Pharacogenetic effects of dopamine transporter gene polymorphisms on response to chlorpromazine and clozapine and on extrapyramidal syndrome in schizophrenia

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Abstract

A number of studies have investigated the effectiveness of the dopamine transporter (SLC6A3) Gene as an antipsychotic target. However, the focus has mainly been on a 40-bp variable number of a tandem repeat (VNTR) in the 3′-region and results have been inconsistent. To fully evaluate SLC6A3 as a therapeutic antipshycotic target we investigated association of the gene with responses to chlorpromazine and clozapine and with chlorpromazine-induced extrapyramidal syndrome (EPS) in the Chinese schizophrenia population. Six polymorphisms across the whole region of this gene were analyzed, namely rs2652511 (T-844C) and rs2975226 (T-71A) in the 5′-regulatory region, rs2963238 (A1491C) in intron 1, a 30-bp VNTR in intron 8, rs27072 and the 40-bp VNTR in the 3′-region. We found that the polymorphic marker, rs2975226, showed significant association of allele and genotype frequencies with response to clozapine (allele-wise: adjusted p = 0.00404; genotype-wise: adjusted p = 0.024), and that patients with the T allele had a better response to the drug. The haplotype block constructed from the first three markers near the 5′-region showed significant association with response to clozapine (for haplotype T-T-A: p = 0.0085; for haplotype C-A-C: p = 0.0092). We did not identify any significant association of the six genetic variants or haplotypes with EPS after Bonferoni correction. Our findings suggest that the 5′-regulatory region of SLC6A3 plays an important role in response to clozapine and that its role in EPS needs to be replicated in a large-scale well designed study.

Introduction

Chlorpromazine and clozapine are benchmark typical and atypical antipsychotic agents which are frequently used as standards in antipsychotic drug trials (Carpenter and Koenig, 2008). Like other typical antipsychotics, chlorpromazine works primarily by blocking dopamine D2 receptors and this action is commonly believed to be responsible for controlling the symptoms of schizophrenia. The main pharmacological activities of clozapine include balanced serotonin and dopamine antagonism (Ase et al., 1999).

Variations in individual clinical response to antipsychotic agents and to antipsychotic-induced side effects have been widely reported (Xu et al., 2008, Zhang et al., 2007, Potkin et al., 2003, Kirchheiner et al., 2004, Kirchheiner et al., 2006, Arranz et al., 2000) .and much of this variability has been attributed to genetic factors (Kirchheiner et al., 2004, Arranz et al., 2000, 2007, 2008). Previous studies have indicated that the human dopamine transporter (SLC6A3) plays a critical role in controlling the spatial and temporal domains of dopamine neurotransmission through the accumulation of extracellular dopamine, and is also the major site of the action of psychostimulant drugs (Zhang et al., 2007, Mehler-Wex et al., 2006, Haddley et al., 2008). Although SLC6A3 does not bind chlorpromazine and clozapine directly, increased dopaminergic release and neurotransmission are known to cause psychotic disturbances, and antipsychotic drugs that block dopamine receptors are thought to relieve psychotic symptoms (Ase et al., 1999). For this reason, it has been suggested that patient response to antipsychotic drugs and side effects of the drugs vary according to SLC6A3 function.

The SLC6A3 gene is mapped on chromosome 5p15.3 with 15 exons separated by 14 introns spanning more than 65 kb. The protein-coding portion begins within exon 2 and ends near the beginning of exon 15 (Bannon et al., 2001). This coding region presents strong conservation and the polymorphisms within the coding region represent either silent nucleotide changes or rare conservative amino acid substitutions (Grünhage et al., 2000), indicating that individual differences in SLC6A3 expression must arise from regulatory sequences. Analyses of the SLC6A3 gene indicate a strong core promoter extending from − 251 to + 63 in the genomic sequence and suggest the presence of repressor elements in the 5′-upstream region and within intron 1 (Zhang et al., 2007, Greenwood & Kelsoe, 2003). Positive association of the single nucleotide polymorphism (SNP) A-67T with schizophrenia and bipolar disorder has been reported (Khodayari et al., 2004, Keikhaee et al., 2005, Talkowski et al., 2008). A 40-bp variable number of a tandem repeat (VNTR) polymorphism within the 3′-untranslated region (NCBI, dbSNP: rs28363170) has been widely studied in a number of dopamine-related neuropsychiatric disorders, including Parkinson's disease (PD), attention-deficit/hyperactivity disorder (ADHD), schizophrenia and bipolar disorder (Laucht et al., 2007, Yang et al., 2007, Waldman et al., 1997, Gamma et al., 2005). Of six studies of association between the 40-bp VNTR within the 3′-untranslated region of the SLC6A3 gene and response to atypical antipsychotics or antipsychotics-induced EPS in schizophrenia (Zhang et al., 2007, Szekeres et al., 2004, Kim et al., 2005, Lafuente et al., 2007, Güzey et al., 2007, Segman et al., 2003), five produced negative findings (Zhang et al., 2007, Szekeres et al., 2004, Kim et al., 2005, Lafuente et al., 2007, Segman et al., 2003). However, to date, no efficacy or side effect studies have investigated other polymorphisms. Recently another 30-bp VNTR located in intron 8 of the SLC6A3 gene has been reported to confer a functional effect in terms of altered gene expression and a robust association with ADHD and cocaine dependence (Brookes et al., 2006, Genro et al., 2008, Franke et al., 2008, Guindalini et al., 2006).

To systematically evaluate the importance of SLC6A3 both in the therapeutic response to chlorpromazine and clozapine and in chlorpromazine-induced EPS in the Chinese schizophrenia population, we performed three independent genetic analyses of polymorphisms across the whole region of the SLC6A3 gene. Six potential functional polymorphisms were investigated , namely rs2652511 (T-844C) and rs2975226 (T-71A) in the 5′-regulatory region, rs2963238 (Intron 1: A1491C) in the intron 1 region, a recently reported 30-bp VNTR in the intron 8 region, and rs27072 and the well-studied 40-bp VNTR in the 3′-region, 3 of which had been used to analyze the therapeutic response to risperidone in one of our previous publications (Zhang et al., 2007).

Section snippets

Patients and psychiatric assessment

In this study, a total of 320 unrelated schizophrenia patients (166 males and 154 females; mean ± SD age: 42.0 ± 13.1 years) were recruited from the Shanghai Mental Health Center based on the following inclusion criteria: (1) They had no physical complications or other psychiatric disorder; (2) Nothing in their medical history suggested that antipsychotics treatment would be ineffective; (3) All were inpatients and had been diagnosed according to the criteria of the Diagnostic and Statistical Manual

Clinical profile

Demographic and clinical characteristics of the patients eligible for analysis are shown in Table 1. The patients were divided into different groups based on the treatment outcomes. No significant differences were found between treatment outcome subgroups within each of the two antipsychotic treatment groups, based on either age, age of onset, number of hospitalizations, average applied dosage, plasma clozapine levels, plasma norclozapine levels or plasma chlorpromazine levels (P > 0.05). This

Discussion

Established association studies featuring SLC6A3, schizophrenia and antipsychotic treatment response have primarily focused on the 40-bp VNTR polymorphism in the 3′-untranslated region. Such studies have shown inconsistent results and a recent meta-analysis actually ruled out the possibility of association of this VNTR with schizophrenia (Gamma et al., 2005). Additionally, few studies on association of this VNTR with response to antipsychotics or antipsychotics-induced extrapyramidal symptom in

Acknowledgements

This work was supported by grants (2006AA02A407, 2009AA022701, 2010CB529600, 2007CB914703, 2007CB947300), the Shanghai Municipal Commission of Science and Technology Program (09DJ1400601), and the Shanghai Leading Academic Discipline Project (B205). We are grateful to all the participants as well as to the psychiatrists and mental health workers who participated in this project.

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    Mingqing Xu, Qinghe Xing, and Sheng Li contributed equally to this work.

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