Pharmacogenomics of Thiopurine S-Methyltransferase: Clinical Applicability of Genetic Variants

Sequence variability among individual human genomes has become a key resource for modern medicine in the search for genetic markers affecting disease susceptibility, disease manifestation and response to treatment. Genetic markers have been used for years as an indispensable tool for the diagnosis and follow-up of a number of diseases. They are also used as prognostic and predictive markers. Their application as pharmacogenetic markers is especially important.


Introduction
Sequence variability among individual human genomes has become a key resource for modern medicine in the search for genetic markers affecting disease susceptibility, disease manifestation and response to treatment. Genetic markers have been used for years as an indispensable tool for the diagnosis and follow-up of a number of diseases. They are also used as prognostic and predictive markers. Their application as pharmacogenetic markers is especially important.
Pharmacogenetics is referred to as the study of the variations in a DNA sequence as related to drug efficacy and toxicity. It began with studying differences among individuals. However, as it developed, it became clear that genetic differences between populations should also be taken into account. Following great progress in understanding the molecular basis of health and disease, pharmacogenetics has evolved into pharmacogenomics, a much newer discipline which can be described as the whole-genome application of pharmacogenetics. More precisely, pharmacogenomics is the study of variations of DNA and RNA characteristics as related to drug response.
Genetic variability can affect various aspects of drug therapy: disposition of the drug (pharmacokinetics), efficacy of the drug (pharmacodynamics) and adverse drug reactions (ADRs). Genetic factors are estimated to account for 15-30% of inter-individual differences in drug metabolism and response.
The ultimate goal of pharmacogenetic testing is to aid physicians in the prescription of the appropriate medication at the correct dose prior to the initiation of the therapy. This would lead to minimizing adverse events and toxicity and maximizing efficacy by excluding those who are unlikely to benefit (non-responders) or who may be harmed (adverse responders).
Here, we provide an overview of the genetic variants of thiopurine S-methyltransferase (TPMT) gene that influence inter-individual dosing of thiopurine drugs, to highlight a tangible benefit of translating genomic knowledge into clinical practice.

Pharmacogenetics
The role of genetics in response to drugs was first predicted by Sir Archibald Garrod in the early 1930s (Garrod 1931). Pharmacogenetics, as it is known today, originated as a new scientific discipline in the late 1950s by the merging of two older ones: pharmacology and genetics. Pharmacogenetics examines the role of inherited individual differences in response to drugs. It is a branch of science that explains variability in response to drugs and genetic basis as the cause of this variability. Initially, the focus was on individual human differences, but over time the area of interest of this science extended to genetic differences between populations. Many professionals from this field deal exclusively with humans, but this science has applied its principles to all living organisms that are able to respond to a drug or other chemicals.
Research in the field of pharmacogenetics is being developed into two main directions: first, the identification of specific genes and their products that are associated with various diseases and that could represent targets for new therapeutics; and second, the identification of genes and allelic variants of genes that might influence the response to already existing drugs (Wolf et al., 2000).

Human genetic variations
In 2003, after more than a decade, the Human Genome Project was completed. It was clear that the information obtained from the Project had the potential to forever transform healthcare and that genome-based medicine, frequently called personalized medicine, is the future of healthcare. Ever since, the main goal was achieving faster and cheaper sequencing of the whole human genome. The key advantage is the possibility to identify very rare or new, "private" genetic variants. Among a patient's personal genome data, the most important data is about inter-individual genetic differences.
More than ninety-nine percent of the DNA sequence is identical among individuals. The remaining DNA is responsible for genetic diversity (Kidd et al., 2004). Polymorphisms are common genetic variations in the human genome. They represent sequence variations that occur with a frequency >1% in the general population. The most studied polymorphisms are SNPs (single nucleotide polymorphisms). They are distributed over the whole genome. The number of SNPs is estimated to range from 0.3 to 1 SNP per 100 base pairs (bp). Besides SNPs, there are other important classes of polymorphisms, such as VNTRs (variable number of tandem repeats, polymorphic sequence containing 20-50 copies of 6-100 bp repeats), STRs (short tandem repeats, a subclass of VNTR in which repeat unit consists of only 2-7 nucleotides) and CNPs (copy number polymorphisms, variation in the number of copies (CNV) of a DNA sequence in the > 1 kb size range, which are common and widely distributed in the human genome).
The totality of these genetic variations found in an individual, a variome, should carry an answer about inborn diseases, compliance with drug therapies and other processes -all specific to that individual. However, in order to be fully understood and finally translated into the everyday clinical practice, variome data needs to be adequately interpreted. The most important approach of interpretation is to correlate genetic variation with clinical data. www.intechopen.com

Thiopurine S-methyltransferase (TPMT)
One of the best examples of the application of pharmacogenetics in clinical practice is the discovery that different individual responses to purine antagonists as therapeutics are caused by individual variations in thiopurine S-methyltransferase (TPMT) enzyme activity (Weinshilboum et al., 1980). Patients who have reduced TPMT enzyme activity can develop toxic effects after the application of standard doses of these drugs (Weinshilboum et al., 1980). On the other hand, there are patients in whom the activity of this enzyme is extremely high and they do not respond to standard doses of drugs (Weinshilboum et al., 1980). The characterization of mutations within the TPMT gene enabled the explanation of these interindividual differences in enzyme activity. Consequently, the goal of pharmacogenetics, the individualization of therapy, becomes a step closer. The characterization of mutations in TPMT gene is also a model system that illustrates how knowledge in the field of pharmacogenetics is successfully used in clinical practice.

Thiopurine drugs
Antimetabolites or structural analogs are compounds analogous to natural cell compounds such as folic acid, purines and pyrimidines. The mechanism of their action is based on the fact that they replace natural metabolites in the biochemical processes of cells. Antimetabolites have the greatest impact on the biochemical pathways that are involved in the metabolism of nucleotides and nucleic acids. Purine antagonists as thiopurine drugs have been widely used in medical practice for over 50 years. The structural analogues of purines are 6-mercaptopurine (6-MP), 6-thioguanine (6-TG) and azathioprine (AZA) (Coulthrad et al., 2005).
Thiopurine drugs are indicated for the treatment of various diseases. 6-MP and 6-TG are mainly used in the treatment of hematologic malignancies, such as acute leukemia and lymphoma in children and adults. In childhood acute lymphoblastic leukemia (ALL), 6-TG is primarily used in the induction phase, and 6-MP in the consolidation phase of ALL therapy. The immunosuppressive drug AZA is the drug of choice in the treatment of inflammatory bowel diseases, rheumatoid arthritis, autoimmune hemolytic anemia, systemic lupus erythematosus, as well as in transplantation medicine.
Thiopurine drugs were synthesized in 1951 (Elion 1986). It was shown that newly synthesized drugs inhibit the use of natural purines and act upon the reduction of some tumors in rats (Elion 1967). Soon the activity of these drugs was confirmed in childhood ALL. At that time, the prognosis of this disease was extremely poor. The discovery that 6-MP can lead to the complete remission of childhood ALL, resulted in the approval of the use of these drugs in medical practice by the U.S. Food and Drug Administration in 1953 (Burchenal et al., 1953). AZA was introduced in therapy later, in 1963, after its successful use in kidney transplantation (Murray et al., 1963).
The basic principle of how thiopurine drugs act is the inhibition of many pathways in nucleic acid biosynthesis. Consequently they prevent proliferation of cells involved in determination and amplification of the immune response, causing suppression of the immune system. Thiopurine drugs are also used in cancer treatment (Katzung 2004). An important biochemical feature of cancer cells is excessive synthesis of nucleic acids. Thiopurine drugs are able to stop this synthesis, and thus prevent the division of neoplastic cells (Katzung 2004). Anticancer and www.intechopen.com immunosuppressive activity is accomplished through the incorporation of thioguanine nucleotides, metabolic products of thiopurine drugs, into DNA.
Thiopurine drugs are inactive in their original form. They are precursors of the active drug, so-called pro-drugs, and they have to be metabolized first in order to exhibit cytotoxic, therapeutic effect (Lennard 1992). The first step is the non-enzymatic degradation of AZA to 6-MP and imidazole group. 6-MP and 6-TG go through metabolic changes prior to the expression of their cytotoxic effects. After metabolic conversion, 6-MP and 6-TG are incorporated into the DNA and RNA molecules as thioguanine nucleotides (6-TGN) (Bertino 1991
Thiopurine drugs are inactivated in the organism by oxidation (mediated by aldehyde oxidase (AO, EC 1.1.3.13) and xanthine oxidase (XO, EC 1.1. 3.22)) and methylation (mediated by thiopurine S-methyltransferase (TPMT, EC 2.1.1.67)), reactions which are needed to prevent high drug concentrations and adverse drug-related events. XO and AO produce metabolites that have little or no cytotoxic effect. XO activity in hematopoietic tissue is very low, almost insignificant. This is the reason why the main pathway of thiopurine drug inactivation goes through the TPMT enzyme (Remy 1963).
The influence of TPMT enzyme activity on cytotoxicity induced by thiopurine drugs was first documented in 1987 (Van Loon et al., 1987).

TPMT allozymes
Thiopurine S-methyltransferase is a cytosolic monomeric enzyme that catalyzes Smethylation of heterocyclic aromatic sulfhydryl compounds, and consequently, partial inactivation of immunosuppressive thiopurine medications. The molecular weight of TPMT protein is 28.18 kDa and it consists of 245 amino acids. The natural substrate for TPMT is unknown, although this enzyme is expressed in nearly all human tissues (Weinshilboum et al., 1978). Structural and biochemical analyses of TPMT protein revealed the existence of certain protein variants with altered activity. In some individuals, TPMT enzyme activity is significantly decreased or increased compared to the normal TPMT activity level.
One of the first studies of TPMT activity in red blood cells determined the distribution of TPMT activity to be trimodal. Namely, it was found that approximately 90% of individuals express high TPMT activity. These individuals are referred to as high methylators. Intermediate methylators represent approximately 10% of the population. Low or undetectable TPMT activity is reported in 0.3% individuals (Weinshilboum et al., 1980). This study initially identified the hereditary nature of the TPMT deficiency in humans. Trimodal frequency distribution of TPMT activity corresponds to monogenic co-dominant inheritance. Additionally, ultra-high methylators have been observed (Spire-Vayron de la Moureyre et al., 1999; Roberts et al., 2008). It has been shown that these different TPMT allozymes are defined by certain TPMT gene polymorphisms.

Genetic variants in TPMT gene
Human TPMT gene (NG_012137, NM_000367, GeneBank: 7172 or MIM: 187680) was discovered using a ″classical″ molecular biology strategy. Firstly, TPMT enzyme from kidneys was purified and a partial amino acid sequence was obtained (Van Loon et al., 1982). This information served for the successful cloning of human TPMT cDNA (Honchel et al., 1993).
Initially, on chromosome 18q.21.1 the pseudogene for TPMT has been discovered, containing a similar sequence to the TPMT gene (Lee et al., 1995). The human gene for TPMT was cloned and mapped on the short arm of chromosome 6, at the position 6p22.3 (Szumlanski et al., 1996). TPMT gene comprises a region of 34 kb and 10 exons, 8 of which encode TPMT protein. Krynetski

Functional characterization of TPMT allozymes
The functional characterization and expression analysis in human cells . The molecular mechanism that leads to the reduction of TPMT activity was studied in the most common TPMT genetic variants. Expression studies of TPMT*2 and TPMT*3A alleles showed that both alleles are about 100 times less expressed than wild type, TPMT*1 allele . Also, the expression of TPMT*2 and TPMT*3A allelic variants was not in correlation with the activities of TPMT*2 and TPMT*3A proteins. The mechanism of accelerated degradation of TPMT*2 and TPMT*3A proteins is responsible for the reduced level of TPMT proteins and thereby for the reduced catalytic ability of enzymes . More detailed studies have confirmed that in the accelerated degradation of TPMT*3A protein, through a ubiquitin-mediated system, the molecular chaperones from the family of heat shock proteins are involved (Wang et al., 2003).

Population-specific distribution of TPMT variant alleles
Pharmacogenetics is generally focused on inter-individual differences in drug metabolism and on variations in response to drugs. The frequency of pharmacogenetic markers studied so far, is different between certain racial and ethnic groups. Historically, the practical application of pharmacogenetic achievements, i.e., the individualization of therapy, has been based on studies conducted on Caucasians. With time, other ethnic groups have been included in clinical trials, and it became clear that responses to drug therapy may depend on www.intechopen.com ethnic background (Relling et al., 2011a). Thus, if the metabolism of drugs varies among different ethnic groups, then the pharmacogenetic data of one population cannot be extrapolated to another one without prior assessment. Knowledge of the pharmacogenetic differences between populations can be of great importance for the pharmaceutical industry.
The distribution of clinically relevant TPMT alleles is population specific (Spire-

Methodology for TPMT phenotype and genotype testing
The TPMT phenotype and genotype can be defined in several ways. Phenotypic analysis of TPMT enzyme activity could be performed by radiochemical activity assays (McLeod et al., 1995;Weinshilboum et al., 1978), or an assay based on high performance liquid chromatography (HPLC) method (Kroplin et al., 1998). Genotyping is performed using PCR- Methods based on PCR are used to detect the most common TPMT variant alleles that lead to reduced TPMT activity. These analyses are helpful in identifying individuals with a high risk of developing potentially fatal hematologic toxicity caused by thiopurine drugs. Measurement of TPMT enzyme activity was, until recently, very expensive and relatively inaccessible to patients. A concordance of more than 95% exists between actual TPMT enzyme activity and the prediction of its activity based on detection of TPMT variant alleles (McLeod et al., 2000; Schwab et al., 2001). Therefore, molecular genetic analysis represents a quick and efficient method to identify patients at risk for toxicity and adverse effects-free guidance of the therapy.
Commercially available genetic tests change over time. Many of them include only the *2, *3A, *3B and *3C alleles. There is no doubt that sequencing of the TPMT gene remains the most accurate, although expensive method.

Clinical applicability of TPMT genetic variants: Individualization of thiopurine therapy
All patients with decreased TPMT activity are at risk of hematologic toxicity owing to the accumulation of high levels of 6-thioguanine nucleotides (Weinshilboum 2003). Thiopurinewww.intechopen.com induced myelosuppression can result in increased morbidity, hospitalization and/or treatment discontinuation (Leung et al., 2009;Ugajin et al., 2009). Myelosuppression increases an individual's risk of developing an infection and sepsis (Campbell et al., 2001;Posthuma et al., 1995;Connell et al., 1993;Schütz et al., 1993). The incidence of mild leukopenia is approximately 5-25% (Gurwitz et al., 2009). Rare, but severe leukopenia can develop suddenly and unpredictably in approximately 3% of patients (Carter et al., 2004). A 27-year analysis showed that AZA contributed to the incidences of myelosuppression in 5% of patients (Connell et al., 1993). Over an 18-year period, 2% of patients with IBD experienced 6-MP-induced leukopenia that resulted in hospitalization (Present et al., 1989). The incidence of myelosuppression occurred more frequently during the first eight weeks after treatment initiation, and was more likely to occur with a higher drug dose (Present et al., 1989;Lewis et al., 2009).
Consequently, thiopurine drug dose reduction is necessary to avoid toxicity (Weinshilboum et al., 1980). Therefore, it is of great importance to determine TPMT status before initiating thiopurine therapy (

Guidelines for thiopurine dosing based on TPMT genotype
The Clinical Pharmacogenetics Implementation Consortium (CPIC), as a part of the National Institutes of Health's Pharmacogenomics Research Network, developed the first guideline for the dosing of thiopurines based on TPMT genotype (updates at http://www.pharmgkb.org) (Relling et al., 2011b).
Dose adjustments based on TPMT genotype have reduced thiopurine induced adverse effects without compromising the desired antitumor and immunosuppressive therapeutic effects in several clinical settings (Relling et al., 2011a).
Although the information on TPMT genotype is recommended rather than required as part of thiopurine drug treatment, some groups (Relling et al., 2011a;Gurwitz et al., 2009;Relling et al., 2010) advocate testing for TPMT status prior to initiating thiopurine therapy, so that starting dosages can be adjusted accordingly. This is very important, since, if one starts with low doses in all patients in order to avoid severe toxicity, in the minority with a TPMT defect, one risks disease progression during the period of upward dosage titration (Sandborn 2001

From TPMT pharmacogenetics to TPMT pharmacogenomics
It is worth noting that there is no clear boundary between the low and intermediate, or intermediate and high methylators. Even individuals within the same ″methylation″ group show different enzymatic activity, and these differences are genetically determined (Vuchetich et al., 1995). Also, there are many patients with wild type TPMT who develop www.intechopen.com toxicity. All this leads to the conclusion that the association of a particular genetic variation with adverse drug effects could be softened or enhanced by the other genetic variations present in the same individual that influence other processes (drug absorption, transportation, metabolism, TPMT gene transcription and consequently the abundance of TPMT protein). Moreover, the effects and clinical relevance of genetic variants in another gene, encoding an enzyme involved in mercaptopurine metabolism (inosine-triphospatepyrophosphatase, ITPA), on mercaptopurine pharmacogenetics has been demonstrated (Stocco et al., 2010).
Finally, a myriad of other genetic factors, which influence interactions between thiopurines and other drugs, could play an important role in the final TPMT activity phenotype.
Therefore, more comprehensive study of the modifying role of different genetic factors in TPMT pharmacogenetics, will in time lead to our understanding of controversial results published in our era. The new era of pharmacogenomics will bring more consistent and reliable guidelines for thiopurine dosing based on patient's genotype.
In recent years, the term pharmacogenomics is more and more present alongside the term pharmacogenetics. Due to rapid technological development and the great success of the human genome sequencing project, the variations and interactions of multiple genes, rather than variations in individual genes, have been recognized as the cause of diverse responses to drugs (O'Brien et al., 1999;Kennedy et al., 2003). Genotyping methods, the application of microarrays and GWAS analyses (Genome Wide Association Studies) that are used in pharmacogenomics, provide an insight into a number of individual genes at one time, their possible interactions and changes in their expression. The final goal of pharmacogenomics is the individualization of therapy in accordance with a patient's genotype and gene expression profile. Thus, by using the appropriate therapeutics and adequate doses without side effects, the cheapest, fastest and the most efficient treatment for patients would be achieved. Under these conditions, patients would not be faced with complications, time and money for additional drugs and hospital days would not be wasted for treatment of complications, while the primary disease progresses. Unfortunately, widely available pharmacogenomic tests for particular diseases still do not exist. The individualization of therapy in medical practice, if at all implemented, is conducted based on pharmacogenetic achievements, by testing polymorphisms in a single gene.

Conclusions
Although pharmacogenetics is one of the most promising fields of biomedicine, only a few pharmacogenetic markers have been introduced in routine clinical practice. Among them are genetic variants in the TPMT gene which can be used for determination of the cause of unusual therapeutic response in patients treated with thiopurine drugs.
TPMT genotyping is recommended prior to initiating thiopurine therapy by several groups and consortia, so that starting dosages can be adjusted accordingly. Doses customized on the basis of TPMT status reduce the likelihood of acute myelosuppression without compromising disease control.
Nowadays, TPMT genetic testing comprise the analysis of DNA sequence at each of the important single-nucleotide polymorphism in the TPMT gene associated with altered level of enzyme activity.

www.intechopen.com
However, the understanding of a single genetic variation is far more complex when it is put in the context of other genetic variations. In that sense, the future of personalized medicine is heading towards variomics -the study of the overall genetic variations found in an individual.
Although the knowledge of pharmacogenomics is incomplete and still in expansion, evidence presented in this chapter show that up to date knowledge can already be used for more successful, personalized patient treatment. There is no doubt that pharmacogenomics together with gene therapy will change the future of medicine and will steadily pave the path to personalized medicine.

Acknowledgment
This work has been funded by a grant No. III 41004, Ministry of Education and Science, Republic of Serbia.