Transmission disequilibrium test and haplotype analysis of the MCP-1
–2518G/A polymorphism with Tourette syndrome in Chinese Han
trios
Running title: Gene studies on Tourette syndrome in Chinese
Meijian Wang a, Yingying Yin b, Wei Ren c,d, Xiuhai Wang a, and Shiguo Liu c,d,e,*
a
Department of Biology, Medical School, Qingdao University, Qingdao, China
b
Department of Psychiatry, Medical College, Qingdao University, Qingdao, China
c
Shandong Provincial Key Laboratory of Metabolic Disease, the Affiliated Hospital of
Medical College, Qingdao University, Qingdao, China
d
Institute of Clinical Research, the Affiliated Hospital of Medical College, Qingdao
University, Qingdao, China
e
Genetic Laboratory, the Affiliated Hospital of Medical College, Qingdao University,
Qingdao, China
*
Corresponding author: Dr. Shiguo Liu
The Affiliated Hospital of Medical College,
Qingdao University,
16, Jiangsu Road, Qingdao, 266003 China.
Tel.: +86 53281912912; Fax: 86 53281912916;
E-mail: lsgpumc@126.com.
Received: 19 April 2011
Revised: 16 June 2011
Accepted: 12 July 2011
1
Abstract. The purpose of this study was to investigate the association between MCP-1 –
2518G/A polymorphism and Tourette syndrome (TS) in Chinese Han population. We
collected 324 Chinese Han individuals from 108 TS nuclear families from Chinese Han
population and employed polymerase chain reaction and restriction enzyme digestion for
determining the genotype of MCP-1–2518G/A polymorphism. The data were analyzed using
the transmission disequilibrium test the genotype haplotype relative risk and haplotype-based
haplotype relative risk. No biases were observed in the transmission of either of the two
alleles (χ2 = 0.016, df = 1, P = 0.898) to the affected probands in the total sample. The
analyses of transmission disequilibrium test, haplotype relative risk and haplotype-based
haplotype relative risk did not indicate any associations at MCP-1 –2518G/A with TS. Our
results suggest that MCP-1 –2518G/A is not associated with susceptibility of Tourette
syndrome in Chinese Han population.
Keywords: Tourette syndrome, MCP-1, transmission disequilibrium test, haplotype relative
risk
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1. Introduction
Tourette syndrome (TS) is a childhood-onset chronic neuropsychiatric disorder
characterized by multiple motor and phonic tics that wax and wane over time [1]. Once
thought to be rare, with prevalence estimates ranging from 1:20,000 to 1:2,000 [2], it has
recently been observed that, in school-age populations, the prevalence may be as high as 1%
among boys [3]. Most of the patients with TS also have other comorbid psychiatric disorders,
such as obsessive-compulsive disorder and attention deficit hyperactivity disorder. Family
studies [4-6] demonstrated that TS is familial, and twin studies provided evidence that genetic
factors are important in its transmission [7,8]. However, until now, no common vulnerability
genes, which clearly associated with the disorder have been reported.
In spite of hereditary factor, postinfectious autoimmunity was also reported to
participate in the etiology of TS [9]. The direct evidence was enhanced activity of T cell and
natural killer cells in peripheral blood [10-12] of TS patients. Other studies demonstrating
increased frequency of streptococcal infections and sinusitis in the patients also implied some
form of immune deficiency [13,14]. Cytokines are generally known as chemical messengers
between immune cells, and they play a crucial role in mediating inflammatory and immune
responses. They are particularly relevant to immune-related disorders such as infection,
allergy, autoimmune diseases, and cancer [14]. According to some studies that have
investigated the action of cytokines in patients with TS, cytokines can cross the blood-brain
barrier (BBB) and influence complex brain functions, suggesting cytokines may play a crucial
role in the pathogenesis of TS [15,16].
As an important cytokine, monocyte chemoattractant protein-1 (MCP-1) is a
chemokine that leads to monocyte migration and accumulation towards inflammatory foci and
is associated with neuro-inflammatory conditions of diverse etiologies (hypoxia, tumor,
infection, epilepsy) [17]. Its deficiency protects against inflammation in brain [18]. The major
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source of MCP-1 are perivascular astrocytes [19]. MCP-1 increases the BBB permeability and
attracts leukocytes across BBB, possibly allowing migration of auto-antibodies in serum of
TS patients to the area of basal ganglia [20-25]. Recently, MCP-1 and its receptor CCL2 were
also shown to mediate differentiation of neural stem cells into neurons, astrocytes and
oligodendrocytes via the interaction with the BBB endothelium [26], as well as the
differentiation of adult subventricolar zone-derived progenitor cell migration following
striatal cell death [27]. Notably, the prior study of cytokines with TS detected increased serum
MCP-1 compared to controls during illness exacerbation [28].
A biallelic polymorphism (A/G) at position -2518 of the MCP-1 gene has been
described. Cells from individuals with the GG or AG genotypes produce more MCP-1 than
those obtained from individuals with the AA genotype, which suggests an influence of this
polymorphism on transcriptional activity [29]. We hypothesized that the –2518G/A
polymorphism MCP-1 could be associated with TS in Chinese Han population. Therefore, the
present study was conducted to make clear whether the functional variants of the –2518G/A
in the MCP-1 is association with TS in a Chinese Han population.
2. Materials and methods
2.1. Patient selection
A total of 108 patients including their parents in the study were recruited from Child
Healthcare Department of the Affiliated Hospital of Qingdao University Medical College and
Linyi People's Hospital. TS cases were comprised of 25 females and 83 males outpatients,
aged between 5 and 18 years. All probands were diagnosed independently by two experienced
psychiatrists according to the DSM-IV criteria and the TS Classification Study Group. The
protocol was approved by the Ethics Committees of Qingdao University Medical College
Hospital. Patients were given informed consent. If the subjects were children or disabled
persons the informed assent would be issued by the parents or guardians. Subjects were
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excluded if they presented with unclear diagnosis and incomplete medical record data.
2.2. Genotyping
Five mL of blood were drawn from each subject into a vacutainer containing sodium
citrate. The genomic DNA was extracted using a commercially available kit (TIANamp
Blood DNA kit; Beijing, China) according to standard methods. The polymorphism at –
2518G/A in the MCP-1 gene was amplificated by polymerase chain reaction from genomic
DNA using the forward primer 5′-CTG ATC CAG GAT GAA AAT TTG G-3………., and
the reverse primer 5′-CCC ATG GCA ACT TGA GAG CTG G-3′………. One µliter (10 ng)
of genomic DNA was added to 99 mL of amplification buffer containing 20 mM Tris (pH
8.3), 50 mM KCl, 1.5 mMMgCl2, 0.001% gelatin, 0.2 mM each of dATP, dCTP, dGTP,
dTTP, 50nM of each primer, and 2.5 U of Taq DNA polymerase (AmpliTaq, Perkin-Elmer,
Norwalk, CT). The DNA was amplified by cycling at 94 °C for 1 min, 55 °C for 1 min, and
72 °C for 1.5 min. After 40 cycles, the reaction was extended for an additional 10 min at 72
°C. Then PCR product was digested with the restriction enzyme pvuII (10U per reaction) at
37 °C overnight and was electrophoresed in 2.5% agarose gels. Position -2518 of the distal
regulatory region is located within a unique PvuII restriction site in the 930 bp DNA segment
of the distal regulatory region sequenced in these studies. This restriction site is intact if G is
at position -2518. The ethidium bromide-stained gel shows that PvuII digests the 930 bp DNA
segment from G/G homozygous individuals into 708 and 222 bp fragments. DNA from A/A
homozygous individuals does not cut with PvuII. DNA from G/A heterozygous individuals
shows the expected fragments at 930, 708, and 222 bp. 100 bp DNA size markers are shown
in the last lane. In order to confirm genotype, several subjects were selected for DNA
sequencing techniques.
2.3. Statistical analysis
All data analyses were carried out using the Statistical Package for Social Sciences
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(Version 12.0 for Windows; SPSS, Inc., Chicago, IL, USA). For all data of 108 TS trios, the
Hardy-Weinberg equilibrium of the genotype distribution was tested using the homogeneity
Chi-square test. A family-based study was performed to assess genetic association by means
of haplotype relative risk (HRR) and transmission disequilibrium test (TDT) statistics. In
order to increase efficiency of test, we performed analysis by haplotype-based haplotype
relative risk (HHRR).
3. Results
3.1. Hardy-Weinberg equilibrium check
Genotype distributions of TS trios are in accordance with Hardy-Weinberg
equilibrium (χ2 <3.84, df = 1, P >0.05). –2518G/A polymorphism of MCP-1 showed no
significant differences in the genotype and allele distributions between patient groups and
parent groups. (χ2 = 2.38, P = 0.122; χ2 = 1.29, P = 0.255) (Table 1).
3.2. TDT test in the overall population
The analysis of association of polymorphisms in MCP-1 –2518G/A with TS was
carried out by TDT. Each parent can be summarized by the transmitted and the nontransmitted allele. Summarizing data were in the table 2. We did not detect biased
transmission of alleles from parents to their affected offspring in our informative samples in –
2518G/A polymorphisms (χ2 = 0.55, df = 1, P = 0.458).
3.3. HRR test
We used the HRR test for the association analysis. First, we used the genotype relative
risk, which compares the genotype in the affected offspring with the control genotype derived
from non-transmitted parental chromosomes (Table 3). We did not detect biased transmission
of genotype from parents to their affected offspring in our specimens in MCP-1 –2518G/A
polymorphism (HRR=1.054; 95% confidence interval: 0.558--1.993; df = 1; χ2 = 0.026. P =
0.871). In order to increase efficiency of test, we performed analysis by HHRR, and still no
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association was found (HHRR = 1.16; 95% confidence interval: 0.80--1.69; df = 1; χ2 = 0.59.
P = 0.44).
4. Discussion
Over the years, many researchers have made the observation that TS tends to be a
inflammatory disorder [9-12]. An indirect evidence was provided by imaging studies which
demonstrated both increases and decreases in volumes of nuclei and white matter structures of
TS cases [30], because these imaging results have been reported to be closely associated to
streptococcal infection [31]. Subsequently, Astrid et al. [32] also found a significantly
increased expression of MCP-1 in TS cases. MCP-1 is a critical chemokine closely associated
with neuro-inflammatory conditions of various etiologies while its deficiency protects against
inflammation in brain [24]. It can take part in a series of immune-related disorders such as
infection [33], allergy [34], autoimmune diseases [35], and cancer [36]. Recent study
indicated that interacting with the BBB endothelium, MCP-1 also took part in mediating
differentiation of neural stem cells into neurons, astrocytes and oligodendrocyte [25], and the
differentiation of adult subventricolar zone-derived progenitor cell migration after striatal cell
death [26], which indicated a intimate association between neurodevelopmental and neuroregenerative processes with inflammatory mediators.
We assumed that MCP-1 could increase the risk of TS and performed an association
study between TS and the –2518G/A polymorphism of MCP-1. We researched the association
using case-control study method based on nuclear family. Family-based linkage/association
studies have gained popularity, because they control for spurious associations between disease
and specific marker alleles due to population admixture. This method eliminates no
consistency of genetic background and avoids false positive result and phenomenon of
layered group structure. Unfortunately, no significant excess transmissions was found in the –
2518G/A of MCP-1 by our analyses, that was inconsistent with our hypothesis. There are
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several reasons for the negative conclusion. First, it is still possible that the G/A allele of the
polymorphism in MCP-1 may influence gene expression levels, whereas the increase of the
serum MCP-1 in TS patients isn't due to the alternative haplotype. The activation of the MCP1 may be caused by other physiologic malfunction or pathways prior to the death processes of
the patients. Moreover, the genetic heterogeneity of -2518G/A may be so high that we are not
able to dissect it by means of association studies alone, even with very large sample sizes.
It is important to note that this is the first report of relationship between MCP-1 2518G/A, and TS by family-based association study. As our results do not confirm an
involvement of the polymorphism in conferring susceptibility to TS, further studies and
different strategies are needed to better disentangle the underlying genetic architecture of TS.
Acknowledgements
We thank all probands for the participation. This work was supported by the National
Basic Research Program of China (2007CB511905), the National Infrastructure Program of
Chinese Genetic Resources (2006DKA21300).
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Table 1
Genotype distribution and relative allele frequencies of Mcp-1 –2518G/A gene in Chinese
patients with Tourette syndrome (n = 108) and parents
Patients
Parents
Genotype frequency
(%)
GG
GA
AA
22 (0.20) 62 (0.57)
27 (0.22)
44 (0.20) 110 (0.54) 56 (0.26)
Allele frequency
(%)
A
110 (0.50)
56 (0.26) 228 (0.53)
Hardy-Weinberg
equilibrium
χ2
P
2.38
0.122
1.29
0.255
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Table 2
The results of transmission disequilibrium test analysis
Transmitted allele
G
A
Total
Untransmitted allele
G
A
A = 44
B = 62
C = 54
D = 56
Y = 98
Z = 118
Transmission disequilibrium test = (b-c) 2/(b+c) = 0.55, P = 0.458.
Total
W = 106
X = 110
2N = 432
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Table 3
The results of haplotype relative risk analysis
Genotype of case
GG, GA
AA
Total
Genotype of control
GG GA AA
A = 84
B = 24
C = 83
D = 25
Y = 169
Z = 49
Total
W = 108
X = 108
N = 216
χ2 = 0.026, P = 0.871, haplotype relative risk = WZ/XY = 1.054, 95% confidence interval:
0.558 -1.993