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Genetic variation in the 5-HT3 receptor gene: No association with schizophrenia or drug response
127
XI. Genetics A. B. C. D.
Candidate Genes Other Molecular Studies Correlated Phenotypes Miscellaneous
A. Candidate Genes A POLYMORPHISM IN THE PROMOTER REGION OF THE DOPAMINE D2 RECEPTOR GENE (DRD2) AND DRUG RESPONSE: ASSOCIATION STUDIES M.J. Arranz, J, M u n r o , T. Li, D.A. Collier, G. Kirov, R.W. Kerwin
Department of P,s3'ehologieal Medicine, Institute o[' Psychiato,, London SE5 8AF, UK The dopamine hypothesis of schizophrenia is supported by strong evidence which shows alterations of the dopaminergic system in schizophrenia patients. Classical antipsychotics display high affinities for dopamine receptors, specifically for the dopamine D2 receptors, in contrast with atypical antipsychotics. However, a recent study (Pilowsky et al., 1997) found that the atypical antipsychotic clozapine shows similar levels of occupancy of D2 receptors than typical antipsychotics in the frontal cortex. Dopamine receptor genes are strong candidates for involvement in the aetiology of schizophrenia and clinical treatment of the illness. However, analysis of a variety of polymorphisms in the dopamine receptor genes have not provided strong associations, with the exception of a small relative risk associated with the dopamine D3 receptor gene (DRD3). In addition several studies have failed to find association between genetic variants in dopamine receptor genes and clinical response to antipsychotics (Shaikh, 1995). Recently, a polymorphism in the promoter region of the dopamine D2 receptor gene (DRD2). a deletion of a cytosine at position -141, was reported to be associated with schizophrenia (Arinami et al., 1997). We tried to replicate these results in a sample of 143 clozapine treated patients and 100 controls. However, no significant differences were found between the patient and control groups, and between patients who responded to the treatment and patients who failed to respond. These results suggest that it is unlikely that genetic variation in the D2 receptor gene plays a major role in the aetiology of schizophrenia or in clinical response to treatment.
FURTHER EVIDENCE FOR ASSOCIATION BETWEEN POLYMORPHISMS IN THE 5-HT2A RECEPTOR GENE AND CLOZAPINE RESPONSE M.J. Arranz, J. M u n r o , D.A. Collier, G. Krov, P. Sham, J. Z h a o , R.W. Kerwin
Department o( Po'chological Medieine, Institute o[ Psyehiato,, London SE5 8AF, UK Serotonin receptors are targeted by a variety of antipsychotic drugs, particularly those termed atypical antipsychotics such as clozapine. We hypothesised that genetic variation in these receptors could affect clinical response to drugs targeting them by altering the receptor binding affinities, function or expression. In support of this hypothesis, we found association between two polymorphisms in the 5-HT2A receptor gene, 102-T/C and His452Tyr, and clinical response in a sample of clozapine treated patients (Arranz et al., 1995, 1996). The CI02 and Tyr452 variants were more frequent among patients who did not respond satisfactorily to clozapine. Several groups tried to replicate the association between 102-T/C and clozapine response but failed to find statistically significant differences. Significant association between His452Tyr and clozapine response was also found by Badri and collaborators, although two other studies failed to find significant differences. To clarify these results, meta-analyses including all published studies and an independent sample collected at the Institute were performed. Significant associations were found between clozapine response and 102-T/C genotype ()~2=9.05, p=0.011 and allele frequencies ( 7.2 = 7.13, p = 0.008, O.R. = 1.36). This association was stronger when considering allele C102 recessive (Z2= 8.49, p=0.004, O.R.= 1.64). Strong association was also observed between allele Tyr452 and poor response to clozapine (f2= 6.97, p=0.008, O.R.=1.69) and between homozigosity for Tyr452 and poor clozapine response ( Z 2 = 4.25, p = 0.04, O.R. = 5.55). These results provide further evidence of the importance of the 5-HT2 receptors as therapeutic targets and of the influence of genetic variation in determining clinical response to antipsychotics.
GENETIC VARIATION IN THE 5-HT3 RECEPTOR GENE: NO ASSOCIATION WITH SCHIZOPHRENIA OR DRUG RESPONSE M.J. Arranz, J. M u n r o , D.A. Collier, G. Kirov, R.W. Kerwin
Department ~]'Psychoh~gieal Medicine, Institute o! P,~Tehiato,, London SE5 8AF, UK Serotonin ( 5-HT ) receptors are strong candidates for involvement in mental disorders such as depression, eating disorders, sexual behaviour and schizophrenia and are targets of atypical antipsychotic drugs. 5-HT3 is the only type of serotonin receptors which is ligand-gated ion channel. 5-HT3 receptors
128
are candidate targets for the treatment of anxiety, depression, schizophrenia and dementia (Greenshaw, 1993). In addition, there is evidence suggesting that 5-HT3 receptors play a role in the therapeutic action of clozapine (Wang et al., 1994; Hermann et al., 1996). We hypothesised that genetic variation in 5-HT3 receptor may be involved in the aetiology of schizophrenia and in determining clinical response to clozapine. To test this hypothesis we screened the 5-HT3 receptor gene for mutations using standard SSCP techniques. Two novel polymorphisms, 178-T/C and 1596-A/G, were detected after screening 50% of the coding region. These polymorphisms were genotyped in a sample of 169 clozapine treated patients, including responders and non-responders, and 102 controls. However, no association was found between these allelic variants and schizophrenia or clozapine response. These results suggest that it is unlikely that genetic variants in this receptor gene are involved in the aetiology of schizophrenia or in determining drug response. However, the rest of the coding region and the promoter region of the gene have yet to be investigated.
SEROTONIN TRANSPORTER GENE POLYMORPHISM AND SCHIZOPHRENIA: AN ASSOCIATION STUDY F. Bonnet-Brilhault, C. Laurent, F. Thibaut, D. C a m p i o n , M. Martinez, M. Petit, J. Mallet Groupe de recherche sur la schizophr(;nie, CH du Rouvray, 76301 Sotteville les Rouen, France Abnormalities in monoamine metabolism, including serotonin metabolism, have been implicated in the pathophysiology of schizophrenia and the response to atypical neuroleptics corroborates this implication. Serotonin transporter protein (SERT) allows neurons to retrieve serotonin that has been released into a synapse and is a site of action for several drugs with central nervous system effects. Therefore the SERT gene is a strong candidate locus for aetiological involvement in schizophrenia. Ogilvie et al. (1996) reported a significant association between a variable-number-tandem repeat polymorphism in intron 2 of this gene and unipolar depression. We analysed this VNTR polymorphism for allelic and genotypic association with schizophrenia using 105 DSMIII-R schizophrenic patients and 114 controls all from Normandy. No significant differences in the distributions of alleles (Z== 1.47, dr=2, NS) or genotypes (;(-'=2.66, d[=3, NS) between schizophrenic patients and controls were found. Our data do not support a major role for the SERT gene in the etiology of schizophrenia. However, because studies of serotonin dysfunction as a possible pathogenic mechanism in schizophrenia have produced conflicting results, further investigations are needed.
ASSOCIATION OF THE BAN I DIMORPHIC SITE AT THE HUMAN CYTOSOLIC PHOSPHOLIPASE A2 GENE WITH SCHIZOPHRENIA K.H. Lee ~, C.N. R a m c h a n d , J. Wei, S.D. Telang, G.K. Vankar, S. Shah, M. Peet University Department qf Psychiatry. Northern General Hospital. Herries Road, She~eld $5 7A U, UK There is convincing evidence that phospholipid metabolism is abnormal in patients suffering from schizophrenia. Cytosolic phospholipase A2 (cPLA2) has been shown to be elevated in schizophrenia. We have screened polymorphic sites at the human cPLA2 gene using restriction fragment length polymorphism (RFLP) analysis. Thirty six DSM IV schizophrenic patients and twenty-seven unrelated healthy subjects were investigated. The sequence spanning from 5'-flanking region to 3' end of the first intron of cPLA2 gene (EMBL accession No U11239) was amplified by using a PCR with a pair of primers. The PCR products were respectively digested with the following restriction enzymes: Taq I, Rsa I, Hae Ill, Hha I, BamH I, Pst 1, Dra I, Alu I, Msp I and Ban I. Ban I digestion gave a dimorphic site with two individual alleles, AI and A2. The alMic frequencies were 0.305 (AI) and 0.694 (A2) in patients and 0.481 (A1) and 0.536 (A2) in controls (Z2=4.05, d f = l . p=0.44). The goodness-of-fit test demonstrated that genotypic frequencies among both patients and controls were in Hardy-Weiberg equilibrium. Moreover schizophrenic patients had an excess of A2/A2 homozygotic genotype as compared with controls. (Z2-5.05, dr= 1, p = 0.025). These results suggest that schizophrenia may involve a recessive locus in linkage disequilibrium with the Ban I dimorphic site at the human cPLA2 gene.
GENETIC DIFFERENCES IN PHOSPHOLIPASE A2 IN CONTROL SCHIZOPHRENIC POPULATION
AND
C.N. R a m c h a n d ~, J. Wei, K.H. Lee, S.D. Telang, L.J. Parekh, M. Peet UniversiO' Department q/Psychiatl3', Northern General Hospital, Herries Road. Sheff4eld $5 7A U, UK Optimal performance of receptors, enzymes, ionic channels and the proteins involved in transduction mechanisms requires a specific three dimensional conformation. This not only depends on the amino acid sequence of the proteins, but also on the composition of the environment, cell membranes which are predominantly made up of phospholipids. Recent studies have shown that membrane phospholipid composition as well as Calcium independent phospholipase A2 is altered in schizophrenia. Based on these evidence Hudson et al. investigated the
XI. Genetics A. B. C. D.
Candidate Genes Other Molecular Studies Correlated Phenotypes Miscellaneous
A. Candidate Genes A POLYMORPHISM IN THE PROMOTER REGION OF THE DOPAMINE D2 RECEPTOR GENE (DRD2) AND DRUG RESPONSE: ASSOCIATION STUDIES M.J. Arranz, J, M u n r o , T. Li, D.A. Collier, G. Kirov, R.W. Kerwin
Department of P,s3'ehologieal Medicine, Institute o[' Psychiato,, London SE5 8AF, UK The dopamine hypothesis of schizophrenia is supported by strong evidence which shows alterations of the dopaminergic system in schizophrenia patients. Classical antipsychotics display high affinities for dopamine receptors, specifically for the dopamine D2 receptors, in contrast with atypical antipsychotics. However, a recent study (Pilowsky et al., 1997) found that the atypical antipsychotic clozapine shows similar levels of occupancy of D2 receptors than typical antipsychotics in the frontal cortex. Dopamine receptor genes are strong candidates for involvement in the aetiology of schizophrenia and clinical treatment of the illness. However, analysis of a variety of polymorphisms in the dopamine receptor genes have not provided strong associations, with the exception of a small relative risk associated with the dopamine D3 receptor gene (DRD3). In addition several studies have failed to find association between genetic variants in dopamine receptor genes and clinical response to antipsychotics (Shaikh, 1995). Recently, a polymorphism in the promoter region of the dopamine D2 receptor gene (DRD2). a deletion of a cytosine at position -141, was reported to be associated with schizophrenia (Arinami et al., 1997). We tried to replicate these results in a sample of 143 clozapine treated patients and 100 controls. However, no significant differences were found between the patient and control groups, and between patients who responded to the treatment and patients who failed to respond. These results suggest that it is unlikely that genetic variation in the D2 receptor gene plays a major role in the aetiology of schizophrenia or in clinical response to treatment.
FURTHER EVIDENCE FOR ASSOCIATION BETWEEN POLYMORPHISMS IN THE 5-HT2A RECEPTOR GENE AND CLOZAPINE RESPONSE M.J. Arranz, J. M u n r o , D.A. Collier, G. Krov, P. Sham, J. Z h a o , R.W. Kerwin
Department o( Po'chological Medieine, Institute o[ Psyehiato,, London SE5 8AF, UK Serotonin receptors are targeted by a variety of antipsychotic drugs, particularly those termed atypical antipsychotics such as clozapine. We hypothesised that genetic variation in these receptors could affect clinical response to drugs targeting them by altering the receptor binding affinities, function or expression. In support of this hypothesis, we found association between two polymorphisms in the 5-HT2A receptor gene, 102-T/C and His452Tyr, and clinical response in a sample of clozapine treated patients (Arranz et al., 1995, 1996). The CI02 and Tyr452 variants were more frequent among patients who did not respond satisfactorily to clozapine. Several groups tried to replicate the association between 102-T/C and clozapine response but failed to find statistically significant differences. Significant association between His452Tyr and clozapine response was also found by Badri and collaborators, although two other studies failed to find significant differences. To clarify these results, meta-analyses including all published studies and an independent sample collected at the Institute were performed. Significant associations were found between clozapine response and 102-T/C genotype ()~2=9.05, p=0.011 and allele frequencies ( 7.2 = 7.13, p = 0.008, O.R. = 1.36). This association was stronger when considering allele C102 recessive (Z2= 8.49, p=0.004, O.R.= 1.64). Strong association was also observed between allele Tyr452 and poor response to clozapine (f2= 6.97, p=0.008, O.R.=1.69) and between homozigosity for Tyr452 and poor clozapine response ( Z 2 = 4.25, p = 0.04, O.R. = 5.55). These results provide further evidence of the importance of the 5-HT2 receptors as therapeutic targets and of the influence of genetic variation in determining clinical response to antipsychotics.
GENETIC VARIATION IN THE 5-HT3 RECEPTOR GENE: NO ASSOCIATION WITH SCHIZOPHRENIA OR DRUG RESPONSE M.J. Arranz, J. M u n r o , D.A. Collier, G. Kirov, R.W. Kerwin
Department ~]'Psychoh~gieal Medicine, Institute o! P,~Tehiato,, London SE5 8AF, UK Serotonin ( 5-HT ) receptors are strong candidates for involvement in mental disorders such as depression, eating disorders, sexual behaviour and schizophrenia and are targets of atypical antipsychotic drugs. 5-HT3 is the only type of serotonin receptors which is ligand-gated ion channel. 5-HT3 receptors
128
are candidate targets for the treatment of anxiety, depression, schizophrenia and dementia (Greenshaw, 1993). In addition, there is evidence suggesting that 5-HT3 receptors play a role in the therapeutic action of clozapine (Wang et al., 1994; Hermann et al., 1996). We hypothesised that genetic variation in 5-HT3 receptor may be involved in the aetiology of schizophrenia and in determining clinical response to clozapine. To test this hypothesis we screened the 5-HT3 receptor gene for mutations using standard SSCP techniques. Two novel polymorphisms, 178-T/C and 1596-A/G, were detected after screening 50% of the coding region. These polymorphisms were genotyped in a sample of 169 clozapine treated patients, including responders and non-responders, and 102 controls. However, no association was found between these allelic variants and schizophrenia or clozapine response. These results suggest that it is unlikely that genetic variants in this receptor gene are involved in the aetiology of schizophrenia or in determining drug response. However, the rest of the coding region and the promoter region of the gene have yet to be investigated.
SEROTONIN TRANSPORTER GENE POLYMORPHISM AND SCHIZOPHRENIA: AN ASSOCIATION STUDY F. Bonnet-Brilhault, C. Laurent, F. Thibaut, D. C a m p i o n , M. Martinez, M. Petit, J. Mallet Groupe de recherche sur la schizophr(;nie, CH du Rouvray, 76301 Sotteville les Rouen, France Abnormalities in monoamine metabolism, including serotonin metabolism, have been implicated in the pathophysiology of schizophrenia and the response to atypical neuroleptics corroborates this implication. Serotonin transporter protein (SERT) allows neurons to retrieve serotonin that has been released into a synapse and is a site of action for several drugs with central nervous system effects. Therefore the SERT gene is a strong candidate locus for aetiological involvement in schizophrenia. Ogilvie et al. (1996) reported a significant association between a variable-number-tandem repeat polymorphism in intron 2 of this gene and unipolar depression. We analysed this VNTR polymorphism for allelic and genotypic association with schizophrenia using 105 DSMIII-R schizophrenic patients and 114 controls all from Normandy. No significant differences in the distributions of alleles (Z== 1.47, dr=2, NS) or genotypes (;(-'=2.66, d[=3, NS) between schizophrenic patients and controls were found. Our data do not support a major role for the SERT gene in the etiology of schizophrenia. However, because studies of serotonin dysfunction as a possible pathogenic mechanism in schizophrenia have produced conflicting results, further investigations are needed.
ASSOCIATION OF THE BAN I DIMORPHIC SITE AT THE HUMAN CYTOSOLIC PHOSPHOLIPASE A2 GENE WITH SCHIZOPHRENIA K.H. Lee ~, C.N. R a m c h a n d , J. Wei, S.D. Telang, G.K. Vankar, S. Shah, M. Peet University Department qf Psychiatry. Northern General Hospital. Herries Road, She~eld $5 7A U, UK There is convincing evidence that phospholipid metabolism is abnormal in patients suffering from schizophrenia. Cytosolic phospholipase A2 (cPLA2) has been shown to be elevated in schizophrenia. We have screened polymorphic sites at the human cPLA2 gene using restriction fragment length polymorphism (RFLP) analysis. Thirty six DSM IV schizophrenic patients and twenty-seven unrelated healthy subjects were investigated. The sequence spanning from 5'-flanking region to 3' end of the first intron of cPLA2 gene (EMBL accession No U11239) was amplified by using a PCR with a pair of primers. The PCR products were respectively digested with the following restriction enzymes: Taq I, Rsa I, Hae Ill, Hha I, BamH I, Pst 1, Dra I, Alu I, Msp I and Ban I. Ban I digestion gave a dimorphic site with two individual alleles, AI and A2. The alMic frequencies were 0.305 (AI) and 0.694 (A2) in patients and 0.481 (A1) and 0.536 (A2) in controls (Z2=4.05, d f = l . p=0.44). The goodness-of-fit test demonstrated that genotypic frequencies among both patients and controls were in Hardy-Weiberg equilibrium. Moreover schizophrenic patients had an excess of A2/A2 homozygotic genotype as compared with controls. (Z2-5.05, dr= 1, p = 0.025). These results suggest that schizophrenia may involve a recessive locus in linkage disequilibrium with the Ban I dimorphic site at the human cPLA2 gene.
GENETIC DIFFERENCES IN PHOSPHOLIPASE A2 IN CONTROL SCHIZOPHRENIC POPULATION
AND
C.N. R a m c h a n d ~, J. Wei, K.H. Lee, S.D. Telang, L.J. Parekh, M. Peet UniversiO' Department q/Psychiatl3', Northern General Hospital, Herries Road. Sheff4eld $5 7A U, UK Optimal performance of receptors, enzymes, ionic channels and the proteins involved in transduction mechanisms requires a specific three dimensional conformation. This not only depends on the amino acid sequence of the proteins, but also on the composition of the environment, cell membranes which are predominantly made up of phospholipids. Recent studies have shown that membrane phospholipid composition as well as Calcium independent phospholipase A2 is altered in schizophrenia. Based on these evidence Hudson et al. investigated the