Evaluation of the Impact of Some Single-Nucleotide Gene Polymorphisms on the Development of Adenomatous Polyps of the Colon in Patients with Irritable Bowel Syndrome

Background/Aims: The number of cases of irritable bowel syndrome is growing worldwide, in which adenomatous polyps can develop as a result of microinflammation of the colonic epithelium. Our study was aimed at the identification of the possible effect of single-nucleotide polymorphisms on the risk of the development of irritable bowel syndrome-related colonic adenomatous polyps. Materials and Methods: The study involved 187 irritable bowel syndrome patients. The single-nucleotide polymorphisms were investigated by the polymerase chain reaction method and DNA was extracted with the phenol-chloroform: interleukin-1β gene-31C/T (rs1143627), -511C/T (rs16944); interleukin-6 gene-174G/C (rs1800795); interleukin-10 gene-592C/A (rs1800872), -819T/C (rs1800871), -1082A/G (rs1800896); Toll-like receptor-2 gene Arg753Gln (rs5743708); Toll-like receptor-4 gene Thr399ile (rs4986791), Asp299Gly (rs4986790); and metalloproteinase-9 gene-8202A/G (rs11697325). The study of polymorphic loci was checked for compliance with the Hardy–Weinberg equilibrium using Fisher’s exact test along with the analyses of the frequency of alleles and the genotypes. Results: The association of diseases with G allele Toll-like receptor-2 gene Arg753Gln (rs5743708) was revealed in irritable bowel syndrome patients with adenomatous polyps of the colon (P < .0006) and AG single-nucleotide polymorphisms s of Toll-like receptor-2 gene (χ2 = 12.78, P < .002); A allele had a protective effect. The AG genotype metalloproteinase-9 gene-8202A/G (rs11697325) polymorphism in irritable bowel syndrome patients with adenomatous polyps of the colon had a protective effect (P < .05). AA genotype interleukin-10 gene-1082A/G (rs1800896) polymorphism in the irritable bowel syndrome patient (χ2 = 33.97, 4.0E-8) can be considered as the risk for adenomatous polyps of the colon in irritable bowel syndrome. Conclusion: G allele Toll-like receptor-2 gene Arg753Gln (rs5743708) and AA genotype interleukin-10 gene-1082A/G (rs1800896) polymorphisms can be the marker of the emergence of adenomatous polyps of the colon concomitant with irritable bowel syndrome.


INTRODUCTION
Currently, the prevalence and incidence of intestinal diseases, including colorectal cancer, are growing worldwide.The number of registered cases of functional bowel disease (FBD) such as irritable bowel syndrome (IBS) [1][2][3] is tending to increase, where hyperplastic changes, including adenomatous polyps of the colon (ADPC), often develop in connection with microinflammation of the epithelium of the colonic mucosa (CM) 4 with high risk of neoplastic transformation. 58][9] In our country, intestinal pathology is tending to grow and most often identified multiple and large polyps are prone to malignancy. 10 Therefore, the detection of ADPC and their timely early removal are the best prevention of colorectal cancer. 11Quite often, ADPC have an asymptomatic course, which leads to a more in-depth approach to the management of IBS patients, since microinflammatory processes and dysbiotic disorders, which are often detected in IBS, 2 can be triggering factors in the emergence of ADPC, contributing to its further progress.
Adenomatous polyps of the colon and IBS multifactorial diseases have many concepts concerning the causes of its emergence.Moreover, the pathogenetic mechanisms of the diseases have similar features, the emergence of which is influenced by hereditary factors, environmental factors, dietary preferences, disorders of the intestinal microbiota, and so on. 2,8Genetic predisposition to the development of ADPC and FBD, being one of the major causes of the development of the disease, can create a negative background for other factors.
3][14] It is reported that SNPs are responsible for the functioning of cytokines in the human body and have the most significant impact on the development of ADPC and IBS.Interleukin (IL)-1β (rs1143627, rs 16944), 4,11 Interleukin IL6 (rs1800795), [13][14][15][16] Interleukin IL10 (rs1800872, rs1800871, rs1800896) 12,13,15,16 are distinguished among the SNPs, which could potentially have an impact on the disease considering their influence on dysbiosis and on changes in the colon epithelium that is caused by deviation of gene expression.
In addition, the emergence of both ADPC and IBS can be influenced by SNPs, which regulate the functioning of Toll-like receptors (TLR).Toll-like receptors are responsible for the body's timely immune response to microbial ligands. 17Some scientists acknowledge the influence of SNP of TLR genes on the IBS development [18][19][20][21] and on the neoplastic changes in colon.In the occurrence of intestinal dysbiosis, which accompanies or precedes the emergence of both IBS and ADPC, in a patient with SNP of TLR, the response of the CM to bacterial and viral microorganisms may be altered and modified, triggering a cascade of inflammatory process, which can be crucial for the further progression of diseases and their organic transformation.Some scientific studies show correlation between the expression of matrix metalloproteinase MMP-9 gene-8202(A/G) (rs11697325) polymorphism and the development of inflammation in CM. 22 Metalloproteinase-9 SNP leads to a variation in the mechanisms of healing of the CM.Besides, it modifies apoptotic changes, proliferation, and differentiation of cells, 11 and also can affect the development of ADPC and initiate the development of low-grade inflammation in CM, 23 particularly in IBS.
Thus, the detection of hereditary predisposition to ADPC and IBS, taking into account SNP, can assist in determining the possible ways of the emergence of ADPC concomitant with FBD at the early stages of the disease, as well as the vectors of disease progression, identifying links in such patients and preventing further neoplastic transformation.Our aim was to identify the possible influence of SNPs on the development of ADPC in IBS patients.

MATERIALS AND METHODS
We have conducted a multicenter open-label observational cohort study in 4 centers in Ukraine for the period from January 2019 to May 2021 and examined 187 patients with different subtypes of IBS.The subjects have been assigned into 2 groups.

DNA Isolation
For genotyping of individuals by the corresponding polymorphisms, samples of total DNA and DNA, isolated from the whole venous blood, have been used.The PCR method was used to isolate polymorphisms and "SNP-Express" (Lytech) diagnostic kits were used for SNP genotyping.Sequence amplification was performed on the Research PCR Thermal Cycler (Corbett, Australia).Subsequently, electrophoresis was performed in 2% agarose gel, stained with ethidium bromide.During the scan, the UV transilluminator was used to analyze the resulting amplification data.To identify the possible influence of genetic polymorphisms on the course and onset of IBS and ADPC concomitant with IBS, we analyzed the frequency of detection of SNPs, which are responsible for the production and functioning of cytokines (Figure 1).

Main Points
The findings of the analysis have shown that patients with IBS, ADPC + IBS, and the control group had all types of SNPs responsible for the production of pro-inflammatory cytokines.
The genetic analysis of IL6-174G/C (rs1800795) polymorphism showed that all types were determined with the same frequency in both group 1 and group 2 patients without a significant difference, compared to the control group.
The genetic analysis of IL10-592C/A (rs1800872) polymorphism showed that it was found with the same frequency in the studied groups of patients.diseases.The emergence of the AA genotype IL10-1082A/G (rs1800896) polymorphism can be considered a marker of the development of ADPC in patients with IBS, in contrast to the GG genotype, which has a protective effect in patients with IBS.
The analysis of the SNP in the ММP-9 gene-8202A/G (rs11697325), TLR2 gene Arg753Gln (rs5743708), TLR4 gene Thr399ile (rs4986791), and Asp299Gly (rs4986790) was made to identify a possible hereditary predisposition to dysregulatory changes in the reparative capacity of the CM, disorders in the microbiota in patients with IBS and ADPC concomitant with IBS (Figure 2).
The genetic analysis of MMP-9 showed that in patients of group 2, AG genotype-8202A/G (rs11697325) polymorphism was detected significantly less frequently (29 (45.3%) patients) (P < .05),compared to group 1 and control group, which may determine the value of this polymorphism in the emergence of ADPC concomitant with IBS, due to the modifying effect on the reparative and inflammatory processes in the CM.
The genetic analysis of TLR4 showed that CC and CG genotype-819T/C (rs1800871) TLR4 gene polymorphisms were detected in all study groups; GG genotype-819T/C (rs1800871) polymorphism of the TLR4 gene was not detected in patients with ADPC concomitant with IBS; no significant difference was found among the study groups.AA genotype in the Asp299Gly (rs4986790) of the TLR4 gene SNP was detected significantly more frequently in both groups: 93 (75.6%) patients with IBS and 47 (73.4%) patients with ADPC concomitant with IBS, compared to intact subjects (P < .01).AG genotype in the TLR4 gene Asp299Gly (rs4986790) polymorphism was also found significantly less frequently in both groups: 27 (21.9%)patients of group 1 and 9 (14.1%)patients of group 2, compared to the control group [51 (72.9%) subjects] (P < .001).The emergence of the SNP variation of the TLR4 gene Asp299Gly (rs4986790) AA can be the unifying factor, influencing the emergence of diseases, a triggering factor in the development of ADPC in patients with IBS.Detection of heterozygous polymorphism determines the protective effect in patients with IBS on the emergence of adenomatous polyps.
The genetic analysis of TLR2 showed that among the SNPs TLR2 gene Arg753Gln (rs5743708), the AA genotype of polymorphism was found significantly less frequently in To analyze the associations of alleles of SNPs with the development of diseases, the distribution of their frequency was estimated according to the H-W equilibrium.Among the examined patients, the equilibrium conditions H-W (χ 2 test under the condition of df = 1) were performed for all groups except substitutions (P < .05).
In the control group, loss of H-W equilibrium was detected for SNPs -819T/C (rs1800871), -1082A/G (rs1800896) of the IL10 gene, Asp299Gly (rs4986790) of the TLR4 gene, 8202A/G (rs11697325) of the MMP-9 gene, not allowing us to analyze SNP data on the association of the allele frequency with the development of IBS and ADPC concomitant with IBS (Table 1).
Moreover, non-compliance with H-W equilibrium was revealed in patients of both group 1 and group 2, which did not allow us to estimate the association of the diseases with the corresponding SNP, using a multiplicative model of heredity (Table 2).The findings of the study have shown that non-compliance with H-W equilibrium was determined for SNPs: -511C/T (rs16944) of the IL1β gene and -8202A/G (rs11697325) of the MMP-9 gene in patients of group 1; SNPs -31C/T (rs1143627) of the IL1β gene, -174G/C (rs1800795) of the IL6 gene, -819T/C (rs1800871), -1082A/G (rs1800896) of the IL10 gene, Asp299Gly (rs4986790) of the TLR4 gene in patients of group 2. We have analyzed the development of a hereditary predisposition to the diseases using a general model of heredity, which evaluates the association of the diseases according to the frequency of genotypes, in compliance with the above polymorphic variations.
The analysis of compliance with H-W equilibrium in both study groups and control group enabled the estimation of the hereditary predisposition for association with the alleles' frequency with respect to the polymorphisms of the IL10 gene -592C/A (rs1800872), TLR4 gene Thr399ile (rs4986791), and TLR2 gene Arg753Gln (rs5743708) (Table 3).
The findings of the study showed that in patients of group 1, the association of the emergence of IBS was detected for SNPs: with the frequency of the C allele -592C/A (rs1800872) (IL10, χ 2 = 7.78, P < .005),C allele Thr399ile (rs4986791) (TLR4, χ 2 = 7.75, P < .005),G allele Arg753Gln (rs5743708) (TLR2, χ 2 = 14.89,P < .0001).In patients of group 2, the association of emergence of ADPC concomitant with IBS was determined with the frequency of the G allele, SNP of the TLR2 gene Arg753Gln (rs5743708) (χ 2 = 11.92,P < .0006).Detection of genetic risk for patients of both groups, associated with the frequency of the G allele, SNP of the TLR2 gene Arg753Gln (rs5743708), confirms the possible effect of this polymorphic variation on the emergence of ADPC concomitant with IBS and is a risk factor for neoplastic alterations in patients with FBD and can be a prognostic marker in patients with IBS.The emergence of SNP in the IL10 and TLR2 genes confirmed the role of cytokine imbalance and disorders in the inflammatory reactions of the CM in IBS associated with dysbiotic processes and contributed to the deterioration of the  prognosis of the disease, promoting organic changes in the epithelium.
The findings of the study were also confirmed by the data of the general model of heredity on the detection of genetic risk in patients of both group 1 and group 2, which assessed the frequency of genotypes (Table 4).The general model of heredity validated the relationship of frequency of SNP genotypes of IL10-592C/A (rs1800872), TLR4 Thr399ile (rs4986791), and TLR2 Arg753Gln (rs5743708) genes with the tendency to emergence of IBS that is in concordance with the data of the multiplicative model of heredity.
The most significant associative relationship has been found in patients of group 1 with AG genotype of the TLR2 gene Arg753Gln (rs5743708) polymorphism (χ 2 = 12.86, P < .002,OR = 7.41, 95% CI: 1.69-32.53).Moreover, from the TLR2 gene Arg753Gln (rs5743708) polymorphism, the strongest genetic relationship was also defined in patients with ADPC concomitant with IBS: AG genotype (χ 2 = 12.78, P < .002,OR = 10.42,95% CI: 2.28-47.61),which confirmed the special role of this polymorphic variation in the susceptibility to these diseases, which is crucial for early diagnosis of adenomatous polyps as a precancerous disease.
The analysis of genetic risks for the development of IBS and ADPC concomitant with IBS, using the general model of heredity, also revealed associations in the frequency of genotypes of 4 SNP genes that are responsible for the functioning of cytokines (Table 5).

DISCUSSION
We confirmed the influence of genetic SNPs on the development of IBS and the formation of ADPC concomitant with IBS, which is in concordance with the data of other researchers. 16,22,24,25The investigated genetic SNP often had a combined effect on the diseases, affecting both the emergence of IBS and adenomatous polyps concomitant   with IBS.The pronounced influence of polymorphic variations on the development of the diseases responsible for the functioning of pro-inflammatory and anti-inflammatory cytokines, reparative properties of the CM, and stability of the intestinal microbiome has been noted, which is in concordance with the findings of other authors. 12,15,26hu et al 24 report that genetic factors increase the risk of IBS.In another meta-analysis comprising 8 studies with 928 IBS patients, 27 polymorphisms of IL10 gene-1082A/G (rs1800870), -592C/A (rs1800872) were associated with IBS and IL10 gene-819T/C (rs1800871) had no association to IBS.Messaritacis et al 28 hypothesized that the presence of TLR2, TLR4, and TLR9 variants affected gut homeostasis resulting in impairment of TLRs activation, thus leading to inflammation and cancer development and progression.The findings of our studies have reveled association of the disease with the C allele of the IL10 gene-592C/A (rs1800872) and the C allele of the TLR4 gene Thr399ile (rs4986791) polymorphisms in patients with IBS.The genetic risk for the development of the disease was also revealed in patients with IBS who had SNPs: TT genotype of the IL10 gene-819T/C (rs1800871) and CT genotype of the IL1 gene-511C/T (rs16944) were determined according to the analysis of the general model of heredity.In addition, the genetic risk for the disease was established in IBS, when the G allele TLR2 gene Arg753Gln (rs5743708) polymorphism appeared.Associative relationships were also determined from the G allele of the TLR2 gene Arg753Gln (rs5743708) polymorphism and in patients with ADPC concomitant with IBS.In addition, the AA genotype of the TLR2 gene Arg753Gln (rs5743708) polymorphism was significantly less frequently determined in patients of both groups, which confirms the protective effect of the A allele in such patients, and the appearance of G allele can be considered as the prognostic marker of possible formation of adenomatous polyps concomitant with IBS.
Confirmation of the similarity of the mechanisms of influence on IBS and ADPC concomitant with IBS 20,25,28 is the detection of an increased frequency of the AA genotype of the TLR4 gene Asp299Gly (rs4986790) polymorphism and a decrease of the heterozygous variation in the SNP, compared to intact subjects (P < .01).The similarity of the detected changes determined the possibility of the relationship between these pathologies and the possible protective effect of SNP of TLR, which confirms the data of other researchers. 17,22In patients with IBS and intact subjects, a heterozygous variation of the AG genotype of the MMP-9 gene-8202A/G (rs11697325) (P < .05)was significantly more frequently detected, compared to ADPC concomitant with IBS, which can possibly be the marker of susceptibility to disruptive reparative properties of the CM.Furthermore, according to our data, there is a genetic risk for emergence of ADPC in patients with IBS if the AA genotype of the IL10 gene-1082A/G (rs1800896) polymorphism appears, in contrast to the GG genotype, which had a protective effect in patients with IBS and was absent in patients with ADPC.
In conclusion, the findings of our studies have revealed similarity in patients with IBS and ADPC concomitant with IBS with regard to the genetic risk of the diseases.The development of the IBS was associated with the C alleles of the IL10 gene-592C/A (rs1800872) and TLR4 gene Thr399ile (rs4986791) polymorphisms, the frequency of the TT genotype of the IL10 gene-819T/C (rs1800871) and GG genotype of the IL10 gene-1082A/G (rs1800896) polymorphisms and heterozygous CT genotype of the IL1 gene-511C/T (rs16944) polymorphism.In patients with IBS and patients with ADPC concomitant with IBS, the association of the diseases was identified with the G allele TLR2 gene Arg753Gln (rs5743708) polymorphism; the A allele had a protective effect in such patients.The heterozygous AG genotype of the MMP-9 gene-8202A/G (rs11697325) also had a protective effect on the development of ADPC.In addition, in both groups of patients the diseases were associated with the frequency of the SNP variations of the TLR4 gene AA genotype Asp299Gly (rs4986790) polymorphism.Detection of the IL10 gene AA genotype-1082A/G (rs1800896) in a patient with IBS can be considered as a risk for emergence of ADPC concomitant with IBS.Therefore, timely study of the SNPs of the IL10-1082A/G (rs1800896), TLR2 Arg753Gln (rs5743708), TLR4 Asp299Gly (rs4986790), and MMP-9-8202A/G (rs11697325) genes in patients with IBS will promote prediction of the course of the disease, identify the tendency to the formation of ADPC concomitant with IBS and further prevention of disease progression.
Ethics Committee Approval: This study was approved by the Ethics Committee of Ukraine Medical Stomatological Academy (Approval number: 2018/169).
Informed Consent: Written informed consent has been obtained from all the participants involved in this study.

Figure 1 .
Figure 1.Genetic SNPs responsible for functioning of pro-and anti-inflammatory cytokines in patients with IBS and ADPC concomitant with IBS.SNP, single-nucleotide polymorphisms; IBS, irritable bowel syndrome; ADPC, adenomatous polyps of the colon.
group 2 [49 (76.6%) patients] and in IBS [96 (78.1%) patients] (P < .02),compared to the control group.The AG genotype in the TLR2 gene Arg753Gln (rs5743708) polymorphism was more frequently detected in patients with ADPC concomitant with IBS [15 (23.4%)], compared to intact subjects (P < .05)and insignificantly, compared to group 1 [22 (17.9%) patients].The single-nucleotide polymorphism of the TLR2 gene Arg753Gln (rs5743708) GG genotype was not detected in all groups, except for patients with IBS [5 (4.1%) patients].The emergence of AG in the TLR2 gene Arg753Gln (rs5743708) polymorphism in patients with IBS may precede the emergence of ADPC and determine the predisposition to hyperplastic changes in such patients.

Table 1 .
Non-Compliance with the H-W Equilibrium in the Control Group P values indicate the presence of statistical significance.H-W equilibrium, Hardy-Weinberg equilibrium; IL, interleukin; MMP, matrix metalloproteinase; TLR, Toll-like receptors.

Table 2 .
Non-compliance with the H-W Equilibrium in the Study Groups P values indicate the presence of statistical significance.H-W equilibrium, Hardy-Weinberg equilibrium; IL, interleukin; MMP, matrix metalloproteinase; TLR, Toll-like receptors.

Table 3 .
The Multiplicative Model that Identified a Genetic Risk for Sampling Patients with IBS and ADPC Concomitant with IBS P values indicate the presence of statistical significance.ADPC, adenomatous polyps of the colon; IBS, irritable bowel syndrome; IL, interleukin; OR, odds ratio; TLR, Toll-like receptors.

Table 4 .
The Results of Genetic Risk Obtained by the Analysis of the General Model of Heredity in Patients with IBS and ADPC Concomitant with IBS [the Analysis of SNP of the IL10 -

Table 5 .
Associations of the Development of the Diseases with the Frequency of Genotypes, According to the General Model of Heredity