|Year : 2016 | Volume
| Issue : 3 | Page : 129-135
Role of CD134 and FAS and FAS ligand genes polymorphism as biomarkers for disease activity in lupus nephritis: A preliminary egyptian study
Hala I El Gendy1, Mona N Abdel Gawad1, Aml S Nasr2, Elham A Ghoneim1
1 Department of Internal Medicine, Cairo University, Cairo, Egypt
2 Department of Clinical Pathology, Cairo University, Cairo, Egypt
|Date of Web Publication||11-Aug-2016|
Aml S Nasr
Kasr El Eini School of Medicine, Cairo University, Kasr El Eini Street, Cairo
Source of Support: None, Conflict of Interest: None
Objective: To illustrate the role of CD134 and FAS and FAS ligand genes polymorphism as biomarkers for disease activity in Egyptian patients with Lupus Nephritis.
Materials and Methods: Twenty-five patients with biopsy-proven LN, 25 patients with SLE with no evidence of nephritis, and fifty patients matched apparently healthy volunteers. Levels of CD134 were measured using flow cytometry. FAS and FASL gene polymorphisms were detected using polymerase chain reaction-restriction fragment length polymorphism. Furthermore, carotid artery intima-media thickness (IMT) measurements were done.
Results: LN group had highest level of CD134 compared to other two groups, and also higher among SLE compared to controls with highly significant differences in between. Frequency of AA genotype of FASA-670G polymorphism was significantly higher in LN and SLE patients than in controls. The frequency of A allele was statistically higher in LN and in SLE group than in controls. Furthermore, the frequency of CC genotype of C-844T polymorphism of FASL gene was significantly higher in LN and SLE patients than in healthy controls. The frequency of C allele was statistically higher in LN and in SLE group than in controls.
Conclusion: Co-stimulatory molecules on CD4+ T-cells together with FAS, and FASL polymorphisms are associated with disease activity in this preliminary study.
Keywords: CD134, FAS, FAS ligand genes polymorphism, lupus nephritis
|How to cite this article:|
El Gendy HI, Abdel Gawad MN, Nasr AS, Ghoneim EA. Role of CD134 and FAS and FAS ligand genes polymorphism as biomarkers for disease activity in lupus nephritis: A preliminary egyptian study. Indian J Rheumatol 2016;11:129-35
|How to cite this URL:|
El Gendy HI, Abdel Gawad MN, Nasr AS, Ghoneim EA. Role of CD134 and FAS and FAS ligand genes polymorphism as biomarkers for disease activity in lupus nephritis: A preliminary egyptian study. Indian J Rheumatol [serial online] 2016 [cited 2017 Apr 24];11:129-35. Available from: http://www.indianjrheumatol.com/text.asp?2016/11/3/129/187414
| Introduction|| |
Systemic lupus erythematosus (SLE) is an autoimmune rheumatic disorder that is thought to involve disturbances in both innate and adaptive immune mechanisms, including complex interactions between T-lymphocytes, B-lymphocytes, and other antigen-presenting cells (APCs). ,
Lupus nephritis (LN) is one of the most severe complications of SLE and is characterized by the production of nephritogenic autoantibodies and immune complex formation. 
OX40 (CD134) is a membrane-bound member of the tumor necrosis factor (TNF) receptor superfamily found on activated CD4+ T-cells. The ligand for OX40 (OX40 L) is expressed on activated APCs including B-cells, macrophages, endothelial cells, and dendritic cells. 
OX40-OX40 L interaction has been involved in the pathogenesis of autoimmunity. Engagement of OX40 on activated T-cells during antigen-specific T-cell stimulation can rescue effector T-cells from peripheral deletion. This results in a greater number of T-cells surviving the primary immune response and developing into memory T-cells, which may lead to the development of an autoimmune disease if they encounter their specific antigen. 
Expression of CD134 was associated with disease activity and renal involvement, but a functional analysis of these CD134 expressing cells is still lacking. As a member of the TNF superfamily, CD134 (OX40) provides co-stimulatory signals upon ligation to the CD134 Ligand. 
High levels of co-stimulatory molecules were also found in human SLE. The expression of these markers correlated with disease activity as assessed by the SLE Disease Activity Index (SLEDAI) score.  Furthermore, expression of CD134 L has been shown to be upregulated in proliferative LN, suggesting a role for the CD134-CD134 L pathway in its pathogenesis. 
FAS/FAS ligand system is the main extrinsic pathway for the initiation of apoptosis in numerous cells and tissues.  FASL is a member of TNF super-family and initiates the death signal cascade, which results in apoptotic cell death. 
FAS and FASL genes are located on chromosomes 10q24.1 and 1q23, respectively. , Abnormal expression of FASL on lymphocytes and triggering apoptosis has been demonstrated in SLE patients. Several polymorphisms have been recognized in FAS gene, as A to G replacement at nucleotide position -670 (FAS A-670G, rs1800682) in the enhancer region.  The FASL gene C-844T polymorphism is located in the binding site of transcription factor CAAT/enhancer-binding protein β. Several studies investigated the role of FAS and FASL gene polymorphisms in the etiology of SLE; however, the role of FAS and FASL gene polymorphisms in lupus has not been conclusively established.
The aim of this work was to illustrate the role of the CD134, as a simple noninvasive method for monitoring of the disease activity in patients with LN, as well as the role of FAS (FAS A-670G, rs1800682) and FASL (FASL T-844C, rs763110) gene polymorphisms in the etiology of SLE. The novelty of the work comes from that, to our knowledge, it is the first study to evaluate the role of the CD134 and the role of FAS and FASL gene polymorphisms in susceptibility to SLE in Egyptian patients.
| Materials and Methods|| |
This study was performed on patients admitted to Internal Medicine Department, as well as patients from the Rheumatology and Immunology Outpatient Clinic of Kasr Al-Ainy School of Medicine, Cairo University, in the period between 2011 and 2013. Local institutional research board approval as well as oral informed consents was taken from all the participants before the study. Furthermore, the study conformed to the provisions of the World Medical Association's Declaration of Helsinki.
The current study was carried out on 100 cases, divided as 25 patients with biopsy-proven LN (21 females and 4 males), 25 patients with SLE but with no evidence of nephritis (20 females and 5 males), as well as 50 age- and sex-matched apparently healthy volunteers (10 males and 40 females). The patients with SLE were diagnosed according to the American College of Rheumatology (ACR) 1987 criteria for the diagnosis and classification of SLE, and the patients with LN were diagnosed according to the ACR 1986 criteria for the classification of LN.
The main exclusion criteria included patients with mixed connective tissue disease.
Criteria by which mixed connective tissues diseases were excluded are the presence of Raynaud's phenomenon, swollen fingers or hands, presence of anti U1-ribonucleoprotein, SLE-like symptoms, scleroderma-like symptoms, and polymyositis-like symptoms.
All the subjects included in the study were subjected to:
Full history taking, detailed clinical examination including neuropsychiatric manifestations, also vascular disorders including arterial and venous disorders, routine laboratory investigations including complete blood picture, erythrocyte sedimentation rate (ESR), urine analysis, 24 h urinary proteins, liver function tests, kidney function tests, and CD134 by flow cytometry, FAS (FAS A-670G, rs1800682) and FASL (FASL T-844C, rs763110) gene polymorphisms using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP).
Patients were subjected tolipid profile: Cholesterol and triglycerides, immunological profile: Antinuclear antibody, anti-double-stranded DNA (Anti-ds DNA), C3, C4, lupus anticoagulant, anticardiolipin, renal biopsy only for patients with LN as well as carotid duplex for measurement of carotid intima-media thickness (CIMT), power Doppler sonography were performed using a HDI 5000 system. A linear transducer was used; at 7-12.5 MHz examination was performed according to standardized EULAR (European league against rheumatism) method.
The carotid artery IMT was selected in specific as an accurate noninvasive method that allows for the assessment of arterial wall thickness and degree of plaque, its important role in the detection of the presence of atherosclerotic disease in humans and also to track the regression, arrest of progression, or progression of atherosclerosis, which is considered as a reliable marker for evaluation of the presence of atherosclerosis in SLE patients.
Flow-cytometry for CD134
Concerning the flow cytometric measurements, 2 ml of venous blood was obtained and preserved in ethylenediaminetetraacetic acid (EDTA) vacutainers for measurement of OX40 expression on peripheral blood CD4+ T-lymphocytes by flow cytometry (Coulter EPICS XL). Samples were analyzed within 6 h. Staining (double-color surface staining) of 100 μL of each sample was done using 10 μL of each of cells was incubated with anti-CD134-PE (Becton-Dickinson) together with anti-CD4-FITC (Becton-Dickinson). Fluorescence intensity was determined using the FAC scan from Becton-Dickinson. The tubes were then incubated in the dark at room temperature for 15 min. Erythrocytes were lysed using ammonium chloride lysing solution (Al-Gomhoreyya CA, Egypt). After two washes with phosphate-buffered saline (PBS), the cells were resuspended in PBS for flow cytometric analysis. Negative isotype-matched controls were included with each sample to determine the nonspecific binding of the monoclonal antibodies. The autofluorescence region was adjusted. The sample tube was introduced; the fraction of cells or particles coated by monoclonal antibody was determined inside the gated population and assessed in a histogram. For each specimen, gating was constructed on CD4+ CD134+ cells and measuring CD4+ CD134+ percent within the gate using isotypic controls of the corresponding fluorochromes in accordance with the manufacturer instructions (Beckman-Coulter Inc., Brea, CA, USA; Affymetrix eBioscience, CA, USA). Isotypic controls were used to define the threshold for positive staining cells.
FAS (FAS A-670G, rs1800682) and FASL (FASL T-844C, rs763110) gene polymorphisms
A volume of 3 ml of venous blood was obtained and preserved in EDTA vacutainers. Genomic DNA was extracted from the whole blood using DNA extraction kit (QIAamp Blood Kit, Catalog number 51106; Qiagen Inc., Valencia, CA, USA) according to the manufacturer's instructions.
FAS and FASL polymorphisms were identified by the PCR-RFLP method. For FAS (FAS A-670G, rs1800682), the following primers were used Forward: 5'-CTACCTAAGAGCTATCTACCGTTC-3', Reverse: 5'-GGCTGTCCATGTTGTGGCTGC-3'.  For FASL (FASL T-844C, rs763110), the following primers were used Forward: 5'-CAGCTACTCGGAGGCCAAG-3', Reverse: 5'-GCTCTGAGGGGAGAGACCAT-3'. 
PCR was performed in a 25 μL final volume which contained 20 pmol of each primer, 0.1 mmol of dNTP (Fermentas), 0.3 μg of genomic DNA, 1.5 mmol/L of MgCl 2 , 2.5 μL of 10XPCR buffer, and 1.5 unit of Taq DNA polymerase (Fermentas), according to the following protocol: Initial denaturation at 95°C for 5 min; 30 cycles of denaturation at 95°C for 1 min, annealing for 1 min at 61°C for FAS A-670G and 63°C for FASL T-844C polymorphisms; extension at 72°C for 2 min; and final extension at 72°C for 5 min. The PCR products (20 μL) were incubated with 5 units of MvaI (New England Biolabs) at 61°C for FAS (FAS A-670G, rs1800682) and BsrDI (New England Biolabs) at 63°C for FASL (FASL T-844C, rs763110) and digested products were separated by electrophoresis on a 2.5% agarose gel and visualized by ethidium bromide staining. The size of the amplified product was read with the use of a DNA ladder of different molecular weights (Fermentas, NoLimits™ 50 bp DNA Fragment, catalog number SM1441).
Concerning the FAS (FAS A-670G, rs1800682) gene polymorphism, the wild genotype (AA) produces a single band at 223 bp (site of cleavage), the heterozygous genotype (AG) gives 3 bands (223, 189 and 44 bp), and the presence of homozygous mutation (GG) gives 2 bands at 189 and 44 bp.
Concerning the FASL (FASL T-844C, rs763110) gene polymorphism, the wild genotype (TT) produces a single band at 401 bp (site of cleavage), the heterozygous allele (TC) gives 3 bands (401,223 and 168 bp), and the presence of homozygous mutation (CC) gives 2 bands at 223and 168 bp.
| Results|| |
Analysis of data was done by IBM computer using SPSS (Statistical Program for Social Sciences version 12) as follows: Quantitative variables were expressed as mean, standard deviation (SD), or median and range when appropriate, qualitative variables were expressed as percentage, Chi-square test was used to compare qualitative variables,. Unpaired t-test was used to compare two groups as regard parametric variables, and Mann-Whitney test was used instead of unpaired t-test in nonparametric data. Correlation between various variables was done using Pearson's moment correlation equation for linear relation. P < 0.05 was considered statistically significant and people value <0.01 to be highly statistically significant.
The current study was carried out on 25 patients with biopsy-proven LN, 25 patients with SLE but with no evidence of nephritis, 50 age- and sex-matched apparently healthy volunteers.
Demographic and clinical data of the patients and controls are summarized in [Table 1].
There were no statistically significant differences between the studied groups as regard demographic and clinical data except for the presence of neuropsychiatric manifestations. Laboratory data of the patients and controls are summarized in [Table 2].
There were statistically significant differences between the studied groups as regard hemoglobin, creatinine, and lactate dehydrogenase. There were highly statistically significant differences between the studied groups as regard ESR, C-reactive protein (CRP), urea, uric acid, and 24 h urine albumin, but no statistically significant differences between the two studied groups regarding the CIMT.
Results of some markers related to disease activity (C3, C4, Anti-ds DNA, and SLEDAI) and CD134 are summarized in [Table 3].
|Table 3: Comparison between the studied groups as regard CD134 and some laboratory parameters |
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This table shows that LN group had highest level of CD134 compared to other two groups, and also higher among SLE compared to controls with highly significant differences in between. There were no statistically significant differences in C3, C4, Anti-ds DNA, and SLEDAI in patients with LN compared to other groups.
Correlation between CD134 versus different variables among LN and systemic lupus groups is summarized in [Table 4].
|Table 4: Correlation between CD134 versus different variables among lupus nephritis and systemic lupus groups |
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In the LN group, there was statistically significant positive correlation versus ESR, CRP, and CIMT and statistically significant negative correlation versus C4. On the other hand, there was no significant correlation versus other variables.
In systemic lupus group, there was statistically significant positive correlation versus ESR, uric acid, and CIMT.
The results of FAS (FAS A-670G, rs1800682) and FASL (FASL T-844C, rs763110) are summarized in [Table 5].
|Table 5: Genotype and allele frequencies of FAS A-670G and FASL C-844T polymorphisms in patients and controls |
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The frequency of the AA genotype of FASA-670G polymorphism was significantly higher in LN (40%) and SLE patients (28%) than in controls (20%). The frequency of A allele was statistically higher in LN (46%) and in SLE group (44%) than in controls (42%).
Furthermore, the frequency of CC genotype of C-844T polymorphism of FASL gene was significantly higher in LN (56%) and SLE patients (52%) than in healthy controls (HC) (44%).
The frequency of C allele was statistically higher in LN (48%) and in SLE group (48%) than in controls (46%) whereas for the T-allele in LN (22%) and in SLE group (24%) than in controls (28%) which were statistically significant [Table 6] and [Table 7].
|Table 6: Distribution of the studied nephritis group as regard renal biopsy results |
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| Discussion|| |
Nephritis is a major cause of morbidity and mortality in patients with lupus. Nephritis occurs in approximately 50% of lupus patients , but rates vary significantly. 
The presence of autoantibodies is a requirement for the development of LN.  Antibodies to dsDNA/nucleosomes are most closely linked with development of nephritis. 
The main aim of work was to measure the levels of CD134 on CD4 positive T-lymphocytes as well as FAS and FASL gene polymorphisms to determine their role in the pathogenesis of SLE and LN and to compare the results with the HC as well as to determine the capability to use CD134 as a novel and noninvasive marker in studying patients with LN. The need for a reliable marker for LN such as CD134 is highly important, whereas renal biopsy is the gold standard for diagnosis and assessment of nephritis in patients with lupus, it is an invasive procedure that cannot be performed serially for monitoring purposes. The carotid artery IMT was selected in specific as an accurate noninvasive method that allows for the assessment of arterial wall thickness and degree of plaque, its important role in the detection of the presence of atherosclerotic disease in humans and also to track the regression, arrest of progression, or progression of atherosclerosis, which is considered as a reliable marker for evaluation of the presence of atherosclerosis in SLE patients.
Our results showed that LN group had highest level of CD134 compared to other two groups, and also higher among SLE compared to controls with highly significant differences in between.
Our results were in agreement with Farres et al., 2011,  who reported that the percentage of CD4+ T-lymphocytes expressing OX40 was significantly higher in SLE patients than in controls, and in patients with LN than in those without but differ from our results in that OX40 expression correlated positively with serum creatinine levels.
Furthermore, our results were in agreement with Dolff et al., 2010,  who reported that expression of CD134 on the interleukin (IL-17) producing T-cell subset was higher in SLE than in HC. In addition, patients with LN expressed higher levels of CD134+ on CD3+ IL-17+ cells as compared to HC (72.69 ± 11.54% vs. 51.45 ± 16.58%, P = 0.006). Furthermore, renal biopsies of LN patients showed infiltration of CD134+ T-cells.
Furthermore, our results were in agreement with Patschan et al., 2006,  who reported that SLE patients showed an increased frequency of peripheral CD4+ T-cells expressing high levels of CD80, CD86, and CD134 compared to HC. There was an increased presence of CD134+ CD4+ cells in SLE patients with LN.
Our results were also consistent with those of Abo-Elenein et al., 2010,  who reported that expression of CD134 on peripheral CD4+ and CD8+ T-cells was increased in SLE group compared to the control group.
However, our results were different from those of Aten et al., 2000,  who had reported that the difference between SLE and control subjects did not reach statistical significance regarding CD134 level. He attributed these results to the small number of the patients included in the study.
The frequency of the AA genotype of FASA-670G polymorphism was significantly higher in LN (40%) and SLE patients (28%) than in controls (20%).
Furthermore, the frequency of CC genotype of C-844T polymorphism of FASL gene was significantly higher in LN (56%) and SLE patients (52%) than in HC (44%).
Our results were in agreement with Moudi et al., 2013,  who reported 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 (P = 0.03). The frequency of -670A allele was significantly higher in SLE patients than in controls too (58% vs. 49%, P = 0.03). The -844CC genotype frequency was significantly higher in SLE patients than in HC, and the risk of SLE was 2.8-fold greater in these subjects (P = 0.01).
Our results were also in agreement with the study of Chen et al., 2005,  who reported that 844C/C genotype is associated with lupus susceptibility.
In consistent with present study, Kanemitsu et al. reported that the A allele of FAS A-670G but not G-1377A was significantly associated with SLE.  In contrast to our study, Arasteh et al. reported no different allelic distributions at position -670 between patients and controls in an Iranian population. However, they observed higher GG genotype and G allele distribution at position -1377 in SLE patients. In addition, they found higher soluble FAS and FAS ligand levels in the patient group and lower amounts of serum anti-SSB/La in patients with the -670GG genotype. 
Although several studies are performed to investigate the effect of FASL gene mutations on SLE susceptibility, to date there are only a few published reports about the association between C-844T polymorphism and SLE. ,,
In 2003, Wu et al. found SNP at -844 within the FASL promoter region (T-allele in addition to the C) in normal populations with an allele frequency of 0.82 for African-Americans. In addition, they reported increased expression of FASL in individuals with -844CC genotype that could enhance autoimmunity risk. 
| Conclusion|| |
From the results of our preliminary study, we can conclude that co-stimulatory molecules on CD4+ T-cells together with FAS (FAS A-670G, rs1800682) and FASL (FASL T-844C, rs763110) gene polymorphisms are associated with renal disease and disease activity in patients with SLE. However, further studies on a larger group of patients are needed to confirm this.
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Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7]