A method for mapping intralocus interactions influencing excessive alcohol drinking

Abstract

Excessive alcohol (ethanol) consumption is the hallmark of alcohol use disorders. The F1 hybrid cross between the C57BL/6J (B6) and FVB/NJ (FVB) inbred mouse strains consumes more ethanol than either progenitor strain. The purpose of this study was to utilize ethanol-drinking data and genetic information to map genes that result in overdominant (or heterotic) ethanol drinking. About 600 B6 x FVB F2 mice, half of each sex, were tested for ethanol intake and preference in a 24-h, two-bottle water versus ethanol choice procedure, with ascending ethanol concentrations. They were then tested for ethanol intake in a Drinking in the Dark (DID) procedure, first when there was no water choice and then when ethanol was offered versus water. DNA samples were obtained and genome-wide QTL analyses were performed to search for single QTLs (both additive and dominance effects) and interactions between pairs of QTLs, or epistasis. On average, F2 mice consumed excessive amounts of ethanol in the 24-h choice procedure, consistent with high levels of consumption seen in the F1 cross. Consumption in the DID procedure was similar or higher than amounts reported previously for the B6 progenitor. QTLs resulting in heightened consumption in heterozygous compared to homozygous animals were found on Chrs 11, 15, and 16 for 24-h choice 30% ethanol consumption, and on Chr 11 for DID. No evidence was found for epistasis between any pair of significant or suggestive QTLs. This indicates that the hybrid overdominance is due to intralocus interactions at the level of individual QTL.

Figure 1

Time course of EtOH drinking procedures. Mice were first acclimated to drinking from graduated drinking tubes, then offered a series of EtOH concentrations in tap water versus tap water for 24-h per day, and then tested in a single-bottle followed by a two-bottle choice drinking in the dark (DID) procedure. Note: Length of bars is not entirely to scale.

Figure 2

EtOH consumption and preference in a 24-h two-bottle choice procedure are greater in female than male F2 mice from the reciprocal cross of the C57BL/6J (B6) and FVB/NJ (FVB) inbred strains. Shown are means ± SEM for (A) EtOH consumption, (B) EtOH preference and (C) water consumption, when multiple concentrations of EtOH were offered versus water for 4 consecutive days at each EtOH concentration in the order listed along the x-axis. Means are the average of days 2 and 4, the last days after an EtOH tube position change for each concentration. Some error bars are hidden by the symbols. n = 303 female and 302 male; * p < 0.05, ** p < 0.01, *** p < 0.001 for the comparison of female and male mice at the indicated concentration.

Figure 3

Frequency distributions for consumption of EtOH offered versus water. Data are expanded from those summarized in Fig 2. The black curves show the predicted normal distributions. Actual female (left panels) and male (right panels) data are shown as histograms. Listed in each panel are Chi-Square test results for the comparison of predicted normal and actual distributions.

Figure 4

EtOH consumption in single-bottle and two-bottle choice drinking in the dark (DID) procedures is greater in female than male B6FVBF2 mice, and EtOH intake is reduced when water is offered as an alternative source of fluid. Shown are means ± SEM for EtOH consumption during 2-h periods on five consecutive days (days 22–26 in Fig 1). EtOH was offered for 4 total hours on days 25 and 26. *** p < 0.001 for the comparison of data for the sexes combined on day 26 versus all means for the sexes combined on days 22–25 (Note: because there was no interaction of sex and day, effects for each sex were not analyzed across days).

Figure 5

Consumption of 30% EtOH in a two-bottle 24-h choice procedure is associated with QTL on chromosomes 9 and 11. Shown are LOD scores from the genome-wide QTL analysis indicating suggestive (bottom dashed line) and significant (top dashed line) associations of genetic markers with the average amount of 30% EtOH consumed on days 14 and 16. Chromosome number is listed along the x-axis.

Figure 6

Additive (a; shown in grey) and dominance (d; shown in black) values plotted separately for the same markers and intervals as the LOD scores shown in Fig 6 for two-bottle choice 24-h 30% EtOH consumption. Significant QTLs were those for which a or d exceeded the absolute value of 0.204 and 0.287, respectively. Based on the magnitude of a alone, QTLs emerged as significant on mid Chr 9 and distal Chr 12. Based on the magnitude of d alone, QTLs emerged as significant on mid Chr 11, mid Chr 15, and mid to distal Chr 16.

Figure 7

Results of dominance deviation QTL mapping in B6FVBF2 mice for 24-h, two-bottle choice 30% EtOH consumption (g/kg/d). Shown are mean ± SEM EtOH consumption values for mice homozygous B6, heterozygous B6FVB or homozygous FVB for markers significantly associated with excessive EtOH consumption in the heterozygous animals. Overdominance was evaluated for statistical significance by testing whether d was significantly greater than a as described in Methods. This comparison was significant for all three regions shown. Please note that the genotypes shown are at a specific marker, not between markers (interval analysis) as shown in Fig 6.

Figure 8

Locations of genetic markers and 2 LOD confidence intervals for QTL associated with heterozygote overdominant 30% EtOH consumption on chromosomes 11, 15 and 16. Shown along the lengths of the chromosomes are the SNPs used for genotyping and their locations in b.

Figure 9

The magnitude of d (dominance deviation) on Chr 11 for all five of the DID measures and the 24-h, two-bottle choice measure (30% EtOH consumption; g/kg/d). For all six measures, a significant overdominant QTL was seen that conferred higher EtOH consumption, preference or blood EtOH concentration in the heterozygotes compared to either homozygote class.