NFKB1 −94 Insertion/Deletion ATTG Polymorphism Contributes to Risk of Systemic Lupus Erythematosus

Abstract

Growing evidence has shown that nuclear factor-κB (NF-κB) plays a key role in the initiation and progression of systemic lupus erythematosus (SLE) pathogenesis. A common polymorphism (−94 insertion/deletion ATTG, rs28362491) in the promoter region of NFKB1 gene was identified as functional. The −94del ATTG allele exhibited loss of binding to nuclear proteins and resulted in reduced promoter activity. We investigated the association between NFKB1 −94 insertion/deletion ATTG polymorphism and risk of SLE. A total of 224 SLE patients and 256 control subjects were genotyped using a polymerase chain reaction–polyacrylamide gel electrophoresis strategy and DNA sequencing. We found that the ATTG₁/ATTG₂ genotype was associated with a significantly decreased risk of SLE (odds ratio=0.52, 95% confidence interval: 0.32–0.87, p=0.012). This finding indicates that the −94 insertion/deletion ATTG polymorphism may play pivotal roles in the development of SLE in the Chinese population. Further studies with larger sample size are warranted to confirm this finding, especially in different populations.

Introduction

S ystemic lupus erythematosus (SLE) is a chronic inflammatory autoimmune disorder that can affect any part of the body and cause inflammation and damage in joints, skin, kidneys, heart, lungs, blood vessels, or the brain. The incidence of SLE varies in different ethnicities, with an estimation of 40/100,000 among Northern Europeans and more than 200/100,000 among blacks (Johnson et al., 1995). SLE affects 10 times more common in women than men, especially in female patients between the ages of 15 and 50 years (Cervera et al., 2003; Rahman and Isenberg, 2008). Several lines of evidence have shown that environmental factors, such as infectious agents, diet, toxins/drugs, and physical/chemical agents, may create a predisposition toward SLE (Mok and Lau, 2003). In addition to environmental factors, genetic factors, such as single-nucleotide polymorphisms, may also play a role in the development of SLE (Kyogoku et al., 2004; Warchol et al., 2009; Xue et al., 2009). However, the underlying cause of SLE is not fully known.

Nuclear factor-κB (NF-κB) regulates genes required for both the innate and adaptive immune responses (Ouaaz et al., 2002; Saccani et al., 2003; Zanetti et al., 2003; Tan et al., 2005). In mammals, there are five members in the NF-κB family: p50/p105, p52/p100, p65/RelA, RelB, and c-Rel. Among them, the major form is a heterodimer of the p50/p105 and p65/RelA subunits that are encoded by NFKB1 and RelA genes, respectively (Chen et al., 1999). Recent work has shown that impaired NF-κB signaling may be involved in the pathogenesis of human autoimmune diseases, including SLE (Oikonomidou et al., 2006; Kurylowicz and Nauman, 2008).

A functional polymorphism (−94 insertion/deletion ATTG, rs28362491) in the promoter region of NFKB1 gene was identified in 2004 (Karban et al., 2004). The presence of the −94del ATTG allele exhibited the loss of binding to nuclear proteins and resulted in reduced promoter activity (Karban et al., 2004). Previously, a wide range of studies have been done to explore the association between the −94 insertion/deletion ATTG polymorphism and the susceptibility to autoimmune and inflammatory diseases (Karban et al., 2004; Borm et al., 2005; Mirza et al., 2005; Oliver et al., 2005; Orozco et al., 2005; Glas et al., 2006; Martinez et al., 2006; Kurylowicz et al., 2007; Dieguez-Gonzalez et al., 2009). However, the results were conflicting. For example, Karban et al. (2004) reported that the −94del ATTG allele was associated with an increased risk of ulcerative colitis in an American population, whereas Mirza et al. (2005) failed to replicate the association in a British population. To date, no study has been carried out to investigate the association between the −94 insertion/deletion ATTG polymorphism and risk of SLE in a Chinese population. The purpose of this study was to determine the potential association of NFKB1 −94 insertion/deletion ATTG polymorphism with the occurrence of SLE.

Materials and Methods

Study population

Two hundred twenty-four unrelated SLE patients and 256 controls were selected consecutively from China-Japan Union Hospital of Jilin University between September 2007 and August 2010 (Table 1). Patients with SLE (21 males and 203 females) had a mean (standard deviation) age of 31.2 (10.9) years. The diagnosis of SLE was established according to the American College of Rheumatology (ACR) criteria for the classification of SLE (Tan et al., 1982; Hochberg, 1997). The clinical characteristics of the patients were recorded based on the criteria: 50.9% patients having malar rash, 21.9% patients having discoid rash, 18.3% patients having serositis, 19.2% patients having oral ulcers, 66.1% patients having arthritis, 51.8% patients having photosensitivity, 12.5% patients having hematologic disorder, 61.6% patients having renal disorder, 80.8% patients having antinuclear antibodies, 89.7% patients having immunologic disorder, and 6.7% patients having neurologic disorder. Control subjects consisted of 256 healthy volunteers (35 males and 221 females) from a routine health survey. The mean age of the control group was 32.9 (10.0) years. All the controls were frequency-matched to cases according to age, sex, and residential area. Control subjects with symptoms of SLE or other autoimmune diseases, such as fever, malar butterfly rash, photosensitivity, erythra, and edema, were excluded. The study was approved by the hospital ethics committee and all the participants provided informed consent.

Table 1.

Characteristics of Study Subjects

Clinical features	Controls	Cases	p-Value
Age (years, mean±SD)	32.9±10.0	31.2±10.9	0.08
Male/female	35/221	21/203	0.14
ACR criteria (%)
Malar rash	—	114 (50.9)
Discoid rash	—	49 (21.9)
Serositis	—	41 (18.3)
Oral ulcers	—	43 (19.2)
Arthritis	—	148 (66.1)
Photosensitivity	—	116 (51.8)
Hematologic disorder	—	28 (12.5)
Renal disorder	—	138 (61.6)
Antinuclear antibodies	—	181 (80.8)
Immunologic disorder	—	201 (89.7)
Neurologic disorder	—	15 (6.7)

SD, standard deviation; ACR, American College of Rheumatology.

Determination of genotypes

Genomic DNA was extracted from EDTA-anticoagulated peripheral blood samples with a DNA isolation kit (Bioteke, Beijing, China). The −94 ATTG polymorphism was determined using a polymerase chain reaction (PCR)–polyacrylamide gel electrophoresis method. The PCR primers were synthesized as previously described (Zhou et al., 2010). PCR was performed in a total volume of 10 μL, including 1.0 μL of 10X PCR buffer, 1.2 μL dNTPs, 0.6 μL MgCl₂, 0.1 μL each primer, 100 ng of genomic DNA, and 0.3 U of Taq DNA polymerase. The PCR conditions were 94°C for 4 min, followed by 32 cycles of 30 s at 94°C, 30 s at 64°C, and 30 s at 72°C, with a final elongation at 72°C for 10 min. The amplified products were separated by a 6% polyacrylamide gel and stained with 1.0 g/L argent nitrate, producing 154 bp band for allele ATTG₁ and 158 bp band for allele ATTG₂. To verify the genotyping results, amplified DNA samples were sequenced, and the results were 100% concordant.

Statistical analysis

Distributions of demographic and clinical features were compared between cases and controls using the χ ² test and the Student's t-test when appropriate. Allele and genotype distributions of the −94 ATTG polymorphism in NFKB1 gene were obtained by direct counting and Hardy–Weinberg equilibrium was tested by the χ ² test. Odds ratio (OR) and 95% confidence intervals (CI) were used to estimate the effects of the −94 ATTG polymorphism on SLE risk. All data analyses were carried out using SPSS statistical software package version 11.5 (SPSS, Chicago, IL). Statistical significance was assumed at the p≤0.05.

Results

The genotype and allele frequencies of the −94 ATTG polymorphism between SLE cases and control subjects are listed in Table 2. The genotype frequency in both cases and controls were in agreement with the Hardy–Weinberg equilibrium. The ATTG₁/ATTG₂ genotype of the −94 ATTG polymorphism in NFKB1 gene was associated with a significantly decreased risk of SLE when compared with the ATTG₁/ATTG₁ genotype (OR=0.52, 95% CI: 0.32–0.87, p=0.012). However, the ATTG₂ allele frequency in SLE patients was not significantly different from that in the control group (OR=0.84, 95% CI: 0.65–1.08, p=0.18). When stratified according to clinical characteristics (i.e., gender and ACR criteria), no significant association was observed between the −94 ATTG polymorphism and the clinical characteristics (data not shown).

Table 2.

Distribution of the −94 ATTG Polymorphism in NFKB1 Gene Between Systemic Lupus Erythematosus Cases and Controls

−94 ins/del ATTG	Controls n=256 (%)	Cases n=224 (%)	OR (95% CI)	p-Value
Genotypes
ATTG₁/ATTG₁	37 (14.5)	51 (22.8)	1.0 (reference)
ATTG₁/ATTG₂	119 (46.5)	86 (38.4)	0.52 (0.32–0.87)	0.012
ATTG₂/ATTG₂	100 (39.1)	87 (38.8)	0.63 (0.38–1.05)	0.08
Alleles
ATTG₁	193 (37.7)	188 (42.0)	1.0 (reference)
ATTG₂	319 (62.3)	260 (58.0)	0.84 (0.65–1.08)	0.18

CI, confidence interval; OR, odds ratio.

Discussion

In this hospital-based case–control study, we investigated the association between the −94 insertion/deletion ATTG polymorphism in the promoter region of NFKB1 gene and risk of SLE in a Chinese population. We found that the ATTG₁/ATTG₂ genotype, but not the ATTG₁ allele, was associated with a significantly decreased risk of SLE. Even though the exact mechanism was unclear, this finding suggests that the functional polymorphism (−94 insertion/deletion ATTG, rs28362491) may play a pivotal role in the development of SLE in the Chinese population. QUANTO software was used to calculate the power of this study. Our study had 81% power to detect an effect with an OR of 1.7 for the NFKB1 −94 insertion/deletion ATTG polymorphism assuming α=0.05, two-sided test, and a dominant model.

NF-κB was originally discovered as a transcription factor present in activated B-cells with binding to the enhancer of the kappa light chain of immunoglobulin (Sen and Baltimore, 1986). NF-κB plays critical roles in the etiology of autoimmune and inflammation diseases, although no direct evidence of knockout or mutation of NF-κB-related genes resulting in spontaneous development of autoimmunity and inflammation was observed (Barnes and Karin, 1997; Tak and Firestein, 2001; Li and Verma, 2002). Loss of p105 precursor displayed an increased susceptibility to bacterial infections and exhibited chronic inflammation in mice (Ishikawa et al., 1998). RelB-deficient animals presented an impaired cellular immunity (Weih et al., 1995), and c-Rel-deficient mice were resistant to autoimmune inflammation (Hilliard et al., 2002).

The NFKB1 gene encoding the p50/p105 subunits is located on chromosome 4q23-q24 in humans (Mathew et al., 1993). In 2004, Karban et al. identified a common insertion/deletion polymorphism in the promoter region of NFKB1 (−94ins/delATTG) using the family-based association test and the pedigree disequilibrium test. They found that the ATTG₁ allele was more frequent in non-Jewish ulcerative colitis cases than that in non-Jewish controls (Karban et al., 2004). Subsequently, several epidemiological studies were conducted in diverse ethnic populations to evaluate the association between the −94ins/delATTG polymorphism and risk of autoimmune and inflammation diseases (Karban et al., 2004; Borm et al., 2005; Mirza et al., 2005; Oliver et al., 2005; Orozco et al., 2005; Glas et al., 2006; Martinez et al., 2006; Kurylowicz et al., 2007; Dieguez-Gonzalez et al., 2009). Borm et al. (2005) confirmed the association in a Dutch population. Conflicting results, however, were reported in other studies (Mirza et al., 2005; Oliver et al., 2005). Oliver et al. (2005) and Mirza et al. (2005) failed to find any association between the −94ins/delATTG polymorphism and ulcerative colitis in Spanish and British populations. Moreover, Orozco et al. (2005) did not replicate the association between the −94ins/delATTG polymorphism and SLE in a Spanish population. We found that the ATTG₁/ATTG₂ genotype had a decreased risk of SLE in a Chinese population. It is likely that the discrepancy is due to variation in different ethnic groups. Studies suggest an association between the NFKB1 −94ins/delATTG polymorphism and certain autoimmune and inflammatory diseases in Asian populations, but not in Caucasian populations (Zou et al., 2011). Our finding, therefore, seems to be plausible. Nevertheless, further functional analysis of NFKB1 −94ins/delATTG polymorphism and SLE risk is needed.

The role of NF-κB in SLE has been extensively studied. Patients with SLE have increased plasma levels of B-cell activating factor (BAFF), which is a known activator of noncanonical NF-κB signaling pathway. Elevated serum levels of BAFF were also observed in murine models of SLE with higher circulating BAFF indicating greater disease activity (Gross et al., 2000). Moreover, T cells isolated from SLE patients were defective in T-cell receptor-mediated activation of NF-κB signaling. One possible reason for this defect is the absence of the p65 subunit in lupus T cells (Wong et al., 1999). Lack of the p65 that contains a DNA-binding domain failed to stimulate the NF-κB transcription (Ballard et al., 1992). Another possible reason for this defect is the reduced ability of NF-κB to bind DNA in SLE (Oikonomidou et al., 2006). Given the crucial roles of NF-κB pathway in the initiation and progression of SLE pathogenesis, the result that the ATTG₁/ATTG₂ genotype of NFKB1 −94ins/delATTG polymorphism was associated with a decreased risk of SLE is reasonable.

There are some limitations in this study. The statistical power may not be sufficient because of the relatively small sample size.Also, detailed information of the effect of gene–environment interaction on the susceptibility to SLE was not available.

In conclusion, this is the first study to investigate an association between the NFKB1 −94ins/delATTG polymorphism and SLE in a Chinese population. We found that the ATTG₁/ATTG₂ genotype was associated with a significantly decreased risk of SLE. Further studies with larger number of SLE patients are warranted to confirm this finding, especially in different populations.

Footnotes

Disclosure Statement

The authors declare that they have no competing interests.

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