Clinical Gastroenterology and Hepatology
Volume 4, Issue 1 , Pages 21-28, January 2006

Pharmacogenomics in Inflammatory Bowel Disease

  • Laurence J. Egan

      Affiliations

    • Department of Pharmacology, National University of Ireland, Galway, Ireland
    • Corresponding Author InformationAddress requests for reprints to: Laurence J. Egan, MD, NUIG Clinical Pharmacology Unit, Clinical Science Institute, University College Hospital, Galway, Ireland; fax: + 353 91 495572.
    • ,
  • Luc J.J. Derijks

      Affiliations

    • Department of Clinical Pharmacy, Máxima Medical Center, Veldhoven, The Netherlands
    • ,
  • Daniel W. Hommes

      Affiliations

    • Department of Gastroenterology and Hepatology, Academic Medical Center, Amsterdam, The Netherlands

published online 30 December 2005.

Article Outline

Inherited variations in the nucleotide sequence of genes influence how individual patients respond to drugs. Most commonly, clinically significant genetic variations consist of single nucleotide polymorphisms (SNPs) within genes that affect drug disposition or drug targets. Up to now, relatively few clinically important examples of inherited traits that affect drug responses have been studied in detail. However, one of the well-characterized examples is highly relevant to inflammatory bowel disease therapeutics, that of thiopurine methyltransferase pharmacogenetics. Individuals with 2 normal alleles of the gene encoding thiopurine methyltransferase metabolize and clear thiopurines such as azathioprine and 6-mercaptopurine rapidly. Individuals with 1 normal and 1 variant allele are intermediate, whereas those with 2 variant alleles clear thiopurines very slowly. Intermediate and slow metabolizers are predisposed to have high active thiopurine drug levels and develop bone marrow suppression. Genomic era technology permits determination of large numbers of SNPs in large numbers of individuals. This capability is allowing the field of pharmacogenomics to become one of the most productive interfaces in translational biomedical research at present. By using high-throughput SNP genotyping, combined with careful phenotypic characterization of disease, pharmacogenomic research carries the potential of identifying individual biomarkers that predict the relative likelihood of benefit or risk from a therapeutic intervention. If this promise can be realized, pharmacogenomics will deliver the opportunity for personalized medicine.

Abbreviations used in this paper:  AZA, azathioprine , IBD, inflammatory bowel disease , ITPA, inosine triphosphate pyrophosphatase , 6-MP, 6-mercaptopurine , NAT, arylamine N-acetyltransferases , SNP, single nucleotide polymorphism , 6-TGN, 6-thioguanine nucleotides , TPMT, thiopurine methyltransferase

 

The benefits and adverse effects of drugs vary greatly among individual patients. Many factors such as age, gender, disease severity, and comorbidities affect the differential responses to therapies. Inherited traits also significantly affect patients’ responses to drugs. For decades it has been known that the degree of variation in response to a drug is much less in an individual person (or between identical twins) than between a group of people.1 Pharmacogenetics refers to the study of the effect of inheritance on individual variation in drug responses. This field of study was spawned in the last century when it was discovered that the concentration of certain drugs in patients varied greatly, and that this trait was often inherited in a mendelian fashion.2 Once drug metabolizing enzymes were discovered and molecularly cloned, it became clear that the observed variation in certain drug levels was often attributable to polymorphisms in the genetic sequences encoding those enzymes that metabolize the drug.3 The genomic revolution of the past 20 years brought the potential to characterize a patient’s likelihood of response to a drug on the basis of DNA sequencing, the field of pharmacogenomics.4 The value of pharmacogenomics lies in the potential to individualize drug therapy. In this review, we set forth the molecular mechanisms underpinning pharmacogenomics, review examples relevant to inflammatory bowel disease (IBD) to date, discuss how these examples affect therapeutic decision making, and look to the future for pharmacogenomics in IBD.

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Molecular Mechanisms of Pharmacogenomics 

Single nucleotide polymorphisms (SNPs), simple nucleotide variations in DNA sequence scattered throughout the genome, constitute the most common genetic basis for variation in drug responses between individuals. Pharmacologically significant SNPs are usually found within the coding regions of the gene but also occasionally in the promoter or intronic regions of the gene. Coding region SNPs can change the RNA sequence transcribed from that gene, resulting in translated proteins that are altered in length or in their amino acid sequence. At the biochemical level, variant low activity alleles usually are associated with the production of proteins that have reduced stability rather than reduced enzymatic activity as the explanation for the decreased activity.5 SNPs in genes encoding drug metabolizing enzymes affect drug concentration, so-called pharmacokinetic mechanisms of inherited variation in drug responses. In contrast, SNPs in genes encoding drug targets cause variations in pharmacodynamics.

Pharmacokinetic Mechanisms 

The most widely known and best studied examples of clinically relevant pharmacogenetic factors are those in which individuals inherit a trait that affects the concentration of a drug. In these situations, germline DNA sequences for genes encoding drug metabolizing enzymes vary. Individuals might possess 2 “normal” (wild-type) alleles, 2 variant (mutant) low activity alleles, or 1 of each. Those with 2 normal alleles metabolize the drug rapidly, resulting in low drug levels (extensive metabolizers), whereas those with 2 variant alleles metabolize the drug more slowly, resulting in higher drug levels (poor metabolizers) (Figure 1). Individuals with 1 normal and 1 variant allele have intermediate drug levels (Figure 1). Many of the isoforms of cytochromes P450, the chief enzymes responsible for phase I biotransformation of xenobiotics including drugs, possess functionally significant polymorphisms (some examples are given in Table 1). Usually lower drug levels are associated with poorer therapeutic responses, whereas higher drug levels are often associated with the occurrence of concentration-related side effects (Figure 1). For drugs that are metabolically activated by polymorphically expressed enzymes such as codeine metabolism to morphine by the 2D6 isoform of cytochrome P450, variant alleles are associated with low levels of the active metabolite and consequently poorer therapeutic responses. It is important to note that the frequency of variant alleles of many of the drug metabolizing enzymes varies considerably between racial groups. For example, CYP2C19 variants are up to 10 times more prevalent in Asian than in white populations.6

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  • Figure 1. 

    Mechanisms of pharmacogenetics. Pharmacokinetic factors (left) affect the concentration of drug at the active site. In the example, an individual inherits 2, 1, or none of the wild-type (WT) alleles of the TMPT gene, along with none, 1, or 2 of the mutant (Mut) inactive alleles. This provides TPMT activity that is normal, intermediate, or low (absent), which is accompanied by low, intermediate, or high active thiopurine drug levels. Therefore, AZA or 6-MP active metabolite levels are greater in the presence of mutant alleles, with a much greater chance of toxicity. Pharmacodynamic factors (right) do not affect drug concentration but often affect the expression or activity of the drug’s target, in this example gefitinib, which inhibits the epidermal growth factor receptor (EGFR). Certain tumors, such as lung cancer and possibly colon cancer, select for mutant EGFR that is constitutively overactive, which provides a growth or survival advantage to those cells. EGFR inhibitor produces a therapeutic effect in the presence of the mutant overactive EGFR, whereas WT EGFR, which has little constitutive activity, is not greatly affected by the inhibitor.

Table 1. Selected Polymorphically Expressed Drug Metabolizing Enzymes
EnzymeSubstrateFunctionEffect of variant allelesOutcome of variant alleles
TPMT6-MPInactivation of drugDecreased inactivationIncreased toxicity
CYP 2C19OmeprazoleInactivation of drugDecreased inactivationIncreased efficacy
CYP 2D6CodeineActivation of pro-drugDecreased activationDecreased efficacy
CYP 2C9WarfarinInactivation of drugDecreased inactivationIncreased efficacy and toxicity

CYP, cytochrome P450.

Another important pharmacokinetic cause of inherited variation in drug response lies in sequence polymorphisms of genes encoding drug transporters. The inheritance of low activity variant alleles is associated with low rates of drug transport. For example, in the case of the P-glycoprotein multi-drug cellular efflux transporter, low activity alleles elevated the concentration of drug at active sites, increasing effects that might be beneficial or potentially harmful.7

Pharmacodynamic Mechanisms 

Novel therapeutics are usually developed to target a specific enzyme, cell surface receptor, or channel. Variation in the expression or structure of that target can affect the therapeutic responsiveness of the targeting drug. In many cases, functionally significant SNPs are present in the germline DNA of the target gene.8 In other cases, cancer cells might somatically mutate the gene for the drug target or amplify the number of copies of the gene.9, 10 Pharmacodynamic mechanisms such as these are unassociated with drug concentration differences but instead reflect the extent to which the drug effectively interacts with its therapeutic target (Figure 1). For example, mutant epidermal growth factor receptors in lung cancer result in constitutive overactivity of the receptor, which provides growth and survival advantage to the cells. The ability of an inhibitor of this receptor, gefitinib, to produce therapeutic benefit in lung cancer is confined to those patients whose tumors possess mutant, overactive epidermal growth factor receptors.11, 12, 13

Well-characterized pharmacodynamic mechanisms of variation in drug responses are much less numerous than pharmacokinetic mechanisms. However, a great deal of current research is directed at the analysis of sequence variations in drug targets, and it is likely that in coming years, there will be further elucidation of more important examples of pharmacodynamic causes of variation in therapeutic responsiveness.

Mechanisms Unrelated to Drug Concentration or Target Expression 

A third group of mechanisms underlying inherited variation in responses to drugs involves polymorphisms of genes encoding disease-modifying proteins that neither affect drug concentration nor alter the drug target. For example, long-QT syndrome caused by variant alleles of certain cardiac ion channels predisposes to torsades de pointes induced by drugs such as the prokinetic agent cisapride and the antihistamine terfenadine.14, 15

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Inflammatory Bowel Disease–Specific Pharmacogenomics 

Thiopurine Therapy 

Thiopurine methyltransferase pharmacogenomics 

Azathiopurine (AZA) and 6-mercaptopurine (6-MP) are often used in clinical practice as maintenance treatment to prevent clinical relapse in IBD patients.16, 17, 18, 19 AZA is a pro-drug that is converted to 6-MP through a non-enzymatic step by sulfhydryl-containing compounds such as glutathione present in erythrocytes and other tissues (Figure 2). Subsequently, 6-MP is metabolized by thiopurine methyltransferase (TPMT) to 6-methylmercaptopurine or by xanthine oxidase to 6-thiouric acid.20 Alternatively, 6-MP is further metabolized to 6-thioguanine nucleotides (6-TGN) via a multistep enzymatic pathway beginning with hypoxanthine phosphoribosyltransferase followed by inosine triphosphate pyrophosphatase (ITPA) and guanosine monophosphate synthetase. The enzyme activities of TPMT and ITPA are under genetic control, and the clinical relevance of existing genetic polymorphism in TPMT and ITPA is much debated.

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  • Figure 2. 

    Pathways of thiopurine metabolism. The positions of 2 polymorphically expressed enzymes, TPMT and ITPA, are shown. 6-TIMP, 6-thio inosine monophosphate; 6-TIDP, 6-thio inosine diphosphate; 6-TITP, 6-thio inosine trinophosphate; 6-TG, 6-thioguanine (nucleotides). Arrows indicate a pathway of metabolism and bar indicates inhibition of purine production.

The gene encoding TPMT is located on chromosome 6 (6p22.3) and contains 10 exons. Two wild-type alleles (TPMT*1 and *1S) and 20 mutant alleles (TPMT*2, *3A, *3B, *3C, *3D, *4, *5, *6, *7, *8, *9, *10, *11, *12, *13, *14, *15, *16, *17, *18) responsible for TPMT deficiency have been described (Figure 3).21, 22 These alleles are characterized by 1 or more SNPs in the open reading frame sequences of the TPMT gene. In addition, several intronic mutations and mutations outside the open reading frame exist. Recently, a variable number tandem repeat within the TPMT promoter region of the TPMT gene has also been reported to modulate levels of TPMT activity.23, 24 Enhanced degradation of TPMT proteins encoded by certain mutant alleles has been proposed as mechanisms for lower TPMT protein and catalytic activity. Thus, whereas the degradation half-life of TPMT*1 is 18 hours, TPMT*2 and TPMT*3A have a markedly decreased half-life of 15 minutes.25 The distribution of TPMT mutant alleles differs significantly among ethnic populations. TPMT*3A (3.2%–5.7%) is the most occurring mutant allele in white populations, followed by TPMT*2 (0.2%–0.5%) and TPMT*3C (0.2%–0.8%), accounting for the vast majority (>95%) of mutant alleles.22, 26, 27, 28, 29 In Asian and African populations, however, TPMT*3C is the most frequent mutant allele.30, 31, 32

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  • Figure 3. 

    TPMT genotypes. The black and white boxes represent translated and untranslated exons, respectively. The gray boxes represent translated exons on mutant alleles containing an SNP, which is captioned below the relevant exon.

There is a strong correlation between TPMT genotype and phenotype.22, 33 The resulting frequency distribution of TPMT activity in white populations is trimodal. Approximately 89% of the population are homozygous for the wild-type allele (homozygous TPMTH) and consequently have high enzyme activity (13.50 ± 1.86 U/mL RBC), 11% inherit 1 wild-type allele and 1 mutant allele and have intermediate levels of enzyme activity (7.20 ± 1.08 U/mL RBC), whereas 1 in 300 subjects has 2 mutant alleles and low or no detectable enzyme activity at all.34, 35 TPMT activity is high in children compared with adults; among adults it is higher in men than in women and higher in smokers than in non-smokers.22, 36, 37 Also, TPMT activity significantly increases during thiopurine treatment as a result of enzyme induction.20

Inosine triphosphate pyrophosphatase 

ITPA catalyzes the pyrophosphohydrolysis of ITP to inosine monophosphate, and deficiency leads to abnormal accumulation of ITP (Figure 2), which is not known to be associated with any defined pathology.38 In ITPA-deficient patients treated with AZA or 6-MP, accumulation of 6-thio-ITP is suggested.39 The gene encoding ITPA is located on chromosome 20, and 5 SNPs have been identified so far, of which 3 (G138A, G561A, G708A) are silent, and 2 (C94A, IVS2 + A21C) are associated with decreased ITPA activity.40 C94A is the most frequently occurring SNP. Homozygotes for the C94A missense mutation have no enzyme activity at all, and IVS2 + A21C homozygotes have approximately 60% of normal ITPA activity. It is estimated that approximately 6% of the population are C94A heterozygotes, and they possess 22.5% of normal enzyme activity.40 In one series of IBD patients, the C94A SNP was significantly associated with influenza-like symptoms, rash, and pancreatitis but not with leukopenia.39 However, there is currently insufficient evidence to recommend ITPA testing in clinical practice.

Methods available for thiopurine methyltransferase/inosine triphosphate pyrophosphatase assessment 

Reliable polymerase chain reaction–based methods have been developed for detecting the major inactivating mutations at the human TPMT locus.27, 41, 42 In brief, leukocyte DNA is amplified and digested by specific restriction enzymes. The resulting DNA fragments are analyzed by gel electrophoresis, and the identified SNPs yield a specific TPMT genotype. Alternatively, TPMT phenotype can be determined by measuring TPMT activity on the basis of the in vitro conversion of 6-MP to 6-methyl-MP.37 The identification of SNPs in the ITPA gene is done in a similar way as TPMT genotyping by using a different set of primers and restriction enzymes.40 ITPA activity can be determined by measuring the in vitro conversion of ITP to inosine monophosphate by a chromatographic method.43 Phenotyping might be more relevant in some cases, because there is a large variation in TPMT activity among individuals with the same genotype. Moreover, as mentioned above, phenotype can alter as a result of thiopurine therapy and the use of concomitant medication, whereas genotype cannot. On the other hand, phenotyping might occasionally lead to misclassification of a patient’s TPMT status, for example, after a blood transfusion. In the United States, TPMT genotyping is available through Prometheus Laboratories (San Diego, CA), and TPMT enzyme activity testing is available through Prometheus Laboratories and Mayo Medical Laboratories (Rochester, MN). Serum level determinations of 6-TGN and 6-MMP are also offered by Prometheus Laboratories.

Thiopurine methyltransferase pharmacogenomics in inflammatory bowel disease 

Patients with 1 or 2 mutant TPMT alleles have lower than normal TPMT activity, leading to elevated 6-TGN concentrations during thiopurine therapy. In IBD patients, this is significantly associated with an increased risk for the development of bone marrow suppression, but only during the early weeks after initiating thiopurine therapy.34 Among patients already established on chronic AZA/6-MP therapy, TMPT genotype does not predict the occurrence of bone marrow suppression. Conversely, some wild-type patients with very high TPMT activity, so-called ultra-methylators, develop suboptimal 6-TGN concentrations, which have been associated with treatment failure.44 In a retrospective study with 106 IBD patients, TPMT genotype predicted phenotype, which correlated to drug efficacy and toxicity; intermediate TPMT activity was associated with an increased risk of AZA toxicity (odds ratio, 5.4), whereas high activity (>14 U/mL RBC) predicted treatment failure (odds ratio, 0.21).28 In a prospective study with 67 patients with rheumatic disease, 6 patients were heterozygous for mutant TPMT alleles, of which 5 discontinued therapy within the first month of AZA treatment because of reduced total leukocyte counts.45 In another study with 113 IBD patients, it was demonstrated that during the initial 4 months, lower TPMT activities correlated with low neutrophil counts.46 In several other studies, no or very poor correlation was found between TPMT status and thiopurine toxicity. In a study with 41 leukopenic patients with Crohn’s disease treated with AZA, only 27% of the patients had 1 or 2 mutant TPMT alleles.47 Myelosuppression was more often caused by other factors, such as viral infections, drugs interfering with AZA metabolism (eg, allopurinol and mesalamine) or directly causing bone marrow suppression (eg, trimethoprim-sulfamethoxazole, captopril, and metronidazole). In the majority of cases, however, no obvious cause of the myelosuppression was apparent, although the lag time to the first appearance of myelosuppression was longer in patients with wild-type alleles. Similar results were found in another study with 56 IBD patients; a slight trend for more frequent TPMT mutations in patients with adverse reactions on AZA or 6-MP was reported, although not statistically significant.48

Clinical utility of thiopurine methyltransferase testing 

At present, the clinical value of TPMT genotyping or phenotyping lies in the prediction of early drug toxicity in patients who have not been previously exposed to AZA or 6-MP. Thus, patients with mutant alleles or low enzyme activity are predisposed to develop leukopenia during the initial 6 weeks after starting AZA/6-MP therapy. Currently, there is insufficient evidence to recommend TPMT testing in patients already established on AZA or 6-MP. Moreover, there is no well-established rationale to use TMPT as a guide to adjust or optimize AZA/6-MP dose to enhance therapeutic efficacy. How should patients with low TMPT activity be managed when initiating AZA/6-MP therapy? In one study, patients with low TPMT activity could be safely managed on lower (<2.0 mg/kg) AZA doses; patients with normal TPMT status received AZA in an initial dose of 2–2.5 mg·kg−1·day−1 compared with 1–1.5 mg·kg−1·day−1 for patients with intermediate enzyme activity, and neither developed acute leukopenia.49 In general, a 50% starting dose, that is, 1–1.5 mg·kg−1·day−1 AZA or 0.75 mg·kg−1·day−1 6-MP, is advocated in patients with heterozygous TPMT alleles (intermediate TPMT activity).50 Some recommend an initial dose reduction to 33% of the standard dose in heterozygotes.51 It is generally recommended that homozygous mutants should not receive thiopurines at all for their IBD. Recently, however, 3 case-reports demonstrated that TPMT deficiency does not preclude thiopurine therapy and hence offers a further option for homozygous mutants; these patients were safely treated with 0.16–0.29 mg/kg AZA daily, which is approximately 10% of standard dose.52

Although TPMT testing is recommended before initiating AZA or 6-MP therapy for IBD to decrease the risk of leukopenia, many centers do not have access to these tests. In the absence of TPMT determination, many clinicians have adopted the approach of rigorous blood count monitoring during the initial weeks of AZA or 6-MP therapy. With this approach, the development of leukopenia could be detected before clinical consequences if the blood count is checked twice weekly for the first 2 weeks and then every week until 6 or 8 weeks of therapy have been completed. However, this approach is empiric, and little published evidence supports its safety.

Mesalamine Therapy 

Mesalamine is widely used in both Crohn’s disease and ulcerative colitis. The enzymes arylamine N-acetyltransferases (NAT) metabolize a range of hydrazines and arylamines, including mesalamine.53 There are 2 NAT isozymes in human beings, NAT1 and NAT2, both of which are now known to be polymorphic.54 Both NAT1 and NAT2 activities have been described in intestine, and these enzymes catalyze the N-acetylation of mesalamine.55 NAT genes are encoded on chromosome 8p22. Polymorphism in NAT activity is primarily due to SNPs in the coding region of NAT genes. NAT polymorphisms have been associated with increased risk for bladder cancer and colorectal cancer, although the latter is still controversial.56 It has been suggested that NAT polymorphisms are associated with different responses to mesalamine therapy, but a study that examined NAT1 and NAT2 polymorphisms in ulcerative colitis patients found no association with therapeutic responses to mesalamine.55

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A Promise of Pharmacogenomics: Individualized Drug Therapy for Inflammatory Bowel Disease 

Up to now, relatively few examples of pharmacogenetics have had meaningful clinical relevance, and if it were not for the importance of thiopurine medications and TPMT polymorphisms in Crohn’s disease and ulcerative colitis, pharmacogenetics would not have any established role in IBD therapeutics. Pharmacogenomics is expected to become an essential part of the patient-physician encounter in the future. Our patients will provide a sample of blood from which DNA will be extracted and analyzed for a battery of polymorphisms in numerous genes. On the basis of this “genetic profile,” it is likely that we will choose the optimum medical therapies for that patient, choices that will maximize the likelihood of beneficial outcomes while minimizing the risk of adversity.

In reality, the majority of currently established genetic polymorphisms that influence drug therapy are monogenic traits, such as those found in TPMT. However, it is certain that polymorphisms in genes whose products affect drug absorption, distribution, metabolism, elimination, targets, and disease modifiers combine in complex ways to influence therapeutic responsiveness. To understand in the fullest possible sense the impact of inheritance on drug responses, we need to study the interacting effects of these multiple genetic polymorphisms or haplotypes.56 The translation of this knowledge into clinically meaningful data will require very careful genotype-phenotype studies.

Additional hurdles that need to be overcome if pharmacogenomic medicine is to become a reality include greater willingness on the part of the pharmaceutical industry and regulatory agencies to incorporate pharmacogenomic factors into the drug development process. Currently, in an effort to avoid market segmentation, most pharmaceutical companies search for blockbuster drugs that are influenced very little by genomics.57 The massive costs of drug development (average $800 million per drug approval) combined with very low success rates (only 1 in 5 compounds entering clinical development succeed in gaining approval) weigh heavily against the commercial viability of multiple “niche” drugs that are only successful in individuals possessing a certain genetic makeup. To this end, physicians must be educated in the appropriate use of pharmacogenomic information, and patients must also be educated and be prepared to allow use of genetic information for the benefit of their health. Critical in this regard is the protection of patients so that genetics are used solely for their benefit and not for discrimination.

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PII: S1542-3565(05)00995-X

doi:10.1016/j.cgh.2005.10.003

Clinical Gastroenterology and Hepatology
Volume 4, Issue 1 , Pages 21-28, January 2006