Genetic Variation in the REL Gene Increases Risk of Behcet’s Disease in a Chinese Han Population but That of PRKCQ Does Not

Genome-wide association studies (GWAS) and candidate gene studies have identified the REL and PRKCQ genes as risk loci for various autoimmune diseases. The purpose of the present study was to investigate the association of the REL and PRKCQ genes with Behcet’s disease (BD) in a Chinese Han population. A case-control study was conducted on three single nucleotide polymorphisms (SNPs), rs13031237, rs702873, and rs842647 of the REL gene and three SNPs (rs4750316, rs11258747, and rs947474) of the PRKCQ gene using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in a total of 623 BD patients and 1,074 healthy controls. Multiple variables were assessed, including age, sex distribution, and extra-ocular findings. In the present study, the frequencies of rs842647 GG genotypes and rs842647 G alleles were significantly higher in patients than in controls and those of the rs842647 AG genotypes were lower in patients than in controls [GG genotype: Bonferroni corrected P-value for gender adjustment (Pca) = 0.0074, odds ratio (OR) = 1.63; G allele: Pca = 0.0072, OR = 1.57; AG genotype: Pca = 0.024, OR = 0.63, respectively]. No statistically significant differences in the frequencies of rs702873, rs13031237, rs4750316, rs11258747, and rs947474 between BD patients and controls were observed. Stratification analysis indicated that the REL rs842647 polymorphism was associated with BD patients with skin lesions. No significant association of the other five SNPs between BD patients with other extra-ocular findings, including genital ulcer, arthritis, and positive pathergy test results was found. The REL rs842647 polymorphism may be a susceptibility factor for BD pathogenesis and skin lesions, which indicate that c-Rel may be involved in the pathogenesis and skin lesions of BD through the NF-κB pathway.


Introduction
Behcet's disease (BD) is a refractory multisystem immune-mediated disease with recurrent episodes of uveitis, multiform skin lesions, oral aphthae, and genital ulceration [1]. Although the etiology of BD remains poorly defined, it is generally believed, as for many autoimmune or auto-inflammatory disorders, that genetic variants triggered by certain environmental factors contribute to its development. Genome-wide association studies (GWAS) and candidate gene studies have identified the predominant BD susceptibility loci within the major histocompatibility complex (MHC) class I region, which include human leukocyte antigen-B51 (HLA-B51) and HLA-A26 [2][3][4][5]. However, these genes only partially explain the genetic risk of BD, suggesting that other non-MHC genes remain to be discovered. Recently, GWAS established an association between the IL10, IL23R-IL12RB2, STAT4, and GIMAP genes and BD [3,4,6]. Candidate gene association studies have involved searches for additional BD genes, including the GIMAP gene in European patients, the NFKB1 and NFKBIA genes in Turkish patients, and the CD40 gene in the Chinese patients [7][8][9]. However, the genes that have been associated to BD do not fully explain its pathogenesis, and no study focusing on the identification of a novel BD susceptibility loci has been conducted.
The nuclear factor-κB (NF-κB) signal transduction pathway plays a crucial role in T-cell development in autoimmunity disorders and inflammation diseases. The REL gene is located on chromosome 2 and encodes for c-Rel, which is a member of the NF-κB family. c-Rel is expressed solely in mature hematopoietic cells and plays a critical role in T-cell development, antigen-presenting cell function, and the CD40 signaling pathway by the formation of proinflammatory cytokines and regulating the expression of genes. Previous studies have shown that Rel knockout mice do not develop autoimmune diseases [10,11]. In addition, the PRKCQ and CD40 genes are involved in the c-Rel signal pathway, thus suggesting that these genes share a common disease pathway. Based on the important roles that REL and PRKCQ genes play in the pathology of autoimmune diseases, the association between polymorphisms in the REL and PRKCQ genes and BD was investigated. Here, three REL SNPs (rs13031237, rs702873, and rs842647) and three PRKCQ SNPs (rs4750316, rs11258747, and rs947474) were selected as candidate risk variants of BD. The present study detected higher frequencies of the rs842647 GG genotype and rs842647 G allele in BD patients. Stratified analysis also showed an association between the rs842647 G allele polymorphism and skin lesions in BD patients.

Ethics Statement
Prior to enrolling in the present study, all subjects, including BD patients and controls, provided their written, informed consent. In the case of the pediatric BD patients, their parents provided written and informed consent. All procedures were in according to the tenets of the Declaration of Helsinki.

Study Population
A total of 623 BD patients with ocular involvement and 1,074 age-and geographic-areamatched normal controls with no history of any ocular or autoimmune disease were enrolled in the present study, and all patients and controls were of Han Chinese ethnicity. All blood samples were obtained from the First Affiliated Hospital of Chongqing Medical University (Chongqing, China) and Zhongshan Ophthalmic Center, Sun Yat-sen University (Guangzhou, China) from April 2005 to December 2013. BD was diagnosed according to the criteria of the International Study Group for BD [12]. The clinical characteristics of BD patients are summarized in Table 1.

Genomic DNA Extraction
Peripheral blood samples were gathered from all study participants by venipuncture. Genomic DNA extraction was obtained from peripheral blood of controls and patients using the QIAmp DNA Blood Mini Kit (QIAGEN Inc., Hilden, Germany) and was reserved at -80°C until analysis.

Genotyping and Quality Control
REL polymorphisms (rs13031237, rs702873, and rs842647) and PRKCQ polymorphisms (rs4750316, rs11258747, and rs947474) were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Amplification of the target DNA fragment of REL and PRKCQ genes was conducted by PCR using the primers designed using Primer Pre-mer5.0 and restriction enzymes presented in Table 2. Each PCR reaction was conducted in a 10-μl reaction mixture containing 5 μL of a Go Taq 1 Green Master Mix (Promega Corporation, Madison, WI, USA), 20 pmol of each primer, and 0.2 μg of genomic DNA. The conditions were as follows: initial denaturation at 95°C for 5 min, followed by 35 cycles of denaturation at 95°C for 30 s, annealing at different temperatures (62°C for rs13031237, rs842647, rs702873, and rs4750316, 60°C for rs947474 and rs11258747) for 40 s, extension at 72°C for 30 s, and a final extension at 72°C for 3 min. PCR products of the rs13031237, rs842647, rs702873, rs947474, rs11258747, and rs4750316 polymorphisms were digested with 2 U of Csp6I (Thermo Fisher Scientific Inc., Ontario, Canada), HpyCH4 III (Thermo Fisher Scientific Inc., Ontario, Canada), BsiWI (New England BioLabs, Inc., Ontario, Canada), DdeI (Thermo Fisher Scientific Inc., Ontario, Canada), Mwol (New England BioLabs, Inc., Ontario, Canada) and  HpyCH4 III (Thermo Fisher Scientific Inc., Ontario, Canada) restriction enzymes (Table 2) in a 10-μL reaction mixture for 12-16 h. Digestion products were separated via electrophoresis on a 4.5% agarose gel and stained with GoldView (SBS Genentech Beijing, China). Genotypes were evaluated in a masked fashion, and analysis of all ambiguous samples was repeated. Moreover, 10% of the samples were double-checked to validate the results of the PCR-RFLP using direct sequencing (Sangon Biotech, Co., Ltd., Shanghai, China).

Statistical Analysis
Statistical analysis was conducted using SPSS version 17.0 for Windows (SPSS Inc., Chicago, IL, U.S.). Hardy-Weinberg equilibrium (HWE) was conducted by using the χ 2 test. Genotype and allele frequencies were evaluated by direct counting and were compared between patients and controls using the χ 2 test or Fisher's exact test. Logistic regression analysis was performed to assess the influence of gender on the association of the polymorphisms to this disease and to assess the association of the tested SNPs to the extra-ocular findings. All statistical tests were two-sided, and statistical significance was set at P < 0.05.

Results
No statistically significant differences in age distribution between BD patients  Table 1.
The distribution of genotype frequencies of the six tested SNPs in the control group conformed to HWE (P > 0.05). The genotype and allele frequencies of the three tested REL polymorphisms and PRKCQ polymorphisms in the patients and controls are shown in Table 3. In the present study, BD patients showed a higher frequency of the rs842647 GG genotype (83.63%) than that observed in healthy controls (75.79%) [Bonferroni corrected P value (P c ) = 0.0036, OR = 1.63]. However, BD patients showed a lower frequency for the rs842647 AG genotype (15.25%) than the controls (22.25%) (P c = 0.011, OR = 0.63). The G allele frequency of the rs842647 was statistically significantly higher in BD patients (91.25%) than in healthy controls (86.92%) (P c = 0.0034, OR = 1.57). To evaluate the influence of gender on the association of rs842647 to the REL gene, we conducted a binary logistic regression analysis and found that differences remained significant after a binary logistic regression analysis (GG genotype: P c a = 0.0074, AG genotype: P c a = 0.024, G allele: P c a = 0.0072, respectively). No differences in the frequencies of rs702873, rs13031237, rs4750316, or rs947474 between BD patients and controls were observed. The associations of the 6 SNPs with clinical characteristics of BD, including genital ulcer, skin lesions, arthritis, and positive pathergy tests were then investigated. The frequencies of GG genotype and G allele of rs842647/REL were significantly higher in patients with skin lesions than in controls (GG genotype: P c a = 0.029, G allele: P c a = 0.034, respectively) ( Table 4, S1 Table). No significant association of the other five SNPs was detected between BD patients with those clinical findings and controls.

Discussion
The present study investigated the association of REL polymorphisms (rs13031237, rs702873, and rs842647) and PRKCQ polymorphisms (rs4750316, rs11258747, and rs947474) with BD in a Han Chinese population. The results indicated that the frequencies of the GG genotype and G allele of rs842647 were positively associated with BD patients in this Han Chinese population. No association with BD for the two tested SNPs polymorphisms (rs702873, rs842647) in the REL gene or the three tested SNPs polymorphisms (rs4750316, rs11258747, and rs947474) in PRKCQ gene in the Han Chinese population was detected.
In the present study, the REL and PRCKQ genes were selected as candidates for the recently reported association of the REL and PRCKQ gene with other autoimmune diseases.
The three SNPs in the REL gene that were tested in the present study, namely, rs13031237, rs702873, and rs842647, have been associated with RA [13], SLE [14], and celiac disease [15]. Because BD is considered as an immune-mediated disease and thus may share similar genetic risk factors, rs13031237, rs702873, and rs842647 of REL were selected as candidate SNPs in the present study. The SNPs rs4750316, rs11258747, and rs947474 in the PRKCQ gene, which encodes a protein kinase C isoform involved in NF-κB activation and T-helper cell subset differentiation, were selected as SNP candidates based on their associations with RA, T1DM, and psoriasis. The following efforts were made for quality control. First, non-Han individuals were excluded to avoid interference from differences in genetic ancestry, and the genotype distribution of controls and patients conformed to the HWE. In addition, the diagnostic criteria of patients were performed in strict accordance with previously described basis. Then, 10% of the samples were randomly chosen for sequencing to validate the genotype results using PCR-RFLP, and the results were determined to be absolutely in agreement with those of the first genotyping results. The intronic rs842647 GG polymorphism in the REL gene has been previously reported as a risk factor for potential CD in Southern Italy populations, and the expression of the c-REL gene was higher in Marsh 0 potentials CD patients than in controls [16]. Similar to the findings of previous studies, a positive association between the GG genotype and G allele of rs842647 with BD was detected, which suggests that the rs842647 GG genotype is a common predisposing factor for BD and potential CD. The SNP rs13031237 T allele has been identified as a risk factor for RA in UK cohorts and SLE in Chinese population [13,17]. The rs702873 G allele was earlier identified as risk factor for psoriasis in populations in the U.K. and Ireland [18]. No association between intronic SNP rs13031237 and rs702873 SNP at REL and susceptibility to BD was detected in the Han Chinese population. In the case of the other three tested SNPs, rs4750316, rs11258747, and rs947474 in the PRKCQ gene, no association with BD patients in the Han Chinese population was detected. These results were not consistent with those of previous studies, including those covering the association of PRKCQ rs4750316 with RA in European populations and that of rs947474 and rs11258747 with T1D in British cohorts [19][20][21]. These difference may be because BD, which is known as an immune-related disease, may have distinct underlying pathogenic mechanisms that are influenced by distinct genetic loci compared with RA and T1D.
The relationship between the clinical characteristics of patients with REL and PRKCQ polymorphisms was also assessed. Those clinical characteristics included genital ulcers, skin lesions, arthritis, and positive pathergy tests. The rs842647 GG genotype and G allele was associated with an increased risk for skin lesions in BD patients. These findings indicate that the REL rs842647 polymorphism is associated not only with the occurrence of disease but also with the clinical characteristics of disease. No relationship between any of the other five tested SNP polymorphisms or clinical characteristics of patients was observed.
BD is generally regarded as a T cell-mediated disease attributed by a Th1, Th17, and Th22 immune responses and their respective cytokines such as IFN-γ, TNF-α, IL-17, IL22, and IL-23 [22]. NF-κB has been indicated to play a crucial role in the pathogenesis of BD via the regulation of apoptosis-related factors, as well as contribute to the resistance of T cells to apoptosis [23]. Moreover, c-Rel, a unique member of the vertebrate NF-κB family, has recently been shown to be required for the development of Treg cells, Th1 cells, and Th17 cells in EAE [10,24], as well as for the production of IL23 subunit by the dendritic cells. In addition, the PRKCQ gene, which encodes a protein kinase C isoform, a key regulator of TCR-mediated NF-κB activation pathway, is associated with RA and T1D susceptibility. The current study showed that the REL gene was associated with BD susceptibility, whereas the PRKCQ was not, suggesting that the NF-κB signaling pathway gene, REL, and not PRKCQ plays an essential role in BD pathogenesis.
The current study has a few limitations. The results have identified the association between REL and PRKCQ polymorphisms with subjects from a Han Chinese population. Future replication studies that include other ethnic groups should therefore be performed. Furthermore, the BD patients in the present study were predominantly male. Sex-matched samples are thus needed to confirm the results in BD. In addition, it is not yet known whether the observed REL rs842647 polymorphism has a biological function in REL in relation to BD pathogenesis, as well as contributes to BD pathogenesis. The fact that NF-κB signaling pathway has a role in the pathogenesis of BD may provide new ways of managing this eye disease.
In conclusion, the results of the current study indicate that REL rs842647 may influence susceptibility to BD and that REL may be involved in the BD pathogenesis and clinical features. No association between any of the other tested SNPs with BD in the Han Chinese population was detected.