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No association between bipolar affective disorder and a serotonin receptor (5-HT2A) polymorphism
Psychiatry Research 70 Ž1997. 65]69
No association between bipolar affective disorder and a serotonin receptor Ž 5-HT2A. polymorphism Brigitte Mahieu aU , Daniel Souery a , Olivier Lipp a , Karin Mendelbauma , Geert Verheyen c , Viviane De Maertelaer b , Christine Van Broeckhovenc , Julien Mendlewicz a a
Department of Psychiatry, Uni®ersity Clinics of Brussels, Erasme Hospital, Free Uni®ersity of Brussels, 808 Route de Lennik, B-1070 Brussels, Belgium b I.R.I.B.H.N., Statistical Unit, Free Uni®ersity of Brussels, Brussels, Belgium c Laboratory of Neurogenetics, Flanders Interuni®ersity Institute of Biotechnology (VIB), Born-Bunge Foundation (BBS), Uni®ersity of Antwerp (UIA) Department of Biochemistry, Antwerp, Belgium Received 8 April 1996; revised 6 January 1997; accepted 4 March 1997
Abstract The serotonergic system is implicated in the pathogenesis of affective disorders. In particular, the role of the postsynaptic 5-hydroxytryptamine Žserotonin. type 2 receptor Ž5-HT2. has been documented by several studies. The 5-HT2A receptor gene located on chromosome 13 Ž13q14-21. can be considered a candidate gene for bipolar affective disorder ŽBPAD.. We tested association between a 5-HT2A receptor DNA variant and BPAD using a case-control design. Eighty-three BPAD patients and 129 unrelated normal controls, carefully matched for sex and geographical origin, were studied. Allele and genotype frequencies as well as homo]heterozygote distribution at the 5-HT2A receptor polymorphism were compared between the two groups. No significant allelic or genotypic associations were observed. There was no significant difference for homo]heterozygote distribution between the two groups. These preliminary results may indicate that in our sample the 5-HT2 receptor polymorphism studied is unlikely to play a role in the genetic susceptibility to BPAD. Q 1997 Elsevier Science Ireland Ltd. Keywords: Candidate gene; Serotonin transmission; Allelic association
1. Introduction The role of the serotonergic system in the pathogenesis of depression has been extensively U
Corresponding author. Tel.: q32 2 5554315; fax: q32 2 5554515.
documented ŽMaes and Meltzer, 1995.. Serotonergic transmission is mediated by post-synaptic receptors and serotonin Ž5-hydroxytryptamine: 5HT. transporter protein that mediates the uptake of 5-HT at the presynaptic level. Serotonin synthesis is rate-limited by the availability of tryptophan and its carrier-mediated brain uptake. The implication of postsynaptic 5-HT2 receptors in affective disorders has been supported by
0165-1781r97r$17.00 Q 1997 Elsevier Science Ireland Ltd. All rights reserved. PII S0165-1781Ž97. 03028-X
66
B. Mahieu et al. r Psychiatry Research 70 (1997) 65]69
data consistent with the hypothesis of 5-HT2 receptor up-regulation Žincreased number, affinity or responsivity. in depression ŽCharney and Delgado, 1992.. Studies on blood cells have shown higher 5-HT2 receptor binding in platelets of major depressed patients in comparison with controls ŽBiegon et al., 1987; Arora and Meltzer, 1989.. Post-mortem studies reported an increase of 5-HT2 receptor binding sites in the prefrontal cortex of depressed patients who died from suicide or natural causes ŽYates et al., 1990; Arango et al., 1992.. Electroencephalographic sleep measures stressed that the increase in slow-wave sleep ŽSWS., usually observed after blockade of 5-HT2 receptors by antagonists, is lower in depressed patients than in normal controls ŽSharpley et al., 1990; Staner et al., 1992.. Finally, several pharmacological studies have shown that chronic treatment with tricyclic antidepressants, monoamine oxidase inhibitors and some but not all selective serotonin reuptake inhibitors leads to down-regulation in the number of 5-HT2 receptor binding sites in the brain ŽCowen, 1990; Leysen, 1992.. An interesting observation is that the time course of this down-regulation is similar to the time for a clinical response in depressed patients following antidepressant treatment. This 5-HT2 receptor down-regulation induced by antidepressants could play a role in their therapeutic action ŽLeysen and Pauwels, 1990.. Taken together, these observations support the up-regulation of the 5-HT2 receptors hypothesis in depression ŽMaes and Meltzer, 1995.. Parallel to these developments, the implication of genetic risk factors in the pathogenesis of affective disorders has been confirmed by traditional methods such as twin, adoption and family studies ŽMendlewicz, 1994.. However, with these methods, it is difficult to specify genetic variables involved or the exact mode of transmission. Linkage Žcosegregation of alleles in families. and association Želevated allele frequencies in patient versus control populations. studies are used to identify the genes involved. Association strategy is interesting in complex diseases such as bipolar affective disorder ŽBPAD. since this non-parametric method does not require the knowledge of genetic parameters Žmode of transmission, penetrance, and gene frequency.. Another advantage
of association studies is the possibility to detect genes of minor effect. However, the principal limitation of this approach is related to the possible spurious association between a genetic marker and the disease which can result from variable allele frequencies between cases and controls occurring when the two populations are ethnically different Žpopulation stratification .. The haplotype relative risk ŽHRR. strategy, using parental data for the control sample, reduces this type of bias. The HRR method selects the non-transmitted alleles from the cases’ parents as the control sample. Several candidate genes Žloci potentially involved in the etiology of the disease. such as genes involved in catecholamine metabolism have already been tested in BPAD in numerous linkage andror association studies Žfor review, see Souery et al., 1996.. For the reasons mentioned above, the 5-HT2A receptor gene can beconsidered as a candidate gene in BPAD. A 5-HT2A receptor polymorphism has been identified within the coding Žexonic. region of the gene located on chromosome 13 Ž13q14-21. ŽWarren et al., 1993.. Both alleles at this polymorphism ŽTrC at position 102. encode for the same protein Žsilent mutation.. Lack of association between this polymorphic variant of the 5-HT2A receptor and BPAD patients in a Spanish sample of 88 patients and 113 controls has been reported recently ŽGutierrez ´ et al., 1995.. From the available data to date, it is impossible to specify the exact role of the 5-HT2A receptor gene in BPAD. It is therefore of interest to further investigate this candidate gene in BPAD. The purpose of the present study is to test for association between the 5-HT2A receptor polymorphism ŽT102C. and BPAD. This association study was conducted with particular attention to stratification bias. In order to limit this bias, patients and controls were carefully matched for geographical origin. 2. Methods 2.1. Sample Eighty-three unrelated BPAD patients Ž41
B. Mahieu et al. r Psychiatry Research 70 (1997) 65]69
males and 42 females. and 129 normal controls Ž63 males and 66 females. were ascertained for the present study. The BPAD patients included 61 bipolar I and 22 bipolar II. The patients were recruited from admissions to the inpatient or outpatient units of the department of psychiatry of Erasme Hospital and from the probands of families included in previous linkage studies. The controls were recruited among the staff of the hospital or medical students via an announcement and among subjects from families included in previous linkage studies Ž25 controls unrelated to the proband and no spouses were included.. The case-control design for association study was applied since, for most patients included in the study, parents were not available Žinterview and blood sampling. for the HRR method. All patients and controls were interviewed using the Schedule for Affecti®e Disorders and Schizophrenia-Lifetime Version ŽSADS-LA. ŽEndicott and Spitzer, 1978. and diagnoses were established according to the Research Diagnostic Criteria ŽRDC. ŽSpitzer et al., 1978.. The subjects of the control group did not present any positive personal or familial history Žassessed by the Family History Research Diagnosis Criteria. of psychiatric disorders. Informed consent was obtained from patients and controls. The patients were carefully matched with controls for sex and geographical origin. All the patients and controls are of western European origin having Belgian ancestry. The mean age Ž"S.D.. was not significantly different between the groups of patients Ž46.0" 14.0 years. and controls Ž44.3" 15.0 years. Ž Ps 0.57.. In the patient group, the mean age at onset was 28.0" 10.5 years and the mean number of affective episodes was 3.3" 2.4 depressive episodes and 4.3 " 3.5 manic episodes. Among the BPAD patients, 13 have attempted suicide at least once in the course of their affective disorder. 2.2. DNA analysis Genomic DNA was isolated from peripheral blood leukocytes using standard salting out procedures. The polymorphism in 5-HT2A was identified by the polymerase chain reaction ŽPCR. fol-
67
lowed by restriction enzyme digestion. Standard PCR was carried out in a 25-m l volume containing 100 ng genomic DNA, 200 m M of each dNTP, 1.25 mM MgCl 2 , 50 pmol of each primer and 0.2 units Goldstar DNA polymerase ŽEurogentec.. Published primer sequences were used ŽWarren et al., 1993.. After an initial denaturation step at 948C for 2 min, 30 cycles were performed: denaturation at 948C for 1 min, annealing at 608C for 1.5 min and extension at 728C for 2 min. An additional final extension step was performed at 728C for 5 min. Twenty microlitres of the PCR product were digested overnight at 378C with 0.1 unitsrm l of M spI in a total volume of 25 m l. Digestion products were visualised by ethidium bromide staining after electrophoresis in a 3% agarose gel. 2.3. Statistical analysis Patients’ and controls’ characteristics were compared using the Student t-test for independent samples Žtwo-tailed.. The analysis of allelic association consisted of comparing allele and genotype frequencies for the investigated marker between patients and controls. The homozygote] heterozygote distribution was also compared. The observed frequencies were recorded into a contingency table; comparisons were performed with the chi-square test. 2.4. Power On the basis of EPI-INFO ŽDean et al., 1991., the power to detect a difference at the 0.05 level was evaluated to be about 60% in our study, estimating a frequency of disease allele in the BPAD population at 10% and at 2% in the control population ŽWalsh et al., 1992. for a sample size of 89 patients and 123 controls. 3. Results We investigated the possible association of 5HT2A receptor DNA variants ŽTrC at position 102. and BPAD. In this sample of 83 BPAD patients and 129 healthy controls pairwise matched for sex and geographical origin, no significant differences were found for allele Ž x 2 s 0.00; d.f.s 1; Ps 1.00. and genotype Ž x 2 s 0.9;
B. Mahieu et al. r Psychiatry Research 70 (1997) 65]69
68
Table 1 Allele and genotype frequencies and homozygote]heterozygote distribution for the 5-HT2A polymorphism in BPAD patients and control subjects Controls Žn s 129.
BPAD Patients Žn s 83.
115Ž44.6. 143Ž55.4.
74Ž44.6. 92Ž55.4.
GenotypesUU Ž%. 1]1 1]2 2]2
26Ž20.2. 63Ž48.8. 40Ž31.0.
14Ž16.9. 46Ž55.4. 23Ž27.7.
Homo-heteroUUU Ž%. Homo Hetero
66Ž51.2. 63Ž48.8.
37Ž44.6. 46Ž55.4.
AllelesU Ž%. 1 2
Frequencies expressed in Ž%. for alleles, genotypes and homo-heterozygote distribution. Number for each allele represents the total number of times alleles are observed Ž n = 2.. U UU UUU Ps 1.00: Ps 0.64: Ps 0.35. BPAD: Bipolar Affective Disorder
d.f.s 2; Ps 0.64. frequencies and homozygote] heterozygote distribution Ž x 2 s 0.88; d.f.s 1; P s 0.35. at the 5-HT2A receptor polymorphism between BPAD patients and controls ŽTable 1.. Genotypes for both populations were in HardyWeinberg equilibrium.
interest to test for association between the 5HT2A receptor gene and subgroups of BPAD patients. Presence of suicidal behaviour could be considered in this approach. Indeed, suicidal patients seem to present a particularly disturbed serotonergic system ŽGhanshyam et al., 1995.. In conclusion, our findings in this case-control association study suggest noassociation between the 5-HT2A receptor polymorphism ŽT102C. and BPAD. This result may indicate that this genetic marker is unlikely to play a major role in genetic susceptibility of BPAD but has to be interpreted in light of the low power of the sample, since we cannot exclude a false negative result. Before definitively excluding an implication of the 5HT2A receptor gene in the etiology of BPAD, other polymorphisms of the gene should be studied in larger samples and with other methodologies Žlinkage, HRR.. Acknowledgements This work was supported by the Association for Mental Health Research ŽAMHR., the European Community Biomed Grant ŽGrant No CT 921217., the National Fund for Scientific Research ŽNFSR. and a concerted action of the Flemish Ministry of Education. The authors would like to thank Geert Raes and An De Bruyn for valuable help with the DNA analyses.
4. Discussion References These results may indicate that in our sample, the 5-HT2A receptor polymorphism studied is not directly involved in the pathogenesis of BPAD. This finding is in agreement with a previous association study between the same marker and BPAD in a Spanish sample ŽGutierrez et al., 1995.. How´ ever, these data have to be interpreted with caution before they can be replicated in larger and more powerful samples using other methodologies Ži.e. HRR and linkage methods.. Moreover, before excluding 5-HT2A receptor gene implication in BPAD, other variants of this gene should be investigated, and in particular, functional ones. Genetic and phenotypic heterogeneity could also explain lack of association. It would be of great
Arango, V., Underwood, M.D. and Mann, J.J. Ž1992. Alterations in monoamine receptors in the brain of suicide victims. J Clin Psychopharmacol 12, 8S]12S. Arora, R.C. and Meltzer, H.Y. Ž1989. Increased serotonin 2 Ž5-HT2. receptor binding as measured by 3 H-lysergic acid diethylamide Ž 3 H-LSD. in the blood platelets of depressed patients. Life Sci 44, 725]734. Biegon, A., Weizman, A., Karp, L., Ram, A., Tiano, S. and Wolff, M. Ž1987. Serotonin 5-HT2 receptor binding on blood platelets } a peripheral marker for depression. Life Sci 41, 2485]2492. Charney, D.S. and Delgado, P.L. Ž1992. Current concept of the role of serotonin function in depression and anxiety. In: Langer, S.Z., Brunello, N., Racagni, G. and Mendlewicz, J. ŽEds.., Serotonin Receptor Subtypes: Pharmacological Significance and Clinical Implication. Karger series, Basel, pp. 89]104.
B. Mahieu et al. r Psychiatry Research 70 (1997) 65]69 Cowen, P.J. Ž1990. A role for 5-HT in the action of antidepressant drugs. Pharmacol Ther 46, 43]51. Dean, J., Dean, A., Burton, A. and Dicker, R. Ž1991. Epi Info Version 5.01a. Centers for Disease Control, Atlanta. Endicott, J. and Spitzer, R.L. Ž1978. A diagnostic interview: the schedule for affective disorders and schizophrenia. Arch Gen Psychiatry 35, 837]862. Ghanshyam, N.P., Subhash, C.P., Yogesh, D., Rajiv, P.S., Philip, G.J. and John, M.D. Ž1995. Platelet serotonin-2A receptors: a potential biological marker for suicidal behavior. Am J Psychiatry 152, 850]855. Gutierrez, B., Arranz, M., Fananas, L., Valles, ´ ` V., Guillamat, R., van Os, J. and Collier, D. Ž1995. 5HT2A receptor gene and bipolar affective disorder. Lancet 346, 969. Leysen, J.E. and Pauwels, P. Ž1990. 5-HT2 receptors, role and regulation, the neuropharmacology of serotonin. Ann NY Acad Sci 600, 183]193. Leysen, J.E. Ž1992. 5-HT2 receptors: location, pharmacological, pathological and physiological role. In: Langer, S.Z., Brunello, N., Racagni, G. and Mendlewicz, J. ŽEds.., Serotonin Receptor Subtypes: Pharmacological Significance and Clinical Implication. Karger series, Basel, pp. 31]43. Maes, M. and Meltzer, H.Y. Ž1995. The serotonin hypothesis of major depression. In: Bloom, F.E. and Kupfer, D.J. ŽEds.., Psychopharmacology: The Fourth Generation of Progress. Raven Press, New York, p. 940. Mendlewicz, J. Ž1994. The search for a manic depressive gene: from classical to molecular genetics. Prog Brain Res 100, 225]259.
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Sharpley, A.L., Gregory, C.A., Solomon, R.A. and Cowen, P.J. Ž1990. Slow wave sleep and 5-HT2 receptor sensitivity during maintenance tricyclic antidepressant treatment. J Affect Disord 19, 273]277. Souery, D., Papadimitriou, G.N. and Mendlewicz, J. Ž1996. New genetic approaches in affective disorders. In: Papadimitriou, G.N. and Mendlewicz, J. ŽEds.., Genetics of Mental Disorders Part II: Clinical Issues. Bailliere’s Clinical ` Psychiatry, International Practice and Research, Bailliere Tindall, London. Vol. 2, No 1, pp. 1]13. Spitzer, R.L., Endicott, J. and Robins, E. Ž1978. Research diagnostic criteria: rationale and reliability. Arch Gen Psychiatry 35, 773]782. Staner, L., Kempenaers, C., Simonnet, M.P., Fransolet, L. and Mendlewicz, J. Ž1992. 5-HT2 receptor antagonism and slow wave sleep in major depression. Acta Psychiatr Scand 86, 133]137. Yates, M., Leake, A., Candy, J.M., Fairbairn, A.F., McKeith, I.G. and Ferrier, I.N. Ž1990. 5-HT2 receptor changes in major depression. Biol Psychiatry 27, 489]96. Walsh, C., Hicks, A., Sham, P., Castle, D., Hunt, N., Clements, A., Sander, T., Murray, R., Darlison, M.G. and Gill, M. Ž1992. GABA A receptor subunit genes as candidate genes for bipolar affective disorder } an association analysis. Psychiatr Genet 2, 239]247. Warren, J.T. Jr., Peacock, M.L., Rodriguez, L.C. and Fink, J.K. Ž1993. An M spI polymorphism in the human serotonin receptor gene ŽHTR2.: detection by DGGE and RFLP analysis. Hum Mol Genet 3, 338.
No association between bipolar affective disorder and a serotonin receptor Ž 5-HT2A. polymorphism Brigitte Mahieu aU , Daniel Souery a , Olivier Lipp a , Karin Mendelbauma , Geert Verheyen c , Viviane De Maertelaer b , Christine Van Broeckhovenc , Julien Mendlewicz a a
Department of Psychiatry, Uni®ersity Clinics of Brussels, Erasme Hospital, Free Uni®ersity of Brussels, 808 Route de Lennik, B-1070 Brussels, Belgium b I.R.I.B.H.N., Statistical Unit, Free Uni®ersity of Brussels, Brussels, Belgium c Laboratory of Neurogenetics, Flanders Interuni®ersity Institute of Biotechnology (VIB), Born-Bunge Foundation (BBS), Uni®ersity of Antwerp (UIA) Department of Biochemistry, Antwerp, Belgium Received 8 April 1996; revised 6 January 1997; accepted 4 March 1997
Abstract The serotonergic system is implicated in the pathogenesis of affective disorders. In particular, the role of the postsynaptic 5-hydroxytryptamine Žserotonin. type 2 receptor Ž5-HT2. has been documented by several studies. The 5-HT2A receptor gene located on chromosome 13 Ž13q14-21. can be considered a candidate gene for bipolar affective disorder ŽBPAD.. We tested association between a 5-HT2A receptor DNA variant and BPAD using a case-control design. Eighty-three BPAD patients and 129 unrelated normal controls, carefully matched for sex and geographical origin, were studied. Allele and genotype frequencies as well as homo]heterozygote distribution at the 5-HT2A receptor polymorphism were compared between the two groups. No significant allelic or genotypic associations were observed. There was no significant difference for homo]heterozygote distribution between the two groups. These preliminary results may indicate that in our sample the 5-HT2 receptor polymorphism studied is unlikely to play a role in the genetic susceptibility to BPAD. Q 1997 Elsevier Science Ireland Ltd. Keywords: Candidate gene; Serotonin transmission; Allelic association
1. Introduction The role of the serotonergic system in the pathogenesis of depression has been extensively U
Corresponding author. Tel.: q32 2 5554315; fax: q32 2 5554515.
documented ŽMaes and Meltzer, 1995.. Serotonergic transmission is mediated by post-synaptic receptors and serotonin Ž5-hydroxytryptamine: 5HT. transporter protein that mediates the uptake of 5-HT at the presynaptic level. Serotonin synthesis is rate-limited by the availability of tryptophan and its carrier-mediated brain uptake. The implication of postsynaptic 5-HT2 receptors in affective disorders has been supported by
0165-1781r97r$17.00 Q 1997 Elsevier Science Ireland Ltd. All rights reserved. PII S0165-1781Ž97. 03028-X
66
B. Mahieu et al. r Psychiatry Research 70 (1997) 65]69
data consistent with the hypothesis of 5-HT2 receptor up-regulation Žincreased number, affinity or responsivity. in depression ŽCharney and Delgado, 1992.. Studies on blood cells have shown higher 5-HT2 receptor binding in platelets of major depressed patients in comparison with controls ŽBiegon et al., 1987; Arora and Meltzer, 1989.. Post-mortem studies reported an increase of 5-HT2 receptor binding sites in the prefrontal cortex of depressed patients who died from suicide or natural causes ŽYates et al., 1990; Arango et al., 1992.. Electroencephalographic sleep measures stressed that the increase in slow-wave sleep ŽSWS., usually observed after blockade of 5-HT2 receptors by antagonists, is lower in depressed patients than in normal controls ŽSharpley et al., 1990; Staner et al., 1992.. Finally, several pharmacological studies have shown that chronic treatment with tricyclic antidepressants, monoamine oxidase inhibitors and some but not all selective serotonin reuptake inhibitors leads to down-regulation in the number of 5-HT2 receptor binding sites in the brain ŽCowen, 1990; Leysen, 1992.. An interesting observation is that the time course of this down-regulation is similar to the time for a clinical response in depressed patients following antidepressant treatment. This 5-HT2 receptor down-regulation induced by antidepressants could play a role in their therapeutic action ŽLeysen and Pauwels, 1990.. Taken together, these observations support the up-regulation of the 5-HT2 receptors hypothesis in depression ŽMaes and Meltzer, 1995.. Parallel to these developments, the implication of genetic risk factors in the pathogenesis of affective disorders has been confirmed by traditional methods such as twin, adoption and family studies ŽMendlewicz, 1994.. However, with these methods, it is difficult to specify genetic variables involved or the exact mode of transmission. Linkage Žcosegregation of alleles in families. and association Želevated allele frequencies in patient versus control populations. studies are used to identify the genes involved. Association strategy is interesting in complex diseases such as bipolar affective disorder ŽBPAD. since this non-parametric method does not require the knowledge of genetic parameters Žmode of transmission, penetrance, and gene frequency.. Another advantage
of association studies is the possibility to detect genes of minor effect. However, the principal limitation of this approach is related to the possible spurious association between a genetic marker and the disease which can result from variable allele frequencies between cases and controls occurring when the two populations are ethnically different Žpopulation stratification .. The haplotype relative risk ŽHRR. strategy, using parental data for the control sample, reduces this type of bias. The HRR method selects the non-transmitted alleles from the cases’ parents as the control sample. Several candidate genes Žloci potentially involved in the etiology of the disease. such as genes involved in catecholamine metabolism have already been tested in BPAD in numerous linkage andror association studies Žfor review, see Souery et al., 1996.. For the reasons mentioned above, the 5-HT2A receptor gene can beconsidered as a candidate gene in BPAD. A 5-HT2A receptor polymorphism has been identified within the coding Žexonic. region of the gene located on chromosome 13 Ž13q14-21. ŽWarren et al., 1993.. Both alleles at this polymorphism ŽTrC at position 102. encode for the same protein Žsilent mutation.. Lack of association between this polymorphic variant of the 5-HT2A receptor and BPAD patients in a Spanish sample of 88 patients and 113 controls has been reported recently ŽGutierrez ´ et al., 1995.. From the available data to date, it is impossible to specify the exact role of the 5-HT2A receptor gene in BPAD. It is therefore of interest to further investigate this candidate gene in BPAD. The purpose of the present study is to test for association between the 5-HT2A receptor polymorphism ŽT102C. and BPAD. This association study was conducted with particular attention to stratification bias. In order to limit this bias, patients and controls were carefully matched for geographical origin. 2. Methods 2.1. Sample Eighty-three unrelated BPAD patients Ž41
B. Mahieu et al. r Psychiatry Research 70 (1997) 65]69
males and 42 females. and 129 normal controls Ž63 males and 66 females. were ascertained for the present study. The BPAD patients included 61 bipolar I and 22 bipolar II. The patients were recruited from admissions to the inpatient or outpatient units of the department of psychiatry of Erasme Hospital and from the probands of families included in previous linkage studies. The controls were recruited among the staff of the hospital or medical students via an announcement and among subjects from families included in previous linkage studies Ž25 controls unrelated to the proband and no spouses were included.. The case-control design for association study was applied since, for most patients included in the study, parents were not available Žinterview and blood sampling. for the HRR method. All patients and controls were interviewed using the Schedule for Affecti®e Disorders and Schizophrenia-Lifetime Version ŽSADS-LA. ŽEndicott and Spitzer, 1978. and diagnoses were established according to the Research Diagnostic Criteria ŽRDC. ŽSpitzer et al., 1978.. The subjects of the control group did not present any positive personal or familial history Žassessed by the Family History Research Diagnosis Criteria. of psychiatric disorders. Informed consent was obtained from patients and controls. The patients were carefully matched with controls for sex and geographical origin. All the patients and controls are of western European origin having Belgian ancestry. The mean age Ž"S.D.. was not significantly different between the groups of patients Ž46.0" 14.0 years. and controls Ž44.3" 15.0 years. Ž Ps 0.57.. In the patient group, the mean age at onset was 28.0" 10.5 years and the mean number of affective episodes was 3.3" 2.4 depressive episodes and 4.3 " 3.5 manic episodes. Among the BPAD patients, 13 have attempted suicide at least once in the course of their affective disorder. 2.2. DNA analysis Genomic DNA was isolated from peripheral blood leukocytes using standard salting out procedures. The polymorphism in 5-HT2A was identified by the polymerase chain reaction ŽPCR. fol-
67
lowed by restriction enzyme digestion. Standard PCR was carried out in a 25-m l volume containing 100 ng genomic DNA, 200 m M of each dNTP, 1.25 mM MgCl 2 , 50 pmol of each primer and 0.2 units Goldstar DNA polymerase ŽEurogentec.. Published primer sequences were used ŽWarren et al., 1993.. After an initial denaturation step at 948C for 2 min, 30 cycles were performed: denaturation at 948C for 1 min, annealing at 608C for 1.5 min and extension at 728C for 2 min. An additional final extension step was performed at 728C for 5 min. Twenty microlitres of the PCR product were digested overnight at 378C with 0.1 unitsrm l of M spI in a total volume of 25 m l. Digestion products were visualised by ethidium bromide staining after electrophoresis in a 3% agarose gel. 2.3. Statistical analysis Patients’ and controls’ characteristics were compared using the Student t-test for independent samples Žtwo-tailed.. The analysis of allelic association consisted of comparing allele and genotype frequencies for the investigated marker between patients and controls. The homozygote] heterozygote distribution was also compared. The observed frequencies were recorded into a contingency table; comparisons were performed with the chi-square test. 2.4. Power On the basis of EPI-INFO ŽDean et al., 1991., the power to detect a difference at the 0.05 level was evaluated to be about 60% in our study, estimating a frequency of disease allele in the BPAD population at 10% and at 2% in the control population ŽWalsh et al., 1992. for a sample size of 89 patients and 123 controls. 3. Results We investigated the possible association of 5HT2A receptor DNA variants ŽTrC at position 102. and BPAD. In this sample of 83 BPAD patients and 129 healthy controls pairwise matched for sex and geographical origin, no significant differences were found for allele Ž x 2 s 0.00; d.f.s 1; Ps 1.00. and genotype Ž x 2 s 0.9;
B. Mahieu et al. r Psychiatry Research 70 (1997) 65]69
68
Table 1 Allele and genotype frequencies and homozygote]heterozygote distribution for the 5-HT2A polymorphism in BPAD patients and control subjects Controls Žn s 129.
BPAD Patients Žn s 83.
115Ž44.6. 143Ž55.4.
74Ž44.6. 92Ž55.4.
GenotypesUU Ž%. 1]1 1]2 2]2
26Ž20.2. 63Ž48.8. 40Ž31.0.
14Ž16.9. 46Ž55.4. 23Ž27.7.
Homo-heteroUUU Ž%. Homo Hetero
66Ž51.2. 63Ž48.8.
37Ž44.6. 46Ž55.4.
AllelesU Ž%. 1 2
Frequencies expressed in Ž%. for alleles, genotypes and homo-heterozygote distribution. Number for each allele represents the total number of times alleles are observed Ž n = 2.. U UU UUU Ps 1.00: Ps 0.64: Ps 0.35. BPAD: Bipolar Affective Disorder
d.f.s 2; Ps 0.64. frequencies and homozygote] heterozygote distribution Ž x 2 s 0.88; d.f.s 1; P s 0.35. at the 5-HT2A receptor polymorphism between BPAD patients and controls ŽTable 1.. Genotypes for both populations were in HardyWeinberg equilibrium.
interest to test for association between the 5HT2A receptor gene and subgroups of BPAD patients. Presence of suicidal behaviour could be considered in this approach. Indeed, suicidal patients seem to present a particularly disturbed serotonergic system ŽGhanshyam et al., 1995.. In conclusion, our findings in this case-control association study suggest noassociation between the 5-HT2A receptor polymorphism ŽT102C. and BPAD. This result may indicate that this genetic marker is unlikely to play a major role in genetic susceptibility of BPAD but has to be interpreted in light of the low power of the sample, since we cannot exclude a false negative result. Before definitively excluding an implication of the 5HT2A receptor gene in the etiology of BPAD, other polymorphisms of the gene should be studied in larger samples and with other methodologies Žlinkage, HRR.. Acknowledgements This work was supported by the Association for Mental Health Research ŽAMHR., the European Community Biomed Grant ŽGrant No CT 921217., the National Fund for Scientific Research ŽNFSR. and a concerted action of the Flemish Ministry of Education. The authors would like to thank Geert Raes and An De Bruyn for valuable help with the DNA analyses.
4. Discussion References These results may indicate that in our sample, the 5-HT2A receptor polymorphism studied is not directly involved in the pathogenesis of BPAD. This finding is in agreement with a previous association study between the same marker and BPAD in a Spanish sample ŽGutierrez et al., 1995.. How´ ever, these data have to be interpreted with caution before they can be replicated in larger and more powerful samples using other methodologies Ži.e. HRR and linkage methods.. Moreover, before excluding 5-HT2A receptor gene implication in BPAD, other variants of this gene should be investigated, and in particular, functional ones. Genetic and phenotypic heterogeneity could also explain lack of association. It would be of great
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