Association Study Of IL-6Gene Single Nucleotide Polymorphisms, Serum IL-6Level And Systemic Lupus Erythematosus

Background Systemic lupus erythematosus (SLE) is a systemic autoimmune diseasewhich is characterized by a diverse array of autoantibody production, immunecomplexes deposition and complement activation, resulting in multiple system andorgan involvement. SLE predominantly affects women (at a9:1ratio), particularlyduring childbearing years. The etiology of SLE remains unclear. Genetic susceptibility,environmental factors, sexual hormone disorder and immune dysfunction are thought tocontribute to disease development.Cytokines play a pivotal role in the pathogenesis of SLE through participating indiferentiation, maturation, and activation of various immune cells. In autoimmunediseases, cytokines may not only be involved in the generation of the aberration ofimmune regulation, but also in the local inflammatory processes that ultimately lead totissue destruction. There is growing evidence to confirm that many cytokines areinvolved in the pathogenic of SLE, such as interleukin-6(IL-6), IL-12, IL-10, IL-17,IL-18, interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α). IL-6is a pleiotropiccytokine which have ability to promote the proliferation of T lymphocytes and inducethe maturation of B lymphocytes into plasma cells and augment the immunoglobulinsecretion. It has been confirmed that IL-6rs1800796polymorphism was associated withSLE in several foreign studies. However, the relationship between IL-6rs1800796polymorphism and SLE in Chinese population has not been reported.Thus, our study adopted case-control design to detect association of IL-6genepolymorphisms, serum IL-6level and SLE by the TaqMan SNP Genotyping Assay kitand ELISA. The accomplishment of this study would help to further reveal the role ofIL-6in the pathogenesis of SLE and develop new potential therapeutic targets. PartⅠAssociation study of IL-6gene single nucleotide polymorphisms andsystemic lupus erythematosusObjective Compare the differences of allele and genotype frequencies about SNP IL-6rs1800796between SLE patients and healthy controls, to explore the association of IL-6gene polymorphisms with susceptibility to SLE and to explore the relationship betweenthe polymorphisms and clinical manifestations.Methods A total of672SLE patients were recruited from Department of Rheumatologyand Immunology of Anhui Provincial Hospital and the First Affiliated Hospital of AnhuiMedical University. All SLE patients fulfilled the1997revised American College ofRheumatology (ACR) classification criteria.576age and sex-matched healthy controlswere recruited from medical examination center and postgraduate students of AnhuiMedical University.596female and76male were in SLE groups (mean age35.17±11.37) and506female and70male were in control group (mean age35.82±12.41).The demographic and clinical data of all the subjects were collected by self-designedquestionnaire. After obtaining the informed consent, blood samples from all studiedsubjects were collected. After extracting DNA, the genotyping of the rs1800796wasdetermined by TaqMan allele discrimination assay on the7300real-time polymerasechain reaction (PCR) system. Excel2003software was used to establish the databaseand type clinical information. SPSS10.01software was used for statistical analysis. Thechi-square test or Fisher’s exact test was applied to compare the distribution of alleleand genotype frequencies between SLE patients and healthy controls. Stata10.1software was used to Hardy-Weinberg equilibrium (HWE) test. Statistical significancedefined as P value <0.05.Results (1) In SLE groups, the success rate of genotyping for rs1800796was99.11%(591female,75male）. In controls, the success rate of genotyping for rs1800796was 98.78%(499female,70male). No deviations from HWE were detected in either theSLE group or the healthy controls.(2) Association of rs1800796and SLE The genotype frequencies of CC, CG, GGwas55.0%,37.2%,7.8%in SLE groups and53.6%,39.2%,7.2%in controls,respectively. The allele frequencies of C, G were73.6%,26.4%in SLE group and73.2%,26.8%in controls. The genotype distribution of SLE group and controls was notsignificantly different (CG vs. GG：OR=0.877,95%CI=0.560-1.372, P=0.565; CCvs. GG: OR=0.946,95%CI=0.611-1.464, P=0.804；CG+CC vs. GG: OR=0.917,95%CI=0.599-1.403, P=0.689). For allele distribution, no significant differenceswere found either (C vs. G：OR=1.019,95%CI=0.852-1.219, P=0.833).(3) The allele C frequency of rs1800796was lower in patients with serositis thanpatients without serositis (OR=0.530,95%CI=0.308-0.910, P=0.019). However, nosignificant differences were found in other clinical features, such as malar rash, discoidrash, photosensitivity, oral ulcers, arthritis, lupus encephalopathy and lupus nephritis.The results indicated that the allele frequency of rs1800796was not associated with theseven common clinical manifestations of SLE mentioned above.Conclusion Our results revealed that the rs1800796polymorphisms in IL-6gene werenot related to the development SLE in Chinese Han population. C allele of rs1800796might be a protective factor for patients with serositis. Part Ⅱ Association study of IL-6serum level andsystemic lupus erythematosusObjective Compare the serum expression of IL-6in SLE patients and healthy controls,patients with and without nephritis, active and inactive SLE patients, to evaluate theassociation of serum IL-6level and laboratory results and clinical profiles and toexplore the role of IL-6in the pathogenesis and development of SLE.Method A total of88SLE patients (85female and3male) were recruited fromDepartment of Rheumatology and Immunology of Anhui Provincial Hospital and theFirst Affiliated Hospital of Anhui Medical University. Diagnosis of SLE was establishedby the presence of four of more American College of Rheumatology (ACR) diagnosticcriteria.86(84female and2male) age-and sex-matched normal controls wererecruited from medical examination center and postgraduate students of Anhui MedicalUniversity. The demographic and clinical data of all the subjects were collected byquestionnaire. Disease activity was evaluated using the SLE Disease Activity Index(SLEDAI) score. All subjects gave their written consent to participate before study.Blood samples from all studied subjects were collected and sera were obtained from3ml of whole blood and stored at-80oC until tested. Specific enzyme linkedimmunosorbent assay (ELISA) kits was used to detect the concentration of IL-6. Forcomparing the means between different groups, the two independent samples t test wasused. The data were analyzed by SPSS10.01software. Probability level <0.05intwo-tailed test was used as a criterion of significance.Results(1) Comparisons of serum IL-6level between different groups There were nosignificant differences of serum IL-6level between SLE patients and normal controls ((37.18±11.80) pg/mL vs.(40.41±10.78) pg/mL, t=-1.879, P=0.062), active andinactive SLE patients ((36.99±11.37) pg/mL vs.(37.50±12.63) pg/mL, t=0.914, P=0.846)). Significant differences of serum IL-6level were found between patients withand without nephritis ((40.88±10.66) pg/mL vs.(34.40±11.68) pg/mL, t=-2.646, P=0.010)).(2) Correlations between serum IL-6level and SLEDAI We did not findassociation of serum IL-6level and SLEDAI in SLE patients (rs=0.101, P=0.350).(3) Association of serum IL-6level and clinical, laboratory data of SLE patientsCompare the level of serum IL-6in different groups. No significant association wasfound between serum IL-6level and all the clinical profiles (P>0.05). As for laboratoryfeatures, serum IL-6level was significantly associated with anti-ds-DNA antibodies (t=-2.365, P=0.020), complement (t=-2.836, P=0.006), ESR (t=-2.628, P=0.010),proteinuria (t=-1.996, P=0.049).Conclusion In summary, our results showed that no significant differences of serumIL-6level was found between patients with SLE and normal controls, patients with andwithout nephritis, active and inactive SLE patients. However, we detected significantassociation of serum IL-6level with some of clinical and laboratory features.