Alcoholism: From Genotypes to Genes and Back Again

1
Alcoholism From Genotypes to Genes (and Back
Again)

• Carol A. Prescott, Ph.D.
• University of Southern California
• cprescot_at_usc.edu

2
Overview

• Definitions
• Epidemiology
• Consequences
• Genetic Epidemiology
• Search for Susceptibility Genes

3
DSM-IV Criteria for Alcohol Dependence

• 3 or more of the following, lasting gt1 month
• withdrawal or drinking to avoid withdrawal
• tolerance
• inability to quit despite wanting to
• loss of control once start drinking
• continued use despite medical consequences
• drinking interferes with functioning
• time spent drinking to exclusion of important
  life activities

4
Epidemiology of Alcohol Dependence

• Lifetime prevalences (among drinkers)
• Males 8 -12
• Females 3 - 4
• 1-yr prevalence in EU (Wittchen Jacobi, 2005)
• Males 5.6
• Females 1.3
• 7.2 Million

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Consequences of Alcoholism

• Formal Treatment only 10 of affected
• Success rate 30
• Personal Costs
• Impaired functioning
• Accidents, Health problems
• Societal Costs (billions annually)
• Important comorbidity tobacco, depression (in
  top 10 of DALYs)

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A Growing Problem

• Finland 50 of males aged 30-49 binge gt1x/mo
• (WHO, 1993)
• Russia 500K deaths annually (Lancet 2006)
• Poland Sharp increase in alcohol-related
  mortality since mid-1980s (Wojtyniak et al 2005)
• UK 31 increase in per capita consumption
  between 1980 and 2000 (Hall, 2005)

7
Consequences of Moderate Alcohol Use

• Personal Benefits
• - stress reduction
• - cardioprotection
• - social lubrication
• Societal Benefits
• - tax revenue
• - employment

8
Genetic Epidemiology

• Adoption studies
• Rates of alcoholism higher in adopted offspring
  of alcoholic biological parents than
  non-alcoholic biological parents

9
Risk of alcoholism among adoptees with and
without biological alcoholic parents
Studies of male adoptees Studies of
female adoptees
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Genetic Epidemiology

• Adoption studies
• Rates of alcoholism higher in adopted offspring
  of alcoholic biological parents than
  non-alcoholic biological parents
• Twin studies
• Resemblance for alcoholism higher in MZ twin
  pairs than DZ pairs

11
Genetic and environmental proportions of variance
in alcoholism from studies of male twins
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Genetic and environmental proportions of variance
in alcoholism from studies of female twins
Clinical sample cotwin followup
Volunteer registry personal interview
Population registry archival diagnosis
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Alcoholism A Complicated Complex Disorder

• Important behavioral component
• Cultural influences
• Common (phenocopies)
• Benefits of moderate use
• Clinical heterogeneity
• Etiological heterogeneity
• Specific vs general risk

14
Estimated Common and Specific Genetic Variance
for Psychiatric and Substance Use
Disorders(Males and Females Combined)
A1
A2
28
2
11
lt1
1
34
3
42
5
31
1
14
29
6
Generalized Anxiety
Phobia
Alcohol Dependence
Drug Abuse/ Dependence
Adult Antisocial Behavior
Major Depression
Conduct Disorder
0
0
5
14
3
0
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As
As
As
As
As
As
As
Kendler, Prescott, Myers Neale, Arch Gen
Psychiatry, 60 929-937, 2003
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Alcoholism A Complicated Complex Disorder

• Important behavioral component
• Cultural influences
• Common (phenocopies)
• Benefits of moderate use
• Clinical heterogeneity
• Etiological heterogeneity
• Specific vs general risk
• Different influences for different stages of
  alcohol involvement

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Stages of alcohol involvement
17
Genetic influences on stages of alcohol
involvement
18
Irish Affected Sib-Pair Study of Alcohol
Dependence (IASPSAD)

• Background Design
• Results
• Genome Scan
• Psychometric Follow-up
• Molecular Follow-up

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Collaborators

• Irish Collaborators
• Dermot Walsh Irish Health Research Board,
  Dublin
• Diana Patterson Shaftsbury Square Hospital,
  Belfast

• Study Design / Data Collection
• Kenneth Kendler
• Patrick Sullivan
• Molecular Genetics
• Brien Riley
• Gursharan Kalsi
• Jeff Alexander
• Jen Vittum
• Statistical Analysis
• Po-Hsiu Kuo
• Edwin van den Oord
• Michael Neale
• Todd Webb
• John Myers

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Other Acknowledgments

• Project Support
• Irish Health Research Board
• Ros Moran
• Carol Cronin
• Queens Hospital, Belfast
• F. Anthony ONeill
• Virginia Commonwealth Univ.
• Stacey Garnett
• Jill Opalesky
• Indrani Ray
• Cheryl Smith
• Rebecca Ortiz
• NIAAA
• Robert Karp
• Project Consultants
• David Goldman, NIAAA
• John Crabbe, Oregon Health Sciences

• Project Management
• Lisa Halberstadt, U.S.
• Margaret Devitt, Dublin
• Victor Robinson, Belfast
• Interviewers
• Craig Barton, John Cosgrove, Michael Crossan,
  Sara Dineen, Phil Gavigan, Claire Killeen,
  Deirdre King, Siobhan McHugh, Amanda Mullan,
  Eileen Murphy, Brian O'Malley, Bernie Purcell
• Blood collection genotyping
• Irish Blood Transfusion Service, Dublin
• Northern Ireland Blood Transfusion Service,
  Belfast
• Irish Gardai, Dublin
• deCODE Genetics, Iceland
• Funding
• NIAAA R01-AA-11408

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IASPSAD Study Design
Probands ascertained Interview
DNA N591 (M364, F227)
Affected siblings referred Interview
DNA N610 (M413, F197)
733 sib pairs (sibship size 2-8)
Parents contacted Brief Interview
DNA N213 (M82, F131)
Control Groups Fully Screened n
72 Self-Screened n 800
Prescott et al., Alc Clin Exp Res, 2005
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Study Inclusionary Criteria

• DSM-IV Alcohol Dependence
• gt1 affected siblings
• All four grandparents born in Ireland or UK
  (England, Scotland or Wales)

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Proband Sibling Assessment

• DSM-IV Alcohol Dependence
• Semi-Structured Assessment of the Genetics of
  Alcoholism (SSAGA, Bucholz et al., 1989)
• Treatment records
• Other Clinical Measures
• Nicotine Dependence
• Other substance use, abuse dependence
• Antisocial personality disorder
• Major depression
• Drinking expectancies
• Subjective response (SRE, Schuckit et al. 1997)
• Family history of alcoholism
• DNA
• Blood 85, Buccal cell 15

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Measurement Issues

• Clinical Diagnosis
• Validity (convergent)
• Clinical characteristics
• Reliability (inter-rater)
• Sibling reports
• Genotyping
• Genotyping error (0.3 in 17 repeat Ss)
• Population substructure

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Distribution of DSM-IV Alcohol Dependence
criteria in IASPSAD probands and siblings
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Genotyping and Data Analysis

• 4 cM autosomal genome scan (deCODE, 1081 markers)
  
• 474 families informative for linkage
• 700 sib pairs (all possible pairs)
• Outcomes
• AD DSM-IV Alcohol dependence
• SX DSM-IV AD symptom count (range 3-7)
• Linkage analysis
• NPL - Merlin (Abecasis et al., 2003)
• empirical p-values by bootstrapping

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Genome-wide scan results for DSM-IV Alcohol
Dependence
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Suggestive Linkage Regions for Alcohol Dependence
(NP LOD gt1.0)
Dick et al. 2002 (alc factor) Bergen et al.
2003 (max drinks)
Bergen et al. 2003 (max drinks)
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Genome-wide Scan results for DSM-IV Alcohol
Dependence and AD Symptoms
CH4
Prescott, Sullivan, Webb, Vittum, Patterson,
Thiselton, Devitt, Halberstadt, Robinson,
Neale, van den Oord, Walsh, Riley Kendler,
Molecular Psychiatry, 2006
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IASPSAD Chromosome 4 Linkage Results
Peak LOD 4.59 (plt.000002)
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Chromosome 4 Linkage to AD Symptoms
SW American Indians AD - Long et al.
1998 COGA (US multiplex families) symptoms
- Reich et al. 1998 max drinks - Saccone et
al. 2000 alc response - Schuckit et al. 2001
severity - Corbett et al. 2005 Mission American
Indians severity - Ehlers et al. 2004
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Follow-Up Work

• Psychometric
• Where is the AD - SX difference coming from?
• What else can we learn from linkage studies?

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IASPSAD Chromosome 4 Linkage Results
Peak LOD 4.59 (plt.000002)
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Comparing the Alcohol Dependence (AD) andSymptom
Count (SX) Linkage Evidence
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Symptom Dropping Analyses NPL LOD Scores for AD
Symptom count and Symptom count - Withdrawal
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NPL LOD Scores for Symptom Dropping Analyses
ADSX
medical consequences lack of control restricted
activities withdrawal failed to
quit binging tolerance
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Linkage Analysis Strategies

• Bivariate models Alcohol dependence with
  comorbid disorders

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Ch 7 linkage to alcohol dependence and major
depression from COGA study
Wang et al, Human Molec Genet 2004
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Bivariate linkage results for Nicotine Dependence
and Alcohol Dependence in IASPSAD
Sullivan et al, under review
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Linkage Analysis Strategies

• Bivariate models Alcohol dependence with
  comorbid disorders
• Components of alcohol dependence
• Tolerance
• Withdrawal
• Initial Sensitivity

42
Ch 1 Linkage Results for Ethanol Sensitivity and
Tolerance In IASPSAD
Kuo et al, under review
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Linkage Study Conclusions

• Strong evidence for linkage of AD symptom count
  to chromosome 4
• Replicates findings from 3 independent samples
• Psychometric follow-up implicates tolerance
  binge drinking
• Bivariate and component analyses suggest
  additional regions (and complex mechanisms)

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Follow-Up Work

• Psychometric
• Molecular
• Which genes?

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Association Study Strategy

• Multi-stage design -- minimize False Discovery
  Rate (van den Oord et al., 2003)
• Stage 1 Screening all candidate genes with
  case-control analyses
• select sample size adequate to detect genes with
  10 effect size at plt.10 N310 in each group
• Stage 2 Internal replication candidates which
  pass Stage 1 tested using remaining sample
  (Ngt800)
• Stage 3 Replication in independent sample

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IASPSAD Chromosome 4 Linkage Results
ADH cluster (1a,1b,1c,4,5,6,7)
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Role of ADH in ethanol metabolism
alcohol dehydrogenase (ADH) ADH1B ADH1C
aldehyde dehydrogenase (ALDH) ALDH2
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Association Studies of Alcohol Dehydrogenase
(ADH)

• 27 SNP markers identified in 7 ADH genes
• Unrelated Case-Control association design
• Stage 1
• 328 cases (probands affected siblings)
• 328 screened population controls
• Stage 2 underway
• Single-marker analyses
• Two markers significant (plt.01, plt.06)
• Haplotype analyses

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Block structure of ADH genes in IASPSAD sample
5 4
6 1a 1b
1c 7
from Haploview program
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Haplotype association results block 4 ADH7
CA haplotype is protective for Alcohol
Dependence OR 1.20
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Association Work in Progress

• Ch 4 follow-up
• Additional markers within ADH-7
• 300 SNPs within Ch4 linkage region
• GWA using pooled samples
• Functional candidates
• 133 alcoholism-susceptibility genes (1536 SNP)
• GABA cluster on Ch 5 (Kalsi et al., 2005)
• Candidates from animal models (mouse, C elegans,
  drosophila)

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Hangover gene

• hangover involved in the development of alcohol
  tolerance in drosophila (Scholz et al Nature
  2005)
• Human ortholog ZNF699 (Ch19)
• 1061 cases, 609 controls from IASPSAD
• 7 SNPs by FP-TDI

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ZNF699 association with Alcohol Dependence(Riley
et al. submitted)

• 4 of 7 SNPs associated
• Single marker plt.001
• Haplotype plt.00008 OR 2.2
• Expression studies in postmortem brain (n34)
• Individuals with risk haplotype mRNA expression
  significantly reduced (plt.02) in dorso-lateral
  prefrontal cortex
• Possible mechanism
• Down-regulation of transcriptional repressor
  facilitates neural changes in response to ethanol
  exposure

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Conclusions

• Replicated susceptibility loci for alcohol
  dependence
• Distinct genes underlie different aspects of
  dependence, including tolerance, sensitivity and
  withdrawal
• Results from clinical studies, animal models and
  genetic epidemiology continue to guide the search
  for susceptibility loci

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