- Email: [email protected]
Exclusion of close linkage of bipolar disorder to the dopamine D3 receptor gene in nine Australian pedigrees
Journal of Affectice Disorders, 27 (1993) 213-224 0 1993 Elsevier Science Publishers B.V. All rights reserved
213 01650327/93/$06.00
JAD 00976
Exclusion of close linkage of bipolar disorder to the dopamine D, receptor gene in nine Australian pedigrees Philip Mitchell a, Brent Waters a, Christina Vivero b, Fie Le b, Jennifer Donald ‘, a, Lars Lannfelt d, Pierre Sokoloff e, John Shine b Michele Tully a, Karen Campedelli and Lisa Selbie b a School of Psychiatry, University of New South Wales, Sydney, Australia, b Garvan Institute of Medical Research, St Vincent’s Hospital, Sydney, Australia, ’ School of Biological Sciences, Macquarie University, Sydney, Australia, d Department of Geriatric Medicine, The Karolinska Institute, Huddinge University Hospital, Huddinge, Sweden and ’ Unite de Neurobiologie et Pharmacologic (U. IO9 INSERM), Centre Paul Broca, 2ter rue d’Alesia, 75014, Paris, France (Received 1 September 1992) (Revision received 3 December 1992) (Accepted 8 December 1992)
Summary The recently cloned dopamine D, receptor (DRD3) gene is of potential relevance to the aetiology of bipolar disorder because of an almost exclusive expression in limbic tissue, the region of the brain putatively responsible for control of emotion. We therefore aimed to determine whether bipolar disorder in nine pedigrees (with 171 members) was linked to this receptor gene, which has been mapped to chromosomal region 3q 13.3. Linkage of bipolar disorder and recurrent depression to the DRD3 gene was tested using a series of autosomal dominant and recessive models with varying penetrance levels. Additionally, linkage was examined using a series of levels of definitions of affective illness (ranging from bipolar I alone to all affective disorders). Close linkage to the DRD3 gene was strongly excluded using each model and definition, and these conclusions persisted when a wide range of rates of ‘sporadic’ (non-genetic) presentations of illness were incorporated in the analysis.
Key words:
Bipolar
disorder;
Dopamine
D, receptor;
Introduction “The cause of the malady we must seek, as it appears, essentially in morbid predisposition. I could demonstrate (hereditary taint) in about 80 percent of the cases observed in Heidelberg.” Kraepelin,
E. (1921)
‘Manic
Depressive
Insanity
and
Para-
noia’ (p. 75)
Linkage;
Chromosome
3
Although there is undoubted evidence for significant genetic transmission of bipolar disorder (Gershon, 19901, segregation analyses have been unable to determine the particular mode of trans-
Correspondence to; Philip Mitchell, Mood Disorders Unit, Prince Henry Hospital, Little Bay, N.S.W., 2036, Australia.
214
mission of this condition (Cox et al., 1989). Nonetheless, linkage analyses offer the potential for identifying the gene or genes involved, provided care is taken in considering a range of models and genetic parameters. There are two major approaches to linkage studies using DNA markers. The first, ‘genome scanning’ - the systematic analysis of linkage of multiple markers covering large regions of the genome - was used to some extent in the Amish study of Egeland et al. (1987). Their original report of linkage of bipolar disorder to chromosoma1 region 11~15 markers was not replicated in other studies (Hodgkinson et al., 1987; DeteraWadleigh et al., 1987; Gill et al., 1988; Kelsoe et al., 1989; Wesner et al., 1990; Mitchell et al., 1991; Mendlewicz et al., 1991). Association studies using DNA polymorphisms at the tyrosine hydroxylase gene from this same chromosomal region have been inconsistent, with one positive report (Leboyer et al., 1990), but three negative findings (Todd and O’Malley, 1989; Korner et al., 1990; Gill et al., 1991). The subsequent development of many more markers on the genetic linkage map of the human genome (Stephens et al., 1990) has enabled genome scanning to be done more completely. Pakstis et al. (1991) have reported preliminary findings from the ongoing genome scanning study of the large Old Order Amish pedigree. They estimated that they had excluded from linkage approximately 23% of the autosomal genome. Similarly, Berritini et al. (1992) have excluded linkage to most regions of chromosomes 1, 2, 5, 8, 11, 12, 13, 15, 17, 18 and 22 in a study of 21 bipolar pedigrees. An alternative strategy is the ‘candidate gene’ approach, which investigates the linkage of specific genes (for example, particular receptors or enzymes) which may have theoretical relevance to the particular disease of interest. Examples of this approach have included recent studies testing for the linkage of bipolar disorder to the D, and D, dopamine receptors (Byerley et al., 1990; Holmes et al., 1991; Mitchell et al., 1992). These investigations have been consistent in failing to demonstrate linkage to these particular receptors, and were corroborated by a similarly negative association study (Noethen et al., 1992).
In this paper we have tested for linkage between the recently cloned dopamine D, receptor (DRD3) gene (Sokoloff et al., 1990) and bipolar disorder, in an enlarged sample of nine pedigrees. As we argued in our previous publication (Mitchell et al., 1992), dopamine receptor genes are of potential relevance to bipolar disorder for several reasons. Firstly, a role of dopamine in bipolar disorder has been suggested by the ability of dopamine agonistic agents such as L-dopa, amphetamine and bromocriptine to induce mania (Silverstone and Romans-Clarkson, 1989). Secondly, three of the medications used in the treatment of this condition have significant effects on central dopamine activity. Lithium reduces dopaminergic turnover, and also effects G proteins (Avissar and Schreiber, 1992). The dopamine D, receptor is a G protein-coupled receptor, though it is unclear which G protein subunits couple to this receptor subtype (Sokoloff et al., 1992). Carbamazepine alters the growth hormone response to the dopaminergic agonist apomorphine (Elphick et al., 1990). The antipsychotics (which are effective in the acute management of mania) block dopamine receptors. The dopamine D, receptor is of particular potential interest with regard to bipolar disorder because of its almost exclusive expression in limbit structures, i.e., that region of the brain putatively responsible for the control of emotion. Although studies of the expression of this receptor in human brain tissue are yet to be published, investigations of rat brain (Bouthenet et al., 1991; Mengod et al., 1992) are consistent in demonstrating mRNA expression almost entirely limited to limbic areas (islands of Calleja, anterior nucleus accumbens, medial mamillary bodies, hippocampus and the bed nucleus of the stria terminalis). There is no expression of the genes for this receptor in the pituitary gland, unlike dopamine D, receptors. The rat and human dopamine D, receptor genes were both cloned in 1990 (Sokoloff et al., 1990; Giros et al., 1990). The human DRD3 gene is localised in chromosomal region 3q 13.3 (Le Coniat et al., 1991). Studies in both rat (Sokoloff et al., 1990) and human (Sokoloff et al., 1992) cell lines have demonstrated that dopamine has a much higher affinity for D, than D, receptors.
215
families (01 and 02) with three generations of illness and a large number of affected individuals. These families have been described in our previous publications (Mitchell et al., 1991; 1992). In the second phase (1990-1992) we identified and fully assessed a further 22 suitable families (03241, seven of whom had genotyping for this particular study. All available relatives were interviewed using standardised instruments to derive life time Research Diagnostic Criteria (RDC) diagnoses using the strict NIMH criteria for depression which require at least one month duration of symptoms, and functional impairment or incapacity. Families 01 and 02 were assessed using the Schedule of Affective Disorders and Schizophrenia-Lifetime Version (SADS-L), and families 03-24 were assessed with the Composite International Diagnostic Interview (CIDI) (Robins et al., 1988). The CID1 was developed from the Diagnostic Interview Schedule (DIS) from which Feighner, RDC, and DSM-III diagnoses could be derived - and incorporates all the mood disorder items of that instrument. The CID1 allows the ability to apply multiple diagnostic systems (RDC, DSM-III, DSM-III-R and ICD-10) to linkage analysis, a capacity recommended in the McArthur Foundation Workshop on Linkage in Affective Disorder (Merikangas et al., 1989). In conjunction with the structured interviews, all marrying-in individuals were routinely ques-
Most antipsychotics demonstrate high affinities at the D, receptor, though all appear to be more potent at the D, receptor. These findings suggest that the D, receptor may be potentially involved in the aetiology of psychotic disorders such as bipolar disorder or schizophrenia. Methods Case ascertainment and diagnosis We sought families with a large number of relatives, a single disease source, illness over two or three generations, a large number of affected family members (at least three affected, with a minimum of two bipolar patients) and large sibships. Families were identified through the Mood Disorders Unit, Prince Henry Hospital, Sydney; the New South Wales Depressive and Manic Depressive Association; local psychiatric units; and from responses to an article in a national women’s magazine. Details of pedigrees were recorded using the method described by Thompson (Thompson et al., 1979). Families were initially assessed using the Family History-RDC (FH-RDC) method of Andreasen (Andreasen et al., 1977) with interviews performed with both the proband and another informative family member. In the first pedigree ascertainment phase of the study (1987-1989) we identified two large
PEDIGREE
40 11 Fig.
1. Pedigree
39 11
05. Circles = females;
. . T
05
35 11 squares
33 11
26 11
’
41 12
45 11
= males. Full hatch = bipolar disorder, disorder. Age. DRD3 gene alleles.
42 11
36 11
35 11'
half hatch = recurrent
major
depressive
216
PEDIGREE 11
24 12
20 12 Fig. 2. Pedigree
11. (as Fig. 1).
PEDIGREE 17
75
77
61
63
28 1 1 Fig. 3. Pedigree
17. (as Fig. 1).
217
PEDIGREE
20
12
11
11
c;‘, Y
12
L;! o 31
1 1
Fig. 4. Pedigree
tioned about any family history of psychiatric disorder, to ensure unilateral descent of illness in the pedigrees. Hospital and other medical records were obtained. Final diagnoses were made using the Yale-NIMH ‘Best Estimate’ Diagnosis Consensus guidelines which are assigned from FHRDC and structured interview derived RDC diagnoses, and medical records. Consensus ‘bestestimate’ diagnoses were made after independent evaluation of all available interviews and records by P.M. and B.W.. Diagnoses were made blind to laboratory investigations. Summarized details of the nine families involved in this study are included in Table 3. The new seven pedigrees are outlined in Figures l-7.
20. (as Fig. 1).
Genetic markers DNA extraction. This was carried out as described in our previous paper (Mitchell et al., 1992). Human dopamine D, receptor polymorphism. For analysis of the polymorphism Bali in the coding region of the DRD3 receptor gene, the polymerase chain reaction was used to amplify the region spanning the BaZI polymorphic digestion site, as described by Lannfelt et al. (1992). The PCR reaction was carried out with the following primers synthesized on an Applied Biosysterns DNA Synthesizer: 5’-GCT CTA TCT CCA ACT CTC ACA-3’, situated before the first ATG of the D, receptor gene and 5’-AAG TCT ACT
PEDIGREE 22
Fig. 5. Pedigree
22. (as Fig. 1).
218
PEDIGREE
23
11
11
11
11
11
Fig. 6. Pedigree
CAC CTC CAG GTA-3’, located in intron 1. Genomic DNA (100 ng) was amplified using a Perkin-Elmer Cetus programmable PCR machine in a 25 ~1 reaction in 50 mM KCl, 10 mM Tris-HCl, pH 8.3, 1.5 mM MgCl,, 5 pmol of each primer, 100 ,uM of each dNTP, 0.5 mM dithiothreitol, 0.15 ~1 (1.5 PCi) t3’P]dCTP (NEN/Dupant) and 0.01% gelatin and 1.5 ~1 AmpliTaqTaq polymerase (Cetus). Samples were overlayed with
11
11
11
23.(as Fig. 1).
approx. 50 ~1 mineral oil and then heated to 95°C for 8 min to denature. The PCR program consisted of 35 cycles as follows: 94°C for 1 min, 56°C for 1 min, and 72°C for 1 min. The samples were then incubated at 72°C for 8 min. To check the efficiency of the reaction, aliquots of the amplified samples were electrophoresed on 1% agarose. An aliquot (3 ~11 of the amplified product was cleaved with 1 unit/sample of BufI restriction
PEDIGREE 24
22 12
24
25
18
26
27
25
21
12
22
22
11
Fig. 7. Pedigree
18
24. (as Fig. 1).
219 TABLE
1
Parameters
of the five genetic Age-specific
Model I II III IV V
models
(applied
to both dominant
and recessive
penetrance
Age: 15-19
20-29
30-39
40+
0.12 0.14 0.16 0.18 0.20
0.30 0.35 0.40 0.45 0.50
0.45 0.53 0.60 0.68 0.75
0.60 0.70 0.80 0.90 1.00
Dopamine
II
III
IV
V
Sporadic rate
0.035 0.035 0.035 0.035 0.035
0.005 0.005 0.005 0.005 0.005
Linkage analysis Linkage analyses were undertaken by calculating lod scores using the computer program LIPED (Ott, 1974). Lod scores > 3 have been considered conventionally to be indicative of linkage and
2 D, receptor
Model
I
Disease allele frequency
frequencies approximated those observed in our sample of 49 unrelated (marrying-in) individuals. .The genotypes of members of families are shown in Figs. 1-7.
enzyme (Promega). Samples were then subjected to electrophoresis on 3% agarose for 2 h at approx. 80 mAmps. The gel was dried and autoradiographed. Two alleles for the dopamine D, receptor gene with Bali were observed, i.e., 1 (304 bp) and 2 (206 + 98 bp), as described in Lannfelt et al., (1992). Published allele frequencies - i.e., 0.67 for allele 1, and 0.33 for allele 2 - (Lannfelt et al., 1992) were used in the linkage analyses. These
TABLE
models)
gene LOD
scores.
Recombination
Autosomal
dominant
inheritance.
Nine families
combined
fraction
0.00
0.05
0.10
0.20
0.30
0.40
M F
- 0.12 - 1.90
- 0.99 - 1.22
- 0.08 - 0.82
- 0.04 - 0.40
0.00 -0.18
0.00 - 0.06
C
-2.12
- 1.31
-0.90
- 0.44
-0.18
- 0.06
M F
-0.15 - 2.34
-0.10 - 1.31
-0.10 - 0.92
- 0.05 - 0.38
- 0.01 -0.17
0.00 - 0.06
C
- 2.49
- 1.41
- 1.02
- 0.43
-0.18
- 0.06
M F
-0.15 -2.81
-0.12 - 1.39
- 0.09 - 0.87
- 0.05 - 0.37
- 0.01 -0.17
0.00 + 0.01
C
- 2.96
- 1.51
- 0.96
- 0.42
-0.18
+0.01
M F
-0.18 - 3.56
-0.13 - 1.48
-0.10 - 1.06
- 0.06 - 0.35
- 0.02 -0.13
0.00 - 0.05
C
- 3.74
- 1.61
- 1.16
- 0.41
-0.15
- 0.05
M F
-0.16 - 4.53
-0.13 - 1.14
- 0.08 - 0.67
- 0.03 - 0.26
-0.02 -0.11
0.00 - 0.04
C
- 4.69
- 1.27
- 0.75
-0.29
- 0.13
- 0.04
M = male; F = female;
C = combined.
220
scores < -2 indicate rejection of linkage at that recombination fraction, though Ott (1989) has suggested that a score > 3.6 should be required to establish linkage where there is a maximization of the lod score using a series of diagnostic schemes and penetrances. Analyses were performed for both autosomal dominant and recessive transmission models, with varying levels of penetrance. Lad scores were calculated separately for female and male recombination and then combined. We tested for linkage using five sets of parameters which were applied to both dominant and recessive genetic models (Table 1). For Model I, a maximum penetrance of 0.60 was used as a conservative representation of the usual penetrance of this disorder. With model V, a maximum penetrance of 1.0 was chosen to specifically reflect the high density of illness in the families under study. Models II-IV represent intermediate levels of penetrance, i.e., 0.70-0.90. The age of onset curve was as described in our previous studies (Mitchell et al., 1991; 1992). The frequency of the disease allele was taken as 0.035 (Detera-Wadleigh et al., 1987) and the penetrance for the non-susceptible genotype (the ‘sporadic rate’) was set at 0.005 (Detera-Wadleigh et al., 1987). For the linkage analyses only subjects of at least 15 years of age were included.
TABLE
D, receptor
Family
Total Number at risk interviewed
Total
(a) Linkage of b’~po1ar d’lsord er and recurrent major depression to the DRD3 gene - autosomal dominant model Combined lod scores for the nine families, using each of the five models, are outlined in Table 2. Close linkage to the DRD3 gene was excluded at a theta (recombination fraction) of 0.00 for each model, from I (maximum penetrance = 0.60) through to V (maximum penetrance 1.0). Table 3 details the lod scores for each individual family, using model IV (which reflects the apparent high degree of penetrance observed in these families). Different gene frequencies of the disease allele (from 0.015 to 0.035) were tested. Lad scores were more negative when the disease allele frequency was assumed to be 0.015, but there was no significant change overall in the results. For example, using model IV, close linkage was still only excluded at a theta of 0.00, though the lod score decreased to -4.52. As in our previous studies (Mitchell et al., 1991; 1992), we examined for the effect of variable rates of sporadic (nongenetic) presentations of illness. We tested a range of sporadic rates varying from 0.005 to 0.1 using model IV (penetrance 0.9). The exclusion of linkage to the DRD3 gene was minimally sen-
3
Dopamine
01 02 05 11 17 20 22 23 24
Results
gene LOD scores by family (model Affected individuals interviewed
Generations
BP
UP
68 16 13 9 9 16 16 13 11
5 5 6 4 4 4 4 3 4
6 _ _ _ 1 2 1 _ 2
171
37
12
3 3 2 3 3 3 3 3 3
IV) Recombination
fraction
0.00
0.05
0.10
0.20
0.30
0.40
+ 0.25 - 1.67 + 0.06 - 1.13 +0.15 - 0.21 +0.13 0.00 - 1.32
+ 0.23 - 0.56 + 0.06 - 0.61 +0.12 -0.17 +0.11 0.00 - 0.79
+0.20 - 0.33 + 0.04 - 0.40 +0.10 - 0.13 +0.10 0.00 - 0.56
+ 0.15 -0.13 + 0.02 -0.19 + 0.07 - 0.07 + 0.05 0.00 -0.31
+0.10 - 0.04 + 0.01 - 0.08 + 0.05 - 0.03 +0.01 0.00 -0.17
+ 0.05 - 0.01 0.00 - 0.02 + 0.02 - 0.01 - 0.01 0.00 - 0.07
- 3.74
- 1.61
- 1.16
-0.41
-0.15
- 0.15
221 TABLE
4
Dopamine
D, receptor
Diagnostic
threshold
A. BIPOLAR
B. BIPOLAR
gene LOD
scores
diagnostic
Recombination
I
I + II
C. BIPOLAR DISORDER AND RECURRENT MDD
M = male; F = female; * Model IV
at various
thresholds.
Dopamine
II
III
IV
V
*
0.00
0.05
0.10
0.20
0.30
0.40
M F
-0.41 - 2.92
-0.32 - 1.52
- 0.25 - 1.06
-0.12 - 0.54
- 0.05 - 0.23
- 0.01 - 0.08
C
- 3.33
- 1.84
- 1.31
-0.66
- 0.28
- 0.09
M F
- 0.38 - 3.93
-0.31 - 2.08
- 0.24 -1.41
-0.12 - 0.74
- 0.05 - 0.32
-0.01 -0.11
C
- 4.31
- 2.39
- 1.65
-0.86
- 0.37
-0.12
M F
- 0.18 - 3.56
-0.13 - 1.48
-0.10 - 1.06
- 0.06 - 0.35
- 0.02 -0.13
0.00 - 0.05
C
- 3.74
- 1.61
- 1.16
- 0.41
-0.15
- 0.05
C = combined
scores.
sporadic rate of 0.10 Clod score - 1.13 at theta = 0.00). These findings indicate that the apparent clear exclusion of linkage to the DRD3 gene was not likely to have been due to a significant rate of
5 D, receptor
Model
I
combined
fraction
sitive to varying the sporadic rate. Linkage was excluded at theta 0.00 at sporadic rates of 0.005 (lad score = -3.741, 0.01 (- 3.49) and 0.05 ( - 2.12). Linkage was not excluded at an assumed
TABLE
Nine families
gene LOD scores. Recombination
Autosomal
recessive
inheritance.
Nine families
combined
fraction
0.00
0.05
0.10
0.20
0.30
0.40
M F
- 1.47 -0.61
-0.84 -0.40
- 0.52 - 0.32
- 0.22 -0.16
- 0.08 - 0.08
- 0.02 - 0.03
C
- 2.08
- 1.24
- 0.84
- 0.38
-0.16
- 0.05
M F
- 1.74 - 0.69
- 0.91 - 0.47
- 0.56 - 0.35
- 0.23 - 0.19
- 0.07 -0.11
- 0.02 - 0.03
C
- 2.43
- 1.38
- 0.91
- 0.42
-0.18
- 0.05
M F
- 2.05 - 0.85
- 0.98 - 0.53
- 0.62 - 0.35
- 0.26 -0.18
- 0.09 - 0.09
- 0.02 - 0.04
C
- 2.90
- 1.51
- 0.97
- 0.44
-0.18
- 0.06
M F
- 2.28 - 1.04
- 1.01 - 0.54
- 0.62 - 0.44
- 0.26 - 0.22
- 0.09 -0.11
- 0.03 - 0.03
C
- 3.32
-1.55
- 1.06
- 0.48
- 0.20
- 0.06
M F
- 2.60 - 1.56
- 1.14 - 0.74
- 0.71 - 0.48
- 0.34 - 0.30
-0.10 -0.11
- 0.04 - 0.02
C
-4.16
- 1.88
- 1.19
- 0.64
- 0.21
- 0.06
M = male; F = female;
C = combined.
222
sporadic presentations of illness. (It should noted that ’ lod scores for the DRD3 gene mained negative for all sporadic rates tested).
be re-
(b) Linkage to the DRD3 gene using other thresholds of diagnosis for affective disorder (Table 4) autosomal dominant model Using model IV, linkage between the DRD3 gene and diagnostic groups defined by the following thresholds of illness was examined in all nine families: (A) bipolar I disorder; (B) bipolar I and II; and (C) bipolar I, II and recurrent major depression. When a narrow definition of the disorder was used, ill subjects who did not meet the criteria for affected were defined as unaffected. Linkage of each of these varying diagnostic thresholds to the DRD3 gene was significantly excluded. For thresholds A and C (bipolar I; bipolar disorder plus recurrent major depression) linkage was excluded at a theta of 0.00, while for threshold B (bipolar I and II), linkage was excluded at a theta of 0.05. (c) Linkage of bipolar disorder and recurrent major depression to the DRD3 gene - autosomal recessive model (Table 5) Combined lod scores for the nine families examining for linkage under the assumption of autosomal recessive inheritance, with different sets of parameters, are outlined in Table 5. As with the assumption of dominant inheritance, linkage was excluded under each model I-V at a theta of 0.00. Discussion This study found a clear exclusion of close linkage between the dopamine D, receptor gene and bipolar disorder. This result indicates that a central role of DRD3 in the aetiology of this condition is unlikely. However, with the exception of diagnostic threshold B (bipolar I and II disorders), this study could not confidently exclude linkage to adjacent genes in the region 3q 13.3. The validity of our result excluding linkage to the dopamine D, receptor gene was strengthened by a number of particular features of the study. Firstly, close linkage was excluded at a series of different thresholds (levels of definition) of affec-
tive status, as indicated in Table 4. Although it is now generally accepted that the ‘bipolar diagnostic spectrum’ (Gershon, 1990) includes schizo-affective, bipolar I and II, unipolar and cyclothymic disorders, testing at various levels is necessary because of both the diagnostic instability of bipolar 11 diagnoses and the possibility of non-genetic or sporadic presentations of depression in these families (Merikangas et al., 1989). In view of this latter problem we only included unipolar depressed subjects who fulfilled the strict NIMH/ Yale criteria for unipolar depression (Gershon and Goldin, 1989). Mis-classification of the sporadic cases as affected significantly reduces ability to demonstrate linkage (Martinez, 1989). Secondly, the finding of exclusion of close linkage was consistent at a wide range of penetrances, i.e., 60 to 100%. We tested for linkage under assumptions of both autosomal dominant and recessive inheritance, as there is currently no consensus concerning the mode of transmission of bipolar disorder. Thirdly, the lack of even suggestive evidence of linkage in any of the nine families, argues against the likelihood of genetic heterogeneity affecting the results with this particular gene marker. However, as only three of these families were informative for this particular marker, we cannot exclude definitively the possibility that a mutation of the dopamine D, receptor gene accounts for a proportion of families with bipolar disorder. Finally, we undertook the conservative strategy of testing for high rates of ‘sporadic’ presentations of illness. Varying the sporadic rate did not significantly affect our conclusion of exclusion of close linkage for the DRD3 gene, although the lod scores were not significant with a high sporadic rate of 0.10 (which is unlikely for this disorder). However, sporadic rates of 0.01 or 0.05 are potentially consistent with the clinical situation, particularly when those with recurrent major depression are included in the diagnostic threshold. In conclusion, after testing a range of single gene models, we have confidently excluded linkage between bipolar disorder and the dopamine D, receptor gene. This finding, in conjunction with our previous report excluding linkage to the dopamine D, and D, receptor genes (Mitchell et al., 1992), suggests that a mutation in the genes
223
for these dopamine receptors is unlikely to play a central aetiological role in bipolar disorder. These results, however, cannot exclude the theoretical possibility that there may be a mutation (or mutations) in the dopamine D, receptor gene which plays an important aetiological role, but which occurs as part of an interaction with mutations in other genes and/or environmental factors. However, the reduced penetrance of some of the genetic models used would allow for the effects of interaction with other genes or environmental factors, provided the dopamine D, receptor gene has a major effect.
Acknowledgements This study was supported by the Australian National Health and Medical Research Council. We are grateful to MS Christina Vivero and Mr. Mark Graham for performing the DNA extraction and Southern hybridization analyses; Ms. Anna Zournazi for technical assistance; and Mrs. Zora Vuckovic for preparation of the manuscript.
References Andreasen, N.C., Endicott, J., Spitzer, R.L. and Winokur, Cl. (1977) The family history method using diagnostic criteria: Reliability and validity. Arch. Gen. Psychiatry 34, 12291235. Avissar, S. and Schreiber, G. (1992) The involvement of guanine nucleotide binding proteins in the pathogenesis and treatment of affective disorders. Biol. Psychiatry 31, 435-459. Berrettini, W., Detera-Wadleigh S.D., Goldin, L.R., Ferraro, T.N., Martinez, M., Hsieh, W.T., Hoehe, M., Nurnberger, J.I., Alexander, R.C. and Gershon E.S. (1992) Linkage studies of bipolar illness (Abstract). Biol. Psychiatry 31, 131A. Bouthenet, M-L., Souil, E., Martres, M-P., Sokoloff, P., Giros, B. and Schwartz, J-C. (1991) Localization of dopamine D, receptor mRNA in the rat brain using in situ hybridization histochemistry: comparison with dopamine D, receptor mRNA. Brain Res. 564, 203-219. Byerley, W., Leppert, M., O’Connell, P., Mellon, C., Holik, J., Lubbers, A., Reimherr, F., Jenson, S., Hill, K., Wender, P., Grandy, D., Litt, M., Lalouel, J.M., Civelli, 0. and White, R. (1990) D2 dopamine receptor gene not linked to manic-depression in three families. Psych. Genet. 1, 55-62. Cox, N., Reich, T., Rice, J., Elston, R., Schober, J. and Keats, B. (1989) Segregation and linkage analyses of bipolar and
major depressive illness in multigenerational pedigrees. J. Psychiat. Res. 23, 109-123. Detera-Wadleigh, S.D., Berrettini, W., Goldin, L.R., Boorman, D., Anderson, D. and Gershon, E.S. (1987) Close linkage of c-Harvey-ras 1 and insulin gene to affective disorder is ruled out in three North American pedigrees. Nature 325, 806-808. Egeland, J.A., Gerhard, D.S., Pauls, D.L., Sussex, J.N., Kidd, K.K., Allen, C., Hostetter, A.M. and Housman, D.E. (1987) Bipolar affective disorders linked to DNA markers on chromosome Il. Nature 325, 783-787. Elphick, M., Yang, J-D. and Cowen, P.J. (1990) Effects of carbamazepine on dopamine and serotonin-mediated neuroendocrine responses. Arch. Gen. Psychiatry 47, 135-140. Gershon, E. (1990) Genetics. In: F.K. Goodwin, K.R. Jamison (Eds.), Manic-depressive Illness. Oxford University Press, New York. Gershon, E. and Goldin, L. (1989) Linkage data on the affective disorders in an epidemiologic context. Genet. Epidem. 6, 201-209. Gill, M., McKeon, P. and Humphries, P. (1988) Linkage analysis of manic depression in an Irish family using H-ras 1 and INS DNA markers. J. Med. Genet. 25, 634-637. Gill, M., Castle, D., Hunt, N., Clements, A., Sham, P. and Murray R.M. (1991) Tyrosine hydroxylase polymorphisms and bipolar affective disorder. J. Psychiat. Res. 25, 179184. Giros, B., Martres, M-P., Sokoloff, P. and Schwartz, J-C. (1990) cDNA cloning of the human dopaminergic D, receptor and chromosome’ identification. CR Acad. Sci. (Paris) 311, 501-508. Hodgkinson, S., Sherrington, R. and Gurling, M. (1987) Molecular genetic evidence for heterogeneity in manic depression. Nature 325, 805-806. Holmes, D., Brynjolfsson, J., Brett, P., Curtis, D., Petursson, H., Sherrington, R. and Gurling, H. (1991) No evidence for a susceptibility locus predisposing to manic depression in the region of the dopamine CD,) receptor gene. Br. J. Psychiatry 158, 635-641. Kelsoe, J.R., Ginns, E.I., Egeland, J.A., Gerhard, D.S., Goldstein, A.M., Bale, S.J., Pauls, D.L., Long, R.T., Kidd, K.K., Conte, G., Housman, D.E. and Paul, S.A. (1989) Reevaluation of the linkage relationship between chromosome llp loci and the gene for bipolar affective disorder in the old order Amish. Nature 342, 238-243. Korner, J., Fritze, J. and Propping, P. (1990) RFLP alleles at the tyrosine hydroxylase locus: no association found to affective disorders. Psychiat. Res. 32, 275-280. Kraepelin, E. (1921) Manic Depressive Insanity and Paranoia. (Transl. Barclay, M). Churchill Livingstone, Edinburgh. Lannfelt, L., Sokoloff, P., Martes, M-P., Pilon, C., Giros, B., Jonsson, E., Sedvall, G. and Schwartz, J-C. (1992) Amino acid substitution in the dopamine D, receptor as a useful polymorphism for investigating psychiatric disorders. Psychiat. Genet. (In press). Leboyer, M., Malafosse, A., Boularand, S., Campion, D., Gheysen, F., Samolyk, D., Henriksson, B., Denise, E., Des Lauriers, A., Lepine, J-P., Zarifian, E., Clergert-Darpoux,
224 F. and Mallet, J. (1990) Tyrosine hydroxylase polymorphisms associated with manic-depressive illness. Lancet 335, 1219. Le Coniat, M., Sokoloff, P., Hillion J., Martres M-P., Giros, B., Pilon, C., Schwartz J-C. and Berger, R. (1991) Chromosomal localization of the human D, dopamine receptor gene. Hum. Genet. 87, 618-620. Martinez, M., Khlat, M., Leboyer, M. and Clerget-Darpoux, F. (1989) Performance of linkage analyses under misclassification error when the genetic model is unknown. Genet. Epid. 6, 253-258. Mengod, G., Villaro, M.T., Landwehrmeyer, G.B., MartinezMir, M.I., Niznik, H.B., Sunahara, R.K., Seeman, P., O’Dowd, B.F., Probst, A. and Palacios, J.M. (1992) Visualization of dopamine D,, D, and D, receptor mRNAs in human and rat brain. Neurochem. Int. 20, 33S-43s. Merikangas, K.R., Spence, A. and Kupfer, D.J. (1989) Linkage studies of bipolar disorder: Methodologic and analytic issues. Arch. Gen. Psychiatry 46, 1137-1141. Mitchell, P., Waters, B., Morrison, N., Shine, J., Donald, J. and Eisman, J. (1991) Close linkage of bipolar disorder to chromosome 11 markers is excluded in two large Australian pedigrees. J. Affect. Disord. 21, 23-32. Mitchell, P., Selbie, L., Waters, B., Donald, J., Vivero, C., Tully, M. and Shine, J. (1992) Exclusion of close linkage of bipolar disorder to dopamine D, and Dz receptor gene markers. J. Affect. Disord. 25, 1-12. Noethen, M.M., Erdmann, J., Korner, J., Lanczik, M., Fritze, J., Fimmers, R., Grandy, D.K., O’Dowd, B. and Propping, P. (1992) Lack of association between dopamine D, and D, receptor genes and bipolar affective disorder. Am. J. Psychiatry 149, 199-201. Ott, J. (1974) Estimation of the recombination fraction in human pedigrees: Efficient computation of the likelihood for human linkage studies. Am. J. Hum. Genet. 28, 525529. Ott, J. (1985) Analysis of human genetic linkage. Johns Hopkins University Press, Baltimore. Ott, J. (1989) Linkage analysis-potential problems introduced by the practice of maximizing the lod score over a wide range of parameters including disease classification (Ab-
stract). Proceedings of First World Congress on Psychiatric Genetics. Pakstis, A.J., Kidd, J.R., Castiglione, C.M. and Kidd, K.K. (1991) Status of the search for a major genetic locus for affective disorder in the Old Order Amish. Hum. Genet. 87, 475-488. Robins, L.N., Wing, W., Wittchen, H.U., Helzer, J.E., Babor, T.F., Burke, J., Farmer, A., Jablenski, A., Pickens, R., Regier, D.A., Sartorius, N. and Towle, L.H. (1988) The Composite International Diagnostic Interview: An epidemiological instrument suitable for use in conjunction with different diagnostic systems and in different cultures. Arch. Gen. Psychiatry 45, 1069-1077. Silverstone, T. and Roman+Clarkson, G. (1988) Bipolar affective disorder: Causes and prevention of relapse. Br. J. Psychiatry 154, 321-335. Sokoloff, P., Giros, B., Martres, M-P., Bouthenet, M-L. and Schwartz, J-C. (1990) Molecular cloning and characterization of a novel dopamine receptor (D,) as a target for neuroleptics. Nature 347, 146-151. Sokoloff, P., Andrieux, M., Besancon, R., Pilon, C., Martres, M-P., Giros, B. and Schwartz, J-C. (1992) Pharmacology of human D, receptor expressed in a mammalian cell line: compairson with D, receptor. Eur. J. Pharmacol. 225, 331-337. Stephens, J.C., Cavanaugh, M.L., Gradie, MI., Mador, M.L. and Kidd, K.K. (1990) Mapping the human genome: Current status. Science 250, 237-244. Thompson, W.D., Kidd, J.R. and Weissman, M.M. (1979) A procedure for the efficient collection and processing of pedigree data suitable for genetic analysis. J. Psychiat. Res. 15, 291-303. Todd, R.D. and O’Malley, K.L. (1989) Population frequencies of tyrosine hydroxylase restriction fragment length polymorphisms in bipolar affective disorder. Biol. Psychiatry 25, 626-630. Wesner, R.B., Scheffner, W., Palmer, P.J., Crowe, P.R. and Winokur, G. (1990) The effect of co-morbidity and penetrance on the outcome of linkage analysis in a bipolar family. Biol. Psychiatry 27, 241-244.
213 01650327/93/$06.00
JAD 00976
Exclusion of close linkage of bipolar disorder to the dopamine D, receptor gene in nine Australian pedigrees Philip Mitchell a, Brent Waters a, Christina Vivero b, Fie Le b, Jennifer Donald ‘, a, Lars Lannfelt d, Pierre Sokoloff e, John Shine b Michele Tully a, Karen Campedelli and Lisa Selbie b a School of Psychiatry, University of New South Wales, Sydney, Australia, b Garvan Institute of Medical Research, St Vincent’s Hospital, Sydney, Australia, ’ School of Biological Sciences, Macquarie University, Sydney, Australia, d Department of Geriatric Medicine, The Karolinska Institute, Huddinge University Hospital, Huddinge, Sweden and ’ Unite de Neurobiologie et Pharmacologic (U. IO9 INSERM), Centre Paul Broca, 2ter rue d’Alesia, 75014, Paris, France (Received 1 September 1992) (Revision received 3 December 1992) (Accepted 8 December 1992)
Summary The recently cloned dopamine D, receptor (DRD3) gene is of potential relevance to the aetiology of bipolar disorder because of an almost exclusive expression in limbic tissue, the region of the brain putatively responsible for control of emotion. We therefore aimed to determine whether bipolar disorder in nine pedigrees (with 171 members) was linked to this receptor gene, which has been mapped to chromosomal region 3q 13.3. Linkage of bipolar disorder and recurrent depression to the DRD3 gene was tested using a series of autosomal dominant and recessive models with varying penetrance levels. Additionally, linkage was examined using a series of levels of definitions of affective illness (ranging from bipolar I alone to all affective disorders). Close linkage to the DRD3 gene was strongly excluded using each model and definition, and these conclusions persisted when a wide range of rates of ‘sporadic’ (non-genetic) presentations of illness were incorporated in the analysis.
Key words:
Bipolar
disorder;
Dopamine
D, receptor;
Introduction “The cause of the malady we must seek, as it appears, essentially in morbid predisposition. I could demonstrate (hereditary taint) in about 80 percent of the cases observed in Heidelberg.” Kraepelin,
E. (1921)
‘Manic
Depressive
Insanity
and
Para-
noia’ (p. 75)
Linkage;
Chromosome
3
Although there is undoubted evidence for significant genetic transmission of bipolar disorder (Gershon, 19901, segregation analyses have been unable to determine the particular mode of trans-
Correspondence to; Philip Mitchell, Mood Disorders Unit, Prince Henry Hospital, Little Bay, N.S.W., 2036, Australia.
214
mission of this condition (Cox et al., 1989). Nonetheless, linkage analyses offer the potential for identifying the gene or genes involved, provided care is taken in considering a range of models and genetic parameters. There are two major approaches to linkage studies using DNA markers. The first, ‘genome scanning’ - the systematic analysis of linkage of multiple markers covering large regions of the genome - was used to some extent in the Amish study of Egeland et al. (1987). Their original report of linkage of bipolar disorder to chromosoma1 region 11~15 markers was not replicated in other studies (Hodgkinson et al., 1987; DeteraWadleigh et al., 1987; Gill et al., 1988; Kelsoe et al., 1989; Wesner et al., 1990; Mitchell et al., 1991; Mendlewicz et al., 1991). Association studies using DNA polymorphisms at the tyrosine hydroxylase gene from this same chromosomal region have been inconsistent, with one positive report (Leboyer et al., 1990), but three negative findings (Todd and O’Malley, 1989; Korner et al., 1990; Gill et al., 1991). The subsequent development of many more markers on the genetic linkage map of the human genome (Stephens et al., 1990) has enabled genome scanning to be done more completely. Pakstis et al. (1991) have reported preliminary findings from the ongoing genome scanning study of the large Old Order Amish pedigree. They estimated that they had excluded from linkage approximately 23% of the autosomal genome. Similarly, Berritini et al. (1992) have excluded linkage to most regions of chromosomes 1, 2, 5, 8, 11, 12, 13, 15, 17, 18 and 22 in a study of 21 bipolar pedigrees. An alternative strategy is the ‘candidate gene’ approach, which investigates the linkage of specific genes (for example, particular receptors or enzymes) which may have theoretical relevance to the particular disease of interest. Examples of this approach have included recent studies testing for the linkage of bipolar disorder to the D, and D, dopamine receptors (Byerley et al., 1990; Holmes et al., 1991; Mitchell et al., 1992). These investigations have been consistent in failing to demonstrate linkage to these particular receptors, and were corroborated by a similarly negative association study (Noethen et al., 1992).
In this paper we have tested for linkage between the recently cloned dopamine D, receptor (DRD3) gene (Sokoloff et al., 1990) and bipolar disorder, in an enlarged sample of nine pedigrees. As we argued in our previous publication (Mitchell et al., 1992), dopamine receptor genes are of potential relevance to bipolar disorder for several reasons. Firstly, a role of dopamine in bipolar disorder has been suggested by the ability of dopamine agonistic agents such as L-dopa, amphetamine and bromocriptine to induce mania (Silverstone and Romans-Clarkson, 1989). Secondly, three of the medications used in the treatment of this condition have significant effects on central dopamine activity. Lithium reduces dopaminergic turnover, and also effects G proteins (Avissar and Schreiber, 1992). The dopamine D, receptor is a G protein-coupled receptor, though it is unclear which G protein subunits couple to this receptor subtype (Sokoloff et al., 1992). Carbamazepine alters the growth hormone response to the dopaminergic agonist apomorphine (Elphick et al., 1990). The antipsychotics (which are effective in the acute management of mania) block dopamine receptors. The dopamine D, receptor is of particular potential interest with regard to bipolar disorder because of its almost exclusive expression in limbit structures, i.e., that region of the brain putatively responsible for the control of emotion. Although studies of the expression of this receptor in human brain tissue are yet to be published, investigations of rat brain (Bouthenet et al., 1991; Mengod et al., 1992) are consistent in demonstrating mRNA expression almost entirely limited to limbic areas (islands of Calleja, anterior nucleus accumbens, medial mamillary bodies, hippocampus and the bed nucleus of the stria terminalis). There is no expression of the genes for this receptor in the pituitary gland, unlike dopamine D, receptors. The rat and human dopamine D, receptor genes were both cloned in 1990 (Sokoloff et al., 1990; Giros et al., 1990). The human DRD3 gene is localised in chromosomal region 3q 13.3 (Le Coniat et al., 1991). Studies in both rat (Sokoloff et al., 1990) and human (Sokoloff et al., 1992) cell lines have demonstrated that dopamine has a much higher affinity for D, than D, receptors.
215
families (01 and 02) with three generations of illness and a large number of affected individuals. These families have been described in our previous publications (Mitchell et al., 1991; 1992). In the second phase (1990-1992) we identified and fully assessed a further 22 suitable families (03241, seven of whom had genotyping for this particular study. All available relatives were interviewed using standardised instruments to derive life time Research Diagnostic Criteria (RDC) diagnoses using the strict NIMH criteria for depression which require at least one month duration of symptoms, and functional impairment or incapacity. Families 01 and 02 were assessed using the Schedule of Affective Disorders and Schizophrenia-Lifetime Version (SADS-L), and families 03-24 were assessed with the Composite International Diagnostic Interview (CIDI) (Robins et al., 1988). The CID1 was developed from the Diagnostic Interview Schedule (DIS) from which Feighner, RDC, and DSM-III diagnoses could be derived - and incorporates all the mood disorder items of that instrument. The CID1 allows the ability to apply multiple diagnostic systems (RDC, DSM-III, DSM-III-R and ICD-10) to linkage analysis, a capacity recommended in the McArthur Foundation Workshop on Linkage in Affective Disorder (Merikangas et al., 1989). In conjunction with the structured interviews, all marrying-in individuals were routinely ques-
Most antipsychotics demonstrate high affinities at the D, receptor, though all appear to be more potent at the D, receptor. These findings suggest that the D, receptor may be potentially involved in the aetiology of psychotic disorders such as bipolar disorder or schizophrenia. Methods Case ascertainment and diagnosis We sought families with a large number of relatives, a single disease source, illness over two or three generations, a large number of affected family members (at least three affected, with a minimum of two bipolar patients) and large sibships. Families were identified through the Mood Disorders Unit, Prince Henry Hospital, Sydney; the New South Wales Depressive and Manic Depressive Association; local psychiatric units; and from responses to an article in a national women’s magazine. Details of pedigrees were recorded using the method described by Thompson (Thompson et al., 1979). Families were initially assessed using the Family History-RDC (FH-RDC) method of Andreasen (Andreasen et al., 1977) with interviews performed with both the proband and another informative family member. In the first pedigree ascertainment phase of the study (1987-1989) we identified two large
PEDIGREE
40 11 Fig.
1. Pedigree
39 11
05. Circles = females;
. . T
05
35 11 squares
33 11
26 11
’
41 12
45 11
= males. Full hatch = bipolar disorder, disorder. Age. DRD3 gene alleles.
42 11
36 11
35 11'
half hatch = recurrent
major
depressive
216
PEDIGREE 11
24 12
20 12 Fig. 2. Pedigree
11. (as Fig. 1).
PEDIGREE 17
75
77
61
63
28 1 1 Fig. 3. Pedigree
17. (as Fig. 1).
217
PEDIGREE
20
12
11
11
c;‘, Y
12
L;! o 31
1 1
Fig. 4. Pedigree
tioned about any family history of psychiatric disorder, to ensure unilateral descent of illness in the pedigrees. Hospital and other medical records were obtained. Final diagnoses were made using the Yale-NIMH ‘Best Estimate’ Diagnosis Consensus guidelines which are assigned from FHRDC and structured interview derived RDC diagnoses, and medical records. Consensus ‘bestestimate’ diagnoses were made after independent evaluation of all available interviews and records by P.M. and B.W.. Diagnoses were made blind to laboratory investigations. Summarized details of the nine families involved in this study are included in Table 3. The new seven pedigrees are outlined in Figures l-7.
20. (as Fig. 1).
Genetic markers DNA extraction. This was carried out as described in our previous paper (Mitchell et al., 1992). Human dopamine D, receptor polymorphism. For analysis of the polymorphism Bali in the coding region of the DRD3 receptor gene, the polymerase chain reaction was used to amplify the region spanning the BaZI polymorphic digestion site, as described by Lannfelt et al. (1992). The PCR reaction was carried out with the following primers synthesized on an Applied Biosysterns DNA Synthesizer: 5’-GCT CTA TCT CCA ACT CTC ACA-3’, situated before the first ATG of the D, receptor gene and 5’-AAG TCT ACT
PEDIGREE 22
Fig. 5. Pedigree
22. (as Fig. 1).
218
PEDIGREE
23
11
11
11
11
11
Fig. 6. Pedigree
CAC CTC CAG GTA-3’, located in intron 1. Genomic DNA (100 ng) was amplified using a Perkin-Elmer Cetus programmable PCR machine in a 25 ~1 reaction in 50 mM KCl, 10 mM Tris-HCl, pH 8.3, 1.5 mM MgCl,, 5 pmol of each primer, 100 ,uM of each dNTP, 0.5 mM dithiothreitol, 0.15 ~1 (1.5 PCi) t3’P]dCTP (NEN/Dupant) and 0.01% gelatin and 1.5 ~1 AmpliTaqTaq polymerase (Cetus). Samples were overlayed with
11
11
11
23.(as Fig. 1).
approx. 50 ~1 mineral oil and then heated to 95°C for 8 min to denature. The PCR program consisted of 35 cycles as follows: 94°C for 1 min, 56°C for 1 min, and 72°C for 1 min. The samples were then incubated at 72°C for 8 min. To check the efficiency of the reaction, aliquots of the amplified samples were electrophoresed on 1% agarose. An aliquot (3 ~11 of the amplified product was cleaved with 1 unit/sample of BufI restriction
PEDIGREE 24
22 12
24
25
18
26
27
25
21
12
22
22
11
Fig. 7. Pedigree
18
24. (as Fig. 1).
219 TABLE
1
Parameters
of the five genetic Age-specific
Model I II III IV V
models
(applied
to both dominant
and recessive
penetrance
Age: 15-19
20-29
30-39
40+
0.12 0.14 0.16 0.18 0.20
0.30 0.35 0.40 0.45 0.50
0.45 0.53 0.60 0.68 0.75
0.60 0.70 0.80 0.90 1.00
Dopamine
II
III
IV
V
Sporadic rate
0.035 0.035 0.035 0.035 0.035
0.005 0.005 0.005 0.005 0.005
Linkage analysis Linkage analyses were undertaken by calculating lod scores using the computer program LIPED (Ott, 1974). Lod scores > 3 have been considered conventionally to be indicative of linkage and
2 D, receptor
Model
I
Disease allele frequency
frequencies approximated those observed in our sample of 49 unrelated (marrying-in) individuals. .The genotypes of members of families are shown in Figs. 1-7.
enzyme (Promega). Samples were then subjected to electrophoresis on 3% agarose for 2 h at approx. 80 mAmps. The gel was dried and autoradiographed. Two alleles for the dopamine D, receptor gene with Bali were observed, i.e., 1 (304 bp) and 2 (206 + 98 bp), as described in Lannfelt et al., (1992). Published allele frequencies - i.e., 0.67 for allele 1, and 0.33 for allele 2 - (Lannfelt et al., 1992) were used in the linkage analyses. These
TABLE
models)
gene LOD
scores.
Recombination
Autosomal
dominant
inheritance.
Nine families
combined
fraction
0.00
0.05
0.10
0.20
0.30
0.40
M F
- 0.12 - 1.90
- 0.99 - 1.22
- 0.08 - 0.82
- 0.04 - 0.40
0.00 -0.18
0.00 - 0.06
C
-2.12
- 1.31
-0.90
- 0.44
-0.18
- 0.06
M F
-0.15 - 2.34
-0.10 - 1.31
-0.10 - 0.92
- 0.05 - 0.38
- 0.01 -0.17
0.00 - 0.06
C
- 2.49
- 1.41
- 1.02
- 0.43
-0.18
- 0.06
M F
-0.15 -2.81
-0.12 - 1.39
- 0.09 - 0.87
- 0.05 - 0.37
- 0.01 -0.17
0.00 + 0.01
C
- 2.96
- 1.51
- 0.96
- 0.42
-0.18
+0.01
M F
-0.18 - 3.56
-0.13 - 1.48
-0.10 - 1.06
- 0.06 - 0.35
- 0.02 -0.13
0.00 - 0.05
C
- 3.74
- 1.61
- 1.16
- 0.41
-0.15
- 0.05
M F
-0.16 - 4.53
-0.13 - 1.14
- 0.08 - 0.67
- 0.03 - 0.26
-0.02 -0.11
0.00 - 0.04
C
- 4.69
- 1.27
- 0.75
-0.29
- 0.13
- 0.04
M = male; F = female;
C = combined.
220
scores < -2 indicate rejection of linkage at that recombination fraction, though Ott (1989) has suggested that a score > 3.6 should be required to establish linkage where there is a maximization of the lod score using a series of diagnostic schemes and penetrances. Analyses were performed for both autosomal dominant and recessive transmission models, with varying levels of penetrance. Lad scores were calculated separately for female and male recombination and then combined. We tested for linkage using five sets of parameters which were applied to both dominant and recessive genetic models (Table 1). For Model I, a maximum penetrance of 0.60 was used as a conservative representation of the usual penetrance of this disorder. With model V, a maximum penetrance of 1.0 was chosen to specifically reflect the high density of illness in the families under study. Models II-IV represent intermediate levels of penetrance, i.e., 0.70-0.90. The age of onset curve was as described in our previous studies (Mitchell et al., 1991; 1992). The frequency of the disease allele was taken as 0.035 (Detera-Wadleigh et al., 1987) and the penetrance for the non-susceptible genotype (the ‘sporadic rate’) was set at 0.005 (Detera-Wadleigh et al., 1987). For the linkage analyses only subjects of at least 15 years of age were included.
TABLE
D, receptor
Family
Total Number at risk interviewed
Total
(a) Linkage of b’~po1ar d’lsord er and recurrent major depression to the DRD3 gene - autosomal dominant model Combined lod scores for the nine families, using each of the five models, are outlined in Table 2. Close linkage to the DRD3 gene was excluded at a theta (recombination fraction) of 0.00 for each model, from I (maximum penetrance = 0.60) through to V (maximum penetrance 1.0). Table 3 details the lod scores for each individual family, using model IV (which reflects the apparent high degree of penetrance observed in these families). Different gene frequencies of the disease allele (from 0.015 to 0.035) were tested. Lad scores were more negative when the disease allele frequency was assumed to be 0.015, but there was no significant change overall in the results. For example, using model IV, close linkage was still only excluded at a theta of 0.00, though the lod score decreased to -4.52. As in our previous studies (Mitchell et al., 1991; 1992), we examined for the effect of variable rates of sporadic (nongenetic) presentations of illness. We tested a range of sporadic rates varying from 0.005 to 0.1 using model IV (penetrance 0.9). The exclusion of linkage to the DRD3 gene was minimally sen-
3
Dopamine
01 02 05 11 17 20 22 23 24
Results
gene LOD scores by family (model Affected individuals interviewed
Generations
BP
UP
68 16 13 9 9 16 16 13 11
5 5 6 4 4 4 4 3 4
6 _ _ _ 1 2 1 _ 2
171
37
12
3 3 2 3 3 3 3 3 3
IV) Recombination
fraction
0.00
0.05
0.10
0.20
0.30
0.40
+ 0.25 - 1.67 + 0.06 - 1.13 +0.15 - 0.21 +0.13 0.00 - 1.32
+ 0.23 - 0.56 + 0.06 - 0.61 +0.12 -0.17 +0.11 0.00 - 0.79
+0.20 - 0.33 + 0.04 - 0.40 +0.10 - 0.13 +0.10 0.00 - 0.56
+ 0.15 -0.13 + 0.02 -0.19 + 0.07 - 0.07 + 0.05 0.00 -0.31
+0.10 - 0.04 + 0.01 - 0.08 + 0.05 - 0.03 +0.01 0.00 -0.17
+ 0.05 - 0.01 0.00 - 0.02 + 0.02 - 0.01 - 0.01 0.00 - 0.07
- 3.74
- 1.61
- 1.16
-0.41
-0.15
- 0.15
221 TABLE
4
Dopamine
D, receptor
Diagnostic
threshold
A. BIPOLAR
B. BIPOLAR
gene LOD
scores
diagnostic
Recombination
I
I + II
C. BIPOLAR DISORDER AND RECURRENT MDD
M = male; F = female; * Model IV
at various
thresholds.
Dopamine
II
III
IV
V
*
0.00
0.05
0.10
0.20
0.30
0.40
M F
-0.41 - 2.92
-0.32 - 1.52
- 0.25 - 1.06
-0.12 - 0.54
- 0.05 - 0.23
- 0.01 - 0.08
C
- 3.33
- 1.84
- 1.31
-0.66
- 0.28
- 0.09
M F
- 0.38 - 3.93
-0.31 - 2.08
- 0.24 -1.41
-0.12 - 0.74
- 0.05 - 0.32
-0.01 -0.11
C
- 4.31
- 2.39
- 1.65
-0.86
- 0.37
-0.12
M F
- 0.18 - 3.56
-0.13 - 1.48
-0.10 - 1.06
- 0.06 - 0.35
- 0.02 -0.13
0.00 - 0.05
C
- 3.74
- 1.61
- 1.16
- 0.41
-0.15
- 0.05
C = combined
scores.
sporadic rate of 0.10 Clod score - 1.13 at theta = 0.00). These findings indicate that the apparent clear exclusion of linkage to the DRD3 gene was not likely to have been due to a significant rate of
5 D, receptor
Model
I
combined
fraction
sitive to varying the sporadic rate. Linkage was excluded at theta 0.00 at sporadic rates of 0.005 (lad score = -3.741, 0.01 (- 3.49) and 0.05 ( - 2.12). Linkage was not excluded at an assumed
TABLE
Nine families
gene LOD scores. Recombination
Autosomal
recessive
inheritance.
Nine families
combined
fraction
0.00
0.05
0.10
0.20
0.30
0.40
M F
- 1.47 -0.61
-0.84 -0.40
- 0.52 - 0.32
- 0.22 -0.16
- 0.08 - 0.08
- 0.02 - 0.03
C
- 2.08
- 1.24
- 0.84
- 0.38
-0.16
- 0.05
M F
- 1.74 - 0.69
- 0.91 - 0.47
- 0.56 - 0.35
- 0.23 - 0.19
- 0.07 -0.11
- 0.02 - 0.03
C
- 2.43
- 1.38
- 0.91
- 0.42
-0.18
- 0.05
M F
- 2.05 - 0.85
- 0.98 - 0.53
- 0.62 - 0.35
- 0.26 -0.18
- 0.09 - 0.09
- 0.02 - 0.04
C
- 2.90
- 1.51
- 0.97
- 0.44
-0.18
- 0.06
M F
- 2.28 - 1.04
- 1.01 - 0.54
- 0.62 - 0.44
- 0.26 - 0.22
- 0.09 -0.11
- 0.03 - 0.03
C
- 3.32
-1.55
- 1.06
- 0.48
- 0.20
- 0.06
M F
- 2.60 - 1.56
- 1.14 - 0.74
- 0.71 - 0.48
- 0.34 - 0.30
-0.10 -0.11
- 0.04 - 0.02
C
-4.16
- 1.88
- 1.19
- 0.64
- 0.21
- 0.06
M = male; F = female;
C = combined.
222
sporadic presentations of illness. (It should noted that ’ lod scores for the DRD3 gene mained negative for all sporadic rates tested).
be re-
(b) Linkage to the DRD3 gene using other thresholds of diagnosis for affective disorder (Table 4) autosomal dominant model Using model IV, linkage between the DRD3 gene and diagnostic groups defined by the following thresholds of illness was examined in all nine families: (A) bipolar I disorder; (B) bipolar I and II; and (C) bipolar I, II and recurrent major depression. When a narrow definition of the disorder was used, ill subjects who did not meet the criteria for affected were defined as unaffected. Linkage of each of these varying diagnostic thresholds to the DRD3 gene was significantly excluded. For thresholds A and C (bipolar I; bipolar disorder plus recurrent major depression) linkage was excluded at a theta of 0.00, while for threshold B (bipolar I and II), linkage was excluded at a theta of 0.05. (c) Linkage of bipolar disorder and recurrent major depression to the DRD3 gene - autosomal recessive model (Table 5) Combined lod scores for the nine families examining for linkage under the assumption of autosomal recessive inheritance, with different sets of parameters, are outlined in Table 5. As with the assumption of dominant inheritance, linkage was excluded under each model I-V at a theta of 0.00. Discussion This study found a clear exclusion of close linkage between the dopamine D, receptor gene and bipolar disorder. This result indicates that a central role of DRD3 in the aetiology of this condition is unlikely. However, with the exception of diagnostic threshold B (bipolar I and II disorders), this study could not confidently exclude linkage to adjacent genes in the region 3q 13.3. The validity of our result excluding linkage to the dopamine D, receptor gene was strengthened by a number of particular features of the study. Firstly, close linkage was excluded at a series of different thresholds (levels of definition) of affec-
tive status, as indicated in Table 4. Although it is now generally accepted that the ‘bipolar diagnostic spectrum’ (Gershon, 1990) includes schizo-affective, bipolar I and II, unipolar and cyclothymic disorders, testing at various levels is necessary because of both the diagnostic instability of bipolar 11 diagnoses and the possibility of non-genetic or sporadic presentations of depression in these families (Merikangas et al., 1989). In view of this latter problem we only included unipolar depressed subjects who fulfilled the strict NIMH/ Yale criteria for unipolar depression (Gershon and Goldin, 1989). Mis-classification of the sporadic cases as affected significantly reduces ability to demonstrate linkage (Martinez, 1989). Secondly, the finding of exclusion of close linkage was consistent at a wide range of penetrances, i.e., 60 to 100%. We tested for linkage under assumptions of both autosomal dominant and recessive inheritance, as there is currently no consensus concerning the mode of transmission of bipolar disorder. Thirdly, the lack of even suggestive evidence of linkage in any of the nine families, argues against the likelihood of genetic heterogeneity affecting the results with this particular gene marker. However, as only three of these families were informative for this particular marker, we cannot exclude definitively the possibility that a mutation of the dopamine D, receptor gene accounts for a proportion of families with bipolar disorder. Finally, we undertook the conservative strategy of testing for high rates of ‘sporadic’ presentations of illness. Varying the sporadic rate did not significantly affect our conclusion of exclusion of close linkage for the DRD3 gene, although the lod scores were not significant with a high sporadic rate of 0.10 (which is unlikely for this disorder). However, sporadic rates of 0.01 or 0.05 are potentially consistent with the clinical situation, particularly when those with recurrent major depression are included in the diagnostic threshold. In conclusion, after testing a range of single gene models, we have confidently excluded linkage between bipolar disorder and the dopamine D, receptor gene. This finding, in conjunction with our previous report excluding linkage to the dopamine D, and D, receptor genes (Mitchell et al., 1992), suggests that a mutation in the genes
223
for these dopamine receptors is unlikely to play a central aetiological role in bipolar disorder. These results, however, cannot exclude the theoretical possibility that there may be a mutation (or mutations) in the dopamine D, receptor gene which plays an important aetiological role, but which occurs as part of an interaction with mutations in other genes and/or environmental factors. However, the reduced penetrance of some of the genetic models used would allow for the effects of interaction with other genes or environmental factors, provided the dopamine D, receptor gene has a major effect.
Acknowledgements This study was supported by the Australian National Health and Medical Research Council. We are grateful to MS Christina Vivero and Mr. Mark Graham for performing the DNA extraction and Southern hybridization analyses; Ms. Anna Zournazi for technical assistance; and Mrs. Zora Vuckovic for preparation of the manuscript.
References Andreasen, N.C., Endicott, J., Spitzer, R.L. and Winokur, Cl. (1977) The family history method using diagnostic criteria: Reliability and validity. Arch. Gen. Psychiatry 34, 12291235. Avissar, S. and Schreiber, G. (1992) The involvement of guanine nucleotide binding proteins in the pathogenesis and treatment of affective disorders. Biol. Psychiatry 31, 435-459. Berrettini, W., Detera-Wadleigh S.D., Goldin, L.R., Ferraro, T.N., Martinez, M., Hsieh, W.T., Hoehe, M., Nurnberger, J.I., Alexander, R.C. and Gershon E.S. (1992) Linkage studies of bipolar illness (Abstract). Biol. Psychiatry 31, 131A. Bouthenet, M-L., Souil, E., Martres, M-P., Sokoloff, P., Giros, B. and Schwartz, J-C. (1991) Localization of dopamine D, receptor mRNA in the rat brain using in situ hybridization histochemistry: comparison with dopamine D, receptor mRNA. Brain Res. 564, 203-219. Byerley, W., Leppert, M., O’Connell, P., Mellon, C., Holik, J., Lubbers, A., Reimherr, F., Jenson, S., Hill, K., Wender, P., Grandy, D., Litt, M., Lalouel, J.M., Civelli, 0. and White, R. (1990) D2 dopamine receptor gene not linked to manic-depression in three families. Psych. Genet. 1, 55-62. Cox, N., Reich, T., Rice, J., Elston, R., Schober, J. and Keats, B. (1989) Segregation and linkage analyses of bipolar and
major depressive illness in multigenerational pedigrees. J. Psychiat. Res. 23, 109-123. Detera-Wadleigh, S.D., Berrettini, W., Goldin, L.R., Boorman, D., Anderson, D. and Gershon, E.S. (1987) Close linkage of c-Harvey-ras 1 and insulin gene to affective disorder is ruled out in three North American pedigrees. Nature 325, 806-808. Egeland, J.A., Gerhard, D.S., Pauls, D.L., Sussex, J.N., Kidd, K.K., Allen, C., Hostetter, A.M. and Housman, D.E. (1987) Bipolar affective disorders linked to DNA markers on chromosome Il. Nature 325, 783-787. Elphick, M., Yang, J-D. and Cowen, P.J. (1990) Effects of carbamazepine on dopamine and serotonin-mediated neuroendocrine responses. Arch. Gen. Psychiatry 47, 135-140. Gershon, E. (1990) Genetics. In: F.K. Goodwin, K.R. Jamison (Eds.), Manic-depressive Illness. Oxford University Press, New York. Gershon, E. and Goldin, L. (1989) Linkage data on the affective disorders in an epidemiologic context. Genet. Epidem. 6, 201-209. Gill, M., McKeon, P. and Humphries, P. (1988) Linkage analysis of manic depression in an Irish family using H-ras 1 and INS DNA markers. J. Med. Genet. 25, 634-637. Gill, M., Castle, D., Hunt, N., Clements, A., Sham, P. and Murray R.M. (1991) Tyrosine hydroxylase polymorphisms and bipolar affective disorder. J. Psychiat. Res. 25, 179184. Giros, B., Martres, M-P., Sokoloff, P. and Schwartz, J-C. (1990) cDNA cloning of the human dopaminergic D, receptor and chromosome’ identification. CR Acad. Sci. (Paris) 311, 501-508. Hodgkinson, S., Sherrington, R. and Gurling, M. (1987) Molecular genetic evidence for heterogeneity in manic depression. Nature 325, 805-806. Holmes, D., Brynjolfsson, J., Brett, P., Curtis, D., Petursson, H., Sherrington, R. and Gurling, H. (1991) No evidence for a susceptibility locus predisposing to manic depression in the region of the dopamine CD,) receptor gene. Br. J. Psychiatry 158, 635-641. Kelsoe, J.R., Ginns, E.I., Egeland, J.A., Gerhard, D.S., Goldstein, A.M., Bale, S.J., Pauls, D.L., Long, R.T., Kidd, K.K., Conte, G., Housman, D.E. and Paul, S.A. (1989) Reevaluation of the linkage relationship between chromosome llp loci and the gene for bipolar affective disorder in the old order Amish. Nature 342, 238-243. Korner, J., Fritze, J. and Propping, P. (1990) RFLP alleles at the tyrosine hydroxylase locus: no association found to affective disorders. Psychiat. Res. 32, 275-280. Kraepelin, E. (1921) Manic Depressive Insanity and Paranoia. (Transl. Barclay, M). Churchill Livingstone, Edinburgh. Lannfelt, L., Sokoloff, P., Martes, M-P., Pilon, C., Giros, B., Jonsson, E., Sedvall, G. and Schwartz, J-C. (1992) Amino acid substitution in the dopamine D, receptor as a useful polymorphism for investigating psychiatric disorders. Psychiat. Genet. (In press). Leboyer, M., Malafosse, A., Boularand, S., Campion, D., Gheysen, F., Samolyk, D., Henriksson, B., Denise, E., Des Lauriers, A., Lepine, J-P., Zarifian, E., Clergert-Darpoux,
224 F. and Mallet, J. (1990) Tyrosine hydroxylase polymorphisms associated with manic-depressive illness. Lancet 335, 1219. Le Coniat, M., Sokoloff, P., Hillion J., Martres M-P., Giros, B., Pilon, C., Schwartz J-C. and Berger, R. (1991) Chromosomal localization of the human D, dopamine receptor gene. Hum. Genet. 87, 618-620. Martinez, M., Khlat, M., Leboyer, M. and Clerget-Darpoux, F. (1989) Performance of linkage analyses under misclassification error when the genetic model is unknown. Genet. Epid. 6, 253-258. Mengod, G., Villaro, M.T., Landwehrmeyer, G.B., MartinezMir, M.I., Niznik, H.B., Sunahara, R.K., Seeman, P., O’Dowd, B.F., Probst, A. and Palacios, J.M. (1992) Visualization of dopamine D,, D, and D, receptor mRNAs in human and rat brain. Neurochem. Int. 20, 33S-43s. Merikangas, K.R., Spence, A. and Kupfer, D.J. (1989) Linkage studies of bipolar disorder: Methodologic and analytic issues. Arch. Gen. Psychiatry 46, 1137-1141. Mitchell, P., Waters, B., Morrison, N., Shine, J., Donald, J. and Eisman, J. (1991) Close linkage of bipolar disorder to chromosome 11 markers is excluded in two large Australian pedigrees. J. Affect. Disord. 21, 23-32. Mitchell, P., Selbie, L., Waters, B., Donald, J., Vivero, C., Tully, M. and Shine, J. (1992) Exclusion of close linkage of bipolar disorder to dopamine D, and Dz receptor gene markers. J. Affect. Disord. 25, 1-12. Noethen, M.M., Erdmann, J., Korner, J., Lanczik, M., Fritze, J., Fimmers, R., Grandy, D.K., O’Dowd, B. and Propping, P. (1992) Lack of association between dopamine D, and D, receptor genes and bipolar affective disorder. Am. J. Psychiatry 149, 199-201. Ott, J. (1974) Estimation of the recombination fraction in human pedigrees: Efficient computation of the likelihood for human linkage studies. Am. J. Hum. Genet. 28, 525529. Ott, J. (1985) Analysis of human genetic linkage. Johns Hopkins University Press, Baltimore. Ott, J. (1989) Linkage analysis-potential problems introduced by the practice of maximizing the lod score over a wide range of parameters including disease classification (Ab-
stract). Proceedings of First World Congress on Psychiatric Genetics. Pakstis, A.J., Kidd, J.R., Castiglione, C.M. and Kidd, K.K. (1991) Status of the search for a major genetic locus for affective disorder in the Old Order Amish. Hum. Genet. 87, 475-488. Robins, L.N., Wing, W., Wittchen, H.U., Helzer, J.E., Babor, T.F., Burke, J., Farmer, A., Jablenski, A., Pickens, R., Regier, D.A., Sartorius, N. and Towle, L.H. (1988) The Composite International Diagnostic Interview: An epidemiological instrument suitable for use in conjunction with different diagnostic systems and in different cultures. Arch. Gen. Psychiatry 45, 1069-1077. Silverstone, T. and Roman+Clarkson, G. (1988) Bipolar affective disorder: Causes and prevention of relapse. Br. J. Psychiatry 154, 321-335. Sokoloff, P., Giros, B., Martres, M-P., Bouthenet, M-L. and Schwartz, J-C. (1990) Molecular cloning and characterization of a novel dopamine receptor (D,) as a target for neuroleptics. Nature 347, 146-151. Sokoloff, P., Andrieux, M., Besancon, R., Pilon, C., Martres, M-P., Giros, B. and Schwartz, J-C. (1992) Pharmacology of human D, receptor expressed in a mammalian cell line: compairson with D, receptor. Eur. J. Pharmacol. 225, 331-337. Stephens, J.C., Cavanaugh, M.L., Gradie, MI., Mador, M.L. and Kidd, K.K. (1990) Mapping the human genome: Current status. Science 250, 237-244. Thompson, W.D., Kidd, J.R. and Weissman, M.M. (1979) A procedure for the efficient collection and processing of pedigree data suitable for genetic analysis. J. Psychiat. Res. 15, 291-303. Todd, R.D. and O’Malley, K.L. (1989) Population frequencies of tyrosine hydroxylase restriction fragment length polymorphisms in bipolar affective disorder. Biol. Psychiatry 25, 626-630. Wesner, R.B., Scheffner, W., Palmer, P.J., Crowe, P.R. and Winokur, G. (1990) The effect of co-morbidity and penetrance on the outcome of linkage analysis in a bipolar family. Biol. Psychiatry 27, 241-244.