Correlation study between Interleukin-17 Gene Polymorphisms and risk of development of recurrent aphthous ulcer in Han Chinese Population

Objective Interleukin-17 (IL-17) is a pleiotropic cytokine which plays important role in the inammatory diseases. Methods: Polymorphisms of IL-17A rs2275913 and IL17F rs763780 were measured in 125 RAU cases and 116 healthy controls. The polymerase chain reaction-restriction fragment length was measured. The genotype distribution and disease risk, and its’ relationship with RAU severity was analyzed. Results: RAU risk were related with polymorphism of IL-17 gene at rs2275913 site after adjusting BMI, sex, age, smoking and drinking status (AA vs. GG: odds ratio (OR), 1.624; 95% condence interval (CI), 1.125–2.250; P = 0.030; A allele vs. G allele: OR, 1.192; P = 0.037; 95% CI, 1.012–1.404;). In addition, the rs763780 variant genotypes (TC and CC) and C allele also have higher relevance to RAU compared with subjects who bears the TT genotype (TC vs. TT, OR: 1.312; P = 0.039; 95% CI: 1.017–1.692; CC vs. TT, OR: 2.812, P = 0.006, 95% CI: 1.338-5.909; C allele Vs. T allele, OR:1.413, P=0.002, 95% CI:1.141-1.751). We also found serum IL-17 levels were greatly higher in RAU patients compared with controls (P = 0.001), and serum IL17 concentration is correlated with IL17 polymorphism. Conclusion: Our research polymorphisms of IL-17 gene might related to the high-risk of RAU occurrence.


Introduction
Recurrent oral ulceration (RAU) is also known as recurrent aphthous stomatitis, with characters of recurrent episodes of oral ulcers. Reports indicated that RAU affects about 20% of populations [1,2]. The etiology of RAU is multifactorial, genetic, immune, nutritional, and microbial factors and local trauma might contribute to the occurrence. In addition, family history of RAU is con rmed to be a risk factor for RAU [3].
The etiology of RAU is not well demonstrated clearly, previous studies indicated that in ammatory factors play an key role in the development of RAU [4,5]. Borra et al revealed that cytokine genes in peripheral blood mononuclear cells, such as IL-2, TNF-α and IL-6, contribute to the development of RAU [6]. Other research indicated that IL-10 mRNA levels decreased in RAU patients, which suggests a failure of suppressing in ammatory reaction to oral mucosa in immune system [7].
So far, few studies reported the association between polymorphism of in ammatory genes and risk of RAU. The polymorphisms of IL-1β -3954 C/T (rs1143634), IL-6-174G/C (rs1800795), IL-10-1082A/G (rs1800896), and IL-10-819C/T (rs1800871) have been reported correlated with risk of RAU in a Chinese population [1]. While no association study about IL-17 polymorphisms and RAU was reported. IL 17 play an important role in adaptive and innate immune systems. Five con rmed receptors (IL-17RA-RD and SEF) and six members (IL-17A-F) [8,9] of IL 17 have been identi ed so far. Among them, IL-17A and IL-17F are secreted by Th17 cells, and composed a distinct lineage of CD4 + effector cells [10]. As a pro-in ammatory cytokine, IL-17 could trigger the release of chemokines, cytokines, antimicrobial peptides, and matrix metalloproteinases (MMPs) from mesenchymal and myeloid cells [11]. On the other hand, IL-17 is expressed in synovial tissues, and could participate in the process of cartilage breakdown and synovial in ltration in RAU through inducing the release of chemokines from chondrocytes [12]. IL-17 also augments nitric oxide production via nuclear factor kappa B activation in RAU cartilage [13].
Compelling studies indicated that genetic polymorphisms of IL-17 are related to the susceptibility of a scope of in ammation-related diseases, including ulcerative colitis, gastric cancer, breast cancer, and rheumatoid arthritis [14][15][16][17]. So far, there is no relative report about association of IL-17 polymorphism with RAU in the publicly available Genome database, we hypothesize that IL-17 is a potential risk factor for RAU, and aim to explore the correlation between the polymorphisms of IL-17A and IL-17F with risk of RAU among Chinese population. The polymorphs of IL-17A rs2275913 and IL-17F rs763780 with the risk factors of RAU were evaluated in our recruited subjects. This case-control study was designed to explore the relationship of these two SNPs with the morbidity and severity of RA.

Subjects
A total of 125 patients who were diagnosed of ROU were enrolled. The diagnostic criteria were horizontal range of oral ulcer being covered with yellow pseudomembrane, surrounding hyperemia, and with central sag and obvious causalgia, ulcer having cyclicity and being self-limiting and being at differential phase. Inclusion criteria included: (1) ≥ 6-month of regularly recurrent episodes of oral aphthous ulcer; (2) at least two ulcers per month within 6 months; The subjects from control group was con rmed with no history of ROU and matched with cases group with sex and age. The gender and age distribution in the RAU and healthy control group were shown in Table 1. Informed consent was obtained from all subjects. The protocol of study was approved by the Ethics Committee of the second hospital of Hebei Medical University. Study participants 125 Chinese patients with RAU (mean age ± s.d. = 39.5 ± 12.9 years) were recruited, 62 was men and 63 was women, 6 were smokers. All subjects were assessed by an oral medicine specialist, the presence of aphthous ulcers were con rmed by them.
Blood samples were also obtained from 116 age and sex-matched healthy Chinese controls. Their RAU status con rmed by medical records and inquiries of patients, there is still possible that a small proportion subjects suffered from RAU. This might weaken the identi ed strength of associations. In our study, only six smokers were recruited to the patient group while 10 smokers were recruited in healthy control, which make it impossible to evaluate the effect of smoking on any gene associations.
The detailed demographic characteristics of the study population was listed in Table 1. Comparison showed there was no signi cant difference in mean age, sex, BMI and smoking status between the 2 groups, indicating subjective matching. However, it seems that alcohol drinking could increase the risk of RAU occurrence (P = 0.032, Table 1).
An allele of rs2275913 and C allele of rs763780 increase the risk of RAU The distribution of each allele and genotype is shown in Table 2. Both SNPs were within the Hardy-Weinberg equilibrium. As for the genotype and allele frequencies in rs2275913 polymorphism, we observed a remarkably difference between RAU patients and healthy controls ( Table 2). In addition, all subjects carrying AA genotype have signi cantly higher risks of RAU compared with GG genotype (P < 0.05). That is subjects with the A allele were more likely to get RAU compared with subjects bearing the G allele (OR, 1.1192; 95% CI, 1.012-1.404; p = 0.037). In addition, analysis also indicated the higher risk of AA genotype and A allele mainly existed in female sub-population (P < 0.05), but not in male population (p > 0.05, Table 2). We also observed similar results about rs763780 polymorphism. There was a signi cant difference in the genotype and allele frequencies between RAU patients and control subjects, as demonstrated in Table 2. In addition, C allele rather than T allele increases the risk of developing RAU (Table 2). Furthermore, our study indicated that C allele caused higher risk than in T allele in female subgroup (P = 0.004). As for the male population, the CC genotype and C allele did not induce signi cant higher risk for RAU. Dominant and recessive model also indicated the similar results that A allele of rs2275913 and C allele of rs763780 increase the risk of RAU.
IL-17 polymorphism is related to RAU severity RAU severity was evaluated based on number of lesions in one patient. In the current study, we classi ed into two sub-groups, less than three lesions and ≥ three lesions in RAU patients. The distribution of SNP among RAU severity was listed in Table 3. Results showed there is a signi cant difference between all the SNPs and alleles in different severity-groups, which suggests that these two IL-17 polymorphisms are risk factors for RAU severity. Serum IL-17A/F levels are higher in RAU patients The median serum concentration of IL-17A was 2.38 ± 0.14 pg/ mL and 4.31 ± 0.27 pg/ mL in healthy controls and RAU patients, respectively (Table 1). IL-17A serum levels in RAU patients were higher than healthy controls (P = 0.0221, Table 1). The serum levels of IL-17F in RAU patients were also higher than healthy controls (P = 0.0314, Table 1). Further, for the average serum concentration of IL-17A and IL-17F in each genotype, there was signi cant difference among all 3 genotypes for these two SNPs (Table 4), which indicated that A allele in rs2275913 and C allele in rs763780 possibly increased the IL17A and IL17F secretion respectively in RAU patients. Previous studies indicated that IL-17/IL23 pathway and TH17 cells play important roles in pathology of in ammation-related diseases [18]. Several meta-analysis demonbstrated the relationship between gene polymorphisms of IL-17A (rs2275913) and IL-17F (rs763780) and the risk of in ammatory diseases, including periodontitis, rheumatoid arthritis (RA), and in ammatory bowel disease [19]. Research also reported polymorphism of IL17A (rs2275913) is associated with the occurrence of rheumatic heart disease in south Indian population [20], and a variant of IL17F (rs763780) may participated in the development of necrotizing enterocolitis [21]. All these researches suggest that IL17 polymorphism may be associated with immune mediated diseases widely.
A previous studies indicated that increasing of IL-17 A/F are effective in the pathogenesis of minor aphthous, particularly in the ulcerative stage [22]. No study reported the relationship between rs2275913 (IL-17A SNP) and rs763780 (IL-17F SNP) with risk of RAU. Our study might be rst time to investigate the involvement of IL-17 gene polymorphisms in RAU and whether these polymorphisms are correlated with serum levels of IL-17A/F. So far, there is no enough evidence to demonstrated whether the 2 SNPs affect the IL-17 secretion in the human plasma. Present results from Table 4, these two SNPs seem to in uence the plasma concentration of IL-17A/F in serum. An allele in rs2275913 and C allele in rs763780 possibly increased the plasma concentration of IL17A and IL17F respectively, which provides the explanation that these 2 alleles increase the risk of RAU (Table 2). Our results suggested that the polymorphism of IL-17A rs2275913 has a signi cant impact on the risk of RAU. In addition, subjects carrying the rs2275913 A allele are at a higher risk of developing RAU as compared with subjects carrying the G allele. Furthermore, rs763780 C allele, was also related to an increased risk of developing RAU greatly. Further assessment of the effect of IL-17 polymorphisms on RAU risk was strati ed by sex, and we also nd increased risk ratio in both male and female patients' subgroups, but no statistical signi cance was found due to limited population, more sample size is still need to further revealed the underlying mechanisms of this association.
Higher average serum concentrations of IL17A/F have been found in RAU patients when it was compared with healthy controls. Analysis of in ammatory cells, such as Th17, some strongly positive IL-17A + cells ae seen in the vascularized super cial connective tissue of human oral mucosa, and aggravate RAU. Cytokine genes such as IL-17A/F are associated with development and function of Th17 cell. Therefore, IL17 polymorphism regulated Th17 cell population is another possible mechanism that involved in RAU-associated in ammatory response.
There are some other limitations except for the small sample size population in 0ur study. The population of study was con ned to Han Chinese, the ndings might not apply to other population. It would of great signi cance if RAU patient from other ethnic population is studied. In addition, we only investigated 2 SNPs in the IL-17 gene. If more SNPs would be de ned for the occurrence of RAU, it will also make our research more signi cance. More importantly, allele frequency differences between cases and controls after population strati cation could cause spurious associations in disease studies due to systematic ancestry differences, and lead to false positive results [23]. Therefore, more detailed statistical analysis is need to make our results more accurately.
In a conclusion, our results demonstrated that functional polymorphisms of IL-17 are correlated with the risk of RAU signi cantly. The variant alleles rs2275913 AA and rs763780 CC might lead to the increased risk of RAU. Elevated serum IL-17 levels correlated with increased risk of RAU signi cantly, which taking together might facilitate de ning high risk subjects to prevent the initial development of RAU.

Declarations
Ethics approval and consent to participate The protocol of study was approved by the Ethics Committee of the second hospital of Hebei Medical University.

Consent for publication
Informed consent was obtained from each patient for their medical data using and consent for publication.

Availability of data and material
The relevant raw data will be freely available to any scientist wishing to use them for non-commercial purposes, without breaching participant con dentiality.

Competing interests
None.

Funding
None.
Authors' contributions