Association Study Of RIP2Gene Polymorphisms, Serum RIP2Level And Systemic Lupus Erythematosus

Background Systemic lupus erythematosus (SLE) is a systemic autoimmune disease,it is characterized by a diverse array of autoantibody production, immune complexesdeposition and complement activation, resulting in multiple system and organinvolvement. The etiology of SLE remains unclear, genetic susceptibility, environmentalfactors, sexual hormone disorder and immune dysfunction are thought to contribute todisease development.Dysfunction of innate immunity and adaptive immunity plays an importment rolein the immune pathogenesis. Multiple immune signaling pathways have been reportedto participate in the immune pathogenesis of SLE, including NF-κB signaling pathway,Toll-like receptor signaling pathway, JAK/STAT signaling pathway, MAPK signalingpathway, PI3-AKT signaling pathway, Th17signaling pathway and so on. Thesesignaling pathways interaction might regulate abnormally, resulting in the immunedysregulation.Our previous study has found that the level of RIP2mRNA in SLE patients wassignificantly elevated compared to the healthy controls using case-control design andReal-time PCR assay to detect differentially expressed genes in Toll-like receptorsignaling pathway. This result indicated that RIP2might take part in the progress ofSLE.RIP2, also known as RIPK2, RICK, CARDAK, is a key serine/threonine kinase insignal transduction pathway. RIP2mediates innate and adaptive immune responsesinduced through multiple receptors, such as Toll-like receptor and NOD-like receptorand so on, resulting in the production of inflammatory cytokines. Several studies have identified that RIP2might be associated with autoimmune diseases, such as Crohn'sdisease, multiple sclerosis. However, so far no study has reported the association ofRIP2and SLE at home and abroad.Thus, on the basis of the content above, our study adopted case-control design todetect association of RIP2gene polymorphisms, serum RIP2level and SLE by theTaqMan SNP Genotyping Assay kit and ELISA. The accomplishment of this studywould help to further reveal the role of RIP2in the pathogenesis of SLE and developnew potential therapeutic targets. Objective To compare the differences of allele and genotype frequencies about twoSNPs (rs16900617, rs16900627) of RIP2between SLE patients and normal controls,and to explore the association of RIP2polymorphisms with susceptibility to SLE.Methods590SLE patients were recruited from Department of Rheumatology andImmunology of Anhui Provincial Hospital and the First Affiliated Hospital of AnhuiMedical University. All SLE patients fulfilled the1997revised American College ofRheumatology classification criteria.660normal controls were recruited from healthyvolunteers. The demographic and clinical data were collected by self-designedquestionnaire. After obtaining the informed consent, blood samples from all studiedsubjects were collected. The genotypes of the rs16900617and rs16900627SNPs weredetermined by the TaqMan SNP Genotyping Assay kit using an ABI7300Real-Timepolymerase chain reaction (PCR) system.SPSS10.01software was used for statistical analysis. The chi-square test orFisher's exact test was applied to compare the distribution of allele and genotype frequencies between SLE patients and normal controls. Stata7.0software was used toHardy-Weinberg equilibrium test. Haplotype analysis was assessed using SHEsissoftware. P values were calculated based on two-sided tests, with a statisticalsignificance defined as P value <0.05.Results(1) Association of rs16900617and SLE The genotype frequencies of AA, AG, GGwere86.4%,13.4%,0.2%in SLE patients and77.9%,20.3%,1.8%in controls. Theallele frequencies of A, G were93.1%,6.9%in SLE patients and88.0%,12.0%incontrols. There were significant differences between SLE patients and controls aboutgenotype (AG vs. AA: OR=0.59,95%CI0.44-0.81, P=0.001; GG vs. AA: OR=0.08,95%CI0.01-0.65, P=0.006; AG+GG vs. AA: OR=0.55,95%CI0.41-0.75, P <0.001) and allele frequencies (G vs. A: OR=0.54,95%CI0.41-0.72, P <0.001).The allele frequencies of A, G were94.0%,6.0%and the genotype frequencies ofAA, AG, GG were88.0%,12.0%,0%in lupus nephritis (LN) patients. They were93.2%,6.8%,86.6%,13.2%,0.2%respectively in non-LN patients. There were nosignificant differences between LN patients and non-LN patients about genotype (AGvs. AA: OR=0.90,95%CI0.52-1.55, P=0.698; GG vs. AA: OR=1.003,95%CI0.997-1.008, P=1.000; AG+GG vs. AA: OR=0.88,95%CI0.51-1.52, P=0.651) andallele frequencies (G vs. A: OR=0.87,95%CI0.52-1.48, P=0.612).(2) Association of rs16900627and SLE The allele frequencies of A, G were80.7%,19.3%and the genotype frequencies of AA, AG, GG were64.6%,32.2%,3.2%in SLEpatients. They were84.2%,15.8%,72.3%,23.9%,3.8%respectively in controls. Therewere significant differences between SLE patients and controls about genotype (AG vs.AA: OR=1.51,95%CI1.17-1.93, P=0.001; AG+GG vs. AA: OR=1.43,95%CI1.13-1.82, P=0.003) and allele frequencies (G vs. A: OR=1.28,95%CI1.04-1.58, P =0.019).The allele frequencies of A, G were77.1%,22.9%and the genotype frequenciesof AA, AG, GG were58.4%,37.3%,4.2%in LN patients. They were82.2%,17.8%,67.5%,29.5%,3.1%respectively in non-LN patients. There were significant differencesabout allele (G vs. A: OR=1.37,95%CI1.00-1.87, P=0.046) and genotypefrequencies (AG+GG vs. AA: OR=1.47,95%CI1.02-2.13, P=0.039) between LNpatients and non-LN patients.(3) Haplotype analysis Haplotypes were constructed with the two variants(rs16900617, rs16900627) and the frequencies of haplotypes were analyzed. The resultsrevealed that two haplotypes, AG and GA, were significantly associated with SLE (OR=1.37,95%CI1.11-1.70, P=0.003; OR=0.60,95%CI0.45-0.79, P=0.0003).(4) Association of RIP2gene polymprphisms and clinical features of SLE patients Nosignificant association was detected between the distribution of allele and genotypefrequencies of RIP2gene polymorphisms and clinical manifestation, such as arthritis,malar rash, oral ulcer and so on (P>0.05).Conclusion There are significant differences between SLE patients and normalcontrols regarding the SNPs of rs16900617, rs16900627and haplotypes constructedwith the two variants. The RIP2gene polymorphisms may be associated withsusceptibility to SLE in the Chinese population. Objective To compare the serum RIP2levels between patients with SLE and normalcontrols, patients with and without nephritis, active and inactive SLE patients. Toanalyze the association of serum RIP2level with laboratory results and clinical profiles.Additionally, to explore the role of RIP2in the occurrence and development of SLE.Methods80SLE patients were in-and out-patients recruited from Department ofRheumatology and Immunology of Anhui Provincial Hospital and the First AffiliatedHospital of Anhui Medical University. Diagnosis of SLE was established according tothe1997revised criteria of American College Rheumatology.76normal controls wererecruited from healthy volunteers. Patients and controls were age-, sex-matched. Thedemographic and clinical data were collected by questionnaire. Disease activity wasevaluated using the SLE Disease Activity Index (SLEDAI) score. After obtaining theinformed consent, blood samples from all studied subjects were collected. Serum RIP2levels were detected by Enzyme-linked Immunosorbent Assay (ELISA). The data wereanalyzed by SPSS10.01software. All P values were calculated based on two-sided tests,with a statistical significance defined as P value <0.05.Results(1) Comparisons of serum RIP2level between different groups There were nosignificant differences of serum RIP2level between patients with SLE and normalcontrols (2.725(1.901,3.876) ng/mL vs.2.627(1.906,3.309) ng/mL, Z=-1.009, P=0.313), patients with and without nephritis (2.684(1.906,3.529) ng/mL vs.2.970(1.897,4.258) ng/mL, Z=-1.353, P=0.176), active and inactive SLE patients (2.813(2.038,3.876) ng/mL vs.2.022(1.491,3.825) ng/mL, Z=-1.550, P=0.121). (2) Correlations between serum RIP2level and SLEDAI Correlation analysis betweenserum RIP2level and SLEDAI showed no significant association (r=0.094, P=0.409).(3) Association of serum RIP2level and clinical, laboratory data of SLE patientsSignificant association was found between serum RIP2level and optic nerve injury (Z=-2.096, P=0.023). As for laboratory features, serum RIP2level was significantlyassociated with anti-RNP antibodies (Z=-2.469, P=0.014), ESR (Z=-2.218, P=0.027) and CRP (Z=-1.967, P=0.049).Conclusion In conclusion, this study showed no significant differences of serum RIP2level between patients with SLE and normal controls, patients with and withoutnephritis, active and inactive SLE patients. However, we detected significant associationof serum RIP2level with part of clinical and laboratory features.