Identification of Candidate Genes for Alcohol Preference by Expression Profiling of Congenic Rat Strains

Genes Contributing to alcoholism are difficult to identify due to both the genetic complexity of alcoholism and environmental variability among alcoholic subjects. While various techniques can be utilized to identify possible molecular and physiological patterns associated with alcoholism, localizing the primary genetic determinants for the risk to develop alcoholism has proven to be difficult. Nevertheless, several genes have been associated with a risk for alcoholism in studies that have been replicated, e.g. GABRA2 (Covault et al., 2004; Edenberg et al., 2004; Fehr et al., 2006; Lappalainen et al., 2005), ADH4 (Edenberg et al., 2006; Guindalini et al., 2005; Luo et al., 2005a), and CHRM2 (Luo et al., 2005b; Wang et al., 2004). It is evident, however, that there are other genes that also influence the risk for alcoholism. Selective breeding of rodents for alcohol-related traits, followed by quantitative trait locus (QTL) analysis has located genomic regions in which variation affects the phenotype. However, finding the specific genes within such regions is difficult. A strategy of making congenic animal models carrying a single QTL, and then using microarray analysis to determine which genes are altered in expression, should provide a powerful approach toward gene identification (Hitzemann et al., 2004; Hoffman and Tabakoff, 2005; Spence et al., 2005). Using this combined approach dramatically decreases the number of genes that are expected to be differentially expressed because the 2 strains are identical except for the QTL sequences, thereby increasing the probability to identify differentially expressed genes contributing to a specific phenotype such as alcohol preference. The alcohol-preferring (P) and alcohol-nonpreferring (NP) rat lines were developed, through bidirectional selective breeding, from a randomly bred, closed colony of Wistar rats, on the basis of alcohol preference in a 2-bottle choice paradigm (Li et al., 1991). Alcohol-preferring rats display the phenotypic characteristics considered necessary for an animal model of alcoholism (Cicero, 1979; Li et al., 1993). Subsequently, inbred P (iP) and NP (iNP) strains were established; these inbred strains maintain highly divergent alcohol consumption scores (Carr et al., 1998). Owing to the physiological and genetic similarity between humans and rats, iP and iNP rats can be studied to identify important genetic factors that might influence alcoholic predisposition in humans. A highly significant QTL that influenced alcohol preference was identified on chromosome 4 (with a maximum lod score of 9.2) in a cross between iP and iNP rats; suggestive QTLs were identified on chromosomes 3 and 8 (Bice et al., 1998; Carr et al., 1998). The chromosome 4 QTL acts in an additive fashion and accounts for approximately 11% of the phenotypic variability. Owing to the strong association between the chromosome 4 QTL region and alcohol preference, this approximately 25 cM region is likely to harbor at least 1 gene that directly contributes to alcohol preference. Reciprocal congenic strains in which the iP chromosome 4 QTL interval was transferred to the iNP (NP.P) and the iNP chromosome 4 QTL interval was transferred to the iP (P.NP) exhibited the expected effect on alcohol consumption of the strain that donated the chromosome 4 QTL interval (Carr et al., 2006). This indicates that the chromosome 4 QTL region is, in part, responsible for the disparate alcohol consumption observed between the iP and iNP rats. The objective of the present study was to identify genes in the chromosome 4 QTL interval that might be responsible for the divergent alcohol preference of the iNP and iP strains. Because the chromosome 4 QTL interval is the only region that differs between the 2 strains, analyzing only the probe sets in this interval focuses on the cis-regulated genes, and increases power by reducing the number of comparisons. Analysis of all the probe sets allows detection of both cis-regulated and trans-regulated genes, but at the expense of testing more probe sets [and therefore a higher false discovery rate (FDR) and lower power]. Discrete brain regions were analyzed for expression differences between the homozygous NP.P and the iNP background strain, to detect differences that might be localized to 1 region. Expression data from the 5 brain regions of each animal were then combined to obtain an average expression level for analysis of genes that may differ across multiple regions; combining the 5 regions in a single animal reduces technical variation and should increase power to detect differences that are in the same direction but that may fall below significance in individual regions. These complementary analyses should allow us to better understand the inherent differences in gene expression between strains that differ in alcohol preference as a result of genetic differences in a single chromosomal region.