IL17A (rs2275913 G>A) and IL17F (rs2397084 T>C) Gene Polymorphisms: Relation to Psoriasis Risk and Response to Methotrexate


 Background The relation of IL17 polymorphisms to psoriasis risk and response to methotrexate has not been previously studied in Egyptians. Objectives To study the relation of IL17A (rs2275913 G>A) and IL17F (rs2397084 T>C) polymorphisms to psoriasis risk and assess their predictive role as regards response to methotrexate.Patients & Methods IL17A (rs 2275913) and IL17F (rs 2397084) polymorphisms were evaluated in 100 healthy subjects and 100 patients with chronic plaque psoriasis by real time-PCR. Patients were given methotrexate weekly intramuscularly (0.6mg/kg) for 12 weeks. Results IL17F TT genotype was more frequent in patients (87%) than controls (68%), while TC genotype was more frequent in controls (32%) than patients (13%). TT genotype was associated with increased risk of psoriasis whereas the TC allele was associated with a decreased risk. There was no significant difference regarding IL17A GG, GA and AA genotype frequencies between patients and controls. PASI ≥75% was achieved in 22 patients (73.3%) with the TT genotype and 8 patients (26.7%) with TC genotype (p=0.019). Conclusion IL17F (rs2397084 T>C) TT genotype could be considered a susceptibility marker in Egyptian patients. Psoriatic patients with TT genotype and T allele of IL17F (rs2397084 T>C) are likely to show a better response to methotrexate.


Introduction
Psoriasis is a multigenic, T-cell mediated immunological disease. (Georgescu et al. 2019) [1] Beside TNFα and IL-23, growing body of evidence from literature supports the role of the newer the IL-17 family in the pathogenesis and treatment outcome of psoriasis. (Puscas et al. 2019) [2] Interleukin 17 family comprises six structurally related cytokines (IL17A-F), the most important members of which are IL-17A, followed by IL-17F. They are believed to play a central role in psoriasis pathogenesis through their pro-in ammatory effect on keratinocytes and neutrophils. (Bialecka et al. 2016) [3] Methotrexate has been used as a standard monotherapy for treating psoriasis for decades and still continues to be used by virtue of its effectiveness, affordability and relative safety. Besides, its antiproliferative effect, it is believed to exert an anti-in ammatory effect through its effect on in ammatory cells and cytokines. Methotrexate has been shown to reduce IL17 expression and serum levels in psoriasis and rheumatoid arthritis patients. (Li et al. 2012) [4] However, variation in patients' response to methotrexate still remains incompletely understood. (Warren et al. 2009) [5]The genetic markers to predict the response to methotrexate treatment are not rmly established and there is paucity of pharmacogenetic studies in literature. It is possible that certain gene polymorphisms involved in the pathogenesis of psoriasis may allow to select patients likely to respond to methotrexate. (Warren et al. 2009) [5] IL-17A and IL-17F genes are located on chromosome 6p12. (Park et al. 2005; Prieto-Perez et al. 2015) [6,7] Several IL17 gene polymorphisms have been described in literature and were associated with risk of developing several autoimmune, in ammatory and infectious diseases. Single nucleotide polymorphisms (SNPs) for IL17A (rs2275913: A > G) and IL17F (rs2397084: T > C, rs11465553: G > A, rs763780: T > C) genes were shown to affect amino acid sequences. (Bialecka et al. 2016) [3] The implications of IL17 polymorphism to psoriasis susceptibility in general and in Egyptian patients however is not yet fully established. The relation of IL17 polymorphism to response to methotrexate has not been previously studied.
We aimed to study the association of IL17A (rs2275913 G > A) and IL17F (rs2397084 T > C) gene polymorphisms to psoriasis risk in a cohort of Egyptian patients and evaluate any predictive role to response to methotrexate.

Patients
This prospective study was conducted on 200 subjects, divided into two groups; group I of 100 healthy control individuals and group II composed of 100 patients with chronic plaque psoriasis. All study subjects were recruited from attendants of the dermatology outpatient clinic of Alexandria Main University Hospital from September 2016 to April 2018. A written informed consent was obtained from all subjects. The study protocol followed the International Ethical Guidelines of the 1975 Declaration of Helsinki and was approved by the local institutional ethical committee. Inclusion criteria were patients with chronic plaque psoriasis of either sex, aged more than 16 years of age with PASI score >10%.
We excluded pregnant and lactating women, patients with severe skin infection, lung diseases, chronic liver failure, leukopenia, aplastic anemia, neoplastic diseases and those with a history of allergy to methotrexate.
All patients were subjected to thorough personal, family and drug history taking and clinical evaluation of onset, and duration of psoriasis. A complete general physical examination was performed and a local dermatologic examination including grading of the disease severity according to the Psoriasis Area and Severity Index (PASI) score, before the start of medication and 12 weeks after treatment with intramuscular methotrexate 0.6mg/kg/week was conducted. PASI 75% was calculated at the end of the study.

Methods
Two mL of whole blood were withdrawn aseptically using sterile vacutainer K2 EDTA tubes. Samples were transferred to the lab immediately and stored at -20 c till the time of DNA extraction DNA Extraction, ampli cation and quanti cation: (Hebron HR 2009) [8] QIAamp DNA Blood Mini Kit (Qiagen, USA) was used to extract genomic DNA was extracted from PMC according to the manufacturer's instructions. The quantity and purity of the DNA was assessed by the NanoDrop 2000 (Thermo Scienti c, USA).
The IL17A (rs 2275913) and IL17F (rs 2397084) SNPs genotyping was performed using the 5′ nuclease Allelic discrimination assays. The PCR reaction mix contained 10 μL TaqMan® Universal PCR Master Mix (Applied biosystems, USA), 20 ng DNA/reaction, 1 μL of TaqMan® SNP Genotyping Assay 20x (Assay ID: C_3219460_20 and C_15903863_10 respectively) and DNAase free water to a nal reaction volume of 20 μL. Thermal cycling pro le was conducted using Rotorgene Q real-time PCR system (Qiagen, Germany) as follows: initial AmpliTaq Gold enzyme activation at 95 °C for 10 min, and 40 denaturation cycles at 95°C for 15 seconds and annealing/extension for 1 minute at 60 °C.

Statistical analysis of the data
Data were fed to the computer and analyzed using IBM SPSS software package version 20.0 (Armonk, NY: IBM Corp). Qualitative data were described using number and percent. The Kolmogorov-Smirnov test was used to verify the normality of distribution. Quantitative data were described using range (minimum and maximum), mean, standard deviation and median. Signi cance of the obtained results was judged at the 5% level. The used tests were chi-square test for categorical variables to compare between different groups and sher's exact or monte carlo correction for chi-square when more than 20% of the cells have expected count less than ve. Odd ratio (OR) was used to calculate the ratio of the odds and 95% con dence interval of an event occurring in one risk group to the odds of it occurring in the non-risk group.

Results
Demographic characteristics of the studied groups: Genotype and allele frequency of IL17A polymorphism (rs2275913 G>A) in the control and patients groups: There was no signi cant difference regarding GG genotype frequency between patients (70%) and controls (74%) (p=0.146). GA genotype frequency was more frequently observed in psoriasis patients (21%) than controls (10%). The AA genotype was observed in 16% of controls and 9% of patients (p=0.146).
The G allele was observed in 79% and 80.5% of the control and patients groups respectively, while the A allele was observed in 21% and 19.5% of the controls and patients respectively. There was no signi cant difference between patients versus control subject group regarding both G and A allele frequencies (p=0.759). (Table 1) Genotype and allele frequency of IL17F polymorphism (rs 2397084 T>C) in the control and patients groups: Genotype TT was signi cantly more frequently observed in the patients group (87%) than the control group (68%), while genotype TC was signi cantly more frequent in controls (32%) than patients (13%). (p=0.005). TT genotype was statistically associated with increased risk of psoriasis occurrence (OR=3.148) whereas the TC allele was associated with a decreased susceptibility to develop psoriasis (OR=0.318). (Table 1) There was a signi cant difference between patients and control groups regarding both T and C allele frequency where the T allele was signi cantly more observed in psoriatic patients (93.5%) than in controls (84%), and the allele C was more frequent in controls (16%) than patients (6.5%) (p=0.009). (Table 1) The T allele was found to be signi cantly associated with increased risk of psoriasis (OR=2.740) whereas the C allele was statistically associated with reduced risk to develop psoriasis (OR=0.365). (Table 1)

Discussion
Psoriasis is a chronic immune-mediated in ammatory disease that affects about 2%-4% of the population. Etiopathogenesis involves multifactorial environmental and polygenic factors. The aim of this study was to study the association of IL17A (rs2275913 G>A) and IL17F (rs2397084 T>C) polymorphisms to the risk of developing psoriasis in a cohort of Egyptian patients with chronic plaque psoriasis and asses the presence of a possible predictive role to response to methotrexate.
A positive family history of psoriasis was signi cantly higher in our cohort of patients than controls suggesting a signi cant genetic background. Most of our patients were of the early onset psoriasis type.
Naldi L et al (Naldi L 2011) [13] similarly reported a signi cantly higher family history of psoriasis among their psoriatic patients compared to the healthy controls. Early onset psoriasis (also referred to as type I) is known to start before 40 years of age, with peak onset at 16-22 years of age in opposition to lateonset psoriasis (also termed type II psoriasis) that starts at or after age 40 years, with a peak age of onset between 57 and 60 years. (Gladman DD 2005) [14] Early onset psoriasis is strongly associated with class I HLA alleles and patients usually have a family history. In contrast, type II psoriasis is more commonly sporadic with an unclear genetic background. (Schmitt-Egenolf M 2000) [15] In the present study, a signi cant reduction of PASI score was observed 12 weeks after methotrexate therapy. Haider et al (Haider et al. 2014) [16] reported a 66% reduction of PASI score after 8 weeks of subcutaneous weekly methotrexate therapy in their patients. Methotrexate continues to be an affordable effective treatment option for patients with moderate to severe chronic plaque psoriasis whose condition requires systemic therapy or who failed to respond to topical treatment alone. Its therapeutic effect can be partly explained by inhibition of DNA synthesis by competitive inhibition of dihydrofolate reductase, accounting for its antiproliferative effect. However, this is not the sole mechanism of its therapeutic effect in psoriasis. A further anti-in ammatory effect through induction of immune cell apoptosis and inhibition of T-cell activation has been suggested by invitro studies. (Meephansan et al. 2011) [17] Methotrexate was shown to reduce the numbers of T cells and monocytes in the skin and decrease expression of adhesion molecules. (Rentenaar et al. 2004) [18] Furthermore, methotrexate therapy was shown to be associated with reduction of serum interleukin-17 and 23 levels in psoriasis patients. (Elghandour et al. 2013) [19] It was suggested that methotrexate suppresses IL17 mRNA expression thereby decreasing IL-17 production. (Li et al. 2012) [4] A signi cant percentage of our patients achieved a PASI 75 response. Similarly, Warren et al (Warren et al. 2017) [20] reported a 41% of patients achieving PASI 75 response. The slightly higher response can be attributed to the longer course of 16 weeks of methotrexate therapy.
We report that IL-17F (rs2397084 T>C) TT genotype was associated with increased risk of psoriasis occurrence whereas the TC allele was associated with a decreased risk to develop psoriasis. The T allele was also signi cantly associated with increased risk of psoriasis whereas the C allele was statistically associated with reduced risk to develop psoriasis. This suggests that IL17F (rs2397084 T>C) polymorphism could serve as a marker of susceptibility to develop psoriasis in our Egyptian patients. This is could be explained by the fact that IL17F (rs2397084 T>C) gene polymorphism has been shown to change amino acid sequences. (Bialecka et al. 2016) [3] Bialecka et al (Bialecka et al. 2016) [3] reported no association between IL17F (rs2397084 T>C) polymorphisms and psoriasis susceptibility in a Polish population. This is probably a re ection of racial or ethnic differences.
Our observed higher frequency of IL17A (rs2275913 G>A) GG and GA genotypes among our psoriasis patients than controls and opposingly higher frequency of AA genotype in controls was insigni cant. The G allele was more frequently observed in patients while the A allele was more frequent in controls. However, no signi cant difference was observed. We suggest that IL17A (rs2275913 G>A) polymorphism cannot be regarded as a susceptibility marker among Egyptian patients. However, this observation should be veri ed by other studies involving larger number of patients is needed before reaching such conclusion.
Kim et al (Kim et al. 2017) [21] similarly reported no association between IL17A (rs2275913 G>A) polymorphism and psoriasis in a Korean population. Bialecka et al (Bialecka et al. 2016) [3] reported no association between IL17A (rs2275913 G>A) polymorphisms and psoriasis susceptibility in a Polish population.
In the present study, TT genotype of IL17F (rs2397084 T>C) polymorphism and T allele were associated with a better treatment response (PASI 75) to methotrexate compared to the TC genotype and carriers of the C allele. Similarly, Bialecka et al (Bialecka et al. 2016) [3] observed that carriers of the C allele of IL17F (rs2397084 T>C) polymorphism needed a greater number of NB-UVB phototherapy sessions to respond compared to carriers of the T allele.
We report that IL17A (rs2275913 G>A) polymorphism genotypes GG, GA, AA and the haplotypes G and A were not related to treatment response to methotrexate. This was in agreement to the observations reported by Bialecka et al (Bialecka et al. 2016) [3] that IL17A (rs2275913 G>A) polymorphisms were not related to PASI score changes after topical vitamin D3 analogue and NB-UVB phototherapy.
To the best of our knowledge, this is the rst report that IL17F (rs2397084 T>C) polymorphism could be regarded as a marker of susceptibility to psoriasis in Egyptian patients with the TT genotype representing an increased susceptibility to develop psoriasis and the TC genotype presenting decreased risk to develop psoriasis. IL17A (rs2275913 G>A) polymorphism cannot be regarded as a marker of susceptibility to psoriasis in Egyptian patients. Further, psoriatic patients with TT genotype and T allele of IL17F (rs2397084 T>C) polymorphism are likely to show a better therapeutic response to the commonly used methotrexate treatment than the TC genotype and carriers of the C allele. We did not observe any relation of IL17A (rs2275913 G>A) polymorphism genotypes and alleles to treatment response to methotrexate.

Declarations
Ethics approval and consent to participate : Our research complies with the guidelines for human studies and was conducted ethically in accordance with the World Medical Association Declaration of Helsinki. Approval of the local institutional ethics committee (IRB 00012098) was obtained (approval number 0304835). Written informed patients consents were taken.
Patients consent for publication: consents for publication were obtained from the patients.

Con ict of interests:
none Funding: none Availability of data and materials: The analyzed data sets are available from the corresponding author upon reasonable request.
Author contributions: