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Genetics of Alcohol Response Addiction Medicine State of the Art Conference October, 2003 Ray White Alcoholism • Many definitions – Precision important for specific studies – Working definition: • alcohol craving has become encompassing drive • Individual is losing, or has lost, job, family, health • Economic and personal costs astronomical – 10s of thousands of traffic deaths each year – Affect not only individual, but family, bystanders… • Cultural context Genetics and Molecular Etiology • Cell and molecular biology describe molecular pathology of disease state • Often cannot distinguish cause from effect or consequence of disease – Important to understand chain of causality in order to intervene – Genetics can define components that are “sufficient” to cause disease • APC gene • Genetics can identify etiologic components not seen by cell, molecular biology analyses – APC gene – Molecular mechanism – Therapeutic targets Alcoholism Has Genetic Component Twin Pair Concordance Rates Twin Group #Pairs Prevalence % S.E. Concordance % S.E. MZF 932 5.3 0.6 29.5 6.1 DZF 534 6.4 0.7 16.7 6.1 MZM 396 21.2 1.7 56.1 4.6 DZM 231 24.2 2.0 33.3 5.7 DZUS 592 M 26.1 1.7 59.5 8.1 14.0 2.8 F Heath et al, Psych Med 27:1381 (1997) 6.4 0.9 Genes for Alcoholism How do we find them? • Positional cloning with families – – Global scan for disease susceptibility genes (+) All genes in region become candidates * M1 * M1 * * * Ascertain family cluster * M1 * M1 * M1 * M1 * M1 * M1 * M1 Expand into pedigree Analyze by linkage * M1 Positional Cloning with Families * M1 * M1 * M1 * M1 * M * * 1 M1 M1 * * * M1 M1 M1 13 13 12 12 11 11 11 11 12 12 21 21 22 22 23 23 24 24 25 25 • Global scan for disease susceptibility genes (+) • All genes in region become candidates L5.63 L5.63 M1 L5.79,71,63 L5.79,71,63 MCC MCC 500 500 kb kb 5 mb DP1 DP1 SRP14 100 100 kb kb SRP19 30-40 30-40 mb mb CHROMOSOME 5 260 260 kb kb APC L5.48 L5.48 * * Genes for Alcoholism Positional Cloning with Families Reich et al. Amer. J. Med Gen 81:207 (1998) Genes for Alcoholism COGA Replica Set Faroud et al, Alcohol: Clin and Exp Res 24:933 (2000) Genes for Alcoholism Linkage Mapping in Families: Conclusions and Caveats • Significant linkage findings strongly support genetic component to alcoholism • Broad peaks – Many genes implicated – 20 to 40 genes in most regions – More data does not solve problem • Replication uncertain – Peaks lower, breadth maintained • Only rarely has led to gene identification in complex diseases – – But often in rare, syndromic diseases Genes for Alcoholism Associations in Populations SNP1 Dvariant SNP2 SNP3 High frequency marker SNPs (alleles @ 10% - 50%) • Experiment – Genotype SNPs in cases and controls • Expected outcome – Some SNPs show increased frequency among cases – Association of SNP haplotypes identifies chromosome region carrying mutation (Dvariant) causing disease susceptibility Genes for Alcoholism Functional Candidates: Associations in Populations • 5 variants – Individuals with family history; % Alcoholism Diagnosis as function of genotype – 5-HT Transporter, • LL–57% SL–21% SS-12.5% ; p=0.04 – 5-HT2A T102C, – 5-HT2A TYR, – 5-HT2C CYS-SER, – GABAAa6 • Pro/Pro-23% Pro/Ser-71% ; p=0.02 Schuckit, M. A., C. Mazzanti, et al. (1999). Biol Psychiatry 45(5): 647-51 Genes for Alcoholism Association in Populations: Conclusions and Caveats • Common SNP markers may miss variants that are low frequency (1% - 2%) • Most interesting variants may be in the low frequency class Individual Response to Alcohol Challenge • Broad range of response among individuals • Not explained by pharmacokinetics – Response largely independent of blood alcohol levels – Response ranges from extreme sensitivity to “hollow leg” • Response measured by questionnaire (euphoric feeling…) and by body sway index Level of Response Heritable Schuckit, M.A., et al., A genome-wide search for genes that relate to a low level of response to alcohol. Alcohol Clin Exp Res, 2001. 25(3): p. 323-9. Key Finding: Level of Response Predicts Risk of Alcoholism • Entering college students with family history of alcoholism – No strong personal alcohol history – Tested with questionnaire and physiological measures – Followed for extended period • Low response seen in – 40% of children of alcoholics J Stud Alcohol. 1998 Sep;59(5):485-94. Biological, psychological and environmental predictors of the alcoholism risk: a longitudinal study. Schuckit MA – 10% of family history negative controls • Individuals with a low response to alcohol – 4-fold more likely to become alcoholic – Drink more, hang out with heavy drinking groups • May explain much of inherited alcoholism risk Level of Response Mapping Schuckit, M.A., et al., A genome-wide search for genes that relate to a low level of response to alcohol. Alcohol Clin Exp Res, 2001. 25(3): p. 323-9. Multipoint Sib-Pair Linkage - Chromosomes 1, 21 Sibs from Lower Third of FIRST 5 (SRE) Chromosome 1 Schuckit, M.A., et al., A genome-wide search for genes that relate to a low level of response to alcohol. Alcohol Clin Exp Res, 2001. 25(3): p. 323-9. Chromosome 21 Mapping Studies: Alcoholism vs Response Level COGA Alcoholism Study Chromosome 1 COGA Level of Response Chromosome 1 Mapping Response Level Genes II Wilhelmsen, K.C., et al., Alcohol Clin Exp Res, 2003. 27(7): p. 10417. Positional Cloning: Not Working in Complex Disease • Too many candidates in linkage • Too much heterogeneity – Locus heterogeneity often lethal for linkage and association approaches – Allelic heterogeneity often kills association • Association needs common variants • New approaches needed - Candidate Genes – Sequencing for rare variants – Isoallelic cohorts for phenotypic characterization Excellent Candidate Genes for Alcohol Response • Positional Candidates – in chromosomal locations that are implicated in disease • Functional Candidates – in pathways that are implicated in disease process; e.g. (cAMP/PKA) • Model system Candidates – found in animal models to impact alcohol response • Best candidates meet all three criteria Genes for Alcoholism Functional Candidates Slide: Anastasia Constantinescu Genes for Alcoholism Functional Candidates A2R AC Gs cAMP Ca Ca RII Nuclear CREB CRE PKA envelope * Ca cAMP inducible genes A Mouse Model: PKA RIIb Knockout Consumption of ethanol by mutant mice lacking the RII subunit of PKA (RII / ) and wild-type control mice (RII +/+) maintained on a 129 SvJ × C57BL/6 hybrid background. a, Consumption (grams per kilogram) of a 20% ethanol solution. b, Consumption (grams per kilogram per day) at each ethanol solution (8-d average). c, Ethanol preference ratios (volume of ethanol consumed/total fluid consumed) as a measure of relative ethanol preference. All values reported as mean ± SEM. ANOVAs indicated that the RII / mice drank significantly more ethanol than RII +/+ mice. RII / versus RII +/+, *p < 0.05. Thiele, Todd E., Willis, Brandon, Stadler, Julia, Reynolds, James G., Bernstein, Ilene L., McKnight, G. Stanley J. Neurosci. 2000 20: 75- Measures of acute sensitivity to the sedative effects of ethanol, consumption of nonalcoholic tastants, and plasma ethanol levels (mean ± SEM). a, Time to regain the righting reflex (minutes) after injection of ethanol (4.0 gm/kg; i.p.). b, Consumption (milliliters per kilograms per day) of solutions containing either sucrose (Suc) or quinine (Qui). c, Plasma ethanol concentration (milligrams per deciliter) either 1 or 3 hr after ethanol injection (4.0 gm/kg; i.p). ANOVAs indicated that RII / mice recovered from ethanol-induced sedation significantly sooner than RII +/+ mice. On the other hand, RII / and RII +/+ mice did not differ significantly in consumption of nonalcoholic tastants or plasma ethanol levels. RII / versus RII +/+, *p < 0.05 Chromosome 7 Mapping PKA RIIb Candidate Genes II: Ethanol Consumption by PKCe -/- Mice Reduced responding for ethanol-reinforced lever presses in PKC / mice compared with PKC +/+ mice. (A) Total number of ethanolreinforced lever presses in a 16-h period, averaged across 8 weeks of testing. PKC / mice (open bars) demonstrated a significantly lower total number of lever presses than PKC +/+ mice (filled bars; t = 2.8, P< 0.05). (B) Total number of ethanol-reinforced lever presses following two different durations of ethanol deprivation. PKC / mice demonstrated a significant reduction in total number of lever presses following 104 h ethanol deprivation compared with PKC +/+ mice [F (1,23)genotype = 7.4, P< 0.05]. In addition, only PKC +/+ mice demonstrated a significant reduction in total number of lever presses following 104 vs. 32 h ethanol deprivation [F (1,23)duration = 21.6, P< 0.01]. (C) Number of ethanol reinforcers per / mice following 104 h ethanol bout were reduced in PKC deprivation compared with wild-type controls [F (1,23)genotype = 5.0, P< 0.05], and decreased as a function of duration of ethanol deprivation period [F (1,23)duration = 9.0, P< 0.05] only in PKC +/+ mice. (Inset) Number of ethanol self-administration bouts did not differ among genotypes, but number of bouts decreased as a function of duration of ethanol deprivation period in both genotypes [F (1,23)duration = 27.3, P< 0.01]. (D) Rate of lever pressing for ethanol reinforcement was significantly reduced in PKC / mice compared with wild-types [F (1,23)genotype = 21.9, P< 0.01]. Similarly, rate of lever pressing decreased as a function of duration of ethanol deprivation period [F (1,23)duration = 8.4, P< 0.05] only in PKC +/+ mice. Data are expressed as mean ± SEM. *Significantly different from 104 h deprivation period. Significantly different from wild-type controls Choi, D. S., D. Wang, et al. (2002). J Neurosci 22(22): 9905-11 Genetics of Genes Candidates • Goal is discovery of phenotypes associated with variants in interesting genes • Candidates emerge from – functional analysis – biochemical pathways – Model organisms • Problem: Since most interesting variants <1%-2% frequency, usually see only one example – Risky to draw conclusions from one example – Need an isoallelic cohort -analogous to mouse knockouts – 25 – 50 subjects who carry same rare allele Genetics of Genes: Bottom-Up Driven by Gene Activity – Phenotype is the Object of Discovery • Functional candidates – Many pathways offer exciting candidates – Ultimately a “Genetics of the Genes” Positional Cloning:Top-Down Phenotype-driven Pathway to Novel Candidate Genes • Mapping reduces the complexity >100 fold – Primary mapping – Secondary mapping by association with variants identified in affected sample sets – Functional candidates within mapped region • Functional candidates – Many pathways now offer exciting candidates – Ultimately a “Genetics of Genes” Next Generation Positional Cloning: Gene Mapping in Superfamilies • Central Problem: Most allele carriers for complex disease susceptibility alleles do not display phenotype – Low, moderate penetrance alleles need bigger window to ascertain families – Cannot extend families by following footprints of disease • Superfamilies provide bigger window, reveal footprints of low, moderate penetrance alleles – > 1,000 family members • Superfamilies display multiple aspects of phenotype – Expect, and are seeing, extensive overlap among cancers Superfamilies Utah Population Databases 50,000 Families, each founded by a pioneer couple Many kinships >1,000 Pedigrees recorded in computerized database Advantages Reduces heterogeneity Founder logic – one locus, one allele Reveals dominant alleles with reduced penetrance Superfamilies Provide Identity-By-Descent Mapping • One gene, one variant brought in by founding pioneer couple • Power for high resolution mapping • Pairwise testing shows IBD segment 10mb – 20mb Co-Aggregation of Cancers in Superfamilies Co-aggregating Disease Outcome Disease Breas t Breast Colon Lun g Melanom Ovar a y Pancrea s Prostate 2.4 1.8 0.7 3.5 0.2 2.7 1.2 0.3 0.4 2.2 2.7 1.1 0.7 1.1 3.5 0.9 1.4 4.2 4.0 2.4 Colorectal 3.0 Lung 3.5 1.8 Melanom a 1.7 0.5 1.3 Ovarian 10.5 0.9 1.8 1.4 Pancreas 0.5 3.7 1.5 1.5 4.3 Prostate 2.2 1.8 1.3 1.4 0.6 Odds ratios for SF at “high-risk” for Outcome Disease given that they are classified as “high-risk” for Co-aggregating Disease. If OR is significantly different from 1.0, number is in light mustard-biege. 8.3 0.8 Affected Individuals within Kinship Identical-by-Descent at Susceptibility Locus • Will see association through genome scan with STR markers A Portion of a Very Large Utah Family Conserved Chromosomal Segment Size D5S1393 112.2 mb D5S1346 118.3 mb D5S2501 122.4 mb D5S2051 123.4 mb D5S421 APC D5S656 APC Chromosome 5 D5S2065 D5S659 D5S1720 124.3 mb 124.8-125.1 mb 125.7 mb 126.7 mb 127.2 mb 127.5 mb D5S494 D5S639 130.8 mb 131.0 mb ATA24E05 131.8 mb D5S592 132.7 mb D5S615 139.0 mb D5S1391 141.3 mb