Genetic polymorphisms of C-type lectin receptors in Behcet’s disease in a Chinese Han population

C-type lectin receptors (CLRs) have been demonstrated to be involved in several autoimmune diseases. The role of CLRs in Behcet’s disease (BD) is unknown and thus was the purpose of this study. A two-stage association study was carried out and a total of 766 BD patients and 1674 healthy controls were recruited. Genotyping of 14 SNPs of 13 genes in CLRs was carried out by iPLEX Gold genotyping or polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. The expression of mannose binding lectin 2 (MBL2) and killer cell lectin like receptor C4 (KLRC4) was measured by Real-time PCR. Significantly increased frequencies of the A allele as well as AA genotype of rs1800450 in MBL2 (Pc = 2.50 × 10−6, OR = 1.494; Pc = 2.24 × 10−6,OR = 2.899; respectively) and TT genotype of rs2617170 in KLRC4 (Pc = 2.53 × 10−6, OR = 1.695) and decreased frequencies of GG genotype of rs1800450 (Pc = 1.56 × 10−3, OR = 0.689) and C allele as well as CC genotype of rs2617170 (Pc = 2.05 × 10−9,OR = 0.664; Pc = 1.20 × 10−5, OR = 0.585; respectively) were observed in BD. Two variants, p.Gly54Asp (rs1800450) and p.Asn104Ser (rs2617170) affect MBL2 and KLRC4 protein stability and expression. Our study demonstrates that the MBL2/rs1800450 and KLRC4/rs2617170 are susceptibility factors for BD in a Chinese Han population.

Behcet's disease (BD) is a well-known multisystem vasculitis, characterized by recurrent uveitis, oral ulcerations, genital ulcerations and typical skin lesions 1 . It often occurs in young adulthood and causes serious disability and significant visual impairment. BD is more frequent among populations along the "silk route" from the Mediterranean Basin to East Asia 2 . Although the pathogenesis of BD is not yet exactly known, it has been hypothesized that autoimmunity and genetic factors are responsible for this disease 3 . Recent studies have implicated that Human Leukocyte Antigen (HLA) and non-HLA genes seem to collectively contribute to the genetic background causing this disorder among different populations [4][5][6][7][8][9][10][11][12] . Most of the non-HLA genes such as TNFAIP3, IL23R,-IL12RB2, IL10, CCR1, STAT4, KLRC4, ERAP1, FUT2, and IL12A have been reported with genome-wide significance whereas NOS3 and JAK1 were reported with study-wide significance.
C-type lectin receptors (CLRs) are a large group of extracellular Metazoan proteins expressed on immune cells that have been classified as pattern recognition receptors (PRRs) which play an important role in the binding of pathogens via their surface carbohydrate structures. CLRs not only play a pivotal role in the process of anti-inflammatory immune responses but also in the maintenance of host immune-homeostasis 13 . Growing evidence suggests that various members of CLRs are associated with severe immune mediated diseases like juvenile idiopathic arthritis (JIA) 14 , type 1 diabetes (T1DM) 15 , systemic lupus erythematosus (SLE) 16 , rheumatoid arthritis (RA) 17 and multiple sclerosis (MS) 18 . It was recently shown that patients with BD had significantly lower median serum mannose-binding lectin (MBL) levels compared to healthy controls 19 . Whether gene polymorphisms of CLRs are associated with the susceptibility to BD is not yet known and was therefore the purpose of our study. We identified two variants, p.Gly54Asp (rs1800450) in MBL2 and p.Asn104Ser (rs2617170) in KLRC4, to contribute to the risk of developing BD.
The Influence of MBL2/rs1800450 and KLRC4/rs2617170 on gene mRNA transcription and cytokine production. In order to find a biological explanation for the association of BD with MBL2/ rs1800450 and KLRC4/rs2617170, the mRNA expression of MBL2 as well as KLRC4 of healthy genotyped individuals was measured in their PBMCs. We also evaluated whether different genotypes of MBL2/rs1800450 and KLRC4/rs2617170 could influence the production of cytokines important in the development of BD such as IFN-γ, IL-6, IL-8, IL-1β, IL-10 and TNF-α. These experiments were performed in healthy individuals to eliminate confounding effects such as the inflammatory status and immunosuppressive drug effects in our BD patient group.
Real-time PCR data demonstrated that the mRNA level of MBL2/rs1800450 in GG carriers was remarkably higher than AG carriers (P = 0.019) (Fig. 1). We did not test AA carriers since the frequency of this genotype is very low (<3-4%). The mRNA expression of KLRC4/rs2617170 in CC carriers showed a significant increase compared to CT/TT individuals (Fig. 2, P < 0.001).
The effect of MBL2 and KLRC4 genotype on cytokine production was tested in LPS treated PBMCs isolated from genotyped healthy controls. ELISA was applied to test the concentration of IFN-γ, IL-6, IL-8, IL-1β, IL-10 as well as TNF-α in the 72 hr cell culture supernatants. LPS stimulated PBMCs from GG genotype MBL2/rs1800450 carriers secreted higher amount of INF-γ, IL-6 and IL-8 than AG carriers (P = 0.002; p = 0.009; p = 0.005; respectively) (Fig. 3a,b,c). Compared to CC (P = 0.002; P = 0.004) carriers, an elevated secretion of IL-8 and IL-10 was observed in TT KLRC4/rs2617170 (Fig. 4a,b). No effect of the various rs1800450 and rs2617170 genotypes on the release of other cytokines could be detected. (Figs 3d,e,f and 4c,d,e).

Discussion
In this study we show that gene polymorphisms of MBL2 encoding rs1800450 and KLRC4 encoding rs2617170 are associated with BD. Furthermore, the two SNPs were found to affect their gene expression. mRNA expression of MBL2 and KLRC4 were higher in individuals with the GG(BD-protective) genotype of rs1800450 and CC(BD-protective) genotype of rs2617170 as compared to the other genotype carriers. Additionally, INF-γ, IL-6 and IL-8 production by stimulated PBMCs from GG genotype carriers of rs1800450 and IL-8, IL-10 production by stimulated PBMCs from CC genotype carriers of rs2617170 were increased. C-type lectin receptors (CLRs), often containing the C-type lectin-like domain (CTLD), are a large family of extracellular proteins 13 . CLRs can bind carbohydrate through CTLD and activate different signaling pathways, which induce the expression of specific cytokines ultimately affecting T cell subtype polarization 20 . Recent findings also showed that CLRs are vital in immune homeostasis, which can induce both pro-inflammatory and anti-inflammatory immune responses 20 . BD is a multifactorial autoinflammatory disease and the interactions between susceptibility genes and environmental factors may affect susceptibility 3 . Several studies suggest that the expression of some members of CLRs are significantly different between BD cases and healthy individuals, such as the increased CD94 expression in BD patients 21 and decreased mannose-binding lectin (MBL) concentration as compared to healthy controls 18 . Moreover, previous reports showed that CLRs are involved in the development of certain autoimmune diseases such as JIA, T1MD, SLE, RA, MS [14][15][16][17][18] . Based on these studies, we assumed that CLR genetic polymorphisms might also be associated with BD. To validate this hypothesis, we examined the association of polymorphisms of CLRs in BD patients and found a strong association between 2 SNPs, rs1800450 in the MBL2 gene and rs2617170 in KLRC4, with BD in a Chinese Han population. The fact that CLRs play vital roles in the innate immune response against microbial pathogens strengthens the view that BD is caused by an aberrant response against environmental stimuli 20 .    MBL2 belongs to the C-type collectin family, and plays a potential role in innate immunity. Many studies showed that a low or high serum MBL level is involved in several immune mediated diseases (e.g., RA, Crohn's disease, Sjögren disease and diabetic retinopathy [22][23][24]. Immune defense function of MBL is associated with its serum level and oligomeric type 23 . Five SNPs of the MBL2 gene, including three structure variants, codon 52 (rs5030737), 54 (rs1800450), 57 (rs1800451) and two promoter variants, −550 (rs11003125) and −221(rs7096206) are thought to be responsible for reducing MBL2 serum levels and influencing the formation as well as the stability of oligomeric MBL2 [25][26][27] . However, others didn't find any association between genetic polymorphisms of MBL2 and BD susceptibility 28 . In our study, we confirmed that MBL2 is a predisposing gene for BD in a Chinese Han population. Sample selection bias and different genetic backgrounds may explain the observed discrepancy between studies. We did not measure MBL2 levels in serum of our BD patients or controls, since the patients were often treated with immunosuppressive drugs, which may influence the serum concentration of MBL2. Further studies are needed to address this issue.
NKG2F encoded by the KLRC4 gene is a recently described member of the NKG2 family receptors, and its function has not been examined in detail 29 . This receptor can activate NK cells following the binding with its ligand DAP12 30 . A recent GWAS has shown that rs2617170 of KLRC4 is associated with BD in Turkish and Japanese patients 31 . However, it has not yet been reported in the Chinese population. Our results indicate that only the rs2617170 association (C allele: Pc = 2.05 × 10 −9 , OR = 0.664; CC genotype: Pc = 1.20 × 10 −5 , OR = 0.585) exceeds the threshold for genome-wide significance (P < 5e-08), Our study confirms the results of a previous GWAS regarding the association of KLRC4/rs2617170 with BD 32 . Interestingly, the C allele of rs2617170 was associated with disease risk in this GWAS study 31 , whereas the C allele had a higher frequency in the controls as compared to the BD patients in our study, and would therefore seem to be associated with disease protection. On the other hand, Dixon et al 32 . have reported that rs2617170 is a significant eQTL for KLRC4 expression, and the C allele is associated with reduced KLRC4 gene expression. This is in contrast with our findings where we showed that the C allele is associated with higher gene expression. The reasons for these discrepancies may be due to different ancestral backgrounds of the subjects investigated and this issue clearly deserves further study. Until now, the role of KLRC4 in BD has not received much attention. It has been demonstrated that the stimulation of IL-2 and IL-15 led to an up-regulation of KLRC4 on NK cells 30 . Other groups have reported that IL-15 levels were elevated in serum, cerebrospinal fluid, and aqueous humor from patients with BD [33][34][35] . Further experiments are needed to unravel the functional role of KLRC4 variants on BD pathogenesis. It is interesting to point out that we observed a lower frequency of CC (30.4%) and a higher frequency of CT of rs2617170 in our healthy control group (47.7%) as compared to data reported in the Asian population as shown in the NCBI Resource (42.2% and 35.6%, respectively). However, our results are similar to a previous report on rs2617170 genotype frequencies 36  Our study has a number of limitations. Firstly, since we only chose the loci with known associations between CLRs and various autoimmune or auto inflammatory diseases, it cannot be excluded that other SNPs in CLRs may have an association with BD. Detailed sequence analysis should be carried out to investigate the potential involvement of other rare variants of these factors in BD development. Secondly, our BD patients were Chinese Han patients recruited from an ophthalmic department and all had uveitis. Not all patients with BD have uveitis and depending on their complaints will see different medical departments. Further studies including BD patients from other medical departments (e.g., dermatology, rheumatology, stomatology) and other populations are therefore required to confirm our results and to investigate whether the observed associations are not only confined to the subpopulation of BD patients with uveitis. Due to sample size we also did not investigate whether subgrouping of our patients according to clinical features had an effect on the CLR gene associations. Last but not least, although our study identified rs1800450 of MBL2 and rs2617170 of KLRC4 as possible risk factors contributing to the susceptibility for BD, the exact mechanism how these variants affect the disease pathogenesis are not yet exactly clarified and await further study.
In summary, our study confirmed that MBL2/rs1800450 and KLRC4/rs2617170 polymorphisms affect disease susceptibility in the Chinese Han population. Further studies are needed to reveal the crucial role of the CLRs pathways in the pathogenesis of BD.

Materials and Methods
Study population. All BD uveitis patients (n = 766) and healthy individuals (n = 1674) included in the present study (n = 2440) were ethnic Han Chinese, recruited from the First Affiliated Hospital of Chongqing Medical University from May 2008 to August 2015. The diagnostic criteria of BD strictly followed the International Study Group for BD 37 . Controls were matched for age, geographic origin and ethnicity with BD patients. A case-control study including two phases was performed. In the first phase, 388 BD patients and 742 healthy individuals were included. and in the second phase, 378 BD cases and 932 controls were recruited. The major clinical symptoms in the recruited BD cases are clarified in Table 1 In the first stage, all SNPs in our study (except rs1800450) were genotyped by the MassARRAY platform (Sequenom, USA) and iPLEX Gold Assay. The PCR reaction was carried out by the Gene Amp PCR System 9700 instrument (ABI, Foster City, CA, USA). MassARRAY Assay design software was used to design the primers (Table 3). Experimental data were analyzed through SpectroTYPER software (version 4.0; Sequenom). Rs2617170 in the second stage was performed by the TaqManH SNP Genotyping Assay in the 7500 Real-Time PCR system (Applied Biosystems, USA). The results were examined through TaqManH Genotyper Software. Rs1800450 was genotyped by the PCR-RFLP method.
Real-time PCR. Total RNA extraction from PBMCs was performed using the TRIzol (Invitrogen, San Diego, California, USA) method. RNA was reverse transcribed into cDNA with a Takara transcriptase kit (Takara, Dalian, China). The assays were carried out on an ABI 7500 real-time system with the following primers (KLRC4: 5′-GGAATGACAAGACATATCACTG-3′and 5′-GTCAGTTGAATACTACACAGACT-3′; MBL2: GCAAACAGAAATGGCACGTAT and AGAGGCCTGGAACTTGACA). The expression level was measured by the 2 −ΔΔCt method. Statistical analysis. The differences between BD cases and healthy individuals with regard to allele and genotype frequencies were analyzed by the chi-square (χ2) test with SPSS17.0 statistical software package (ver-sion17.0, SPSS, Chicago, IL). Hardy-Weinberg equilibrium was examined by the SHEsis website.

Measurement of cytokines by
Scientific RepoRts | 7: 5348 | DOI:10.1038/s41598-017-05877-x For multiple comparisons, the Bonferroni correction was used to adjust P values to corrected P values (Pc) according to the number of performed analyses. A p c < 0.05 was viewed as significant. Expression of KLRC4, MBL2 and cytokine levels among three genotype groups was tested by the non-parametric Mann-Whitney test or student t test, with P < 0.05 (Two-tailed) taken as being statistically significant.  Table 3. Primers applied in the analysis of iPLEX Gold genotyping in the CLR related genes.