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Genetic polymorphism: (i) individual handling of drugs (ii) inherited susceptibility to disease

Genetic polymorphism: (i) individual handling of drugs (ii) inherited susceptibility to disease. Much individuality in drug response is inherited (polymorphism). PHARMACOGENETICS. Identify specific genes associated with specific diseases and that may be targets for new drugs.

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Genetic polymorphism: (i) individual handling of drugs (ii) inherited susceptibility to disease

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  1. Genetic polymorphism:(i) individual handling of drugs (ii) inherited susceptibility to disease

  2. Much individuality in drug response is inherited (polymorphism) PHARMACOGENETICS Identify specific genes associated with specific diseases and that may be targets for new drugs Identify polymorphic genes that mediate response to drugs

  3. Menu • Metabolism of drugs- phase 1 and 2 reactions • what is polymorphism? • Examples of polymorphic genes relevant in drug metabolism • polymorphism and disease susceptibility

  4. Polymorphism • polymorphism= the inheritance of genes in different forms termed alleles alleles have different DNA sequences • polymorphic locus: the frequency of the most common allele is less than 99%. 1 allele in 100 alleles 100 alleles =50 people 1 person in 50 (2%) is heterozygous

  5. Important points • every conceivable type of variation in DNA has been identified • some genes demonstrate many alleles (>50). Some of these alleles may have a high population frequency

  6. Examples of polymorphic variation gene allele change/ phenotype GSTM1 GSTM1*0 gene deleted: no enzyme GSTM1*A G519: active GSTM1*B C519: active GSTM1*1X2 gene duplicated: high activity GSTM3 GSTM3*A wild type: active GSTM3*B 3bp deletion in intron 6: linkage dysequilibrium with M1*A CYP2D6 CYP2D6*4 G/A intron 3/exon 4: splice site defect, inactive enzyme TNF-alpha TNF*A substitution in the promotor region at -308

  7. Polymorphism: • is common (probably all genes show allelic variation) • BUT does it matter? (outside celebrated examples such as cystic fibrosis, haemoglobin)

  8. Individual variation in response to drugs is a substantial clinical problem 1 in 15 British hospital admissions is due to adverse drug reactions In the US, 106,000 patients die and 2.2 million are injured each year by adverse reactions to prescribed drugs.

  9. Drug metabolism • Drugs are usually lipophilic. This allows them to cross membranes and enter cells. • Lipophilic compounds are difficult to remove from the body. • Enzymatic transformation of drugs into hydrophilic, inactive metabolites is usually necessary.

  10. Detoxication reactions often involve phase 1 and phase 2 steps Phase 1 reactions add or expose a functional group through oxidative reactions: N-dealkylation O-dealkylation hydroxylation N-oxidation S-oxidation deamidation

  11. The cytochrome P-450 mono-oxygenase system is largely responsible for catalysing phase 1 reactions. • Complex supergene family: at least 40 enzymes expressed in human tissues. • CYP1A2, 3A4, 2C9, 2C19, 2D6, 2E1 exert a major role in drug metabolism. • Enzymes located on smooth endoplasmic reticulum ARE RESPONSIBLE FOR THE METABOLIC ELIMINATION OF MOST DRUGS CURRENTLY USED IN MEDICINE

  12. Examples of phase 1 reactions N-dealkylation RNHCH2 RNH2 +CHO2 codeine theophylline aliphatic RCH2CH2 RCHCH2cyclosporine hydroxylation tolbutamide OH R R R Aromatic hydroxylation phenytoin O OH

  13. Detoxication reactions often involve phase 1 and phase 2 steps Phase 2 reactions are conjugations with: glucuronic acid sulphate acetate amino acids reduced glutathione that result in water-soluble molecules that are inactive and easily excreted.

  14. Phase 2 reactions involve an extensive enzymology • Glucuronidation: uridine 5’-diphosphate glucuronyltransferase • Methylation: catechol O-methyltransferase histamine N-methyltransferase thiopurine methyltransferase • Sulphation: sulphotransferases • Glutathione: glutathione S-transferases

  15. Example of phase 2 reaction sulphation O ROH + 3’-phosphoadenosine 5’phosphosulphate O S OH R O + 3’-phosphoadenosine 5’phosphate Eg: acetaminophen

  16. Cytochrome P450 CYP2D6: debrisoquine hydroxylase • Strongly expressed in liver • gene located on chromosome 22 • many drugs are substrates for CYP2D6: amitriptyline, clozapine, haloperidol propanolol, amiodarone, flecainide highly polymorphic with more than 50 allelic variants identified

  17. CYP2D6 polymorphic variants Relatively common gene inactivating mutations: CYP2D6*4 splice site variants (GA transition at intron 3/exon 4) CYP2D6*3 base pair deletion in exon 5 CYP2D6*5 gene deletion homozygotes for these (and other rarer) mutant alleles are PM (poor metabolisers) and comprise about 10% of European populations

  18. Further CYP2D6 polymorphic variants • Amplification of the entire CYP2D6 gene with up to 13 copies is found: ultra rapid individual. • Found in 1.5% Scandinavians, 7% Spaniards, 20% Ethiopians • Affected people metabolise CYP2D6 substrates so quickly that a therapeutic effect cannot be obtained at conventional doses. Nortriptyline: CYP2D6 PM individual requires 10-20 mg/day CYP2D6 ultra rapid individual requires 500mg/day

  19. Thiopurine methyltransferase • potentially important polymorphism • responsible for the metabolism of anti-tumour agents, 6-mercaptopurine, 6-thioguanine • polymorphism associated with difficulty in achieving effective dose of these drugs in children with leukaemia • children with TPMT deficiency show severe haematopoietic toxicity when exposed to drugs like 6-mercaptopurine.

  20. Should patients be tested for specific polymorphisms? • Not generally available at present. • likely to become more available since: (a) particularly sensitive individuals may avoid serious adverse reactions. (b) can avoid giving drugs to patients who cannot benefit from them. • Trials in psychiatric patients are underway. • Technology may allow a detoxication DNA chip that screens for all relevant polymorphisms.

  21. What is the true function of drug metabolising enzymes? • Cytochrome P450 gene family is believed to be the product of an ancestral gene formed about 3 billion years ago. • Possible that P450s are the result of evolution of plants producing toxins and animals evolving enzymes to detoxify these chemicals.

  22. Genetic susceptibility to most diseases appears to be due to multiple genes that interact with each other and the environment.

  23. environment general population susceptibility genes SET1 high environment low genetic risk susceptibility genes SET2 less environment more genetic risk susceptibility genes SET3 even less environment even more genetic risk susceptibility genes SET4 low environment high genetic risk

  24. Problems unknown: • number of patient subgroups • number of susceptibility sets • number of genes in a susceptibility set • how genes interact within a susceptibility set- perhaps two/three genes critical and a variable number of others modify their effect.

  25. Assume SET1 comprises 3 genes genes 1 2 3 alleles a, b x, y m,n risk genotypes: aa xx mm ? effects of the aa/xx/mm combination influenced by other genes susceptibility genes SET1 susceptibility genes SET2 susceptibility genes SET3 susceptibility genes SET4 ? completely different genes and arrangement of genes

  26. Why have we identified so few genuine epistatic effects? • there is no basis for predicting epistatic effects- need to examine all possible 2-way, 3-way etc genotype combinations. problems for statistical analysis (multiple testing) • many genotypes are found in low frequency: genotype aa found in 20% of cases and genotype xx found in 30% of cases aa/xx interaction present in only 6% of cases

  27. Disease group General population Modifier genes SET G1 SET B1 Susceptibility genes SET 1 Modifier genes SET G2 SET B2 Susceptibility genes SET 2 environment Modifier genes SET G3 SET B3 Susceptibility genes SET 3 Modifier genes SET G4 SET B4 Susceptibility genes SET 4 GOOD OUTCOME BAD

  28. Susceptibility genes: compare gene frequencies in affected and unaffected subjects. case-control and/or family studies Modifier genes: compare gene frequencies in cases with different outcomes; young/old, good/bad outcome studies in cases

  29. Selection of candidate genes Problematical: there are 50,000 genes use whatever information is avalable: functional chromosomal location but: allele frequencies will determine patient numbers

  30. Selection of candidate genes: functional approach • atopy • bronchial hyper-responsiveness • detoxication of environmental irritants • detoxication of reactive oxygen species and their oxidised lipid and DNA by-products • recruitment of inflammatory cells • cytokines determining Th1/Th2 response • eicosanoid production • tissue remodelling (growth factors)

  31. Selection of candidate genes: positional approach • chromosome 5q: regulation of IgE, pro-inflammatory cytokines, beta-adrenergic receptor • chromosome 6: HLA, TNF-alpha • chromosome 11q: high-affinity IgE receptor • chromosome 12q: IFN-gamma, nitric oxide synthase • chromosome 13: IgE levels • chromosome 14: T cell antigen receptor, NFkB

  32. Biological vs Statistical significance p Odds ratio Change n= <0.05 1.1 55%® 57% >7100 <0.05 2.5 55%® 75% >94 <0.05 5.0 55%® 86% >39 <0.05 15 55%® 95% >22 Statistical significance Biochemically interesting ?clinically significant Useful for Medical Screening/Diagnosis

  33. alpha mu theta pi zeta sigma kappa omega The glutathione S-transferase supergene family ancestral GST gene Chrom 6p 1p 22 q 11q 14 q 4q ND 10q Genes A1-A4 M1-M5 T1,T2 P1 Z1 S1 K1 O1 Allelic yes yes yes yes yes ? ? ? Gene products expressed in cytosol

  34. . O2- Superoxide dismutase (Cu,Zn-SOD, Mn-SOD) Hydroxyl radical (OH.) H2O2 DNA Catalase, Glutathione peroxidase Lipid DNA hydroperoxides H2O Lipid hydroperoxides a, m, q, p class Glutathione S-Transferases Detoxified products

  35. GSTP1 is associated with asthma symptoms with an OR that indicates a strong biological impact. Question: What do I do now? Answer: Confirm results in a separate patient cohort

  36. Occupational asthma 104 unrelated Italian Caucasians occupationally exposed to toluene isocyanate detailed clinical history CE Mapp et al Dept Environmental Medicine and Public Health, University of Padova, Italy

  37. Italian occupational asthma cases:GSTP1 Val/Val frequency in asthmatics and non-asthmatics with >10 years exposure 60.0% 54.2% 41.7% 39.6% 33.3% Non-asthmatic 30.0% Asthmatic 25.0% 6.3% 0.0% Ile/Ile Val/Val Ile/Val GSTP1 genotype

  38. What do we do now? (i) identify further genes to build up susceptibility sets and identify which biochemical pathways have the greatest impact on phenotype. (ii) in vitro studies to determine the mechanism of gene/phenotype associations.

  39. Molecular epidemiology can identify associations between genes and disease phenotypes GSTP1 Val/Val confers protection in allergic and occupational disease. DOES THIS MAKE BIOLOGICAL SENSE? GSTP1 is located on a hotspot region, chromosome 11q Chronic inflammation is a prominent feature of both asthma types, in vitro GSTP1 substrates include ROS by-products.

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