| BackgroundSystemic lupus erythematosus (SLE) is a prototypic systemic autoimmune disease characterized by a diverse array of autoantibody production, complement activation and immune complex deposition, causing tissue and organ damage. Up to nowadays, although there have been a lot of hypothesises about the etiology and pathogenesis of SLE such as the immunology theory, the genetics theory and theory of the interaction between environment and genetics, the exact development mechanisms of SLE are elusive. exploration into the disease mechanisms might lead to the development of improved therapeutic approaches.P2X purinoceptor 7 is a protein that in humans is encoded by the P2RX7 gene. The P2X7 receptor belongs to a family of ion channels gated by extracellular ATP, but unlike other P2X receptors it is largely restricted to haematopoietic cells. The P2X7 receptor has been proposed to play a role in a variety of immune functions including the secretion of leaderless cytokines and the shedding of the lymphocyte homing receptor CD62L. As P2X7 deficient mice exhibit resistance to antibody-induced arthritis and impaired CD62L shedding and IL-1βsecretion, stimulation of this receptor is proinflammatory suggesting a potential role in autoimmune disease. Stimulation of the proinflammatory haematopoietic P2X7 receptor results in IL-1βsecretion, in high rates of PCD and in CD62L shedding. Moreover, the gene encoding human P2X7 is located within a region 12q24, recently identified and confirmed in Hispanic and European-American Families as a lupus susceptibility locus, designated SLEB4.Above all, that P2X7R might play an important role in the pathogenesis of SLE, however, studies on P2X7R and SLE has so far been limited, according to the latest achievements in this field, our group put forward assumption: P2X7R gene may be susceptibility loci in the Chinese Han SLE population, meanwhile, there are differences of expression in SLE patients and healthy controls, and also P2X7R SNPs polymorphisms are associated with LN, characters of clinical and laboratory. Further reaserching on the involvement of P2X7R in SLE may shed new light on disease mechanisms and development of new treatment paradigms.ObjectiveAssess the role of the polymorphism of P2X7R in the development of SLE in Chinese subjects, and analyze it's associations with disease manifestations, laboratory characteristics. And to explore the association between genetics and the expression of P2X7R.MethodsSLE patients were recruited at the Department of Rheumatology of Auhui Provincial Hospital and First Affiliated Hospital, Anhui Medical University. Diagnosis of SLE was established according to the 1997 revised criteria of American College Rheumatology (ACR). Disease activity was evaluated using the SLE Disease Activity Index (SLEDAI) score. Healthy volunteers were included as normal controls, all of them were without evidence of rheumatologic conditions. A total of 684 SLE patients and 410 healthy control were genotyped SNPs using TaqMan assays with ABI 7300 system.The demographic and clinical data were collected by questionnaire or hospital records. Before study, all subjects gave their written consent to participate. Venous blood from all studied subjects was collected in tube.16 SLE patients, 14 normal voluntary controls were selected. By employing flow cytometry, the percentage of P2X7R+ cells, CD19+P2X7R+ cells, CD4+P2X7R+cells, in the peripheral blood was tested, while the relation of its changes with P2X7R genetic polymorphisms .We genotyped SNPs using TaqMan assays with ABI 7300 system, and analyzed by Plink 1.03 software.The data were analyzed by SPSS 13.00 software (SPSS, Chicago, IL, USA).Results1. Association between P2X7R gene single nucle polymorphism with SLETo SNP rs1718119, genotype frequencies for AA,AG and GG were 0.670%,18.928% and 80.402% in the SLE group and 3.430%,27.704% and 68.865% in the control group, respectively. Allelic frequencies for A and G were 10.134%,89.866% and 17.282%,82.718% in the SLE group and control group, respectively. Significant differences were exhibited in the allelic distributions between them, the association was significant for the G allele of rs1718119, the odds ratio was(OR:1.853,95% CI:1.420-2.417), the association was significant for the GG genotype of rs1718119, the odds ratio was 5.977 (95% CI:1.929-18.517) . No deviations from HWE were observed in either the patients with SLE or the controls(χ~2=0.917,P=0.338;χ~2=0.364,P=0.546); Genetic model was additve model.To SNP rs3751143, genotype frequencies for AA,AC and CC were 59.900%,35.940% and 4.160% in the SLE group and 60.302%,34.673% and 5.025% in the control group, respectively. Allelic frequencies for A and C were 77.870%,22.130% and 77.638%,22.362% in the SLE group and control group, respectively. There was no significant differences were exhibited in the allelic distributions betweeen them(P>0.05), No deviations from HWE were observed in either the patients with SLE or the controls in this polymorphism(χ~2=1.101,P=0.294;χ~2=0.001,P=0.977);To SNP rs2230911, genotype frequencies for CC,CG and GG were 67.613%,29.382% and 3.005% in the SLE group and 68.766%,28.463% and 2.771% in the control group, respectively. Allelic frequencies for C and G were 82.304%,17.696% and 82.997%,17.003% in the SLE group and control group, respectively. There was no significant differences exhibited in the allelic distributions between them(P>0.05), No deviations from HWE was observed in either the patients with SLE or the controls in this polymorphism(χ~2=0.045, P=0.832 ;χ~2=0.029 ,P=0.865);The linkage disequilibrium were significant between rs1718119, rs3751143 and rs2230911.Haplotype analysis used SHEsis software, seven haplotypes AAC,AAG,ACC,GAC,GAG,GCC,GCG,AAC were significant between SLE group and healthy control group.2. Association between P2X7R gene single nucle polymorphism with LNTo SNP rs1718119, genotype frequencies for G/G,A/G and A/A were 77.368%,21.579% and 1.053% in the SLE with LN group and 81.818%,17.690% and 0.491% in the SLE without LN group, respectively. Allelic frequencies for G and A were 88.158%,11.842% and 90.663%,9.337% in the SLE with LN group and SLE without LN group, respectively. There was no significant differences exhibited in the allelic distributions between them. To SNP rs3751143, genotype frequencies for AA,AC and CC were 61.053%,34.737% and 4.211% in the SLE with LN group and 59.367%,36.496% and 4.136% in the SLE without LN group, respectively. Allelic frequencies for C and A were 22.384%,77.616% and 21.579%,78.421% in the SLE with LN group and SLE without LN group, respectively. There was no significant differences exhibited in the allelic distributions between them.To SNP rs2230911, genotype frequencies for CC,CG and GG were 73.016%,25.397% and 1.587% in the SLE with LN group and 65.122%,31.220% and 3.659% in the SLE without LN group, respectively. Allelic frequencies for C and G were 85.714%,14.286% and 80.732%,19.268% in the SLE with LN group and SLE without LN group, respectively. There was significant differences exhibited in the allelic distributions between them(C vs G, OR,1.432, 95%CI, 1.023-2.005).3. Association between P2X7R gene single nucle polymorphism with clinic characteristics of SLE patientsAssociation of P2X7R SNPs with SLE analyzed by primary symptom stratification: there was significant association between rs1718119 and mouth sores(χ~2=9.894, P=0.006), rs2230911 and mouth sores(χ~2=12.780,P =0.002) and pleurisy(χ~2=7.311, P=0.028).Association of P2X7R SNPs with SLE analyzed by main clinical feature stratification: there was significant association between rs1718119 and mouth sores(χ~2=9.455, P=0.007), rs2230911 and mouth sores(χ~2=10.329,P =0.006); rs2230911 and abnormality of nervous system(χ~2=6.235,P =0.049). Association of P2X7R SNPs with SLE analyzed by items of subphenotype stratification: there was significant association between rs3751143 and optic nerve injury(χ~2=5.541, P=0.019), rs2230911 and degraded platelet (χ~2=5.909,P =0.015)and degraded leucocyte(χ~2=7.642,P=0.006).4. Association between P2X7R gene single nucle polymorphism with the level of P2X7R expression.There was signifcant difference of the percentage of P2X7R+ cells between rs1718119 GA and GG genotype. There was no signifcant difference of the level of P2X7R expression between genotypes of rs3751143 and rs2230911. There was no signifcant difference of the level of P2X7R expression between SLE patients and controls with the same genotype.Conclusion1) This study demonstrated a significant association between variants in P2X7R and SLE in Chinese populations, and the polymorphisms of rs1718119 was significantly with genetic susceptibility to SLE patients; AAC haplotype were significant between SLE group and healthy control group; The linkage disequilibrium were significant between rs1718119, rs3751143 and rs2230911. P2X7R may be susceptibilty loci in the Chinese Han SLE Population.2) P2X7R SNPs polymorphisms were associatied with phenotypes of SLE, such as dental ulcer, pleurisy, lupus nephritis, optic nerve injury, et al.3) There was signifcant difference of the percentage of P2X7R+cells between rs1718119 GA and GG genotype.
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