Genetic Association of Interkeukin-21 and Interleukin-23 Receptor Polymorphisms With Systemic Lupus Erythematosus in Iranian Population

doi:10.21203/rs.3.rs-924037/v1

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Genetic Association of Interkeukin-21 and Interleukin-23 Receptor Polymorphisms With Systemic Lupus Erythematosus in Iranian Population

https://doi.org/10.21203/rs.3.rs-924037/v1

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Objective: T helper 17 (Th17) is believed to play a crucial role in pathophysiology of systemic lupus erythematosus (SLE). Our study aimed to detect the potential association of IL-21 and IL-23R polymorphisms with susceptibility to SLE.

Method: Two hundreds and ten patients with SLE and the same number of matched controls were genotyped by polymerase chain reaction to detect two polymorphisms of IL-21 and one polymorphism of IL-23 receptor (IL-23R). Clinical data was collected from patients’ archives.

Results: IL-21 polymorphisms (rs4833837 C/T and rs2055979 G/T) did not show any association with the disease susceptibility (p= 0.54 and p= 0.49, respectively). Comparison of patients with rs2055979GG and T allele carriers showed higher frequency of rs2055979GG genotype in patients with pleurisy compared to the ones without pleurisy (p=0.004). In addition, categorization of IL-23R rs10889677 to GG and T allele carriers (TT+TG) showed an association between TT+TG genotypes and susceptibility to SLE (p=0.0002). Moreover, a significant association was found between the inheritance of rs10889677GG+TG genotypes and presence of mucosal ulcers in SLE patients (p=0.01).

Discussion: The results showed that the lower IL-21 producer rs2055979GG genotype was associated to pleurisy. Lower IL-21 serum levels in patients with rs2055979GG genotype may lead to more frequent pulmonary infections and an increased risk of pleurisy. Association of high+ intermediate IL-23R producer genotypes (rs10889677TT+TG) with SLE may be explained by possible overactivation of Th17 cells. Higher activity of Th17 cells may also explain the over representation of mucosal ulcer in patients with high producer genotype of IL-23R.

Rheumatology

SLE

IL-21

IL-23R

Single nucleotide polymorphisms

Systemic lupus erythematosus (SLE) is an autoimmune disease involving various organs in the body with a significant mortality and morbidity. SLE is characterized by a relapsing remitting course and production of several autoantibodies against host cell’s nuclear components. Still not clearly understood, the disease etiology in believed to be the outcome of impaired interaction between genes and environment (1). Abundant studies have confirmed the role of genetic background in SLE development. The disease does not follow a simple Mendelian inheritance rule; thus a polygenic inheritance model has been suggested (2). Due to the inflammatory nature of SLE, current studies are focusing on finding abnormal immune responses to the triggers. Cytokines are of the major actors of inflammation which regulate the proliferation, maintenance, differentiation and apoptosis of immune cells (3). IL-21 is one of the newly investigated cytokines in pathogenesis of autoimmune diseases. Mostly produced by CD4 + cells, IL-21 has various immune regulatory functions including augmenting T cells proliferation, increasing the activity of natural killer cells and induction of B cell differentiation to plasma and memory cells resulting in increased antibody production (4). According to previous studies, some polymorphisms of IL-21 predispose the individual to autoimmunity (5). Of interest, patients with active SLE have higher serum concentration of IL-21 and blockade of IL-21 receptor in murine models results in decreased disease activity (6–7). Therefore, single nucleotide polymorphisms (SNPs) in the IL-21 gene may be associated with susceptibility to SLE.

Another interesting cytokine in the pathogenesis of autoimmune diseases is IL-23. This cytokine is produced by the activated dendritic cells and its main function is to stimulate the production of IL-17 by Th17 cells. The release of IL-17 embarks an inflammatory response by production of several other pro-inflammatory mediators (8). The IL-23/IL-17 axis is a well-known pathway necessary for maintaining chronic inflammatory states such as bacterial infections and autoimmune disorders. Over expression of IL-23R has been reported in some autoimmune diseases and allergic diseases (9, 10). In addition, several IL-23R gene polymorphisms have been associated with increased risk of various autoimmune diseases (11–12).

Considering the importance of IL-21 and IL-23 in Th17-related immune responses and development of SLE (13), the association of the two IL-21 gene polymorphisms (rs4833837 C/T and rs2055979 G/T) as well as rs10889677 G/T polymorphism in the IL-23R gene with SLE was investigated in the present study.

Patients and controls

SLE patients were enrolled from Shiraz Medical School related rheumatology clinics including Hafez and Motahari centers. Two hundred and ten patients and the same number of healthy controls were selected. Patients were confirmed cases of SLE by the laboratory tests and certified physicians and all met the new SLICC criteria (14). Patients were reviewed based on the most recent follow up and their self-reported questionnaires and notes of certified rheumatologists. Healthy controls were recruited from blood donors of blood transfusion units. Control cases had no history of known autoimmune, inflammatory diseases, and cancer. Patients and controls were matched for age, gender and ethnicity. The female/male ratio was 11.35/1 in both groups. All subjects signed an inform consent regarding blood sampling and access to medical records before enrolling in the study. The study was approved by the ethics committee of Shiraz University of Medical Sciences.

Genotyping

Genotyping was performed on genomic DNA isolated from peripheral leukocytes. DNA was extracted by using the salting-out method in Autoimmune Diseases Research Centre of Shiraz University of Medical Sciences and was stored at -21 ℃ until use. The international HapMap project databank was used to get access to the nucleotide sequence of the DNA segments containing the desired SNPs and the appropriate primers were designed by the Primer 3 software and were ordered for the PCR process (Table 1). To genotype IL-21 rs2055979 G/T, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was used. The amplification reaction was done by using 2.5 µl of PCR buffer (1.8 mM MgCl2), 1 unit of Taq polymerase, 2 µl of 2.5 mmol/L dNTPs and 20 pmol/L of primers with 50 ng of DNA. The PCR program was designed as following, 5 minutes heating at 94°C, denaturation for 30 cycles of 30 seconds at 94°C, annealing for 30 seconds at 61°C and elongation for 30 seconds at 72°C. The extension time lasted for 5 minutes at 72°C at the end of the cycles. The next step, we added the restriction enzyme Hin1II to the PCR products for digestion. The digestion process lasted for 16 hours at 37°C. The digested PCR products were stained by 2% ethidium bromide and were run on 1.5% agarose gel at 100 V for 70 minutes. Results were interpreted using a UV illuminator machine. Genotypes were determined by comparing the DNA segment sizes (Fig. 1-A). To genotype IL-21 rs4833837 C/T and IL-23R rs10889677 G/T we used allele specific PCR method. The first round PCR program was set as previously stated. After the amplification, 2 µl of a 1:50 dilution of the first round PCR mixture was used for the second PCR round, which needed two tubes for the amplification of wild and variant type alleles using different primers (Table 1.). The PCR products are shown in Fig. 1B-C.

Table 1

Primer sequences designed for the study
SNP	left Primer Sequence	Right primer sequence	Genotyping method
IL-21 rs4833837 C/T	5’-TTA GTT GCG CCT TCT GAA Aa-3’ 5’-TTA GTT GGG CCT TCT GAA Ag-3’	5’-TGT AAT GCA CGA CAT TGC AG-3’	Allele specific PCR
IL-21 rs2055979 G/T	5’-GCT CTG AAC CCA AAC ACT CTC-3’	5’-ACA GCC AGG AAA CTC TGG AA-3’	PCR-RFLP using Hin1II as restriction enzyme
IL-23R rs10889677 G/T	5’-TTT TAG CCA TTC TTC TGC CTc-3’ 5’-TTT TAG CCA TTC TTC TGC CTa-3’	5’-TTT TAG CCA TTC TTC TGC CTa-3’	Allele specific PCR

Statistical Analysis

Genotypes and alleles frequencies were compared between patients and controls and between patients classified based on clinical manifestations using the chi-square test. Association of genotypes with continuous parameters (such as age at disease onset) was analyzed by ANOVA. All the tests were performed two-tailed with confidence interval (CI) and a p-value < 0.05 was regarded as the level of statistical significance. SPSS version 11.5 (SPSS Inc., Chicago, IL, USA) was used for statistical analysis.

Two SNPs within IL-21 gene were genotyped in 210 SLE patients and matched controls. None of the selected SNPs showed significant association with susceptibility to the disease in our study (Table 2). The minor allele frequency of the rs4833837C/T was 20.2% in patients compared to 18.6% in matched controls (p = 0.54) while for rs2055979G/T was 46.9% in patients and 49.3% in controls (p = 0.49%).

Of interest, analysis of the rs10889677G/T genotypes in IL-23R gene showed a significant association with susceptibility to the disease (p < 0.0001; Table 3). After classification of cases and controls based on their gender, the difference remains significant in females (Table 3). Of interest, after categorization of females into rs10889677GG genotype and rs10889677T allele carriers (TT + TG genotypes), it was revealed that part of the observed difference in female patients and controls is due to the higher frequency of rs10889677T allele carriers in female patients compared to controls (84.4% and 67.9%, respectively; p < 0.0001; Table 3). Of interest, the frequency of heterozygote rs10889677TG genotype was also significantly higher in female patients compared to female controls (55.4% and 34.2%, respectively; p < 0.0001).

Table 2. Allele and genotype frequencies of IL-21 rs4833837 and rs2055979 in SLE patients and control

Table 3. Allele and genotype frequencies of rs10889677 in SLE patients and control

Furthermore; the medical records of the patients were reviewed for different clinical manifestations and their associations with the three selected SNPs were investigated. While there were no significant association between IL-21 rs4833837C/T and involvement of different organs in SLE patients, IL-21 rs2055979G/T was associated with pulmonary involvement (p = 0.05). Furthermore, after stratification of patients into IL-21 rs4833837TT genotype and G allele carriers (GG + GT), it was revealed that the frequency of GG + GT genotypes is significantly higher in patients with pleurisy compared to those without it (100% vs 24.4%, respectively; p = 0.004).

The same analysis for IL-23R rs10889677G/T showed a significant association between this polymorphism and the presence of mucosal ulcers (p = 0.001). In further analysis, patients were categorized into TT and TG + GG or TG and TT + GG genotypes. The results showed that the association between rs10889677 and mucosal ulcers is a result of higher expression of rs10889677TT genotype in patients without mucosal ulcers compared to those patients with mucosal ulcers (40.5% and 10.5%, respectively; p = 0.01) or lower frequency of rs10889677TG genotype in patients without mucosal ulcers compared to ones with mucosal ulcers (38.8% and 84.2%, respectively; p = 0.0002; Table 4). Furthermore, we found a trend towards significant association between rs10889677 genotype distribution and neurological symptoms in SLE patients (p = 0.064; Table 4). After patients classification based on their allele inheritance it has been cleared that rs10889677TT genotype was associated with resistance to neurological involvement in the patients (p = 0.025; Table 4).

Table 4. IL-23R rs10889677 genotypes distribution in patients with different clinical presentations.

rs10889677

Mucosal ulcers

----------------------------

Yes No

N(%) N(%)

Neurologic symptoms

--------------------------------

Yes No

N(%) N(%)

*P1 *p2

Genotypes:

1(5.3%) 25(20.7)

16(84.2) 47(38.8)

2(10.5) 49(40.5)

5(25) 21(22.3)

12(60) 34(36.2)

3(15) 39(41.5)

0.001 0.064

0.11 0.79

0.0002 0.05

0.01 0.025

Alleles:

20(52.6) 145(59.9)

18(47.4) 97(40.1)

18(45) 112(59.6)

22(55) 76(40.4)

0.39 0.09

IL-21 is an inflammatory cytokine known for its role in activating both humoral and cell mediated immune responses, most notably the differentiation of B cells to plasma cells (4). Various in vivo and in vitro studies in human and animal models have revealed the possible role of IL-21 in initiation and maintenance of inflammation (15). Of interest, serum concentration of IL-21 is reported to be significantly higher in active SLE patients compared to healthy population (6). Furthermore, some genetic variations in the IL-21 gene were associated with susceptibility to SLE (16). Zhou,et al. and Zhang,et al. found associations of certain haplotypes containing rs4833837 with psoriasis and Hashimoto’s thyroiditis (17, 18). However, neither of the rs4833837 alleles was significantly associated with higher serum levels of IL-21 (17, 18). Previously, Hughes, et al. reported that the C allele was the risk allele in SLE patients of European descent but such association was not seen in African American patients (19). In our study, the minor allele (C) frequency of rs4833837 was 20.2% in south Iranian patients and 18.6% in matched controls and no statically remarkable association of this SNP with SLE was found (p = 0.54). The inconsistent findings might result from differences in genetic make-up between African American and Iranian population as well as the complicated interaction of various genes and environmental triggers in pathogenesis of SLE. Lan, et al. reported a significant association between rs2055979T allele and increased levels of IL-21 and susceptibility to SLE in Chinese population (20). However, this association was not repeated in a study on 1318 American patients (21). Also, in the current study no significant association was found between the inheritance of rs2055979T allele and development of SLE (p = 0.49). However, classification of patients into rs2055979GG genotype holders and T allele carriers (GT + TT) showed an increased frequency of GG genotype in patients with pleurisy compared to the ones without it (100% vs 24.4%, respectively; p = 0.004). This finding is in favor of the risk role of the G allele or the protective role of T allele in developing pleurisy. The base of this association is not clear at the present time. However, given the fact that infections are among the most important inducers of pleurisy and IL-21 plays an important role in antibody production, it can be hypothesized that SLE patients with low IL-21 producer genotype (rs2055979GG) may be prone to pulmonary infections and pleurisy due to the their lower ability of IL-21 production.

IL-23 is a pro-inflammatory cytokine which embarks a cascade of inflammatory factors via activation of IL-17, a well-known mediator of inflammation (8). Consequently, the association of IL-23R gene variations with disorders of the immune system has been studied in several studies (22–23). So far, none of the SNPs in the IL-23R gene was reported to be associated with the susceptibility to SLE in the Hungarian, Korean and Chinese patients (24–26). rs10889677 is located in the 3'-UTR region of the IL-23R gene. 3'-UTR region is not translated to proteins but has regulatory functions and influences gene expression and mRNA stability (27). It has been reported that rs10889677T allele increases the mRNA production and results in an overall increase in IL-23R production (28). Of interest, in contrast to previous studies, the result of the present study showed an association between rs10889677T allele carriers (GT + TT genotypes) and susceptibility to SLE (p = 0.0002), which is consistent with the enhancing effect of rs10889677T allele on IL-23R mRNA production. To our surprise, this association was only detected in the studied females. This gender biased association may be related to the difference in hormonal factors between various genders. However, it should be considered that the number of the male patients in the present study was rather small (a total of 16 males) and further studies are needed before taking any conclusion. In addition, the results of the present study showed a significant association between inheritance of high producer rs10889677T allele carriers (GT + TT) and the presence of mucosal ulcers in SLE patients (p = 0.01). Inheritance of rs10889677T allele carriers may predispose patients to the development of mucosal lesions through enhancing IL17/IL23 axis activity. This inflammatory axis is profoundly active during active phases of SLE (29), when the patients experience more oropharyngeal lesions. Unexpectedly, our results showed an increased frequency of rs10889677TT genotype in patients without neurologic involvement compared to the ones with neurologic manifestations (41.5% vs 15%, respectively; p = 0.025). Considering the importance of Th17-related cytokines in the development of immune-associated neurological diseases (30), the basis for this association is not clear to us and indicates the need for further researches in this area.

The result of the present study showed that rs2055979 in the IL-21 gene is associated with pleurisy development in SLE patients while this SNP is not associated with SLE development. Furthermore, we found that rs10889677 in the IL-23R gene is associated with susceptibility to SLE as well as some clinical manifestations in the studied SLE patients. It is worth mentioning that these preliminary findings need further studies on larger SLE populations to come into a conclusive conclusion.

Polymerase Chain Reaction (PCR), Systemic Lupus Erythematosus (SLE), Single Nucleotide Polymorphism (SNP)

Ethics approval and consent to participate

The study involving a human participant to report was approved by the ethics committee of the department of Immunology at Shiraz University of Medical Sciences. Written consent was obtained from the patients.

Consent for publication

Written informed consent was obtained from the patients for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor of this journal.

Availability of data and material

Not applicable.

Competing interests

The authors declare no conflict of interest.

Funding

The Work was funded by Shiraz University of Medical Sciences.

Authors' contributions

MAN, EL and SSH were the clinical rheumatologists who followed the patients and gathered the clinical data. MRM was the immunology PHD candidate who performed the laboratory tests. EKS was the professor of immunology who supervised the study in all stages. NA was the student of the research committee and took part in data analysis and preparation of the manuscript. All authors reviewed the paper and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Acknowledgement

Not applicable.

Conflict of Interest

There is no conflict of interest to be declared regarding the manuscript

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Genetic Association of Interkeukin-21 and Interleukin-23 Receptor Polymorphisms With Systemic Lupus Erythematosus in Iranian Population

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Abstract

Figures

Introduction

Methods

Patients and controls

Genotyping

Statistical Analysis

Results

Discussion

Conclusions

Abbreviations

Declarations

References

Status:

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