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GCRC Summer Student Research Seminars

GCRC Summer Student Research Seminars. Genetics in Clinical Research Jon Covault, MD, PhD. July 8, 2008. Genetic modes of action. Major effect genes (dominant or recessive) which acting alone produce significant risk

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GCRC Summer Student Research Seminars

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  1. GCRC Summer Student Research Seminars Genetics in Clinical Research Jon Covault, MD, PhD. July 8, 2008

  2. Genetic modes of action • Major effect genes (dominant or recessive) which acting alone produce significant risk • Oligogenic – simultaneous presence of multiple disease related genes required • Genetic heterogeneity – subtypes of illness result from different disease gene(s) / illness in different populations associated with different sets of disease genes • Gene / environment interactions –risk due disease locuc is modified by environmental exposures / releasers. • Epigenetics – alterations of chromatin in response to environment or development that establish and propagate individual patterns of gene expressions from the same genome. Alter activity levels of genes not their function.

  3. Genetics of complex disorders • Epidemiological and genetic data are most consistent with oligogenetic patterns of inheritance. In a given individual, multiple susceptibility genes, interact with each other and with environmental exposures. • Complex disorders appear to be genetically heterogeneous. Different families (and populations) vary in the constellation of specific susceptibility genes active as well as modification of penetrance by the individual genetic setting • Gene x environment interactions are important and poorly understood

  4. Use of indirect genetic markers for disease associated genetics DNA is inherited as chromosomes, not as individual genes, producing linkage between markers on each chromosome – e.g. between trait or disease causing loci and non-functional signpost markers Collins, Science 278:1580 (1997)

  5. SNPs – a key tool for genetic association studies of human traits Single nucleotide polymorphisms (SNPs; e.g. GCATTTCG vs. GCAGTTCG) - the most common genetic variation and easiest to measure. Occur approximately every 1,000 bases your DNA sequence is 99.9% identical to the person sitting next to you! Human genome database contains over 3 million SNPs - a key genetic tool

  6. Human “genome” • Prototypical composite sequence of 3 billion base pairs provides map of physical location of known and putative genes (based on the presence of gene like features- an open reading frame, typical splice site sequences etc.) • Catalog of known sequence variations based on increasing sample of subjects sequenced

  7. Popular genome browser- genome.ucsc.edu Example of human genome annotation- www.genome.ucsc.edu

  8. Genetic linkage – haplotype blocks • Genetic recombination during meiosis continuously reshuffles the genetic deck between paired chromosomes. • Recombination hot spots produce block segments of DNA which are the basic unit of inheritance within a population. • SNPs (single nucleotide polymorphisms) within such a haplotype block are highly predictive of each other. • Although most SNPs are silent, they provide convenient tags to mark chromosomal segments – nearby silent as well as functional polymorphisms are inherited as a group.

  9. a2 g1 |D’| plot of genetic linkage SNP markers identify adjacent GABRG1 and GABRA2 haplotype blocks

  10. HapMap.org – Haplotype block data / Tag SNPs help focus genotyping

  11. Sources of DNA Blood – 15 ug / ml blood OraGene Saliva Kit – 40 ug Scope mouth rinse- 4 ug

  12. TaqMan 5’nuclease quantitativefluorescent probe PCR assay

  13. Homozygote Vic/Vic Heterozygote Vic/Fam Homozygote Fam/Fam TaqMan Allelic discrimination

  14. - WT - Heterozygote - Homozygote - No DNA SNP genotypes are relatively easy to assay: TaqMan allelic discrimination using fluorescent oligonucleotide probes Asn allele – fam “A” probe Asp allele –vic “G” probe

  15. Haplotype Relative Risk Allele-sharing Types of Genetic Studies Association Linkage analysis

  16. Chromosome 4 and Alcohol Dependence- Collaborative Study on the Genetics of Alcoholism (COGA) • Alcohol dependence risk loci mapped to several chromosomes including chromosome 1, 4, 7 and 11 in COGA and NIAAA studies using linkage methods • The chromosome 4 linkage regions include a GABAA receptor gene cluster

  17. GABA(A) receptors and the pharmacology of alcohol • Many of alcohol’s behavioral effects have long been thought to be mediated by GABAergic neurotransmission: • Disruption of motor coordination • Anti-anxiety effect • Sedation • Benzodiazepines enhance GABA signaling via GABA(A) receptor ion channels and mimic many of the effects of alcohol

  18. SNP mapping of chromosome 4 GABA(A) cluster in COGA sample for association in multiply affected alcoholic families 1/6 30/49 0/6 0/7 # alcohol dependence associated SNPs / tested SNPs DNA sequencing revealed no aa coding variants in GABRA2 Edenberg et al., (2004) Am J Hum Genet 74:705

  19. 5’-GABRG1 SNPs show greater association w/ AD compared with 3’-GABRA2 SNPs Combined MATCH / CT EA case/control sample AD=1,099; Control = 535 Covault et al. 2007 Neuropsychopharmacology (in press)

  20. Replications of the association of GABRA2/GABRG1 and alcohol dependence • Multiply affected, family based - Edenberg et al. 2004 • Case/control Connecticut - Covault et al. 2005 • Case/control Russian - Lappalainen et al. 2005 • Case/control German - Fehr et al. 2006 • Case/control 10 site national sample – Covault et al. 2007

  21. Project MATCH: Matching Alcoholism Treatment to Client Heterogeneity (NIAAA 1997) • Multi-center (N=10) randomized clinical trial of three types of psychosocial treatment for alcoholism • Cognitive Behavioral Therapy (CBT) • Motivational Enhancement Therapy (MET) • Twelve Step Facilitation (TSF)

  22. Project MATCH drinking data • Pre-treatment alcohol use • Avg. 15.1 drinks per drinking day • Abstinent on 3 of 10 days • SAS Proc GENMOD used to generate probabilities of any drinking and heavy drinking (>4 drinks for women and >5 for men) on each of 450 days during the 12-week treatment and 12-month follow-up period.

  23. Project MATCH and GABRA2 • Daily drinking reports during the 3-month treatment and 12-month post-treatment periods were examined in relation to GABRA2 rs279858 genotype • Carriers of the exon 5 (K132K) alcohol risk G-allele (n=547) compared with for A-allele homozygotes (n=265) • No differences between genotype groups in pre-treatment drinking patterns or psychological matching characteristics

  24. Project MATCH outcomes and GABRA2 • G-allele carriers drank more frequently during the 15-month observation period • Any drinking OR=1.26 (CI 95% = 1.12-1.43) • Heavy drinking OR=1.94 (CI 95% = 1.71-2.21) • Additionally, there was an interaction of treatment type by genotype • A-allele homozygotes – TSF>MET>CBT • G-allele carriers – no distinction in outcome by treatment type.

  25. GABRA2 AD risk-allele vs. GABRG1 and MATCH outcome – frequency of any drinking GABRA2 GABRG1

  26. Trophic factors Axon guidance markers Neurotransmitter receptors Neurotransmitter metabolism Immune regulators Prenatal (second trimester) viral exposure Perinatal anoxia / infection Childhood / developmental environment Life events / stressors Nature and Nurture Gene - Environment Interactions

  27. Chronic psychosis and adolescent cannabis use: COMT val/val subjects at increased risk cannabis OR=10.9 Caspi et al. 2005 Biol Psych

  28. COMT and cannabis: Gene by environment interaction • No association of genotype and prevalence of adolescent cannabis abuse (24,23 and 27%) • No COMT interaction observed for psychosis when cannabis exposure onset after age 21 • Genetic risk of COMT may only be evident in the setting of co-morbid risks such as cannabis

  29. Serotonin transporter: 5-HTT • Key component of synaptic 5-HT clearance • Target of SSRI anti-depressant medications • Encoded by SLC6A4 gene at ch17q12 • 5-HTT gene-linked polymorphic region in SLC6A4 promoter (5-HTTLPR) is one of the most widely studied variations in behavioral neuroscience • The 5-HTTLPR VNTR (14-repeat “short” vs. 16-repeat “long”) confers differential promoter activity with 16-repeat having 2-fold higher activity

  30. Gene by environment interactions: 5-HTT and life stress Monkeys with serotonin transporter gene “s-allele” and exposed to early life stress, have altered stress response patterns as adults including increased alcohol consumption under experimental conditions. Does life stress interact with the analogous serotonin transporter variation in humans to affect drinking behavior? Drug use behavior?

  31. Does 5HTTLPR genotype associate with behavior relevant to alcohol use?

  32. Longitudinal daily process examination of college student life. Alcohol Research Center, H. Tennen, PI • 400 undergraduate UConn college students enrolled in a longitudinal study of daily life • Baseline surveys about life past year life experiences, personality inventory, alcohol use, alcohol expectations • Daily collection of life experiences and alcohol use for 30 days each school year using a web based survey • Scope mouthwash collection for isolation of salivary DNA

  33. *** * Interaction of past year life stress and 5-HTTLPR in alcohol use by college students S/S L/S L/L *** p=0.001 vs. l/l * p=0.05 vs. l/l Covault et al (2006) Biol Psych in press

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