Alcoholism is a complex disease with both genetic and environmental risk factors. To identify genes that affect the risk for alcoholism, we systematically ascertained and carefully assessed individuals in families with multiple alcoholics. Linkage and association analyses suggested that a region of chromosome 4p contained genes affecting a quantitative endophenotype, brain oscillations in the beta frequency range (13-28 Hz), and the risk for alcoholism. To identify the individual genes that affect these phenotypes, we performed linkage disequilibrium analyses of 69 single-nucleotide polymorphism (SNPs) within a cluster of four GABA(A) receptor genes, GABRG1, GABRA2, GABRA4, and GABRB1, at the center of the linked region. GABA(A) receptors mediate important effects of alcohol and also modulate beta frequencies. Thirty-one SNPs in GABRA2, but only 1 of the 20 SNPs in the flanking genes, showed significant association with alcoholism. Twenty-five of the GABRA2 SNPs, but only one of the SNPs in the flanking genes, were associated with the brain oscillations in the beta frequency. The region of strongest association with alcohol dependence extended from intron 3 past the 3' end of GABRA2; all 43 of the consecutive three-SNP haplotypes in this region of GABRA2 were highly significant. A three-SNP haplotype was associated with alcoholism, with P=.000000022. No coding differences were found between the high-risk and low-risk haplotypes, suggesting that the effect is mediated through gene regulation. The very strong association of GABRA2 with both alcohol dependence and the beta frequency of the electroencephalogram, combined with biological evidence for a role of this gene in both phenotypes, suggest that GABRA2 might influence susceptibility to alcohol dependence by modulating the level of neural excitation.

Linkage evidence indicated that gene(s) located on chromosome 4q, in the region of the alcohol dehydrogenase (ADH) genes, affected risk for alcoholism. We genotyped 110 single nucleotide polymorphisms (SNPs) across the seven ADH genes and analyzed their association with alcoholism in a set of families with multiple alcoholic members, using the pedigree disequilibrium test. There was strong evidence that variations in ADH4 are associated with alcoholism: 12 SNPs were significantly associated. The region of strongest association ran from intron 1 to 19.5 kb beyond the 3' end of the gene. Haplotype tag SNPs were selected for the block in the ADH4 gene that provided evidence of association and subsequently used in association analysis; the haplotype was significantly associated with alcoholism (P=0.01) There was weaker evidence that variations in ADH1A and ADH1B might also play a role in modifying risk. Among African-Americans, there was evidence that the ADH1B*3 allele was protective.

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A microfabricated 384-lane capillary array electrophoresis device is developed and utilized for massively parallel genetic analysis. The 384 capillary lanes, arrayed radially about the center of a 200-mm-diameter glass substrate sandwich, are constructed using scalable microfabrication techniques derived from the semiconductor industry. Samples are loaded into reservoirs on the perimeter of the wafer, separated on the 8-cm-long poly(dimethylacrylamide) gel-filled channels, and detected with a four-color rotary confocal fluorescence scanner. The performance and throughput of this bioanalyzer are demonstrated by simultaneous genotyping 384 individuals for the common hemochromatosis-linked H63D mutation in the human HFE gene in only 325 s. This lab-on-a-chip device thoroughly exploits the power of microfabrication to produce high-density capillary electrophoresis arrays and to use them for high-throughput bioanalysis.

Opioid receptors and their endogenous peptide ligands play important roles in the reward and reinforcement of drugs such as heroin, cocaine, and alcohol. The binding of dynorphins to the j-opioid receptor has been shown to produce aversive states, which may prevent the development of reinforcement. We genotyped SNPs throughout OPRK1, encoding the j-opioid receptor, and PDYN, which encodes its ligand prodynorphin, in a group of 1860 European American individuals from 219 multiplex alcohol dependent families. Family-based analyses demonstrated associations between alcohol dependence and multiple SNPs in the promoter and 3 0 end of PDYN, and in intron 2 of OPRK1. Haplotype analyses further supported the association of PDYN. Thus, variations in the genes encoding both the j-opioid receptor and its ligand, OPRK1 and PDYN, are associated with the risk for alcohol dependence; this makes biological sense as variations in either should affect signaling through the j-opioid system.