From QTL to candidate gene: a genetic approach to alcoholism research

Abstract

A major focus of research in alcohol-related disorders is to identify the genes and pathways that modulate alcohol-seeking behavior. In light of this, animal models have been established to study various aspects of alcohol dependence. The selectively bred alcohol-preferring (P) and -nonpreferring (NP) lines were developed from Wistar rats to model high and low voluntary alcohol consumption, respectively. Using inbred P and NP strains, a strong QTL (LOD-9.2) for alcohol consumption was identified on rat chromosome 4. To search for candidate genes that underlie this chromosomal region, complementary molecular-based strategies were implemented to identify genetic targets that likely contribute to the linkage signal. In an attempt to validate these genetic targets, corroborative studies have been utilized including pharmacological studies, knock-out/transgenic models as well as human association studies. Thus far, three candidate genes, neuropeptide Y (Npy), alpha-synuclein (Snca), and corticotrophin-releasing factor receptor 2 (Crhr2), have been identified that may account for the linkage signal. With the recent advancements in bioinformatics and molecular biology, QTL analysis combined with molecular-based strategies provides a systematic approach to identify candidate genes that contribute to various aspects of addictive behavior.

Fig. 1

An illustration of a stepwise approach to target and attempt to validate candidate genes associated with a heritable trait. Following selective breeding and inbreeding, QTL mapping strategies were applied to the inbred P and NP strains in order to identify chromosomal regions that are correlated with alcohol consumption, the quantitative trait of interest. To target specific genes of interest, molecular-based strategies were implemented to screen for genetic targets that map to the chromosome 4 QTL region and display differences in gene expression and DNA sequence polymorphism. These complementary molecular-base strategies were utilized to identify and prioritize genetic targets for hypothesis-driven research. In an attempt to validate and further characterize the genes of interest, genetic (e.g., knockout, transgenic), pharmacological interventions (e.g., targeting associated receptors, enzymes and or transporters), and human association studies can provide corroborative evidence, ultimately defining the relevance of each candidate gene to alcohol-seeking behavior.

Fig. 2

A QTL for alcohol consumption identified on rat chromosome 4 in iP and iNP rats. Boxes and arrows illustrate the basic methodologies that were incorporated to conduct QTL analysis in the P and NP strains. Using bidirectional selective breeding, the alcohol-preferring (P) and –nonpreferring (NP) rat strains were developed from a closed colony of Wistar rats (top box). The inbred P (iP) and NP (iNP) strains were later developed from the P and NP strains using brother-sister mating for over 20 generations. To perform QTL analysis, the iP and iNP strains were crossed to generate iP x iNP F1 animals, and these F1 animals were subsequently bred to generate the iP x iNP F2 population. In the F2 animals, free choice alcohol consumption (g/kg/day) and alcohol preference (v/v) were measured, and polymorphic microsatellite markers were genotyped. A genome screen was then performed to identify chromosomal regions that segregate with the alcohol consumption phenotype. Linkage analysis detected a highly significant QTL (lod score = 9.2) for alcohol consumption on rat chromosome 4. Thus far, three candidate genes have been identified that underlie the peak of this linkage signal including neuropeptide Y (Npy), α-synuclein (Snca), corticotrophin releasing factor receptor 2 (Crhr2).