SLE is not an independent disease, but a complex disease with heterogeneous sub-phenotypes. Currently, a large number of evidence suggests that the pathogenesis of SLE has a strong genetic basis. Studies on SNPs polymorphisms in SLE have shown that patients with different races and regions may have different types of genetic mutations. However, studies of multiple members in the same family have shown that patient-specific SNPs in families may be a major causative factor for SLE.[9] Therefore, based on the genetic screening of SLE family, we selected further verification in the sporadic cases of SLE patients. Compared with previous reports, this study focused on the association of rs15672 and rs12640056 gene polymorphisms involved in NF-κB signaling pathway with SLE susceptibility and clinical characteristics. Some characteristics of family specific SNPs in the Southwest Chinese SLE patients were notable.
IKBKE gene is localized on chromosomal locus 1q32 and encodes inhibitor of κB kinase ε (IKKε) light chain, which participates in NF-κB signaling pathway.[10] Inflammatory cytokines like TNF-α, IL-1, IL-6 and IFN-γ stimulated the up-regulated expression of IKBKE.[11] IKBKE was phosphorylated under the regulation of the kinase complex. In addition, the lack of IKKε actived tumor necrosis factor (TNF) which was relate to activation NF-κB signaling pathway. As a result, IKBKE/TBK1 (TRAF family member-associated NF-κB activator-binding kinase 1, TBK1) mediated Lys63-linked polyubiquitination and activated the NF-κB pathway.[12] Previous studies have indicated that IKBKE rs1539241, rs12142086 and rs2151222 were associated with NF-κB pathway.[10] GWAS study showed that IKBKE rs2297550 polymorphism was correlated with SLE.[13]
BANK1 gene is localized on chromosomal locus 4q24 and encodes a B-cell specific scaffold protein. BANK1 binds to BLK gene, promoting inositol 1, 4, 5-trisphosphate and its receptors and activates downstream genes. Polymorphism of BANK1 has been shown to be associated with susceptibility to systemic lupus erythematosus.[14] Recent studies have shown that Bank1 and NF-κB subunit 1 played critical regulatory role in antinucleolar antibodies in mercury-induced autoimmunity in mice.[15] However, the association of IKBKE and BANK1 gene polymorphisms of SLE in the Han population of southwest China remained unclear. Based on this background, this study investigated the association of IKBKE rs15672 and BANK1 rs12640056 gene polymorphisms and susceptibilities of SLE in Han population of southwest China.
Our results from Table 2 showed that A allele at the IKBKE rs15672 locus was 1.25 times the risk of developing SLE in the population carrying the G allele and T allele at the BANK1 rs12640056 locus was 0.78 times the risk in the population carrying the C allele. The results suggested that rs15672 A allele was a candidate risk allele while rs12640056 T allele was a protective allele for SLE in the Chinese Han population. It was worth noting that heterozygous mutation at rs15672 and rs12640056 accounted for higher proportions in SLE compared with homozygous mutation. rs15672 and rs12640056 were located in the 3' untranslated region (UTR), and the mutation of this site could further regulate the expression of gene by regulating the transcriptional function of mRNA.[16] The above data supported the significance of regional SNP gene polymorphisms related to the transcriptional regulatory function of gene.
We also attempted to explore the relationship between serum cytokines and organ injury in the dominant model. The results showed that serum levels of IL-6, granzyme B, IL-4, IL-18 and IL-33 in rs15672 A allele were significantly different from that of GG genotype. Compared with CC genotype, rs12640056 T allele only showed a decrease in IL-33. Recent study showed that CD95 and fas-associated via death domain (FADD) could enhance lipopolysaccharide (LPS)-induced NF-κB responses by activating macrophages and cause increased production of IL-6.[17] Another study showed that IL-6 enhanced the activity of the promoter of miR-34a by activating the NF-κB/p65 signaling pathway, and further attenuated the expression of forkhead box P3 (Foxp3) by targeting the 3' UTR.[18] Granzyme B was derived from cytotoxic lymphocytes and cytoplasmic granules released by natural killer cells. It mediated apoptosis by activating caspase. CD137 agonist enhanced the secretion of granzyme B by CD8+T cells, which was activated by NF-κB nuclear translocation.[19] One study showed that SLE patients had a significant decreased percentage of granzyme B in B cells with disease activity and lupus nephritis.[20] IL-4 was a cytokine produced by activated T lymphocytes. It was involved in B cell proliferation and differentiation and stimulated the production of immunoglobulin IgE by B cells. Bach2-/-mice model showed that transcription factor Bach2-deficient follicular helper T (Tfh) cells were skewed toward the IL-4 producing subset, which induced IgG1 and IgE transformation of B cells.[21]
As an inflammatory cytokine, IL-18 can induce the synthesis of interferonγ(IFNγ). Studies supported the role of IL-18 in promoting organ injury of SLE. IL-18 mRNA was positively correlated with SLEDAI and anti-dsDNA antibody.[22] Studies on IL-18 gene polymorphisms and SLE organ injury showed that IL-18 (-1297C) were associated with renal involvement, and CC haplotype was associated with serositis.[23] Our study showed that IL-18 of rs15672 A allele was significantly higher than that of GG genotype. The correlation between IKBKE gene and IL-18 needs further confirmation. Recent studies on IL-33 in SLE have shown significant differences between IL-33, C-reactive protein (CRP) and erythrocyte regulatory rate (ESR).[24] Studies have shown that haplotype rs1929992G and rs7044343T were both risk factors for SLE.[25] In this study, we found that IL-33 level was significantly lower in TT genotype of rs12640056. These results suggested that the relationship between rs12640056 polymorphisms and IL-33 may also be significant in the pathogenesis of SLE.
We also found that the carriers of rs15672 A allele might be involved in the development of clinical laboratory characteristics and organ injury in SLE. Compared with the wild group, the proportion of arthritis and serositis was increased, while the proportion of neuropathy was significantly decreased. Laboratory results showed the increased proportion of IgM and anti-SSB and the decreased proportion of C3 and anti-RIB. Studies have shown that C3 in active SLE patients was significantly lower than that in inactive SLE patients. The ratio of complement fission products iC3b and serum C3 in patients with SLE was correlated with SLE activity.[26] Another study showed that the activation product of C3, such as iC3b/C3dg in patients with SLE was significantly higher than that in healthy individuals.[27] This study showed that rs15672 A allele mutation was a risk factor for SLE and was associated with the decline level of C3 in SLE patients. A variety of autoantibodies and inflammatory factors in the blood of NPSLE patients can cause damage to the blood-brain barrier. Anti-ribosomal P protein (anti-RIB) antibody could bind to neuronal surface protein, causing calcium flow and neuronal apoptosis, further leading to nervous system damage.[28] The meta-analysis supported the possibility of anti-RIB in the pathogenic role of neuropsychiatric SLE (NPSLE).[29] We found that the percentage of anti-RIB and proportion of neuropathy in rs15672 A allele were decreased in this study.
Anti-SSB antibody is one of the common autoantibodies in the serum of SLE patients. A retrospective study showed a positive rate of secondary Sjögren syndrome (SLE-sSS) in adult SLE was 23%.[30] Further evaluation showed that anti-SSB antibody was associated with alopecia. In anti-SSB positive SLE patients, the incidence of alopecia, serositis and secondary Sjögren's syndrome (sSS) was higher than the negative group.[31] We found that the percentage of anti-SSB and proportion of serositis in rs15672 A allele were increased in this study. It was also worth noting that the incidence of sSS was relatively high. At the same time, systemic inflammatory state with higher levels of proinflammatory cytokines was found in SLE-sSS subgroup.[30] Therefore, the relationship between gene polymorphisms and SLE organ injury should be subdivided into SLE subgroups on the basis of expanding the sample size.
IgM autoantibodies appeared earlier than IgG in humoral immunity and regulated immune function in cardiovascular disease by identifying lipoproteins exposed during apoptosis and oxidative epitopes on phospholipid cell membranes.[32] IgM-antibodies against phosphorylcholine (anti-PC) was significantly reduced in SLE patients with cardiovascular disease (CVD) and atherosclerosis risk.[33] One study showed that IgM anti-cardiolipin and IgM anti-dsDNA were significantly higher in patients without renal disease. IgM anti-PC was also higher in patients with low disease activity.[34] In this study, the IgM of rs15672 A allele and rs12640056 T allele were higher than the wild groups. However, there was no difference in lupus nephritis and pericarditis between different groups. Previous study had shown that the rate of rheumatoid factor (RF) in SLE patients was high.[35] In this study, the incidence of arthritis was significantly increased in the rs15672 A allele, which suggested that IgM might be correlated with arthritis in SLE patients. We also noted that the diagnostic criteria for arthritis were derived from the medical records in this study. We should collect more relevant imaging data to describe the pericarditis more specifically.
In summary, we identified two loci, rs15672 and rs12640056, associated with SLE risk in Chinese Han population. IKBKE rs15672 gene polymorphism was related to SLE susceptibility and cytokine. rs15672 A allele increased the risk of SLE. It could increase the risk of arthritis and serositis, but reduce the risk of neuropathy. It was also associated with the high expression of serum cytokines IL-4 and IL-6. BANK1 rs12640056 polymorphism was also associated with SLE susceptibility and cytokine, but not with the organ injury of SLE. rs12640056 T allele could reduce the risk of SLE and was associated with low expression of serum cytokine IL-33.