|Year : 2021 | Volume
| Issue : 1 | Page : 22-27
Toll-like receptor gene polymorphisms in Indian patients with inflammatory bowel disease
Kirankumar Baskaran1, Srinivasan Pugazhendhi2, Balakrishnan Ramakrishna3
1 Department of Gastrointestinal Sciences, Christian Medical College, Vellore; SRM Medical College Hospital and Research Centre, Chennai, Tamil Nadu, India
2 Department of Gastrointestinal Sciences, Christian Medical College, Vellore, Tamil Nadu, India; Kansas University Medical Center, Kansas City, USA
3 Department of Gastrointestinal Sciences, Christian Medical College, Vellore; Institute of Gastroenterology, SRM Institutes for Medical Science, Chennai, Tamil Nadu, India
|Date of Submission||04-Aug-2020|
|Date of Decision||05-Aug-2020|
|Date of Acceptance||05-Aug-2020|
|Date of Web Publication||04-Dec-2020|
SRM Institutes for Medical Science, Chennai, Tamil Nadu
Source of Support: None, Conflict of Interest: None
Background: Defective innate immune recognition of gut microbes may be one reason for the genesis of inflammatory bowel disease (IBD). Genetic defects in IBD show considerable variation between populations. This study evaluated three single-nucleotide polymorphisms (SNP) in the toll-like receptor (TLR) genes in patients with IBD and healthy controls. Methods: DNA isolated from peripheral blood of 200 IBD patients (100 Crohn’s disease and 100 ulcerative colitis) and 100 healthy controls were subjected to polymerase chain reaction and restriction enzyme digestion for three SNPs in TLR4 (D299G, rs4986790), TLR5 (392 stop codon, rs5744168) and TLR9 (-1486 T/C, rs187084) genes, and genotypes assigned according to the restriction fragment length polymorphism pattern. Results: Minor allele frequencies for the 3 polymorphisms were not significantly different between IBD patients and healthy controls. The TLR4 rs4986790 minor allele (G) frequency was 27/200 in controls compared to 48/400 in IBD (odds ratio [OR] 0.87, 95% confidence interval [CI] 0.52–1.44, P = 0.6007). The TLR5 rs5744168 minor allele (T) frequency was 19/200 in controls compared to 45/400 in IBD (OR 1.20, 95% CI 0.63–2.12, P = 0.5132). The TLR9 rs187084 minor allele (C) frequency was 71/200 in controls compared to 129/400 in IBD (OR 0.88, 95% CI 0.61–1.26, P = 0.4261). Conclusion: These three polymorphisms in the TLR genes were not associated with IBD in the Indian population.
Keywords: Crohn’s disease, genetics, innate immunity, ulcerative colitis
|How to cite this article:|
Baskaran K, Pugazhendhi S, Ramakrishna B. Toll-like receptor gene polymorphisms in Indian patients with inflammatory bowel disease. Gastroenterol Hepatol Endosc Pract 2021;1:22-7
|How to cite this URL:|
Baskaran K, Pugazhendhi S, Ramakrishna B. Toll-like receptor gene polymorphisms in Indian patients with inflammatory bowel disease. Gastroenterol Hepatol Endosc Pract [serial online] 2021 [cited 2021 Jan 16];1:22-7. Available from: http://www.ghepjournal.com/text.asp?2021/1/1/22/302218
| Introduction|| |
Inflammatory bowel disease (IBD) is caused by an abnormal immune reaction to commensal microbiota in the gastrointestinal tract in a genetically predisposed host., The three players in the genesis of IBD thus are a genetic predisposition that is not well defined in most patients, largely unknown microbes residing in the gastrointestinal tract, and an abnormal immune driven inflammation. The gastrointestinal tract behaves as a primary lymphoid organ. The luminal microbiota of the gastrointestinal tract interacts with innate immune cells in the intestine through pattern recognition receptors, in turn influencing the differentiation of T-lymphocytes to different lineages of T-helper cells.,, Cytokines released as part of the effector immune responses contribute to inflammation in the intestine. Microbial molecules are recognized by pattern recognition receptors on the cell surface or in the cytoplasm of cells, chief of which are the toll-like receptors (TLR) and the NOD-like receptors (NLR).
The earliest IBD mutations identified were in the NOD2 gene., These mutations affect innate immune responses to commensal bacteria of the gastrointestinal tract leading to excessive production of proinflammatory cytokines and inflammation., Intracellular recognition of bacterial products such as lipopolysaccharide (LPS) and peptidoglycan derived from Gram-negative and positive bacteria, respectively, is facilitated by the Nod2 protein, which is a cytosolic pattern recognition receptor. Subsequent activation of nuclear factor ĸB triggers an inflammatory response. However, while NOD2 gene mutations are common in Western IBD patients, they are quite uncommon in Indian IBD patients., Several of the other genes that have been associated with IBD in Indian patients also affect the innate immune system recognition and handling of bacteria and innate effector response, including the ATG16 L1 gene, IRGM gene, and the TNFSF15 genes.,,
TLR are cell surface molecules that recognize a variety of pathogen-associated molecular patterns. Altered TLR signaling has been associated with a number of inflammatory and infectious diseases. TLR4 is a transmembrane protein, which plays a vital role in the recognition of bacterial LPS. Intestinal epithelial cells, macrophages, and dendritic cells express TLR4 during intestinal inflammation. TLR5 detects flagellin from bacteria, and polymorphisms in this gene have been described to be protectively associated with systemic lupus erythematosus. TLR9 senses cytosine guanosine nucleotides of bacterial DNA, leading to attenuation of intestinal inflammation.
We undertook this case–control study to assess the frequency of specific polymorphisms in the TLR4 (D299G), TLR5 (392stop), and TLR9 (-1486T/C) genes in healthy controls and patients with IBD.
| Methods|| |
This study included a total of 200 IBD patients (100 with CD and 100 with UC), and 100 unrelated healthy controls. Participants were recruited from patients attending the IBDs and Gastroenterology Clinics of the Department of Gastrointestinal Sciences in Christian Medical College, Vellore. Inclusion criteria were the age of 18 years or above and diagnosis of IBD (ulcerative colitis or Crohn’s disease) based on standard consensus criteria specified by the Indian Society of Gastroenterology Task Force on IBD., Patients with proven intestinal or extra-intestinal tuberculosis and those who refused consent to participate were excluded from the study. Controls were recruited from unrelated healthy individuals, without gastrointestinal symptoms, attending the outpatient clinic for health check-ups. As there may be regional differences in the prevalence of genetic polymorphisms, the patients and controls were matched for the region and compared accordingly. Patients were investigated, and clinical data were recorded according to the usual clinical protocol followed in the Department. Informed written consent was obtained from all participants for the genetic analyses. The consent forms and protocol were approved by the Ethics Committee of the Christian Medical College, Vellore (IBD on treatment/or naïve cases, on probiotics, rifaximin, etc.).
A volume of 5 ml samples of venous blood were collected in EDTA-coated Vacutainer tubes. DNA was isolated from whole blood using the salting-out method, and the yield and quality checked. Three single nucleotide polymorphisms (SNPs) were evaluated – TLR4 -896A/G (D299G) polymorphism, TLR5392 stop (C1174T) polymorphism, and TLR 9 -1486T/C polymorphism, using primers that have been described previously.,, These polymorphisms are called rs4986790, rs5744168, and rs187084, respectively. Three separate polymerase chain reactions (PCRs) were used to amplify the target segment of the respective genes, followed by enzymatic digestion and gel electrophoresis for restriction fragment length polymorphism analysis. The PCRs were done as per the following protocol after the initial standardization of the conditions in a gradient PCR. PCRs were performed using 1x Taq DNA Polymerase Master Mix Red, which was composed of 0.2 units/ml AmpliqonTaq DNA Polymerase, the NH4+ buffer system, 0.4 mM dNTPs, 1.5 mM magnesium chloride and 200nM of forward and reverse primer in 20 ml reaction volume. The PCR conditions were optimized by performing gradient PCR. The PCR conditions involved initial denaturation at 95°C for 5 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing for 30 s, initial extension at 72°C for 30 s, and a final extension at 72°C for 10 min. The annealing temperatures varied for each of the three SNPs. The forward and reverse primers, annealing temperature, and amplicon length are shown in [Table 1]. The PCR products were electrophoresed on 2% agarose gel to check the amplification. Amplified products were digested with the appropriate restriction enzyme in a reaction volume of 20 ml at 37°C in a water bath for 16 h. The digestion patterns were obtained by resolving the digestion products on 2% agarose gel electrophoresis, and the digestion pattern was captured using gel documentation system. The restriction enzyme used and the fragment lengths obtained for each SNP are shown in [Table 1]. For the TLR4 polymorphism, the wild type was AA (amplicon remained undigested, 249 bp), and the polymorphic gene was GG. For the TLR 5 polymorphism, TT was the wild type (digested by the restriction enzyme), and the mutant was CC. For the TLR9 polymorphism, the wild type was TT (not digested by the enzyme), while the mutant was CC.
|Table 1: Specific polymerase chain reaction primers and restriction enzymes for each single nucleotide polymorphism|
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Allele and genotype frequencies in IBD patients and in controls were compared for association with the disease using the Fisher’s exact test.
| Results|| |
The demographic details of the control and the IBD participants are shown in [Table 2].
The PCR product and restriction enzyme patterns for the 3 SNPs are shown in [Figure 1], [Figure 2], [Figure 3]. The restriction patterns for each participant were used to assign the genotype as homozygous wild type, homozygous mutant, or heterozygous. The gene counts and the allele counts for each SNP are shown in [Table 3] and [Table 4], respectively. As is clear from these tables, there was no significant difference in the frequency of these polymorphisms between healthy controls and IBD patients. The tables also show that the subgroups of UC and CD had nearly identical counts of these SNPs. The minor allele frequencies (MAFs) for the three SNPs were calculated. For the TLR4 rs4986790 polymorphism, the minor allele (G) frequency was 0.135 in controls compared to 0.120 in IBD. For the TLR5 rs5744168 polymorphism, the minor allele (T) frequency was 0.095 in controls compared to 0.1125 in IBD. For the TLR9 rs187084 polymorphisms, the minor allele (C) frequency was 0.355 in controls compared to 0.3225 in IBD. There was no significant difference between patients with Crohn’s disease and ulcerative colitis.
|Figure 1: The restriction digestion pattern for D299G polymorphism. The lanes 1, 2, 4, 7 displayed wild type digestion pattern (AA) while the lanes 3, 5, 6, 8 represented partial digestion corresponding to heterozygous pattern (AG). Lane 9 showed complete digestion pattern analogous to mutant homozygote (GG). The smallest fragment size 26 bp could not be seen. Marker lane corresponds to pUC19 DNA/MspI marker|
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|Figure 2: The restriction digestion pattern for392stop polymorphism. The digestion pattern was same in all the samples showing wild type genotype (CC) with the exception of lane 4 and 7 displaying heterozygous pattern (CT) due to partial digestion. Marker lane corresponds to pUC19 DNA/MspI marker|
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|Figure 3: The restriction digestion pattern for-1486T/C polymorphism. The lanes 2, 4, 5, 6 displayed wild type digestion pattern (TT) while the lanes 1, 3, 7, 8 represented partial digestion corresponding to heterozygous pattern (TC). Lane 9 showed no digestion analogous to mutant homozygote (CC). Marker lane corresponds to pUC19 DNA/MspI marker|
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|Table 3: Genotype counts of toll-like receptor 4, toll-like receptor 5 and toll-like receptor 9 polymorphisms in controls, Crohn's disease and ulcerative colitis patients|
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|Table 4: Allele frequencies in controls and inflammatory bowel disease patients at the three polymorphic sites that were tested in this study|
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| Discussion|| |
This short report essentially did not find any link between the three TLR polymorphisms tested and IBD, either Crohn’s disease or ulcerative colitis. The major significance of this report is that it provides MAFs for each of the three polymorphisms in the Indian population.
The role of the innate immune system in the pathogenesis of IBD is well established. The cytosolic receptor NOD2 is the pattern recognition receptor that has been most clearly associated with IBD genesis. TLRs are important in innate immune recognition of gut microbes. TLR4 polymorphisms impair the innate immune response to LPS. Upregulated TLR4 expression has been described in IBD, both CD and UC. Franchimont et al. in a Belgian population reported a two-fold elevation in allele frequency of TLR4 D299G polymorphism in both Crohn’s disease and ulcerative colitis Henckaerts et al., again in the Belgian population, observed nearly identical increased frequency of TLR4 D299G in both forms of IBD compared with controls (5%) The TLR4 D299G polymorphism was more common in Crohn’s disease compared to controls both in Greek and German populations., However, studies in the Dutch, southern Italians, and Tunisians, did not find any association between this TLR4 polymorphism and IBD.,, Studies in the Asian populations, including Japanese, Chinese, and Koreans, did not find an association of this polymorphism with IBD.,,, On the other hand, in a fairly small study of 139 Indian patients and 176 controls, Sivaram et al. found that the TLR4 D299G polymorphism was significantly more common in patients with ulcerative colitis and was associated with increased expression of mRNA for TLR4. TLR4 and TLR5 are known to be expressed much more in the colon than in the small intestine and therefore it is possible that we may have observed an association in our study also if we had selected purely colonic IBD.
The stop codon variant in TLR5 has been reported to have a protective association with CD as substantiated by the marked reduction in the frequency of TLR-5 stop allele (T), with only 0.9% of CD patients being positive for this allele compared with unaffected relatives (5.4%), unrelated controls (6.5%) and UC patients (6.0%) in a Jewish population. This was in direct contrast with our report, where we did not identify any significant difference in the stop codon allele frequency between cases and controls. Limited data also exist for an association between the TLR9 polymorphism and IBD. The CC genotype of TLR-9 -1486T/C polymorphism was reported to be significantly more common in Japanese UC patients (41.7%) compared to controls (21.3%).
In summary, our study detected no significant association of TLR 4 (D299G), TLR 5 stop codon and-1486 T/C gene polymorphisms with IBD in our population. This is in contrast to studies mainly from the Western populations indicating a role for these polymorphisms in subsets of patients with IBD.,,,,, The study was limited by small sample size. However, this study provides important information on the MAF for these 3 SNPs in the Indian population. The highest MAF was for the TLR9 polymorphism (around 0.35) and to detect an odds ratio of 0.8 with an error of 5% and study power of 80% we will require to study 1500 controls and 1500 IBD patients/for the other polymorphisms where the MAF is around 0.1, and the necessary sample sizes will be more than 3500 controls and an equal number of IBD patients. It seems unlikely, therefore, that the TLR polymorphisms studied here contribute significantly to the genesis of IBD in our population.
| Conclusions|| |
Three common polymorphisms in the TLR genes were not associated with IBD in the Indian population in this limited study. Replication studies will be useful to validate this conclusion.
The study was approved by the institutional Ethics Committee of the Christian Medical College, Vellore. All studies were conducted in accordance with the relevant guidelines and regulations.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3], [Table 4]