IFI44L Gene Promoter is Differentially Methylated in Iranian Patients with Systemic Lupus Erythematosus and Rheumatoid Arthritis.

Systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) are multisystemic autoimmune diseases with multifactorial nature. Considering limitations of the current conventional serological tests for diagnosis of these diseases, researchers strive to nd new and more valid biomarkers. The methylation level of interferon-induced protein 44-like (IFI44L) promoter was evaluated in 69 patients with SLE, 61 patients with RA, and 71 healthy subjects. Quantitative methylation of the promoter region of IFI44L gene was measured in DNA extracted from peripheral blood mononuclear cells (PBMCs) with methylation-quantication endonuclease-resistant DNA (MethyQESD) method. Our ndings revealed a substantial hypomethylation of IFI44L promoter in SLE and RA patients compared with healthy volunteers (mean: 60.36%±64.54%, 47.59%±30.34%, and 89.17%±76.96%, respectively; P SLE = 0.018, P RA <0.001). In comparison between SLE and RA patients with control group, IFI44L promoter methylation had a sensitivity of 84/06% and 93/65% and specicity of 32/39% and 29/58, respectively. The promoter methylation level was not meaningfully different between SLE and RA patients (P > 0.05). Moreover, our analysis revealed that the methylation level of IFI44L promoter was not statistically signicantly different between SLE disease activity and renal involvements (P > 0.05). While RA patients with a higher concentration of CRP had a lower DNA methylation level (P = 0.012). The methylation level of IFI44L promoter was lower in PBMCs of Iranian patients with SLE and RA than that control group. Furthermore, DNA methylation level of IFI44L promoter had a negative correlation with RA disease activity. However, there was not a signicant association between clinical characteristics of SLE.

. Hence, considering the very wide spectrum of discovered serological proteins, researchers strive to nd new and more valid biomarkers for a better diagnosis and management of these diseases.
Numerous studies have revealed that the disrupted gene expression pro les in immune cells are involved in the pathogenesis and represent pro-in ammatory phenotype in RA and SLE diseases. Speci cally, dysregulated DNA methylation patterns were well-known as a central contributor to autoin ammatory diseases including RA and SLE [19,20]. In this way, several studies with work on alteration of DNA methylation in these autoimmune diseases hope to reach a biomarker set for diagnosis and monitoring.
One of these genes is Interferon-induced protein 44-like (IFI44L). Although the exact function of this gene is unknown but studies demonstrated that expression of this gene is upregulated in SLE and RA patients which this upregulation was attributed to hypomethylation of IFI44L [21][22][23][24][25]. For the rst time, in two different studies, Chen and Zhao et al proposed that DNA methylation alteration of IFI44L is a promising biomarker for the diagnosis of SLE and RA with high speci city and sensitivity [24,26]. Therefore, we intended to investigate the utility of IFI44L methylation level in peripheral blood mononuclear cells (PBMCs) as a biomarker for the diagnosis of these diseases. Furthermore, in this study, we compared the difference between methylation levels of this gene in RA and SLE patients to evaluate the ability of this factor in distinguishing between two different diseases in the Iranian population.

Materials And Method:
Study Populations: In this case-control study, we enrolled 63 RA patients and 69 SLE patients according to diagnostic criteria created by the European League Against Rheumatism (EULAR) and the American College of Rheumatology (ACR) (2019). Besides, 71 age and gender-matched apparently healthy voluntaries were included as a control group. SLE and RA patients were recruited from rheumatology clinics and inpatient wards at Al-Zahra hospital, the biggest a liated hospital of Isfahan University of Medical Sciences. All healthy subjects in the control group had no symptoms or personal and family history of RA and SLE, or other autoimmune and immune-mediated conditions. This study was approved by the Isfahan University of Medical Sciences research ethics committee and all the voluntaries provided written informed consent.
Demographic and clinical presentation data of all subjects were collected. These data were sex, age, blood pressure (systolic blood pressure (SBP) and diastolic blood pressure (DBP)), height and weight to calculate body mass index (BMI, calculated as weight [kg] divided by height [m] squared), family history of RA, SLE and other autoimmune disorders and clinical manifestations such as the presence of cutaneous manifestations, neurological disorders, hematological abnormalities, oral ulcer, arthritis, and renal involvement. Likewise, laboratory characteristics such as C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), anti-dsDNA antibodies, complement component 3 (C3), complement component 4 (C4), white blood cell (WBC) count, hemoglobin, creatinine, platelet count test (PLT), blood urea nitrogen (BUN), fasting blood sugar (FBS), triglyceride (TG), high-density lipoprotein (HDL), and low-density lipoprotein (LDL) were documented. Ultimately, about 5 ml of venous blood was collected into EDTA anticoagulant tubes from each individual and stored at − 20°C for further processing.
The yield, purity, and suitability of DNA for the MethyQESD (methylation-quanti cation of endonucleaseresistant DNA) method was evaluated by spectrophotometry and agarose gel electrophoresis.

DNA methylation assessment:
Methylation of the IFI44L gene in both healthy and case groups was assessed by the MethyQESD method. This technique is a combination of methylation-sensitive digestion and real-time polymerase chain reaction (RT-PCR). The amount of methylated DNA that resists digestion by the methylationsensitive endonuclease Hin6I is determined by RT-PCR and is calibrated using a reference DNA that remains uncut. In detail, this method is a combination of two processes: the rst is restriction digestion of template DNA by two sets of methyl sensitive (Hin6I) enzymes, which is called "Methylation Quanti cation Digestion (MQD)". Hin6I does not affect methylated GCGC but was used to cut unmethylated GCGC regions. The methyl insensitive enzymes (XbaI & DraI), referred to as "Methylation-Independent Calibrator Digestion (CalD)", digest separately before RT-PCR ampli cation and their recognition sites are not present within the amplicon and were used to digest total DNA. The second process is the quantitation of the Hin6I digested template using real-time PCR in which XbaI and DraI Statistical analysis: The MedCalc statistical software version 10.2.0 (MedCalc Software bv, Ostend, Belgium) was used for statistical analyses to assess the percentage of methylation of the IFI44L gene in the cases and control groups. Because of the abnormal distribution of IFI44L in both groups, Mann-Whitney U-test was used to compare this variable between cases and controls. A receiver operating characteristic (ROC) curve analysis was used to evaluate the areas under the ROC curve (AUC). This established the best cutoff values for % DNA methylation for diagnosing SLE and RA and then sensitivity and speci city were calculated. For demographic, clinical, and laboratory characteristics, P values were calculated by Student's t-test, Chi-square, or Mann-Whitney test. The signi cance level was set at P < 0.05.

Results:
Demographic and laboratory characteristics: In this case-control study, we investigated 69 SLE patients (15 males and 54 females with mean age of 43.72 ± 13.41) and 63 subjects as RA patients (23 males and 40 females with mean age of 42.54 ± 11.80). The healthy control group consisted of 71 subjects (18 males and 53 females with mean age of 45.08 ± 12.32). The mean age of onset in the SLE and RA patient groups was 24.81 ± 10.80 and 40.31 ± 11.10, respectively. Table 1 shows the characteristics of the patients and healthy controls. Clearly, SLE and RA patients had higher BMI compared with the control group (P > 0.001). Likewise, there was a signi cant difference between the SLE patients and the control group in terms of systolic blood pressure (SBP) and diastolic blood pressure (DBP) (P < 0.05). However, there was not any difference between the RA patients and the control group in terms of SBP and DBP (P > 0.05).   tests, ESR, CRP, and creatinine were signi cantly higher in case groups than healthy controls (P < 0.05). While serum concentration of hemoglobin was obviously higher in the control group than in cases (P > 0.001). Furthermore, in SLE patients, the concentration of anti-dsDNA antibodies was signi cantly higher than in controls, while C3 and C4 levels were expressively higher in controls than in patients (P > 0.001).
On the other hand, white blood cell count, FBS, HDL, LDL, and TG were not markedly different between two groups of patients and healthy individuals (P > 0.05). The laboratory characteristics of patients (SLE and RA) and healthy controls are presented in Table 2.

IFI44L methylation analysis:
Based on our ndings, the mean IFI44L promoter methylation level in the SLE group was 60.36%±64.54% and in RA patients was 47.59%±30.34%, while in the control group was 89.17%±76.96%. Differences between the average percentages of methylation between RA and control groups were obviously signi cant (P > 0.001). Similarly, the difference between SLE and control groups was noticeably signi cant (P = 0.018). Although the average percentage of methylation in SLE groups was higher compared with RA subjects, this difference was not statistically signi cant (P = 0.144). Figure 1 demonstrates the comparison of IFI44L promoter methylation level between cases (RA and SLE patients) and the healthy control group. Additionally, the ROC analyses showed that the diagnostic power of the IFI44L promoter methylation level for RA was 0.  (Fig. 2).
Our analysis about the association between IFI44L promoter methylation level and clinical characteristics revealed that there was no statistically signi cant difference between laboratory parameters associated with SLE disease activity (P > 0.05). For instance, the DNA methylation level of IFI44L promoter in SLE subjects with renal involvement (54.25%±36.41) was lower compared to patients without renal involvement (69.31% ±91.58), however, it was not statistically signi cant (Table 3). Furthermore, the results of this study demonstrated that RA patients in whose serum concentration of CRP was ≥ 6 (mg/l) had a lower methylation level in the IFI44L promoter (38.42%±28.29) than those patients with CRP < 6 (mg/l) (39.42%±28.29) (P = 0.012). Discussion: Considering the limitations of the current conventional serological test for diagnosing SLE and RA, researches to identify new biomarkers that enable the development of new diagnostic tools with high sensitivity and speci city are imperative. The goal of the current study was to compare the DNA methylation level of the IFI44L promoter and its application in the diagnosis of SLE and RA patients as well as evaluate the association between this genetic factor with clinical characteristics of these disorders. Until 2016, the role of IFI44L in the pathogenesis of autoimmune diseases was unclear. Luo et al in 2016 reported that induction of IFI44L leads to upregulation of in ammation via activating the TBK1/IRF3 pathway [28]. Newly, some studies demonstrated that this gene also is involved in the negative regulation of innate immune response after induction of viral infections [29,30]. In this way, a body of works revealed that the expression of this gene is upregulated in peripheral blood and immune cells of RA and SLE patients [21,22,31] In summary, in this study, we indicated that promoter methylation of IFI44L in PBMCs of Iranian SLE and RA patients was obviously lower compared with healthy individuals. However, regarding sensitivity and speci city of the methylation level of IFI44L promoter, this biomarker has low power in distinguishing RA and SLE from healthy subjects. In this work, probably, some limitations in the statistical validity of our results such as small population size exist, so further similar studies with larger sample size would help to con rm the suggested correlations. Moreover, we propose that this factor should be evaluated in the other autoimmune diseases to con rm the speci city of DNA methylation and also clarify the role of IFI44L promoter methylation in the pathogenesis of these diseases.

Declarations
Authors' contributions: All authors were involved in whole work Manuscript has been seen and approved by all the authors.
Con ict of interest: Authors don't have any con ict of interest and we hope that you will nd our manuscript acceptable for publication in this Journal. Comparison of interferon-induced protein 44-like (IFI44L) promoter methylation level between patients with Systemic lupus erythematosus and Rheumatoid arthritis and healthy controls. * P value < 0.05

Figure 2
The receiver operating characteristic (ROC) curves of the methylation levels in patients with rheumatoid arthritis (A) and systemic lupus erythematosus (B) compared with healthy controls.