Association of IL-17A -197G/A (rs2275913) Single Nucleotide Polymorphism and Rheumatoid Arthritis in Sudanese Patients

Objectives: Rheumatoid arthritis (RA) is a chronic inammatory autoimmune disease. The aim of this study is to determine the association of IL-17A -197G/A polymorphism with RA in Sudanese Patients. A descriptive cross-sectional study was conducted between March and December, 2018. Clinical and demographic data of the study participants were collected and analysed. All statistical tests were considered as statistically signicant when p < 0.05. Results: The study population included 266 participants, 166 (63.1%) were females [mean age 41.4 ± 15.5 years] and 97 (36.9%) were males [mean age 36.2 ± 16.0 years]. Of the 266 participants, 31% (85/266) were RA cases and 69% (181/266) were healthy controls. Prevalence of IL-17A genotypes among the study population was 52.6% (140/266) were AG heterozygote genotype, 38.4% (102/266) were AA homozygote genotype, and 9.0% (24/266) were GG homozygote genotype. Correlation of IL-17A genotypes was negatively statistically signicant based on participants clinical status, and family history of RA, Pearson’s correlation [r = -0.392, P value 0.001] and [r = -0.226, P value 0.001], respectively, while positively statistically signicant with gender, Pearson’s correlation [r = 0.140, P value 0.023]. Based on the duration of RA, no statistically signicant correlation was observed, Pearson’s correlation [ r = -0.138, P value 0.207]. The correlation of the different IL-17A genotypes was negatively statistically signicant based on participants clinical status; case and control, and family history of RA, Pearson’s correlation [r = -0.392, pvalue 0.001] and [r = -0.226, p-value 0.001], respectively. While correlation of IL-17A genotypes was positively statistically signicant with participant gender, Pearson’s correlation [r = 0.140, p-value 0.023]. Whereas based on the duration of RA, no statistically signicant correlation was observed, Pearson’s correlation [r = -0.138, p-value 0.207] (Table 3).


Introduction
Rheumatoid arthritis (RA) is an autoimmune disease characterized by a chronic in ammatory response that affects various part of the human body. At late stages, RA leads to deformities of the joints and bones causing severe pain [1][2][3][4][5]. Certain factors were hypothesized to be involved in the induction and the development of RA; however, the most acceptable theory for the development of the disease is a multifactorial theory that involve the interaction of environmental and genetic background of patients and render them more susceptible to RA [6]. The disease severity is varied from one population to another; African populations, especially Sudanese had more severe form of the disease comparing to other countries [7].
The microenvironment is very critical for the disease progression and it usually impact treatment outcomes, in RA the affected site show an adherence of several types of in ammatory cells including polymorph nuclear cells and macrophages as well as T cells [8]. Growing body of evidence indicates the central role of T cells in the pathophysiology of RA; as the patients carrying Human Leukocytes antigen (HLA) -DRB1 are more susceptible to disease development [9][10][11][12].
Recently, a newly discovered subset of T Helper cells which is known as TH 17 was reported to play a crucial role in host defense toward several infectious agents [13,14]. TH 17 cells form a family of cytokines which consist of IL-17A, IL-17B, IL-17C, IL-17D, IL-17E, and IL-17F [15,16]. Recent study demonstrated the exact role of IL-17 in RA, the level of IL-17 expression, and the producing cells in the synovial uid in RA patients showed increase in IL-17 level in the patients with RA [17]. Also, a correlation between IL-17 levels on serum and synovial uid with various RA activity markers such as erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), and rheumatoid factor (RF) [18]. Interestingly, in the IL-17 gene there are several polymorphisms have been detected that may in uence the expression of IL-17 [19,20]. In this study we aimed to investigate the association between IL-17A -197G/A (rs2275913) polymorphism and RA among Sudanese patients.

Study Design and Study Population:
A descriptive cross-sectional hospital-based study was conducted at Zain medical center in Khartoum state, Sudan, from February 2017 to December 2018. Three ml of blood were collected into EDTA blood containers from each participant voluntary enrolled in our study after reading and signing the informedconsent, parents/guardians agreed and signed on the behalf of their children in case of an underage patient. Personal interviews with each participant/ his signatory guardian prior to the blood donation to obtain the demographics and clinical data of participants, including age, gender, locality, tribe, family history, and the duration of disease. All those who attended to the hospital for follow-up for RA were giving a similar chance to participate in the study. Also, apparently healthy co-patient individuals regardless of their age and gender were included as a control group. All participants diagnosed with autoimmune diseases other than RA were excluded from the study.
DNA extraction and IL-17A Genotyping: The total genomic DNA was extracted from blood samples using QIAamp DNA Blood Mini Kit (Qiagen, Germany). Extracted DNA was checked for quality using nanophotometer (Implen, Germany) and stored at − 20 °C until IL-17A genotyping. IL-17A genotyping was done using polymerase chain reaction and restriction fragment length polymorphism techniques (PCR-RFLP). The primers used for amplifying the IL-17A gene were adopted from Mohamed et al., [19]. PCR conditions were consisted of a pre-denaturation step of 4 minutes at 94 °C and 40 cycles each of 30 seconds denaturation at 94 °C, 30 seconds annealing at 55 °C and 30 seconds elongation at 72 °C. This was followed by a post-elongation step of 7 minutes at 72 °C. After obtaining PCR amplicons, fragmentation was done using XagI endonuclease according to manufacturer's instructions (Roche, Germany). Restriction fragments were loaded on 3% agarose gel and visualized using UV-transilluminator (Sigma Aldrich, Germany).

Statistical analysis:
Data were analyzed using the Statistical Package for the Social Sciences (SPSS v20). Fisher's exact test was used to test the differences in genotypes frequency. Pearson's χ2 test was used for the correlation of IL-17A genotypes with the study variables. A P value < 0.05 was considered statistically signi cant.

Characteristics of the study population
The study population included 266 participants, of them 166 (63.1%) were females [mean age 41.4 ± 15.5 years] and 97 (36.9%) were males [mean age 36.2 ± 16.0 years]. The study participants overall, aged between 1 and 85 years with average of 40 years. The participants were classi ed into 85 (31.2%) cases and 181 (68.8%) were controls. Based on age grouping, the age groups of 21-40 years followed by 41-60 years were the most frequent age groups; with 106 (39.8%) and 101 (38.0%) participants, respectively. Whereas, the frequency of the remaining age groups was 35 (13.2%) for 1-20 years, 23 (8.6%) for 61-80 years, and 1 (0.4%) for the age group of more than 80 years.
Among the studied population, those were from Arab ethnicity were the most frequent; 109 (41.0%). Also, those with a previous history of RA were 27 (10.2%), whereas those with no family history constituted 239 (89.8%). Based on duration of RA among the case group, the most frequent age group was 41-60 years; 53 (62.3%). And the duration of RA among the case group included 42 (49.4%) were having RA for less than 5 years, 29 (34.1%) for 5-10 years, and 7 (8.2%) for each of 11-15 years and 15-20 years (Table 1).

Discussion
Investigation of IL-17A polymorphisms among Sudanese population to determine the frequency distribution of the different IL-17A genotypes among healthy individuals [19]. in this study we investigated the frequency distribution of IL-17A genotypes among Sudanese patients diagnosed with RA and to determine the genetic susceptibility to RA in comparison to healthy individuals. The results of IL-17A genotypes distribution showed that the three genotypes were more prevalent in females compared to males, and among Sudanese Arab ethnic group. This result was similar to previous report, in which IL-17A polymorphism was signi cant among Sudanese Arab ethnic group [19]. As well as, the signi cant distribution of IL-17A genotypes among those with family history of RA, this could be attributed to the role of IL-17A SNPs inheritance to the descendants [6,19]. This was also supported by the negatively signi cant correlation of IL-17A with the family history of RA.
In this study, IL-17A genotypes showed a signi cant association to be linked with RA. This result is agreeing with a previously published systematic review including Fourteen studies and comprising more than three thousand patients with RA and more than two thousand healthy individuals to evaluate the relationship between serum level of IL-17 cytokine among RA patients and the possible SNPs that might be associated with the disease severity [21]. Their results showed a positive correlation between IL-17 level and RA compared to the control populations and they found some polymorphisms including IL-17A (rs2275913) was linked to increase the susceptibility toward developing the disease [21]. However, in a previous report investigated IL-17A (rs2275913) and the susceptibility to RA, the results showed that there were no statistically signi cant associations between IL-17A genotypes and the possibility of developing RA among Polish population [4]. And also, in a study conducted in a Tunisian population, IL-17A polymorphisms did not show any signi cant association with RA prevalence [22].

Conclusion
This study is the rst study in Sudan established the association between IL-17A (rs2275913) polymorphisms and susceptibly to RA. These ndings appeal for further research in various settings in Sudan to investigate the exact role of IL-17 in immunopathology and disease severity among Sudanese RA patients.

Limitations
Analysis of further SNPs that found in the IL-17A gene could also be linked to RA.
Functional analysis should be done to establish the effect of this SNP on the level of IL-17 and correlated the cytokine level with each genotyping and linked it to the outcome of the disease.