Genetic Association Study Principles:

Andrew C. Heath

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)

Case-Control Study

• Usually the most powerful design, but need to address possible ‘population stratification’ effects

ALCOHOL METABOLISM

ALCOHOL ACETALDEHYDE ACETATE

AlcoholDehydrogenase

(ADH)

AldehydeDehydrogenase

(ALDH)

Higuchi Data -- Japanese Alcoholics

and Controls: ALDH2 locus

(Higuchi, 1994)

Controls Alcoholics

(N=461) (N=655)

Locus Genotype (%) (%)

ALDH2 *1 / *1 58 88

*1 / *2 35 12

*2 / *2 7 0p < .001

Higuchi -- Changes in ALDH2*1/*2

Frequency in Japanese Alcoholics

(Higuchi, 1994)

1979 1986 1992

(N=400) (N=400) (N=500)

ALDH2 *2 / *2 0.0 0.0 0.0

*1 / *2 2.5 8.0*** 13.0**

*1 / *1 97.5 92.0 87.0

i.e. protective effect of a single *2 allele is being diminished

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 Alcoholicsand Controls: ADH1B locus

(in those who are ALDH2*1/*1 homozygotes)Population

Controls Alcoholics

ADH1B *2 / *2 58.1% 35.8%

*2 / *1 34.7% 33.7%

*1 / *1 7.3% 30.4%

Higuchi Data -- Genetic effects on alcoholconsumption levels in a community sample

Average monthly alcohol consumption(ml pure alcohol)

Genotype MEN WOMEN

ALDH2*1/*1 1054.7 104.9

ALDH2*1/*2 390.9 46.6

ALDH2*2/*2 94.1 3.3

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)

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

FREQUENCIES OF GENES INFLUENCINGALCOHOL METABOLISM

High Risk Japanese EuropeanLocus Allele Ancestry Ancestry

ALDH2 ALDH2*1 76% 100%

ADH1B ADH1B*1 25% 95%

ADH1C ADH1C*2 6% 45%

NOTE: Predicted magnitude of effects is ALDH2*1 >> ADH1B*1 >>

ADH1C*2.

Table 2. Lifetime DSM-III-R Alcohol Dependenceby ADH1B (“ADH2”)

Type: Australian twin panel

ADH2*11 ADH2*12

Male affected 36 1

Male unaffected 101 18

Female affected 24 3

Female unaffected 144 7

Whitfield et al., 1998

But how do we know this is THE gene?

• 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

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%).

POPULATION STRATIFICATIONHypothetical Example

Falsely infer that A1 allele is risk-factor for Roman Catholicism.

OR = 2.28, 95%CI 1.39 - 3.73

NO ASSOCIATION NO ASSOCIATION

SWEDISH ANCESTRY (N=200) IRISH ANCESTRY (N=200)

NOT A1 alleleA1 allele

NOTROMAN

CATHOLICROMAN

CATHOLIC

NOTROMAN

CATHOLICROMAN

CATHOLIC

16218

90%

182

10%

3515

25%

10545

75%

70%

30%

90%

10%

NOTROMAN

CATHOLICROMAN

CATHOLIC

19733

12347

NOT A1 alleleA1 allele

MINGLED IN U.S. POPULATION (N=400)

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)

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

TRANSMISSION DISEQUILIBRIUM TEST

CONSISTENT WITHPREDICTION

AGAINSTPREDICTION

MOTHER

ADH2*1 / *2

FATHER

ADH2*1 / *1

MOTHER

ADH2*1 / *2

FATHER

ADH2*1 / *1

ADH2*1 / *1

ALCOHOLIC

ADH2*2 / *1

ALCOHOLIC

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 1 3 4 5 7 8 10 11 20 21

Transmitted 3 0 22 0 1 0 0 0 0 2

Not Transmitted 0 4 0 4 3 4 1 1 2 9

Allele

Population stratification: solution 3: Genomic Control methods