Association Study Of Polymorphisms,mRNA Expression Of FAS And FASLG With Systemic Lupus Erythematosus

Background Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease,characterized by autoantibody production, immune complex formation, and subsequentmultiple organ damage. The pathogenic mechanisms of SLE remain unclear. Bothenvironmental initiating elements and genetic background play crucial roles in thepathogenesis of SLE. Immune function abnormalities of SLE are associated with thedisorder of apoptosis. Recent studies have indicated that the dysfunction of cellsapoptosis and deficient clearance of apoptotic cells could contribute to the pathogenesisof SLE.Apoptosis is also called programmed cell death (PCD), is death of a cell in anyform, mediated by an intracellular program. In1972, Kerr first brought out the conceptof apoptosis. It is also involved in inducing regulation development, maintainhomeostasis and neoplasms. Peripheral tolerance is broken down in SLE. So, apoptoticcells as the primal immune source stimulate autoimmue systems leading to abnormalactivation and proliferation of autoreactive lymphocyte. Many signal pathways areinvolved in regulation of cell apoptosis, one of which is Fas/FasL pathway.Fas (CD95/APO-1) is a45kD type I transmembrane glycoprotein that belongs tothe tumor necrosis factor or neuronal growth factor receptor superfamily (TNFR/NGFR). It is expressed in many cells, including activated T/B lymphocytes, naturalkillers, and thymocyte cells. The human FAS gene has been mapped to chromosome10q24.1or10q23and spans25kb of the chromosome. It has nine exons (25bp to>1.44kb) and eight introns. Fas ligand (FasL), a trimeric type II membrane protein, belongs to the TNF receptor family, which is mainly expressed on activated T cells.FASLG gene consists of four exons and spans8kb on chromosome1q23. Fas/FasLsystem induces the death signal cascade leading to apoptosis, which palys importantroles in the maintenance of immune tolerance and prevention autoimmune disease.Observations in mouse models of SLE demonstrated that mice with Fas (MRL/lpr)or FasL (gld) mutations developed lymphadenopathy with accumulation ofauto-antibodies leading to spontaneous autoimmune diseases.In recent years, studies have indicated that the dysfunction of Fas-mediated isassociated with SLE. Previous research found an increase of early aoptosis rate ofPBMCs maong SLE, and Fas protein is up-regulated in our study teams. SLE is acomplex genetic disease. Genetic background plays crucial roles in the pathogenesis ofSLE. Many genes have found to be associated with SLE susceptibility in differentpopulations.Although FAS/FASLG genes have been shown to play important roles in SLE,little is known about FAS/FASLG gene polymorphisms and expressions of SLE inChinese Han population. Thus, our study adopted case-control design to assess thatFAS/FASLG gene correlated with the development of SLE through geneticsusceptibility and mRNA expression. We analyzed the relationship among mRNAexpression and single-nucleotide polymorphisms (SNPs) of the FAS/FASLG genes, andthe main clnical and laboratory features in SLE. Besides, we also used a Meta-analysismethod to assess the risk of FAS and FASLG genes polymorphisms for SLE.Part â…  Association Study of FAS and FASLG Polymorphismswith Systemic Lupus ErythematosusObjective To explore FAS (SNPs rs2234767and rs1800682) and FASLG genes (SNPrs763110) whether or not confer susceptibility to SLE, and to investigate associationbetween the polymorphisms and the main clnical and laboratory features in SLE, besides, to assess the risk of FAS and FASLG genes polymorphisms for SLE using aMeta-analysis method.Methods A total of552patients with SLE were recruited from the First AffiliatedHospital of Anhui Medical University and Anhui Provincial Hospital. All patientsfulfilled the1997revised criteria of the American College of Rheumatology for theclassification of SLE. A total of718healthy blood donors were included as controls, allof whom were excluded from SLE or other autoimmune diseases. Demographic dataand clinical data were collected by questionnaire. EDTA anti-coagulated venous bloodsamples were collected from all participants. Genomic DNA was extracted fromperipheral blood lymphocytes. Genotyping was performed by TaqMan SNP assay.Haplotype analysis was assessed using SHEsis software. A Meta-analysis method waspeformed to assess the association between polymorphisms and SLE using Stata10.0software. The probability level as <0.05in two-tailed test was considered statisticallysignificant.Results(1) HWE testNo deviations from HWE were observed in the controls in each polymorphism.(For SNP rs2234767, controls,2=1.235,P=0.266ï¼›cases,2=4.451,P=0.035; For SNPrs1800682, controls,2=3.280,P=0.070ï¼›cases,2=5.134,P=0.023; For SNP rs763110,controls,2=1.666,P=0.197ï¼›cases,2=1.074,P=0.300)(2) Association analysis of FAS gene and SLETo SNP rs2234767, genotype frequencies for G/G,A/G and A/A were46.6%,40.4%and13.0%in the case group,40.3%,44.8%and14.9%in the control group,respectively. Case-control comparison revealed a significant association between SLEand the minor allele A at SNP rs2234767(P=0.033). Significant differences ingenotypic distribution were also found in SLE patients and controls (AG vs GG, P=0.041). To SNP rs1800682, genotype frequencies for A/A,A/G and G/G were39.7%,42.9%and17.4%in the case group,35.4%,45.4%and19.2%in the controlgroup, respectively. And no significant differences were either showed in the allelicdistributions betweeen them (P>0.05).(3) Association analysis of FASLG gene and SLETo SNP rs763110, genotype frequencies for C/C,C/T and T/T were59.2%,34.4%and6.3%in the case group,58.9%,34.5%and6.5%in the control group, respectively.And no significant differences were either showed in the allelic distributions betweeenthem (P>0.05).(4) FAS gene haplotype analysisStrong linkage disequilibrium (LD) was found between the two SNPs (D'=0.937,r2=0.708). We analysed the haplotypes of FAS gene and determined three mainhaplotype: AG, GA and GG. Analysis of the haplotypes revealed that the haplotype GAwas significantly associated with SLE (P=0.039).(5) Association of FAS and FASLG gene polymorphisms and clinical featuresWhen we analysed FAS and FASLG gene polymorphisms and the main clinicalfeatures, we did not find association of clinical features with three SNPs in SLE patients(P>0.05).(6)The results of Meta-analysisIn the meta-analysis, available studies including our data were combined up to totaleight studies. The results showed significant association between rs2234767, rs1800682and rs763110polymorphisms and SLE (P<0.05).Conclusion In summary, FAS rs2234767polymorphism may contribute to SLEsusceptibility in the Chinese population. The haplotype GA constructed with rs2234767and rs1800682was significantly associated with SLE. The results of meta-analysisshowed significant association between the three SNPs and SLE in differentpopulations. Part â…¡ Association Study of FAS and FASLG mRNA expressionwith Systemic Lupus ErythematosusObjective To compare the expression of FAS and FASLG mRNA in SLE patients andhealthy controls, LN and SLE without LN, active and inactive SLE. Combined withclinical and laboratory data, the relationship with their mRNA levels were furtheranalyzed.Methods A total of35patients with SLE and34controls were randomly selected fromsamples of Partâ… . Individual disease activity was quantified using the systemic lupuserythematosus disease activity index (SLEDAI) scores. RNA were extracted fromperipheral blood lymphocytes. The lever of mRNA expression in PBMC was detectedby relative quantitation PCR. For comparing the median between different groups, thenonparametric Mann-Whitney two sample tests were used. For the correlation analysisbetween FAS and FASLG mRNA and SLEDAI, Spearman's rank correlation coefficientwas used. The probability level as <0.05in two-tailed test was considered statisticallysignificant.Results(1) FAS mRNA expressions in PBMCFAS mRNA expressions in PBMC were increased in SLE patients compared withhealthy controls (P=0.011). Higher FAS mRNA expression was also found in the activeSLE patients when compared to inactive counterparties (P=0.045). However, there is nosignificant difference between LN patients and non-LN SLE patients (P>0.05).(2) FASLG mRNA expressions in PBMC There was no significant difference regarding FASLG mRNA levels between patientswith SLE and healthy controls, active SLE patients and inactive ones (P>0.05).However, higher FASLG mRNA expression was found between SLE patients withnephritis and those without nephritis (P=0.015).(3) Association of FAS/FASLG mRNA expressions and clinical featuresAs for clinical and laboratory features, SLE patients with facial erythema and fevershowed increased expression level of FAS mRNA (P=0.041, P=0.016, respectively).And, SLE patients with rash and facial erythema indicated higher expression level ofFASLG mRNA (P=0.023, P=0.029, respectively). However, there is no significantdifference between FAS/FASLG mRNA expressions and laboratory features (P>0.05).(4) Association of FAS/FASLG polymorphisms and mRNA expressionsWe analysed both FAS gene (SNP rs2234767and SNP rs1800682) and the FASLG gene(SNP rs763110) polymorphisms with mRNA expression among69participants. There isno significant difference between genetypes of the three SNPs and mRNA expression(P>0.05).Conclusion In summary, FAS mRNA expressions in PBMC were increased in SLEpatients compared with healthy controls. Higher FAS mRNA expression was also foundin the active SLE patients when compared to inactive patients. We detected significantassociation of FAS and FASLG mRNA expression with part of clinical features. Noassociation was found between the FAS (rs2234767, rs1800682)/FASLG (rs763110) andmRNA expression.