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Mendelian Randomization
A deconfounding method
Dr. Jørn Olsen
Epi 200B
February 23 & 25, 2010
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.
Violent death/cancer
Se-cholesterol
Cause
Confounding
Reverse causation?
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’.
This argument assumes that the APOE gene is
randomly allocated according to the laws of Mendel
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 = green
P-generation
Gametes
B/B
b/b
B
b
F1 generation
Gametes
F2 generation
BB/bb
B/b
homozygotes
Random process
B/b
B/b
Bb
Bb
B/B, B/b, b/B
b/b
Yellow
Green
3
1
Heterozygotes
Random process
Independent assortment:
The alleles of one pair of genes segregate independently of
other pairs of genes during gametogenesis
P generation
B/B A/A
Yellow, round
B = yellow b = green
b/b a/a
Green, wrinkled
A = round a = wrinkled
Gametes
BA
ba
F1
B/b A/a
Yellow/round
B/b A/a
Yellow/round
Gametes
BA Ba bA ba
BA Ba bA ba
F2
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)
Gametes
BA
Ba
bA
ba
BA
B/B A/A
B/B A/a
B/b A/A
B/b A/a
Ba
B/B A/a
B/B a/a
B/b A/a
B/b a/a
bA
B/b A/A
B/b A/a
b/b A/A
b/b A/a
ba
B/b A/a
B/b a/a
b/b A/a
b/b a/a
9 : 3 : 3 :1
Mendel’s Experiment - visit to home page
APOE +
APOE -
High Se-chol
Low Se-chol
If the APOE gene is randomized then we will not
expect an association between the genotype and
cancer under Ho
G
Genotype
IP
D
Low cholesterol
Cancer
G = gene
IP = intermediate phenotype
D = disease
If IP  D is confounded or due to reverse causation
D  IP, then there should be no G D association.
Low cholesterol
cancer
confounders
Cancer
low cholesterol
IP
G
D
G --- D
not present in
population at risk
Observations
Folate
NTD
Confounding?
Folate
NTD
C
C
Randomized trial
MRC-trial + trial in Hungary
Folate
Vitamins
High risk women
NTD
0
0
N00
D00
0
+
N0+
D0+
+
0
N+0
D+0
+
+
N++
D++
( D+ 0 + D++ ) / ( N +0 + N + + )
PR =
= 0.3
( D00 + D0+ ) / ( N 00 + N 0+ )
Could we have avoided the NTDs caused by low folate intake?
Mechanism
Folate
Homocystein
NTD
Genotype
MTHFR (TT)
Homocystein
NTD ?
If TT, Tt, tt are associated with different levels of
homocystein, one would expect these genotypes
to carry different risks for NTD
MTHFR (TT)
Homocystein
NTD
Since these genotypes are randomized at birth,
other correlates of homocystein are not expected
to be associated with the genotypes
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.
Alcohol and CVD
Most studies show the following:
CVD
alcohol drinks/day
0
6
Observation
Alcohol
CVD
Confounding
Alcohol
CVD
Diet
Smoking
Exercise
Etc.
All populations have slow and fast metabolizers of alcohol
guided by genetic factors
ALCOHOL
1
2
ACETALDEHYDE
ACETIC ACID
Slow metabolizers at step 1 = High level of alcohol
Slow metabolizers at step 2 = High level of acetaldehyde
Genotype
Slow metabolisers
But
Slow metabolisers
CVD
Alcohol
CVD ?
Genotype influence phenotype
Fixed alcohol intake: slow metabolisers
CVD
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.
Relative risks of myocardial infarction
according to the genotypes
Variable
ADH3 Genotype
P value
11
1  2
2 2
Patients
161 (51)
184 (46)
51 (13)
Controls
279 (36)
361 (47)
130 (17)
Matched
1.0
0.90 (0.69-1.17)
0.72 (0.50-1.05)
0.09
Multivariate
1.0
0.81 (0.61-1.09)
0.64 (0.43-0.98)
0.03
Multivariate,
with adjustment for
1.0
0.83 (0.62-1.11)
0.65 (0.43-0.99)
0.04
No. Of subjects (%)
Relative risk (95% CI)
alcohol consumption
Multivariate relative risk of myocardial infarction
according to the genotypes and the level of daily
alcohol consumption
1.2
1
0.8
Relative risk of
myocardial 0.6
infarction
0.4
y1
y2
y3
0.2
0
< 1 Drink/day > 1 Drink/day
ALDH2
Alcohol
Acetaldehyde
ALDH2•1•1
fast
ALDH2•2•2
slow
Acetic acid
Alcohol
esophageal cancer
Alcohol (not carcinogenic in animal models)
Confounding, modification of carcinogens – tissue
damage
Metabolite – acetaldehyde?
Elimination of acetaldehyde – ALDH2 enzyme
A point mutation in ALDH2
ALDH2 x 2 x 2
Allele and inability to metabolize acetaldehyde
Alcohol
ALDH2 x 2 x 2: 18 times higher peak
ALDH2 x 1 x 2: 5 times higher peak than
ALDH2 x 1 x 1
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
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
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
Plasma fibrinogen correlates with MI
A common mutation in G
fibrinogen levels.
A the -fibrinogen gene is associated with high
The G/A genotype correlated with plasma fibrinogen in men and in premenopausal women or post-menopausal women treated with HRT
ANOVA
p < 0.001
3,8
ANOVA
p < 0.001
****
3,6
*
3,4
****
G/G
3,2
G/A
3
A/A
2,8
2,6
2,4
GENOTYPE
Women
n = 4,889
Men
n = 3,972
If fibrinogen is a cause of MI, the G/A or A/A
genotype should have high risk of MI.
Without ischemic
heart disease
No. of women
With ischemic heart
disease
3937
174
G/G
0.63 (2467)
0.58 (101)
G/A
0.34 (1323)
0.40 (70)
A/A
0.04 (147)
0.02
Genotype
A-allele
0.21
No. of men
(3)
0.22
2915
315
G/G
0.64 (1877)
0.65 (204)
G/A
0.31 (913)
0.31 (98)
A/A
0.04 (124)
0.04 (13)
Genotype
A-allele
0.20
C-reactive protein (CRP) is a maker of systemic
inflammation.
CRP
High Blood Pressure (HBP)
- many studies show association
but could be
HBP
CRP – reverse causation
Or
CRP
C1
C2
HBP
Most studies have
Measurement errors of confounders
(obesity, smoking, social factors)
Most studies rely on cross-sectional data; inverse
causation
GG, GC, CC; G associated with high levels of CRP
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.
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.
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.
Alcohol
Acetaldehyde
Eliminated
Alcohol Dehydrogenates
ALDH2
ALDH 2x2x2 Homozygotes 18 times higher peak
acealdehyde levels
ALDH 2x1x2 Heterozygoter 5 times
ADH 2x1x1 Homozygotes
Acetaldehyde cause nausea, headache etc. that
makes drinking alcohol unpleasant-lowers the
exposure
Is alcohol in high levels causing hypertension?
Alcohol
BP
Diet
Obesity
Stress
Physical activity
ALDH2
Alcohol
CI..CN
BP
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
Alcohol intake in matter for males:
ALDH 2x1x1 20-30g /day
ALDH 2x1x2 10-15g /day
ALDH 2x2x2 0-2g /day
Or for ALDH 2x2x2 versus
ALDH 2x1x1 for hypertension in males
Difference in systolic
and diastolic blood
pressure for ALHD2x2x2
versus ALDHD 2x1x1
Hypothesis
A high level of intrauterine exposure to
testosterone increases the risk of autism / ADHD
(Simon –Cohen)
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
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?
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
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.
Problems
Population stratification/genetic confounding:
Different ethnic groups may have different genotypes
and different disease risks.
Linkage disequilibrium – association between genes
because they are located close together on the
chromosome.
Gene 1 and Gene 2 associated-are in linkage
disequilibrium:
Gen 2
Gene1
Conf
Exp
Disease
Is Gene 1 a useful instrumental variable for Exp?
Is Gene1 still a useful instrumental variable for
Exp?
Gene 2
Gene 1
Cont
Exp
Disease
Conf
Maternal
FTO
Maternal obesity
offspring obesity
Offspring FTO
Can maternal FTO be used as an instrumental variable for
Maternal obesity to study offspring obesity?
Canalization: environmental adaptation to a
specific genotype like for ALDH2
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.
Mendelian Randomization
 First described as such by Katan (Lancet 1986; i: 50781).
 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
Mendelian randomization in case-parent design
Mother
Genotype
aa´
Offspring
Genotype
aa
Father
Genotype
aa´
Offspring
Genotype
a´a´
Allele that is inherited
from each parent is
randomly determined
Offspring
Genotype
aa´
Offspring
Genotype
a´a
The case-parent triad design
aa’
aa
aa’
Parents
Cases
a’ more frequent in cases than expected
from Mendel laws (probability in this case
0.50)
Will a gene with a specific polymorphism (GX)
cause disease?
If 4 parents look like this
GX/G
GX/G
: ¾ of offspring expected to have the SNP
GX/G
G/G
: ½ of offspring expected to have the SNP
GX/ GX
G/G
: All will have the SNP
G/G
G/G
: None will have the SNP
2.25 of their 4 affected children are expected to have the GX
if the SNP correlate with the disease.
This expectation be tested, but a larger sample is needed.