Association analysis between polymorphisms in the dopamine D2 receptor (DRD2) and dopamine transporter (DAT1) genes with cocaine dependence

Abstract

Genetic research on cocaine dependence (CD) may help clarify our understanding of the disorder as well as provide novel insights for effective treatment. Since dopamine neurotransmission has been shown to be involved in drug reward, related genes are plausible candidates for susceptibility to CD. The dopamine receptor D₂ (DRD2) protein and dopamine transporter (DAT1) protein play regulatory roles in dopamine neurotransmission. The TaqI A single-nucleotide polymorphism (SNP) in the DRD2 gene and the 3′ variable number tandem repeat (VNTR) polymorphism in the DAT1 gene have been implicated in psychiatric disorders and drug addictions. In this study, we hypothesize that these polymorphisms contribute to increased risk for CD. Cocaine-dependent individuals (n = 347) and unaffected controls (n = 257) of African descent were genotyped for the polymorphisms in the DRD2 and DAT1 genes. We observed no statistically significant differences or trends in allele or genotype frequencies between cases and controls for either of the tested polymorphisms. Our study suggests that there is no association between the DRD2 and DAT1 polymorphisms and CD. However, additional studies using larger sample sizes and clinically homogenous populations are necessary before confidently excluding these variants as contributing genetic risk factors for CD.

Cocaine dependence (CD) is characterized by compulsive cocaine seeking and continued drug use despite adverse consequences. The profound loss of behavioral control is the hallmark of cocaine addiction and contributes to the high risk of relapse. Currently, treatment options for this devastating addiction are limited and there are no FDA approved pharmacological treatments available. While the interplay between genetic and environmental factors underlying CD is not fully understood, studies have estimated that approximately two thirds of an individual’s risk for developing CD is heritable [26,27]. Therefore, identifying genetic susceptibility factors for CD may provide important new insights into its pathophysiology and eventually may lead to effective therapies. Nevertheless, identifying genetic risk factors is difficult due to the complex mode of inheritance as well as clinical and genetic heterogeneity of cocaine-dependent individuals and strong environmental influences. Furthermore, associated genes may only contribute a small portion to the overall risk.

Genes involved in dopamine neurotransmission are biologically plausible candidate genes for CD since dopamine pathways play a major role in drug reward [8,25,28]. Specifically, genes for dopamine receptors and transporters are logical targets for study since they are directly responsible for transmitting dopamine-mediated brain signals. Several studies have investigated associations between CD and variants in the dopamine receptor D₃ gene (DRD3) and variants in the dopamine receptor D₄ gene (DRD4). Results of these studies were equivocal with both positive [7,33] and negative [2,6] association reports. This inconsistency is due at least in part to small sample sizes as well as the complex genetic and clinical nature of CD.

The dopamine receptor D₂ gene (DRD2) encodes an inhibitory dopamine receptor subtype. The striatopallidal medium spiny neurons, cells involved in psychostimulant reward pathways, predominantly express the D₂ dopamine receptor subtype [18,19,24,30,45,54]. Hence, variations in the DRD2 gene may affect dopamine signaling via the striatopallidal pathway and consequently increase susceptibility to CD. While many single-nucleotide polymorphisms (SNPs) spanning the DRD2 gene are cataloged, the TaqI A SNP (rs1800497) has been shown to directly affect dopamine binding with the D₂ receptor [48]. Furthermore, the polymorphism has been implicated previously in drug addictions such as heroin dependence [23,39] and alcoholism [4]. Therefore, the TaqI A SNP in the DRD2 gene might be a biologically functional variant underlying susceptibility to CD.

Another plausible susceptibility gene for CD is the dopamine transporter gene (DAT1). The DAT1 protein mediates dopamine reuptake from the synaptic cleft into the presynaptic terminals, regulating the duration and intensity of dopaminergic signaling. Like DRD2, DAT1 is expressed in the striatal neuroanatomical region, which is implicated in cocaine reward [3,10–12,37,50,51]. Many polymorphisms across the DAT1 gene have been cataloged. The variable number tandem repeat (VNTR) polymorphism in the 3′ region of DAT1 consists of a 40-bp repetitive sequence. One study reported that the 10-repeat allele (10R) enhances the expression of the DAT1 protein [14] while another study claimed that the 9-repeat allele (9R) enhanced the DAT1 expression [35]. Although the specific results of each study conflicted, both reports suggest that the DAT1 3′ VNTR polymorphism affects DAT1 expression, consistent with subsequent findings that the DAT1 VNTR is associated with drug addictions such as methamphetamines [22] and alcoholism [46].

Both DRD2 and DAT1 have been investigated in CD, and positive associations have been found among Caucasian and Brazilian populations [20,36]. In this study, we tested the hypothesis that the DRD2 TaqI A SNP and the DAT1 3′ VNTR give rise to protein variants that increase susceptibility to CD among individuals of African descent.

Blood samples for DNA isolation from cocaine-dependent individuals of African-American decent (n = 347; 72% male, mean age: 43) were collected during clinical studies of CD at the University of Pennsylvania Treatment Research Center. Subjects were at least 18 years of age. All were assessed with the Structured Clinical Interview for DSM Disorders (SCID) and urine drug screens were obtained. All patients had a clinical diagnosis of CD as defined by DSM-IV. Family history was not obtained and ethnicity was determined by self-report. All psychiatric axis I disorders except alcohol dependence/abuse and nicotine dependence were used as exclusion criteria. In addition, participants were excluded if they had a history of a seizure disorder (except cocaine-induced seizures) or a severe medical illness, including a history of AIDS (but not merely of HIV+ status). Individuals currently being treated with psychotropic medications were also excluded. Blood samples from control persons of African-American descent (n = 257; 29% male, mean age: 40) were collected at the University of Pennsylvania, Thomas Jefferson University, and the National Institute of Mental Health Genetics Initiative (www.nimhgenetics.org). Control individuals were screened for history of substance use disorders or other psychiatric illness. They were not assessed with a urine drug screen and ethnicity was determined by self-report. Subjects with a history of substance dependence or a history of major psychiatric illness were excluded from this study [5]. Genomic DNA was extracted from peripheral leukocytes within obtained blood samples using standard methods. All protocols were approved by the Institutional Review Boards at Thomas Jefferson University and the University of Pennsylvania, and all subjects provided written informed consent before blood sample collection.

The DRD2 gene is located on chromosome 11q23. DRD2 contains 9 exons and spans 65,683 bp (Ensembl Human Exon View accession ENSG00000149295). The TaqI A polymorphism (rs1800497) is located 10,541 bp downstream of the termination codon in the 3′-flanking region of DRD2 gene. SNP genotyping was performed using Applied Biosystems Inc. (ABI) ‘Assays-on-demand’ as per manufacturer protocol. Quality control was maintained by genotyping 10% duplicates for cases and controls. The DAT1 gene is located on chromosome 5p15.3. DAT1 contains 15 exons and spans 52,638 bp (Ensembl Human Exon View accession ENSG00000142319). VNTR genotyping was performed using protocols previously described [29].

Genotype and allele frequencies were compared between groups using χ² contingency analysis. A two-tailed type I error rate of 5% was chosen for the analysis. Our sample size had reasonable power to detect a disease association at a P-value less than or equal to 0.05, assuming an odds ratio of 1.5 and a minor allele frequency of 30% (99% for a log-additive mode of inheritance, 92% for a dominant, and 56% for a recessive mode of inheritance). Power analysis was performed using the Quanto program [15].

None of the genotype distributions deviated significantly from those expected by Hardy–Weinberg equilibrium for cases or controls. Single marker analysis for the DRD2 TaqI A SNP did not yield evidence for association on the genotypic level or allelic level (Table 1). Genotyping success rates were 98.59% and 99.23% for cases and controls, respectively. The concordance rate was 100% with respect to the 10% of samples that were genotyped twice for quality control. Allelic frequencies were consistent with those reported in the Hapmap database for Yoruba in Ibadan, Nigeria.

Table 1.

Genotype and allele frequencies of the TaqI polymorphism in the DRD2 gene.

SNP	Sample	n	Genotype frequency			P	Allele frequency	P
rs1800497			C/C	C/T	T/T		f(C)
	Cocaine	347	0.433	0.436	0.132	0.800	0.650	0.852
	Controls	257	0.451	0.409	0.140		0.656

No association was found between the DAT1 VNTR polymorphism and CD on a genotypic or allelic level (Table 2). Additional genotypic analysis by clustering cases and controls into groups of individuals with a long genotype (10/11, 9/11, 10/12) and short genotype (6/10, 7/9, 7/10, 9/9, 10/10), as suggested by others [23], did not reveal any association (data not shown). Genotyping success rates were 94.60% and 96.90% for cases and controls, respectively. The concordance rate was 100% with respect to the 10% of samples that were genotyped twice for quality control.

Table 2.

Genotype and allele frequencies of the DAT1 40-bp VNTR polymorphism.

	Cocaine n = 339		Control n = 245		P
Genotype a
2/2	3	(0.9%)	6	(2.4%)	0.131
2/9	4	(1.2%)	2	(0.8%)
2/10	10	(2.9%)	5	(2.0%)
6/9	0	(0.0%)	1	(0.4%)
6/10	0	(0.0%)	3	(1.2%)
7/9	1	(0.3%)	2	(0.8%)
7/10	4	(1.2%)	5	(2.0%)
8/8	1	(0.3%)	0	(0.0%)
8/9	3	(0.9%)	6	(2.4%)
8/10	16	(4.7%)	7	(2.9%)
8/12	0	(0.0%)	1	(0.4%)
9/9	16	(4.7%)	8	(3.3%)
9/10	90	(26.5%)	64	(26.1%)
9/11	2	(0.6%)	2	(0.8%)
10/10	184	(54.3%)	131	(53.5%)
10/11	2	(0.6%)	0	(0.8%)
10/12	1	(0.3%)	0	(0.0%)
10/13	1	(0.3%)	0	(0.0%)
11/11	1	(0.3%)		(0.0%)
Allele
2 repeats	20	(2.9%)	19	(3.9%)	0.272
6 repeats	0	(0.0%)	4	(0.8%)
7 repeats	5	(0.7%)	7	(1.4%)
8 repeats	21	(3.1%)	14	(2.9%)
9 repeats	132	(19.5%)	93	(19.0%)
10 repeats	492	(72.6%)	348	(71.0%)
11 repeats	6	(0.9%)	4	(0.8%)
12 repeats	1	(0.1%)	1	(0.2%)
13 repeats	1	(0.1%)	0	(0.0%)

^aGenotypes are presented as number of repeats observed for both alleles of DAT1 gene.

Dopaminergic brain systems have been implicated to play a major role in drug reward [25], thus making genes involved in these circuits plausible candidates for influencing susceptibility to substance use disorders. In fact, several genes coding for the dopaminergic system have been investigated in CD including genes for the dopamine receptor D2 (DRD2) [16,34,36,40] the dopamine receptor D3 (DRD3) [6,7,13,34], the dopamine receptor D4 (DRD4) [2], the dopamine transporter (DAT) [17,20,41] and the catechol-O-methyl-transferase (COMT) gene [31]. The results of all these studies have been conflicting with some positive reports and some negative findings, possibly due to small sample sizes and the complex genetic nature of CD. In this study we investigated whether polymorphisms the DRD2 and DAT1 genes confer risk to CD. DRD2 and DAT1 play a direct role in striatal dopamine signaling. Furthermore, polymorphisms in these genes have been associated with psychiatric disorders and drug addiction. In particular, the TaqI A SNP in the DRD2 gene and the 3′ VNTR polymorphism in the DAT1 gene convey functional polymorphisms and have been associated with drug addictions. Therefore, we hypothesized that the DRD2 TaqI A SNP and the DAT1 3′ VNTR increase susceptibility to CD among individuals of African descent.

Our data suggests that these polymorphisms are not involved in risk for CD in this cohort of cases and controls since single marker analysis did not yield evidence in support of an association; however, additional markers are needed for complete gene coverage in order to comprehensively rule out a role of these genes in CD. Furthermore, our study had limited statistical power due to the small sample size to detect risk alleles that contribute small effects to the overall disorder. This limited power could explain our negative results. Additional association studies with larger sample sizes should be conducted to test for the DRD2 and DAT1 polymorphisms, which may contribute small effects to susceptibility to CD. An increased sample size would also provide stronger statistical power to investigate a DRD2–DAT1 gene–gene interaction, which was performed in previous psychiatric genetic studies [23,44]. Our limited sample size provided us no statistical power to detect gene–gene interaction, but it would be interesting for future studies with larger sample sizes to investigate whether dopaminergic pathway genes show interaction in individuals with CD. On the other hand, it must also be considered that larger sample sizes may increase genetic heterogeneity and contribute to undetected population stratification, which would negatively impact the interpretation of association analysis [32]. Unaccounted differences in population structure can create spurious associations in and of themselves and lead to inaccurate interpretation of results [43]. The possibility of unaccounted differences in population structure is especially relevant to analyses involving individuals of African descent since there is substantial genetic heterogeneity among African-Americans [38,42,49,53]. Genomic controls and/or the utilization of family-based association studies may control for these stratification issues [1,9,47]. Another limiting factor in our study was clinical heterogeneity. When conducting genetic association studies, the clinical phenotype might add additional heterogeneity which could obscure an association or lead to a false positive finding. Patients with co-morbid alcohol dependence/abuse and nicotine dependence were not excluded from our cocaine-dependent population. While all patients were diagnosed with CD in accordance with the DSM-IV criteria, co-morbid alcohol and nicotine use might have differed between patients. It has been shown that genetic factors play a role in nicotine dependence [52] and alcoholism [21], thus perhaps shared genetic factors contribute to all substance use disorders. Hence, unaccounted clinical heterogeneity within a population may have exacerbated genetic heterogeneity among the cocaine cases and may have led to false negative or positive findings. Although all patients were diagnosed according to DSM-IV criteria and the diagnosis of CD was supported by urine drug screen data, the control subjects were assessed using semi-structured interviews but did not undergo urine drug testing. While drug testing is useful in establishing a diagnosis, it might not be useful for the assessment of controls since it does not rule out past exposure or substance use. Unreported or minimized substance abuse in the control population is thus an important limitation that needs to be considered; however, even under the assumption that the control group had 1% of undetected CD cases, assuming the general prevalence rate of CD in the control population, this factor might have only minor impact when comparing cocaine cases to the group of controls.

In summary, our results do not support an association between the TaqI A polymorphism in the DRD2 gene or the 3′ VNTR in the DAT1 gene and CD in individuals of African descent. However, additional studies using larger sample sizes and clinically homogenous populations are necessary before excluding these variants as contributing genetic risk factors for CD.