Association between the Intron 8 VNTR Polymorphism of the DAT1 Gene and Crack Cocaine Addiction

Abstract

Background:
This study aims to compare allele and genotype frequencies of a 30-bp variable number of tandem repeats (VNTR) polymorphism of the DAT1 gene, located at intron 8, between adult crack cocaine users and nonaddicted individuals. Due to its involvement in drug addiction, this gene is a good candidate for molecular studies.

Methods:
A cross-sectional sample of 239 current adult crack abusers or dependents from in- and outpatient clinics and 211 control individuals was collected in Brazil. They were evaluated using ASRS, ASI-6, WAIS-III, and MINI assessments. DNA samples extracted from whole blood were genotyped for the intron 8 VNTR in DAT1.

Results:
Logistic regression analysis was performed and controlled for gender, age, ethnicity, educational level, and comorbidities of clinical interest (generalized anxiety disorder, suicide risk, major depressive episode, and attention deficit/hyperactivity disorder). This analysis showed that the 6R6R genotype was associated with crack cocaine addiction (OR = 1.844; CI = 1.101-3.089; p = 0.020).

Conclusions:
Our results are consistent with the role of DAT1 in the neurobiology of drug addiction. Nevertheless, the study of other genes, environmental factors, and their interactions is also important to gain a broader understanding of this condition.

Full text

Original Paper
Neuropsychobiology 2017;75:141–144
Association between the Intron 8 VNTR
Polymorphism of the DAT1 Gene and
Crack Cocaine Addiction
Anderson R. Stolf a Diana Müller b Jaqueline B. Schuch a, b
Gláucia C. Akutagava-Martins b Luciano S.P. Guimaraes a Claudia M. Szobot a, c
Ricardo Halpern d Felix H.P. Kessler a Flavio Pechansky a Tatiana Roman b
a Center for Drug and Alcohol Research (CPAD), Hospital de Clínicas de Porto Alegre (HCPA), Unidade Álvaro Alvim,
Porto Alegre, Brazil; b Department of Genetics, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil;
c Child and Adolescent Psychiatric Service (SPIA), Hospital de Clínicas de Porto Alegre (HCPA), Porto Alegre, Brazil;
d Federal University of Health Sciences of Porto Alegre, Porto Alegre, Brazil
Received: January 24, 2017
Accepted after revision: November 8, 2017
Published online: January 12, 2018
Anderson Ravy Stolf
Center for Drug and Alcohol Research, Hospital de Clínicas de Porto Alegre
Universidade Federal do Rio Grande do Sul, Rua Álvaro Alvim, 400
Porto Alegre, RS 90420-020 (Brazil)
E-Mail andersonstolf @ gmail.com
© 2018 S. Karger AG, Basel
E-Mail karger@karger.com
www.karger.com/nps
DOI: 10.1159/000485128
Keywords
Crack cocaine · Dopamine transporter · DAT1 gene ·
Genetics · Risk factors · Association
Abstract
Background: This study aims to compare allele and geno-
type frequencies of a 30-bp variable number of tandem re-
peats (VNTR) polymorphism of the DAT1 gene, located at in-
tron 8, between adult crack cocaine users and nonaddicted
individuals. Due to its involvement in drug addiction, this
gene is a good candidate for molecular studies. Methods: A
cross-sectional sample of 239 current adult crack abusers or
dependents from in- and outpatient clinics and 211 control
individuals was collected in Brazil. They were evaluated us-
ing ASRS, ASI-6, WAIS-III, and MINI assessments. DNA sam-
ples extracted from whole blood were genotyped for the in-
tron 8 VNTR in DAT1. Results: Logistic regression analysis
was performed and controlled for gender, age, ethnicity,
educational level, and comorbidities of clinical interest (gen-
eralized anxiety disorder, suicide risk, major depressive
episode, and attention deficit/hyperactivity disorder). This
analysis showed that the 6R6R genotype was associated
with crack cocaine addiction (OR = 1.844; CI = 1.101–3.089;
p = 0.020). Conclusions: Our results are consistent with the
role of DAT1 in the neurobiology of drug addiction. Never-
theless, the study of other genes, environmental factors, and
their interactions is also important to gain a broader under-
standing of this condition. © 2018 S. Karger AG, Basel
Introduction
One of the main concerns of crack cocaine use is its
higher addiction potential compared to other forms of
cocaine administration. This difference may be attributed
to a shorter time between administration and the percep-
tion of drug effects by the subject, given that delivery
speed of cocaine to its target regions in the brain seems to
play a pivotal role in its potential for abuse and depen-
dence [1]. Cocaine binds and blocks the dopamine trans-
A.R. Stolf and D. Müller contributed equally to this article.
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Neuropsychobiology 2017;75:141–144
142
DOI: 10.1159/000485128
porter (DAT) protein, leading to modification of brain
function and neurotransmission. DAT is the primary reg-
ulator of dopaminergic function and mediates both the
intensity and duration of dopaminergic signaling [2]. The
lifetime risk of crack cocaine use ranges from 1.4 to 4.1%,
being higher in the USA compared to Brazil [3, 4]. In
terms of effects and consumption patterns, there are dif-
ferences between users who inhale cocaine and those who
smoke crack cocaine. In addition, there are few specific
studies involving crack cocaine users. Moreover, it is not
clear whether the neurobiological model of cocaine ad-
diction can also be applied to the dependence on crack
cocaine.
Substance dependence is a complex disorder and prob-
ably results from interactions between environmental
factors and several genes with small and cumulative ef-
fects [5]. The overall heritability estimate for cocaine de-
pendence is approximately 70%, indicating a strong ge-
netic influence [6]. Moreover, twin studies showed a sim-
ilar pattern of genetic and environmental risk factors for
psychoactive substance dependence according to sex. In
this sense, the heritability estimates are not different in
males and females [7]. Despite this, a possible influence
of sex on the susceptibility to the disorder cannot be ruled
out. To date, genetic association studies on cocaine de-
pendence have focused on the gene that encodes DAT
(DAT1 or SLC6A3). The 40-bp variable number of tan-
dem repeats (VNTR) polymorphism located at the 3′-un-
translated region has been extensively evaluated, and
both positive and negative findings on substance depen-
dence have been described so far [2, 8–13]. This polymor-
phism has also been investigated in crack cocaine subjects
[11], including a previous report from our group [12]. For
instance, Guindalini et al. [13] found that this polymor-
phism was associated with addiction only in haplotypes
including other markers such as the intron 8 VNTR, and
functional analysis suggested that cocaine could alter and
decrease DAT1 expression, but only when the 6R allele
was present [13].
In this way, a second DAT1 polymorphism that ap-
pears to influence crack cocaine addiction is the VNTR
located at intron 8. This variant is generated by the rep-
etition of a 30-bp sequence, and 7 different alleles have
been described, where the alleles consisting of 5 and 6
repeats (5R and 6R, respectively) are the most common.
Recently, the 6R allele was identified as a possible risk fac-
tor for addiction in a sample of cocaine, crack cocaine,
and heroin addicts from Colombia. Isaza et al. [14] did
not observe an association between the 5R5R genotype
and other genotypes, and covariates such as educational
level, marital status, and family history of drug addiction
were included in their analyses. This VNTR has also been
studied in Brazil, but only in subjects with inhaled-co-
caine dependence. In this study, evidence was found of an
association between cocaine dependence and the 6R al-
lele, which conferred a small but significant effect [13].
Therefore, given that the 6R allele appears to have an
effect on crack cocaine addiction, it is a good candidate
for further studies. The present study investigated the as-
sociation between the DAT1 intron 8 VNTR and crack
cocaine addiction in Brazilian subjects.
Materials and Methods
Sample
This study was approved by the Ethics Committee of the Hos-
pital de Clínicas, Porto Alegre, Brazil (approval No. 100201). The
sample included 239 crack cocaine addicts and 211 control indi-
viduals. The crack cocaine abuse/dependence diagnosis and psy-
chiatric comorbidities were based on a semistructured question-
naire, the Mini International Neuropsychiatric Interview (MINI).
The Addiction Severity Index version 6 (ASI-6) was used to assess
the severity of dependence, the cubes and vocabulary subtests from
the Wechsler Intelligence Scale III edition (WAIS-III) were used
for IQ estimation, and the Adult ADHD Self-Report Scale (ASRS)
was used for attention deficit/hyperactivity disorder (ADHD)
screening. Control subjects were recruited from the community
and were evaluated for drug use using the Alcohol, Smoking and
Substance Involvement Screening Test. Subsequently, these indi-
viduals were required to test negative for cocaine by a urine test.
Control subjects were also evaluated using the MINI, ASRS, WAIS,
and ASI-6 tests to collect demographic data. More detailed infor-
mation on the subjects can be obtained in our previous article,
which used the same sample [12].
Genotyping
DNA was extracted from whole blood using a high-salt pre-
cipitation method [15]. The 30-bp VNTR polymorphism at intron
8 of DAT1 was genotyped by conventional PCR according to Sano
et al. [16].
Statistical Analyses
Allele and genotype frequencies were obtained for each group
(cases and controls). Logistic regression analysis was performed to
evaluate the association of the DAT1 intron 8 polymorphism with
crack cocaine addiction. Genotypes of DAT1 intron 8 were grouped
into 2 categories: 6R6R and all other genotypes detected in our
study. Covariates were defined based on empirical and statistical
(p < 0.2) criteria and included as factors in the logistic regression
model. This analysis included all individuals and was adjusted by
gender, age, ethnicity, educational level, and comorbidities (gener-
alized anxiety disorder, suicide risk, major depressive episode, and
ADHD). Continuous variables were compared between cases and
controls by either a Student t test or a Mann-Whitney test; categor-
ical variables were compared by a χ2 test or a Fisher exact test. A 5%
two-tailed level of significance was accepted for all analyses. All
analyses were performed using the software SPSS version 20.
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DAT1 Gene and Crack Cocaine
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Neuropsychobiology 2017;75:141–144
DOI: 10.1159/000485128
Results
A total of 239 cases and 211 controls were evaluated.
Most cases were male (90.3%), approximately 30 years
old, and had significantly more psychiatric comorbidities
than the controls did, including current depressive disor-
der (29.1 vs. 6.0%, p = 0.011, cases vs. controls), suicide
risk (58.8 vs. 13.9%, p < 0.001), ADHD (45.2 vs. 15.6%,
p < 0.001), and generalized anxiety disorder (22.2 vs.
5.2%, p = 0.044).
DAT1 intron 8 VNTR was in Hardy-Weinberg equi-
librium in both European and African-American ethnic
groups and also in both cases and controls. Alleles 5R and
6R, and the 5R6R and 6R6R genotypes, were the most fre-
quent in both ethnic groups. In the control group, allele
(European descent: 71% – 5R allele, 27.4% – 6R allele,
African descent: 67.9% – 5R allele, 32.1% – 6R allele, p =
1.000) and genotype frequencies (European descent:
49.6% – 6R6R genotype, 40.7% – 5R6R genotype, African
descent: 42.9% – 6R6R genotype, 50% – 5R6R genotype,
p = 0.999) were similar in all ethnicities. However, there
were significant differences between cases of African and
European descent. Subjects of African descent (63.4% –
6R allele; 38.5% – 6R6R genotype) had a lower frequency
of the 6R allele (p = 0.004) and 6R6R (p = 0.012) genotype
compared to subjects of European descent (77.8% – 6R
allele; 62.1% – 6R6R genotype).
Association analysis, including all subjects, showed
that the 6R6R genotype was associated with crack cocaine
addiction (OR = 1.844; CI = 1.101–3.089; p = 0.020) com-
pared to other genotypes (Table 1). Concerning an allelic
approach, we did not find any association with crack co-
caine dependence (p = 0.065).
Discussion
In this original study, we describe an association be-
tween the 6R6R genotype of the intron 8 VNTR of the
DAT1 gene and crack cocaine addiction. In a previous
study that evaluated this polymorphism in crack cocaine
users, an association with the 6R allele was observed, al-
though no association with any genotypes was detected
[14]. However, the subjects investigated in this study had
comorbid opioid addiction. Another study detected a
similar association between the 6R allele and addiction in
Brazilian cocaine users [13]. Therefore, our data reinforce
the importance of the DAT1 intron 8 VNTR polymor-
phism in crack cocaine addiction.
Previous evidence showed a putative role of DAT1 in-
tron 8 VNTR on crack cocaine addiction. For instance,
Guindalini et al. [13] showed that the presence of the 6R
allele was associated with a small reduced expression of
DAT1 when compared to the presence of the 5R allele,
especially when cocaine was added in the cell culture. In
addition, twin and family studies showed that genetic
factors are important in the susceptibility of addiction
and other mental disorders [6, 7]. In this sense, it is pos-
sible that the DAT1 association with crack cocaine addic-
tion might involve and be influenced by the effects of
different genetic variants. Furthermore, dopaminergic
neurons play a fundamental role in the brain reward sys-
tem, which is associated with addiction and related phe-
notypes, emphasizing the necessity for more translation-
al studies integrating molecular and clinical approaches.
Further investigations combining findings from differ-
ent genetic studies could lead to a better understanding
of the influence of this locus on crack cocaine addiction
as a whole, including possible dose-effect properties, in-
Table 1. Logistic regression analysis of the DAT1 intron 8 variable number of tandem repeats and comorbid phe-
notypes in relation to crack cocaine susceptibility (cases n = 209, controls n = 186)
OR 95% CI p value
Intron 8 (6R6R)11.844 1.101–3.089 0.020
Generalized anxiety disorder 2.552 1.024–6.361 0.044
Suicide risk 5.519 2.965–10.271 <0.001
Major depressive episode 3.527 1.328–9.365 0.011
Attention deficit/hyperactivity disorder 3.563 1.885–6.735 <0.001
OR, odds ratio; CI, confidence interval. The logistic regression model was adjusted for sex (male as reference)
(p = 0.010, OR = 0.162, 95% CI = 0.041–0.644), age (p = 0.010, OR = 0.162, 95% CI = 0.041–0.644), ethnicity
(non-European as reference) (p = 0.10, OR = 0.313, 95% CI = 0.129–0.757), and educational level (p = 0.813,
OR = 1, 95% CI = 1–1). ¹All other genotype groups were used as the reference.
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DOI: 10.1159/000485128
fluencing the amount of drug required to achieve its re-
inforcing effects.
Despite these exciting results, our conclusion is re-
stricted due to the lack of molecular studies on crack co-
caine addiction, specifically those concerning this partic-
ular variant. Although our sample size was greater than
those in other studies, e.g., 200 [11] and 220 subjects [14],
it may not be sufficient to detect the small genetic effects
that are typically observed in multifactorial diseases. Nev-
ertheless, power calculation was performed for case-con-
trol analysis considering an expected OR from 1.25 to 1.5,
which is in accordance with previous evidence for this OR
average in complex disorders [17, 18]. In this sense, our
study has a power ranging from 75 to >99%. In addition,
we must consider differences in the ethnic composition
of cases and controls. The proportion of subjects of Afri-
can descent in the cases (21.8%) was significantly differ-
ent from controls (13.4%; p = 0.01). Even though ethnic-
ity was included in the logistic regression models as a co-
variate, genetic variability that can exist for one marker in
different ethnicities may have influenced the results.
Considering the complex, multifactorial nature of this
phenotype, we hypothesize that the study of other genes,
environmental factors, and their interactions is impor-
tant to gain a broader understanding of the etiology of
this condition. In the future, the application of this knowl-
edge may help to tailor more effective prevention and
treatment strategies, and to improve the quality of life for
patients and their families.
Acknowledgments
We thank everyone who collaborated with the Center for Drug
and Alcohol Research team during this study, all health units and
their teams where cases were identified, and the Matias Velho
community where the controls were recruited.
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