Interleukin 12 (IL12B), interleukin 12 receptor (IL12RB1) and interleukin 23 (IL23A) gene polymorphism in systemic lupus erythematosus

E. Sánchez, S. Morales, L. Paco1, M. A. López-Nevot1, C. Hidalgo2, J. Jiménez-Alonso2, B. Torres3, M. A. González-Gay4, J. L. Callejas5, N. Ortego-Centeno5, J. Sánchez-Roman6, M. F. González-Escribano3 and J. Martín

Instituto de Parasitología y Biomedicina López-Neyra, CSIC, 1 Servicio de Inmunología and 2 Servicio de Medicina Interna, Hospital Virgen de las Nieves, Granada, 3 Servicio de Inmunología, Hospital Virgen del Rocío, Seville, 4 Servicio de Reumatología, Hospital Xeral-Calde, Lugo, 5 Servicio de Medicina Interna, Hospital Clínico San Cecilio, Granada and 6 Servicio de Medicina Interna, Hospital Virgen del Rocío, Seville, Spain.

Correspondence to: J. Martín, Instituto de Parasitología y Biomedicina ‘López-Neyra’, CSIC Parque Tecnológico de Ciencias de la Salud, Avenida del Conocimiento s/n 18100-Armilla (Granada), Spain. E-mail: martin{at}ipb.csic.es


    Abstract
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Objective. The aim of this study was to assess the possible association between the interleukin-12B (IL12B) and interleukin-12 receptor beta 1 (IL12RB1) gene polymorphisms with systemic lupus erythematosus (SLE). In addition, we have undertaken a systematic search for genetic variants of interleukin 23 (IL23A).

Methods. The study was conducted on 559 SLE patients and 603 ethnically matched healthy controls. Genotyping of the IL12B [IL12Bpro and IL12B 3' untranslated region (UTR)] and IL12RB1 (641A->G, 1094T->C and 1132G->C) polymorphisms was performed with polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and PCR-fluorescent methods, whereas IL23A genetic variants were realized with direct sequencing.

Results. No statistically significant differences in the distribution of the IL12B and the IL12RB1 genotypes and alleles were observed when comparing SLE patients and control subjects. Additionally, no differences in the genotype and allele distribution were found when SLE patients were stratified according to the presence or absence of lupus nephritis. Despite an extensive analysis in 30 individuals, variations located in the exons and in the 5' and 3' UTR regions of IL23A gene were not found in any case.

Conclusions. These results suggest that polymorphisms located in IL12B, IL12RB1 and IL23A genes may not play a relevant role in the susceptibility or severity of SLE in the Spanish population.

KEY WORDS: Systemic lupus erythematosus (SLE), Interleukin-12 (IL-12), Interleukin-12 receptor (IL-12R), Interleukin-23 (IL-23), Polymorphism, Susceptibility


    Introduction
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Systemic lupus erythematosus (SLE) is a systemic autoimmune disease with a complex pathogenesis involving multiple genetic and environmental contributions [1]. The disease is characterized by autoantibody production and inflammatory manifestation such as nephritis, vasculitis, arthritis and lymphadenopathy [2, 3]. Therefore, genetic polymorphisms of inflammatory mediators and immunological modulators, such as cytokines, are attractive genetic factors that may predispose to the development of SLE [4, 5].

Interleukin-12 (IL-12) is a pro-inflammatory cytokine that induces the production of interferon-{gamma} (IFN-{gamma}), favours the differentiation of T helper 1 (Th1) cells and it is a connecting point for innate and adaptive immunity [6]. IL-12 is a heterodimer formed by a 35 kDa chain (p35 or IL-12{alpha}) and a 40 kDa chain (p40 or IL-12ß). IL-12ß is encoded by the IL12B gene located on 5q31–33 and because of its pro-inflammatory and immunoregulatory activities it might be an important functional candidate gene for autoimmune diseases such as SLE. In addition, excessive IL-12 production has been found in patients with SLE [7–9]. Differences observed in IL-12 production between SLE patients and controls could be caused by variations in the regulatory regions of the IL12B gene. Two polymorphisms which may have a biological significance have been previously found in IL12B gene: an insertion/deletion of 4 bp in the promoter region and an A->C single nucleotide polymorphism (SNP) located in the 3' untranslated region (UTR) at position 1188 [10, 11].

The biological activities of IL-12 are mediated through high-affinity binding to the IL-12 receptor (IL-12R), which is composed of two subunits: IL-12Rß1, encoded by IL12RB1 and IL-12Rß2, encoded by IL12RB2 [6]. Three SNPs have been described in IL12RB1 (641A->G, 1094T->C and 1132G->C), causing three missense variants (Q214R, M365T, G378R) [12], which may influence IL-12-induced signalling [13].

Interleukin-23 (IL-23) is a recently discovered cytokine that bears structural and functional resemblance to IL-12. IL-23 is a heterodimer composed of a subunit identical to IL-12 p40 and a novel IL-12 p35-related protein, p19. IL-23 shares its receptor structure and signalling pathways with IL-12. IL-23 affects IFN-{gamma} production by T and natural killer (NK) cells, activates memory T cells and stimulates Th1-cell responses. IL-23 may enhance inflammation through stimulating the production of pro-inflammatory cytokines [14, 15]. Therefore, IL-23 may have important implications for the pathogenesis of chronic inflammatory diseases such as SLE.

The aim of this work was to examine the possible influence of the previously described IL12B and IL12RB1 gene polymorphisms on the development of SLE and lupus nephritis, and also to undertake a systematic search for common genetic polymorphisms in the IL23A gene and to analyse the possible influence of these putative variants in the development and outcome of SLE.


    Patients and methods
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Subjects
A total of 559 SLE patients, meeting the American College of Rheumatology (ACR) criteria for SLE [16], were recruited from four Spanish hospitals: Hospital Virgen de las Nieves and Hospital Clínico San Cecilio (Granada), Hospital Virgen del Rocio (Seville) and Hospital Xeral-Calde (Lugo). The control group consisted of 603 healthy individuals from the same geographical areas. Both patient and control groups were Spanish Caucasians matched for age and sex. Eighty-seven per cent of the SLE patients were women, the mean age of SLE patients at diagnosis was 43±13.3 yr, the mean age at onset was 32±15 yr. The SLE clinical manifestations studied were articular involvement, renal effects, cutaneous lesions, haematopoietic alterations, neurological disease and serositis. All study subjects included in this study gave written informed consent. We obtained approval for the study from all local ethical committees of the corresponding hospitals.

Genotyping
IL-12 promoter insertion/deletion and 3' UTR 1188 SNP
Genomic DNA was isolated from anticoagulant-treated peripheral blood mononuclear cells (PBMCs) using standard methods. Genotyping of the IL12B promoter was performed by a polymerase chain reaction (PCR)-based method as previously described [11]. Briefly, forward primer was labelled at its 5' end with the fluorescent dye 6-FAM and the lengths of the fragments were analysed in an ABI PRISM 3100 genetic analyser using Genescan 672 software (Applied Biosystem, Foster City, CA, USA). The 3' UTR genotypes were determined using a PCR-restriction fragment length polymorphism (RFLP)-based method as previously described [17]. DNA samples were amplified using the forward primer 5'-TTCTATCTGATTTGCTTTA-3' and the reverse primer 5'-TGAAACATTCCATACATCC-3'. The presence of the A->C polymorphism in the 3' UTR of the gene generates a TaqI site. PCR products were digested overnight at 65°C, and 3' UTR alleles were detected after separation on ethidium bromide-stained 4% agarose gels. Samples showing one 233 bp band were typed as homozygous AA, samples displaying 165 bp and 69 bp bands were typed as homozygous CC, and samples exhibiting 233 bp, 165 bp and 69 bp bands were typed as heterozygous.

IL12RB1 641A->G, 1094 T->C and 1132 G->C polymorphisms
The polymorphisms within the IL12RB1 gene were genotyped as previously described [18]. Briefly, forward primer 5'-AGCCAGGACTTGAACTGAGG-3' and reverse primer 3'-TTTCTAATGCCTGCCCTGT-5' were used to amplify exon 7 where the polymorphic position 641 is found. PCR products were digested with PvuII (641A->G). Exon 10, containing polymorphisms located in 1094 and 1132 positions, was amplified using the forward primer 5'-CCCTGTAGGGTCAGGGGTA-3' and the reverse primer 3'-CAACACCTCTCTGGGCCTTA-5'. PCR products were digested with NlaIII and HpaII to type 1094T->C and 1132G->C, respectively. The digested PCR products were analysed by electrophoresis on 3% agarose gels.

Search for polymorphisms in the IL23 gene
The detection of genetic variants in the IL23A gene was performed by direct sequencing. We analysed a fragment of 4.5 kb of the IL23A gene, including the promoter region (1000 bp upstream of the start codon), four exons, three introns and the 3' UTR (1000 bp) of 30 healthy controls. The primers designed to amplify each of the exons and 5' and 3' UTR regions of the IL23A gene are listed in Table 1. PCR products were purified using the Millipore Montage PCR kit (Millipore Corporation, Bedford, MA, USA) This purified DNA was sequenced using the ABI PRISM 3100 genetic analyser (Applied Biosystem), and sequence data were analysed using two different sequence editor software suites: Chromas version 1.43 and DNASIS MAX version 2.0.


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TABLE 1. IL23A primer sequences and expected PCR product sizes

 
Statistical analysis
Allele and genotype frequencies of IL12B and IL12RB1 were obtained by direct counting. Statistical analysis, to compare allele and genotype distributions, was performed by the {chi}2 test. Odds ratios (OR) and 95% confidence intervals (95% CI) were calculated according to Wolf 's method. using the Statcalc program (Epi Info 2002; Centers for Disease Control and Prevention, Atlanta, GA, USA). A P value of less than 0.05 was considered statistically significant. We used the UNPHASED software created for case–control analysis of haplotypes. Statistical power was estimated using Quanto 0.5 software (Department of Preventive Medicine, University of Southern California, CA, USA).


    Results
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
IL12B typing
Table 2 shows the allele and genotype distribution of the IL12B promoter and the IL12B 3' UTR polymorphisms in 559 Spanish SLE patients and 603 controls. In both patient and control groups, genotype and allele frequencies did not deviate significantly from those expected from the Hardy–Weinberg equilibrium. No significant differences in the allele and genotype frequencies of the IL12B promoter and IL12 3' UTR variants were found between SLE patients and controls.


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TABLE 2. Allele and genotype frequencies of IL12B polymorphisms and IL12RB1 haplotypes in SLE patients and healthy controls

 
In addition, available clinical characteristics of patients with SLE were analysed for possible association with the different alleles or genotypes of the IL12B polymorphisms. No correlation was observed between IL12B variants and the following variables: sex, age at onset, articular involvement, cutaneous lesions, haematopoietic alterations, neurological disease and serositis (data not shown). Of interest, the frequency of homozygous individuals for the IL12 3' UTR A allele was slightly increased among patients with lupus nephritis compared with patients without it, although this difference did not reach statistical significance (data not shown).

IL12RB1 typing
Genotyping of IL12RB1 SNPs A641G, T1094C and G1132C was performed in a group of 559 SLE patients and 603 controls (Table 2). Results of this study confirm a complete linkage disequilibrium among these polymorphisms in our population and allows us to establish two haplotypes: haplotype 1 Q214–M365–G378 and haplotype 2 R214–T365–R378 [13]. No statistically significant differences were observed when the IL12RB1 haplotype distribution was compared between SLE patients and healthy controls. In addition, no statistically significant differences were observed in the distribution of IL12RB1 haplotypes between SLE patients with and without lupus nephritis (data not shown) or with regard to other SLE clinical manifestations (data not shown).

IL23A polymorphism screening
We analysed the IL23A gene to search for variants. Despite an extensive analysis in 30 individuals no sequence variations in any of the exons or 5' and 3' UTR regions were found. Indeed, neither of the IL23A polymorphisms previously described in the SNP database (http://www.ncbi.nlm.nih.gov/SNP/) could be confirmed. This is in concordance with the low frequency reported for these SNPs, indicating that the IL23A gene is highly conserved in humans.


    Discussion
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
The present study constitutes the first attempt to analyse the IL12B and IL12RB1 polymorphisms in SLE, but provided no evidence for an association of the polymorphisms investigated in these genes with predisposition to SLE or with the development of lupus nephritis. This lack of association of the polymorphisms tested with SLE cannot be due to a lack of statistical power, because our sample size had 90% power for IL12 3' UTR and IL12RB1 and 80% for IL12B promoter to detect the effect of a polymorphism, conferring an OR of 1.5 at the 5% significance level. However, our study was underpowered to detect a smaller effect. The genotype frequencies of the IL12B promoter and 3' UTR polymorphisms in our population were comparable with those described in other European and Australian populations [17, 19, 20].

Although a primary role for the IL12B 3' UTR gene polymorphism in type 1 diabetes (T1D) has been proposed [21], several large case–control and familial studies have failed to replicate the association between the IL12B 3' UTR SNP and T1D [22–24]. Similarly, a lack of association with the IL12B gene has been observed in a range of autoimmune diseases, such as coeliac disease [20], multiple sclerosis [25] and Crohn's disease [26], suggesting that the IL12B gene has a negligible effect on susceptibility to autoimmune diseases. In addition to the lack of agreement in the role that the IL12B 3' UTR may play in the susceptibility to autoimmune diseases, the biological significance of this polymorphism has been called into question. While several studies observed differences in IL12B gene expression regarding the IL12B 3' UTR genotypes [21, 26], other authors have not observed any correlation between IL12B genotype and IL-12 expression [23, 27].

To further test the involvement of the IL-12 pathway in susceptibility to SLE, a gene encoding another component of this pathway, IL12RB1, was chosen for analysis. We found no association between the IL12RB1 (641A->G, 1094T->C and 1132G->C) gene polymorphisms and susceptibility to SLE and development of lupus nephritis. Our findings are in agreement with others reporting no association of these polymorphisms and T1D [18, 28]. As previously reported, these three IL12RB1 polymorphisms were in a high linkage disequilibrium forming two major haplotypes in our population [12]. The haplotype frequency of these IL12RB1 polymorphisms was similar to that described in other Caucasian populations [18]. Further analysis to observe functional differences between IL12RB1 haplotypes is important to establish their significance in Th1-mediated immune diseases. We cannot excluded the possibility that other genes in the IL-12 pathway that are not in linkage disequilibrium with the alleles tested may contribute to the development of SLE.

Having found no association with these polymorphisms in IL12B and IL12RB1 genes and the pathogenesis of SLE, we considered a new member of the IL-12-related cytokine family, IL-23, as a candidate for involvement in SLE. However, no SNPs were found among 30 samples from healthy donors, suggesting that an important role for an IL23A polymorphism in SLE is unlikely. Our results are in concordance with the reported low frequency of the IL23A SNPs in other populations of European ancestry (see http://www.ncbi.nlm.nih.gov/SNP/ and http://www.innateimmunity.net/IIPGA2/PGAs/InnateImmunity). In conclusion, our data do not provide a direct role for the tested polymorphisms in IL12B and IL12RB1 genes in predisposition to SLE nor in the development of lupus nephritis in our population. In addition, we have confirmed the high level of conservation of the IL23A gene.


    Acknowledgments
 
This work was supported by grant SAF03-3460 from Plan Nacional de I+D+I, and in part by the Junta de Andalucía, grupo CTS-180. We thank Ma Paz Ruiz for her excellent technical assistance.

The authors have declared no conflicts of interest.


    References
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 

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Submitted 9 March 2005; revised version accepted 29 April 2005.



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