Neuroscience Letters 453 (2009) 210–213 Haplotype analysis confirms association of the serotonin transporter (5-HTT)gene with schizophrenia in the Han Chinese population Chuwen Lin , Wei Tang , Jianxin Hu , Linhan Gao , Ke Huang , Yifeng Xu ,Guang He , Peiji Liang , Guoyin Feng , Lin He , Yongyong Shi a Bio-X Center, Shanghai Jiao Tong University, Shanghai, Chinab Institute of Neuropsychiatric Science and Systems Biological Medicine, Shanghai Jiao Tong University, Shanghai, Chinac Changning Institute of Mental Health, the Bio-X Center Hospital, Shanghai, Chinad Institute for Nutritional Sciences, SIBS, Chinese Academy of Sciences, Shanghai, Chinae Shanghai Institute of Mental Health, Shanghai, Chinaf School of Life Science and Technology, Shanghai Jiao Tong University, Shanghai, China Serotonin transmission has long been suspected as being involved in the pathogenesis of schizophre- nia. 5-HTT is a promising candidate gene for schizophrenia due to its critical role in regulating serotonin Received in revised form 10 February 2009 transmission and role in the mechanism of the atypical antipsychotic drugs. A common polymorphism STin2 VNTR in the 5-HTT gene has been extensively investigated in the genetic association studies, butthe results are conflicting. Meanwhile, the SNPs of the 5-HTT gene have been much less explored. We therefore conducted a case-control study of the association between STin2 VNTR and three tagging SNPs in 5-HTT and schizophrenia in the Han Chinese population based on a cohort of 329 schizophrenic patients and 288 control subjects. No association was found in the single locus, but haplotype-based anal- yses revealed significant association between two haplotypes with schizophrenia even after Bonferroni correction (P = 0.00000538 and 0.011).
2009 Elsevier Ireland Ltd. All rights reserved.
Schizophrenia (OMIM #181500) is a chronic, debilitating psychotic extensive interaction with other neurotransmitter systems espe- disorder, affecting 0.5–1.0% of the population worldwide with dev- cially the glutamate system In the molecular level, many astating consequences for affected individuals and their families second-generation antipsychotic drugs such as clozapin, olanzapin, genetic contribution to the etiology of schizophrenia has quietapine are 5-HT2 antagonists, whereas 5-HT receptor agonists been shown to be upward of 80%, however the pathogenesis is com- are psychotogenic n addition to acting as a neurotransmitter plex and the specific factors that give rise to the disorder remain in mature brain, 5-HT has a role in neural development Serotonin transporter (5-HTT, SLC6A4) is a promising candidate Serotonin (5-HT) transmission has long been suspected of being gene for schizophrenia. 5-HTT is the major regulator of sero- involved in the pathogenesis of schizophrenia ostmortem tonin concentration in the synaptic cleft, terminating the action brain tissue analysis and cerebrospinal fluid studies have identified of serotonin by uptaking serotonin from brain synapses into the a deficit of 5-HT function in the cortex of patients with schizophre- presynaptic neuron 5-HTT gene is mapped to chromo- nia plays an important role in many physiological some 17q11.1–q12, within the 17p11–q25 region which has been processes which are disturbed in schizophrenics, including cog- reported to be linked with schizophrenia Dysregulation of nition, mood, perception and attention Furthermore, it has 5-HTT has been reported in various complex behavioral traits andpsychiatric disorders abnormal expression of 5-HTT has been reported in the brains of schizophrenic patients And postmortem studies have shown decreased 5-HTT affinity in Corresponding author at: Institute for Nutritional Sciences, SIBS, Chinese the hippocampus in schizophrenics y, 5-HTT is the Academy of Sciences, 294 Taiyuan Road, Shanghai 200031, China.
Tel.: +86 21 62822491; fax: +86 21 62822491.
primary target of SSRIs which are used as a first-line treatment for ∗∗ Corresponding author at: Institute of Neuropsychiatric Science and Systems Biological Medicine, Changning Institute of Mental Health, the Bio-X Center Hospital, A lot of genetic association analyses between two common poly- 299 XieHe Road, Shanghai 200042, China. Tel.: +86 21 62822491; morphisms of 5-HTT have been carried out in different populations, E-mail addresses: (L. He), (Y. Shi).
5-HTTLPR is a 44-bp insertion/deletion polymorphism in the 5 reg- 1 These authors contributed equally to this work.
ulatory region, involving two major alleles, namely ‘S’ (short) and 0304-3940/$ – see front matter 2009 Elsevier Ireland Ltd. All rights reserved.
doi: C. Lin et al. / Neuroscience Letters 453 (2009) 210–213 Fig. 1. Diagram of the genomic structure of 5-HTT gene and the positions of the markers studied in the present work.
‘L’ (long) allele. STin2 VNTR is a 17-bp variable-number tandem- atric Association). The diagnosis was checked and verified by two repeats (VNTR) polymorphism, located in intron 2 and involving independent senior psychiatrists who reviewed the psychiatric case two major alleles, STin2.10 and STin2.12, which respectively cor- records. Written informed consent was obtained from either the respond to 10- and 12-repeat units of the 17-bp VNTR. Additional participants or participants’ relatives, after the procedure had been low frequency alleles have been identified including 7- and 9-repeat units. Functional studies on this polymorphism have shown that the Beside STin2, we included SNPs from the dbSNP database amount of 5-HTT protein is significantly higher in cells carrying the 12-repeat allele when activated by morphogens in devel- oping mouse brains STin2 acts as a transcriptional regulator in an Haploview ver. 2.05 software ee intronic and non-functional allele-dependent way many studies on the association SNPs, rs2054847, rs140700 and rs2020942 were expected to belong between 5-HTTLPR and STin2 and schizophrenia were reported, the to the STin2-containing haplotype block oreover, tagger SNP results have been conflicting recent progress analysis by Haploview showed they were among the tagger SNPs is a meta-analysis on these two polymorphisms finding signifi- of this block. Thus these three SNPs were chosen for the present cant association between STin2 VNTR and schizophrenia, while study. The positions of the chosen markers were shown in 5-HTTLPR polymorphism showed no association more recent Genomic DNA was prepared from venous blood using the stan- study failed to detect positive association between STin2 VNTR and dard phenol/chloroform method. For genotyping of STin2 VNTR, schizophrenia, but it showed that the haplotype consisting STin2 primers were: forward: 5 -FAM- TCAGTATCACAGGCTGCGAG-3 ; VNTR was associated with schizophrenia, while 5-HTTLPR did not reverse: 5 - TGTTCCTAGTCTTACGCCAGTG-3 . PCR were conducted belong to the same haplotype block efore, the haplotype in a final volume of 5 ␮l consisting of 10 ng DNA, 0.1 ␮l 10 ␮M block containing STin2 VNTR emerges as a candidate for risk factor primers, and 1 U HotStart Taq polymerase (Qiagen). MegaBACE 1000 capillary electrophoresis (Amersham Biosciences, Piscataway, On the other hand, the associations between single nucleotide NJ) were used to discern the genotype of 10- and 12-repeat of the polymorphisms (SNPs) of 5-HTT and schizophrenia have been much less explored compared with STin2 VNTR and 5-HTTLPR.
SNP genotyping was performed using ligase detection reaction.
In the present study, we attempt to evaluate the role of 5-HTT Fluorescence-labeled primer was employed to detect the genotype in the pathogenesis of schizophrenia using a case-control approach of the (GT)n repeat using MegaBACE 1000 capillary electrophoresis.
involving 329 patients and 288 healthy subjects drawn from the CLUMP 2.2 was used to compare the allelic, genotypic fre- Han Chinese population. STin2 VNTR and three SNPs of 5-HTT gene quencies and haplotype distribution between cases and controls (rs2054847, rs140700, and rs2020942) were investigated.
erroni correction was used for multiple testing. Analysis 329 unrelated schizophrenic patients (203 males, 126 females, of Hardy–Weinberg equilibrium and linkage disequilibrium were mean age 49.54 ± 11.48) and 288 unrelated healthy subjects (159 males, 129 females, mean age 37.02 ± 8.16) were recruited in this study. All the subjects were from Shanghai and were Han Chinese in All genotype frequencies of the three SNPs and STin2 VNTR were origin. All of the controls were interviewed to exclude any history in Hardy–Weinberg equilibrium (P > 0.05, We observed no of psychiatric disorder. Subjects with schizophrenia were strictly significant deviation in allelic or genotypic frequencies of any single diagnosed according to the criteria of DSM-IV (American Psychi- one for the four markers examined For the allele frequen- Table 1
Allelic and genotypic frequencies of four markers among schizophrenic patients and normal controls.
a Chi-square values or empirical P-values obtained using CLUMP (10,000 simulation).
c Test for departure from Hardy–Weinberg expectation in case and control groups using SHEsis.
C. Lin et al. / Neuroscience Letters 453 (2009) 210–213 with schizophrenia, suggesting that the 5-HTT region contributing Estimation of linkage disequilibrium among the four markers.
genetic risk to schizophrenia lay in the haplotype block containing STin2. This also strengthened the data of the meta-analysis In this study, we did not detect statistically significant associ- ation between STin2 VNTR and schizophrenia (P = 0.07) as did the meta-analysis, and this suggests that some allele combinations may constitute a risk genetic background, rather than any individual allele. Considering the P-value was <0.1% and the power was 66.0%at OR = 1.5, however, it could not be concluded that the lack of sig-nificant association is a negative result. It should be noted that a cies, the P-value was 0.94 and the odds ratio (OR) was 0.99 for both trend of increase in STin2 allele 12 frequencies was observed in rs2020942 and rs2054847; the P-value was 0.18 and the OR was schizophrenic patients tent with the result of the meta- 0.68 for rs140700; the P-value was 0.07 and the OR was 1.48 for analysis. This is also reminiscent of the studies of Liu et al. and STin2 VNTR. As to the genotype frequencies, the P-value of both Tsai et al. which reported marginal association of STin2 VNTR with rs2020942 and rs2054847 was 1.00, the P-value for rs140700 was schizophrenia in Chinese population, with an odds ratio of approxi- 0.42, and the P-value for STin2 VNTR was 0.10. The power of both mately 2.0 odds ratio of STin2 VNTR in our study is 1.48, rs2054847 and rs140700 was 78.9%, power of rs140700 was 30.8%, and this discrepancy may be also due to difference in sample size.
and power of STin2 VNTR was 66.0% at the level of OR = 1.5.
Thus, a larger sample size may be required for a conclusive result on ws the pair-wise linkage disequilibrium (LD) among association between STin2 VNTR and schizophrenia. Finally, while each pair of markers. The three SNPs are in strong LD (D ≥ 0.7), the meta-analysis reported increased STin2 allele 12 frequencies in and in particular rs2020942 and rs2054847 were in complete LD schizophrenic patients, our study found two haplotypes were asso- (D = 1.0, R2 = 1.0), which is inconsistent to the genotype data in ciated with schizophrenia, the one containing allele 12 (P = 0.011) HapMap database. Whereas LD between SNPs and microsatellites and the one containing STin2 allele 10 (P = 5.38 × 10−5). Since haplo- was much weaker (D = 0.571 and 0.29).
type analysis has taken into consideration the frequencies of single A three-marker haplotype analysis was conducted with alleles, the difference between single allele and haplotype together rs2054847, rs140700 and STin2 VNTR, since rs2020942 was in with different sample size may result in this discrepancy.
total LD with rs2054847. wed the estimated haplotype rs2054847 and rs2020942 turned out to be in total LD, which was frequencies in both groups, with Bonferroni corrected P-values.
inconsistent with the LD data in the HapMap database, suggesting Globally, P = 4.67 × 10−6 after Bonferroni correction, indicating a they belonged to one big LD block which may span across 3 region significant association. The haplotype A-G-10 (#4 in of 5-HTT gene including STin2 VNTR. But the LD between these displayed a strong association with schizophrenia (Bonferroni cor- two SNPs with STin2 VNTR was not as high as expected, although rected P = 5.38 × 10−5), carried by about 8.4% of the schizophrenic rs2020942 is localized near to STin2 polymorphism (and patients versus 1.8% of controls. In addition, The 5# haplotype A- rs2054847 was predicted to be in the same LD block with STin2 G-12 is also significantly associated with schizophrenia (Bonferroni corrected P-value = 0.011), carried by about 69.9% of patients versus One recent neurochemical hypothesis on the etiology of schizophrenia has implicated a primary cortical hypoglutamater- No other significant associations were observed.
gia that enhances subcortical hyperdopaminergia and cortical In this study, we carried out a case-control study of the associ- hypodopaminergia In our view, 5-HT may contribute signif- ation between four markers in 5-HTT and schizophrenia based on icantly to the circuit alteration due to its extensive interaction subjects drawn from the Han Chinese population. While no single with other neurotransmitter systems especially the glutamate sys- locus associations was observed, we found that the frequencies of tem. The physiological basis for this view is the role of 5-HT two haplotypes were significantly different between the patients in the extensive biological processes which are relevant to the positive schizophrenic symptoms (such as hallucination), the neg- Our results support 5-HTT as a risk factor for schizophrenia in ative symptoms (such as diminished emotional expression), and the Han Chinese population. This is consistent with some but not the cognitive symptoms (such as impaired long-term memories).
all previous reports. A study on a Japanese cohort investigating four Importantly, 5-HT is also involved in the early brain development SNPs of 5-HTT as well as 5-HTTLPR and 5-HTTVNTR, found no asso- 5-HT neuronal network is among the earliest to develop ciation with schizophrenia However, a meta-analysis found in embryogenesis, where 5-HT may influence neurogenesis, neu- highly significant association between STin2 VNTR and schizophre- ronal apoptosis, and synaptic plasticity The involvement of nia from 12 population-based association studies hat is more, 5-HT system components in the therapeutic action of antipsychotic a recent association study by Zaboli et al. on Caucasians revealed drugs supports a role of 5-HT system in pathogenesis of schizophre- a significant association between a haplotype consisting of STin2 nia Consistently, other components of 5-HT system in VNTR and schizophrenia erestingly, we repeated the result addition to 5-HTT were also reported to be in association with of Zaboli et al. that the STin2-containing haplotype was associated schizophrenia The critical role of 5-HTT in regulating 5-HT Table 3
Estimated haplotype frequencies in the case-control subjects.
P = 0.000000467
a Haplotypes with a frequency <3% in both group are not shown.
b P-value Bonferroni corrected for multiple testing. Significant P-values (<0.05) are in boldface.
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