GENDIA

Pharmacogenetics

A: Choosing the Right Medication: Drug Specificity

B: Choosing the Right Dose: Drug Efficacity and Toxicity

1. Phase I Enzymes

2. Phase II Enzymes

3. Genetic Variation in Activity of Phase I and Phase II Enzymes

4. Examples

GENDIA

GENDIA (www.GENDIA.net), a global network of genetic diagnostic laboratories, offers the largest panel of genetic diagnostic tests worldwide. More than 800 genetic tests are performed either in-house or in one of the GENDIA laboratories. By providing straightforward access to this large panel of tests, GENDIA aims to assist geneticists, medical doctors, health professionals, hospitals, and clinics to effectively manage the clinical needs of their patients.

GENDIA has a general website with information on the network and his tests (www.GENDIA.net), and also special websites for specific tests such as paternity tests (www.paternity.be) or pharmacogenetic tests (www.PHARMACO-GENDIA.net).



Pharmacogenetics

People vary in their response to the same medicine. Consequently very few medicines are effective for everyone, and it has been estimated that less than 20 % of the medication prescribed is effective.
On the other hand, many drugs may cause adverse drug reactions (ADR) leading in some cases to death. In the US alone more than 100.000 patients dye because of ADR each year, thereby making ADR to the fourth leading cause of death, representing 5 % of all hospital admissions.
Some of the variation between individuals in response to drugs is due to differences in their genetic make-up (genotype). Pharmacogenetics is the study of the genetic variation that affects response to medicines both in terms of response (efficacy) and side effect (ADR). The term "Pharmacogenomics" is not distinctly differentiated from pharmacogenetics, but implies the examination of a very large number of genes or even whole genomes to identify genetic differences in the response to medication. Usually pharmacogenomic studies are performed in clinical trials, whereas pharmacogenetic tests are performed in individual treatment of patients.
Using genetic information to predict response to medicines is an important step in the development of ‘personalised’ medicine, or ‘the right medicine, for the right patient, at the right dose’.



A: Choosing the Right Medication: Drug Specificity

Some diseases, notably cancers, specifically develop in cells with an altered genetic constitution (mutation). Specific tests to detect these mutations can confirm the presence or genetic origin of the tumor (diagnostic test), but also guide treatment and detect residual cancer cells during or after treatment (follow up test).
Over the last decade major advances have been made in the elucidation of the genetic mechanisms involved in the development of cancer :deregulated protein tyrosine kinase activation has been found to cause many types of leukemias, lymphomas and solid tumors.
Over 40 chromosomal rearrangements (including different chromosome translocations, deletions, duplications, or amplifications) have been identified that lead to activation of more than 10 different tyrosine kinases, resulting in various malignancies. These tyrosine kinases are part of signal transduction pathways regulating cell division. these tyrosine kinases

Pharmacogenetic tests determining the presence of a mutated and activated tyrosine kinase are therefore essential in the treatment as only these patients will respond to the treatment with the TKI.
The most prominent examples of such pharmacogenetic tests are given below.

1. BCR-ABL and Gleevec
c-Abl is a nonreceptor tyrosine kinase that contributes to several leukemogenic fusion proteins, including BCR-ABL. The BCR-ABL fusion gene is associated with leukemia. It is generated by the Philadelphia chromosome translocation between chromosomes 9 and 22, creating a chimeric oncogene in which the BCR and c-ABL genes are fused. The product of this oncogene, BCR-ABL, has elevated ABL tyrosine kinase activity and transforms hematopoietic cells to cancer cells. It is found in more than 90% of patients with Philadelphia positive (Ph+) chronic myeloid leukemia (CML) and in 20-30% of those with Ph+ acute lymphoblastic leukemia (ALL).
The first example of targeted therapy with a specific tyrosine kinase inhibitor (TKI) is imatinib-mesylate (Gleevec). Gleevec is a small molecule drug that inhibits the Abelson (ABL), ARG (ABL-related gene), stem cell factor receptor (KIT), and platelet-derived growth factor receptor A and B (PDGFRA and PDGFRB) tyrosine kinases. The excellent clinical results obtained with Gleevec in CML have completely changed the therapeutic approach to this disease, and Gleevec is now the gold standard for treatment of newly diagnosed CML,and is being evaluated in a series of other tumors.
Mutations in ABL that arise as secondary mutations in previously sensitive leukemias harboring an activating mutation, are associated with the emergence of acquired resistance to Gleevec. Mutations in ABL exons 4-10, mainly the T315I mutation, are present in a subset of cases with acquired resistance to Gleevec. To circumvent acquired resistance to secondary ABL mutations irreversible inhibitors of the BCR-ABL fusion gene are curently being developed.

2. HER2/NEU and Herceptin
The human epidermal growth factor receptor-2 gene ERBB2 is a proto-oncogene encoding a 185-kD transmembrane glycoprotein HER-2 (or HER-2/NEU), which is a member of the EGFR family of growth factor receptor tyrosine kinases regulating cell growth, cell survival, and differentiation.
HER-2 is expressed in a relatively wide range of epithelial tissues in normal conditions, whereas overexpression of HER-2 has been found in many human cancers. Sufficiently high levels of overexpression result in spontaneous receptor dimerization and activation, in the absence of any ligand.
HER-2 overexpression occurs in 20-30% of invasive breast carcinomas, in 40-50% of ductal carcinoma in situ and in almost 100% of Paget’ s disease of the nipple. HER-2 overexpression is associated with a poor prognosis on one hand, and response to specific forms of treatment on the other hand. The latter includes hormonal treatment, chemotherapy, and treatment with Herceptin (trastuzumab), a monoclonal antibody targeted against HER-2. Accurate determination of HER-2 overexpression status is required for appropriate use of this targeted therapy and is currently analysed by immunohistochemistry (IHC) on tissue sections and/or in situ hybridisation (ISH or FISH) on interphase nuclei of tumor cells.

3. EGFR and Iressa
Epidermal growth factor receptor (EGFR) signaling is triggered by the binding of growth factors, such as epidermal growth factor (EGF), resulting in the dimerization of EGFR molecules or heterodimerization with other closely related receptors, such as HER2/neu. Autophosphorylation and transphosphorylation of the receptors through their tyrosine kinase domains leads to the recruitment of downstream effectors and the activation of proliferative and cell-survival signals.
A cluster of somatic mutations in the kinase domain (exons 18-21) of the EGFR gene are observed in about 10 % of patients with non-small cell lung cancer (NSCLC). The mutations lead to activation of EGFR signaling and lung tumor.The patients with EGFR mutations demonstrate remarkable responses to TKIs of EGFR such as gefitinib (Iressa) and erlotinib (Tarceva).
Mutations in EGFR that arise as secondary mutations in previously sensitive NSCLCs harboring an activating EGFR mutation, are associated with the emergence of acquired resistance to gefitinib (Iressa) and erlotinib (Tarceva). The T790M mutation in EGFR is present in a subset of cases with acquired resistance. To circumvent acquired resistance to secondary EGFR mutations irreversible inhibitors of the EGF receptor are being developed.

4. KIT and Gleevec
The stem cell factor receptor (KIT) tyrosine kinase signalling pathway is essential for development of erythrocytes, melanocytes, germ cells, mast cells and interstitial cells of Cajal (ICCs). Activating KIT mutations result in development of tumors from mast cells (mastocytosis, mast cell leukaemia), and ICCs (gastrointestinal stromal tumors or GISTs). Mastocytosis and mast cell leukaemia are mainly associated with KIT mutations in exon 17 at amino acid position 816. Whereas a subset of GISTs has mutations in PDGF receptor alpha (PDGFRA), approximately 80% of GISTs harbor activating mutations in KIT.
Tumors with activating KIT mutations are a good target for the TKI imatinib-mesylate (Gleevec).
Mutations in KIT that arise as secondary mutations in previously sensitive tumors harboring an activating KIT mutation, are associated with the emergence of acquired resistance to Gleevec. The T670I mutation in KIT is present in a subset of cases with acquired resistance. To circumvent acquired resistance to secondary KIT mutations irreversible inhibitors of KIT are being developed.

5. PDGFRA and Gleevec

Growth, survival and differentiation of many cells are regulated by the interaction between growth factors and their receptors such as the platelet-derived growth factor receptor alpha (PDGFRA). Activating mutations in exons 12 and 18 of the PDGFRA gene cause a subset of gastrointestinal stromal tumors (GISTs).
Also eosinophilia-associated chronic myeloproliferative disorders (hypereosinophilic syndrome, chronic eosinophilic leukemia) are characterized by activating mutations of the PDGFRA gene. The PDGFRA activation in chronic myeloproliferative disorders usually results from an interstitial deletion involving chromosome 4q12 and the CHIC2 locus leading to fusion of the FIP1L1 gene to the PDGFRA gene. This PDGFRA fusion gene is a constitutionally activated tyrosine kinase that transforms hematopoietic cells resulting in eosinophilia-associated chronic myeloproliferative disorders. The FIP1L1-PDGFRA fusion is also present in systemic mast cell disease with eosinophilia.
Gleevec not only inhibits ABL, KIT, but also PDGFRA and PDGFRB tyrosine kinases, and is therefore used in GISTs and eosinophilia-associated chronic myeloproliferative disorders

6. PDGFB and Gleevec
A small proportion of patients with chronic myeloproliferative diseases and eosinophilia have constitutive activation of the gene for platelet-derived growth factor receptor beta (PDGFRB), which encodes a receptor tyrosine kinase. The gene is located on chromosome 5q33, and the activation is usually caused by a t(5;12)(q33;p13) translocation associated with a fusion between the PDGFRB gene and the ETV6 (TEL) gene on chromosome 12p13. Also other fusion genes of the PDGFRB gene with other genes (p53, NIN, HIP1, H4/D10S170 , myomegalin, CEV14, etc) can lead to activation of PDGFRB and chronic myeloproliferative diseases with eosinophilia.
Gleevec is used in chronic myeloproliferative diseases as it inhibits both PDGFRB and PDGFRA kinases in these tumors.

7. FLT3
Fms-like tyrosine kinase 3 (FLT3) encodes a receptor tyrosine kinase for which activating mutations have been identified in a proportion of acute myelogenous leukemia (AML) patients. FLT3 is the most commonly mutated gene in AML, and is constitutively activated by acquired mutation in approximately 30%–35% of AML. In 20%–25% of cases of AML, there are internal tandem duplications (ITD) of a small number of amino acid residues in the juxtamembrane domain of FLT3, and in 10 % there are activating mutations in other FLT3 domains (mainly in exon 14). These mutations activate the FLT3 kinase activity constitutively, and result in increased cellular proliferation and viability. AML patients with FLT3 mutations have a poor prognosis. This progress had led to the development of small molecules that specifically inhibit the abnormally activated FLT3 kinase.



B: Choosing the Right Dose: Drug Efficacity and Toxicity

More than hundred different genes can modify the function of drugs, whereas more than 30 genes are involved in the metabolism of drugs.
In order to facilitate renal or hepatobilliary excretion of a drug different liver enzymes make the lipophilic drug hydrophilic, and thus easier to excrete. The enzymes involved in drug catabolism and elimination are:

1: Phase I Enzymes

The lipophilic drugs are either oxidatively, reductively or hydrolytically modified. Different enzyme systems perform these reactions, including the cytochrome p450 system, alcohol dehydrogenases, epoxide hydroxylases, and flavin  monooxygenases. Phase 1 enzymes include:

• Cytochrome p450 (CYP)
• Alcohol dehydrogenases (ADH)
• Epoxide hydroxylases (EPXX)
• Flavin monooxygenases (FMO)

2: Phase II Enzymes

Phase II enzymes include:

• Catechol-o–methyltransferase (COMT)
• Glutathione s-transferases (GST)
• Sulfonyl transferases (SULT)
• N-acetyl transferase type 2 (NAT2)
• Thiopurine methyltransferases (TPMT)
• Uridine diphosphate-glucoronyl transferases (UGT)

3: Genetic Variation in Activity of Phase I and Phase II Enzymes

Variation in the enzyme activity of phase 1 or 2 enzymes caused by mutations in the respective genes leads to poor, intermediate, fast or ultrafast breakdown and excretion of many drugs. In poor metabolisers the drug is eliminated from the body at a slower rate due to inactivating mutation(s) in the corresponding gene : the drug accumulates in the body, which can cause adverse drug reactions (toxicity). To a lesser content intermediate metabolisers are also prone to toxicity (ADR). Ultrafast metabolisers eliminate the drug from the body at an increased rate by the increased amounts of enzyme caused by activating mutations: consequently, the efficacity of the medication in ultrafast metabolisers is reduced, and they require significantly increased concentrations of the medicine to achieve the desired pharmacological effect.

4: Examples

Cytochrome P450
Cytochrome P-450 comprises a large superfamily of many enzymes that play an important role in the metabolism of endogenous substrates, environmental toxins, carcinogenic substances and a variety of pharmaceuticals. These enzymes hydrophilise lipophilic molecules in so-called Phase I reactions. The cytochrome P-450 superfamily represents the most important enzymes in drug metabolism.
The 3 cytochrome enzymes (CYP) mainly involved in variation in response to medication are CYP2D6, CYP2C19, and CYP2C9. CYP2D6 is of particular interest because it is involved in the metabolism of many common drugs, and variation in enzyme activity is great due to many mutations leading to a high number of poor, intermediate or ultrafast metabolisers, the percentage of which is different in different ethnic groups.

CYP2D6
CYP2D6 is an enzyme found in the human liver, which is involved in the metabolism of approximately 25% of all medicines that are currently prescribed, including some beta-blockers, tricyclic anti-depressant and antipsychotic medicines. It is difficult to predict how a particular person will respond to a given dose of these medicines, in part due to the amount of variation in the CYP2D6 gene (over 70 alleles have been identified).
Approximately 10% of the Caucasian population has a genetic variant that results in reduced activity of the CYP2D6 enzyme: they are ‘poor metabolisers’. 7% have multiple copies of the CYP2D6 gene, arranged in tandem, so that these individuals metabolise the relevant medicines very quickly: they are ultrafast metabolisers. Thirty-five percent are intermediate metabolisers, and at risk of ADRs when taking multiple drugs.
Drugs that CYP2D6 metabolizes include Prozac, Zoloft, Haldol, Metoprolol, Tagamet, Tamoxifen, Paxil, Effexor, Hydrocodone, Amitriptyline, Claritin, Cyclobenzaprine, Allegra, Dytuss, Tusstat, Rythmol. CYP2D6 is also responsible for activating the pro-drug codeine into its active form and the drug is therefore inactive in CYP2D6 poor metabolizers.

CYP2C9
Approximately 10% of the Caucasian population are poor CYP2C9 metabolisers. Drugs metabolized by CYP2C9 include Coumadin (Warfarin), Viagra, Amaryl, Isoniazid, Sulfa, Ibuprofen, Amitriptyline, Dilantin, Hyzaar, Tetrahydrocannabinol, Naproxen. Warfarin is one of the most difficult to manage drugs : one in four or five people taking warfarin will have an adverse drug reaction, many of them serious requiring transfusions and hospitalisation.

CYP2C19
Approximately 3% of the Caucasian population are poor CYP2C19 metabolisers, due to mutations (mainly alleles 2 and 3), but up to 20% of Asians is a poor metaboliser. CYP2C19 is associated with the metabolism of Carisoprodol, Diazepam, Dilantin, Premarin, and Prevacid.

TPMT and Thiopurine toxicity
Thiopurines-TP (azathioprine-AZA, 6-mercaptopurine-6MP, and thioguanine-6TG) have a well-established role as immunosuppressive agents in a variety of chronic inflammatory conditions, haematological neoplasia and in transplant rejection. One important route for the metabolism of these agents is methylation, mediated by thiopurine methyltransferase (TPMT). However, there is a wide variation in the concentration of TPMT due to inactivating mutations in the TPMT gene (alleles 1, 2, 3a, 3c) leading to TMPT deficiency resulting in TP toxicity. TP toxicity leads to cessation of therapy in 9-25% of patients, with the most serious adverse drug reaction being myelosuppression that can be fatal. Ten percent of the population are heterozygous for inactivating TPMT mutations and have approximately 50% of normal activity, whilst 1 in 300 are completely deficient and at prone for serious TP toxicity.

DPD and 5-Fluoro Uracil toxicity
5-Fluorouracil (5-FU) remains one of the most widely used chemotherapeutic agents for the systemic treatment of cancers of the gastrointestinal tract, breast, and head and neck.
Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme in the pathway of uracil and fluorouracil catabolism. Deficiency of DPD caused by homozygous mutations in the DPD gene can lead to an autosomal recessive monogenic disorder dihydropyrimidinuria, which is characterised by various anomalies. DPD deficiency can also cause adverse drug reactions in patients treated with fluorouracil. Fluorouracil toxicity is characterised by leukopenia, thrombocytopenia, hair loss, diarrhea, fever, marked weight loss, and neurologic symptoms.
The most frequent DPD mutation leading to fluorouracil toxicity is an intron 14 splice site mutation IVS14+1g-a (allele 2a), but also other DPD mutations (alleles 3, 7, 8, 9, 10) can lead to fluorouracil toxicity. The frequency of heterozygotes / homozygotes has been estimated to be as high as 3% in the normal population, necessitating the determination of the presence of DPD mutations before prescribing fluorouracil.

UGT1A1 and Irinotecan toxicity
Irinotecan has exhibited clinical activities against a broad spectrum of carcinomas by inhibiting DNA topoisomerase I. However, severe and unpredictable dosing-limiting toxicities (mainly myelosuppression with leukopenia and severe diarrhea) hinder its clinical use. Glucuronidation is the main metabolic pathway of irinotecan, and mutations in UDP-glucuronosyltransferase (UGT1A1) affects this glucuronidation.
UGT1A1 is also responsible for conjugating bilirubin, and >30 UGT1A1 mutations have been known to decrease the enzyme activity, leading to constitutional unconjugated jaundice, Crigler-Najjar or Gilbert’s syndrome One is a 2-bp insertion (TA) in the TATA box in the UGT1A1 promoter (normal: (TA)6TAA), resulting in the sequence (TA)7TAA (UGT1A1*28
allele). Patients either heterozygous or homozygous for UGT1A1*28 also have a significant risk for severe toxicity by irinotecan.

N-Acetyltransferase (NAT-2) and Isoniazid toxicity
The variability in the metabolism of isoniazid, a tuberuclostatic, was already described over 40 years ago. N-Acetyltransferase-2 (NAT2) conjugates an acetyl group to various pharmaceuticals in a phase II reaction. The phenotype (slow or fast acetylator) is determined by mutations in NAT2 gene. Inactivating mutations lead to the slow acetylator phenotype, whereas activating mutations cause the ultrafast acetylator phenotype. Toxic effects in slow acetylators include peripheral neuropathy in isoniazid therapy, hypersensitivity to sulphonamides.