Association between IL-35 Gene Single Nucleotide Polymorphisms and Systemic Lupus Erythematosus in Egypt

INTRODUCTION

SLE is defined as a chronic, relapsing, and intermittent autoimmune disorder which affects mainly women. It affects multiple organ systems and is distinguished by a wide range of clinical manifestations. Arthritis, cutaneous affection, nephritis, and vasculitis are the most common manifestations. ¹

It has been debated whether SLE is a single disease with numerous phenotypes or whether it is a common feature shared by many separate diseases with diverse pathogenic processes. This debate has arisen because of the high degree of heterogeneity in SLE. ²

In Egypt, the prevalence of SLE was estimated in a national cross-sectional study in 2021 to be 6.1 for 100000 populations and it is more prevalent in females (11.3/100000 females) while in males (1.2/100000) ³

In the United States, for example, documented cases  ranging from 20 to 150 cases per 100,000 individuals, with the highest prevalence reported in

North America, and the frequency looks to be growing as the disease becomes more widely recognized and survival rates improve. When compared to those of other racial or ethnic groups, people of African, Hispanic, or Asian heritage had a higher incidence rate of SLE and more organs affection in the United States. ⁴

Patients with severe and moderate SLE had significantly lower numbers of regulatory T cells (Treg) when compared to healthy controls, and the ratio of CD4 ⁺ CD25 effector T cells to CD4 ⁺ CD25 high CD127 Treg in severe and moderate SLE patients was significantly higher when compared to healthy controls, the researchers found. Patient's with moderate to severe SLE had significantly decreased gp130 expression on the CD4⁺ Th cell surface and on the CD14⁺ monocyte surface when compared to healthy controls, according to the findings. gp130 expression on CD4⁺ T cells and the proportion of Tregs were shown to be positively related with one another, while being negatively associated with the SLEDAI, which stands for Systemic Lupus Erythematosus Disease Activity Index. ⁵

Interleukin (IL)-35 is a member of the IL-12 family. It was found by Niedbala and Collison in 2007 and is composed of the interleukin-12 chain p35 and the interleukin-27 chain Epstein-Barr virus-induced gene 3 (Ebi3), which are joined by a disulfide bond. It is created by the immunological system of the human body.⁶ When the EBV virus infects B lymphoblastoid cells, the expression of EBI3 is upregulated. ⁷ IL-35 may have a role in the development of SLE by directly inhibiting effector T cell growth, particularly that of Th17 cells, and so delaying the onset of inflammatory responses, as well as by indirectly decreasing effector T cell development. Moreover, serum levels of interleukin-35 (IL-35) were found to be significantly higher in lupus patients compared to healthy subjects ⁸, and IL-35 has been shown to significantly alleviate lupus flare-ups and renal affection, as well as increased proinflammatory cytokines (IFN- γ, TNF- α, and IL-6 and IL-17a) in plasma ⁸.

These findings confirm that the interleukin-35 (IL-35) gene plays a critical role in the development and progression of SLE, but patients from the previous studies were all Chinese which recommend to study the effect of this polymorphism in different ethnic groups, in this study, we examine the association between Il-35 gene polymorphism and occurance of SLE in Egypt.

PATIENTS AND METHODS

Thirty (30) female patients with SLE who had already been diagnosed (Group 1) according to the six diagnostic criteria from Systemic Lupus International Collaborating Clinics (SLICC)⁹ for SLE and they were followed were included in this observational cross-sectional study, as well as twenty (20) healthy subjects who were age and sex-matched to the cases (Group 2) and had no history of autoimmune diseases. After informed consent was acquired, patients and a control group were enrolled in the study.

Inclusion Criteria for patients:

Already diagnosed with SLE.

Patients were selected from an outpatient clinic in their regular follow up.

All of  the control idividuals are characterized by the following:

a. does not meet any of the criteria for being classified as having SLE;

 b. neither the individual nor any near relatives have a history of autoimmune disorders.

The demographic profiles of the participants, as well as their clinical characteristics as mentioned in table 2, were acquired from the patients. The Medical Scientific Research Ethics Committee of Al-Azhar University gave its approval for this research project.

Specimen collection

Specimens from patients and control subjects were collected from Al-Azhar University hospitals; 2 mL was drawn from venous blood then added to EDTA tubes with stopper, then DNA extraction was done using a whole blood extraction kit manufactured by Hangzhou Bioer Technology Company Limited. Lot Number: 20201103.

Specimen storage

Specimens(DNA extract) were then capped and stored at -80° C  till the time of the assay.

Genotyping for IL-35 gene

Table 1: SNP details, Rstriction enzyme and sequence of primers used in the study

Table 1 shows the primer sequences, restriction enzyme utilised, and restriction digestion pattern for each allele.

The polymorphism of EBI3 (rs4740) was investigated using the PCR-based (RFLP) technique.

The restriction enzyme used was manufactured by New England Biolabs incorporation Ipswich, MA, USA. cat no: R0619S, we also used a 50 bp ladder for the gel electrophoresis step.

 Statistical Analysis

Continuous data, such as age, were displayed as mean ± SD to simplify the display. In the case of qualitative data, such as sex, it was supplied in the form of percentages. For numerical data Student t-test was used. For caategorical data. The Chi-square test was used. P-value at ≤ 0.05 was considered significant, which is the smallest possible value. The statistical analysis was done using IBM Statistical Package for Social Science (SPSS) software Version 21 for Windows.

RESULTS

Demographic data

Table 2: Age and clinical characteristics of two groups.

Regarding the age of SLE patients and healthy controls, there were no statistically significant differences between the two stdied groups (t = 0.7, p = 0.48). Table (2) also provides a list of the clinical characteristics of SLE patients.

Table 3: Genotypic and allelic frequency of various genotypes in controls or cases

Association between rs4740 SNP and SLE susceptibility

There were no statistically significant differences between SLE patients and healthy controls in the genotyped SNP rs4740 studied and also in allele frequency. (p > 0.05). (See Table 3)

Fig. 1: The electrophoresis of EBI3 rs4740 PCR and restriction digestion products is depicted in Figure 1. Representative RFLP product samples were electrophoresed with ethidium bromide to determine their composition:

heterozygous A/G (lanes 4, 5, 7,8,11, and 12) 98 bp and 388 bp;

homozygous  G/G (lanes 2, 3, 6, and 10) 98 bp and 345 bp;

homozygous  A/A (lane 9) 98 bp and 388 bp.