American Journal of Medical Genetics (Neuropsychiatric Genetics) 88:594–597 (1999)

Brief Research Communication

No Evidence for Involvement of Polymorphisms of
the Dopamine D4 Receptor Gene in Anorexia
Nervosa, Underweight, and Obesity
Anke Hinney,1* Jennifer Schneider,1 Andreas Ziegler,2 Gerd Lehmkuhl,3 Fritz Poustka,4
Martin-H. Schmidt,5 Hermann Mayer,6 Wolfgang Siegfried,7 Helmut Remschmidt,1 and
Johannes Hebebrand1
1
Department of Child and Adolescent Psychiatry, Clinical Research Group, University of Marburg, Germany
2
Institute of Medical Biometry and Epidemiology, University of Marburg, Germany
3
Department of Child and Adolescent Psychiatry, University of Cologne, Germany
4
Department of Child and Adolescent Psychiatry, University of Frankfurt, Germany
5
Department of Child and Adolescent Psychiatry, University of Heidelberg, Germany
6
Children’s Hospital Hochried, Murnau, Germany
7
Obesity Treatment Center Insula, Berchtesgaden, Germany

Family and twin studies suggest a genetic
contribution to the etiology of anorexia ner-
vosa (AN) and obesity. Genes involved in
weight regulation can be considered as can-
didate genes for AN. The dopaminergic sys-
tem has been implicated in weight regula-
tion; previous results had suggested a pos-
sible involvement of the dopamine D4
receptor gene (DRD4). We screened for al-
leles of two different polymorphisms (13-bp
deletion, 48-bp repeat) in the DRD4. For as-
sociation tests, allele frequencies were com-
pared between 109 inpatients with AN, 82
underweight students, and 327 extremely
obese children and adolescents. For applica-
tion of transmission disequlibrium tests
(TDT) we additionally genotyped 57 and 137
trios comprising a patient with AN or an ex-
tremely obese child or adolescent, respec-
tively, and both parents. All genotyping was
performed with polymerase chain reaction
fragment length polymorphism analyses.
None of the association tests or TDT ren-
dered nominal P values below 0.1. An influ-
ence of alleles of the DRD4 on the develop-
ment of AN, underweight, or extreme early
onset obesity was not detected. Am. J. Med.
Genet. (Neuropsychiatr. Genet.) 88:594–597,
1999. © 1999 Wiley-Liss, Inc.
KEY WORDS:
Contract grant sponsor: the Deutsche Forschungsgemein-
schaft.
*Correspondence to: Dr. A. Hinney, Clinical Research Group,
Department of Child and Adolescent Psychiatry, Hans-Sachs-Str.
6, D-35033 Marburg, Germany.
E-mail: hinneya@post.med.uni-marburg.de
Received 26 June 1998; Accepted 13 April 1999
© 1999 Wiley-Liss, Inc.
KEY WORDS: eating disorder; transmission
disequilibrium test; associa-
tion
INTRODUCTION
Evidence from family and twin studies suggests a
genetic contribution to the etiology of anorexia nervosa
(AN) [Hebebrand and Remschmidt, 1995] Thus, Hol-
land et al. [1984] showed proband-wise concordance
rates for AN of 0.71 for monozygotic twins and 0.1 for
dizygotic twins. Heritability estimates based on these
rates ranged from 0.86 to 0.98 [Holland et al., 1988].
Twin studies also suggest a high heritability of the
body mass index (BMI; kg/m2). This effect applies to all
ages and weight ranges. Nonadditive effects were
found to account for more than one-half of the genetic
contribution to the BMI [Stunkard et al., 1990].
The dopaminergic system could be involved in the
pathophysiology of AN [Barry and Klawans, 1976;
Golden and Shenker, 1994] as well as in obesity and in
feeding behavior in general [Terry et al., 1995; Yang et
al., 1996]. The dopaminergic system has also been re-
lated to hyperactivity [Pirke et al., 1993], the distortion
of body image [Barry and Klawans, 1976], and reward
and reinforcement processes [Wise and Rompre, 1989].
Additionally, dopamine deficient (knock-out) mice be-
come hypoactive and stop feeding soon after birth, in-
dicating that dopamine is essential for movement and
feeding [Zhou and Palmiter, 1995].
Genes that are involved in weight regulation can be
considered as candidate genes for AN [Hebebrand and
Remschmidt, 1995]. Recently, an association study per-
taining to the role of a polymorphism in the dopamine
D3 receptor gene (DRD3) in AN rendered a negative
result [Bruins-Slot et al., 1998]. To further investigate
the role of the dopaminergic system in AN and weight
 Dopamine D4 Receptor Gene 595

TABLE I. Allele Distributions of a 13-bp Deletion Polymorphism of the Dopamine D4 Receptor

Gene in Anorexia Nervosa Patients, Underweight Students, and Extremely Obese Children
and Adolescents*
Wild-type 13-bp deletion
Study group number (%) number (%)
Patients with anorexia nervosa (n ⳱ 109) 214 (98.2) 4 (1.8)
Underweight students (n ⳱ 82) 162 (98.8) 2 (1.2)
Extremely obese children and adolescents (n ⳱ 327) 650 (99.4) 4 (0.6)
*Genotype-frequencies are not different from Hardy-Weinberg equilibrium. Association tests (two sided Fisher’s
exact test) for the 13-bp deletion revealed that none of the P values were below 0.1.

regulation, the DRD4 appears as a suitable candidate. Study Groups

Nöthen et al. [1994] described a 13-bp deletion in the
first exon of the DRD4 that causes a frameshift, which
results in a downstream premature stop, and thus
leads to a truncated, nonfunctional DRD4. They iden-
tified one homozygous carrier of this null-allele who
completely lacks DRD4. This individual is extremely
obese with a BMI of 37 kg/m2. Knock-out mice for the
DRD4 display locomoter supersensitivity to ethanol,
cocaine, and methamphetamine and are less active in
the open field than normal littermates [Rubinstein et
al., 1997]. Unfortunately, the body weight of the knock-
out mice was not reported. An additional finding im-
plicating the DRD4 is based on pharmacological evi-
dence. Thus, clozapine, which binds with a high affinity
to the DRD4 [Van Tol et al., 1991] can lead to increased
food consumption followed by weight gain, both of
which are associated with an increased leptin secretion
[Brömel et al., 1998].
The human DRD4 harbors an expressed highly poly-
morphic region in the third exon [Van Tol et al., 1992].
The polymorphism is characterized by a varying num-
ber (2–8 or 10 repeat units) of direct 48-bp repeats,
which are expressed and located in the putative third
cytoplasmatic loop of the receptor. Varying the num-
bers of repeats changes the length, structure, and pos-
sibly the functional efficiency of the receptor, which
makes this gene an interesting candidate for variations
in dopamine-related behaviors [Van Tol et al., 1992;
Asghari et al., 1995]. Accordingly, several studies have
addressed the role of this polymorphism in diverse psy-
chiatric disorders and in novelty-seeking behavior with
ambiguous results [e.g., Sanyal and Van Tol, 1997;
Benjamin et al., 1996; Ebstein et al., 1996; Jonsson et
al., 1997].
In the present study, we investigated both the role of
the 13-bp deletion and the 48-bp repeat polymorphism
of the DRD4 in AN, underweight, and obesity.
Study groups and sample preparations have been de-
scribed previously [Hinney et al., 1997]. Briefly, blood
samples were collected from 109 (5 males) adolescent
or young adult in patients with acute AN (DSM IV
criteria [APA, 1994]; mean BMI ± SD: 14.6 ± 1.7 kg/m2;
mean age: 16.9 ± 3.8 years), 82 (51 males) underweight
students (mean BMI: 18.7 ± 1.1 kg/m2; mean age: 25.5
± 4.2 years), and 327 (146 males) children and adoles-
cents with extreme obesity (mean BMI: 33.2 ± 6.4 kg/
m2; mean age: 14.0 ± 2.4 years). Seventy-three percent
of the young AN patients had the restricting type.
Ninety-nine percent of the obese children and adoles-
cents had an age and gender specific BMI percentile
above 95 as previously determined in a representative
German population sample [Hebebrand et al., 1996].
Among the underweight students, individuals with a
lifetime occurrence of AN or another eating disorder
were excluded from the analyses by screening with an
updated version (kindly provided by Professor
Wittchen, Munich, Germany) of the module for eating
disorders of the Composite International Diagnostic In-
terview [CIDI; World Health Organization, 1990]. The
BMI of the underweight students was below the 15th
percentile [Hebebrand et al., 1996].
Additionally, 57 trios comprising a female patient
with AN and her parents and 137 trios comprising an
extremely obese proband and both parents were geno-
typed for the alleles of the 48-bp repeat of the DRD4.
The frequency of the 13-bp deletion (approximately 1.5
percent) of the DRD4 was too low to perform a trans-
mission disequilibrium test (TDT). Written informed
consent was given by all participants and, in the case of
minors, their parents. The Ethics Committee of the
University of Marburg approved this study.

TABLE II. Allele Distributions of the 48-bp Repeat Polymorphism of the Dopamine D4 Receptor Gene in Anorexia Nervosa
Patients, Underweight Students, and Extremely Obese Children and Adolescents*
DRD4*2R DRD4*3R DRD4*4R DRD4*5R DRD4*6R DRD4*7R DRD4*8R
Study group number (%) number (%) number (%) number (%) number (%) number (%) number (%)
Patients with anorexia
nervosa (n ⳱ 109) 22 (10) 10 (4.6) 143 (65.6) 0 1 (0.5) 39 (17.9) 3 (1.4)
Underweight students
(n ⳱ 82) 13 (7.9) 6 (3.7) 110 (67.1) 2 (1.2) 0 31 (18.9) 2 (1.2)
Extremely obese children and
adolescents (n ⳱ 327) 62 (9.5) 27 (4.1) 443 (67.7) 6 (0.9) 3 (0.5) 108 (16.5) 5 (0.8)
*Association tests (two-sided Fisher’s exact test) for alleles of the 48-bp repeat of the DRD4 gene revealed that none of the P values were below 0.5.
596 Hinney et al.

TABLE III. Transmission Disequilibrium Tests For the Two Most Frequent Alleles (DRD4*4R and DRD4*7) of the 48-bp Repeat
Polymorphism of the Dopamine D4 Receptor Gene in 57 Anorexia Nervosa Patients and in 137 Extremely Obese Children and
Adolescents, Respectively, and Both Parents
DRD4*4R DRD4*7R
Transmitted/nontransmitted,* Transmitted/nontransmitted,*
Study group (transmission rate in %) P value** (transmission rate in %) P value**
Patients with anorexia nervosa 33/28 (0.54) 0.61 21/21 (0.5) 1
Extremely obese children and adolescents 67/65 (0.51) 0.93 32/39 (0.46) 0.48
*Transmitted versus nontransmitted alleles from parents heterozygous for the respective alleles.
**P values according to the two-sided exact asymptotic ␹2 test for transmission disequilibrium.

Molecular Analysis frequencies among the three groups. Allele frequencies

Polymerase chain reaction fragment length analysis
was performed essentially as described [Nöthen et al.,
1994; Lichter et al., 1993]. Briefly, the 13-bp deletion in
the first exon of the DRD4 was amplified and analyzed
according to Nöthen et al. [1994]. The 48-bp repeat
polymorphism in the third exon of the DRD4 was am-
plified with the primers D4-42 and D4-3 according to
Lichter et al. [1993]. Fragment sizes were determined
by comparison to molecular length standards. The nu-
meric designation of each allele refers to the number of
repeats (DRD4*nR).
Statistics
For association studies we genotyped 109 AN pa-
tients, 82 underweight students, and 327 extremely
obese children and adolescents. Fisher’s exact two-
sided test was used to investigate association. Asymp-
totic ␹2 tests for transmission disequilibrium [Spiel-
man et al., 1993] were performed for the two most fre-
quent alleles of the 48-bp repeat (DRD4*4R and
DRD4*7R) of the DRD4, in trios comprising a patient
with AN (n ⳱ 57) or extreme obesity (n ⳱ 137) and
both parents, respectively. We also applied an exact
multiallelic version of the TDT (for marginal homoge-
neity) as proposed by Cleves et al. [1997].
RESULTS
In order to perform association tests we compared
allele frequencies of the 13-bp deletion and all alleles of
the 48-bp repeat of the DRD4 among 109 AN patients,
82 underweight students, and 327 extremely obese
children and adolescents (Tables I and II). None of the
comparisons rendered nominal P values < 0.1.
Additionally, for the two most frequent alleles of the
48-bp repeat (DRD4*4R and DRD4*7R), transmission
disequilibrium tests were performed in 57 trios com-
prising AN patients and both parents and 137 trios
comprising extremely obese children and adolescents
and both parents (Table III). Again, none of the P val-
ues were < 0.4. The multiallelic version of the TDT
rendered P values of 0.74 (for trios comprising a patient
with AN) and 0.52 (for trios comprising an extremely
obese child or adolescent), respectively.
DISCUSSION
To our knowledge, this is the first study pertaining to
polymorphisms in the DRD4 and AN, underweight,
and extreme obesity. We found no differences in allele
were similar to those previously described in other Ger-
man study groups [Nöthen et al., 1994; Chang et al.,
1996; Hebebrand et al., 1997]. Our sample size for pa-
tients with AN well exceeds the number of patients
previously investigated for an association between the
DRD3 polymorphism and this eating disorder [Bruins-
Slot et al., 1998].
Additionally we did not detect transmission disequi-
librium of one of the analyzed alleles in patients with
AN or in probands with obesity. We conclude that a role
of the investigated DRD4 polymorphisms in the etiol-
ogy of AN and obesity appears unlikely. Future genetic
research related to the role of the dopaminergic system
in AN and weight regulation should focus on other can-
didate genes.
ACKNOWLEDGMENTS
We thank the probands and their families for their
participation. This study was supported by the
Deutsche Forschungsgemeinschaft.
REFERENCES
American Psychiatric Association. 1994. Diagnostic and Statistical Manual
of Mental Disorders. 4 ed. American Psychiatric Association: Washing-
ton.
Asghari V, Sanyal S, Buchwaldt S, Paterson A, Jovanovic V, Van Tol HH.
1995. Modulation of intracellular cyclic AMP levels by different human
dopamine D4 receptor variants. J Neurochem 65:1157–1165.
Barry VC, Klawans HL. 1976. On the role of dopamine in the pathophysi-
ology of anorexia nervosa. J Neural Transm 38:107–122.
Benjamin J, Li L, Patterson C, Greenberg BD, Murphy DL, Hamer DH.
1996. Population and familial association between the D4 dopamine
receptor gene and measures of Novelty Seeking. Nat Genet 12:81–84.
Bruins-Slot L, Gorwood P, Bouvard M, Blot P, Ades J, Feingold J, Schwartz
JC, Mouren-Simeoni MC. 1998. Lack of association between anorexia
nervosa and D3 dopamine receptor gene. Biol Psychiatry 43:76–78.
Brömel T, Blum WF, Ziegler A, Schulz E, Bender M, Fleischhaker C, Rem-
schmidt H, Krieg JC, Hebebrand J. 1998. Serum leptin levels increase
rapidly after initiation of clozapine therapy. Mol Psychiatry 3:76–80.
Chang FM, Kidd JR, Livak KJ, Pakstis AJ, Kidd KK. 1996. The world-wide
distribution of allele frequencies at the human dopamine D4 receptor
locus. Hum Genet 98:91–101.
Cleves MA, Olson, JM, Jacobs KB. 1997. Exact transmission-disequi-
librium tests with multiallelic markers. Genet Epidemiol 14:337–347.
Ebstein RP, Novick O, Umansky R, Priel B, Osher Y, Blaine D, Bennett
ER, Nemanov L, Katz M, Belmaker RH. 1996. Dopamine D4 receptor
(DRD4) exon III polymorphism associated with the human personality
trait of Novelty Seeking. Nat Genet 12:78–80.
Golden NH, Shenker IR. 1994. Amenorrhea in anorexia nervosa: neuroen-
docrine control of hypothalamic dysfunction. Int J Eat Disord 16:53–60.
Hebebrand J, Remschmidt H. 1995. Anorexia nervosa viewed as an ex-
treme weight condition: genetic implications. Hum Genet 95:1–11.
Hebebrand J, Himmelmann GW, Heseker H, Schäfer H, Remschmidt H.

1996. Use of percentiles for the body mass index in anorexia nervosa:
diagnostic, epidemiological and therapeutic considerations. Int J Eat
Dis 19:359–369.
Hebebrand J, Nöthen MM, Ziegler A, Klug B, Neidt H, Eggermann K,
Lehmkuhl G, Poustka F, Schmidt MH, Propping P, Remschmidt H.
1997. Nonreplication of linkage disequilibrium between the dopamine
D4 receptor locus and Tourette syndrome. Am J Hum Genet 61:238–
239.
Hinney A, Barth N, Ziegler A, von Prittwitz S, Hamann A, Hennighausen
K, Lentes KU, Heils A, Rosenkranz K, Roth H, Coners H, Mayer H,
Herzog W, Siegfried A, Lehmkuhl G, Poustka F, Schmidt MH, Schäfer
H, Grzeschik KH, Pirke KM, Lesch K-P, Remschmidt H, Hebebrand J.
1997. Serotonin transporter gene-linked polymorphic region: allele dis-
tributions in relationship to body weight and in anorexia nervosa. Life
Sci 61:PL295–303.
Holland AJ, Hall A, Murray R, Russell GF, Crisp AH. 1984. Anorexia
nervosa: a study of 34 twin pairs and one set of triplets. Br J Psychiatry
145:414–419.
Holland AJ, Sicotte N, Treasure J. 1988. Anorexia nervosa: evidence for a
genetic basis. J Psychosom Res 32:561–571.
Jonsson EG, Nöthen MM, Gustavsson JP, Neidt H, Brene S, Tylec A, Prop-
ping P, Sedvall GC. 1997. Lack of evidence for allelic association be-
tween personality traits and the dopamine D4 receptor gene polymor-
phisms. Am J Psychiatry 154:697–699.
Lichter JB, Barr CL, Kennedy JL, Van Tol HH, Kidd KK, Livak KJ. 1993.
A hypervariable segment in the human dopamine receptor D4 (DRD4)
gene. Hum Mol Genet 2:767–773.
Nöthen MM, Cichon S, Hemmer S, Hebebrand J, Remschmidt H,
Lehmkuhl G, Poustka F, Schmidt M, Catalano M, Fimmers R, Körner
J, Rietschel M, Propping P. 1994. Human dopamine D4 receptor gene:
frequent occurrence of a null allele and observation of homozygosity.
Hum Mol Genet 3:2207–2212.
Pirke KM, Broocks A, Wilckens T, Marquard R, Schweiger U. 1993. Star-
Dopamine D4 Receptor Gene 597
vation-induced hyperactivity in the rat: the role of endocrine and neu-
rotransmitter changes. Neurosci Biobehav Rev 17:287–294.
Rubinstein M, Phillips TJ, Bunzow JR, Falzone TL, Dziewczapolski G,
Zhang G, Fang Y, Larson JL, McDougall JA, Chester JA, Saez C, Pugs-
ley TA, Gershanik O, Low MJ, Grandy DK. 1997. Mice lacking dopa-
mine D4 receptors are supersensitive to ethanol, cocaine, and metham-
phetamine. Cell 90:991–1001.
Sanyal S, Van Tol HH. 1997. Review the role of dopamine D4 receptors in
schizophrenia and antipsychotic action. J Psychiatr Res 31:219–232.
Spielman RS, McGinnis RE, Ewens WJ. 1993. Transmission test for link-
age disequilibrium: the insulin gene region and insulin-dependent dia-
betes mellitus (IDDM). Am J Hum Genet 52:506–516.
Stunkard AJ, Harris JR, Pedersen NL, McClearn GE. 1990. The body-mass
index of twins who have been reared apart. N Engl J Med 322:1483–
1487.
Terry P, Gilbert DB, Cooper SJ. 1995. Dopamine receptor subtype agonists
and feeding behavior. Obes Res 4:515S–523S.
Van Tol HH, Bunzow JR, Guan HC, Sunahara RK, Seeman P, Niznik HB,
Civelli O. 1991. Cloning of the gene for a human dopamine D4 receptor
with high affinity for the antipsychotic clozapine. Nature 350:610–614.
Van Tol HH, Wu CM, Guan HC, Ohara K, Bunzow JR, Civelli O, Kennedy
J, Seeman P, Niznik HB, Jovanovic V. 1992. Multiple dopamine D4
receptor variants in the human population. Nature 358:149–152.
Wise RA, Rompre PP. 1989. Brain dopamine and reward. Annu Rev Psy-
chol 40:191–225.
World Health Organization (WHO). 1990. Composite international diag-
nostic interview (CIDI): CIDI interview version 1.0, CIDI-user manual,
CIDI-training manual, CIDI computer programs. Geneva: WHO.
Yang ZJ, Meguid MM, Koseki M, Oler A, Chong C, Boyd J. 1996. Increased
food intake and body weight gain after lateral hypothalamic dopami-
nergic cell implantation. Neuroreport 7:449–453.
Zhou QY, Palmiter RD. 1995. Dopamine-deficient mice are severely hypo-
active, adipsic, and aphagic. Cell 83:1197–1209.