Pharmacogenetics             

INTRODUCTION

Pharmacogenetics deals with pharmacological responses and their modification by hereditary influences. Variation of drug metabolising enzymes represents variations within the chemical defense systems between individuals. These variations also affect susceptibility to infectious diseases like tuberculosis and malaria and aid the survival of populations exposed to toxins or infectious agents.

Pharmacogenetics will give clinicians the tools to predetermine response to pharmacotherapy by looking for specific polymorphisms in cytochrome P450 and other enzymes involved in drug metabolism. Pharmacogenetics also will have an important role in determining or predicting patient response to environmental toxins.

Genetic differences can result in considerable variation in the rate of metabolising a drug. The metabolism may take longer than expected, increasing the risk of side effects. In case of high metabolic rates, the therapeutic effect may be diminished or absent. Metabolic rates depend on the cytochrome P450 and N-acetyltransferase enzymes, and patients are classified as fast or slow metabolises depending on the activity of the level of these enzymes. The best known of the cytochrome P450 enzymes is CYP2D6, which plays a role in the metabolism of several drugs including P-blockers and antidepressants. Slow N-acetyltransferase forms are found in a majority of the population. These enzymes play a role in the metabolism of various drugs like isoniazid used in the treatment of tuberculosis.

Pharmacogenetic effects can be caused by differences in enzymatic conversion rates and by inter-individual variation in the proteins to which the drugs are targeted (target proteins). Genetic differences in receptors can mean variation in drug efficacy from patient to patient. Examples of this include the variable efficacy of salbutamol in asthma and treatment with anti-malHelvetica drugs in some patients with malaria resulting in severe anemia.

Pharmacogenetics can help address why some individuals respond to drugs and others do not. It can also help physicians understand why some individuals require higher or lower dosing for optimum response to a drug. It could potentially tell physicians who will respond to a drug and who will have toxic side effects. Systemic drug concentration is the end result of drugs ingestion absorption, metabolism, clearance and excretion. Much of pharmacogenetics has focused on the mechanisms that control the systemic drug concentration.

Drug metabolising enzymes known to be genetically variable include esterases, transferases, dehydrogenases, oxido- reductases, and the cytochrome P450 group of enzymes. Many of the well-defined pharmacogenetic variants represent Mendelian (monogenic traits). Therefore, the rate of occurrence of such a variant in a population can be defined in terms of an allele frequency. These frequencies differ between racially or ethnically defined populations accounting for geographical differences in drug safety. In addition, multifactorial variation accounts for innumerable differences between individuals as well as between populations. The Table 1 outlines ethnic variation in some pharmacogenetic disorders.

Table 1: Ethnic variation in some pharmacogenetic disorders

An example of pharmacogenetic variability is seen in a genetic defect, which affects the function of a specific enzyme of cytochrome P450, CYP2D6. The P450 system is important for the metabolism of many endogenous compounds and for the detoxification of exogenous substances. This polymorphism affects the oxidative biotransformation of debrisoquine and over 60 other therapeutic agents. The CYPD gene cluster is located on chromosome 22q13.1 and is highly polymorphic in the human population. It may contain 2-4 genes, only one of which (CYP2D6) produces functional enzyme in individuals of the extensive metabolizer (EM) phenotype. Poor metabolizers (PM) possess two of the known 60 mutant CYP2D6 mutant alleles. The clinical importance of genetically variable function is for the response of a given drug or chemical. The significance is the role of CYP2D6 in governing the fate of a compound, its therapeutic window and its use in clinical practice.

Pharmacogenetics is the study of the hereditary basis for differences in a populations’ response to a drug. The same dose of a drug will result in elevated plasma concentrations for some patients and low concentrations for others. Some patients will respond well to the drugs, while others will not. A drug might be toxic to some patients but not to others.

Researchers can identify candidate genes that might influence the effectiveness of a drug and look for polymorphisms that correlate with a certain clinical outcome. Most polymorphisms simply contribute to individual diversity, including a variable affinity for drugs. High-throughput technologies like DNA chips will allow simultaneous analysis of thousands of genes for thousands of people, providing information that could then be correlated with clinical outcomes data. An interesting polymorphism could then be examined in prospective and/or retrospective clinical trials of a drug.

Pharmacogenetics in cardiology

The 2D6 mutation in the group of CYP drug metabolising enzymes is responsible for the metabolism of a large number of cardiac drugs like beta-blockers. Beta-blockers are used for the treatment of both hypertension and congestive heart failure. Poor metabolizers can have two to three-fold higher plasma concentrations and can have a higher rate of side effects like dizziness.

Another example of the importance of pharmacogenetics is the 2C9 enzyme and warfarin. About one percent of Caucasians and Africans are poor metabolizers. Patients that take warfarin and that do not have the particular active gene, 2C9, ought to be on a dose of about five milligrams a week as rather than the normal dose of five milligrams a day.

Pharmacogenetics in neurology

Treatment of Alzheimer’s Disease

There are two major forms of Alzheimer’s disease, familial and sporadic. The sporadic form comprises 85% of all cases worldwide, and 50 to 60% of these cases have been linked to the apolipoprotein gene.

Apolipoprotein E (ApoE) appears to modulate Alzheimer’s pathology. There is a clear association with the number of ApoE4 isoforms a person has and the risk of developing the disease, the age of onset, and the accumulation of brain markers of Alzheimer’s. Two copes of E-4 are linked to an Alzheimer’s disease that starts roughly at 60 years of age. One copy of E-4 produces an Alzheimer’s disease that starts around the age of 75 years old. And for those patients with no copies of E-4, the age of onset is normally around 85 years. For example, on looking at genotype and drug response, the non- ApoE4 subjects responded quite well to a drug called Tacrine, while the ApoE4 subjects did not. Those drugs designed to stimulate the cholinergic system tend to work well in the non- E4 patient, whereas those agents that are non-cholinergic will work in the E4 subject.

Pharmacogenetics in environmental medicine

Human disease is the consequence of both genetic susceptibility and environmental exposure. By identifying the genes and variants that affect the individual response to environmental toxins, we can better predict health risk. People with a polymorphism that makes them more susceptible, however, will have a much higher risk. People with one kind of p53 polymorphism, for example, will have a higher risk of cervical cancer if they get exposed to human papilloma virus.

Environmental carcinogens are metabolically activated or inactivated by metabolizer enzymes like the variants of Cytochrome P450. Some human population studies have also shown that CYP polymorphisms like CYP2D6 are linked to a higher incidence of various cancers. CYP2E1 is a major CYP enzyme and has several known polymorphisms that have been linked to cancers of the lung, stomach, liver, and nasopharynx.

Normal Drug Metabolism

There is a common sequence of events in the normal metabolism of any drug. The drug after being absorbed from the gastrointestinal tract enters into the blood stream and is distributed to various tissues and tissue fluids. The final step of excretion of the drug takes place through organs like the liver, the kidney or the lungs. The drugs undergo biochemical reactions like conjugation, glucuronidation or acetylation to increase their solubility and facilitate their excretion through different channels. Some are completely oxidized to CO₂, which is exhaled through the lungs; others are excreted via kidneys into the urine, or by the liver into the bile and then into feces.

Pharmacokinetics

The aim of drug therapy is to control, cure or prevent disease. To achieve this goal, therapeutic non-toxic levels need to be delivered to the target tissues. Four pathways of drug modification control the speed of onset, duration and intensity of drug action. These pathways include drug absorption, distribution, metabolisation and elimination of the drug. Pharmacokinetics is defined as the quantitative time dependant changes of both the plasma drug concentration and the total amount of drug in the body.

Pharmacokinetics is the study of the metabolism and effects of a particular drug. It involves giving a standard dose of the drug and monitoring its bioavailability and the response to that particular dosage. Several such studies when conducted earlier showed considerable differences in the bioavailability of the same drug in different patients having same phenotype. This is due to variability in response. Using statistical methods this variability shows a form of continuous or discontinuous distribution.

Target Selection

The pharmaceutical industry is concerned with validation of target data that will predict the tolerance and the effectiveness of the drug in question. Existing data of those drugs, which have proved their efficacy in humans, forms the basis of such a study.

Two broad strategies are involved in the identification and expression of genes through their proteins. Two types of terminologies are used for this study. The first is discovery genetics, where disease related genes are identified from human disease populations. The second is discovery genomics where bases of DNA sequences from families of genes are used for screening purposes.

The information on disease susceptibility genes of patients is very important and is relevant to the patient’s genetic contribution to the disease. In order to identify the products of gene expression, it becomes necessary to compare differential metabolisms related to the relevant gene variants with that of a control population. Critical enzymes or receptors associated with the altered metabolism are then used as targets. This helps in the understanding of the role of specific susceptibility gene variants on appropriate cellular metabolisms. With the help of reverse Genetic Engineering, it is again possible to find out the expressed protein sequence from affected tissues or cells from an affected population and compare it with the same data in a healthy population.

With the genomic approach, it becomes necessary not only to validate tissue distribution of the gene, but also correlate the corresponding disease or the clinical indication. On the other hand using the genetic approach, the susceptible gene is automatically validated once the disease related variants are known. Modern research focusing on the relationship of a particular gene with the disease can lead to a greater understanding of pharmacogenetics.

How pharmacogenetics can help in medical practice

Genetic constitution of a person greatly influences the therapeutic responsiveness to drugs. Sometimes a drug or drug combinations may have synergistic effect. Individual and family history is used to indicate susceptibility before subjecting the patient to a drug. Some persons show adverse reaction to a drug, for example anaphylactic shock with penicillin. Some drugs may have a teratogenic effect or malignant type of action or mutagenic effect at a cellular level in certain genotypes.

By applying the results of pharmacogenetic research in clinical practice, physicians will be more confident about the patient’s response to a specific medicine by using information from the patient’s DNA. Polymorphisms in genes encoding P450 enzymes, N-acetyltransferase and other key enzymes in drug metabolism determine the bio availability of the drug in patient’s blood, as a different population shows variation in the metabolism of ethanol, due to polymorphism in the enzyme alcohol dehydrogenase. In future, metabolic screens of genetic variants will be standardized so that it will be possible to have automated read-outs of a persons’ predicted response to any medicine. These DNA based screens will not provide disease specific diagnosis but will help the physician in determining an optimum dose and avoid side effects.

SNP Mapping a Tool for Personalized Genetic Profiling

Single Nucleotide Polymorphisms (SNP’s) can be defined as differences in a single base pair in the DNA sequence that is observed between individuals in a population. It is the simplest form of DNA polymorphism. SNPs are present throughout the human genome with an average frequency of approximately 1 in 1000 base pairs (bp). A SNP fine map will enable disease and drug response phenotypes to be mapped by linkage disequilibrium, which is the non-random association of susceptible disease gene and genetic markers. Linkage disequilibrium mapping is used to identify candidate genetic markers in the vicinity of the gene of interest. It is also now possible to narrow down the large size of the DNA region containing disease susceptible genes from a millions of base pairs to a few thousands. This fine mapping will help a quicker identification of disease susceptibility genes from a large chunk of DNA. For example, polymorphism of the apolipoprotein E (ApoE) gene is the first example of SNP linkage disequilibrium mapping to find out the locus around a known susceptibility gene for Alzheimer’s disease. In 1997, a high-density SNP map for a region of 4 million bases around the ApoE locus on chromosome 19 was constructed. The next goal was to detect a small region of linkage disequilibrium as a susceptible locus associated with Alzheimer’s disease. Moreover by using DNA from patients with Alzheimer’s disease and controls, it is possible to detect those SNP’s in linkage disequilibrium that are associated with the disease.

Today advanced DNA automated systems such as chip- based re-sequencing or microsphere-based analytical methodologies are available. With this it is possible to read out thousands of SNPs automatically. Chip technologies are now available for genotyping hundreds to a few thousand SNP’s. Each chip contains profiles of abbreviated SNP linkage disequilibrium for a number of drugs, which are prescribed in similar clinical indications.

The abbreviated SNP linkage disequilibrium profiles predict a patient’s response to medicines, but they do not specifically test the patient for the presence or absence of a disease gene- specific mutation. In addition they do not provide any disease predictive information about the patient or family members. In simple terms, medicine response profiles measure phenotypic responses to a medicine based on a pattern of inherited factors detected as small regions of linkage disequilibrium. From this abbreviated SNP linkage disequilibrium profiles, it is possible to find out, which SNP is responding to which drug in a specific disease. Similarly analysis of patients with identical disease phenotypes could be used to determine disease heterogeneity. Different SNP linkage disequilibrium profiles of patients with the same disease phenotype could define patterns of disease heterogeneity without necessarily identifying actual genes and alleles involved. As a result of disease heterogeneity, there can be large definable sub-groups of patients suffering with a common phenotype as in Alzheimer’s disease where treatment can be varied. Focusing drug development on such or similar sub-groups with a disease specific diagnosis or a medicine response profile will provide opportunities to develop more medicines for a large proposition of patients with heterogeneous diseases.

Application of SNP mapping technology will help in the development of more effective medicines for clinical use by using abbreviated SNP linkage disequilibrium mapping. Medicine response profile is being identified in the phase II clinical trials. This will further lead to selection of patient groups for drug efficacy in phase III studies. This is likely to make these trials smaller, faster and more efficient. The phase II trials will enable identification of the location of genes contributing to heterogeneous forms of the disease, leading to the discovery of new medicines and additional susceptibility targets.

The application of pharmacogenetics to the delivery of medicines will maximize the value of each medicine. Medicines can then be prescribed to patients in anticipation of therapeutic response and a high probability of efficacy without significant adverse events by considering genetic constitution of patients. Using effective and well-tolerated medicines. The prevention or cure of a disease will be more frequent and it will be treated well in advance before it takes a chronic form. In consequence, the period for hospitalization will also be reduced leading to a decrease in the costs of hospitalization and long-term medical care. Thus genetic methods are useful in differentiating those patients who experience good efficacy and lower significant adverse events in response to a medicine from other patients who fail to respond and develop serious adverse effects. In future, pharmacogenetics will have a stronger impact on medicines and medical practice.

Pharmacogenomics

While pharmacogenetics has to do with individuals’ response to certain drugs, pharmacogenomics is a broader term used to describe the commercial application of genomic technology in drug development and therapy. Pharmacogenetics is probably the study of known polymorphisms and known metabolic enzyme families of known drug targets. It is the role of polymorphisms and candidate genes and drug therapy and toxicity. It will be the discovery of new drug response genes and development of novel molecules to target these genes. After genes are linked with disease pathogenesis, pharmacogenomics will validate targets as appropriate sites of therapeutic intervention. Then scientists will identify or design therapeutic agents that interact with these targets in a way that achieves positive clinical outcome and minimal toxicity. Genetic tests will be used to predict clinical progression, likeliness of therapeutic response, and environmental influences. This will be coupled with drug development that will be rationally based on our understanding of molecular pathogenesis. The role of genes in determining disease susceptibility, progression, complications, and its response to treatment will be equally important. Pharmacogenomics can identify the patients for whom a drug would be safe and effective and eliminate possible drug toxicity at “normal” doses in non-metabolisers.

By taking into account individual genetic makeup, dosages prescribed for individual patients may be adjusted on the basis of DNA polymorphism analysis, hastening recovery and reducing side effects. As pharmacogenetics develops, new drug development may result in drugs that exhibit less variation in their metabolic conversion rates or drugs for which pre-prescription DNA testing is available. The role of the clinical laboratory should be in applying pharmacogenetics to medical care. One role might be to develop genetic profiling strategies to maximize the sensitivity and specificity of tests in predicting phenotypes. Another role might be to reduce the cost of the test and the technical difficulty of the test.

References

Purandarey, H. (2009). Essentials of Human Genetics. Second Edition. Jaypee Brothers Medical Publishers (P) Ltd.