| With the completion of the human genome project, attention is now rapidly shifting towards the study of individual genetic variation. The most abundant source of genetic variation in the human genome is represented by single nucleotide polymorphisms (SNPs), which can account for heritable inter-individual differences in complex phenotypes. Identification of SNPs that contribute to susceptibility to comples diseases will provide highly accurate diagnostic information that will facilitate early diagnosis, prevention, and treatment of human diseases.Systemic lupus erythematosus(SLE) is a prototypic systemic autoimmune disease characterized by a diverse array of autoantibody production, complement activation, immune complex deposition, and tissue and organ damage. Although the etiopathogenesis of SLE remains unclear, SLE has long been appreciated to be a complex interlay of genetic and environmental factors. The complex patterns of inheritance in SLE suggest that multiple genes contribute to the etiology. Several studies have shown that the Fc receptor-like 3 (FCRL3) and signaling lymphocytic activation molecule (SLAM) genes play important role in the pathogenesis of SLE. However, the importance between their genetic polymorphisms and SLE has not been studied in Chinese population. In this study, we investigated the occurrence of polymorphisms within the FCRL3 and SLAM gene, and further validated the biological functions of positive associated SNP loci.We analyzed four SNPs (-169A/G, -110C/T, +358C/G and +1381C/T) of FCRL3 gene and four SNPs (-262A/T, -188A/G, -112C/T and +116A/C) of SLAM gene in a case–control cohort composed of 248 SLE patients and 278 healthy controls using the PCR-RFLP assay. The case-control association studies were analyzed usingχ2 tests for Hardy–Weinberg equilibrium. And 2×2 and 2×3 contingency tables for allele and genotype frequencies were conducted respectively. For the haplotype analysis, pairwise linkage disequilibrium measures were investigated by using linkage disequilibrium analyzer(LDA), haplotype frequencies were estimated and calculated using the PHASE package. To further validate the biological functions of positive associated SNP loci, the functions of the haplotypes(-262A-188T,-262T-188A) in the 5'untranslated region of the SLAM gene in patients with SLE were studied. The promoter activities of the haplotypes on the SLAM gene were evaluated with the dual-luciferase reporter system. EMSA was used for identify nulear protein binding ability between the alleles of -262A/T and -188A/G in SLAM gene.The mRNA and protein expression of SLAM on the peripheral blood monocyte cells of SLE patients with different genotypes were determined by real-time PCR and flow cytometry respectively.The main results and conclusions were summarized as follows:1. Analysis of allelic frequencies revealed no significant difference between SLE patients and control subjects for the FCRL3 gene polymorphisms. We also inferred three common haplotypes (ACCC,GTGT,GCGT) and found no significant difference of haplotype frequencies between SLE patients and controls. The associations of FCRL3 with SLE patients with antinuclear antibody(ANA) were examined, we did not detect any association of FCRL3 genotype with ANA in the SLE patients. These results suggested that genetic variations in FCRL3 were not associated with SLE in Chinese population.2. We evaluated the -262A/T,-188A/G,-112C/T and +116A/C polymorphisms of SLAM gene for associations with SLE in a case-control study. The -262A/T and -188A/G polymorphisms were associated with susceptibility of SLE(p=0.0003,0.002), analysis showed that the two SNPs were in linkage disequilibrium(D'=0.6443). The -262A-188G and -262T-188A haplotypes were associated with SLE(p=0.002,0.003), the -262A-188G haplotype might increase the risk for developing SLE(OR=1.478, 95%CI=1.152-1.897).3. The four different haplotype DNA of SLAM gene were cloned into a luciferase expression plasmid(pGL3-Basic) respectively, then the different vectors were transfected into HeLa cells and Jurkat cells, the reporter activities were measured by using dual luciferase reporter assay. The results showed increased luciferase expression of the four haplotypes, however, the luciferase expression of the -262A-188G haplotype was significant greater than the other three haplotype. This suggested that -262A-188G can significantly increase the expression level of the reporter gene. 4. EMSA showed that there were no different abilities of nuclear protein binding between the two alleles of -262A/T. However, the intensity of binding band was higher for the -188G allele than for the -188A allele. The -188A/G polymorphism altered the binding affinity of nuclear protein.5. In response to phytohemagglutinin(PHA) stimulation, the SLAM mRNA expressions on PBMC of SLE patients were significantly higher in -262AA-188GG homozygous genotype compared with individuals heterozygous and homozygous with other genotypes(p<0.01). The SLAM protein expressions on T cells of SLE patients were significantly higher in -262AA-188GG homozygous genotypes compared with -262A/T-188A/G heterozygous(p<0.05).In conclusion: we find that FCRL3 polymorphisms are not associated with susceptibility to SLE in Chinese population. The -262A/T and -188A/G polymorphisms of SLAM gene are associated with susceptibility of SLE. The haplotype -262A-188G can significantly increase the expression level of the reporter gene. The expression level of mRNA and protein expression of SLAM is significantly higher in SLE patients with -262AA-188GG haplotype. The -188A/G polymorphism alters the binding affinity of nuclear protein. These findings suggest that the -262A-188G haplotype in the SLAM gene promoter contributes to risk of the occurrence SLE by increasing expression of SLAM.
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