American Journal of Medical Genetics (Neuropsychiatric Genetics) 96:678–683 (2000)

Rapid Publication
Long Forms of the Dopamine Receptor (DRD4)
Gene VNTR Are More Prevalent in Substance
Abusers: No Interaction With Functional Alleles of the
Catechol-O-Methyltransferase (COMT) Gene
David J. Vandenbergh,1,4 Lawrence A. Rodriguez,1,5 Elisabeth Hivert,1 Jocelyn H. Schiller,1
Greg Villareal,1 Elisabeth W. Pugh,2 Herbert Lachman,3 and George R. Uhl1*
1
Molecular Neurobiology Branch, Intramural Research Program, National Institute on Drug Abuse,
Baltimore, Maryland
2
Center for Inherited Disease Research, The Johns Hopkins University, Baltimore, Maryland
3
Department of Psychiatry, Albert Einstein College of Medicine, Bronx, New York
4
Current address: Department of Biobehavioral Health and Center for Developmental and Health Genetics,
Pennsylvania State University, University Park, Pennsylvania
5
Current address: Phenogenex, L.L.C., La Jolla, California

Substance abuse is a complex behavior that Keywords: association; drug abuse;

is caused by both environmental and ge-
netic factors. Work to understand the ge-
netic factors has focused on genes related to
dopamine activity because of its critical role
in rewarding and reinforcing behaviors.
The DRD3 and other dopamine receptor
subtypes are expressed in many areas of the
limbic system, and have been the objects of
study for their possible roles in several neu-
ropsychiatric disorders. Interest in variants
of the D4 gene was heightened by reports
that some alleles were more frequent in in-
dividuals who score high on Novelty Seek-
ing, an aspect of personality that may be re-
lated to drug seeking behavior. We now
show that the long form of the DRD4 gene is
more frequent in individuals with high
quantity/frequency of drug use compared to
controls (␹2 = 5.7, df = 1, P = 0.017, odds ratio
= 1.89, CI = 1.1–3.2). There is no difference in
DRD3 allele frequencies in these samples,
and there is no interaction of DRD4 alleles
with those of the catecholamine-o-methyl-
transferase gene (COMT) that we previ-
ously identified to be more frequent in sub-
stance abusers than controls [Vandenbergh,
et al.: 1997: Am. J. Med. Gen. 74:439–442]. Am.
J. Med. Genet. (Neuropsychiatr. Genet.) 96:
678–683, 2000. Published 2000 Wiley-Liss, Inc.†
Contract grant sponsor: NIDA, NIH
*Correspondence to: George R. Uhl, NIDA, National Institute
of Health, Intramural Research Program, P.O. Box 5180, Balti-
more, MD 21224. E-mail: guhl@intra.nida.nih.gov
Received 9 September 1999; Accepted 16 June 2000
Published 2000 Wiley-Liss, Inc. †
This article is a US
Government work and, as such, is in the public domain in the
United States of America.
DRD3; functional allele; be-
havior genetics
INTRODUCTION
Family, twin, and adoption studies suggest that vul-
nerability to drug abuse and dependence is based in
part on genetic factors [reviewed in Uhl et al., 1995].
The complex nature of drug use and abuse also sug-
gests that this disorder is likely to display genetic ar-
chitectures similar to those of other complex disorders
heterogeneously influenced by variants at several gene
loci, some acting largely independently and some act-
ing chiefly in concert with other genes. Understanding
the genetic contributions to substance abuse and the
interactions between genetic and environmental fac-
tors that lead to the disorder may aid in our ability to
more appropriately target prevention and treatment
strategies to individuals who have specific genetic risks
for abusing addictive substances.
Dopaminergic brain systems, especially mesocortico-
limbic systems, can play prominent roles in drug re-
ward [Gardner, 1992]. Polymorphisms in genes that
regulate dopaminergic transmission are thus candi-
dates to contribute to interindividual differences in
vulnerability for abusing addicting drugs [Koob and
Boom, 1988]. The genes encoding the five subtypes of
dopamine receptors (DRD1–5) are especially attractive
candidates to contribute to heritable variation seen in
these behaviors.
The D4 subtype is of special interest. D4 is a member
of the D2-like family of Gi/Go protein coupled receptors
that inhibit cyclic AMP formation when activated by
dopamine. Some of the most interesting DRD4 allelic
variants change the length of the protein’s third intra-

cellular loop due to the presence of a variable number
of tandem repeat polymorphism (VNTR) in the third
exon of the D4 gene. D4 receptors with longer loops can
couple more poorly to G-proteins than those with
shorter loops [Ashgari et al., 1995]. D4 receptors en-
coded by the gene’s various alleles could thus relay sig-
nals initiated by dopamine in different fashions and
provide one avenue for relationships between D4 recep-
tor alleles and complex behaviors. The DRD4 subtype
also has been of particular interest for other reasons.
Its expression is highest in limbic areas of the brain
that are largely innervated by mesolimbic/mesocortical
dopaminergic neurons, such as the frontal cortex,
amygdala, midbrain, and medulla [Van Tol et al.,
1991]. These areas are thought to contribute promi-
nently to several aspects of behavior, temperament,
and personality. DRD4 binds with higher affinity to the
atypical neuroleptic clozapine. Evidence for D4’s role in
drug responsiveness also comes from D4 knockout (KO)
mice in which the D4 gene has been deleted. These
mice show enhanced locomotor responses to ethanol,
cocaine, and methamphetamine in comparison with
their wild-type littermates [Rubenstein et al., 1997].
The DRD3 receptor is another distinct member of the
D2 dopamine receptor subfamily [Sokoloff et al., 1992].
This receptor is expressed in limbic areas such as the
ventral striatal, substantia nigra-ventral tegmental
area, and the Islands of Calleja [Diaz, 1995], although
in a pattern different from the DRD4 gene. A common
polymorphism in the DRD3 gene leads a serine for gly-
cine substitution at its ninth amino acid [Lannfelt et
al., 1992], although the ways in this polymorphism al-
ters DRD3 receptor function are currently unknown.
Markers at both of these dopamine receptor genes’
loci have been tentatively associated with neuropsychi-
atric disorders and personality traits in which dopa-
mine has been implicated. Alleles at the D4 locus have
been tentatively associated with chronic alcoholism in
some [George et al., 1993], but not all populations
[Chang et al., 1997], with Tourette’s syndrome [Grice et
al., 1996], but not with schizophrenia [Daniels et al.,
1994; Sommer et al., 1993]. Long forms of the D4 VNTR
have been associated with novelty seeking in some
[Benjamin et al., 1996; Ebstein et al., 1996], but not in
other studies [Vandenbergh et al., 1997b; Malhotra et
al., 1996]. A population of Israeli methadone clinic pa-
tients with heroin dependence displays an increased
frequency of the long forms of the DRD4 gene [Kotler et
al., 1997], but this result has not extended to all studies
[Gelernter et al., 1997]. The D11S1984 genomic poly-
morphism located near the DRD4 gene on distal chro-
mosome 11 was linked to alcohol abuse and dependence
in Southwest Indians [Long et al., 1998] but not in
another COGA sample [Reich et al., 1998]. More recent
data shows that the Indians display significant linkage
disequilibrium between D11S1984 and this DRD4
marker, suggesting to these authors that the DRD4
locus contributes much of the effect [D. Goldman, per-
sonal communication].
The D3 gene polymorphism has also shown associa-
tions with alcohol dependence. DSMIII-R (Diagnostic
and Statistical Manual of Mental Disorders) defined
cocaine dependence [Freimer et al., 1996], Tourette’s
DRD4 Alleles in Substance Abusers 679
syndrome [Comings et al., 1993], and schizophrenia
[Ebstein et al., 1997 and references therein], although
other reports fail to show such associations [Inada et
al., 1995 and references therein]. An increase in brain
D3 receptor concentrations has been found in sub-
stance abusers who died from cocaine overdoses [Staley
and Mash, 1996]. D3 KO mice show hyperactivity [Ac-
cili et al., 1996] and also enhanced sensitivity to co-
caine and amphetamine effects on locomotion [Xu et
al., 1997].
Because of each of these features, we have studied
the frequency of polymorphisms at the DRD4 and D3
gene loci in association samples of unrelated polysub-
stance abusers and ethnically matched control indi-
viduals free from substantial lifetime use of any addic-
tive substance. These data add to the emerging body of
information that tentatively identifies roles of dopami-
nergic variations in vulnerabilities to substance abuse
and related phenotypes.
METHODS
Research Volunteers
Caucasian research volunteers, 184 substance abus-
ers and 122 controls, were recruited from the National
Institute on Drug Abuse, Addiction Research Center
Clinic (n ⳱ 260), an adjacent hemodialysis unit (n ⳱
4), and a public health facility studying HIV infection
in Baltimore (n ⳱ 42). Most of these subjects’ DNA had
been previously genotyped for COMT (Catechol-o-
methyltransferase) alleles [Vandenbergh et al., 1997a].
Subjects gave informed consent, and lifetime histories
of substance use were assessed using the Drug Use
Survey (DUS) [Smith et al., 1992]. A subset of the vol-
unteers also were administered the Diagnostic Inter-
view Schedule-IIIR (DIS) of the Diagnostic and Statis-
tical Manual of Mental Disorders (DSM III-R)
(American Psychiatric Association, 1987). The DUS as-
sessment consisted of a questionnaire measuring peak
lifetime use on a scale of 0 to 3 for each of 15 classes of
addictive substances. No use of a drug was scored as 0,
experimental or infrequency use as 1, regular or inter-
mediate use as 2, and heavy frequent use as 3. As an
example, use patterns for heroin include a score of 1 for
using ⱕ1 time/week or spending <$30/week; a score of
2 for 2–6 times/week or spending $30–100/week; and a
score of 3 for daily use, typically spending >$100/day. A
composite score of total use of addictive substances was
generated for determining the phenotype used in this
study as described by Smith et al. [1992], which con-
tains a full description of the DUS. The composite score
of 0 indicated no illicit drug use and minimal use of
alcohol, nicotine, or marijuana, whereas a composite
score of 1 allowed moderate use of alcohol, nicotine, or
marijuana and minimal use of other drugs. Volunteers
with a composite score of 0 or 1, and no DSMIII-R di-
agnosis of abuse or dependence served as controls for
comparison to abusers that received composite scores
of 3, indicating heavy use of at least one illicit drug,
often with heavy use of alcohol and nicotine. For fur-
ther description of the DUS and phenotype see Smith
et al. [1992]. The controls also were compared with a
subset of the abusers who had been administered the
680 Vandenbergh et al.
DISIII-R and received a diagnosis of abuse or depen-
dence. Not all individuals from the initial stages of the
study received the DIS due to participation in other
studies at NIDA that did not include the DSM inter-
view.
Substance abuser and control groups were similar in
self-reported ethnicity, but differed modestly in age
(33.7 ± 0.6 vs. 28.6 ± 0.8 years old, respectively). In
secondary analyses, we therefore compare results from
drug abuse groups to only those controls ⱖ25 years old
(n ⳱ 70) rendering the difference in mean age insig-
nificant (32.7 ± 1.0 years old for older controls, t-test P
⳱ 0.381).
D4 Genotyping
D4 genotypes were determined from DNA extracted
from 306 of the volunteers’ peripheral blood by PCR
amplification as described by Lichter et al. [1993].
Forty nanograms of genomic DNA served as a template
in a 20 ␮L reaction containing 20 ␮M forward (5⬘-
CGCGACTACGTGGTCTACTCG-3⬘) and reverse (5⬘-
AGGACCCTCATGGCCTTG-3⬘) primers, 2 mM each
of dATP, dCTP and dTTP, one mM each of dGTP
and 7 deaza-dGTP, 10% DMSO, and 1× PCR buffer
(Perkin-Elmer). One unit of Taq polymerase (Ampli-
Taq, Perkin-Elmer) in 1× PCR biffer was added during
a “hot start” phase at 82°C and the DNA was amplified
for 40 cycles at 95°C for 20 sec, 54°C for 20 sec, and
72°C for 40 sec. The PCR products were separated by
electrophoresis through 4% nondenaturing polyacryl-
amide gels, and sizes were estimated by comparison
with 123 bp marker ladder standards (Gibco-BRL). All
allele sizes were determined independently by two in-
vestigators. In some cases the presence of a heterozy-
gote band was used to find the presence of an allele of
greater than five repeats, since these alleles can am-
plify poorly in the presence of shorter alleles. Long al-
leles were defined as those with six or more copies of
the tandem repeat, and short as those with five or
fewer copies as described by Benjamin et al. [1996] and
Ebstein et al. [1996].
D3 Genotyping
D3 genotypes were determined from 280 of the vol-
unteers by the method of Lannfelt et al. [1992]. Forty
nanograms of genomic DNA was amplified with for-
ward (5⬘-GCTCTATCTCCACATCTCACA-3⬘) and re-
verse (5⬘-AAGTCTACTCACCTCCAGGTA-3⬘) primers
in the recommended conditions for AmpliTaq polymer-
ase (Perkin-Elmer). Polymerase was added after the
reactions reached 94°C and was followed by 35 cycles of
94°C, 40 sec/55°C, 40 sec/72°C, 40 sec. One-half of the
volume (10 ␮L) was digested with 1 unit of MscI for 3
hours at 37°C in the appropriate buffer (Gibco-BRL).
The digested DNA was electrophoresed on a 5% poly-
acrylamide gels. The Al allele generated a 304 bp band
and the A2 206 and 98 bp bands.
Statistical analyses used SPSS and BMDP software
packages. Bivariate analysis was performed to assess
whether DRD4, DRD3, or COMT genotypes in these
same individuals were associated with level of drug use
in males, females, and the combined population. Logis-
tic regression was used to test whether DRD4 and
COMT were independent predictors of level of drug
use, and whether the DRD4 and COMT alleles dis-
played significant interactions. Sample numbers differ
in each analysis because some individuals did not pro-
duce genotypes for all three markers tested. Multiple
attempts to successfully amplify each marker were
made without success. Two possible reasons for the
lack of success are: the difficulty of amplifying the
DRD4 allele which is approximately 70% G+C content
and requires atypical reaction conditions (7-deaza-
dGTP in place of part of the standard dGTP), and deg-
radation of the sample DNA (new DNA could not be
collected from these anonymous samples).
Genotyping the individuals studied revealed the al-
lele and genotype frequencies for controls shown in
Table I, which are similar to those previously reported
in other Caucasian samples [Benjamin et al., 1996; Eb-
stein et al., 1996].
RESULTS
There was a significant excess in the frequency of the
long alleles (ⱖ6 copies) of DRD4 in individuals with
high life time use of illegal addictive substance (n ⳱
184) compared with controls who display no significant
use (n ⳱ 122) [␹2 ⳱ 6.50, df ⳱ 1, P ⳱ 0.007, Fisher’s
exact (one-tail), odds ratio ⳱ 1.9, 95% confidence inter-
val 1.2–3.2] (see Table II). A dichotomous variable ex-
amining only those individuals with a DUS score of 3
(heavy users) to those with a score of 0 or 1 (not/low
users) limited the comparison to the extremes of the
population and minimized the chance of misclassifica-
tion of individuals reporting intermediate drug use
when in fact their use might fall into experimental or
heavy drug use. The increased frequency of long alleles
in abusers is still significant when compared with the
older control (>25 years old) subsample (␹2 ⳱ 7.53, P ⳱
0.004). The differences seen in allele frequencies in the
complete sample are carried completely by the males (n
⳱ 248, ␹2 ⳱ 7.00, P ⳱ 0.006). Female polysubstance
abusers reveal DRD4 genotypes no different from con-
trols (␹2 ⳱ 0.00, P ⳱ 1.0). We also compared allelic
frequencies in controls with a subset of drug abusers
who completed the DIS and achieved a psychiatric di-
agnosis of drug abuse/dependence. A significantly
higher frequency of the long alleles is found in sub-
stance abusers meeting DSMIII-R criteria for sub-
stance abuse or dependence (n ⳱ 38) compared with
the controls, who lack any significant drug use (␹2 ⳱
TABLE I. DRD4 Allele and Genotype Frequencies in Controls
Alleles Frequency
(No. of repeats) n (%) Genotypesa Frequency n (%)
1 25 (10.2) 4,4 65 (53.3)
3 7 (2.9) 4,7 18 (14.7)
4 172 (70.5) 4,R 24 (19.7)
5 4 (1.6) 7,7 5 (4.1)
6 2 (0.8) 7,R 4 (3.3)
7 32 (13.1) R,R 6 (4.9)
10 2 (0.8)
a
Rare alleles (not 4 or 7) were combined and are listed as R in the table.
 DRD4 Alleles in Substance Abusers 681

TABLE II. DRD4 Allele Frequencies in Controls and TABLE IV. Logistic Regression Terms for D4 and COMT
Drug Users
Variable Coeff Std Err Odds ratio 95% conf. int.
Total groupsa Controls Older Controls Users
DRD4 0.61 0.26 1.84 1.10–3.09
Short rep homozygotes 92 (75%) 55 (80%) 113 (61%) COMT 0.67 0.29 1.95 1.11–3.42
Long rep presence 30 (25%) 14 (20%) 71 (39%) constant 0.06 0.15 1.06 0.78–1.44
Malesb
Short rep homozygotes 70 (75%) 39 (81%) 91 (59%)
Long rep presence 23 (25%) 19 (19%) 64 (41%)
that encode D2-family dopamine receptors and drug
c
abuse behaviors. The finding that alleles of the D4 gene
Females are associated with drug abuse vulnerability is of in-
Short rep homozygotes 22 (76%) 16 (76%) 22 (76%)
Long rep presence 7 (24%) 5 (24%) 7 (24%)
terest for two reasons. First, these alleles may alter the
ability of the receptor to couple dopamine binding to
␹ ⳱ 6.5, DF ⳱ 1, P ⳱ 0.012.
a 2
intracellular second messenger systems. This relation-
␹ ⳱ 7.0, DF ⳱ 1, P ⳱ 0.007.
b 2

␹ ⳱ 0, DF ⳱ 1, P ⳱ 1.
c 2
5.67, df ⳱ 1, P ⳱ 0.02). Most, but not all, individuals
were genotyped at this locus owing to the difficulty of
PCR amplification of the DRD4 VNTR, which is G+C
rich. The DRD3 allele and genotype frequencies did not
differ between controls and users. These data are
shown in Table III.
We previously reported that subjects with increased
illicit drug use had an increased frequency of an allele
of COMT gene that encodes as enzyme with relatively
high activity [Vandenbergh et al., 1997a]. We sought
possible interactive effects of the DRD4 and COMT loci
by logistic regression. Both loci have significant main
effects in the model suggesting independent effects for
the loci (shown in Table IV). However, we were not able
to find evidence of an interaction between DRD4 and
COMT in these data. When the interaction term was
forced into the model it was not significant (P ⳱ 0.48).
The model with the interaction term included did not
fit significantly better than the term with only the two
loci (Chi-square ⳱ 0.67, P ⳱ 0.41). The interaction
term was not significant when using either all controls
or only the older controls. DRD4 and COMT alleles
were thus largely independent predictors of polysub-
stance use. The results of this analysis are shown in
Table IV. Logistic regression for DRD3 with DRD4 and
COMT also showed no significant effect for DRD3 and
no significant interactions (not shown).
DISCUSSION
Virtually all addicting drugs stimulate dopamine re-
lease in mesolimbic-mesocortical brain pathways, pro-
viding a convergent pathway for the action of abused
drug classes that initially work at quite different re-
ceptor or transporter sites. The current work asks
whether associations exist between alleles of two genes
TABLE III. DRD3 MscI Allele and Genotype Frequencies in
Controls and Drug Users
Allelea Cont User Genotypeb Cont
A1 absent 18 11 A1/A1 63
A1 present 130 121 A1/A2 67
A2/A2 18
␹ ⳱ 1.1, DF ⳱ 1, P ⳱ 0.29
a 2
␹ ⳱ 1.2, DF ⳱ 2, P ⳱ 0.55
b 2
ships suggests one path for further studies to examine
the effects at a biochemical level. Second, these alleles
are not rare. One might expect that for a complex dis-
order with multiple genes exerting small effects, many
of the “disease” alleles at each gene might be relatively
common. A significant proportion of the control popu-
lation carries long alleles but does not express sub-
stance abuse. These individuals may be protected from
drug abuse for many reasons, both genetic and envi-
ronmental.
Although the difference in D4 allele frequencies in
this study was limited to males, the female sample size
(n ⳱ 58) was too small to draw any definitive conclu-
sions. We are unaware of data suggesting a sex-linked
difference in expression or function of DRD4. It is in-
teresting to note, however, that recent reports of an
excess of the seven-repeat allele in Israeli opiate abus-
ers studied a male population [Kotler et al., 1997].
There were no differences in D3 receptor alleles in
our two groups, which suggests that there is no large
effect of the serine to glycine variation encoded by
these alleles on substance abuse vulnerability. We can-
not exclude the possibility that additional variants
within the D3 gene, or nearby genes, that are not in
linkage disequilibrium with the alleles used herein
may contribute to substance abuse vulnerability.
In a recent report [Vandenbergh et al., 1997a], we
demonstrated that many of the same volunteers de-
scribed here possessed higher frequencies of the COMT
variant that encodes a high activity form of this dopa-
mine degrading enzyme. Tests for the genetic interac-
tion between the D4 and COMT loci, however, provide
no evidence that DRD4 and COMT are not indepen-
dent predictors of level of drug use. Conceivably, vari-
ants at these gene loci could function in different neu-
roanatomical regions, or alter different aspects of the
phenotype that are not revealed by our methods of
characterizing the drug abuse phenotypes of the indi-
viduals studied here.
The positive results described here must be consid-
ered in light of issues raised in any genetic association
study of unrelated individuals and issues relevant spe-
cifically to substance abuse. Our primary phenotypic
characterization is likely to suffer from the limitations
User of self-report accuracy in this population, although we
61 have achieved good concordance between quantity fre-
60 quency and DSMIII-R diagnosis of abuse or depen-
11 dence (␬ ⳱ 0.68, 91% agreement) [Smith et al., 1992].
Our sample was selected based on research volunteer
self-reports of Caucasian ancestry. Both abuser and
682 Vandenbergh et al.
control samples were recruited from the same area.
Both of these methods should reduce the likelihood of
false-positive associations due to racial or ethnic strati-
fication. However, we cannot totally exclude occult eth-
nic stratification in our sample. We report uncorrected
values for statistical significance to minimize the risk
of failure to identify true association for genes with
small effects, although the positive results for the D4
gene are also significant at the P ⳱ 0.05 level if a
Bonferroni correction for multiple testing is applied.
However, further replication of these results in addi-
tional independent samples is likely to provide the
most compelling evidence of the effect of DRD4 func-
tional variants on substance abuse vulnerability. Rep-
lication of these results may lead to improved under-
standing of the biochemical pathways of individuals
with increased susceptibility to drug abuse, enhanced
ability to identify new treatments, and better tailor
prevention and treatment choices to an individual’s
needs.
ACKNOWLEDGMENTS
We are indebted to Brenda Campbell, Miqun Robin-
son, Judy Hess, and Carolo Contoreggi, who provided
help with clinical assessment.
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