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Lack of association between SLC5A7 polymorphisms and Tourette syndrome in a Chinese Han population
Accepted Manuscript Title: Lack of association between SLC5A7 polymorphisms and Tourette syndrome in a Chinese Han population Authors: Hongzai Guan, Wenmiao Liu, Chuanping Gao, Xingfu Wu, Guiju Wang, Shiguo Liu, Jinsheng Liu PII: DOI: Reference:
S0304-3940(17)30688-2 http://dx.doi.org/10.1016/j.neulet.2017.08.041 NSL 33042
To appear in:
Neuroscience Letters
Received date: Revised date: Accepted date:
6-9-2016 14-8-2017 15-8-2017
Please cite this article as: Hongzai Guan, Wenmiao Liu, Chuanping Gao, Xingfu Wu, Guiju Wang, Shiguo Liu, Jinsheng Liu, Lack of association between SLC5A7 polymorphisms and Tourette syndrome in a Chinese Han population, Neuroscience Lettershttp://dx.doi.org/10.1016/j.neulet.2017.08.041 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Lack of association between SLC5A7 polymorphisms and Tourette syndrome in a Chinese Han population Hongzai Guana, Wenmiao Liua,b, Chuanping Gaoc, Xingfu Wud, Guiju Wange, Shiguo Liub* & Jinsheng Liud* a
Department of clinical laboratory diagnosis, Medical College, Qingdao University,
Qingdao, Shandong, China b
Prenatal diagnosis center, the Affiliated Hospital of Qingdao University, Qingdao, Shandong, China
c
Department of Radiology, the Affiliated Hospital of Qingdao University, Qingdao, Shandong, China
d
Department of Pediatrics, Linyi people’s Hospital, Linyi, Shandong, China
e
Child Healthcare Department of Rizhao People’s Hospital, Rizhao, Shandong, China
Hongzai Guan and Wenmiao Liu contribute equally to this work.
Corresponding Authors Shiguo Liu, E-mail: [email protected]. Tel: +86053282911385; Fax: +86053282912016. Jinsheng Liu, E-mail: [email protected]. Tel.: +86 05398056597; Fax: +86 05398056597
Highlights
We have investigated the association between polymorphisms in the solute carrier family 5, member 7 gene (SLC5A7) and Tourette syndrome (TS) in a Chinese Han population.
We combined a family-based analysis with a case–control study which may supply an authentic interpretation for our hypothesis.
SLC5A7 polymorphisms rs1013940, rs2433718, and rs4676169 were genotyped in 401 TS trios and 400 controls. The transmission disequilibrium test (TDT) and haplotype relative risk (HRR) compared genetic distributions of trios, while the chi-square test compared patients and controls.
No significant association was identified between SLC5A7 and TS in a Chinese Han population, indicating this gene may not be the main risk factor.
Abstract Although Tourette syndrome (TS) is a chronic neuropsychiatric disorder whose pathogenesis remains unclear, genetic factors play an important role in the occurrence and development. A variety of studies have been shown that the candidate genes related to cholinergic neurons may be associated with the onset of TS. To investigate the association between the SLC5A7 polymorphisms and Tourette syndrome (TS) in the Chinese Han population, the SNP rs1013940, rs2433718, and rs4676169 were genotyped in 401 TS trios and 400 controls. The transmission disequilibrium test (TDT) and haplotype relative risk (HRR) compared genetic distributions of trios,
while the chi-square test compared patients and controls. However，no transmission disequilibrium was found between the three SLC5A7 SNPs and TS. Therefore, we think that this gene may not be the main risk factor on the onset of TS. However, these results should be further validated in different populations.
Keywords: Tourette syndrome; SLC5A7 gene; Transmission disequilibrium test; Haplotype relative risk; Family-based study;Case–Control study
1. Introduction Tourette syndrome (TS) is a chronic childhood-onset neurodevelopment illness characterized by involuntary multiple motor or vocal tics lasting for at least 1 year [15]. The prevalence of TS has increased from 1% to 3% year by year, and is more common in males than females which the ratio of the incidence is about 3 to 4:1[21]. Most TS individuals have comorbid psychiatric disorders such as attention deficit hyperactivity disorder (ADHD), obsessive–compulsive disorder (OCD), impulsive and self-injurious behaviour [5], which together have severe effects on the patient’s family and society. Although some earlier studies indicated that TS may be the result
of an interaction between genetics, immunology, the environment, and hormonal factors [6, 7, 17], the precise disease aetiology and pathogenesis has remained unknown. However, pedigree and twin studies have confirmed that TS is a polygenic disease with a complex inheritance pattern and obvious genetic predisposition [27]. To date, many candidate genes have been studied such as dopamine receptors D1–D4, dopamine β–hydroxylase (DBH), monoamine oxidase A (MAOA), and serotonin receptor (5-HT2A) [19], but most of these candidate gene associations have not been replicated. The manifestation of TS is also associated with the dysfunction of monoaminergic systems, especially increased dopaminergic activity [26]. Several genes related to cholinergic neurons such as choline O-acetyltransferase (CHAT), solute carrier family 5, member 7 (SLC5A7), solute carrier family 18, member 3 (SLC18A3), and cholinergic receptor, muscarinic 2 (CHRM2) are significantly downregulated in the basal ganglia of TS mouse models [4, 18, 29]. This affects the synthesis of the neurotransmitter acetylcholine (ACh), which is responsible for the control of autonomic functions, learning, memory, and cognition. SLC5A7, also known as the choline transporter 1 gene (CHT1), is located on chromosome 2q12 and is divided into nine exons. It is a Na+ and Cl--dependent high-affinity presynaptic choline transporter that mediates choline uptake into cholinergic nerve terminals, which is a rate-limiting step for ACh synthesis [11]. Mouse experiments previously demonstrated a compensatory mechanism between SLC5A7 and Ach, which suggested that SLC5A7 expression increases to compensate for the dysfunction of other cholinergic neurons to maintain ACh synthesis and to sustain normal cholinergic
signalling [3]. Although the symptoms of TS such as yawning, motion sickness, pain, and sensory tics may be related to the cholinergic system [23], this has not been proven. Taken together, we hypothesised that SLC5A7 may affect the synthesis of ACh and be involved in the pathogenesis of TS. In the present study, therefore, we investigated the potential association between three SLC5A7 single nucleotide polymorphisms (SNPs) and TS in a Chinese Han population. We selected the SNP rs1013940 (located in exon 3), rs2433718 (located in intron 1) and rs4676169 (located in intron 4) in SLC5A7 which are all important tag SNPs in Chinese Han population. Moreover, they are good representatives for SLC5A7 because they are independent inheritance from each other (the linkage disequilibrium tests between the three SNPs are 3%, 17% and 0.1%, respectively). In addition, there is little research on the association between these three SNPs and TS. To evaluate the role of SLC5A7 in TS pathogenesis, we combined a family-based analysis with a case–control study which may supply an authentic interpretation for our hypothesis. 2. Materials 2.1 Subjects We enrolled 401 TS trios and 400 healthy controls from the Affiliated Hospital of Qingdao University and Linyi People’s Hospital, China. Patient diagnoses were made independently by two experienced psychiatrists using criteria of the Diagnostic and Statistical Manual of Mental Disorders, 4th Edition (DSM-IV). The control group we collected has excluded any symptoms of TS and other neuropsychiatric disorders.
Moreover, all matched to the TS patients in relation to gender, geographic region and ethnic origin. As TS is an early onset disorder, age was not taken as a matching factor. All participants or their legal guardians gave their informed written consent, and the Human Ethics Committee of the Affiliated Hospital of Qingdao University approved the study project. 2.2 Genotyping analysis Blood samples were collected from all participants and stored at 20C until analysed. Genomic DNA was extracted from 300 μl peripheral blood leukocytes using a DNA extraction kit (Qiagen, Hilden, Germany). Alleles of SLC5A7 SNPs rs1013940, rs2433718, and rs4676169 were determined by TaqMan allelic discrimination real-time PCR. Taqman probes and primers were designed by Applied Biosystems of Life
Technologies.
For
rs1013940,
forward
and
reverse
primers
5-TGGCCTAGCTTGGGCTCAGGCACCA-3
were and
5-TTGGATATTCTCTTAGTCTGATTTT-3, respectively; for rs2433718, these were 5-GAAATGGGTAGAGGGGGCCGAGGAA-3
and
5-GGCCGCAGGGGGCCGGGAGAGCATC-3, respectively; and for rs4676169, these
were
5-AAACTACCTAAACTCCTGAGACAAC-3
and
5-GTGTTGCATGTTATTCTAAAAGCAC-3, respectively. PCR was conducted in 25 µl reactions mixture，containing 20 × SNP Genotyping Assay 1.25 ul, 2 × PCR Master Mix 12.5 ul, DNA and DNase-free water 11.25 ul. Amplifications were carried out using a C1000TM thermal cycler system with the following conditions: 95C for 3 min, followed by 45 cycles of 95C for 15 s and 60C for 1 min. All steps were
carried out with relevant guidelines and regulations, and the fluorescent signal from VIC- or FAM-labelled probes could be detected in each cycle. The discrimination of genotypes was conducted using Bio-Rad CFX manager 3.0 software. 2.3 Statistical analysis All analyses were performed by the statistical software package SPSS21.0. Hardy–Weinberg equilibrium (HWE) was examined in control groups using the chi-square test. Case–control and family-based studies were used to detect the association between the three SLC5A7 SNPs and TS, and all data were tested by means of haplotype relative risk (HRR) and the transmission disequilibrium test (TDT). To increase the efficiency, we enlarged cases by haplotype-based haplotype relative risk (HHRR). Allelic and genotypic distributions of patients and controls were compared using the chi-square test, with P values <0.05 considered to be significant. 3. Results 3.1 Case–control study The study group consisted of 401 patients (319 males, 82 females; mean age, 8.87 3.24 years) and 400 controls (310 males, 90 females; mean age, 33.00 8.44 years). The distribution of genotypic frequencies for all three SNPs in control groups was consistent with the Hardy-Weinberg equilibrium, suggesting that the population was genetically balanced and could be used for association studies (for rs1013940， χ2=3.457, P=0.063; for rs2433718， χ2=2.171, P=0.141; for rs4676169, χ2=1.012, P=0.314). To estimate whether there was a difference between cases and controls, the
allelic and genotypic distributions of the two groups were compared by the chi-square test which indicated that there was no significant difference (for rs1013940, genotype: χ2=0.622, P=0.733, allele: χ2=0.257, P=0.612, OR=1.079, 95% CI=0.804-1.449; for rs2433718, genotype: χ2=3.405, P=0.182, allele: χ2=3.465, P=0.063, OR=1.211, 95% CI=0.990–1.482; for rs4676169, genotype: χ2=2.908, P=0.234, allele: χ2=2.918, P=0.088, OR=0.830, 95% CI=0.670–1.028). The results are shown in Table 1. 3.2 Family-based study A total of 401 trios took part in the family-based study. No significant statistical significance of allele transfer was determined for the three SNPs (for rs1013940, TDT=0.268, P=0.657, OR=0.728, 95% CI=0.366–1.451; HRR=0.959, χ2=0.111, P=0.739, 95% CI=0.762–1.466; for rs2433718, TDT=0.968, P=0.351, OR=1.120, 95% CI=0.839–1.496; HRR=1.089, χ2=0.341, P=0.559, 95% CI=0.818–1.450; for rs4676169, TDT=3.440, P=0.072, OR=1.027, 95% CI=0.739–1.428; HRR=1.185, χ2=1.441, P=0.230, 95% CI=0.898–1.563). Genotypic and allelic frequencies are presented in Tables 2 and 3. To increase the test efficiency, we enlarged case numbers by HHRR and obtained similar results for these SNPs, which confirmed the absence of transmission disequilibrium in genotypic and allelic frequencies (for rs1013940, HHRR=1.082, χ2=0.276, P=0.599, 95% CI=0.806–1.453; for rs2433718, HHRR=1.104, χ2=0.942, P=0.332, 95% CI=0.904–1.349; for rs4676169, HHRR=0.817, χ2=3.429, P=0.064, 95% CI=0.660–1.012). The results are shown in Table 4.
4. Discussion TS is a complicated hereditary neuropsychiatric disease with complicated clinical manifestations and genetic patterns. All TS patients have an accompanying neurological disorder, and there is evidence that the occurrence of TS may be related to the dual role of genetics and the environment [25]. Nevertheless, the precise disease aetiology and the localisation of susceptibility genes remain unclear. Central nervous system neurotransmitter metabolic disorders have been considered to be related to TS pathogenesis. In recent years, a variety of neuroimaging studies have revealed abnormalities in the central nervous systems of TS patients [13]. The most commonly observed organic abnormality occurs in the basal ganglia, which contains cholinergic neurons and three classes of GABA interneurons [10]. Many studies have reported that cholinergic neurons play an important role in a variety of diseases of the central nervous system, such as primary Parkinson’s syndrome, hyperactivity disorder, and Alzheimer’s disease [22]. Additionally, cholinergic neurons are thought to be decreased in the striatum of TS patients [14]. We therefore supposed that genes related to cholinergic neurons may also be TS susceptibility genes. The main choline transporter of cholinergic neurons is the high affinity protein family, whose main function is to synthesise ACh through the uptake of choline. SLC5A7, mainly located in the basal forebrain, cortex, and other nerve tissue, is one of the representative members involved in the transport of choline, which is typically the major determinant step for ACh production [28]. Ferguson et al. have found that the sensitive transporter of choline and the synthesis of ACh have disappeared totally
in a mouse animal model with a deletion of SLC5A7 [9]. SLC5A7 dysregulation was also shown to lead to difficulties in the release of ACh, which is closely related to cognitive impairment [20]. Moreover, the interruption of cholinergic neuron signals such as SLC5A7 may be an important pathophysiological mechanism of TS and may further interfere with the metabolism[16]. Previous studies have shown that SLC5A7 polymorphisms are involved in the pathogenesis of neuropsychiatric disorders such as ADHD [8], and depression in the case of the rs1013940 polymorphism [12]. Santoro et al. have found SLC5A7 was significantly downregulated in the prefrontal cortex of the spontaneously hypertensive rat, which was a useful animal model to study several behavioral, pathophysiological and pharmacological aspects of schizophrenia and ADHD [24]. In addition，Berry et al. have demonstrated that rs1013940 carriers exhibit reduced activation in right prefrontal cortex in response to attentional demands compared to controls [1]. The other study of Berry was the first to demonstrate a specific impairment in cognitive control associated with the rs1013940 polymorphism. They found the rs1013940 participants were more distraction during everyday life than controls, and their task performance was more severely impacted by the presence of an ecologically valid video distractor (similar to a television playing in the background)[2]. Despite little attention has been given to their association with TS, the role played by cholinergic neurons in the control of disorders of the central nervous system were crucial. On this basis, the cholinergic neurons, involved in its comorbidity, may participate in the pathogenesis of TS as well.
We aimed to clarify the potential associations between SLC5A7 SNPs rs1013940, rs2433718, and rs4676169 and TS in a Chinese Han population. We innovatively combined a family-based association study with classic case–control analysis to avoid the influence of confounding factors and to better reflect the efficacy of the experiment. However, our results revealed no significant differences between patients and controls in either test, suggesting that SLC5A7 may not a susceptibility gene for TS, at least in the current study population. However, our study has a number of limitations, including its focus on a single population and its small sample size. Because polymorphic distributions vary in different races, this association should be validated in different populations. Moreover, because we only investigated the association of three SLC5A7 SNPs, this is not sufficient to represent the entire association with this gene and TS. Therefore, future studies should investigate the association between other SLC5A7 SNPs and TS. To our knowledge, this is the first report of genetic associations between SLC5A7 SNPs and TS in a Chinese Han population. Our study will be of use in the screening and ruling out of potential TS susceptibility genes, clarifying the molecular basis of the disease, and assisting with treatment and drug-design strategies for TS. Acknowledgements We thank all the participants for our study. This work was supported by the National Natural Science Foundation of China (81371499 and 30971586). Statement of Interest None to declare.
Author Contributions SGL and JSL conceived the study, participated in its design and guided drafting of the manuscript. HZG and WML participated in statistical analysis and drafted the manuscript. CPG, XFW and GJW were responsible for the clinical diagnosis and clinical phenotype analysis. All authors read and approved the final manuscript.
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Table 1：The genotypic and allelic frequencies of three genetic loci in two groups.
rs1013940 Group
rs2433718
rs4676169
N CC
CT
TT
C
T
CC
CT
TT
C
T
AA
AG
GG
A
G
Patients
401
9
87
305
105
697
71
184
146
326
476
192
160
49
544
258
Control
400
10
78
312
98
702
59
171
170
289
511
210
154
36
574
226
χ
2
P-value
0.622
0.257
3.405
3.465
2.908
2.918
0.733
0.612
0.182
0.063
0.234
0.088
OR
1.079
1.211
0.830
95%CI
0.804-1.449
0.990-1.482
0.670-1.028
Table 2: TDT test results of three genetic loci in 401 trios. Non-transmitted allele Transmitted
rs1013940
C
rs2433718
C
T
10
95
C
rs4676169
C
T
130
196
A
A
G
393
151
allele
T
88
609
T
177
299
G
185
73
TDT results Χ2
0.268
0.968
3.440
P-value
0.657
0.351
0.072
OR
0.728
1.120
1.027
95%CI
0.366-1.451
0.839-1.496
0.739-1.428
Table 3: The HRR results of three genetic loci in 401 trios. rs1013940
Group
rs2433718
rs4676169
C（＋）
C（－）
C（＋）
C（－）
G（＋）
G（－）
Transmitted allele
96
305
255
146
209
192
Non-transmitted allele
92
309
247
154
192
209
χ2
0.111
0.341
1.441
P-value
0.739
0.559
0.230
OR
1.057
1.089
1.185
95%CI
0.762-1.466
0.818-1.450
0.898-1.563
Table 4: The HHRR results of three genetic loci in 401 trios. Group
rs1013940
rs2433718
rs4676169
C
T
C
T
G
A
Transmitted allele
105
697
326
476
258
544
Non-transmitted allele
98
704
307
495
224
578
χ2
0.276
0.942
3.429
P-value
0.599
0.332
0.064
OR
1.082
1.104
0.817
95%CI
0.806-1.453
0.904-1.349
0.660-1.012
S0304-3940(17)30688-2 http://dx.doi.org/10.1016/j.neulet.2017.08.041 NSL 33042
To appear in:
Neuroscience Letters
Received date: Revised date: Accepted date:
6-9-2016 14-8-2017 15-8-2017
Please cite this article as: Hongzai Guan, Wenmiao Liu, Chuanping Gao, Xingfu Wu, Guiju Wang, Shiguo Liu, Jinsheng Liu, Lack of association between SLC5A7 polymorphisms and Tourette syndrome in a Chinese Han population, Neuroscience Lettershttp://dx.doi.org/10.1016/j.neulet.2017.08.041 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Lack of association between SLC5A7 polymorphisms and Tourette syndrome in a Chinese Han population Hongzai Guana, Wenmiao Liua,b, Chuanping Gaoc, Xingfu Wud, Guiju Wange, Shiguo Liub* & Jinsheng Liud* a
Department of clinical laboratory diagnosis, Medical College, Qingdao University,
Qingdao, Shandong, China b
Prenatal diagnosis center, the Affiliated Hospital of Qingdao University, Qingdao, Shandong, China
c
Department of Radiology, the Affiliated Hospital of Qingdao University, Qingdao, Shandong, China
d
Department of Pediatrics, Linyi people’s Hospital, Linyi, Shandong, China
e
Child Healthcare Department of Rizhao People’s Hospital, Rizhao, Shandong, China
Hongzai Guan and Wenmiao Liu contribute equally to this work.
Corresponding Authors Shiguo Liu, E-mail: [email protected]. Tel: +86053282911385; Fax: +86053282912016. Jinsheng Liu, E-mail: [email protected]. Tel.: +86 05398056597; Fax: +86 05398056597
Highlights
We have investigated the association between polymorphisms in the solute carrier family 5, member 7 gene (SLC5A7) and Tourette syndrome (TS) in a Chinese Han population.
We combined a family-based analysis with a case–control study which may supply an authentic interpretation for our hypothesis.
SLC5A7 polymorphisms rs1013940, rs2433718, and rs4676169 were genotyped in 401 TS trios and 400 controls. The transmission disequilibrium test (TDT) and haplotype relative risk (HRR) compared genetic distributions of trios, while the chi-square test compared patients and controls.
No significant association was identified between SLC5A7 and TS in a Chinese Han population, indicating this gene may not be the main risk factor.
Abstract Although Tourette syndrome (TS) is a chronic neuropsychiatric disorder whose pathogenesis remains unclear, genetic factors play an important role in the occurrence and development. A variety of studies have been shown that the candidate genes related to cholinergic neurons may be associated with the onset of TS. To investigate the association between the SLC5A7 polymorphisms and Tourette syndrome (TS) in the Chinese Han population, the SNP rs1013940, rs2433718, and rs4676169 were genotyped in 401 TS trios and 400 controls. The transmission disequilibrium test (TDT) and haplotype relative risk (HRR) compared genetic distributions of trios,
while the chi-square test compared patients and controls. However，no transmission disequilibrium was found between the three SLC5A7 SNPs and TS. Therefore, we think that this gene may not be the main risk factor on the onset of TS. However, these results should be further validated in different populations.
Keywords: Tourette syndrome; SLC5A7 gene; Transmission disequilibrium test; Haplotype relative risk; Family-based study;Case–Control study
1. Introduction Tourette syndrome (TS) is a chronic childhood-onset neurodevelopment illness characterized by involuntary multiple motor or vocal tics lasting for at least 1 year [15]. The prevalence of TS has increased from 1% to 3% year by year, and is more common in males than females which the ratio of the incidence is about 3 to 4:1[21]. Most TS individuals have comorbid psychiatric disorders such as attention deficit hyperactivity disorder (ADHD), obsessive–compulsive disorder (OCD), impulsive and self-injurious behaviour [5], which together have severe effects on the patient’s family and society. Although some earlier studies indicated that TS may be the result
of an interaction between genetics, immunology, the environment, and hormonal factors [6, 7, 17], the precise disease aetiology and pathogenesis has remained unknown. However, pedigree and twin studies have confirmed that TS is a polygenic disease with a complex inheritance pattern and obvious genetic predisposition [27]. To date, many candidate genes have been studied such as dopamine receptors D1–D4, dopamine β–hydroxylase (DBH), monoamine oxidase A (MAOA), and serotonin receptor (5-HT2A) [19], but most of these candidate gene associations have not been replicated. The manifestation of TS is also associated with the dysfunction of monoaminergic systems, especially increased dopaminergic activity [26]. Several genes related to cholinergic neurons such as choline O-acetyltransferase (CHAT), solute carrier family 5, member 7 (SLC5A7), solute carrier family 18, member 3 (SLC18A3), and cholinergic receptor, muscarinic 2 (CHRM2) are significantly downregulated in the basal ganglia of TS mouse models [4, 18, 29]. This affects the synthesis of the neurotransmitter acetylcholine (ACh), which is responsible for the control of autonomic functions, learning, memory, and cognition. SLC5A7, also known as the choline transporter 1 gene (CHT1), is located on chromosome 2q12 and is divided into nine exons. It is a Na+ and Cl--dependent high-affinity presynaptic choline transporter that mediates choline uptake into cholinergic nerve terminals, which is a rate-limiting step for ACh synthesis [11]. Mouse experiments previously demonstrated a compensatory mechanism between SLC5A7 and Ach, which suggested that SLC5A7 expression increases to compensate for the dysfunction of other cholinergic neurons to maintain ACh synthesis and to sustain normal cholinergic
signalling [3]. Although the symptoms of TS such as yawning, motion sickness, pain, and sensory tics may be related to the cholinergic system [23], this has not been proven. Taken together, we hypothesised that SLC5A7 may affect the synthesis of ACh and be involved in the pathogenesis of TS. In the present study, therefore, we investigated the potential association between three SLC5A7 single nucleotide polymorphisms (SNPs) and TS in a Chinese Han population. We selected the SNP rs1013940 (located in exon 3), rs2433718 (located in intron 1) and rs4676169 (located in intron 4) in SLC5A7 which are all important tag SNPs in Chinese Han population. Moreover, they are good representatives for SLC5A7 because they are independent inheritance from each other (the linkage disequilibrium tests between the three SNPs are 3%, 17% and 0.1%, respectively). In addition, there is little research on the association between these three SNPs and TS. To evaluate the role of SLC5A7 in TS pathogenesis, we combined a family-based analysis with a case–control study which may supply an authentic interpretation for our hypothesis. 2. Materials 2.1 Subjects We enrolled 401 TS trios and 400 healthy controls from the Affiliated Hospital of Qingdao University and Linyi People’s Hospital, China. Patient diagnoses were made independently by two experienced psychiatrists using criteria of the Diagnostic and Statistical Manual of Mental Disorders, 4th Edition (DSM-IV). The control group we collected has excluded any symptoms of TS and other neuropsychiatric disorders.
Moreover, all matched to the TS patients in relation to gender, geographic region and ethnic origin. As TS is an early onset disorder, age was not taken as a matching factor. All participants or their legal guardians gave their informed written consent, and the Human Ethics Committee of the Affiliated Hospital of Qingdao University approved the study project. 2.2 Genotyping analysis Blood samples were collected from all participants and stored at 20C until analysed. Genomic DNA was extracted from 300 μl peripheral blood leukocytes using a DNA extraction kit (Qiagen, Hilden, Germany). Alleles of SLC5A7 SNPs rs1013940, rs2433718, and rs4676169 were determined by TaqMan allelic discrimination real-time PCR. Taqman probes and primers were designed by Applied Biosystems of Life
Technologies.
For
rs1013940,
forward
and
reverse
primers
5-TGGCCTAGCTTGGGCTCAGGCACCA-3
were and
5-TTGGATATTCTCTTAGTCTGATTTT-3, respectively; for rs2433718, these were 5-GAAATGGGTAGAGGGGGCCGAGGAA-3
and
5-GGCCGCAGGGGGCCGGGAGAGCATC-3, respectively; and for rs4676169, these
were
5-AAACTACCTAAACTCCTGAGACAAC-3
and
5-GTGTTGCATGTTATTCTAAAAGCAC-3, respectively. PCR was conducted in 25 µl reactions mixture，containing 20 × SNP Genotyping Assay 1.25 ul, 2 × PCR Master Mix 12.5 ul, DNA and DNase-free water 11.25 ul. Amplifications were carried out using a C1000TM thermal cycler system with the following conditions: 95C for 3 min, followed by 45 cycles of 95C for 15 s and 60C for 1 min. All steps were
carried out with relevant guidelines and regulations, and the fluorescent signal from VIC- or FAM-labelled probes could be detected in each cycle. The discrimination of genotypes was conducted using Bio-Rad CFX manager 3.0 software. 2.3 Statistical analysis All analyses were performed by the statistical software package SPSS21.0. Hardy–Weinberg equilibrium (HWE) was examined in control groups using the chi-square test. Case–control and family-based studies were used to detect the association between the three SLC5A7 SNPs and TS, and all data were tested by means of haplotype relative risk (HRR) and the transmission disequilibrium test (TDT). To increase the efficiency, we enlarged cases by haplotype-based haplotype relative risk (HHRR). Allelic and genotypic distributions of patients and controls were compared using the chi-square test, with P values <0.05 considered to be significant. 3. Results 3.1 Case–control study The study group consisted of 401 patients (319 males, 82 females; mean age, 8.87 3.24 years) and 400 controls (310 males, 90 females; mean age, 33.00 8.44 years). The distribution of genotypic frequencies for all three SNPs in control groups was consistent with the Hardy-Weinberg equilibrium, suggesting that the population was genetically balanced and could be used for association studies (for rs1013940， χ2=3.457, P=0.063; for rs2433718， χ2=2.171, P=0.141; for rs4676169, χ2=1.012, P=0.314). To estimate whether there was a difference between cases and controls, the
allelic and genotypic distributions of the two groups were compared by the chi-square test which indicated that there was no significant difference (for rs1013940, genotype: χ2=0.622, P=0.733, allele: χ2=0.257, P=0.612, OR=1.079, 95% CI=0.804-1.449; for rs2433718, genotype: χ2=3.405, P=0.182, allele: χ2=3.465, P=0.063, OR=1.211, 95% CI=0.990–1.482; for rs4676169, genotype: χ2=2.908, P=0.234, allele: χ2=2.918, P=0.088, OR=0.830, 95% CI=0.670–1.028). The results are shown in Table 1. 3.2 Family-based study A total of 401 trios took part in the family-based study. No significant statistical significance of allele transfer was determined for the three SNPs (for rs1013940, TDT=0.268, P=0.657, OR=0.728, 95% CI=0.366–1.451; HRR=0.959, χ2=0.111, P=0.739, 95% CI=0.762–1.466; for rs2433718, TDT=0.968, P=0.351, OR=1.120, 95% CI=0.839–1.496; HRR=1.089, χ2=0.341, P=0.559, 95% CI=0.818–1.450; for rs4676169, TDT=3.440, P=0.072, OR=1.027, 95% CI=0.739–1.428; HRR=1.185, χ2=1.441, P=0.230, 95% CI=0.898–1.563). Genotypic and allelic frequencies are presented in Tables 2 and 3. To increase the test efficiency, we enlarged case numbers by HHRR and obtained similar results for these SNPs, which confirmed the absence of transmission disequilibrium in genotypic and allelic frequencies (for rs1013940, HHRR=1.082, χ2=0.276, P=0.599, 95% CI=0.806–1.453; for rs2433718, HHRR=1.104, χ2=0.942, P=0.332, 95% CI=0.904–1.349; for rs4676169, HHRR=0.817, χ2=3.429, P=0.064, 95% CI=0.660–1.012). The results are shown in Table 4.
4. Discussion TS is a complicated hereditary neuropsychiatric disease with complicated clinical manifestations and genetic patterns. All TS patients have an accompanying neurological disorder, and there is evidence that the occurrence of TS may be related to the dual role of genetics and the environment [25]. Nevertheless, the precise disease aetiology and the localisation of susceptibility genes remain unclear. Central nervous system neurotransmitter metabolic disorders have been considered to be related to TS pathogenesis. In recent years, a variety of neuroimaging studies have revealed abnormalities in the central nervous systems of TS patients [13]. The most commonly observed organic abnormality occurs in the basal ganglia, which contains cholinergic neurons and three classes of GABA interneurons [10]. Many studies have reported that cholinergic neurons play an important role in a variety of diseases of the central nervous system, such as primary Parkinson’s syndrome, hyperactivity disorder, and Alzheimer’s disease [22]. Additionally, cholinergic neurons are thought to be decreased in the striatum of TS patients [14]. We therefore supposed that genes related to cholinergic neurons may also be TS susceptibility genes. The main choline transporter of cholinergic neurons is the high affinity protein family, whose main function is to synthesise ACh through the uptake of choline. SLC5A7, mainly located in the basal forebrain, cortex, and other nerve tissue, is one of the representative members involved in the transport of choline, which is typically the major determinant step for ACh production [28]. Ferguson et al. have found that the sensitive transporter of choline and the synthesis of ACh have disappeared totally
in a mouse animal model with a deletion of SLC5A7 [9]. SLC5A7 dysregulation was also shown to lead to difficulties in the release of ACh, which is closely related to cognitive impairment [20]. Moreover, the interruption of cholinergic neuron signals such as SLC5A7 may be an important pathophysiological mechanism of TS and may further interfere with the metabolism[16]. Previous studies have shown that SLC5A7 polymorphisms are involved in the pathogenesis of neuropsychiatric disorders such as ADHD [8], and depression in the case of the rs1013940 polymorphism [12]. Santoro et al. have found SLC5A7 was significantly downregulated in the prefrontal cortex of the spontaneously hypertensive rat, which was a useful animal model to study several behavioral, pathophysiological and pharmacological aspects of schizophrenia and ADHD [24]. In addition，Berry et al. have demonstrated that rs1013940 carriers exhibit reduced activation in right prefrontal cortex in response to attentional demands compared to controls [1]. The other study of Berry was the first to demonstrate a specific impairment in cognitive control associated with the rs1013940 polymorphism. They found the rs1013940 participants were more distraction during everyday life than controls, and their task performance was more severely impacted by the presence of an ecologically valid video distractor (similar to a television playing in the background)[2]. Despite little attention has been given to their association with TS, the role played by cholinergic neurons in the control of disorders of the central nervous system were crucial. On this basis, the cholinergic neurons, involved in its comorbidity, may participate in the pathogenesis of TS as well.
We aimed to clarify the potential associations between SLC5A7 SNPs rs1013940, rs2433718, and rs4676169 and TS in a Chinese Han population. We innovatively combined a family-based association study with classic case–control analysis to avoid the influence of confounding factors and to better reflect the efficacy of the experiment. However, our results revealed no significant differences between patients and controls in either test, suggesting that SLC5A7 may not a susceptibility gene for TS, at least in the current study population. However, our study has a number of limitations, including its focus on a single population and its small sample size. Because polymorphic distributions vary in different races, this association should be validated in different populations. Moreover, because we only investigated the association of three SLC5A7 SNPs, this is not sufficient to represent the entire association with this gene and TS. Therefore, future studies should investigate the association between other SLC5A7 SNPs and TS. To our knowledge, this is the first report of genetic associations between SLC5A7 SNPs and TS in a Chinese Han population. Our study will be of use in the screening and ruling out of potential TS susceptibility genes, clarifying the molecular basis of the disease, and assisting with treatment and drug-design strategies for TS. Acknowledgements We thank all the participants for our study. This work was supported by the National Natural Science Foundation of China (81371499 and 30971586). Statement of Interest None to declare.
Author Contributions SGL and JSL conceived the study, participated in its design and guided drafting of the manuscript. HZG and WML participated in statistical analysis and drafted the manuscript. CPG, XFW and GJW were responsible for the clinical diagnosis and clinical phenotype analysis. All authors read and approved the final manuscript.
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Table 1：The genotypic and allelic frequencies of three genetic loci in two groups.
rs1013940 Group
rs2433718
rs4676169
N CC
CT
TT
C
T
CC
CT
TT
C
T
AA
AG
GG
A
G
Patients
401
9
87
305
105
697
71
184
146
326
476
192
160
49
544
258
Control
400
10
78
312
98
702
59
171
170
289
511
210
154
36
574
226
χ
2
P-value
0.622
0.257
3.405
3.465
2.908
2.918
0.733
0.612
0.182
0.063
0.234
0.088
OR
1.079
1.211
0.830
95%CI
0.804-1.449
0.990-1.482
0.670-1.028
Table 2: TDT test results of three genetic loci in 401 trios. Non-transmitted allele Transmitted
rs1013940
C
rs2433718
C
T
10
95
C
rs4676169
C
T
130
196
A
A
G
393
151
allele
T
88
609
T
177
299
G
185
73
TDT results Χ2
0.268
0.968
3.440
P-value
0.657
0.351
0.072
OR
0.728
1.120
1.027
95%CI
0.366-1.451
0.839-1.496
0.739-1.428
Table 3: The HRR results of three genetic loci in 401 trios. rs1013940
Group
rs2433718
rs4676169
C（＋）
C（－）
C（＋）
C（－）
G（＋）
G（－）
Transmitted allele
96
305
255
146
209
192
Non-transmitted allele
92
309
247
154
192
209
χ2
0.111
0.341
1.441
P-value
0.739
0.559
0.230
OR
1.057
1.089
1.185
95%CI
0.762-1.466
0.818-1.450
0.898-1.563
Table 4: The HHRR results of three genetic loci in 401 trios. Group
rs1013940
rs2433718
rs4676169
C
T
C
T
G
A
Transmitted allele
105
697
326
476
258
544
Non-transmitted allele
98
704
307
495
224
578
χ2
0.276
0.942
3.429
P-value
0.599
0.332
0.064
OR
1.082
1.104
0.817
95%CI
0.806-1.453
0.904-1.349
0.660-1.012