The literature contains a plethora of evidence from murine studies that the GABA neurotransmitter and GABAA
receptor are involved in the physiological responses to ethanol (e.g., motor incoordination and sedation) as well as ethanol reinforcement, tolerance, and withdrawal severity (Buck and Hood, 1998
; Grobin et al., 1998
; Hodge et al., 1996
; Hood and Buck, 2000
; Korpi et al., 1998
). These results spurred the COGA researchers to examine the within-family association of four of the genes encoding subunits of the GABAA
receptor. These genes were GABRG1, GABRA2, GABRA4,
corresponding to the GABAA
α4, and GABAA
β1 subunits, respectively. The original analysis of these data (Edenberg et al., 2004
) indicated a significant association between 30 SNPs within GABRA2
and alcohol dependence. This result was replicated in three independent populations using a case-control approach (Covault et al., 2004
; Fehr et al., 2006
; Lapplainen et al., 2005
). The present study was an attempt to replicate the findings of Edenberg et al. (2004)
using another data set consisting of multiplex alcohol-dependent families. No evidence of an association or linkage to the alcohol dependence phenotype and GABRA2
could be found in this sample.
The present failure to replicate the findings of Edenberg et al. (2004)
may have occurred for a number of reasons. First, the power to detect within-family association may have been less than that available in those studies specifically using family-based association analyses (Agrawal et al., 2006
; Edenberg et al., 2004
). The present study may be underpowered because only 63 pedigrees were analyzed. However, post hoc evaluation of power for the FBAT or PDT are infeasible. The complexity of the alcohol dependence phenotype precludes a simple specification of the underlying genetic model that is required for any power evaluation. Also, it is recognized that the COGA data set has a larger number of multiplex pedigrees for analysis than the present data set. However, placing the available power of the present sample in the context of the power of these COGA studies is difficult, because the number of informative families on which these positive COGA results were based was not reported and because power estimates were not available from those analyses.
Although there might have been a lack of power to completely test our hypothesis that these SNPs in the GABRA2 would be associated with disease status within the multiplex families, the absence of a difference in the transmission probabilities from parents to affected offspring and in the frequencies within the discordant pairs suggests that it is unlikely that our results would have been statistically significant.
It might be argued that performing haplotype analysis on this data set—as Edenberg et al. (2004)
did with the COGA data set—might have revealed a significant association. Because of complete LD between four of the SNPs chosen for analysis, a haplotype analysis on the two remaining SNPs was not performed. At any rate, such an analysis would only have increased the number of degrees of freedom resulting in loss of statistical power.
PDT and FBAT results do not show any evidence for an association using one SNP. This result is consistent with a previous genome-wide analysis in which no linkage findings of note could be seen on Chromosome 4 (Hill et al., 2004
). Following up on an earlier suggestive linkage finding between the MNS blood group (4q28-31) and alcohol dependence (Hill et al., 1988
), Neiswanger et al. (1995)
used the same sample to perform linkage analyses of the 4q21-31 region. No evidence for linkage was found. In fact, all of the lod scores were zero or negative.
Another possible explanation for disparities between our results and those of COGA, as well as subsequent positive associations based on three case-control studies, may be the result of differing methods of sample selection. Using the disorder that occurs first in the participant’s life as the primary diagnosis, and those occurring at least 1 year or later as the secondary diagnosis, the study was designed so that families were not included if the proband pair or their first-degree relatives had primary diagnoses other than alcohol dependence. Accordingly, the present data set was selected so that minimal primary comorbidity for drug dependence and other psychiatric disorders was introduced. In contrast, more than half (51%) of the genotyped, alcohol-dependent COGA subjects had a comorbid diagnosis of illicit drug dependence that appeared to be a primary diagnosis. In the present sample, alcohol-dependent relatives of probands were less frequently drug dependent (30%) than in the COGA sample (see ). In addition to error variance that may occur by inclusion of drug dependent cases, other comorbidity may cloud interpretation of findings. Comorbid depression, anxiety, and antisocial personality disorder (ASPD) are common conditions (Helzer et al., 1991
) that may influence results. For example, Dick et al. (2006b)
have demonstrated a relationship between GABRA2
and conduct disorder. Because COGA did not exclude any family as a result of comorbid conditions, whereas the present study did limit comorbidity, it may be assumed that depressive and anxiety disorders, as well as ASPD, were less frequent in the present sample.
Taking all of the studies of GABAA
and alcohol dependence together, one might conclude that removing conditions comorbid with alcohol dependence is necessary to find a relationship (Covault et al., 2004
). When depression and drug dependence were removed from the Covault et al. (2004)
sample, the GABRA2
association proved to be even stronger. However, the obverse appears to be the case as well. Some studies find that the presence of comorbid conditions is the only situation in which significant results do occur (Agrawal et al., 2006
; Drgon et al., 2006
; Fehr et al., 2006
). Drgon et al. (2006)
analyzed data for 415 polysubstance abusers and 239 controls, finding a significant relationship with one of six SNPs in the GABRA2
gene (rs279871). However, this relationship was considered to be only modest because no particular allele was found to be associated with polysubstance abuse. The importance of substance abuse other than alcohol is apparent in the work of Agrawal et al. (2006)
. Using the COGA multiplex families, these investigators found no association of any GABAA
SNP with the alcohol dependence phenotype alone (cases with drug dependence removed). Similarly, analysis of alcohol-dependent individuals (n
= 65) and unrelated controls (n
= 112) from the COGA data set found no significant differences for six selected GABRA2
SNPs, including the rs279871 SNP (Drgon et al., 2006
The presence of other psychiatric diagnoses also appears related to whether significant differences between cases and controls are seen. ASPD may be a factor in determining the strength of the gene/dependence relationship. Significant differences between alcohol-dependent cases and controls were seen in individuals with comorbid ASPD, but removal of the comorbid ASPD cases resulted in no significant difference between the groups (Fehr et al., 2006
). These considerations suggest that improving the separation between cases and controls may provide the opportunity for uncovering genetic variation if it exists. Similarly, where no genetic association may be present, removing the comorbidity from cases and controls may uncover true negative findings. Moreover, if several psychiatric disorders have a unique relationship to GABRA2
variation, studies need to be designed to assess the independent effects of each of these disorders. To date, study of the relationship between disorders other than alcohol dependence and GABRA2
has been based on comorbid cases.
Another possibility is that a yet-undiscovered endophenotype that is highly correlated with alcohol dependence may, in fact, be what is contributing to previously reported positive findings. These possible endophenotypes might include lower level of response to alcohol reported for another GABAA
α6 receptor (Schuckit et al., 1999
) or predisposition to anxiety disorders. One endophenotype that has been found to be associated with GABRA2
is the EEG β phenotype (Edenberg et al., 2004
). Additionally, patients with panic disorder have shown a deficit in GABAA
receptors, resulting in enhanced anxiety for threat cues (Crestani et al., 1999
). It is noteworthy that through selection criteria used in the present sample, anxiety disorders were exceptionally low. At any rate, the present analyses did not find evidence favoring a relationship between GABAA
and alcohol dependence in this sample of multiplex families.