Lack
of Association Genes
Between Dopamine and Bipolar Affective
Di and D2 Receptor Disorder
Markus M. N#{246}then,M.D., Jeanette Erdmann, M.Sc., Judith K#{246}rner,M.D., Mario Lanczik, M.D., Jurgen Fritze, M.D., Rolf Fimmers, Ph.D., David K. Grandy, Ph.D., Brian O’Dowd, Ph.D., and Peter Propping, M.D.
Fifty-six for
patients
association
D2 receptor mutant form affective (Am
J
bipolar
with of
restriction
loci. No ofeither
disorder Psychiatry
affective
disorder
significant receptor
urnan dopaminergic neurons are involved in the control of hormone secretion, voluntary movement, and emotional behavior. These effects are mediated by dopamine D1 and D2 receptors that stimulate and inhibit, respectively, the enzyme adenylate cyclase. Dopamine antagonists display antimanic properties and, conversely, dopaminergic drugs can provoke a switch to mania. Thus, an involvement of dopaminengic receptors in the pathophysiology of bipolar affective disorder would seem evident. The human D1 and D2 receptor genes have recently been cloned and assigned to the chnomosomal regions 5q31-34 and 1 1q22-23, respectively (1-5). Sometimes chnomosomal aberrations provide additional clues to where a gene may reside. In a recently reported family, a balanced 9/i 1 chromosomal translocation cosegnegated with bipolar affective disorder; the breakpoint on chromosome 1 1 was at 1 iq22.3 in close proximity to the D2 receptor gene (6). True association may occur for two reasons: 1 ) there is strong linkage disequilibrium between a disease-causing locus and the locus under study (recombination fraction < 1 %), and 2) the marker locus itself contnibutes to the pathogenesis of the illness (7). In a number of complex disorders, association studies have been successfully performed. In narcolepsy, for example, an
ment
of Psychiatry,
and
the Institute
received July 18, 1991; accepted of Human Genetics, the Departfor Medical
Statistics,
many. Supported Copyright
Am
University
of Bonn,
Wilhelmstra1e
31, D-5300
by the Deutsche Forschungsgemeinschaft. © 1992 American Psychiatric Association.
/ Psychiatry
1 49:2,
control
subjects
were
tested D1 and a single bipolar
February
1992
almost 100% association with the human lymphocyte antigen (HLA) DR2 allele was found. In affective disondens, studies focused on blood group and HLA antigens; results were either negative or inconsistent. Recent advances in molecular biology and the increasing availability of polymorphic DNA segments as markers have greatly expanded the potential utility of these markers in association studies. Genes that, by evidence of biochemical on pathophysiological abnormalities in the illness under study, possibly contribute to the etiology of the illness serve as candidate genes. Usually, a candidate gene for the disease is selected and a nearby restriction fragment length polymorphism (RFLP) is used as a tool to compare patients and control subjects. The association approach is different from linkage studies, where cosegregation of a marker trait and the disease is examined in families. A major advantage of association studies is that no assumptions have to be made about the mode of inheritance, penetrance, and age at onset of the disease. Recently, the first association of an RFLP with bipolan affective disorder was reported for the tynosine hydroxylase gene in patients and control subjects in France (8). However this association could not be replicated in a German sample (9). To look for a possible association between RFLP alleles detected by D1 and D2 DNA probes and bipolar affective disorder, we examined a series of unrelated patients with bipolar illness and a group of control subjects.
University
of Bonn; the Department of Psychiatry, University of Wurzburg, Germany; the Vollum Institute, Oregon Health Sciences University, Portland; and the Department of Pharmacology, University of Toronto, Canada. Address reprint requests to Dr. Propping, Institute of Human Genetics,
69 healthy
was not supported. 1992; 149:199-201)
H
Received Feb. 26, 1991; revision Aug. 16, 1991. From the Institute
and
length polymorphism alleles at the dopamine associations were found; thus, the hypothesis that gene is responsible for the phenotype ofpatients with
fragment
Bonn
1, Ger-
METHOD We tested 56 patients with bipolar ascertained at random from admissions nc Clinic, University of Wunzburg, one of the patients were female and
affective disorder to the PsychiatGermany. Thirty25 were male. All
199
DOPAMINE
RECEPTOR
TABLE 1. Dopamine Subjects
GENES
D
AND
BIPOLAR
DISORDER
a nd D2 Re ceptor Gen otypes
and Allele Freque ncies
in Patie nts With Bipolar Aff ective Disorder
D1 Receptor
D2 Receptor
Allele FrequencyL
Genotypea
and Heal thy Control
Allele GenotypeC
.
#{149}
Frequency’
Estimated
Group Patients Control allele bX2017 CB1
and
dX2,105,
(N=56) subjects
Al/Al
Al/Al
I I
13 19
(N=69)
corresponds to absence df=1, p=O.68. B2 refer
to absence
and
of EcoRl presence,
A2/A2 42 49 restriction respectively,
site,
a1
a
SE
0.13 0.15
0.87 0.85
0.03 0.03
and
of TaqI
B1IB2
3 S
B21B2
b1
b2
SE
17
36
0.21
0.79
0.04
26
38
0.26
0.74
0.04
restriction
site
polymorphism.
df=1, p=O.3O.
AND
DISCUSSION
To test for the general involvement of the doparnine D1 and D2 receptor genes in bipolar affective disorder, we determined RFLP genotypes in the patients and the control subjects. Genotypic frequencies were not found to differ significantly from those expected according to the Hardy-Weinberg equilibrium (10). When the allele frequencies of the two test groups
200
B1IB1
A2 to its presence.
suffered from bipolar disorder that had been diagnosed according to the DSM-III criteria. Diagnostic assessments were based on clinical interviews by an expenienced psychiatrist, review of case notes, and family histony data. Twenty-nine patients had family histories of affective disorder. Sixty-nine persons were tested as control probands. They were recruited from among medical students and laboratory staff members. Persons with family histories of psychiatric illness were excluded. The female-tomale ratio in this group was 34:35. All subjects examined (patients and control subjects) were native Germans. All gave informed consent. Venous blood samples, with EDTA as the anticoagulant, were drawn from all probands. Genornic DNA was extracted from buffy coat preparations by salting out with saturated sodium chloride solution and then digested with restriction enzymes. DNA fragments were subjected to electrophoresis on 0.7% aganose gel and transferred to nylon filters. DNA probes were labeled with [a-32P]dCTP by the random primer method and hybridized to the blots. Filters were washed at a final stringency of 0.lxstandand saline citrate at 65 #{176}C and exposed to Kodak XAR-S film with image intensifier for 1-4 days at -70 #{176}C to produce autonadiognaphs. A 3-kilobase (kb) fragment of a human D1 DNA clone containing on a single exon the entire coding region of the D1 receptor detects a 6.8-kb and a 10.5-kb band RFLP in EcoRI digests. An 1 8-kb fragment of a human D2 DNA clone detects a 2.2-kb and a 9.0-kb band RFLP in TaqI digests. This fragment comprises the sequence coding the 63 amino acid residues of the C terminus of the D2 receptor, the 3’ untranslated region of the receptor mRNA, and 3’ flanking sequences.
RESULTS
Estimated
were compared, there were no significant associations between affected status and allele frequencies at the two doparnine receptor loci (table 1 ). The allele frequencies in both of the groups were similar to those reported for unrelated healthy Caucasians (2, 5). For the EcoRI site, the comparison between patients and control subjects, expressed as a relative risk, was 1.16 (p=O.68). The relative risk for the TaqI site was 1.37 (p=O.3O). To demonstrate the power of the study under the given sample size, we included a power calculation. Fixing the allele frequencies for the control subjects at 0.15 for the Al allele of the D1 receptor RFLP and at 0.26 for the Bi allele of D2, the power to detect an association is >0.80 for allele a1 frequencies0.04 (b10.12) and a1 frequencies0.30 (b10.43) in the patient group. Since the genetic load may be higher in patients with family histories of affective disorder, we subdivided the patient group on the basis of having no relative with affective disorder (N=27) or at least one relative with affective disorder (N=29). The allele frequencies of the two dopamine receptor RFLPs for these two groups of bipolar patients were compared to the frequencies in the control group (data not shown). No significant diffenences between the two patient groups and the control group were found. The results of this study do not support the hypothesis that a single mutant form of either the dopamine D1 on the dopamine D2 receptor gene is responsible for the phenotype of all or an appreciable proportion of patients from the general population who are suffering from bipolar affective disorder.
REFERENCES 1. Zhou Q-Y, Grandy DK, Thambi L, Kushner JA, Van Tol HHM, Cone R, Pribnow D, Salon J, BunzowJR, Civelli 0: Cloning and expression of human and rat D1 dopamine receptors. Nature 2.
1990; 347:76-80 Grandy DK, Zhou Q-Y, Allen L, Litt R, Magenis Liii M: A human D1 dopamine receptor gene chromosome S at q35.l and identifies an EcoRl
Hum 3.
Genet
Sunahara
1990; RK,
RE, Civelli 0, is located on RFLP. Am J
47:828-834
Niznik
HB,
Weiner
DM,
Stormann
TM,
Brann
MR, Kennedy JL, Gelernter JE, Rozmahel Seeman P, O’Dowd BF: Human dopamine 4.
by an intronless gene on chromosome Grandy DK, Marchionni MA, Makam
Am
/ Psychiatry
R, Yang Y, Israel Y, D1 receptor encoded S. Nature I 990; 347:80-83 H, Stofko
RE,
Alfano
1 49:2, February
M,
1992
NOTHEN,
S.
Frothingham L, Fischer JB, Burke-Howie AC, Civelli 0: Cloning of the cDNA and dopamine receptor. Proc Nail Acad Sci 9766 Grandy DK, Litt M, Allen L, Bunzow Makam H, Reed L, Magenis RE, Civelli mine
D2 receptor
gene
is located
and identifies a TaqI RFLP. AmJ 6. Smith M, Wasmuth J, McPherson Civelli
7.
Am
0,
Potkin
5, Litt
M:
KJ, Bunzow JR, Server gene for a human D2 USA 1989; 86:9762JR. Marchionni 0: The human
M, dopa-
1 1 at q22-q23
Hum Genet 1989; 45:778-785 JD, Wagner C, Grandy DK, of an
11q22.3-9p22
translocation with affective disorder: proximity of the dopamine D2 receptor gene relative to the translocation breakpoint. Am J Hum Genet Suppl 1989; 45:A220 Bodmer WF: The human genome sequence and the analysis of
/ Psychiatry
1 49:2,
February
1992
genic
England, 8.
on chromosome
Cosegregation
multifactorial
Leboyer
traits,
Disease:
John M,
in Molecular
Ciba
Foundation
Wiley
& Sons,
Malafosse
ERDMANN,
KORNER,
Approaches Symposium
ET AL.
to Human 1 30.
Poly-
Chichester,
1987
A, Boularand
S, Campion
D, Gheysen
F,
Samolyk
D, Henriksson B, Denise E, Des Lauriers A, Lepine J-P, Zarifian E, Clerget-Darpoux F, Mallet J: Tyrosine hydroxylase polymorphisms associated with manic-depressive illness (letter). Lancet 1990; 335:1219 9. N#{246}thenM, K#{246}rner J, Lanczik M, Fritze J, Propping P: Tyrosine hydroxylase polymorphisms and manic-depressive illness (letter). Lancet 1990; 336:575 10. Emery AEH: Methodology in Medical Genetics: An Introduction to Statistical Methods, 2nd ed. London, Churchill Livingstone, 1986
201
of Association Genes
Between Dopamine and Bipolar Affective
Di and D2 Receptor Disorder
Markus M. N#{246}then,M.D., Jeanette Erdmann, M.Sc., Judith K#{246}rner,M.D., Mario Lanczik, M.D., Jurgen Fritze, M.D., Rolf Fimmers, Ph.D., David K. Grandy, Ph.D., Brian O’Dowd, Ph.D., and Peter Propping, M.D.
Fifty-six for
patients
association
D2 receptor mutant form affective (Am
J
bipolar
with of
restriction
loci. No ofeither
disorder Psychiatry
affective
disorder
significant receptor
urnan dopaminergic neurons are involved in the control of hormone secretion, voluntary movement, and emotional behavior. These effects are mediated by dopamine D1 and D2 receptors that stimulate and inhibit, respectively, the enzyme adenylate cyclase. Dopamine antagonists display antimanic properties and, conversely, dopaminergic drugs can provoke a switch to mania. Thus, an involvement of dopaminengic receptors in the pathophysiology of bipolar affective disorder would seem evident. The human D1 and D2 receptor genes have recently been cloned and assigned to the chnomosomal regions 5q31-34 and 1 1q22-23, respectively (1-5). Sometimes chnomosomal aberrations provide additional clues to where a gene may reside. In a recently reported family, a balanced 9/i 1 chromosomal translocation cosegnegated with bipolar affective disorder; the breakpoint on chromosome 1 1 was at 1 iq22.3 in close proximity to the D2 receptor gene (6). True association may occur for two reasons: 1 ) there is strong linkage disequilibrium between a disease-causing locus and the locus under study (recombination fraction < 1 %), and 2) the marker locus itself contnibutes to the pathogenesis of the illness (7). In a number of complex disorders, association studies have been successfully performed. In narcolepsy, for example, an
ment
of Psychiatry,
and
the Institute
received July 18, 1991; accepted of Human Genetics, the Departfor Medical
Statistics,
many. Supported Copyright
Am
University
of Bonn,
Wilhelmstra1e
31, D-5300
by the Deutsche Forschungsgemeinschaft. © 1992 American Psychiatric Association.
/ Psychiatry
1 49:2,
control
subjects
were
tested D1 and a single bipolar
February
1992
almost 100% association with the human lymphocyte antigen (HLA) DR2 allele was found. In affective disondens, studies focused on blood group and HLA antigens; results were either negative or inconsistent. Recent advances in molecular biology and the increasing availability of polymorphic DNA segments as markers have greatly expanded the potential utility of these markers in association studies. Genes that, by evidence of biochemical on pathophysiological abnormalities in the illness under study, possibly contribute to the etiology of the illness serve as candidate genes. Usually, a candidate gene for the disease is selected and a nearby restriction fragment length polymorphism (RFLP) is used as a tool to compare patients and control subjects. The association approach is different from linkage studies, where cosegregation of a marker trait and the disease is examined in families. A major advantage of association studies is that no assumptions have to be made about the mode of inheritance, penetrance, and age at onset of the disease. Recently, the first association of an RFLP with bipolan affective disorder was reported for the tynosine hydroxylase gene in patients and control subjects in France (8). However this association could not be replicated in a German sample (9). To look for a possible association between RFLP alleles detected by D1 and D2 DNA probes and bipolar affective disorder, we examined a series of unrelated patients with bipolar illness and a group of control subjects.
University
of Bonn; the Department of Psychiatry, University of Wurzburg, Germany; the Vollum Institute, Oregon Health Sciences University, Portland; and the Department of Pharmacology, University of Toronto, Canada. Address reprint requests to Dr. Propping, Institute of Human Genetics,
69 healthy
was not supported. 1992; 149:199-201)
H
Received Feb. 26, 1991; revision Aug. 16, 1991. From the Institute
and
length polymorphism alleles at the dopamine associations were found; thus, the hypothesis that gene is responsible for the phenotype ofpatients with
fragment
Bonn
1, Ger-
METHOD We tested 56 patients with bipolar ascertained at random from admissions nc Clinic, University of Wunzburg, one of the patients were female and
affective disorder to the PsychiatGermany. Thirty25 were male. All
199
DOPAMINE
RECEPTOR
TABLE 1. Dopamine Subjects
GENES
D
AND
BIPOLAR
DISORDER
a nd D2 Re ceptor Gen otypes
and Allele Freque ncies
in Patie nts With Bipolar Aff ective Disorder
D1 Receptor
D2 Receptor
Allele FrequencyL
Genotypea
and Heal thy Control
Allele GenotypeC
.
#{149}
Frequency’
Estimated
Group Patients Control allele bX2017 CB1
and
dX2,105,
(N=56) subjects
Al/Al
Al/Al
I I
13 19
(N=69)
corresponds to absence df=1, p=O.68. B2 refer
to absence
and
of EcoRl presence,
A2/A2 42 49 restriction respectively,
site,
a1
a
SE
0.13 0.15
0.87 0.85
0.03 0.03
and
of TaqI
B1IB2
3 S
B21B2
b1
b2
SE
17
36
0.21
0.79
0.04
26
38
0.26
0.74
0.04
restriction
site
polymorphism.
df=1, p=O.3O.
AND
DISCUSSION
To test for the general involvement of the doparnine D1 and D2 receptor genes in bipolar affective disorder, we determined RFLP genotypes in the patients and the control subjects. Genotypic frequencies were not found to differ significantly from those expected according to the Hardy-Weinberg equilibrium (10). When the allele frequencies of the two test groups
200
B1IB1
A2 to its presence.
suffered from bipolar disorder that had been diagnosed according to the DSM-III criteria. Diagnostic assessments were based on clinical interviews by an expenienced psychiatrist, review of case notes, and family histony data. Twenty-nine patients had family histories of affective disorder. Sixty-nine persons were tested as control probands. They were recruited from among medical students and laboratory staff members. Persons with family histories of psychiatric illness were excluded. The female-tomale ratio in this group was 34:35. All subjects examined (patients and control subjects) were native Germans. All gave informed consent. Venous blood samples, with EDTA as the anticoagulant, were drawn from all probands. Genornic DNA was extracted from buffy coat preparations by salting out with saturated sodium chloride solution and then digested with restriction enzymes. DNA fragments were subjected to electrophoresis on 0.7% aganose gel and transferred to nylon filters. DNA probes were labeled with [a-32P]dCTP by the random primer method and hybridized to the blots. Filters were washed at a final stringency of 0.lxstandand saline citrate at 65 #{176}C and exposed to Kodak XAR-S film with image intensifier for 1-4 days at -70 #{176}C to produce autonadiognaphs. A 3-kilobase (kb) fragment of a human D1 DNA clone containing on a single exon the entire coding region of the D1 receptor detects a 6.8-kb and a 10.5-kb band RFLP in EcoRI digests. An 1 8-kb fragment of a human D2 DNA clone detects a 2.2-kb and a 9.0-kb band RFLP in TaqI digests. This fragment comprises the sequence coding the 63 amino acid residues of the C terminus of the D2 receptor, the 3’ untranslated region of the receptor mRNA, and 3’ flanking sequences.
RESULTS
Estimated
were compared, there were no significant associations between affected status and allele frequencies at the two doparnine receptor loci (table 1 ). The allele frequencies in both of the groups were similar to those reported for unrelated healthy Caucasians (2, 5). For the EcoRI site, the comparison between patients and control subjects, expressed as a relative risk, was 1.16 (p=O.68). The relative risk for the TaqI site was 1.37 (p=O.3O). To demonstrate the power of the study under the given sample size, we included a power calculation. Fixing the allele frequencies for the control subjects at 0.15 for the Al allele of the D1 receptor RFLP and at 0.26 for the Bi allele of D2, the power to detect an association is >0.80 for allele a1 frequencies0.04 (b10.12) and a1 frequencies0.30 (b10.43) in the patient group. Since the genetic load may be higher in patients with family histories of affective disorder, we subdivided the patient group on the basis of having no relative with affective disorder (N=27) or at least one relative with affective disorder (N=29). The allele frequencies of the two dopamine receptor RFLPs for these two groups of bipolar patients were compared to the frequencies in the control group (data not shown). No significant diffenences between the two patient groups and the control group were found. The results of this study do not support the hypothesis that a single mutant form of either the dopamine D1 on the dopamine D2 receptor gene is responsible for the phenotype of all or an appreciable proportion of patients from the general population who are suffering from bipolar affective disorder.
REFERENCES 1. Zhou Q-Y, Grandy DK, Thambi L, Kushner JA, Van Tol HHM, Cone R, Pribnow D, Salon J, BunzowJR, Civelli 0: Cloning and expression of human and rat D1 dopamine receptors. Nature 2.
1990; 347:76-80 Grandy DK, Zhou Q-Y, Allen L, Litt R, Magenis Liii M: A human D1 dopamine receptor gene chromosome S at q35.l and identifies an EcoRl
Hum 3.
Genet
Sunahara
1990; RK,
RE, Civelli 0, is located on RFLP. Am J
47:828-834
Niznik
HB,
Weiner
DM,
Stormann
TM,
Brann
MR, Kennedy JL, Gelernter JE, Rozmahel Seeman P, O’Dowd BF: Human dopamine 4.
by an intronless gene on chromosome Grandy DK, Marchionni MA, Makam
Am
/ Psychiatry
R, Yang Y, Israel Y, D1 receptor encoded S. Nature I 990; 347:80-83 H, Stofko
RE,
Alfano
1 49:2, February
M,
1992
NOTHEN,
S.
Frothingham L, Fischer JB, Burke-Howie AC, Civelli 0: Cloning of the cDNA and dopamine receptor. Proc Nail Acad Sci 9766 Grandy DK, Litt M, Allen L, Bunzow Makam H, Reed L, Magenis RE, Civelli mine
D2 receptor
gene
is located
and identifies a TaqI RFLP. AmJ 6. Smith M, Wasmuth J, McPherson Civelli
7.
Am
0,
Potkin
5, Litt
M:
KJ, Bunzow JR, Server gene for a human D2 USA 1989; 86:9762JR. Marchionni 0: The human
M, dopa-
1 1 at q22-q23
Hum Genet 1989; 45:778-785 JD, Wagner C, Grandy DK, of an
11q22.3-9p22
translocation with affective disorder: proximity of the dopamine D2 receptor gene relative to the translocation breakpoint. Am J Hum Genet Suppl 1989; 45:A220 Bodmer WF: The human genome sequence and the analysis of
/ Psychiatry
1 49:2,
February
1992
genic
England, 8.
on chromosome
Cosegregation
multifactorial
Leboyer
traits,
Disease:
John M,
in Molecular
Ciba
Foundation
Wiley
& Sons,
Malafosse
ERDMANN,
KORNER,
Approaches Symposium
ET AL.
to Human 1 30.
Poly-
Chichester,
1987
A, Boularand
S, Campion
D, Gheysen
F,
Samolyk
D, Henriksson B, Denise E, Des Lauriers A, Lepine J-P, Zarifian E, Clerget-Darpoux F, Mallet J: Tyrosine hydroxylase polymorphisms associated with manic-depressive illness (letter). Lancet 1990; 335:1219 9. N#{246}thenM, K#{246}rner J, Lanczik M, Fritze J, Propping P: Tyrosine hydroxylase polymorphisms and manic-depressive illness (letter). Lancet 1990; 336:575 10. Emery AEH: Methodology in Medical Genetics: An Introduction to Statistical Methods, 2nd ed. London, Churchill Livingstone, 1986
201
