Genome-wide studies of linkage disequilibrium have revealed that most of the human genome can be divided into blocks of varying length within which marker to marker linkage disequilibrium is very high. Each haplotype block is separated by recombination sites. There are usually only 3-6 haplotypes with greater than 5% frequency within each block. These haplotypes reflect descent from a single, ancient ancestral chromosome. The main advantage of haplotype methods for linkage and association studies is that these common haplotypes capture most of the information on genetic variation within these regions and the haplotypes can be identified using only a small number of SNPs, usually 3 to 8. Thus haplotype-based case-control studies can detect associations with disease or behavior without having to find and test every single variant in the region. We have genotyped two ethnically diverse population isolates, approximately 500 Finnish Caucasians and 400 Plains American Indians, and one admixed population, 900 African Americans, for genes for alcoholism and anxiety using a custom designed 130 gene, 1350 haplotype-tagging SNP addictions array that also includes 186 ancestry informative markers, using the Illumina GoldenGate platform. The neurotransmitter serotonin (5-HT) has a profound influence on mood, and CNS 5-HT variation has been implicated in alcohol and drug dependence. The African American men showed a haplotype association with alcohol dependence that was driven by a gain of function SNP, rs1176744, Tyr129Ser. Moreover, we showed that although the HTR3B rs1176744 Ser129 gain of function allele predicted alcohol dependence and the low activity triallelic serotonin transporter promoter variant 5-HTTLPR predicted cocaine dependence, the two HTR3B and 5-HTTLPR variants combined synergistically in their effects on alcohol and drug dependence. The mechanism underlying this synergism might be that increased synaptic 5-HT plus increased 5-HT3 receptor responsiveness to 5-HT might result in alterations in dopamine release in the reward pathway that increase vulnerability to addiction (Enoch et al, 2011). The addictions array includes a comprehensive GABAergic gene panel such as the chromosome 4 cluster of GABAA receptor genes that are predominantly expressed in the brain reward circuitry. In both Caucasians and Plains Indians we found GABRG1 haplotype linkage to alcoholism. Moreover, long-distance ancestral haplotypes spanning GABRG1 and GABRA2 were associated with alcoholism;this association was determined by GABRG1 (Enoch et al, 2009). The African Americans had four common GABRA2 haplotypes within the distal haplotype block: two that correspond to the Caucasian and Asian yin-yang haplotypes and two not found in other ethnic groups. One of the unique haplotypes predicted heroin addiction whereas the other haplotype was more common in controls. Moreover, an intron 2 SNP rs11503014, not located in any haplotype block and potentially implicated in exon splicing, was independently associated with addiction, specifically heroin addiction (Enoch et al, 2010). In a related project using RNA-Seq, we investigated differences in GABAergic gene expression in postmortem hippocampus from alcoholics, cocaine addicts and controls and postmortem hippocampus from alcohol-nave, alcohol preferring (P) and non-preferring (NP) rats selectively bred for extremes of alcohol-seeking behavior that also show a general addictive tendency. The pathway-targeted analysis of 25 GABAergic genes encoding proteins implicated in GABA synthesis, metabolism, synaptic transmission and re-uptake showed directionally consistent and biologically plausible overlapping and specific changes. Principal FDR-corrected findings were that GABBR1 was down-regulated in alcoholics, cocaine addicts and P rats with congruent findings in NSF, implicated in GABAB signaling efficacy, potentially resulting in increased synaptiic GABA. GABRG2, encoding the gamma2 subunit required for postsynaptic clustering of GABAA receptors together with GPHN, encoding the associated scaffolding protein gephryin, were both down-regulated in alcoholics and cocaine addicts but were both up-regulated in P rats. There were also expression changes specific to cocaine addicts (GAD1, GAD2), alcoholics (GABRA2) and P rats (ABAT, GABRG3). Congruent findings in human addicts and P rats provide clues to predisposing factors for alcohol and drug addiction and may have therapeutic potential (Enoch et al, 2012). Identical RNA-Seq studies in P rats that have been rendered alcohol dependent (2 bottle choice) are underway. All the above analyses are being conducted for glutamatergic candidate genes. As a follow on from this GABAergic gene expression study we have shown that haplotypes and SNPs in GAD1, the gene that encodes the enzyme responsible for most of GABA synthesis in the brain, were associated with alcohol dependence in the African Americans. Similar analyses are underway for GABBR1, GPHN, GABRG2 and SLC6A1 that encodes GAT1, the neuronal GABA transporter. We have used RNA-Seq data from the publicly available resource 'BrainSpan'to identify global patterns of GABAergic gene expression in healthy adults across 16 brain regions. A factor analysis identified 6 factors that together accounted for 0.86 of the total variance in the expression of 21 key GABAergic genes. Our study has shown that the GABAA gene clusters show both cis and trans correlations of gene expression. Moreover, the factor analysis combined with data from our earlier study (Enoch et al, 2012) revealed that distinct groups of genes, notably those involved in GABA synthesis and synaptic transport and the chr 4 cluster, previously associated with alcohol and drug dependence in humans, are sensitive to the effects of chronic alcohol / cocaine exposure. These findings might have implications for therapeutic targets to combat stress-related craving and relapse (Enoch et al, submitted).
