| Background:Systemic lupus erythematosus(SLE) is a multisystem, inflammatory disorder of autoimmune etiology. Common manifestations may include a diverse array of autoantibody production, immune complexes deposition and complement activation, resulting in multiple system and organ involvement. Single nucleotide polymorphisms(SNPs), are the most common type of genetic variation among people. SNPs can be used to track the inheritance of disease genes within families. Related studies on SLE are working to identify SNPs associated with susceptibility of SLE, which will provide genetic markers for early diagnosis, prevention and treatment of this complex disease.Pellino1(human PELI1 gene, mouse Peli1 gene) is a member of the RING(really interesting new gene) finger E3 ubiquitin ligase family, a key component in many immune regulation pathways. It was demonstrated that Pellino1 is involved in the pathogenesis of inflammatory and autoimmune diseases in many studies. Chang et al. reported that Peli1-deficient mice spontaneously developed lupus that was characterized by renal damage and autoantibody production through dysregulated c-Rel. c-Rel is a member of the NF-κB family of transcription factors with pivotal roles in T-cell activation. Furthermore, Peli1 is required for Toll-like receptor(TLR) 3-stimulated expression of proinflammatory cytokines and B-cell activation. Peli1 also has a critical role in other TLR signaling pathways and regulates activation of NF-κB, through an interaction with IRAK1(interleukin-1 receptor-associated kinase 1, IRAK1), which is also a potential susceptibility gene for the development of SLE. Since Pellino1 expression is critical for many immune regulation pathways, we hypothesized that human PELI1 gene might be involved in the development of SLE. To our knowledge, no previous reports have elucidated the role of PELI1 gene polymorphism in SLE pathogenesis.The IRAK family is defined as intracellular kinases that play a significant role in the innate immune system as they participate in signalling networks of the innate axis of the immune response. IRAK1 was the first member of the IRAK family to be discovered and was initially shown to have a role in IL-1 signalling. SLE related phenotypes disappeared in IRAK1-deficient mice, including Ig M and Ig G autoantibodies, lymphocyte activation and kidney damage, and reversed the excessive activation of dendritic cells. IRAK1 is a critical role in IL-1R/TLR signaling pathways. Pellino1 is able to induce IRAK1 polyubiquitination, and then interacts with TRAF6(TNF receptor-associated factor 6, TRAF6), leading to many important innate immunity and acquired immunity signals to be activated. Concerning IL-17 in SLE, recent data from human and mice clearly support the role of this cytokine and Th17 cells in lupus pathogenesis. IL-17 could promote inflammation in lupus by affecting both cellular and humoral immune responses. Human Th17 dysfunction is closely related to IL-1R/TLR signaling, and IRAK1 can regulate transcription factors which affect Th17 cells number and function, but the exact mechanism remains to be explored.Thus, on the basis of the content above, in the first part of our study, we performed case-control study to detect association of PELI1 gene polymorphisms and SLE by the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF SNPs located at exons. The accomplishment of this study would help to further reveal the role of these two genes in the pathogenesis of SLE and develop new potential therapeutic targets.PartⅠ Association study of PELI1 gene polymorphisms with systemic lupus erythematosusObjective:To investigate the association of PELI1 gene polymorphisms(rs329497, rs329498 and rs10496105) with SLE susceptibility in Chinese Han by a case-control study, and compare the expression of gene in the peripheral blood mononuclear cells(PBMC) of patients and controls.Methods:1. 395 SLE patients were recruited from the Southwest Hospital of the Third Military Medical University in Chongqing and the Affiliated Hospital of Guilin Medical College. All SLE patients fulfilled the 1997 revised American College of Rheumatology classification criteria. 378 normal controls were recruited from healthy volunteers. MALDI-TOF MS was used for genotyping of three SNP loci of the PELI1 gene. The case-control association studies were analyzed using Chi-square(χ2) tests for Hardy-Weinberg equilibrium(HWE). Allele, genotype and haplotype frequencies were compared between the SLE patients and healthy individuals using the χ2 test. The SLE group was further stratified by clinical evidences of nephritis(as described previously). Associations with different genotypes or alleles frequencies were compared using the χ2 test and logistic regression model. P-value < 0.05 was regarded as the level of statistical significance. Data were analyzed with SAS? 9.2 software for Windows(SAS Institute Inc, Cary, NC, USA) and Haploview 4.2(Daly Lab, Cambridge MA).2. The relative expression of PELI1 m RNA and protein in PBMC were measured by real-time PCR and Western blot respectively. Data was analyzed using SPSS13.0 for windows. P-value < 0.05 was regarded as the level of statistical significance.Results:1. Association of three SNPs and SLE1) rs329497: The genotype frequencies of AA, AG, GG were 6.0%, 38.9%, 55.1% in SLE patients and 9.6%, 43.4%, 47.1% in controls. The allele frequencies of A, G were 25.5%, 74.5% in SLE patients and 31.3%, 68.8% in controls. No significant difference was detected between SLE patients and controls about genotype(P > 0.05), but had significant difference about allele frequencies(G vs. A, Pcorrect =0.036, OR =1.33, 95% CI =1.06-1.67).2) rs329498: The genotype frequencies of CC, AC, AA were 8.4%, 39.2%, 52.4% in SLE patients and 10.6%, 42.9%, 46.6% in controls. The allele frequencies of C, A were 28.0%, 72.0% in SLE patients and 32.0%, 68.0% in controls. There were no significant differences in two groups about genotype and allele frequencies(P > 0.05).3) rs10496105: The allele frequencies of A, G were 13.2%, 86.8% and the genotype frequencies of AA, AG, GG were 3.3%, 19.7%, 77.0% in SLE patients. They were 12.6%, 87.4%, 1.1%, 23.0%, 75.9% respectively in controls. It wasn’t found significant differences about genotype and allele frequencies(P > 0.05) between SLE patients and controls.2. Association of three SNPs and lupus nephritis1) rs329497: The genotype frequencies of AA, AG, GG were 5.6%, 42.4%, 52.0% in lupus nephritis patients and 5.8%, 33.3%, 60.8% in controls. The allele frequencies of A, G were 26.7%, 73.2% in lupus nephritis patients and 22.5%, 77.5% in controls.2) rs329498: The genotype frequencies of CC, AC, AA were 6.5%, 43%, 50.5% in lupus nephritis patients and 8.1%, 34.1%, 57.7% in controls. The allele frequencies of C, A were 28.0%, 72.0% in lupus nephritis patients and 25.2%, 74.8% in controls.3) rs10496105: The allele frequencies of A, G were 11.0%, 89.0% and the genotype frequencies of AA, AG, GG were 2.0%, 18.0%, 80.0% in lupus nephritis patients. They were 12.6%, 87.4%, 4.1%, 17.1%, 78.9% respectively in controls.3. Haplotype analysis in SLEHaplotypes were constructed with the three variants(rs329497, rs329498, rs10496105) and the frequencies of haplotypes were analyzed. In SLE patients, the frequency of haplotype A-C-G was lower than the frequency seen in the controls(P = 0.001,OR = 0.63, 95% CI = 0.47-0.83). The haplotypes G-A-G and G-C-G when compared with others were significantly associated with SLE(P=0.008,OR=1.36, 95% CI=1.08-1.69).4. Haplotype analysis in lupus nephritisThe frequency of haplotype G-A-G was 72.7% in lupus nephritis patients and 76.7% in controls. No significant association was detected in the distribution of haplotype frequencies between lupus nephritis patients and controls(P > 0.05).5. The relative of PELI1 m RNA and protein expression of PBMC in SLE patientsThe expression of PELI1 m RNA was increased in PBMC from SLE patients, but there were no significant differences between patients with SLE and normal controls(P > 0.05). And there were no significant differences of protein expression levels between patients with SLE and normal controls(P > 0.05).Conclusion:There are significant differences between SLE patients and normal controls regarding rs329497 and haplotypes constructed with the three variants. The PELI1 gene polymorphisms may be associated with susceptibility to SLE in the Chinese population.PartⅡ Association study of IRAK1 gene polymorphisms with systemic lupus erythematosusObjective:To analyze the association of SLE with six SNPs(rs1059702, rs1059703, rs3027898, rs7061789, rs5945174, rs2239673) in the IRAK1 gene from a Chinese Han population, and explore the biological functions of positive associated SNPs located at exons of IRAK1 gene.Methods:1. MALDI-TOF MS was used for genotyping of six SNP loci of the IRAK1 gene in SLE patients and normal controls. The case-control association studies were analyzed using Chi-square(χ2) tests for Hardy-Weinberg equilibrium(HWE). Allele, genotype and haplotype frequencies were compared between the SLE patients and healthy individuals using the χ2 test. The SLE group was further stratified by clinical evidences of nephritis(as described previously). Associations with different genotypes or alleles frequencies were compared using the χ2 test and logistic regression model. P-value < 0.05 was regarded as the level of statistical significance. Data were analyzed with SAS? 9.2 software for Windows(SAS Institute Inc, Cary, NC, USA) and Haploview 4.2(Daly Lab, Cambridge MA).2. The effects of two non-synonymous SNPs of IRAK1 gene on transcription factors which related to Th17 cells were evaluated with the dual-luciferase reporter system.Results:1. Association of six SNPs and SLE1) Exon regions ①rs1059702: The genotype frequencies of TT, CT, CC were 69.5%, 26.9%, 3.6% in SLE patients and 58.2%, 34.6%, 7.2% in controls. The allele frequencies of T, C were 83.0%, 17.0% in SLE patients and 75.5%, 24.5% in controls. It has significant difference in genotype between two groups(TT vs. CT+CC, Pcorrect = 0.020, OR = 1.64, 95% CI = 1.22-2.20) and allele frequencies(T vs. C, Pcorrect = 0.002, OR = 1.58, 95% CI = 1.23-2.03). ②rs1059703: The genotype frequencies of CC, CT, TT were 71.0%, 25.4%, 3.6% in SLE patients and 59.5%, 33.1%, 7.4% in controls. The allele frequencies of C, T were 83.7%, 16.3% in SLE patients and 76.1%, 23.9% in controls, with significant difference between the two groups about genotype(CC vs. CT+TT, Pcorrect = 0.014, OR = 1.66, 95% CI = 1.23-2.25) and allele frequencies(C vs. T, Pcorrect = 0.001, OR = 1.62, 95% CI = 1.26-2.08).2) 3’UTR rs3027898: The allele frequencies of C, A were 84.0%, 16.0% and the genotype frequencies of CC, AC, AA were 71.3%, 25.4%, 3.3% in SLE patients. They were 77.9%, 22.1%, 61.9%, 32.0%, 6.1% respectively in controls. There was no significant difference in genotype compared with the controls(P > 0.05), but had significant difference about allele frequencies(C vs. A, Pcorrect = 0.013, OR=1.49, 95% CI=1.15-1.93).3) Intron regions ①rs7061789: The allele frequencies of G, A were 84.7%, 15.3% and the genotype frequencies of GG, AG, AA were 72.7%, 24.1%, 3.3% in SLE patients. They were 78.5%, 21.5%, 63.1%, 30.8%, 6.1% respectively in controls. There was significant difference about allele frequencies(G vs. A, Pcorrect = 0.012, OR=1.51, 95% CI=1.17-1.96), but no significant difference about genotype(P> 0.05) between SLE patients and controls. ②rs5945174: The genotype frequencies of GG, AG, AA were 71.9%, 24.8%, 3.3% in SLE patients and 61.9%, 31.7%, 6.3% in controls. The allele frequencies of G, A were 84.3%, 15.7% in SLE patients and 77.8%, 22.2% in controls. It hasn’t be found significant difference about genotype(P > 0.05), but had significant difference about allele frequencies(G vs. A, Pcorrect = 0.006, OR=1.53, 95% CI=1.18-1.98) between two groups. ③rs2239673: The genotype frequencies of CC, CT, TT were 71.9%, 24.8%, 3.3% in SLE patients and 62.6%, 31.3%, 6.1% in controls. The allele frequencies of C, T were 84.3%, 15.7% in SLE patients and 78.2%, 21.8% in controls. No significant difference was detected between SLE patients and controls about genotype(P > 0.05). However, we have found a significant difference about allele frequencies(C vs. T, Pcorrect = 0.014, OR=1.62, 95% CI=1.15-1.93).2. Association of six SNPs and lupus nephritis1) Exon regions ①rs1059702: The genotype frequencies of TT, CT, CC were 67.5%, 29.5%, 3.0% in lupus nephritis patients and 69.7%, 27.1%, 3.3% in controls. ②rs1059703: The genotype frequencies of CC, CT, TT were 69.8%, 27.1%, 3.0% in lupus nephritis patients and 70.5%, 26.2%, 3.3% in controls.2) 3’UTR rs3027898: The genotype frequencies of CC, AC, AA were 69.8%, 27.1%, 3.0% in lupus nephritis patients. They were 70.7%, 26.8%, 2.4% respectively in controls.3) Intron regions ①rs7061789: The genotype frequencies of GG, AG, AA were 70.0%, 27.0%, 3.0% in lupus nephritis patients. They were 73.2%, 24.4%, 2.4% respectively in controls. ②rs5945174: The genotype frequencies of GG, AG, AA were 70.5%, 26.5%, 3.0% in lupus nephritis patients and 71.5%, 26.0%, 2.4% in controls. ③rs2239673: The genotype frequencies of CC, CT, TT were 70.0%, 27.0%, 3.0% in lupus nephritis patients and 72.4%, 25.2%, 2.4% in controls.No significant difference was found between lupus nephritis and controls about genotype and allele frequencies the three SNPs(P > 0.05).3. Haplotype analysis in SLEHaplotypes were constructed with the six variants(rs1059702, rs1059703, rs3027898, rs7061789, rs5945174, rs2239673) and the frequencies of haplotypes were analyzed. In SLE patients, the frequency of haplotype C-A-A-T-T-A was lower than the frequency seen in the controls(P=0.001,OR=0.64, 95% CI=0.49-0.83). The haplotypes T-G-G-C-C-C when compared with others were significantly associated with SLE(P<0.001,OR=1.60, 95% CI=1.24-2.07).4. Haplotype analysis in lupus nephritisThe frequency of haplotype T-G-G-C-C-C was 82.1% in lupus nephritis patients and 15.7% in controls. No significant association was detected in the distribution of haplotype frequencies between lupus nephritis patients and controls(P > 0.05).5. The dual-luciferase reporter system of RORγt, STAT3 and IRF4 had no significant differents(P > 0.05).Conclusion: the six SNPs of IRAK1 gene and haplotypes constructed with the six variants. The IRAK1... |
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