambiguous due to the potential for PCR error to create false heterozygotes.

Figures 2a and b show the inter-SNP LD, in terms of pair-wise r2 values, calculated in our data for common variants (MAF greater than or equal to5%). A block of LD encompasses SNP10, SNP11 and SNP13–21; LD among these 11 SNPs is high (r2=0.83–1.00). Overall, the CES1CES4 region shows three LD blocks. LD across CES2 is lower.

Figure 2.
Figure 2 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Linkage disequilibrium across the candidate genes. (a) CES1 and CES4. (b) CES2. This plot was generated using sequence data for 44 common variants in CES1 and CES4 in 170 individuals. The variants are listed in order of their physical position. Blocks with no value indicate an r2 of 1.0. This figure was generated using Haploview33 with some modifications. The diagrams are not to scale. Numbering of SNPs is according to Table 2.

Full figure and legend (100K)

Hardy–Weinberg equilibrium was tested in Caucasian controls and Asian controls separately, for each of the 6 common SNPs in CES2 and 44 common SNPs in CES1 and CES4. Genotype frequencies of CES2 variants did not deviate significantly from Hardy–Weinberg equilibrium. Seven variants in CES1 and CES4 deviated significantly from Hardy–Weinberg equilibrium (one in Asian controls, six in Caucasian controls; see Supplementary Online Table B). These variants were excluded from all analyses.

Table 3 shows the results of association tests of each common variant in CES1 with INH-induced hepatotoxicity. A small group of individuals with other ethnicity including mixed/unknown was excluded from these analyses, which were adjusted for age and sex. None of the 28 common variants was associated with a significantly increased risk of INH-induced hepatotoxicity. We did observe initially a significant result at P<0.05 in all subjects. However, this P-value did not remain significant after correction for multiple testing. There were no statistically significant SNPs results associated with an increased risk of INH-induced hepatotoxicity in CES2 or CES4 (data not shown).


Table 4 summarizes the results of association tests of haplotypes predicted using the Haplo.stats package38 in the high LD region of CES1, including SNP10, SNP11 and SNP13–21. We compared haplotype frequencies in all cases vs all controls, with adjustment for age and sex. Rare haplotypes (frequency <5%) were combined in the analysis. However, no significant association was observed in all subjects.


Haplotype analysis was also performed for CES2 (data not shown) and for the entire CES1CES4 region (Supplementary online Table C). None of these haplotype-based association tests achieved significance. SNP16 was predicted to be ‘possibly damaging’ to protein function by PolyPhen.39, 40 This variant, however, was not predicted to be functional by SIFT41 (see Supplementary online Table D).

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Discussion

CES can activate prodrugs containing ester linkage and increase the solubility and bioavailability of therapeutic agents.42 They are also involved in the metabolism of illegal drugs such as heroin and cocaine.43 Genetic variants in both CES1 and CES2 genes may contribute to ADRs and increased sensitivity or resistance to drug treatment. For example, an SNP of the CES1 gene has been associated with responsiveness to imidapril,44 an angiotensin-converting enzyme inhibitor used to treat hypertension and congestive heart failure. Rare nonsynonymous variants in CES1 encode enzymes with impaired activities that dramatically alter the pharmacokinetics and drug response to the psychostimulant methylphenidate.45 Several CES2 variants have been shown to be functionally deficient, and some have decreased esterase activities toward the anticancer agent irinotecan.46 CES are clearly important in a variety of pharmacogenetic phenotypes. No studies to date, however, have addressed CES genes in drug-induced hepatotoxicity.

Other genes have been assessed for association with hepatotoxicity induced by INH alone and/or with other drugs, including NAT2, CYP2E1, glutathione S-transferase M1, glutathione S-transferase T1 and HLA variants.47, 48, 49 We have recently carried out a pharmacogenetics study of NAT2 and CYP2E1 on INH-induced hepatotoxicity using the same study population; although no single variant showed a significant association, there was evidence of a trend of increasing hepatotoxicity across the rapid, intermediate and slow acetylator categories of NAT2 genotypes.50

We have assessed the effect of genetic variation in three novel candidate genes for hepatotoxicity, CES1, CES2 and CES4, by systematically cataloguing the variants present in subjects with and without hepatotoxicity, and conducting association tests of SNPs and haplotypes. We found no evidence for association of genetic variation in CES2 with INH-induced hepatotoxicity. There were no common (MAF greater than or equal to5%) nonsynonymous variants in CES2. We observed common SNPs in the upstream region including the 5′ UTR and promoter of CES2, but they were not significantly associated with INH-induced hepatotoxicity.

Six human CES genes have been reported on chromosome 16. CES1 and CES2 are the most extensively studied51; CES4 is less well characterized. We found very high sequence similarity between CES1 and CES4; CES4 is likely to be an inverted duplication of CES1 (Figure 1). Moreover, these genes cluster with CES7 on chromosome 16 and may have arisen from an ancestral duplication event.41 CES4 is a transcribed pseudogene,26 and its mRNA expression level in liver is lower than that of CES1.52 A pseudogene that has arisen by duplication or retroposition might not be subject to natural selection, particularly if the original copy remains functional. Transcribed pseudogenes may also compete with their functional counterparts for transcription factors.53 It is therefore not unreasonable to consider an effect of CES4 (and/or CES1) on INH metabolism and that genetic variation in CES4 could affect the risk of INH-induced hepatotoxicity. We discovered a large polymorphic deletion in the CES4 gene. Sequence data from 85 polymorphisms within the deleted region were used to distinguish nondeleted samples from heterozygous samples. This large deletion, however, was not significantly associated with hepatotoxicity. We identified eight SNPs in CES4 (SNPs 85–92) that are in complete LD (r2=1.00) with each other (Figure 2), in a region that contains putative Sp1 binding sites.54 These SNPs, however, were not significantly associated with INH-induced hepatotoxicity.

SNPs in CES1 were challenging to genotype due to the high similarity between CES1 and CES4. We were able to distinguish the two genes using long PCR and nested PCR. SNP4 is located in a putative CCAAT/enhancer binding protein site in the CES1 promoter region. CCAAT/enhancer binding proteins are expressed in several organs and involved in controlling differentiation-dependent gene expression.54 It is possible that this SNP could have a functional effect on the gene, although it did not appear to affect risk of hepatotoxicity in this study. Moreover, we reported that several SNPs in the 5′ UTR and exon 1 of CES1 are in high LD with each other (Figure 2). Point mutations in the translation initiation or ‘Kozak’ sequence, especially in position from −1 to −5 relative to the start codon, influence translational efficiency.55 SNP15, C(−2)G, alters the Kozak sequence of CES1 (Table 2). Previous studies suggested that a C allele in position −2 of a Kozak sequence enhances translation in vivo by twofold.55 SNP15 is a good candidate for a functional variant in CES1, though the high LD in this region makes it impossible to determine, by genetics alone, which SNP could be exerting an effect. Of the coding region changes in this haplotype, only four were nonsynonymous. SNP16 was classified by PolyPhen39, 40 as ‘possibly damaging’ (see Supplementary Table D) and may warrant further study. Four other SNPs in this haplotype were in the 5′ UTR or promoter region of CES1 and could affect gene regulation. Thus, functional studies56 will be needed to investigate whether any of these SNPs affect CES1 activity.

In conclusion, though SNPs and haplotypes at CES2 and CES1/CES4 were not associated with hepatotoxicity in this study, we identified genetic variants that could affect CES1 function and be relevant to other pharmacogenetic phenotypes. One SNP alters the translation initiation ‘Kozak’ sequence of CES1. Tests for association in a larger sample set will be necessary to determine if genetic variation in CES1 has a role in INH-induced hepatotoxicity. Our systematic characterization of genetic variation in these genes will be useful in other pharmacogenetic studies, including those that address metabolism of therapeutic drugs, such as imidapril, methylphenidate and irinotecan, as well as pharmacogenetics of illicit drugs.

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Conflict of interest

The authors declare no conflict of interest.

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Acknowledgements

This study was funded by a grant from the BC Lung Association to FM and AB-W. AB-W is a senior scholar of the Michael Smith Foundation for Health Research. JH-W was supported in part by an Erwin Schroedinger Fellowship from the Austrian Science Foundation (FWF). VC was supported in part by ‘in it for life’, Vancouver Coastal Health Research Institute. JMFG is a recipient of a Michel Smith Distinguished Scholar Award and a CIHR/BC Lung Scientist Award. SY was supported in part by a Ritsumeikan University International Research Fellowship.

Supplementary Information accompanies the paper on The Pharmacogenomics Journal website