Genetics of Alcoholism

Part II

Ian GizerUniversity of Missouri-Columbia

Columbia, MO, USAgizeri@missouri.edu

Definitions• Chromosomes – threadlike

structures on which individual genes are located

Karyotype of normalhuman male

• Locus (location) and allele (alternative form)

• Centromere, short (p) and long (q) arms

Chromosome #9

ABO locus

p

q

Centromere

(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 proteinINTRON – 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

TTT TCCAAA AGG

UUU UCC

Transcription

Phenylalanine Serine

Translation

Non-transcribed DNA strand

Transcribed DNA strand

mRNA

Amino Acid

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 (non-synonymous) 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 BasicsIBD – 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

Sib 2

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

AC AD BC BD

AC

BCBD

AD

A/B C/D

AC AD BC BDAC 2 1 1 0AD 1 2 0 1BC 1 0 2 1BD 0 1 1 2

Sib 1 Sib 2

Sib 1

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 via IBD

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

0

0.5

1

1.5

2

2.5

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160cM

Lod

Sco

res

Wave 1

Wave 2

Combined

1. Ascertain multiplex alcoholic families

2. Linkage analyses to identify chromosomal regions

3. Association analyses to identify specific genes

allele-sharing among affecteds within a family

Gene A Gene B Gene C

Polydiagnostic interviewElectrophysiological data262 Families, 2282 individuals

LOD score

Williams et al., 1999

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

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

Cardon & Bell, 2001 Nat Rev Genet

Association Analysis

Association• Family Based(transmission

disequilibrium test)

• How often is the risk allele transmitted to an affected child from a parent who is heterozygous (A/a) for the SNP?

A/a a/a A/a aa a/a A/a

A/a A/a a/a A/a

Association• Case/Control Design

• Is the prevalence of the risk allele greater in affected versus unaffected people?

a/a a/a

A/a a/a

A/a A/a

A/a A/a

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 & 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

GABAmajor inhibitory neurotransmitter of the central nervous

system

GABRA2 and AUDs

Edenberg et al., 2004

Region contains:• GABRG1• GABRA2• GABRA4• GABRB1

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

-1

-0.5

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

0 50 100 150 200

chromosome 4 position (cM)

LOD

Symptom CountAlcohol Dependence

ADH cluster (1a,1b,1c,4,5,6,7)

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 Acetate

ADH 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 acetaldehydeADH1C(3)*1 faster to acetaldehydeALDH2*2 slower breakdown acetaldehyde

PROTECTIVE EFFECTS

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)

Edenberg et al., 2006

MacGregor et al., 2009

0

1

2

3-Log

10p

ADH5 ADH4 ADH6 ADH1A

ADH1B

ADH1C ADH7

Alcohol Dependence

Withdrawal

Severity

rs3762894

rs2066702

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 HeritabilitiesDelta (1.5-3.5 Hz) 76%Theta (4-7.5 Hz) 89%Alpha (8-12.5 Hz) 89%Beta (13-25 Hz) 86%

Van Beijsterveldt et al., 1996

Frequency band

Mean h2

Increased BETA Log Power in Alcoholics (F3-C3)

-0.05

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

BETA1 BETA2 BETA3p-values : 0.004 0.007 0.004

log

pow

er

CONTROL (n= 257) ALCOHOLICS (n=271)

Rangaswamy et al., 2002

*Significant for all beta bands, particularly Beta 1 for males, and Beta 2 and Beta 3 for femalesHR=high risk; LR=low risk

Increased BETA Power in offspring of alcoholics

Rangaswamy et al., 2004

Beta 1 Beta 2 Beta 3

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

Discordant stable: One twin has AUD, other does not;Newly discordant: One twin develops AUD, other does not;

20

25

30

Discordant stable Newly Discordant Unaffected

P300

am

plitu

de

Alc

No Alc

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 historypositive

Family historynegative

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 (phenotype-driven)– Inbred strains– Selectively bred strains– Mutagenesis

• Reverse Genetic Approaches (genotype-driven)– Transgenics– Knockouts

QTL mapping

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).

M. Lynne CooperAndrew Littlefield

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?