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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
Dehydrogenase
(ADH)
ALCOHOL
Aldehyde
Dehydrogenase
(ALDH)
ACETALDEHYDE
ACETATE
Higuchi Data -- Japanese Alcoholics
and Controls: ALDH2 locus
Locus
Genotype
Controls
(N=461)
(%)
Alcoholics
(N=655)
(%)
ALDH2
*1 / *1
58
88
*1 / *2
35
12
*2 / *2
7
0
p < .001
(Higuchi, 1994)
Higuchi -- Changes in ALDH2*1/*2
Frequency in Japanese Alcoholics
ALDH2
1979
(N=400)
1986
(N=400)
1992
(N=500)
*2 / *2
0.0
0.0
0.0
*1 / *2
2.5
8.0***
*1 / *1
97.5
92.0
13.0**
87.0
i.e. protective effect of a single *2 allele is being diminished
(Higuchi, 1994)
Higuchi Data -- Japanese Alcoholics
and 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%
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 -- Genetic effects on alcohol
consumption 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 INFLUENCING
ALCOHOL METABOLISM
Locus
High Risk
Allele
Japanese
Ancestry
European
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 Dependence
by 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?
Population Genetics of ADH
gene region
• 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 (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 STRATIFICATION
Hypothetical Example
SWEDISH ANCESTRY (N=200)
NOT
ROMAN
CATHOLIC
162
NOT A1 allele
18
A1 allele
90%
ROMAN
CATHOLIC
18
90%
2
10%
10%
NO ASSOCIATION
IRISH ANCESTRY (N=200)
NOT
ROMAN
ROMAN
CATHOLIC CATHOLIC
35
105
70%
15
45
30%
25%
75%
NO ASSOCIATION
MINGLED IN U.S. POPULATION (N=400)
NOT
ROMAN
CATHOLIC
NOT A1 allele
197
A1 allele
33
ROMAN
CATHOLIC
123
47
OR = 2.28, 95%CI 1.39 - 3.73
Falsely infer that A1 allele is risk-factor for Roman Catholicism.
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
TRANSMISSION DISEQUILIBRIUM TEST
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 nontransmitted allele.
TRANSMISSION DISEQUILIBRIUM TEST
CONSISTENT WITH
PREDICTION
AGAINST
PREDICTION
MOTHER
FATHER
MOTHER
FATHER
ADH2*1 / *2
ADH2*1 / *1
ADH2*1 / *2
ADH2*1 / *1
ADH2*1 / *1
ADH2*2 / *1
ALCOHOLIC
ALCOHOLIC
TRANSMISSION-DISEQUILIBRIUM TEST:
A Medical Genetic Example
Ataxia-Telangiecstasia (AT) in Costa Rica
Transmission
Pattern
1
3
4
5
7
Allele
8 10
Transmitted
3
0
22
0
1
0
Not Transmitted
0
4
0
4
3
4
11
20
21
0
0
0
2
1
1
2
9
2 = 92.91, highly significant by permutation test
(Lange, 1997)
Population stratification: solution
3: Genomic Control methods