Correspondence: Chul Lee, MD, Department of Psychiatry, Kangnam St. Mary' Hospital, The Catholic University of Korea College of Medicine, 505 Banpo-dong, Seocho-gu, Seoul 137-701, Korea
Tel: +82-2-590-2766, Fax: +82-2-536-8744, E-mail: knpsy@catholic.ac.kr

Introduction

Serotonin is related to some of the symptoms that are frequently detected in schizophrenics such as emotional problems, anxiety, obsession, aggression and sleep disturbances¹, and lysergic acid diethylamide has been reported to induce hallucinatory behaviors in normal individuals as well as in schizophrenic patients². The atypical antipsychotic medications have a higher occupancy rate of the serotonin receptor than for the dopamine receptor, and this has brought more attention on the influence of aberrations of the serotonin system for the development of schizophrenia³.
The 5-HTT gene (SLC6A4) is located on chromosome 17q11.1-q12, and it was recently found to be linked to the susceptibility to schizophrenia⁴. The two main 5-HTT gene polymorphisms have been reported as following; a 17bp variable number of tandem repeats polymorphism in the intron 2 region and a 44 bp insertion/deletion polymorphism (5-HTTLPR) in the promotor region⁵. For the latter, the long (l) and short (s) alleles of the 5-HTTLPR polymorphism can differentially modulate the transcription of the 5-HTT gene: the s allele of the 5-HTTLPR polymorphism reduces the transcriptional efficiency of the 5-HTT gene, resulting in decreased 5-HTT expression and 5-HT uptake⁶. The l/l homozygotes were found to have higher rate of 5-HTT mRNA transcription and 5-HT uptake than those subjects having only one copy of the s allele⁷. Recently, some studies have reported that 5-HTTLPR polymorphism was related to the positive symptoms of schizophrenia and the response to clozapine treatment^8,9,10, whilst other studies have not replicated these results^11,12, and this indicates there are conflicted results for the involvement of 5-HTTLPR polymorphism on the development and symptomatology of schizophrenia.
This study was conduced to investigate the possible contribution of 5-HTTLPR polymorphism on the development and clinical symptomatology of patients with schizophrenia.

Methods and Materials

Subjects
One hundred and fifty-two schizophrenic patients and 152 control subjects participated in this study. The diagnosis of schizophrenia was made by consensus between two board-certified psychiatrists according to the Diagnostic and Statistical Manual of Mental Disorders-IV (DSM-IV)¹³. The Structured Clinical Interview for DSM-IV Axis I Disorders-Clinician Version¹⁴ was administered to all the patients. The other available resources such as the patients' clinical course, family information and medical records were also used. Those patients with a diagnosis of other mental or neurological disorders other than schizophrenia were excluded from the study. The family history of schizophrenia in the first-degree and second-degree relatives was collected from the chart review.
The control group consisted of volunteers from the paramedical- and medical staff or they were chosen from students who showed no family or personal history of major mental disorders and neurological diseases. The control group was also evaluated by two board certified-psychiatrists to exclude any control subject with a current or a past history of psychiatric problems. A written informed-consent form was obtained from all the subjects after they were given a full description of the study. The Ethics Committee of the Kangnam St. Mary's Hospital approved this study.

Assessment of the antipsychotic treatment response
The Positive and Negative Syndrome Scale (PANSS)¹⁵ was evaluated to examine the antipsychotic treatment response at the time of admission and at 8 weeks after the administration of antipsychotic medicine. Risperidone (n=71) was the most frequently administered antipsychotic and then olanzapine (n=39), quetiapine (n=17), chlorpromazine (n=13) and haloperidol (n=12) followed in the order of usage. Chlorpromazine equivalent doses were described according to each polymorphism in Table 2. One hundred and thirty-one (86.2%) patients completed the 8-week antipsychotic treatment. Thus, the last data of 21 patients was obtained by the method of the last observation carried forward.

Genotyping with restriction Fragment length polymorphism
The DNA was extracted from whole blood of the patients by using the standard method. The forward and reverse primers were 5'-GGC GTT GCC GCT CTG AAT GC-3' and 5'-GAG GGG ACT GAG CTG GAC AAC CAC-3', respectively. Amplification of the polymorphic region was performed as described previously⁵.
484 bp and 528 bp fragments were determined after comparison with the DNA molecular marker XIV (100 bp ladder, Roche Molecular Biochemicals, Mannheim, Germany). The s and l alleles were assigned to the 484 bp and 528 bp fragments, respectively.

Statistical analyses
Differences in the alleles and the genotype frequencies of the 5-HTTLPR polymorphism between the schizophrenic patients and the controls were calculated using a chi-square test. Hardy-Weinberg equilibrium at each polymorphism was examined for by a goodness-of-fit Chi-square test. To compare the numerical variable such as age and the number of admissions, t-test and univariate analysis of variance were used where appropriate. The Monte Carlo method with the CLUMP program v 1.9 (10,000 simulations)¹⁶ was used when a small cell count was observed in the case of the categorical comparisons.
Multiple regression analysis was applied to detect the possible effects of the 5-HTTLPR polymorphism for its influences on the psychopathology such as the PANSS scores and the antipsychotic treatment response (a response was defined as a 20% decrease of PANSS score after the 8-week antipsychotic treatment compared to the PANSS score at the time of admission).
A P value less than.05 was considered significant. All statistical tests were performed using SPSS v10.0 software (SPSS Inc., Chicago, IL). The power of a sample to detect the differences between the alleles was calculated by considering a two tailed alpha value of 0.05. Using these parameters, the sample was estimated to have a power (0.80) to detect a small to medium effect size (w=0.16), which corresponded to a difference of approximately 15% for the two alleles between the two groups.

Results

Seventy-three (48.0%) of the patients with schizophrenia were male and 69 (45.4%) of the normal controls were male. No difference was present in the gender distribution between the two groups (p=0.730). The average age was older in the patient group (mean±SD, 36.9±11.6 years) than in the control group (32.0±10.7 years) (p<0.05).
  The genotype frequencies of the 5-HTTLPR polymorphism were not different from the expected values of the Hardy-Weinberg equilibrium in the patients group (p=0.931) and in the control group (p=0.333) (Table 1). The 5-HTTLPR polymorphism was not associated with the development of schizophrenia (Table 1). The description of the patients' characteristics, according to genotypes of the 5-HTTLPR polymorphism, is shown in Table 2. Subjects with the 5-HTTL-PR s allele were found to have a more frequently positive familial history [genotypes ss and ls, p=0.026, OR=2.698, 95% CI=1.187-6.116] (Table 2). The 5-HTTLPR polymorphism was not associated with the antipsychotic treatment response as a categorical variable (Table 2). The other clinical variables such as the onset age, a suicide history, the number of past hospital admissions and the duration of illness were not associated with the 5-HTTLPR polymorphism.

Discussion

The association between the 5-HTTLPR polymorphism and schizophrenia has shown mixed results, but the results have mainly been negative^{7,11,12,17,18,19,20,21,22}, and this is also in line with the present findings. This study found an association of the 5-HTTLPR s allele with a positive family history, suggesting that this polymorphism may be indicative of conferring a familial propensity for the development of schizophrenia.
Malhotra et al⁸ have recently reported that the l/l group was related to positive symptoms such as auditory hallucinations, while the other studies have not replicate this result^11,12,20. In relation to the 5-HTTLPR polymorphism with an antipsychotic treatment response, previous studies^9,11 have shown negative results, although it was found that subjects with the s allele had a tendency to be non-responders, and this was also observed in the present study. Ethnic differences for the 5-HTTLPR polymorphism, the small sample size, the methods of the studies and the recruited patients' characteristics may all have contributed to these discrepancies. In addition, a recent study could not detect any differential effect for variants of the 5-HTTLPR polymorphism on the maximum 5-HT uptake in human cells²³.
Different ethnic backgrounds for the polymorphism should be considered when analyzing the results of different studies^11,17. Our control subjects do not represent the Koreans in general, and case-control association studies have inherent pitfalls of stratification. More single nucleotide polymorphisms (SNPs) from the 5-HTT genes should provide more accurate information. Additionally, the multiple comparison issue should also be kept in mind. Finally, we should consider the small size of the subject samples with considering the conventional sample size that is needed in a case-control association study²⁰, and this is related to the possibility of obtaining a false negative finding with regard to the sample size.
These results suggest that the 5-HTTLPR polymorphism offers no significant contribution to the susceptibility for schizophrenia, and it is not associated with the clinical variables, except for family history, at least in the Korean population. Further studies with larger study populations and with assessing other critical SNPs should be performed to consider the biological background of the 5-HTT gene in relation to the pathogenesis of schizophrenia.

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