| Background:Systemic lupus erythematosus (SLE) is a complex autoimmune disease with multiple organ injury and characterized by aberrant T- and B-cell activation, with a result of upregulation of autoantibody production and loss of immunological tolerance to self-nuclear antigens. The etiology of SLE involves the interaction of genetic, epigenetic, hormonal,environmental and immunoregulatory factors. More than 40 susceptible loci that causing SLE have been identified in recent years, by using targeted, genome-wide linkage analysis and genome-wide association studies (GWASs).Solute carrier family 15, member 4 (SLC15A4) is a proton-coupled histidine and oligopeptide cotransporter that contains 12 membrane-spanning domainsincludingL-Ala-g-D-Glu-meso-diaminopimelic Acid (tri-DAP), nucleotide oligomerization domain-1 (NOD1) ligand, and carnosine, together with a proton. The most common expression sites are in brain and immune cells, and especially in plasmacytoid dendritic cells.Through study with SLC15A4-mutant and -deficient mice, the deficiency of SLC15A4 has been linked to injury nucleotide oligomerization domain-1, toll-like receptor 7, and TLR9-dependent cytokines such as type 1 interferon, all of which are found to be essential to the SLE pathogenesis. The lack of SLC15 A4 reduced the production of antibodies in B cells in a mouse lupus model,and SLC15 A4 was found to be essential for the production of IgG2a and IgG2c auto-antibodies, such as anti-DNA antibodies and anti-snRNP. Recent studies showed that SLC15A4 participanted in maintaining the stability of endosomal PH, and is criminal for the innate immune response triggered by TLR7, TLR9, NOD1 and NOD2. In a SLC15A4 defective mouse model, the function of plasmacytoid dendritic cells were impaired and lead to chronic viral infection. To investigate how SLC15A4 contributes to such a result,a research group shown that SLC15A4 was crucial for TLR7-triggered IFN-I and autoantibody productions in B cell with a mouse lupus model. SLC15A4 loss disturbed the endolysosomal pH regulation and probably the v-ATPase integrity, and these changes were associated with disruption of the mTOR pathway, leading to failure of the IFN regulatory factor 7 (IRF7)-IFN-I regulatory circuit. Importantly, SLC15A4's transporter activity was necessary for the TLR-triggered cytokine production. Revealed that SLC15A4-mediated optimization of the endolysosomal state is integral to a TLR7-triggered, mTOR-dependent IRF7-IFN-I circuit that leads to autoantibody production.GWASs have been showed that SLC15A4 is a lupus-associated locus in both Korean and Chinese populations. The first GWAS reported that SLC15A4 was a new predisposing genes for SLE was in 2009, and the authors detected 2 SNPs (rs10847697, rsl385374) significant associated with SLE in a Chinese Han people. Subsequently,a genotype-phenotype analysis showed that there was a significant difference of the SNP rs10847697 of the gene between SLE with discoid rash and SLE with no discoid rash. In addition, another group reported that the SNPs rs10847697 and rs1385374 of SLC15A4 were stronger association with renal disorder involvement in SLE patients in a Caucasian population. In a word, all of these studies foreshadows that the polymorphisms of SLC15A4 have an essential role in SLE pathogenesis.In the present study, we evaluated associations between SLC15A4 polymorphisms and SLE in Southwestern Han Chinese people via comparisons between 355 SLE patients and 375 healthy individuals. In particular, we focused on the association of alleles and genotypes of 18 SNPs with SLE,and a linkage disequilibrium analysis of these SNPs.Objective:The gene SLC15A4 has been reported as contributing to the pathogenesis of SLE, to investigate the association of SLC15A4 polymorphism with SLE susceptibility. We performed a case-control replication study between the SNPs in the SLC15A4 gene and SLE in a Chinese Han population.Methods:1. A case-control study was performed with 355 SLE patients and 375 healthy individuals. Patients were recruited from Department of Dermatology, SouthwestHospital of the Third Military Medical University (Chongqing, China) from January 2008 to March 2013.All patients with SLE had fulfilled the revised diagnostic criteria of the American College of Rheumatology (1997) and excluded other systematic or autoimmune diseases(Hochberg 1997).Control group was collected at the Medical Examination Center of the same hospital during regular health examinations. 16 of the 18 SNPs were detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS).The remaining 2 SNPs (rs7965732,rs3765108) was performed using TaqMan SNP Genotyping Assays on a LightCycler480 real-time polymerase chain reaction (PCR) system.The Hardy-Weinberg equilibrium test was applied to examine the genotype frequency of each SNP, using the exact chi-squared test for both the SLE patients and the healthy controls. The allele,genotype,and haplotype frequency distributions between the SLE patients and healthy individuals were assessed with the chi-squared test and logistics regression model. Linkage disequilibrium blocks of all SNPs were analyzed with Haploview. All the tests were two-sided and P-values < 0.05 were regarded as significant. Data was analyzed with SPSS software for windows (Version 17.0; SPSS, Chicago, IL).2. Quantitative Real-time PCR and Western blot were applied for detecting the expression of SLC15A4 mRNA and protein in PBMC from SLE and controls. Data was analyzed by using SPSS 17.0 software for windows. P-values<0.05 was regarded as significant.Main results:1. Allele frequency distribution of SLC15A4 polymorphism: 5 of the 18 SNPsfrom SLC15A4 were significantly different between SLE and controls. Specifically, the following SNPs were associated with an increased risk of SLE: rs959989 T (OR 1.317, 95%CI 1.027-1.689), rsl385374 T (OR 1.332, 95% CI 1.039-1.707), and rs983492 T (OR 1.295,95% CI 1.001-1.675). Associated with a decreased risk of SLE were: rs12298615 G (OR 0.778,95% CI 0.606-0.998) and rs10847697 G (OR 0.770, 95% CI 0.600-0.986).2.Genotype frequency distribution of SLC15A4 polymorphism: the genotypedistributions of the SLE and control groups were compared using codominant, dominant,and recessive models. The frequency distribution of the AG genotype of rs3765108 was significantly higher than that of the AA genotype (P = 0.019, OR 1.447, 95% CI 1.063-1.970),and the frequency distribution of the AT genotype of rs7308691 was significantly higher than that of the AA genotype (P = 0.049, OR 1.645, 95% C1 1.000-2.705) in codominant model.One SNP (rs1385374, TT+CT cf.CC,P = 0.042, OR 1.1363, 95% CI 1.010-1.840) was significantly associated with increased risk of SLE in the dominant model.3. Association analysis within SLE patients with different disease manifestations: We performed case-only association in subgroups of 325 SLE patients with ACR classification data. Significant association signals were detected for the SNPs rs959989, rs1385374,rs10847697 and rs12298615 in the SLE patients with discoid rash. In addition, rs983492 showed association with renal disorder in the SLE patients.4. Linkage disequilibrium analysis: The linkage disequilibrium analysis of the 18 SNPs was conducted using Haploview; 2 haplotype blocks were presented. Block 1 was 1 kb and has the following 3 SNPs: rs7965732, rs3765108 and rs7308691. Block 2 was 29 kb with 15 SNPs: rs7302634, rs9738216, rs2291350, rs11059915, rs12298615, rs4760592, rs959989,rs959987, rs7311875,rs11059925,rs10847697,rs1385374,rs10847699,rs983492 and rs1552336. The frequencies of the different haplotypes and their association with SLE in block 1 and block 2 were analyzed. Allthe haplotypes that had frequencies <5 were combined to simplify the statistical comparisons. The most common haplotype was set as the reference for the analysis of associations of specific haplotypes with SLE. In block 1, the most common haplotype in the SLE and control groups was AAT (57.3% and 57.2%, respectively) while the other haplotypes had P-values > 0.05 (Table 5). In block 2, the most common haplotype in the SLE and control groups was TTTGGCACAAGCTTC (31.7% and 31.5%, respectively), and the prevalence of the haplotype CCCGAGTCAAATTTC was significantly lower in the SLE patients than in the control group (P = 0.033, OR 1.376, 95% CI 1.026-1.845; Table 6).5. The expression of SLC15A4 mRNA and protein in SLE patients compared with contros: The expression of SLC15A4 mRNA was significant increased in PBMC from SLE patients compared with controls (P<0.05). The protein expression levels was also increased in PBMC from SLE compared to controls, but there were no significant differences between the two groups (P>0.05).In summary, our study provides some clues that help elucidate the role of the SLC15A4 gene in the pathogenesis of SLE in Han Chinese people. Allele frequencies of the 5 SNPs were significantly associated with SLE. The analysis of codominance revealed that the genotype frequencies of rs3765108 AG and rs7308691 AT were significantly higher in the SLE group than the control (P=0.019, 0.049, respectively). Analysis of dominance showed that rs1385374 (TT+CT) carried a higher risk of SLE than (CC) (P = 0.042). One SLC15A4 haplotype (CCCGAGTCAAATTTC) was associated with SLE (P = 0.033). More com prehensive research which includes populations of different ethnicities is warranted in the future. |
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