Genetics of Alcoholism Part II Ian Gizer University of Missouri-Columbia Columbia, MO, USA

Definitions Chromosomes – threadlike structures on which individual genes are located Karyotype of normal human 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 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 TTTTCC AAAAGG UUUUCC Transcription PhenylalanineSerine 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% % 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)

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 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 ACADBCBD AC BC BD AD

A/B C/D ACADBCBD AC2110 AD1201 BC1021 BD0112 Sib 1 Sib 2 Sib 1 Sib 2 H(0): IBD (0) = 25%; IBD (1) = 50%; IBD (2) = 25% H(A): IBD (0) 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 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 AGene BGene C Polydiagnostic interview Electrophysiological data 262 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. Log = 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 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/aa/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 GABA A receptor agonists tend to potentiate the behavioral effects of alcohol, while GABA A receptor antagonists attenuate these effects GABA major 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 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 AlcoholAcetaldehydeAcetate ADHALDH

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 ADH1C(3)*1  faster to acetaldehyde ALDH2*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)

Edenberg et al., 2006

MacGregor et al., 2009

Log10 p ADH5ADH4ADH6ADH1A ADH1B ADH1CADH7 Alcohol Dependence Withdrawal Severity rs rs 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:

EEG Heritabilities Delta ( Hz) 76% Theta (4-7.5 Hz) 89% Alpha ( Hz) 89% Beta (13-25 Hz) 86% Van Beijsterveldt et al., 1996 Frequency bandMean h2

Rangaswamy et al., 2002

*Significant for all beta bands, particularly Beta 1 for males, and Beta 2 and Beta 3 for females HR=high risk; LR=low risk Increased BETA Power in offspring of alcoholics Rangaswamy et al., 2004 Beta 1Beta 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; Discordant stableNewly DiscordantUnaffected P300 amplitude 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 history positive Family history negative

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 Cooper Andrew 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?