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Keywords:

  • schizophrenia;
  • 5-HT3A receptor;
  • clozapine;
  • SNP;
  • genotyping

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION AND PERSPECTIVES
  7. REFERENCES

The serotonin type 3 (5-HT3) receptor (R) belongs to the family of ligand-gated ion channels. It is supposed to play an important role in the pathogenesis of schizophrenia and might also represent an interesting target for the pharmacological treatment of this disorder. In this study, we searched for variations within the 5-HT3A receptor gene which might be specifically associated to schizophrenia. Twenty-nine single nucleotide polymorphisms (SNPs) of 943 schizophrenic patients compared to 2,343 healthy individuals were analyzed. SNPs were selected taking into account previous results on a 5-HT3A receptor domain involved in neuroleptic binding. Dominant logistic and linear regression models were calculated for the phenotypes number of hospitalizations, duration of hospitalization, age at onset and case–control. The data did not show significant associations of any SNP under investigation specific for schizophrenic patients. In conclusion, our study does not support the hypothesis that the 5-HT3A receptor plays a major role in the pathogenesis of schizophrenia. © 2012 Wiley Periodicals, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION AND PERSPECTIVES
  7. REFERENCES

Schizophrenia is a young-adult onset mental disorder which is highly disabling and affects about 1% of the population [Awad and Voruganti, 2008; Ibrahim and Tamminga, 2011]. It is characterized by hallucinations, delusions, cognitive impairment and poverty of emotions accompanied by social withdrawal. A dysregulation of the dopaminergic neurotransmitter system seems to play an important role in the development of schizophrenia [Abi-Dargham and Moore, 2003; Bertolino and Blasi, 2009]. In the treatment of this disorder, antipsychotic drugs are used which mostly antagonize dopamine at the type 2 (D2) receptor in the brain. An exception is the atypical neuroleptic clozapine which shows higher affinity to the D4 receptor [Brunello et al., 1995]. Furthermore, clozapine has been shown to exert antagonistic potency also at other neurotransmitter systems, such as serotonin (5-HT) receptors [Brunello et al., 1995; Rammes and Rupprecht, 2007].

Within the family of serotonin receptors, the 5-HT3 receptor is a pentameric non-selective ligand-gated cation channel [Maricq et al., 1991]. It exists either as homomeric 5-HT3A or as heteromeric 5-HT3AB receptor [van Hooft and Vijverberg, 2000; Hannon and Hoyer, 2008]. Functionally, 5-HT3A homomers appear to be more relevant, because most of the native 5-HT3 receptor complexes do not contain the 5-HT3B subunit [Hussy et al., 1994; Fletcher and Barnes, 1998; Reeves and Lummis, 2006]. Within the central nervous system (CNS), 5-HT3 receptors show high expression in the area postrema, the caudate nucleus, the hippocampus and the amygdala [Barnes and Sharp, 1999]. They modulate dopaminergic activity in mesolimbic and nigrostriatal pathways [Mylecharane, 1996; Barnes and Sharp, 1999].

The competitive antagonism of clozapine at 5-HT3A receptors might contribute to its antipsychotic effects as well as its side effect profile [Rammes et al., 2004]. Moreover, from a therapeutic point of view, it might exemplarily support the serotonin–dopamine hypothesis of schizophrenia [Kuroki et al., 2008]. To investigate the structural requirements of the binding site for clozapine, we recently constructed chimeras with different 5-HT3A receptor sequences of murine and human origin, which we expressed in human embryonic kidney (HEK) 293 cells [Rammes et al., 2009]. These investigations revealed that an extracellular sequence (length 86 aa) close to the transmembrane domain M1 appears to determine the binding affinity of clozapine to a high degree [Rammes et al., 2009].

Individual differences in the primary sequence within the 5-HT3A receptor gene might result in alterations of antipsychotic potency and side effect profile of neuroleptics, which is highly relevant in clinical practice. In this study, we compared the frequencies of genetic variations within the 5-HT3A receptor between schizophrenic patients and healthy volunteers to figure out if there are schizophrenia specific alterations that might in a second step be of relevance for neuroleptic treatment. Furthermore we analyzed if one of the schizophrenia specific phenotypes like number of hospitalizations, duration of hospitalization, age at onset and case–control is associated with one of the genetic variants.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION AND PERSPECTIVES
  7. REFERENCES

Schizophrenic Patients

Nine hundred forty-three patients suffering from schizophrenia were ascertained from the Munich area in Germany (for details see Table I). 71.85% of them were of German descent (i.e., both parents were German), 28.15% were other Caucasians from middle Europe. All patients were classified as schizophrenia according to ICD-10. Detailed medical and psychiatric histories were collected, including the Structured Clinical Interview for DSM-IV (SCID), to evaluate lifetime Axis I and II diagnoses [Spitzer et al., 1992; Williams et al., 1992]. SCID interviews were rated by four physicians and one psychologist. All measurements were double-rated by a senior researcher. Exclusion criteria included a history of head injury or neurological diseases. Furthermore, a patient was not eligible for inclusion if they had been diagnosed with schizoaffective disorder or as an intravenous drug user with a lifetime diagnosis of dependency. All participants of the study were outpatients or stable in-patients.

Table I. Characteristics of Schizophrenic Patients and Healthy Controls
 Schizophrenic patientsHealthy controls
SexTotal (943): male (591) and female (352)Total (2,343): male (1,139) and female (1,204)
Age (years; mean ± SD)37.78 (±11.77)51.78 (±15.57)
Diagnosis
 Paranoid type78.43% 
 Disorganized type14.98% 
 Catatonic type2.66% 
 Undifferentiated3.93% 
Educational level
 Low (secondary school)41.83%25.32%
 Middle (junior high school)25.47%32.69%
 High (general qualification for university entrance)32.70%41.99%
Age of onset (years; mean ± SD)23.67 (±8.69) 
Total number of inpatient admissions (mean ± SD)5.68 (±6.12) 
Total time of inpatient hospitalization (months; mean ± SD)12.89 (±16.20) 
Schizophrenia diagnosis in first-degree relatives19.72% 
Schizophrenia diagnosis in relatives in general29.64% 

Healthy Controls

Unrelated volunteers of German descent (i.e., both parents German) were randomly selected from the general population of Munich, Germany and were contacted by mail (for details see Table I). To exclude subjects with central neurological diseases and psychotic disorders or subjects who had first-degree relatives with psychotic disorders, the following screenings were conducted: First, subjects were initially screened by phone for the absence of neuropsychiatric disorders. Second, detailed medical and psychiatric histories were assessed for both themselves and their first-degree relatives by using a semi-structured interview. Third, if no exclusion criteria were fulfilled, they were invited to a comprehensive interview including the SCID I and SCID II [Spitzer et al., 1992; Williams et al., 1992] to validate the absence of any lifetime psychotic disorder. Additionally, the Family History Assessment Module [Rice et al., 1995] was conducted to exclude psychotic disorders among their first-degree relatives. Furthermore, a neurological examination was made to exclude subjects with current CNS impairment. In the case that the volunteers were older than 60 years, the Mini Mental Status Test [Cockrell and Folstein, 1988] was performed to exclude individuals with cognitive impairment. Finally, 2,343 control subjects were included in the study.

Written informed consent was obtained from all participants of the study after a detailed and extensive description of the study, which was approved by the local ethics committee and carried out in accordance to the ethical standards laid down in the Declarations of Helsinki.

One sample was genotyped with MALDI-TOF (MALDI; 3278 individuals), the other sample was genotyped using the Illumina HumanHap300 BeadChip (GWA; 1,167 individuals). One thousand one hundred fifty-nine of them were part of both samples.

Genotyping (MALDI)

DNA extraction was done with the QIAamp Blood Maxi Kit (QIAamp DNA Blood Midi/Maxi Handbook, Qiagen, Hilden, Germany, 2005). DNA concentration was adjusted using the PicoGreen quantitation reagent (Invitrogen, Karlsruhe, Germany). Single nucleotide polymorphisms (SNPs) were selected from the NCBI SNP database (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=snp), and pubmed publications (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed). Due to previous results by our group on the 5-HT3A receptor [Rammes et al., 2009] SNPs were preferentially selected in chimeric regions 1, 2, and 3. Chimera 1 contained the 5′-untranslated region and the four first exons and introns of the 5-HT3A receptor gene. In this region, four SNPs were genotyped. Chimera 2 (ten SNPs genotyped) covered a large part of intron 5 and the exon 6. Further four exonic SNPs were selected in the chimeric region 3 that encompassed the extracellular sequence close to transmembrane domain M1, which appeared to determine the binding affinity to clozapine [Rammes et al., 2009]. Moreover, haplotype tagging SNPs were selected using the “Tagger” algorithm to cover the haplotype block structure of the 5-HT3A receptor (http://www.broad.mit.edu/mpg/tagger/server.html). Altogether 29 SNPs were included covering ∼14.5 kb of the 5-HT3A receptor gene (Chr11: 113351216–113365953 position, NCBI Genome Built 36.3) with a mean distance of 508 bp per SNP (for details see Table II). DNA (12.5 ng) was genotyped using the iPLEX assay on the Mass ARRAY MALDI-TOF mass spectrometer (SEQUENOM, San Diego, CA). Genotyping call rates in cases and controls were >99%. Allele frequencies were similar to CEU sample frequencies (www.hapmap.org). All SNPs were in Hardy–Weinberg equilibrium (HWE) (P < 0.05).

Table II. Characterization of SNPs Under Investigation
SNP IDChromosome positionMinor alleleMajor alleleMAFFunctionRegiona
  • SNPs that did not enter the analysis (MAF < 0.01) are typed in bold.

  • a

    Chimeras of the 5-HT3A receptor according to Rammes et al. [2009]. In this study, five different receptor chimeras consisting of different murine and human sequences were constructed. These chimeric receptors contained the sequence between the amino terminus (=restriction site HindIII) and restriction site of BstEII (sequence 1) and the sequence between restriction site BstEII and SgrA1 (sequence 2). Sequence 1 and 2 together belong to the extracellular domain and form the ligand binding site. The sequence between restriction site of SgrA1 and the carboxyl-terminal domain (=restriction site XhoI; sequence 3) belongs to the transmembrane and cytoplasmatic domain. Chimeras were a combination of these three different sequences.

rs7942029113329075GA0.190Intergenic 
rs1176754113332860CT0.127Intergenic 
rs1150229113338362CT0.484Intergenic 
rs1176752113348687AC0.078Intergenic 
rs1150226113350751TC0.088Intergenic, near 5′ 
rs1062613113351216TC0.226Exon 1, 5′-UTRChim 1
rs2276302113355350GA0.326Intron 3Chim 1
rs10891611113356623CT0.160Intron 3Chim 1
rs3737457113358909AG0.011Intron 4Chim 1
rs35448518113359306CT0.114Intron 5Chim 2
rs909412113359775GA0.001Intron 5Chim 2
rs11214796113359889CT0.221Intron 5Chim 2
rs1176718113359900TG0.002Intron 5Chim 2
rs1176715113360616TC0.219Intron 5Chim 2
rs897687113361021CT0.002Intron 5Chim 2
rs2846632113361749AG0.053Intron 5Chim 2
rs10160548113361891GT0.328Intron 5Chim 2
rs72466467113361969TG0.001Exon 6, F189LChim 2
rs34327364113361978AG0.021Exon 6, L192LChim 2
rs4938063113362502AGNon-polymorphicExon 7, N253SChim 3, M1
rs35944954113365599TCNon-polymorphicExon 9, S447SChim 3
rs1176713113365635CT0.220Exon 9, L459LChim 3, M4
rs1150219113365953CG0.002Exon 9, 3′-UTRChim 3
rs897685113369319GA0.114Intergenic 
rs1379170113371761AG0.337Intergenic 
rs4938066113390622GA0.335Intergenic 
rs7118530113395335CT0.352Intergenic 
rs12221649113395563CT0.122Intergenic 
rs6589402113407969GT0.230Intergenic 

Genotyping (GWA)

DNA extraction and adjustment of concentration was performed as described above. The samples were genotyped using the Illumina HumanHap300 BeadChip according to the manufacturer's specifications (Illumina, San Diego, CA). Several quality checks for genome wide studies, like check for duplicates, gender or batch clusters were performed [for details, see Stefansson et al., 2008]. SNPs (266,382) passed the quality control with the following filters: (i) individual call rate >95%, (ii) SNP call rate >99%, (iii) minor allele frequency (MAF) > 0.01 deviation from HWE in controls at P > 10−06. For the chromosomal region of interest between the positions Chr11: 113329075–113407969 (NCBI Genome Built 36.3) 13 SNPs were selected. HWE for these 13 SNPs was P > 0.05.

Statistical Analysis

Sociodemographic parameters were tested using Chi-square-, T-, or ANOVA-tests as appropriate. Quality checks for the chip genotypes were done with PLINK (http://pngu.mgh.harvard.edu/∼purcell/plink/). Dominant logistic and linear regression models were calculated in R (www.r-project.org/) for case–control studies and analyses of age at onset, duration and number of hospitalizations, respectively. Duration and number of hospitalizations were log-transformed to meet the assumptions of linear regression. Furthermore age at onset was included as covariate for the analysis of these two phenotypes.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION AND PERSPECTIVES
  7. REFERENCES

Detailed characteristics of schizophrenic patients and healthy controls are provided in Table I. Out of 29 SNPs of the 5-HT3A receptor, only those with MAFs > 0.01 were selected to enter the analysis (Table II). The genotype distribution of all SNPs under investigation did not show any significant deviation according to the Hardy–Weinberg equilibrium (P > 0.05). Four SNPs showed weak statistical association with one of the four phenotypes (rs34327364 to age at onset of disease, rs6589402 to case–control, rs1062613 to duration of hospitalizations in months and rs1176752 to number of hospitalizations) (Fig. 1). However, the analysis revealed no association of any SNP under investigation to schizophrenic patients or rather the three linear phenotypes that survived Bonferroni correction (P < 0.002) (Fig. 1). Furthermore, we tested for association between controls and patients with schizophrenia in first-degree relatives, but also here we found no association.

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Figure 1. Single SNP analysis.

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DISCUSSION AND PERSPECTIVES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION AND PERSPECTIVES
  7. REFERENCES

The identification of specific key mutations in schizophrenia is essential for a better understanding of its pathophysiology and for the improvement of treatment options. Although schizophrenia is basically believed to be a dysfunctional disorder of the dopaminergic system [Sayed and Garrison, 1983; Heinz and Schlagenhauf, 2010], the 5-HT3 receptor has also attracted considerable interest in this context. 5-HT3 receptor antagonists (such as ondansetron) have been shown to exert beneficial effects on schizophrenic symptoms, especially on cognitive impairment and negative symptoms [Adler et al., 2005; Levkovitz et al., 2005; Akhondzadeh et al., 2009]. Moreover, antipsychotic drug response has been associated at least in part with variabilities in 5-HT3 receptor genes which indirectly implies a role of this receptor in the pathogenesis of schizophrenia. For example, Souza et al. [2010] found an association of clozapine response in schizophrenic patients for rs1062613 in the 5-HT3A receptor gene. A number of other studies revealed associations of further 5-HT3A receptor gene polymorphisms with classical, as well as atypical neuroleptic drug response or treatment resistance [Gu et al., 2008; Ji et al., 2008; Schuhmacher et al., 2009].

The recent identification by our group of a sequence within the 5-HT3A receptor which determines the binding affinity of clozapine [Rammes et al., 2009] provided further support that the 5-HT3A receptor might be an interesting target to search for putative mutations involved either in the pathophysiology of schizophrenia or its pharmacological treatment. Therefore we investigated 29 SNPs within the 5-HT3A receptor of 943 schizophrenic patients compared to 2,343 healthy controls. We could not find any significant associations of the SNPs under investigation specific for schizophrenia. Particularly the 5-HT3A receptor domain involved in clozapine binding recently identified by Rammes et al. (Table II, SNP rs4938063) could not be associated to schizophrenia because it was non-polymorphic in our sample. We only found a slight, although not significant association to the duration of hospitalization for this SNP. These results confirm previous investigations by Niesler et al. [2001], which suggested only a minor role of the 5-HT3A receptor in the etiology of schizophrenia due to the lack of sequence variations in this gene. However, the fact that there are no specific genetic variations of the 5-HT3A receptor associated with schizophrenia compared to healthy controls does not rule out an association with clozapine response and/or tolerance within a sample of schizophrenic patients.

The figure represents significance levels (−log10(P-values)) of single SNPs and the respective linkage disequilibrium. Symbols indicate P-values for different phenotypes: Number of hospitalizations (number), duration of hospitalization (duration: sum of all admissions duration), age at onset (aao) and case–control (case/control), respectively. The line represents the significance threshold for P = 0.05. The figure was created with the snp.plotter package in R [Luna and Nicodemus, 2007].

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION AND PERSPECTIVES
  7. REFERENCES