1. Genetic Association Study Principles: Andrew C. Heath

2. UNUSUAL EXAMPLE: Asian Studies of Japanese Alcoholics and Community Controls Work by Higuchi, Murumatsu and colleagues is documenting ways in which genes that influence alcohol metabolism may be associated with differences in alcohol dependence risk or alcohol consumption levels. (Higuchi et al., 1994, Lancet) (Murumatsu et al., 1996)

3. Case-Control Study Usually the most powerful design, but need to address possible ‘population stratification’ effects

4. ALCOHOL METABOLISM ALCOHOL ACETALDEHYDEACETATE Alcohol Dehydrogenase (ADH) Aldehyde Dehydrogenase (ALDH)

5. Higuchi Data -- Japanese Alcoholics and Controls: ALDH2 locus (Higuchi, 1994) ControlsAlcoholics (N=461)(N=655) LocusGenotype(%)(%) ALDH2*1 / *15888 *1 / *23512 *2 / *270 p <.001

6. Higuchi -- Changes in ALDH2*1/*2 Frequency in Japanese Alcoholics (Higuchi, 1994) 197919861992 (N=400)(N=400)(N=500) ALDH2*2 / *20.0 0.0 0.0 *1 / *22.5 8.0***13.0** *1 / *197.592.087.0 i.e. protective effect of a single *2 allele is being diminished

7. From Higuchi’s community data, in individuals who are also ALDH2*1/*1 homozygotes, we may estimate the penetrance of the AHD1B*2/*2 genotype (the low risk genotype) as ~0.07, that of the high risk ADH1B*1/*1 genotype as ~0.29. Higuchi Data -- Japanese Alcoholics and Controls: ADH1B locus (in those who are ALDH2*1/*1 homozygotes) Population ControlsAlcoholics ADH1B*2 / *258.1% 35.8% *2 / *134.7% 33.7% *1 / *17.3%30.4%

8. Higuchi Data -- Genetic effects on alcohol consumption levels in a community sample Average monthly alcohol consumption (ml pure alcohol) Genotype MEN WOMEN ALDH2*1/*11054.7104.9 ALDH2*1/*2390.946.6 ALDH2*2/*294.13.3 From other data, we can estimate that approximately one-third of the variance in alcohol consumption levels in Japanese males is explained by this genetic locus. (Higuchi et al., 1996b)

9. Conclusion: there are genes with powerful effects on behavioral traits waiting to be discovered!

10. FREQUENCIES OF GENES INFLUENCING ALCOHOL METABOLISM High RiskJapaneseEuropean LocusAlleleAncestryAncestry ALDH2ALDH2*176%100% ADH1BADH1B*125%95% ADH1CADH1C*26%45% NOTE: Predicted magnitude of effects is ALDH2*1 >> ADH1B*1 >> ADH1C*2.

11. Table 2. Lifetime DSM-III-R Alcohol Dependence by ADH1B (“ADH2”) Type: Australian twin panel Whitfield et al., 1998

13. But how do we know this is THE gene?

14. See Kidd paper (Osier et al., Am J Hum Genet 71:84-99, 2002) ADH1B “2” allele is occurring on different haplotypes: - East Asian (also ADH1C “1” allele); - Middle Eastern, Ethiopian (also ADH1C “1” allele), rare in N. American European Ancestry (< 5% haplotype frequency). Population Genetics of ADH gene region

15. Population Genetics of ADH gene region (II) ADH1B “3” allele is mainly seen in African American, sub-saharan African populations, but at low frequency (6/1000) in N. Americans of European ancestry ADH1C “2” allele in European ancestry cases occurs on two different haplotypes, the higher frequency haplotype (30%) being rare in East Asians (< 2%), the other occurring at a lower frequency in Europeans (7-15%) except Finns (30%) and seen at slightly higher rate in East Asians (3-10%).

16. POPULATION STRATIFICATION Hypothetical Example Falsely infer that A1 allele is risk-factor for Roman Catholicism. OR = 2.28, 95%CI 1.39 - 3.73 NO ASSOCIATION SWEDISH ANCESTRY (N=200) IRISH ANCESTRY (N=200) NOT A1 allele A1 allele NOT ROMAN CATHOLIC ROMAN CATHOLIC NOT ROMAN CATHOLIC ROMAN CATHOLIC 162 18 90% 18 2 10% 35 15 25% 105 45 75% 70% 30% 90% 10% NOT ROMAN CATHOLIC ROMAN CATHOLIC 197 33 123 47 NOT A1 allele A1 allele MINGLED IN U.S. POPULATION (N=400)

17. HOW DO WE HANDLE POPULATION STRATIFICATION? SOLUTION 1: Make comparisons within (full) sibships, i.e. of siblings who share the same biological mother and father (  same ancestry). 5 DZ twin pairs where one twin was ADH2*1/*1 second twin was ADH2*1/*2 In all 5 pairs, *1/*1 had higher consumption (p =.03) (Whitfield et al., 1998)

18. POPULATION STRATIFICATION - SOLUTION 2 Genetic marker data collected on affected (e.g. alcohol dependent) individuals and both their biologic parents. For heterozygous parents, compare frequency of transmitted allele (i.e. passed from a parent to the affected individual) and non- transmitted allele. TRANSMISSION DISEQUILIBRIUM TEST

19. CONSISTENT WITH PREDICTION AGAINST PREDICTION MOTHER ADH2*1 / *2 FATHER ADH2*1 / *1 MOTHER ADH2*1 / *2 FATHER ADH2*1 / *1 ALCOHOLIC ADH2*2 / *1 ALCOHOLIC

20. TRANSMISSION-DISEQUILIBRIUM TEST: A Medical Genetic Example  2 = 92.91, highly significant by permutation test Ataxia-Telangiecstasia (AT) in Costa Rica (Lange, 1997) Transmission Pattern 13457810112021 Transmitted30220100002 Not Transmitted0404341129 Allele

22. Population stratification: solution 3: Genomic Control methods