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Genetics of Alcoholism
Part II
Ian Gizer
University of Missouri-Columbia
Columbia, MO, USA
[email protected]
Definitions
• Chromosomes – threadlike structures on
which individual genes are located
Karyotype of normal
human male
Chromosome #9
• Locus (location) and
allele (alternative form)
p
• Centromere, short (p)
and long (q) arms
q
Centromere
ABO locus
(9q34.1)
Definitions
• Chromatin: genetic material contained in
chromosomes – DNA & proteins (histones
and nonhistones)
• Euchromatin – less condensed/light bands;
coding DNA
• Heterochromatin – compacted/dark bands,
usually noncoding DNA
Chr 21
UM Bauer
Definitions
•
•
•
•
•
DNA: Deoxy ribonucleic acid
Purine and pyrimidine bases
Purines: Cytosine, Thymine
Pyrimidines: Adenine, Guanine
Double stranded (each strand has
full information content)
• Strands are held together by
(hydrogen) bonds that form
between the nucleotide bases of
the DNA molecule
Adenine (A) <====> Thymine (T)
Guanine (G) <====> Cytosine (C)
Definitions
• Gene: A sequence of DNA (a locus on a
chromosome) that is involved in (“codes
for”) the synthesis of a functional
polypeptide (proteins consist of one or
more polypeptides, which are strings of
amino acids).
Gene Structure
EXON – EX-pressed or coding DNA that is converted into protein
INTRON – IN-active or noncoding DNA that is not converted to protein
Definitions
• Transcription: One of the two DNA strands
is transcribed to a single-stranded nucleic
acid called ribonucleic acid (RNA) RNA has
the same bases as DNA except uracil (U)
substitutes for thymine (T).
• Translation: Conversion of the basic
informational unit of 3 nucleotide bases
(called a codon) into a single amino acid.
Example
Non-transcribed DNA strand
Transcribed DNA strand
TTT
AAA
TCC
AGG
Transcription
mRNA
UUU
UCC
Translation
Amino Acid
Phenylalanine
Serine
Genetic Variation
•
•
•
•
95% - 98% of human DNA does not code
directly for protein.
An estimated 99.8% - 99.9% of our DNA
is common.
But then .1% of 3,000,000,000 = 3 million
differences!
We are interested in these variations and
the transmission and co-aggregation of
these variations with AUDs.
Two major types
• Microsatellite/short tandem repeat (STR): a
stretch of DNA that is sequentially repeated
a variable number of times.
• Can cause disease (e.g. CAG repeat
expansion causes Huntington’s
disease;
• Can also be benign variation;
• Assume it is close to a disease
contributing gene;
Single Nucleotide Polymorphism
• SNPs are single base pair changes that occur
as natural variation in the human genome.
They can code for protein change (nonsynonymous) or not.
Two major methods for identifying
genes associated with AUDs
• Linkage
• Association
Linkage Analysis
AA (BB)
Aa (Bb)
AA (BB)
Aa (Bb)
AA (BB)
AA (BB)
AA (BB)
Aa (Bb)
Aa (Bb)
AA (BB)
LINKAGE
• Basic idea is identity-by-descent (IBD) or
how often does an affected pair of relatives
share the same ancestral DNA. If more
often than expected by chance, then
somewhere near this shared DNA is a gene
that contributes to affection status.
• Need related individuals where multiple
relatives are affected.
• Identifies large stretches of DNA.
Linkage Analysis: The Basics
IBD – An Illustration
A. One allele IBS and one allele IBD.
B. One allele IBS and zero alleles IBD.
C. Two alleles IBS and at least one allele IBD.
IBD Sharing in pairs affected for
disorder
Sib 1
AC
AD
BC
BD
AC
Sib 2
AD
BC
BD
4/16 = 1/4 sibs share BOTH parental alleles IBD = 2
8/16 = 1/2 sibs share ONE parental allele
IBD = 1
4/16 = 1/4 sibs share NO parental alleles
IBD = 0
LINKAGE via IBD
C/D
Sib 2
A/B
Sib 1
Sib 1
AC
AD
BC
BD
AC
2
1
1
0
AD
1
2
0
1
BC
1
0
2
1
BD
0
1
1
2
Sib 2
H(0): IBD (0) = 25%; IBD (1) = 50%; IBD (2) = 25%
H(A): IBD (0) < 25%; IBD (1) > 50%; IBD (2) > 25%
H(A) is evidence for linkage.
Linkage studies of AUDs
• Most prominent is Collaborative Study of
the Genetics of Alcoholism (COGA).
• Has identified many important genetic
regions using STRs and SNPs.
COGA strategy
1. Ascertain multiplex alcoholic families
Polydiagnostic interview
Electrophysiological data
262 Families, 2282 individuals
2. Linkage analyses to identify chromosomal regions
2.5
Wave 1
2
LodScores
Wave 2
1.5
Combined
1
0.5
0
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160
 allele-sharing among affecteds
within a family
cM
3. Association analyses to identify specific genes
Gene A
Gene B
Gene C
LOD score
LOD = Likelihood
of Odds;
LOD of 3.0
means it is 1000
times more likely
than expected by
chance that there
is linkage.
Log101000 = 3
Higher the LOD,
more likely genes
are nearby
Williams et al., 1999
Irish affected sib pair study
Prescott et al., 2006
Problems with Linkage
• Methodological
problems;
• Need BIG sets of
families;
• Home in on a big
chunk of DNA –
possibility of
hundreds of
genes!!!
1 cM (centiMorgan) is approximately equal to
1 Megabase or 1000000 bp!!!!
Genes may be anywhere in the 50cM region
Association Analysis
Cardon & Bell, 2001 Nat Rev Genet
Association
• Family Based(transmission disequilibrium test)
A/a
a/a
A/a
a/a
A/a
A/a
A/a
a/a
aa
A/a
• How often is the risk allele transmitted to an
affected child from a parent who is
heterozygous (A/a) for the SNP?
Association
• Case/Control Design
a/a
a/a
A/a
A/a
A/a
a/a
A/a
A/a
• Is the prevalence of the risk allele greater in
affected versus unaffected people?
Which Genes should I look at?
1. Genes in a linkage region
2. Genes that metabolize alcohol
(candidates)
3. All genes
Genes in the linkage region
GABRA2:
gamma-amino butyric acid receptor A, subunit 2 gene
GABA
major inhibitory neurotransmitter of the central nervous system
• GABA & Alcohol
–
–
–
–
–
–
(Buck, 1996; Grobin et al., 1998)
motor incoordination
anxiolytic effects
sedation
ethanol preference
withdrawal signs
tolerance & dependence
• GABAA receptor agonists tend to potentiate the
behavioral effects of alcohol, while GABAA
receptor antagonists attenuate these effects
GABRA2 and
AUDs
Region contains:
• GABRG1
• GABRA2
• GABRA4
• GABRB1
Edenberg et al., 2004
Many replications…
• Many studies now show an association
between SNPs in GABRA2 and AUDs.
• SNPs are also associated with drug
dependence, nicotine dependence, conduct
problems and antisocial personality
disorder – likely to be general vulnerability
to thrill seeking.
• Replicated in family-based and case-control
studies.
Genes that metabolize alcohol
ADH cluster
(1a,1b,1c,4,5,6,7)
5
4.5
4
Symptom Count
Alcohol Dependence
3.5
3
2.5
LOD 2
1.5
1
0.5
0
0
50
100
150
-0.5
-1
chromosome 4 position (cM)
200
Flushing Response
• Dysphoric effects that occur w/i 15
minutes of drinking:
– Heart palpitation
– Facial reddening
– Nausea, dizziness
• There are large ethnic group differences
in rate of flushing – metabolic not
cultural
Pathway of Alcohol
Metabolism
Alcohol
Acetaldehyde
ADH
Acetate
ALDH
ALDH2 Deficiency
• ADH1B*2, ADH1C*1 code for protein
subunits that have greater enzymatic
activity, suggesting faster conversion to
acetaldehyde
• ALDH2*2 – inactive enzyme, can’t break
down acetaldehyde
– Causes facial flushing, nausea
ADH1B(2)*2  faster to acetaldehyde
PROTECTIVE EFFECTS
ADH1C(3)*1  faster to acetaldehyde
ALDH2*2  slower breakdown acetaldehyde
ADH2*2 less
common in
alcoholics
ADH3*1 less
common in
alcoholics
ALDH2*2 less
common in
alcoholics
Wall et al. (2001)
Wall et al. (2001)
MacGregor et al., 2009
3
Alcohol Dependence
rs3762894
Withdrawal
rs2066702
Severity
-Log10p
2
1
0
ADH5
ADH4
ADH6
ADH1A
ADH1C
ADH1B
ADH7
Gizer et al., 2011
Examine ALL genes
• Called GWAS: Genomewide association
study;
• Saturate the genome with a million SNPs
and then test association with each SNP.
• Maybe find something new!
Treutlin et al., 2009
Bierut et al., 2010
Problems with association studies
1.
Population stratification (only when using
unrelateds) –when an association between a
SNP and AUDs is due to ethnic variation in that
SNP.
2. P-values need to be adjusted for testing many
markers (e.g. 0.05/#markers tested).
3. Replication in other samples.
4. What does the gene/SNP do in the etiology of
AUDs?
ENDOPHENOTYPES
• Inherited mediators;
• Associated with, but not a consequence of,
alcoholism;
• Transmitted in families of alcoholics;
• Present when disorder is not in active phase;
• Heritable;
• Examples: EEG, P300, Subjective
response to alcohol.
Irv Gottesman
Why study EEG for AUDs?
• EEG (Electro-encephal0grams) of waves suggest
that certain EEG activity is associated with risk for
AUDs;
• EEG is heritable;
• In families with AUDs, unaffected relatives of
AUD individuals have distinct EEG patterns;
• EEG pattern is not modified when an individual
goes into recovery;
• EEG is an ENDOPHENOTYPE for AUDs
EEG readings
EEG Waves
• Alpha waves : major rhythm seen in normal relaxed
adults - it is present during most of life especially
beyond the thirteenth year when it dominates the
resting tracing.
• Beta activity : dominant rhythm in patients who are
alert or anxious or who have their eyes open.
• Theta activity abnormal in awake adults but is
perfectly normal in children upto 13 years and in
sleep.
• Delta activity : quite normal and is the dominant
rhythm in infants up to one year and in stages 3 and
4 of sleep.
Ref: http://www.brown.edu/Departments/Clinical_Neurosciences/louis/eegfreq.html
EEG Heritabilities
Frequency band
Mean h2
Delta (1.5-3.5 Hz)
76%
Theta (4-7.5 Hz)
89%
89%
86%
Alpha (8-12.5 Hz)
Beta (13-25 Hz)
Van Beijsterveldt et al., 1996
Increased BETA Log Power in Alcoholics (F3-C3)
0.4
CONTROL (n= 257)
0.35
ALCOHOLICS (n=271)
log power
0.3
0.25
0.2
0.15
0.1
0.05
0
-0.05
p-values :
BETA1
BETA2
BETA3
0.004
0.007
0.004
Rangaswamy et al., 2002
Increased BETA Power in offspring of alcoholics
Beta 1
Beta 2
Beta 3
*Significant for all beta bands, particularly Beta 1 for males, and Beta 2 and Beta 3 for females
HR=high risk; LR=low risk
Rangaswamy et al., 2004
P300
•
•
•
•
•
Event-related potential (ERP)
P300 /oddball task
Subject attends to rarer of two cues
Rarer the event = larger the amplitude
Reflects context/memory updating whereby
current model of environment is updated
with incoming info.
Rangaswamy & Porjesz: P300 amplitude is reduced in alcoholics
Carlson et al., 2004
P300 amplitude
30
Alc
25
No Alc
20
Discordant stable
Newly Discordant
Unaffected
Discordant stable: One twin has AUD, other does not;
Newly discordant: One twin develops AUD, other does not;
Heritable across all levels of alcohol
use
Perlman et al., 2009
Sensitivity to Alcohol: SRE
• Self-rating
of the
effects of
alcohol
(Schuckit
et al, 1997)
Twin Study
(Heath, et al.1999)
Behavioral Sensitivity
(Schuckit, 1984)
Family history
negative
Family history
positive
Schuckit et al., 1994
Problems with Endophenotypes
• Not specific (e.g. P300 amplitude reduction
is also associated with schizophrenia);
• Links between endophenotype and
phenotype maybe unknown;
• Underlying genetic architecture may not be
any less complex;
• Requires special equipment/lab and subject
consent;
Genetic Strategies with Animals
• Forward Genetic Approaches (phenotypedriven)
– Inbred strains
– Selectively bred strains
– Mutagenesis
QTL mapping
• Reverse Genetic Approaches (genotypedriven)
– Transgenics
– Knockouts
Importance of the Mouse
Genome
• Mouse genome (Nature, December 5, 2002):
– 2.5Gb
– ~27,000 – 30,500 genes
• Relationship to human genome:
– ~99% of mouse genes have counterparts (orthologs) in
human
– ~96% of human genes have orthologs in mouse
– Conservation of some non-coding regions
– Synteny – stretches of DNA that are the same in mouse
and human
Alcohol Preference
• % of times in 14-day period animal selects
10% ethanol solution vs. tap water (both a
sweetened with saccharin)
• Marked differences between strains, 0-80%
Selection for
Alcohol Preference
Li et al., 1993
Sleep Time: Loss of Righting Reflex
(LORR)
Markel et al., 1997
Behavioral Examples –NPY
(Theile et al. Nature, 1998)
• Neurotransmitter known to be a potent
stimulator of appetite
• Relevance to alcohol:
– QTL studies of rat preference map to NPY region
– Inbred strain comparisons
• Knock-out (loss-of-function) – increased ETOH
consumption & decreased sleep time
• Transgenic (gain-of-function) – decreased
consumption and increased sleep time
Why we are not animals…
• Animals self administer alcohol and drugs –
so do we – but, often, there is a social
context for alcohol use in humans.
• The motivational model of alcohol use is
strongly linked to continued drinking.
• Environmental modified.
• Rather complex to study in animals.
Drinking motives (Cooper et al.)
• Drinking motives (How often do you drink to …?) stem
from a motivational model of alcohol use – we drink to
achieve a certain socio-cognitive outcome (e.g. drink to
reduce stress and/or drink to fit in with friends);
• Motives have both valence (positive/negative) and source
(internal/external).
• Motives are moderately heritable (Prescott et al., 2004;
Agrawal et al., 2008).
• They share genetic influences with alcohol consumption
(Prescott et al., 2004) – they moderate the genetic links
between personality and alcohol consumption (Littlefield
et al., in prep).
Andrew Littlefield
M. Lynne Cooper
Why do we DRINK?
Kuntsche et al., 2005, Clin Psych Rev
WHY DO WE DRINK?
• Coping motives
– How often do you drink to forget your worries?
• Enhancement Motives
– How often do you drink because you like the
feeling?
• Social Motives
– How often do you drink to be sociable?
• Conformity Motives
– How often do you drink so you won’t be left out?
Prescott et al., 2004
PART II
• Genetic regions have been identified for
alcoholism: chromosomes 2,4,5,7
• Genes: GABRA2, ADH cluster
• GWAS largely unsuccessful
• Endophenotypes replicate results with
AUDs but tend to be generalizable to
externalizing behaviors.
• Animal studies lack context of drinking.
What next for the genetics of
alcoholism?