1. Mendelian Randomization A deconfounding method Dr. Jørn Olsen Epi 200B February 23 & 25, 2010

2. Stanner SA, Hughes J, Kelly CNM, Buttriss J. A review of the epidemiological evidence for the ‘antioxidant hypothesis’. Public Health Nutrition: 2003;7(3):407-422.

3. Violent death/cancer Se-cholesterol Cause Confounding Reverse causation?

4. Katan. Lancet. 1986 Given that ‘the gradient in serum cholesterol levels in the population is associated with a gradient in APOE [genotype]’, under the causal hypothesis we would expect to see a corresponding association between APOE and cancer. The absence of such a genetic association ‘would suggest that the association between low cholesterol and cancer is spurious’.

5. This argument assumes that the APOE gene is randomly allocated according to the laws of Mendel

6. Mendelian Laws, published 1866 The law of segregation: Hereditary traits are determined by pairs of factors, which segregate (separate) during gamete formation and reunite in the zygote. B = yellow b = greenBB/bb P-generationB/Bb/bhomozygotes GametesBb F 1 generationB/bRandom process B/b Heterozygotes GametesB b F 2 generationB/B, B/b, b/Bb/bRandom process YellowGreen 31

7. Independent assortment: The alleles of one pair of genes segregate independently of other pairs of genes during gametogenesis P generationB/B A/A Yellow, round B = yellow b = green b/b a/a Green, wrinkled A = round a = wrinkled GametesB Ab a F1F1 B/b A/a Yellow/round B/b A/a Yellow/round GametesBA Ba bA ba F2F2 9/16 yellow/round (¾ x ¾) 3/16 yellow/wrinkled (¾ x ¼ ) 3/16 green/round (¼ x ¾) 1/16 green/wrinkled (¼ x ¼)

8. GametesBABabAba BAB/B A/AB/B A/aB/b A/AB/b A/a BaB/B A/aB/B a/aB/b A/aB/b a/a bAB/b A/AB/b A/ab/b A/Ab/b A/a baB/b A/aB/b a/ab/b A/ab/b a/a 9 : 3 : 3 :1 Mendel’s Experiment - visit to home page

9. APOE +High Se-chol APOE -Low Se-chol

10. If the APOE gene is randomized then we will not expect an association between the genotype and cancer under H o

11. G = gene IP = intermediate phenotype D = disease GIPD Genotype Low cholesterol Cancer

12. If IP  D is confounded or due to reverse causation D  IP, then there should be no G  D association. Low cholesterolcancer confounders Cancer low cholesterol

13. IP G D G --- D not present in population at risk

14. ObservationsFolate NTD Confounding?FolateNTD C Randomized trial

15. MRC-trial + trial in Hungary FolateVitaminsHigh risk womenNTD 00N 00 D 00 0+N 0+ D 0+ +0N +0 D +0 ++N ++ D ++ PR Could we have avoided the NTDs caused by low folate intake?       ()/() ()/(). DDNN DDNN 0+0 000 0 03

16. MechanismFolate HomocysteinNTD GenotypeMTHFR (TT) HomocysteinNTD ?

17. If TT, Tt, tt are associated with different levels of homocystein, one would expect these genotypes to carry different risks for NTD

18. MTHFR (TT)NTD Homocystein

19. Since these genotypes are randomized at birth, other correlates of homocystein are not expected to be associated with the genotypes

20. Important use of MR could be to eliminate confounding as a cause of associations between E and D in situations where randomization is not an option. Mendelian deconfounding.

21. Alcohol and CVD Most studies show the following: CVD 06 alcohol drinks/day

22. ObservationAlcoholCVD ConfoundingAlcoholCVD Diet Smoking Exercise Etc.

23. All populations have slow and fast metabolizers of alcohol guided by genetic factors ALCOHOLACETALDEHYDE ACETIC ACID Slow metabolizers at step 1 = High level of alcohol Slow metabolizers at step 2 = High level of acetaldehyde 1 2

24. GenotypeSlow metabolisersCVD ButSlow metabolisers AlcoholCVD ? Genotype influence phenotype Fixed alcohol intake: slow metabolisersCVD

25. Alcohol metabolism ADH1 produces two different enzymes:  1 – fast,  2 – slow. If alcohol protects against MI  2 should have low risk given the same intake.

26. Relative risks of myocardial infarction according to the genotypes Variable ADH3 Genotype P value 1111  1  2  2 No. Of subjects (%) Patients161 (51)184 (46)51 (13) Controls279 (36)361 (47)130 (17) Relative risk (95% CI) Matched1.00.90 (0.69-1.17)0.72 (0.50-1.05)0.09 Multivariate1.00.81 (0.61-1.09)0.64 (0.43-0.98)0.03 Multivariate, with adjustment for 1.00.83 (0.62-1.11)0.65 (0.43-0.99)0.04 alcohol consumption

27. Multivariate relative risk of myocardial infarction according to the genotypes and the level of daily alcohol consumption P = 0.04 P = 0.001

28. ALDH2 AlcoholAcetaldehydeAcetic acid ALDH211 fast ALDH222 slow

29. Alcoholesophageal cancer Alcohol (not carcinogenic in animal models) Confounding, modification of carcinogens – tissue damage Metabolite – acetaldehyde?

30. Elimination of acetaldehyde – ALDH2 enzyme A point mutation in ALDH2 ALDH2 x 2 x 2 Allele and inability to metabolize acetaldehyde AlcoholALDH2 x 2 x 2: 18 times higher peak ALDH2 x 1 x 2: 5 times higher peak than ALDH2 x 1 x 1

31. Lewis, Sarah J. and Smith, George Davey. Alcohol, ALDH2, and Esophageal Cancer: A Meta-analysis which Illustrates the Potentials and Limitations of a Mendelian Randomization Approach. Cancer Epidemiol Biomarkers Prev. 2005;14(8):1967-1971

34. Plasma fibrinogen correlates with MI A common mutation in G A the  -fibrinogen gene is associated with high fibrinogen levels. The G/A genotype correlated with plasma fibrinogen in men and in pre- menopausal women or post-menopausal women treated with HRT ANOVAp < 0.001 2,4 2,6 2,8 3 3,2 3,4 3,6 3,8 G/G G/A A/A **** * Plasmafibrinogen (g/L) Women Men n = 4,889 n = 3,972 GENOTYPE

35. If fibrinogen is a cause of MI, the G/A or A/A genotype should have high risk of MI. Without ischemic heart disease With ischemic heart disease No. of women 3937 174 Genotype G/G 0.63 (2467)0.58 (101) G/A 0.34 (1323)0.40 (70) A/A 0.04 (147)0.02 (3) A -allele 0.21 0.22 No. of men2915315 Genotype G/G 0.64 (1877)0.65 (204) G/A 0.31 (913)0.31 (98) A/A 0.04 (124)0.04 (13) A -allele 0.20

36. C-reactive protein (CRP) is a maker of systemic inflammation. CRPHigh Blood Pressure (HBP) - many studies show association but could be HBPCRP – reverse causation

37. Or CRPHBP C 1 C 2

38. Most studies have Measurement errors of confounders (obesity, smoking, social factors) Most studies rely on cross-sectional data; inverse causation

39. GG, GC, CC; G associated with high levels of CRP

40. Smith GC, et al. Association of C-Reactive Protein with Blood Pressure and Hypertension: Life Course Confounding and Mendelain Randomization Tests of Causality. Arterioscler Thromb Vasc Biol. 2005;25:1051-1056.

42. Some studies show that coffee correlates with stillbirth. If caffeine is the culprit we can examine this by studying slow and fast metabolism of caffeine. Slow metabolism should have the highest risk at fixed levels of coffee intake. No studies are large enough to demonstrate this.

43. Alcohol Acetaldehyde Eliminated Alcohol Dehydrogenates ALDH2

44. ALDH 2x2x2 Homozygotes 18 times higher peak acealdehyde levels ALDH 2x1x2 Heterozygoter 5 times ADH 2x1x1 Homozygotes

45. Acetaldehyde cause nausea, headache etc. that makes drinking alcohol unpleasant-lowers the exposure

46. Is alcohol in high levels causing hypertension? Alcohol BP Diet Obesity Stress Physical activity

47. ALDH2 Alcohol BP C I..C N

48. Chen et al. PLOS Medicine 2008 5 (3): e52 Meteanalysis: search word in PubMed and ISI ALDH2, hypertension, blood pressure, cardiovascular disease, heart disease-studies published before 2007 Reference list Meta-analysis guidelines JAMA 2000;283:2008-12

50. Alcohol intake in matter for males: ALDH 2x1x1 20-30g /day ALDH 2x1x2 10-15g /day ALDH 2x2x2 0-2g /day

51. Or for ALDH 2x2x2 versus ALDH 2x1x1 for hypertension in males

52. Difference in systolic and diastolic blood pressure for ALHD2x2x2 versus ALDHD 2x1x1

53. Hypothesis A high level of intrauterine exposure to testosterone increases the risk of autism / ADHD (Simon –Cohen)

54. Twin girls with a twin brother have a higher prenatal exposure than twin girls with a twin sister. We expect autism/ADHD to have genetic as well as environmental causes How can we study it the intrauterine level of androgens play a role

55. Take Dz twins, compare females in FM sets with females in FF sets. FM females should have higher risks How do we use mendelian randomization in this study?

56. Nitsch D., et al. Limits to Causal Inference based on Mendelian Randomization: A Comparison with Randomized Controlled Trails. Am J Epidemiol. 2006;163:397-403. Comparison of RCT (intention to treat) And MR

57. Nitsch D., et al. Limits to Causal Inference based on Mendelian Randomization: A Comparison with Randomized Controlled Trails. Am J Epidemiol. 2006;163:397-403.

58. Problems Population stratification/genetic confounding: Different ethnic groups may have different genotypes and different disease risks.

59. Linkage disequilibrium – association between genes because they are located close together on the chromosome.

60. Gene 1 and Gene 2 associated-are in linkage disequilibrium: Gen 2 Conf Gene1 Exp Disease Is Gene 1 a useful instrumental variable for Exp?

61. Gene 2 Cont Gene 1 Exp Disease Is Gene1 still a useful instrumental variable for Exp?

62. Conf Maternal Maternal obesity offspring obesity FTO Offspring FTO Can maternal FTO be used as an instrumental variable for Maternal obesity to study offspring obesity?

63. Canalization: environmental adaptation to a specific genotype like for ALDH2

64. Limitations Requires well defined and rather strong genetic risk factors with high penetrance. Should not interfere with behaviour of relevance to the exposure under study – unless this exposure could be controlled in the design or analysis. Slow metabolisers of alcohol tend to drink less. Genes involved in detoxification only play a role when the toxic exposure is present (gene-environment interaction). Without the exposure the gene has no function.

65. Mendelian Randomization  First described as such by Katan (Lancet 1986; i: 507- 81).  Is now being explored in situations where we have a genotype that is causally linked to a phenotype.  Smith GD, Ebrahim S. Mendelian randomization: prospects, potentials, and limitations. Int J Epidemiol 2004; 33: 30-42.  Katan MB. Commentary: Mendelian randomization, 18 years on. Int J Epidemiol 2004; 33: 10-11.  Chen et al. PLOS medicine 2008; 5: 461-470

66. Offspring Genotype a´a´ Offspring Genotype aa Offspring Genotype aa´ Offspring Genotype a´a Mother Genotype aa´ Father Genotype aa´ Allele that is inherited from each parent is randomly determined Mendelian randomization in case-parent design

67. The case-parent triad design aa’aaParents aa’Cases a’ more frequent in cases than expected from Mendel laws (probability in this case 0.50)

68. Will a gene with a specific polymorphism (G X ) cause disease?

69. If 4 parents look like this G X /G G X /G: ¾ of offspring expected to have the SNP G X /GG/G: ½ of offspring expected to have the SNP G X / G X G/G: All will have the SNP G/GG/G: None will have the SNP  2.25 of their 4 affected children are expected to have the G X if the SNP correlate with the disease. This expectation be tested, but a larger sample is needed.