Advances in molecular genetics and the prevention and treatment of substance misuse: Implications of association studies of the A1 allele of the D2 dopamine receptor gene
Introduction
Addictive behaviour is influenced by a multitude of biological and environmental factors. Advances in molecular genetic research over recent decades have enabled the deciphering of the entire human genome. The regulatory functions of these identified genetic sequences remain a significant scientific undertaking in addiction research. It has been estimated that the human genome contains 30,000 to 40,000 genes (Claverie, 2001). The process of identifying possible molecular biological markers is complex and requires the examination of exons, which represent gene coding regions, as well as the introns that constitute about 98% of the human genome and are not yet fully understood. Introns, however, are known to be involved in gene expression or “activation” in response to environmental factors that influence cellular function. Polymorphisms in linkage disequilibrium (LD) with these exonic or intronic variants can also influence gene function. The methods used to examine these potential markers with addiction phenotypes include nonparametric linkage analysis, family association studies, genome-wide association studies, and case-control association studies (Burmeister, 1999). The current review examines only the latter.
Our understanding of the underlying neurobiological aspects of addiction and drug reinforcement provides a useful template to focus the scientific efforts of gene identification in genetic association studies. Genetic influences on brain neurotransmitter function are likely to represent the underlying basis of the reinforcing potential of psychotropic substances. Genes associated with dopamine, serotonin, endogenous opioids, anandamide, GABA, and glutamate have been investigated in relation to neurotransmitter synthesis, receptor function, transport, or degradation (see Table 1). Given the key role of brain dopaminergic systems in drug-related reinforcement, considerable interest has focused on polymorphisms of dopamine receptor and transporter genes. Genes associated with dopaminergic function have been examined with regard to all of these drugs of abuse except cannabis.
The association of DRD2 A1+ allelic status (A1A1 and A1A2 genotypes) with alcoholism is consistent with a genetic dopaminergic influence on drug-seeking behaviour (Blum et al., 1990). Some individual studies have not supported this finding (e.g., Blomqvist, Gelernter, & Kranzier, 2000) and have suggested that positive effects may be attributable to population stratification. In general, the association has been robust, although the DRD2 A1+ allele is associated, a range of other substance use disorders, including nicotine (*Comings et al., 1996, *Noble et al., 1994, *Noble et al., 1994, Spitz et al., 1998), stimulant (*Noble et al., 1993, *Persico et al., 1996), and opiate (Lawford et al., 2000) dependence, indicates that it is not a specific risk-factor for alcoholism.
A1+ allelic status is associated with low CNS D2 receptor density, particularly in the striatum and associated structures. An in vitro study using the D2 dopamine receptor ligand (3H) spiperone (Noble, Blum, Ritchie, Montgomery, & Sheridan, 1991) found a significant decrease in the number of D2 dopamine receptors in the brains of A1+ compared to A1− allelic individuals (A2/A2 genotypes). An in vivo positron emission tomography (PET) study using (11C) raclopride found a significant reduction in brain D2 receptor density in the brains of healthy A1+ allelic subjects (Pohjalainen et al., 1998). Other in vitro (Thompson et al., 1997) and in vivo (Jonsson et al., 1999) studies, using (11C) raclopride, found significant associations of the A1+ allele with low D2 dopamine receptor density. Low D2 dopamine receptor density is a potentially important marker of drug reward. Low striatal D2 dopamine receptor density predicts enhanced subjective reinforcement related to intravenous methylphenidate, a D2 dopamine transporter inhibitor (Volkow et al., 2002). Conversely, animal studies have shown that overexpression of D2 dopamine receptors is associated with reduced alcohol self-administration (Thanos et al., 2001).
Given the associations with clinical status and underlying physiological phenotypes of substance misuse risk, the A1+ allele of the DRD2 gene has become one of the most widely studied and controversial polymorphisms in psychological and psychiatric research. The hypothesized association between the A1+ allele and a range of psychological phenotypes has generated over 80 peer-reviewed journal articles since the initial report by Blum et al. (1990). Given the importance of dopamine in the mesolimbic reward pathway, and evidence that A1+ allelic status is related to the use of a variety of substances, it has been hypothesized that the association between the A1+ allele may be with a broad psychological phenotype (‘endophenotype’) that includes substance misuse. Indeed, the association of the DRD2 A1+ allele with both substance use disorders and reduced brain D2 dopamine receptors has led to the hypothesis that the DRD2 is a reward or reinforcement gene (Noble, 1998, Noble, 2000). The “reward deficiency syndrome” (Blum et al., 2000) theory proposes that, due to reduced D2 receptor density, those with the A1+ allelic status are more likely to repeat behaviours that result in enhanced dopamine release to compensate for a chronically low “reward” state. These behaviours include drug and alcohol abuse and a range of other impulsive and addictive behaviours. Consistent with this idea, some studies that have not specifically examined the reward deficiency syndrome have confirmed the association between the A1+ allele and personality factors, such as novelty seeking (Noble et al., 1998).
Given the size of body of research examining the A1 allele, it is timely to review all published studies suitable for meta-analytic evaluation. In undertaking a meta-analysis of DRD2 A1+ allele association studies, several key factors need to be addressed methodologically.
- (i)
Control selection.
Screening procedures for the selection of controls are necessary to ensure control samples are relatively representative of the general population. Screening procedures for selection of controls have varied greatly, with some studies not reporting screening procedures (*Bau et al., 2000, *Gelernter et al., 1991). In general, most studies have excluded drug and alcohol abusers from the control group, while others have also excluded those with a family history of substance abuse (Lawford et al., 1997).
- (ii)
Clinical group phenotype definitions.
In addition to the need for adequate control group screening, the variations in selection and definition of clinical samples has also been a confounding variable. In particular, the definition of problematic substance use has been broadly defined, varying from regular use in many studies of cigarette smoking (*Singleton et al., 1998, *Yoshida et al., 2001) to dependence with complex medical complications resulting from excessive alcohol consumption (Lee et al., 1999). There is a significant subset of studies that have included severity of dependence or associated problems as a grouping variable (*Noble et al., 1994, *Noble et al., 1994), examining less severe and more severe substance users separately.
- (iii)
Ethnic composition of the sample.
As noted, another variable that has introduced potential confounds is population stratification. Estimates of A1 allelic prevalence vary considerably across ethnic groups. Barr and Kidd (1993) reported that the prevalence of the A1 allele among a general sample of Yemenite Jews was 9%, while among a general sample of Muskoke Native Americans, the prevalence was 75%. Following the Barr and Kidd study, most A1 allele association studies have limited inclusion to one ethnic group, most commonly to Caucasians.
- (iv)
Small sample size.
One of the factors making investigation of the association between the A1 allele of the DRD2 gene and substance misuse difficult is the importance of environmental influences and other genes that contribute to development of addictive disorders (Pato, Macciardi, Pato, Verga, & Kennedy, 1993). It has been noted that in some studies reporting nonsignificant findings, the sample sizes obtained may not have been sufficient to detect a statistical effect (Pato et al., 1993).
Cloninger (1991) was the first to attempt a meta-analysis of the association between the A1 allele of the DRD2 gene and alcoholism. Based on the six case-controlled studies that had been conducted at that time, a significant association between the A1 allele and alcoholism was confirmed. However, the summary noted considerable variation across the studies examined. This was explained as indicative of epistasis, with the A1 allele not responsible for ‘causing’ alcoholism as much as having an effect on the causal factors of the disorder. By contrast, Gelernter, Goldman, and Risch (1993) concluded that there was no evidence of an association between the A1 allele and alcoholism. Gelernter et al. concluded that any investigation into the effect of the A1+ allele must involve carefully ethnically matched cases and controls. At the time of their review, only two studies had been conducted with ethnically matched cases and controls. Pato et al. (1993) conducted a meta-analysis on a similar set of studies, but excluded studies without control groups. In contrast to Gelernter et al., they found a significantly increased risk of alcoholism among participants who carried the A1 allele as well as an association between the severity of alcoholism and the prevalence of the A1+ allelic status.
Uhl, Blum, Noble, and Smith (1993) found a significant difference between substance users and controls in the nine studies that had been conducted on the association between the A1+ allele and any drug misuse. However, their polydrug use findings were based on only three studies. Blum et al. (1995) extended their review to studies investigating the association between the A1+ allele and a range of impulsive, addictive, and compulsive behaviours. They found a significant association between the A1+ allele and alcoholism in studies with Caucasian participants. Blum et al. suggested that obesity and pathological gambling might also be associated with the presence of the A1 allele although there were insufficient studies available to assess these relationships using meta-analysis.
Noble (2003) has conducted a large meta-analysis with studies that only involved Caucasian participants to avoid stratification. This analysis examined the frequency of the A1 allele in 1837 alcoholics and 1492 Caucasian controls finding a significantly higher A1 allele frequency in the alcoholics (P=2.14×10−7; Noble, 2003). The Noble (2003) review identified only three studies where the A1+ frequency was lower in alcoholics than in controls. Among studies that separated more severe and less severe alcoholics, the A1+ allele was more prevalent among the more severe alcoholics. The influence of control group screening was also analysed with control groups subjected to screening having a significantly lower prevalence of the A1+ allele than those that were not screened. In addition, Noble analysed the studies that had researched the association between the A1+ allele and illicit drug use among Caucasians. While based on only six studies, the A1+ allele was more prevalent among illicit drug users then controls.
While the stratification issue is acknowledged in a comprehensive meta-analysis, it is recommended that all methodologically appropriate studies should be included, as long as they are statistically comparable (Glass, McGaw, & Smith, 1981). In addition, both the Noble (2003) review and the Gelernter et al. (1993) review used methods of meta-analysis that did not include effect size estimates for each of the studies that were included. Glass et al. (1981) recommends the use of effect sizes for each study as a way of establishing a standardised basis of comparison when conducting meta-analyses.
As noted, several studies have now investigated the association between the A1+ allele and drug abuse other than alcoholism (e.g., Comings et al., 1991, *Lawford et al., 2000, *Noble et al., 1993), as well investigating the association of the A1+ with personality factors (e.g., *Berman et al., 2002, Limosin et al., 2003, *Noble et al., 1998). The current analyses build on prior meta-analyses and reviews by incorporating the entire set of suitable published studies examining the association between the DRD2 gene A1 allele and substance abuse. These analyses focus on the key methodological issues of control selection, clinical severity phenotype, and ethnic composition. In addition, studies of the A1 allele and personality traits were examined given the broad risk that this allele may convey via personality structure.
Section snippets
Method
The meta-analyses were conducted on 55 studies that examined the association between the A1+ allele of the DRD2 gene and substance abuse and on 9 studies related to the A1+ allele and personality. The substance misuse papers were found using Ovid MEDLINE, and Web of Science, 1990 to 2004. The research combined the key words DRD2, dopamine, and substance abuse or alcoholism or dependence. This search yielded 114 results. Using this list and by investigating the references of comprehensive
Results
As shown in Table 2, among studies that did not screen controls for the presence of alcohol or drug use, the A1 allele was significantly more likely to be found in the clinical samples or problem substance use than the control groups (effect=1.425; 95% CI 1.010–2.010, P<.05). Similarly shown in Table 3, Table 4, Table 5, the A1 allele was significantly more likely to be present in the severe clinical groups compared than in control groups (effect=1.675; 95% CI 1.238–2.266, P<.005), as well as
Discussion
The current meta-analyses have confirmed the A1 allele of the DRD2 as a marker of substance use and a particularly strong marker of severe substance misuse. Given that the studies with substance misusing and control groups involved almost 10,000 participants, this is a robust meta-analysis. Personality studies did not confirm a possible endophenotype associated with A1+ allelic status.
More finely grained associations of the A1 allele have been reviewed recently (Noble, 2003), but there were
Acknowledgements
This research was funded by an NH and MRC grant to Professors Young, Dr. Lawford, and Professor Noble, a Faculty of Health Research Grant from The Queensland University of Technology and the Christopher D. Smithers Foundation. The expert assistance of Robyn Johnston in producing this document is acknowledged with thanks.
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