Investigation of TLR2 (-196 to -174del) and TLR9 (T-1486C) Gene Polymorphisms Association with Inflammatory Bowel Diseases

Background: Inflammatory bowel diseases (IBD), Crohn’s disease (CD), and ulcerative colitis (UC) are diseases that result from the combined effects of a predisposing genetic background and several environmental factors, including smoking. Some genes can influence these diseases through genetic inheritance, and their regulation is explained by gene polymorphism. However, Toll-like receptor (TLR) genes have been identified as susceptibility genes for CD and UC. Methods: A case-control study was performed on a Turkish population composed of 105 healthy controls and 79 CD, 77 UC patients genotyped by Allele-specific PCR and PCR–RFLP for TLR9 (T-1486C) and TLR 2 (-196 to -174del) gene. Genotype and allele frequencies of TLR9 (T-1486C) and TLR 2 (-196 to -174del) gene polymorphisms compared to allele frequencies in CD and UC patients. Results: No statistically significant findings were found between the CD, UC patients, and the control group in terms of both genotype distributions and allele frequencies for TLR 9 (T-1486C; rs187084) and TLR 2 (-196 to -174del; rs111200466) gene polymorphisms in a Turkish population (P > 0.05). Conclusion: No association was found between the TLR2 (rs111200466) and TLR 9 (rs187084) gene polymorphisms among IBD patients and the control groups in the Turkish population.


Introduction
Inflammatory Bowel Diseases (IBD) comprises mainly Crohn's Disease (CD) and Ulcerative Colitis (UC), and in between these two diseases is Indeterminate Colitis (IC); these diseases possess specific effects on various regions and layers of the gastrointestinal tract [1].These diseases are known for their chronic inflammatory disorders in the gastrointestinal tract, which have been experimentally defined, and their clinical, pathological, endoscopic, and radiological features have been completely determined [2].IBD has increased tremendously globally over the last decade, especially in the European and Asian populations, and its daily increase has been reported [3,4].In IBD, CD is a disease that causes treatable inflammation in any region of the intestinal epithelium along the gastrointestinal tract [1].Although the clinical findings in CD vary depending

Investigation of TLR2 (-196 to -174del) and TLR9 (T-1486C)
Gene Polymorphisms Association with Inflammatory Bowel Diseases radiological, and biochemical findings of the physical examination ascertained [5].It has been determined that infectious agents, various enteric bacteria, viruses, chlamydia, mycobacteria, and dietary factors are involved in the etiology of CD [6,7].The other variant of IBD, UC, is a disease that causes long-term inflammation in a part of the intestinal epithelium and mainly in the colon of the digestive tract [1].Ulcerative colitis (UC) is a chronic inflammatory disease characterized by mucosal inflammation primarily affecting the colon.The disease is classified based on the anatomical region of the large intestine, ranging from proctitis (limited to the rectum) to pancolitis (involving the entire colon).The exact cause of UC is not fully understood, but it is believed to be influenced by a combination of environmental and genetic factors.UC is more commonly observed in developed countries.[8].It has been suggested that environmental and genetic components affect the prevalence of IBD, such as gastroenteritis, smoking habits, and alcohol addiction.Unaccounted risk factors (e.g., dietary, obesity, antibiotic use, improved hygiene, smoking, etc.) CD incidence Some research shows that the intake of saturated fat and refined sugar alters the gut's microenvironment and increases the risk of developing IBD [3].It has been determined that the most important common feature of UC and CD is a genetic predisposition, and the long duration of these diseases increases the risk of cancer in patients [9,10].It is interesting to know that the signs and symptoms of both diseases are very similar [11,12].These diseases are included in the important colon and rectal diseases group because of the complications and malignant potential they cause in extra-intestinal organs [7].The most valid hypothesis in the pathogenesis of IBD has been expressed as an abnormal inflammatory response in the gastrointestinal tract with the effect of various factors (such as microbial antigen, autoantigen, and smoking) under genetic predisposition.In addition, in Mahid's meta-analysis of 245 studies revealed 22 articles that highlighted the link between IBD and smoking, and it has been shown that smoking affects UC and CD differently [13].It has been shown that this inflammatory response to the protection of the immune system is controlled by suppression.Still, intestinal damage occurs because this inflammation cannot be suppressed in genetically predisposed individuals [12].According to the data obtained because of the expression of surface antigens in a patient's peripheral blood cells with CD and UC, it has been suggested that there are important differences between these two diseases [14,15].In addition, according to the autoimmune hypothesis, it has been reported that autoantigens expressed in the intestinal epithelium of these patients cause different mechanisms that trigger the immune system [16].The genetic predisposition to these diseases was revealed by the determination of mutations in the NOD2/CARD15 gene on the 16th chromosome, which affect most of the population and are generally seen in young adults, especially in the western world [17].It has also been shown that geographical and ethnic factors are also important in the etiology of these diseases [18][19][20].IBD is usually seen in young adulthood in humans, and even if most of the affected individuals are treated, it has been determined that the disease progresses and reoccurs in these individuals, thus turning into a chronic disease [2].It has also been reported that individuals affected by CD have clinical propensity at any age [21].The prevalence of these diseases increases over time, it characterized by an unstable inflammatory response, and it is an autoimmune disease that occurs as a product of a deviation in the autoimmune response to intestinal bacteria or other foreign biological materials [4,22,23].
Inflammatory bowel disease -notably Crohn's disease (CD) and ulcerative colitis (UC) tend to be facilitated by various risk factors, and smoking is chief among them.Research in this matter over the years has provided ample evidence that smokers face a notably higher chance of developing CD compared with their counterparts who do not smoke, and those who are diagnosed with the condition also have more complications.While it is less evident whether smoking induces or worsens UC.Medical analysis so far has been ambiguous.Given such potential hazards, there is an urgent need for smoking cessation measures to provide relief for individuals suffering from these conditions, whereby studies support such recommendations as stated in Targownik's work from 2009 onwards [24].
Genetic polymorphism studies in IBD have also reported that individual variations, SNPs, may contribute significantly to the formation of these diseases.Gene polymorphism studies conducted to determine genetic differences in individuals with IBD have suggested that alleles in some genes in humans may be associated with genetic susceptibility alleles, and one of these genes in polymorphisms were seen in Toll-like receptor (TLR) genes [23].In humans, TLRs have been reported as genes forming a group of transmembrane proteins found in monocyte cells, dendritic cells, and macrophage cells that play a significant role in the innate immune system [25].It has been determined that TLRs are generally expressed by the cell surface of innate immune cells, and they are very important regulators in maintaining the balance between the intestinal commensal bacteria and the mucosal immune system [23].They have also been found to have superior roles as mediators of intestinal inflammatory pathways [26].The ability of TLR gene products to react differently to their ligands is due to single-nucleotide polymorphisms (SNP) occurring in TLR genes, and genetic variations in these genes have been associated with susceptibility to diseases.
Research from different populations and cohorts has shown contradictory results regarding the association between inflammatory bowel disease (IBD) and polymorphisms in the TLR2 and TLR9 genes.Certain TLR2 polymorphisms have been linked in certain studies to an increased risk of IBD, which may have an effect on TLR2 function and microbial identification.Differential clinical symptoms, the location, behavior, and severity of the illness have all been associated with TLR2 variations.TLR9 polymorphisms, such rs5743836 and rs352140, may change TLR9 activity and microbial DNA recognition, and they have been linked to an increased risk of developing IBD.But because of the complexity of the connection between TLR2 and TLR9, further study is required to completely comprehend the processes and therapeutic implications [27].
In This case-control study, aims to investigate TLR 9 (T-1486C) and TLR 2 (-196 to -174del) gene polymorphisms in CD and UC patients, whose clinical features and epidemiology are very similar, as an distinguishing factor in terms of these diseases using PCR and PCR-RFLP method in the Turkish population.

Materials and Methods
The peripheral blood samples used in this investigation were obtained from 156 patients who applied to Nigde State Hospital Gastroenterology Clinic between 2013 and 2015 who were diagnosed with UC (77) and CD (79) clinically, endoscopic, or histopathological, and 105 healthy individuals who had no previous bowel disease as the control group as detailed in Table 1.

Isolation of DNA
Using the DNA isolation kit (QIAGEN GmbH, the control group.This research ascertained the genetic association between the patient and control groups using odds ratios (OR) and 95% confidence intervals (CI).Following a significance test, the same evaluation was carried out once more while accounting for factors such as age, gender, and smoking (Figure 3).The threshold for statistical significance was set at P-value < 0.05 (P-value 0.05).Furthermore, the online computation method developed by Michael H. Court (2005Court ( -2008) ) was used to do the Hardy-Weinberg analysis on the patient and control groups.

Results
The study found significant differences in demographic characteristics between the control group (N=105), CD (N=77) patients, and UC (79) patients.The CD group had a slightly reduced eventuality of developing CD, UC, or IBD compared to the control group, while the UC group had a somewhat higher likelihood.These findings contribute to understanding the connection between demographic factors and the occurrence of CD, UC, and IBD results shown in Figure 3 and Table 1. a significant difference in smoking rates between the CD group and the control group, with a 1.88 odds ratio, indicating a 1.88 times higher number of smokers in the CD group.

Analysis of the TLR9 (T-1486C; rs187084) and TLR2 (-196 to -174 del; rs111200466) gene polymorphisms in CD
The study analyses TLR9 (T-1486C) and TLR2 (-196 to-174 del) gene polymorphisms in DNA samples from 79 CD and 105 control groups.Results show no significant differences in genotype distributions or allele frequencies between CD and control groups (Tables 2, 3).

Analysis of the TLR9 (T-1486C; rs187084) and TLR2 (-196 to -174 del; rs111200466) gene polymorphisms in UC patients
The study evaluated UC patients' and a control group's TLR9 and TLR2 gene polymorphism data.The results showed no significant difference in genotype and allele frequencies between the two groups.The TLR2 gene polymorphism genotype distributions were consistent with the Hardy-Weinberg equation, and no significant difference was observed between the patients and control Maryland, USA), DNA was extracted from 0.2 ml blood samples according to the manufacturer's instructions protocol and eluted in 200µl D.w.The concentration and purity of the extracted DNA were determined using a Nanodrop Spectrophotometer.The samples were then stored in a refrigerator at -20°C until further analysis.Used PCR-RFLP and Allele-Specific PCR (AS_PCR) techniques, the TLR2 (-196 to -174 del; rs111200466) and TLR9 (T-1486C; rs187084) gene polymorphisms were genotyped [28,29].

PCR and RFLP
The PCR conditions were adjusted according to the specifications outlined in the program in Table 1S (S; Supplementary material).Each reaction was conducted in a 25μl volume using 200nM of primers specified in Table 2S.The PCR reactions were prepared with the 2xTaq DNA polymerase Master Mix from (Vivantis).
Subsequently, the PCR products underwent gel analysis.TLR2 genotyping employed a 2% agarose gel, and the PCR fragments were visualized using RedSafe, a safe stain provided by (iNtRON Biotechnology), and visualization utilized (Fusion Fx Imaging System from Vilber Lourmat, utilizing UV light) shown in Figure1S, 2S.TLR9 genotyping, the PCR products digested with the AflII restriction enzyme, separated on a 3% agarose gel as shown in Figure 2, the resulting gel image captured in the documentation system.
As-PCR For TLR2 genotyping, the PCR products were separated on a 3% agarose gel, as shown in Figure 1; the resulting gel image was captured in the documentation system.

Statistical Analysis
The SPSS 22.0 package program was used to analyze the Inflammatory Bowel Disease (IBD) patients and control groups statistically.The age variable underwent normal distribution analysis utilizing the Kolmogorov-Smirnov test as a demographic trait and etiological data.The non-parametric Mann-Whitney U (M-W) test was used since the data did not fit the normal distribution.
Binary logistic regression and Chi-Square (X 2 ) analysis were used to compare the homozygous and heterozygous genotype frequencies as well as the allelic frequencies in the TLR2 (-196 to -174del) and TLR9 (T-1486C) genes between the patient groups (CD, UC) and    The study concluded that the TLR9 and TLR2 gene polymorphisms were consistent with the Hardy-Weinberg equation and did not show any deviation between the two groups (Tables 4, 5).

Discussion
There have been reports of correlations between TLR9 polymorphisms and atherosclerosis, SLE, IBD, and asthma [30].Several researchers have investigated potential connections between TLR9 polymorphisms and IBD because the TLR9 gene's close to associated with Crohn's disease and ulcerative colitis susceptibility [31].Emingil et al. (2007) examined the susceptibility of TLR2 (Arg753Gln and Arg677Trp) and TLR4 (Asp299Gly and Thr399Ile) gene polymorphisms in patients with generalized aggressive periodontitis in the Turkish population.They discovered no significant difference in distribution and allele frequencies (P>0.05)[32].The host's innate immune system plays a critical role Investigation of TLR2 (-196 to -174del)   in the recognition and neutralization of gram-negative bacteria, and the polymorphism in the BPI gene, called Lys216Glu, has been studied in CD and UC patients in the Turkish population.This gene is on the suspect list of IBD pathogenesis.CD and UC have been identified to be related to the BPI (Lys216Glu) polymorphism [33].This study evaluated the susceptibility status of rs187084 polymorphisms in patients with CD and UC.We on purpose chose -196 to -174del for the TLR 2 gene and T-1486C for the TLR 9 gene.Our findings showed that genotypes and alleles of TLR 2 (-196 to -174del) did not substantially differ between the patients and control groups.Similar to our work, Hishida, A. et al. 2010 looked into the association between gastric cancer risk and TLR 2 (-196 to -174del) polymorphism in the Japanese population.They found no evidence of a connection between the two groups [34].It was discovered that there was no statistically significant difference in the genotype and allele frequencies of the TLR 2, TLR  Smoking is a known risk factor in the causation of numerous diseases, particularly cardiac and respiratory conditions.Smoking was first established as a risk factor in developing inflammatory bowel disease (IBD) study [33].Inflammatory bowel disease includes Crohn disease (CD) and ulcerative colitis (UC).İn our result association between smokers in CD and control was a significant P value < 0.01, an increased CD risk, and a reduced UC risk.Smoking was associated with an odds ratio (OR) of 1.884 for CD compared to nonsmokers.Although the precise origin of IBD is unknown, smoking is the most widely studied and accepted behavioral risk factor.Smoking has been implicated as increasing the risk for the development of CD and as a protective factor in UC, with relative consistency in the literature [33,36].
Further, the potential significance of TLR2 (-196 to -174del and Arg753Gln) variations in the pathophysiology of patients was examined in research on type 2 diabetes and its consequences in the Turkish population.There was a significant difference in the frequency of the TLR-2 del/del genotype (P=0.003),ins/del genotype (P=0.009), and del allele (P=0.001) in patients (100 patients) and controls (98 controls) [28].Recent studies have shown that TLR genes are essential in signaling pathogenrelated molecules and endogenous proteins associated with immune activation.The TLR 2 gene, the first human TLR involved in host defense has two exons in 4q32 of the chromosome.The TLR9 gene in humans is in the 3p21.2chromosome region and has two exon numbers.Stimulation of the TLR9 gene by its ligands triggers a signaling pathway common to all TLR genes, activating the transcription factor nuclear factor-kB (NF-kB) and producing pro-inflammatory cytokines.
Research has shown that the carriage of minor TLR2 (-196 to -174) del and TLR3-1377 T alleles appear to exert a significant influence on HNC risk, providing evidence of the involvement of these polymorphisms and their haplotypes as risk factors of HNC.Identifying genetic risk predictors for NPC and LC, the two major HNC types, may improve diagnosis, risk prediction, and clinical care.TLR 9 (T1486C and T123C) seems to influence the progression of malaria under certain genetic models and in specific age groups of people from specific geographical regions.TLR 9 (T1237C) also plays a role in susceptibility to malaria under specific genetic models in children.A case-control study in China found that TLR 9 gene rs187084 polymorphism was positively associated with susceptibility to knee OA, especially among older adults.Balbaloglu et al. (2017) found a correlation between TLR 9 T-1486C gene polymorphism and advanced knee OA in the Turkish population.Still, no significant difference was found between CD and UC patients and the control group [37].Messaritakis et al. (2018) investigated the gene polymorphisms of TLR 2 (-196 to -174del), TLR 4 (Asp299Gly and Thr399Ile), and TLR 9 (T1237C and T-1486C) gene polymorphisms in the risk of development and progression of colorectal cancer in Greece.Conducting advanced genetic studies and confirming these results with a meta-analysis test will contribute more to scientific knowledge development [38].
In conclusion, the present study demonstrates that the TLR2 -196 to -174del/del, TLR9 (T-1486C) polymorphism is no associated with an increased risk of CD, UC and IBD.A significant difference in smoking rates between the CD group and the control group, with a 1.88 odds ratio, indicating a 1.88 times higher number of smokers in the CD group.However, we only investigated the TLR2, TLR9 polymorphism; in two SNPs in a limited region of genes, we need sequencing of whole gene of TLR2 and TLR9.In addition, because the TLR2, TLR9 gene polymorphism may show variations in different ethnic groups, further studies will be needed in a larger and ethnically diverse population to confirm the impact of this gene on the susceptibility of IBD.

Figure 2 .
Figure 2. TLR 9 (T-1486C) Gene Bands Cut with Afl II Enzyme in RFLP Method.The CC genotype did not reflect in this figure, however, TC-represents the three bands: 499,327 and 172.TT-represents two bands: 327 and 172.

Figure 3 .
Figure 3. Demographic Factors and the of CD, UC, and Control

Table 1 .
Demographic Factors and the Occurrence of CD, UC, IBD and Control

Table 2 .
TLR2 (-196 to -174 del) Genotype and Allele Frequencies in the UC and Control Group

Table 4 .
TLR9 (T-1486C) Genotype and Allele Frequencies in UC Patients and Controls 4, and TLR 9 gene polymorphisms in the 182 CD and 188 control

Table 5 .
(Bank et al. 2014)ype and allele frequency in UC patients and controls groups of the New Zealand Caucasian population[35].39SNPs in 26 genes, including TLR 2 and TLR 9, which govern inflammation, were examined in a cohort of 624 patients with CD, 411 patients with UC, and 795 controls in a study by Danish researchers utilizing the candidate gene technique(Bank et al. 2014).It was shown that 16 polymorphisms in 13 genes, including TLR 2 and TLR 9, were related to the risk of CD and/or UC, thereby controlling inflammation.