Joint ACC/CMGS MRCPath Pt 2 Study Day, Nov 2007 Future Impact of Complex Disorders &

1. Joint ACC/CMGS MRCPath Pt 2 Study Day, Nov 2007 Future Impact of Complex Disorders & Pharmacogenetics on Genetic Testing Dr Ian M Frayling Consultant in Genetic Pathology Institute of Medical GeneticsUniversity Hospital of Wales Cardiff ian.frayling@cardiffandvale.wales.nhs.uk

2. On Developments • Developments in place when one is born are accepted as the natural order of things

3. On Developments • Developments in place when one is born are accepted as the natural order of things • Developments in the first third of one’s existence are accepted as new and exciting and something one could make a career in

4. On Developments • Developments in place when one is born are accepted as the natural order of things • Developments in the first third of one’s existence are accepted as new and exciting and something one could make a career in • Developments in the middle third of one’s existence are thought of as against the natural order of things

5. On Developments • Developments in place when one is born are accepted as the natural order of things • Developments in the first third of one’s existence are accepted as new and exciting and something one could make a career in • Developments in the middle third of one’s existence are thought of as against the natural order of things • \,=Q-X%&!>@;##B86 z`zzz……

6. On Developments • Developments in place when one is born are accepted as the natural order of things • Developments in the first third of one’s existence are accepted as new and exciting and something one could make a career in • Developments in the middle third of one’s existence are thought of as against the natural order of things • \,=Q-X%&!>@;##B86 z`zzz…… The Hitch-hiker’s Guide to the Galaxy, Douglas Adams

7. Some fundamentals • Phenotype = genotype + environment • Environment of 1 gene includes 2 x 29,999 others • Evolution by natural selection (change) occurs as a consequence of a struggle for survival (competition) where there are differences (variation) within the system that can be selected (fitness) • The Internet & search engines

8. Complex Disorders

9. Complex Disorders • Are there any simple ones? • Down’s, CF, FraX, HD, cancer, thrombophilia … • What’s Mendelian? • Can clinical variety be predicted? • Is it worth predicting? Should it be?

10. Complex Disorders • Are there any simple ones? • Why variety/complexity? • Metabolic polymorphism • Modifiers • genetic • environmental

11. Complex Disorders • Are there any simple ones? • Why variety/complexity? • Unless you can test for most of the cause of the variety it is probably not worth testing for any of it • [CF and alleles and risk reduction] • BRCA and SNPs

12. Pharmacogenetics • Treatment: pharmacology and therapeutics • Genetic variation that has a bearing on this • Promises to individualise therapy, savings in time, money, adverse events, better therapeutic effect • What genetic tests do we currently do that have an impact on treatment?

13. Pharmacogenetics • Treatment: pharmacology and therapeutics • Genetic variation that has a bearing on this • Promises to individualise therapy, savings in time, money, adverse events, better therapeutic effect • What genetic tests do we currently do that have an impact on treatment?

14. Pharmacogenetics • What genetic tests do we currently do that have an impact on treatment?

15. Pharmacogenetics • What genetic tests do we currently do that have an impact on treatment? • Haemophilia

16. Pharmacogenetics • What genetic tests do we currently do that have an impact on treatment? • Haemophilia • Leukaemia

17. Pharmacogenetics

18. Pharmacogenetics • Treatment: pharmacology and therapeutics • Genetic variation that has a bearing on this • Absorbtion/assimilation • Metabolic • Activation > Action (Receptor binding) • Inactivation • Excretion • SIDE EFFECTS

19. Pharmacogenetics • Where’s the variation? • Absorbtion/assimilation • Metabolic • Activation > Action (Receptor binding) • Inactivation • Excretion

20. Pharmacogenetics • Metabolic • Activation • Phase 1 and Phase 2 metabolism • Prodrugs • P450, GST, NAT • > Action (Receptor binding): Receptors vary • Inactivation • P450, CYP2D6 etc etc • Excretion

21. Gene-Environment Combinations in CRCa • Adenoma risk and Aspirin • CYP2C9 activates, UGT1A6 deactivates: Bigler J, et al. CYP2C9 and UGT1A6 genotypes modulate the protective effect of aspirin on colon adenoma risk Cancer Res 2001;61:3566-9

22. Gene-Environment Combinations in CRCa • Adenoma risk and Aspirin • CYP2C9 activates, UGT1A6 deactivates: Bigler J, et al. CYP2C9 and UGT1A6 genotypes modulate the protective effect of aspirin on colon adenoma risk Cancer Res 2001;61:3566-9

23. Gene-Environment Combinations in CRCa • Adenoma risk and Aspirin • CYP2C9 activates, UGT1A6 deactivates: Bigler J, et al. CYP2C9 and UGT1A6 genotypes modulate the protective effect of aspirin on colon adenoma risk Cancer Res 2001;61:3566-9

24. Pharmacogenetics • Familial Hypercholesterolaemia • LDLR, APOB, PCSK9 • “Acquired” hypercholesterolaemia • PCSK9 variants are associated with: • Increased risk of CHD • & need for more expensive treatment in FH • Decreased risk of CHD • & better response to treatment in FH • GP > [chol] > Hi > genetic testing > report to GP on drug suitability, and cascade testing if FH

25. Pharmacogenetics • SIDE EFFECTS • This is what really scares the drug companies, and the NHS: -£££ • Testing for adverse event susceptibility will probably be the priority – avoiding nasty dangerous expensive events • Testing for drug preference is likely “second phase”: +£££

26. Pharmacogenetics • Who’s going to do this? • Timeliness: m.1555A>G • £61k Rx, £18k pa education • BIG business – 100,000s of tests • Reports to Drs, pharmacists, nurses … • Pathway: GP, generic Rx, blood test, report to pharmacist, specific Rx issued • How much interpretation is needed? • How will it be provided?

27. Tumour testing in Sporadic CRCa • MSI in 20% of sporadic colon* cancers • Theoretical reasons to expect differences in response to chemotherapy MSI+/- • Old studies showed response to BCNU in 20% colon cancers • Large prospective studies in progress

28. Tumour testing in Sporadic CRCa • MSI in 20% of sporadic colon* cancers • Theoretical reasons to expect differences in response to chemotherapy MSI+/- • Old studies showed response to BCNU in 20% colon cancers • Large prospective studies in progress • 2007: MRC K-ras & topo2 IHC • What will happen when a £50 test predicts response to £15k of chemotherapy? • And 15,000 colon cancers pa need testing …

29. Tumour testing in Sporadic CRCa • MSI in 20% of sporadic colon* cancers • Theoretical reasons to expect differences in response to chemotherapy MSI+/- • Old studies showed response to BCNU in 20% colon cancers • Large prospective studies in progress • 2007: MRC K-ras & topo2 IHC • What will happen when a £50 test predicts response to £15k of chemotherapy? • And 15,000 colon cancers pa need testing … • Within 7 calendar days!

30. Future impact of complex disorders and pharmacogenetics • Exiting • Challenging • Potentially great benefit

31. Pharmacogenetics etc • Slides not in the lecture but alluded to …

32. Metabolism of drugs (and other foreign compounds) • Complex system - “DME”s • Evolved to cope with natural organic compounds • Phase I enzymes • Cytochrome P450: CYP • Phase II enzymes • N-acetyl transferases: NAT1, NAT2 • Glutathione S-transferases: GSTM1, GSTP1, GSTT1 • Sulphotransferase: SULT • Others • Microsomal epoxide hydrolase: mEH • Myeloperoxidase: MPO • Great individual gene / gene family variation, even more combinations Houlston RS & Tomlinson IPM. Polymorphisms and colorectal tumor risk. Gastroenterology 2001;121:282-301.

33. N H 2 N N C H 3 IQ N H N O H H N O S O 3 C H PhIP 3 N N H H N O C H 2 3 N N O H R C O S O 2 2 H R C O H 2 7 B[a]P B [ a ] P ( O H ) B [ a ] P ( O H ) 2 4 HETEROCYCLIC AMINES Carcinogen Activation REACTIVE PRODUCTS OF METABOLISM MPO CYP SULT CYP NH+ NAT1 NAT2 CYP C+ AROMATIC AMINES 4-ABP POLYCYCLIC AROMATIC HYDROCARBONS SULT CYP 7,12 DMBA Epoxide mEH CYP. MPO CYP SULT [C+] mEH Adapted from: Williams JA Carcinogenesis 2001;22:209-214.

34. Metabolism • Cytochrome P450: CYP family • Colorectal cancer: • CYP1A1 I462V: OR 7.9 [CI 1.4 - 44] • CYP1A1 6325T/C: no association • Sivaraman L et al. CYP1A1 genetic polymorphisms and in situ colorectal cancer. Cancer Res 1994;54:3692-5 • Houlston R & Tomlinson I. Polymorphisms and colorectal tumor risk. Gastroenterology 2001;121:282-301

35. Metabolism • N-acetyl transferase: NAT2 • 50-60% Caucasians are slow acetylators • Colorectal cancer: • 15 population studies • 4 phenotype - increased risk if rapid acetylators • 11 genotype - no association • overall no association • Houlston R & Tomlinson I. Gastroenterology 2001;121:282-301 • 1 study in HNPCC • MSH2/MLH1 gene carriers more likely penetrant if slow acetylators (P <0.03) Heinimann K et al. Cancer Res 1999;59:3038-40 • Breast cancer: • no association, but not expressed in mammary epithelium!

36. Metabolism • Glutathione S-transferase: GST family • Colorectal cancer: • GSTM1 null • increased risk in 5 studies, only significant in 2 • 1 study: proximal tumour risk, not otherwise confirmed • no relationship with adenoma risk • GSTT1 null • increased risk in 2 studies • nil or decreased risk in 5 studies • GSTP1 105I/V and 114A/V • no relationship • Houlston R & Tomlinson I. Gastroenterology 2001;121:282-301

37. Metabolism • Sulph / fotransferase: SULT family • SULT1A1 R213H: Arg ~65% • Arg allele 10~300x less active at mutagen activation • Glatt H et al. Sulphotransferases: genetics and role in toxicology. Toxicol Lett 2000;112-113:341-348. • Needs exploring

38. Metabolism • Microsomal epoxide hydrolase: mEPHX • Colorectal cancer: • One study • Y113H • Y homozygosity significantly increased risk • 139H/R • no association • problem with population stratification • Harrison DJ et al. Microsomal epoxide hydrolase gene polymorphism and susceptibility to colon cancer. Br J Cancer 1999;79:168-171.

39. Metabolism • Myeloperoxidase: MPO • ex-neutrophils • Lung cancer: • c.-463G/A = loss of SP1 transcription binding site • G:A 3:1 • 4 studies: • 182 cases, 459 controls: OR 0.30 (P = 0.04; cauc.) • 157 cases, 244 controls: OR 0.61 (ns; african) • 323 cases, 437 controls: OR 0.50 • 196 cases, 255 controls: OR 0.47 (P = 0.004) • 93 cases, 121 controls: OR 0.52 (P = 0.02) • Williams JA. Single nucleotide polymorphisms, metabolic activation and environmental carcinogenesis: why molecular epidemiologists should think about enzyme expression. Carcinogenesis 2001;22:209-214.

40. Overall mutation rate is a function of: Environment Pro-carcinogens Pro-carcinogens metabolism excretion Carcinogens Micro- Environment DNA DNA Repair Cell turnover Micronutrients Antioxidants ...

41. Individualisation in CRCa • NSAIDs • Consistent association between NSAID use and reduced risk of CRCa: RR 0.63 • Aspirin is subject to metabolic activation and deactivation

42. Individualisation in CRCa • Adenoma risk and Aspirin • CYP2C9 activates, UGT1A6 deactivates: Bigler J, et al. CYP2C9 and UGT1A6 genotypes modulate the protective effect of aspirin on colon adenoma risk Cancer Res 2001;61:3566-9

43. Individualisation in CRCa • Adenoma risk and Aspirin • CYP2C9 activates, UGT1A6 deactivates: Bigler J, et al. CYP2C9 and UGT1A6 genotypes modulate the protective effect of aspirin on colon adenoma risk Cancer Res 2001;61:3566-9

44. Individualisation in CRCa • Adenoma risk and Aspirin • CYP2C9 activates, UGT1A6 deactivates: Bigler J, et al. CYP2C9 and UGT1A6 genotypes modulate the protective effect of aspirin on colon adenoma risk Cancer Res 2001;61:3566-9

45. DNA Repair

46. DNA Repair • necessary because DNA is subject to damage • dsDNA for more than one reason • very old (conserved) function • only one DNA toolkit • bacterial mutator loci • keeps mutation rate low, but not zero

47. DNA Damage, Repair & Consequences DNA repair Cell cycle arrest Mutagenic survival ? DNA Damage Toxic death Apototic death

48. DNA Damage, Repair & Consequences DNA repair Cell cycle arrest Mutagenic survival ? DNA Damage Toxic death Apototic death All a function of amount of damage, repair, and cell turnover

49. DNA Damage • most mutations due to misincorporation of dNTPs, due to damaged bases • very rarely ‘spontaneous’ • A, C, G & T exist as tautomers, but equilibria very ‘one-sided’ ...

50. Why A, C, G & T?