APO-1 / FAS Promoter (-670 A / G ) Polymorphisms and Risk of Lupus Nephritis in SLE Egyptian Female Patients

Background: Self-immunization in systemic lupus is driven by defective in apoptosis. Fas, is an apoptosis-promoting cell surface receptor. The present study evaluate the possible association between APO-1/FAS Promoter (-670A/G) Polymorphism and sFAS level with susceptibility to lupus nephritis in SLE patients. Design and Methods: This study was performed on 88 female patients with SLE (mean age, 39.82 ± 10.16 years). 82 patients with lupus nephritis (mean age, 42.50 ± 6.65 years). 150 age and sex-matched person served as controls. All participants were genotyped for the APO-1/FAS Promoter (-670A/G) Polymorphism, manifestations and serum sFAS were correlated with the genotypes. Results: Serum sFAS was significantly higher in patients with -670 AA genotype compared to others. (-670A/G) AA genotype frequencies were significantly higher in the lupus nephritis and SLE patients groups compared with the controls and were associated with increased risk for lupus nephritis and SLE development (odds ratio, 4.08 and 1.91 respectively). Conclusions: The APO-1/FAS Promoter (-670A/G) A allele can be used as a genetic marker for lupus nephritis susceptibility in SLE and was associated with high sFAS level.


Introduction
The etiology of SLE is still unknown, but many studies demonstrate association between the disease and genetic variations which are crucial to immunological response [1].
Lupus nephritis is a serious complication of SLE.Several factors have been proposed in the initiation and progression of LN.Two important factors that are suggested are apoptosis imbalance and overproduction of several cytokines [2].
Abnormalities in the apoptosis (programmed cell death) process could be related to development of SLE, and impairment of T or B cells [3].
Soluble FAS (sFAS) belongs to super family of death receptors and plays an important role in apoptotic signaling in different cells [4].FAS gene is located on chromosomes 10q24.1 and consists of nine exons and eight introns [5].
Several single-nucleotide polymorphisms have been identified in the promoter region of the FAS gene, one of the substitutions of A to G at position-670 (FAS-670A/G).This functional polymorphism abolishes the binding site of nuclear transcription element and decreases the binding ability, thus diminishes the promoter activity and decreases the FAS-gene expression [6].
Therefore, the apoptosis genes, Fas and Fas ligand (FasL), could be suitable candidate genes in human SLE and lupus nephritis susceptibility.
The present study aimed to study the possible association between APO-1/FAS Promoter (-670A/G) Polymorphism and sFAS level with susceptibility lupus nephritis in SLE Egyptian female patients.

Statistical Analysis
Results were collected, tabulated, statistically analyzed by IBM personal computer and statistical package SPSS version 16 (SPSS Inc.Chicago, Illinois, USA).All data were expressed as mean ± standard deviation number and percent.A P-value of <0.05 was considered statistically significant.

Results
The results of the present study are presented in Tables 1-3.
Table 1 shows a significant increase of ESR, ANA titre, dsDNA titre, proteinuria percent and sfas level, while there is a significant decrease as regarding hemoglobin level and platelet count in each of SLE and lupus nephritis groups when compared to control group.Also, there is a significant increase in urea and creatinine level when compared the lupus nephritis group to the two other groups, while there is a significant increase of sfas level and proteinuria percent in lupus nephritis group when compared to SLE group, while there is no significant differences as regarding other parameters.
Table 2 shows a significant difference between lupus nephritis group and the other two groups as regarding APO-1/FAS Promoter (-670A/G) genotyping.AA genotype frequency was associated with 4.08 times higher risk of lupus nephritis than control and 1.91 times than SLE group, while AG genotype frequency was associated with 1.76 times higher risk of lupus nephritis than control.Also, A allele frequency is higher in lupus nephritis and it was associated with 4.16 times higher risk of lupus nephritis than control and 1.78 times than SLE group.As regarding SLE group, AA genotype frequency was associated with 2.014 times higher risk of SLE than control, while AG genotype frequency was associated with 1.87 times higher risk of SLE than control.Also, A allele frequency is higher in lupus nephritis and it was associated with 2.33 times higher risk of SLE than control.
Table 3 shows a significant differences among APO-1/FAS Promoter (-670A/G) polymorphisms three genotypes (AA, AG and GG) in patient groups as regarding SLEDA score and sfas level with increase level in AA genotypes than other two genotypes.Also, there is a significant increase in ANA, ds DNA, urea, creatinine, ESR and proteinuria percent and decrease in hemoglobin level and platelet count when AA and AG genotypes compared to GG genotype, while there is no significant differences regarding other parameters.

Discussion
SLE is an chronic inflammatory autoimmune disease involves multiple organs [7].It is widely thought that self-immunization in systemic lupus is driven by defective clearance of dead and dying cells.In lupus patients, large numbers of apoptotic cells accumulate in various tissues including germinal centers [8].
The dysfunction of apoptosis may be a direct consequence of alterations in proteins/genes such as Fas, which   is an apoptosis-promoting cell surface receptor.sFas is a variant of the Fas receptor lacking the transmembrane region.It has been detected in human sera and was found to inhibit apoptosis induced by Fas protein [9].APO-1/Fas gene promoter was subdivided into basal promoter, enhancer region and silencer region.A GC-rich region is present upstream of the transcription start sites [10].
Single nucleotide polymorphism at nucleotide position -670 in the enhancer region, caused by A to G base change (-670G>A), occurs where transcription factor signal transducer and activator of transcription 1 (STAT-1) binds and thus has an effect on the level of APO-1/Fas expression.This substitution also creates MvaI restriction fragment length polymorphism [11].
The aim of this study to evaluate if there is association between APO-1/FAS Promoter (-670A/G) polymorphisms and susceptibility to SLE and lupus nephritis.
In this study there is increase percent of oral ulcer, discoid rash, arthritis, and serositis in AA genotypes.While increase perecent of photosensitivity and malar flush in AG genotypes in SLE and lupus nephritis patients.
In line with this result, Huang et al. reported the association of photosensitivity and oral ulcers with the -670 AA genotype.STAT-1 binding activity was found to be higher in -670A allele of the APO-1/Fas promoter as compared with the -670G allele and thus may be a candidate site for SLE susceptibility [12].The present study showed that, Serum levels of sFas in the patients with lupus nephritis were significantly higher than patients with SLE.Also, levels of sFAS were in SLE patients were significantly higher than controls.and a positive correlation was also shown between sFas and SLEDA score.
The results of this study were in line with other studies Alecu et al., 2000Hao et al., 2006 that demonstrated the impressive action of sFas increase level in LN [13] [14].
In accordance with this study Cheng et al and.Jodo et al stated that sFas levels in the serum are a marker for evaluating SLE disease activity.sFas is present at a high concentration in about 60% of lupus patients.Frequency of positive serum sFas is much greater with high SLE disease activity index scores than with low scores [10] [15].
Telegina et al. described that the oligomeric form of sFas stimulates apoptotic cell death, and that its levels are increased in sera from lupus patients when compared to the monomeric form.The exact function of sFas in the pathogenesis of SLE remains to be established.Patients with SLE show increased levels of sFas when compared to healthy controls [16].
The present study releaved that AA genotype frequency was associated with 4.08 times higher risk of lupus nephritis than control and 1.91 times than SLE group.While, A allele frequency is higher in lupus nephritis and it was associated with 4.16 times higher risk of lupus nephritis than control and 1.78 times than SLE group.Diecker et al., 2004 stated that, deranged clearance of apoptotic cell debris leads to the release of nuclear autoantigens.These autoantigens become clustered in apoptotic blebs that are restricted to sites of free radical generation in apoptotic cells for oxidative modification.These autoantigens when exposed on the surface of dying cells trigger an autoimmune response.Immune complexes of autoantigen/autoantibody bind to the basement membrane of different organs, especially the kidney, where these induce inflammation and may cause lupus nephritis, the most serious manifestation of SLE [17].
Also, in this study, As regarding SLE group, AA genotype frequency was associated with 2.014 times higher risk of SLE than control, While, A allele frequency is higher in lupus nephritis and it was associated with 2.33 times higher risk of SLE than control.This in accordance with Xiang et al. who observed a negative association between the FAS-670 G allele and SLE susceptibility [18].The study of Bita et al. show that the frequency of -670AA genotype was significantly higher in SLE patients than control group and the risk of SLE was 2.1-fold greater in subjects with AA genotype [19].
In contrast to present study Huang et al., Araste et al., Lee et al and Man-Manlu et al. reported no different allelic distributions at position -670 between patients and controls in an Iranian, Korean, Australian and Chinese population respectively [22]- [25].
It can be concluded that The APO-1/FAS Promoter (-670A/G) AA genotype and A allele can be used as a genetic marker for susceptibility of lupus nephritis in SLE patient, associated with high sFAS level and SLE susceptibility in Egyptian female population.

Material and Methods
This study was conducted on 320 female subjects, categorized into three groups, group I (88 SLE patients), group II (82 patients with lupus nephritis), and group III (150 subjects of healthy controls matched for age and sex).All participants were females.The mean age was 39.82 ± 10.16; 42.50 ± 6.65; and 43.76 ± 7.22 years, respectively.None of the healthy controls had a history of rheumatic disease.All patients were selected from Rheumatology and Internal Medicine Departments, Main University Hospital, Shebin El-Kom, Menofia University.
After approval of the local ethical committee and informed consent from each one, patients who were selected scheduled to undergo a sheet was taken to all patients subjected.
Inclusion criteria included patients with an established diagnosis for SLE.All patients were diagnosed according to the criteria of the American College of Rheumatology for SLE [26].LN is defined as clinical and laboratory manifestations that meet ACR criteria, persistent proteinuria > 0.5 gm per day or greater than 3+ by dipstick, and/or cellular casts including red blood cells [RBCs], hemoglobin, granular, tubular, or mixed [27] all patients with lupus nephritis were already diagnosed with renal biopsy in the nephrology unit.Exclusion criteria included all patients with SLE who had other connective tissue diseases or those with suspected mixed connective tissue disease.
The groups were evaluated for clinical (age, sex, constitutional manifestations, fever, malar rash, discoid rash, photosensitivity, oral ulcers, arthritis, pleurisy), laboratory parameters [hemoglobin, white blood cell count, platelets count, blood urea, serum creatinine, erythrocyte sedimentation rate (ESR), ANA, anti-dsDNA, proteinuria] and disease activity.Disease activity was assessed according to the SLE Disease Activity Index (SLEDAI), which is a reliable and validated method for assessment of disease activity in SLE [28].
Sample collection and assay: I-Sampling: (a) Venous blood samples (10 ml) were withdrawn under complete aseptic condition by clean venipuncture and then dispensed into 4 tubes (two EDTA tubes one for complete blood picture and the other for extraction of DNA for PCR-RFLP of APO1 gene, tube with citrate for ESR measurement and the last plain tube for detection of ANA, Anti-dsDNA, soluble fas (sFas) levels and for chemical analysis of urea and creatinine.The samples in the plain tubes were allowed to clot for 30 minutes, then the serum was separated by centrifugation at 4000 rpm for 15 minutes, and the clear serum was separated and stored at −80 until time of assay.
-ESR was determined with the classical Westergren method.
-Anti-dsDNA detected by an indirect solid phase ELISA for the quantitative measurement of IgG class autoantibodies against dsDNA in the serum [30].
-Measurement of serum sFas, using quantikine R and D sFas Immunoassay (USA), which is a solid phase ELISA.This assay employs the quantitiative sandwich enzyme immunoassay technique [31].
-DNA was isolated from whole blood EDTA tube samples using the QIAGEN extraction kit (Hilden, Germany), DNA eluted in buffer AE was stored at -20˚C for PCR-RFLP.
The PCR product of the APO-1/FAS Promoter gene was digested by MvaI restriction enzyme (provided by Fermentas), the reaction conditions were, 6.5 μl nuclease-free water, 2.5 μl 10× buffer, 10 μl of PCR product and 1 μl (2 units) MvaI.The mixture was incubated for 2 hours at 37˚C then 10 μl of the products were loaded into 2% agarose gel containing ethidium bromide for electrophoresis and digestion products were resulted in 233 bp and 99 bp for A/A genotype, 233 bp, 188 bp and 99 bp for A/G genotype and 188 bp and 99 bp for G/G genotype (Figure 1).

Table 1 .
Statistical comparison between the three studied group as regarding demographic data and laboratory findings.

Table 2 .
Statistical comparison among the three studied group as regarding genotyping.

Table 3 .
Statistical comparison between the two patients groups according to genotyping as regarding demographic and laboratory data.