Download Single-nucleotide polymorphisms of toll

Single-nucleotide polymorphisms of toll-like receptor 9 gene and its associated
risk in ulcerative colitis
Running title: TLR9 SNPs in Chinese UC patients
Disclosure
The authors have no conflict of interests, and the work was not supported or funded
by any drug company.
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Abstract
Background with Objective: Ulcerative colitis patients have a high risk to develop
colorectal cancer. Toll-like receptor 9 (TLR9) plays a key role in the innate immune
system in gastroenterological tract and TLR9 polymorphisms could contribute to
development of ulcerative colitis. We carried out this study to determine whether
single nucleotide polymorphisms covering TLR9 gene is associated with Ulcerative
colitis in Chinese Han population.
Methods: Genomic DNA from the peripheral blood of 110 patients with ulcerative
colitis and 112 healthy controls were analyzed for TLR9 rs187084 and rs352140
polymorphisms using PCR-RFLP for risk of ulcerative colitis. All subjects were
recruited from the outpatient department of digestology of the second hospital of
Harbin medical University between January 2010 and January 2012.
Results: There was no evidence showing an association between TLR9 gene rs187084
and rs352140 polymorphisms and ulcerative colitis risk or disease progression.
Conclusion: In this study of Chinese Han patients with ulcerative colitis, there is no
association between TLR9 rs187084 and rs352140 polymorphisms and risk of
ulcerative colitis.
Keywords: Toll-like receptor 9, Single nucleotide polymorphism, Ulcerative colitis,
Innate immunity
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Introduction
Ulcerative colitis patients have a high risk to develop colorectal cancer.
Approximately 16% of such patients will develop either a cancer precursor or colon
cancer over 30 years 1. Clinically, ulcerative colitis is considered as an autoimmune
disease and treated by anti-inflammatory or immunosuppression drugs 2.
Pathologically, ulcerative colitis is a disease with multiple genes involved in the
immune system. To date, a number of studies have been reported on the association of
different gene polymorphisms with development of ulcerative colitis 3.
Toll-like receptors (TLRs) are a family of evolutionarily conserved proteins and play
a key role in the innate immune system by directly recognizing pathogen-derived
elements, including viral and bacterial DNA, lipopolysaccharides, and proteoglycans.
The TLR ability to recognize microbial components and to trigger activation of innate
immunity and/or adaptive immunity in human body makes them excellent candidate
genes to be investigated for its association with various autoimmune diseases,
including ulcerative colitis. TLR9 polymorphisms were shown to associate with
susceptibility to many diseases, including immunodeficiency disorders, Crohn’s
disease, and inflammatory disorders 4. TLR9 was expressed in colorectal cancer and
TLR9 agonist leads to decreased cell survival and induced apoptosis of colon cancer
cells 5. Therefore, our study investigates the association of single nucleotide
polymorphisms of TLR9 gene with ulcerative colitis in Chinese Han patients.
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Method
Study population
After informed consent, patients with unrelated ulcerative colitis (n = 110) were
recruited from Gastroenterology Department in The Second Hospital of Harbin
Medical University, China. Healthy and ethnically matched blood donors (n = 112)
were recruited from the Harbin regions as the controls. All patients were diagnosed as
ulcerative colitis based on clinical, endoscopical, radiological, and histopathological
criteria guided by the cooperated IBD group of Chinese Medical Association
Digestive Branch in 2007. All control subjects were matched with healthy Chinese
Han citizens who visited our hospital between January 2010 and January 2012. 5ml of
blood was collected from the individuals in vacuum EDTA tubes and stored at -20°C.
This study was approved by the Ethical Review Committee of Research in Harbin
Medical University.
Genotyping
Genomic DNA was extracted from EDTA-preserved peripheral blood samples by
using a blood genomic DNA extraction kit (Tiangen, Beijing, China) according to the
manufacturer’s instructions and then stored at -20°C until further use.
A polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP)
method was utilized to detect of TLR9 gene SNPs using markers of rs187084 and
rs352140 according to a previous study 6. After PCR, the products were digested with
restriction enzymes AflII or BstUI and then run on 1.5% agarose gels and visualized
and photographed under UV illumination. The different TLR9 genotypes were
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determined by the number of bands after the digestion of PCR products.
Statistical analysis
To compare the distributions of genotypic frequency between the cases and controls,
x2 test and Fisher’s exact test was performed using SPSS16.0. (SPSS, Chicago, IL,
USA). The Hardy–Weinberg equilibrium was performed for cases and controls. The
odds ratios with 95% confidence interval (CI) were then calculated. Statistical
significance was defined as p<0.05.
Results
The genotypes of these case and control subjects were in Hardy–Weinberg
equilibrium. However, there was no statistically significant deviation of these two
TLR9 gene polymorphisms between the cases and controls. The clinicopathological
characteristics of the patients are shown in Table 1. The data showed that both the
case and control groups were well balanced in terms of age and sex distribution. Male
to female ratio was 1.3:1 and 1.8:1.
Genotype and distribution of allele frequencies in ulcerative colitis patients and
healthy controls are shown in Tables 2, The dominating disease extension of
Ulcerative colitis was proctitis (36.36%)，and the disease scale division was midrange
(53.64%), and none of these two TLR9 SNPs showed any statistically significant
association with ulcerative colitis risk (p>0.05). No significant differences were
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observed between ulcerative colitis patients and healthy controls in frequencies of the
carriership of the alleles rs187084 (x2= 0.625, p = 0.433) and rs352140 (x2= 2.367, p =
0.124). Furthermore, there were no statistically significant differences between the
clinicopathological parameters from the patients and TLR9 gene polymorphisms
(p>0.05).
Discussion
In the present study, we aimed to investigate the association between Ulcerative
colitis and SNP polymorphisms in TLR9 in Chinese Han population. Our current data
showed that there is no association between Chinese Han patients with ulcerative
colitis and TLR9 polymorphisms, which contradicted the data from a Japanese study,
in which TLR9 gene polymorphisms were associated with severity of ulcerative
colitis in Japanese patients, and in their study TLR9-1486CC, 1174GG and 2848AA
increased the risk of UC and TLR9 -1486TT, 1174AA and 2848GG decreased the risk
of UC, although there were no correlations between SNPs and disease phenotype or
TLR9 mRNA expression.
Toll-like receptors (TLRs) are evolutionarily conserved components of the innate
immune system that act to directly recognize pathogen-derived elements, including
viral and bacterial DNA, lipopolysaccharides, and proteoglycans. They provide a
crucial link between the recognition of pathogens by the innate immune system and
subsequent activation of adaptive immunity, inducing maturation of antigen
presenting cells and differentiation of T cells. So they play a central role in innate
immunity. TLR9 (chromosome 3p21.3, GenBank accession number NM_017442) is a
member of the family that recognizes bacterial unmethylated CpG-DNA motifs.
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Previously, TLR9 polymorphisms were associated with various autoimmune or
infectious diseases, including Crohn’s disease. TLR9 is able to recognize
unmethylated CpG oligodeoxynucleotide DNA of bacteria and DNA viruses. The
immunostimulatory effects of CpG-DNA can initiate and/or modulate autoimmunity
by inducing the inflammatory cells and production of cytokines, such as interleukin 6
(IL-6), IL-8, IL-12, and TNF, some of which may be associated with development of
ulcerative colitis and colorectal cancer or for their therapy
7, 8
; TLR9-induced
signaling pathway may play an important role in development of stomach, lung, and
bowel cancers. Ulcerative colitis is a heterogeneous polygenic disease and the
associated studies reveal the susceptibility genes may depend on the patients’ ethnic
background.
So we carried out this study to determine whether single nucleotide
polymorphisms covering the TLR9 are associated with Ulcerative colitis in Chinese
Han population, in order to increase understanding of how TLR9 gene polymorphisms
affect development or progression of Ulcerative colitis.
Our results indicate that none of the two TLR9 SNPs showed a statistically significant
association with Ulcerative colitis risk in our cohort. So TLR9 may be unable to
explain how innate immune system is involved in chronic inflammatory condition
including Ulcerative colitis. As Ulcerative colitis is a heterogeneous polygenic disease,
association studies are expected to reveal various sets of susceptibility genes
depending on the ethnic background of the study populations.
In conclusion, we found no association between TLR9 polymorphisms and risk of
ulcerative colitis in Han Chinese patients. Further studies in different ethnic
population may resolve the role of TLRs polymorphisms in ulcerative colitis
susceptibility.
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Acknowledgment
This work was supported by the Nature Science Foundation of Heilongjiang Province
(D201178) and the research projects of Heilongjiang Provincial Health Department
(2011-094).
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Table 1. TLR-9 rs187084 and rs352140 genotype distributions in cases and controls
Gene (LR9)
UC patients
Controls
(n = 110)
(n = 112)
TT
34 (30.9%)
36 (32%)
TC
58 (52.7%)
48 (42.9%)
CC
18 (16.4)
28 (25%)
T
126 (57.3%)
120 (53.6%)
C
94 (42.7%)
104 (46.4%)
AA
25 (22.7%)
33 (29.5%)
AG
44 (40%)
46 (41.1%)
GG
41 (37.3%)
33 (29.5%)
A
94 (42.7%)
112 (50%)
G
126 (57.3%)
112 (50%)
X2
p value
(<0.05)
rs187084
3.157
0.206
0.615
0.433
1.995
0.369
2.361
0.124
Alleles
rs352140
Alleles
P values < 0.05 are bolded.
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Table 2. Demographic and clinicopathological features of UC patients (Mean ± SD)
Demographic features
UC patients (n=110)
Age in years (Min–Max)
38.64 ± 14.5 (16–65)
Sex (Female/Male)
48/62
Age of diagnosis (Years)
≤40 years
76 (69.09%)
>40 years
34 (30.91%)
Disease location
Proctitis
40 (36.36%)
Left colitis
38 (34.55%)
Right colitis
3 (2.73%)
Pancolitis
29 (26.36%)
Disease severity
Light
38 (34.55%)
Mild
59 (53.64%)
Severe
13 (11.82%)
Disease type
Initial
10 (9.1)
Chronic continuation
9 (8.2)
Chronic recidivation
91 (82.7)
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