Genetic Association Study Principles:

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Genetic Association Study Principles

• Andrew C. Heath

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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)
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Case-Control Study

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

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ALCOHOL METABOLISM
Alcohol Dehydrogenase (ADH)
Aldehyde Dehydrogenase (ALDH)
ALCOHOL ACETALDEHYDE ACETATE
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Higuchi Data -- Japanese Alcoholics and Controls
ALDH2 locus
Controls Alcoholics (N461) (N655)
Locus Genotype () () ALDH2 1 / 1 58 88 1 /
2 35 12 2 / 2 7 0
p lt .001
(Higuchi, 1994)
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Higuchi -- Changes in ALDH21/2 Frequency in
Japanese Alcoholics
1979 1986 1992 (N400) (N400) (N500)
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
(Higuchi, 1994)
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Higuchi Data -- Japanese Alcoholics and Controls
ADH1B locus (in those who are ALDH21/1
homozygotes)
Population Controls Alcoholics
ADH1B 2 / 2 58.1 35.8 2 / 1 34.7
33.7 1 / 1 7.3 30.4
From Higuchis community data, in
individuals who are also ALDH21/1 homozygotes,
we may estimate the penetrance of the AHD1B2/2
genotype (the low risk genotype) as 0.07, that
of the high risk ADH1B1/1 genotype as 0.29.
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Higuchi Data -- Genetic effects on
alcohol consumption levels in a community
sample Average monthly alcohol consumption (ml
pure alcohol)
Genotype MEN
WOMEN ALDH21/1 1054.7 104.9 ALDH21/2 390.9 46.
6 ALDH22/2 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)
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• Conclusion there are genes with powerful effects
  on behavioral traits waiting to be discovered!

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FREQUENCIES OF GENES INFLUENCING ALCOHOL
METABOLISM
High Risk Japanese European Locus Allele Ancestry
Ancestry ALDH2 ALDH21 76 100 ADH1B ADH1B1 25
95 ADH1C ADH1C2 6 45
NOTE Predicted magnitude of effects is ALDH21
gtgt ADH1B1 gtgt ADH1C2.
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Table 2. Lifetime DSM-III-R Alcohol
Dependence by ADH1B (ADH2) Type Australian
twin panel
Whitfield et al., 1998
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But how do we know this is THE gene?

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Population Genetics of ADH gene region

• See Kidd paper (Osier et al., Am J Hum Genet
  7184-99, 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 (lt
  5 haplotype frequency).

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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 (lt 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).

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POPULATION STRATIFICATION Hypothetical Example
SWEDISH ANCESTRY (N200)
IRISH ANCESTRY (N200)
NOT ROMAN CATHOLIC
ROMAN CATHOLIC
NOT ROMAN CATHOLIC
ROMAN CATHOLIC
35 15 25
105 45 75
70 30
162 18 90
18 2 10
90 10
NOT A1 allele A1 allele
NO ASSOCIATION
NO ASSOCIATION
MINGLED IN U.S. POPULATION (N400)
NOT ROMAN CATHOLIC
ROMAN CATHOLIC
NOT A1 allele A1 allele
197 33
123 47
OR 2.28, 95CI 1.39 - 3.73
Falsely infer that A1 allele is risk-factor for
Roman Catholicism.
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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
ADH21/1 second twin was
ADH21/2 In all 5 pairs, 1/1 had higher
consumption (p .03)
(Whitfield et al., 1998)
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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 non-transmitted allele.
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TRANSMISSION DISEQUILIBRIUM TEST
CONSISTENT WITH PREDICTION
AGAINST PREDICTION
MOTHER ADH21 / 2
FATHER ADH21 / 1
MOTHER ADH21 / 2
FATHER ADH21 / 1
ADH21 / 1 ALCOHOLIC
ADH22 / 1 ALCOHOLIC
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TRANSMISSION-DISEQUILIBRIUM TEST A Medical
Genetic Example
Ataxia-Telangiecstasia (AT) in Costa Rica
Allele
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
?2 92.91, highly significant by permutation test
(Lange, 1997)
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Population stratification solution 3 Genomic
Control methods