Analysis of HLA DR, HLA DQ, C4A, FcγRIIa, FcγRIIIa, MBL, and IL-1Ra allelic variants in Caucasian systemic lupus erythematosus patients suggests an effect of the combined FcγRIIa R/R and IL-1Ra 2/2 genotypes on disease susceptibility
© Jönsen et al.; licensee BioMed Central Ltd. 2004
Received: 22 December 2003
Accepted: 16 July 2004
Published: 23 September 2004
Dysfunction in various parts of immune defence, such as immune response, immune complex clearance, and inflammation, has an impact on pathogenesis in systemic lupus erythematosus (SLE). We hypothesised that combinations of common variants of genes involved in these immune functions are associated with susceptibility to SLE. The following variants were analysed: HLA DR3, HLA DQ2, C4AQ0, Fcγ receptor IIa (FcγRIIa) genotype R/R, Fcγ receptor IIIa (FcRγIIIa) genotype F/F, mannan-binding lectin (MBL) genotype conferring a low serum concentration of MBL (MBL-low), and interleukin-1 receptor antagonist (IL-1Ra) genotype 2/2. Polymorphisms were analysed in 143 Caucasian patients with SLE and 200 healthy controls. HLA DR3 in SLE patients was in 90% part of the haplotype HLA DR3-DQ2-C4AQ0, which was strongly associated with SLE (odds ratio [OR] 2.8, 95% CI 1.7–4.5). Analysis of combinations of gene variants revealed that the strong association with SLE for HLA DR3-DQ2-C4AQ0 remained after combination with FcγRIIa R/R, FcγRIIIa F/F, and MBL-low (OR>2). Furthermore, the combination of the FcγRIIa R/R and IL-1Ra 2/2 genotypes yielded a strong correlation with SLE (OR 11.8, 95% CI 1.5–95.4). This study demonstrates that certain combinations of gene variants may increase susceptibility to SLE, suggesting this approach for future studies. It also confirms earlier findings regarding the HLA DR3-DQ2-C4AQ0 haplotype.
KeywordsFcγ receptor HLA interleukin-1 receptor antagonist mannan-binding lectin systemic lupus erythematosus
The genetic contribution to the aetiology of systemic lupus erythematosus (SLE) is high, as is indicated by familial aggregation and a higher concordance rate in monozygotic than dizygotic twins . The major histocompatibility complex (MHC) haplotype HLA DR3-DQ2-C4AQ0 is strongly associated with SLE in Caucasians [2, 3]. The IgG Fc receptors appear to be important in the pathogenesis of SLE, as recently reviewed by Salmon and Pricop . With the allelic variant of R (arginine) instead of H (histidine) on amino acid position 131, the ability of Fcγ receptor IIa (FcγRIIa) to bind IgG2 is diminished . Similarly, an amino acid substitution in position 158 (phenylalanine [F] instead of valine [V]) in the Fcγ receptor IIIa (FcγRIIIa) reduces the IgG1-, IgG3-, and IgG4-binding capacity of the receptor . These variants can result in suboptimal clearance of immune complexes from the circulation, which might contribute to the pathogenesis of immune-complex-mediated manifestations .
Mannan-binding lectin (MBL) is structurally similar to C1q and has the ability to activate the complement cascade through the lectin pathway. Point mutations are found in the structural gene that affect the MBL serum concentration and the stability of MBL complex formation required for efficient complement activation . In the promoter regions, there are two polymorphisms that influence serum concentration, with LX conferring the lowest MBL level, LY a medium level, and HY the highest [8–11]. MBL variant alleles have been suggested as a minor risk factor in susceptibility to SLE in several populations [8, 10, 12]. Interleukin-1 receptor antagonist (IL-1Ra) is a naturally occurring competitive inhibitor of IL-1. The IL-1Ra gene contains a polymorphism in intron 2 consisting of a variable number of copies of an 86-base-pair repeat sequence (two, three, four, five, or six copies) . An association has been found between the IL-1Ra 2 allele and SLE [13, 14]. Multiple genes are involved in the development of SLE, and the relative importance of these genes may vary between populations and with environmental exposure. We investigated common variant alleles involved in the immune response, immune complex clearance, and regulation of inflammation, with the hypothesis that combinations of polymorphic candidate genes could have synergistic effects on disease susceptibility. Therefore, we have analysed polymorphisms in the genes HLA DR, HLA DQ, C4A, FcγRIIa, FcγRIIIa, MBL, and IL-1Ra and their association with the development of SLE.
Materials and methods
Distribution of American College of Rheumatology (ACR) classification criteria in 143 patients with SLE
DNA was extracted by the salting-out method described by Miller and colleagues . Analysis of genetic polymorphism was predominantly performed by polymerase chain reaction (PCR).
HLA DR and DQ alleles were determined with PCR (Olerup SSP™ DQ-DR SSP Combi Tray, Olerup SSP AB, Stockholm, Sweden). However, a minority of the patients had previously been typed with a lymphocytotoxicity test or by restriction fragment length polymorphism as described before . C4A gene deletion was determined by PCR as described by Grant and colleagues , or in a few cases by analysis of restriction fragment length polymorphism and determination of MHC haplotypes . With the presence of a DR3 allele together with a DQ2 and a C4AQ0 allele, due to C4A gene deletion, the subject was considered to have the haplotype HLA DR3-DQ2-C4AQ0, although family studies were not uniformly performed to confirm this assumption.
FcγRIIa gene polymorphism
The genetic polymorphism resulting in amino acid R or H in amino acid position 131 was determined as previously described .
Analysis of FcγRIIIa gene polymorphism
The analysis of the F/V polymorphism was performed essentially as previously described .
MBL gene polymorphism
Variants of MBL due to mutations at codon 52 (D), 54 (B), and 57 (C) in exon 1 of the MBL gene and promotor variants at position -550 (H/L) and -221 (X/Y) were determined by allele-specific PCR amplification, essentially as described before . The wild-type structural allele is designated A, while 0 is a description of the mutant alleles B, C, and D. Based on previously described associations between MBL genotype and serum concentrations, which were confirmed in our 200 healthy controls, the MBL genotypes were divided into three groups. Group 1 (MBL-low) consisted of patients with two structural mutant alleles (0/0) or on one haplotype a structural mutant allele together with another haplotype containing an LX promoter and the wild-type structural allele (ALX/0). Group 2 (MBL-intermediate) consisted of patients with the promoters LX conferring low serum MBL on both haplotypes but with normal structural alleles (ALX/ALX), or, alternatively, haplotypes with one mutant and one wild-type structural allele with a non-LX promoter together with the wild-type allele. Group 3 (MBL-high) included patients with the A/A genotype and at least one non-LX promoter.
IL-1Ra gene polymorphism
Primers: 5'-CTC AGC AAC ACT CCT AT-3'
5'-TTC CAC CAC ATG GAA C-3'
The amplified fragment size depends on the number of repeats (two repeats, designated allele 2; three, allele 4; four, allele 1; five, allele 3; six, allele 5).
Two group comparison tests were performed using the Fisher exact test. Comparisons between multiple groups were made using the χ2 multiple comparison test. Significance was considered when P <0.05. Correction for multiple comparisons was not applied to the results, because the study design consisted in hypothesis testing. The presence of synergistic interaction between genetic variants was investigated by calculating relative excess risk due to interaction (RERI) .
Comparisons of genetic variants in 143 patients with SLE and 200 healthy blood donors
IL-1 Ra 2/2
HLA DR3-DQ2-C4AQ0 / FcγRIIa R/R
HLA DR3-DQ2-C4AQ0 / FcγRIIIa F/F
HLA DR3-DQ2-C4AQ0 / MBL-low
HLA DR3-DQ2-C4AQ0 / IL-1 Ra 2/2
FcγRIIa R/R / FcγRIIIa F/F
FcγRIIa R/R / MBL-low
FcγRIIa R/R / IL-1 Ra 2/2
FcγRIIIa F/F / MBL-low
FcγRIIIa F/F / IL-1 Ra 2/2
MBL-low / IL-1 Ra 2/2
The increasing number of reports on polymorphic genes involved in susceptibility to SLE prompted us to investigate whether a combination of polymorphic candidate genes, tentatively thought to be involved in the pathogenesis of SLE, could further elucidate the genetic basis of the disease. In the present study we found that the combination of the FcγRIIa R/R genotype with the IL-1Ra 2/2 genotype was strongly associated with SLE. Although only a few of the patients had this particular genetic background, the results indicate that certain combinations of susceptibility genes can be of crucial importance. Furthermore, a strong association between the haplotype HLA DR3-DQ2-C4AQ0 and susceptibility to SLE was seen in this study, which is in concordance with the findings of previous studies [2, 22, 24, 25]. The patients and controls studied were all from a homogeneous Caucasian population, although a possible bias exists in the fact that the controls used were blood donors, which principally include only healthy individuals, instead of age-matched controls from the normal population. The distributions of the polymorphic variants in the controls were in agreement with data published by others [13, 26, 27].
There have been ample studies on the association between FcγRIIa and SLE [24, 28–30]. However, the results are somewhat conflicting regarding whether or not the R allele is associated with increased susceptibility to SLE in general or for SLE glomerulonephritis or other clinical manifestations of SLE. In our study, there was no association between either the R allele or the R/R genotype and susceptibility to SLE, with a glomerulonephritis frequency of 27%.
The MBL genotype did not seem to be involved in susceptibility to SLE in our Caucasian cohort. This differs from a finding of a recent meta-analysis in which MBL variant alleles were found to be associated with SLE . Furthermore, in that study the conclusion was drawn that several studies are too small to detect an increased SLE susceptibility dependent on MBL risk alleles, which could also explain the lack of association in our study.
An increased carriage rate of the 2 allele of the IL-1Ra gene has been shown for SLE patients [13, 14]. In our study, the 2/2 genotype in conjunction with the FcγRIIa R/R genotype was associated with SLE. This IL-1Ra genotype is associated with higher IL-1 beta concentrations as well as higher serum IL-1Ra levels [31, 32]. Furthermore, immune complex binding to Fc receptors can influence the production of IL-1Ra , which provides a possibility for a pathogenetic mechanism concordant with the genetic interaction seen in our study. Analyses of disease phenotypes were beyond the scope of this study and will be addressed in future studies. However, there were no apparent associations between the various genotypes and clinical subsets of SLE. Because of the low number of patients included in the study, the results must be interpreted cautiously, and independent confirmation is needed.
Our findings suggest that the combination of the FcγRIIa R/R and IL-1Ra 2/2 genotypes is associated with SLE in Caucasian patients, whereas individually these genotypes do not increase susceptibility to the disease. This finding illustrates that combinations of polymorphic genes may act in concert in the pathogenesis of SLE, a concept that may be instrumental in the analysis of the genetics of SLE as well as providing hypotheses for pathways in the pathogenesis of lupus.
= American College of Rheumatology
= Fcγ receptor IIa
= Fcγ receptor IIIa
= interleukin-1 receptor antagonist
= mannan-binding lectin
= MBL genotype conferring a low/intermediate/high serum concentration of MBL
= major histocompatibility complex
= odds ratio
= polymerase chain reaction
= relative excess risk due to interaction
= systemic lupus erythematosus
We thank Mrs Birgitta Gullstrand and Mrs Gertrud Hellmer for their skilful work with the genetic typing and Jonas Björk, PhD, for valuable statistical aid. The study was supported by grants from the Swedish Rheumatism Association, the Swedish Research Council (grant nos. 13489 and 15092), the Medical Faculty of the University of Lund, Alfred Österlund's Foundation, The Crafoord Foundation, Greta and Johan Kock's Foundation, The King Gustaf V's 80th Birthday Fund, Lund University Hospital and Prof Nanna Svartz' Foundation
- Deapen D, Escalante A, Weinrib L, Horwitz D, Bachman B, Roy-Burman P, Walker A, Mack TMA: Revised estimate of twin concordance in systemic lupus erythematosus. Arthritis Rheum. 1992, 35: 311-318.View ArticlePubMedGoogle Scholar
- Truedsson L, Sturfelt G, Johansen P, Nived O, Thuresson B: Sharing of MHC haplotypes among patients with systemic lupus erythematosus from unrelated Caucasian multicase families: Disease association with the extended haplotype [HLA-B8, SC01, DR17]. J Rheumatol. 1995, 22: 1852-1861.PubMedGoogle Scholar
- Rood MJ, van Krugten MV, Zanelli E, van der Linden MW, Keijsers V, Schreuder GM, Verduyn W, Westendorp RG, de Vries RR, Breedveld FC, et al: Tnf-308A and HLA-DR3 alleles contribute independently to susceptibility to systemic lupus erythematosus. Arthritis Rheum. 2000, 43: 129-134. 10.1002/1529-0131(200001)43:1<129::AID-ANR16>3.0.CO;2-S.View ArticlePubMedGoogle Scholar
- Salmon JE, Pricop L: Human receptors for immunoglobulin G: key elements in the pathogenesis of rheumatic disease. Arthritis Rheum. 2001, 44: 739-750. 10.1002/1529-0131(200104)44:4<739::AID-ANR129>3.3.CO;2-F.View ArticlePubMedGoogle Scholar
- Warmerdam PA, van de Winkel JG, Vlug A, Westerdaal NA, Capel PJ: A single amino acid in the second Ig-like domain of the human Fc gamma receptor II is critical for human IgG2 binding. J Immunol. 1991, 147: 1338-1343.PubMedGoogle Scholar
- Koene HR, Kleijer M, Algra J, Roos D, dem Borne AE, de Hass M: Fc gammaRIIIa-158V/F polymorphism influences the binding of IgG by natural killer cell Fc gammaRIIIa, independently of the Fc gammaRIIIa-48l/R/H phenotype. Blood. 1997, 90: 1109-1114.PubMedGoogle Scholar
- Davies KA, Peters AM, Beynon HL, Walport MJ: Immune complex processing in patients with systemic lupus erythematosus. In vivo imaging and clearance studies. J Clin Invest. 1992, 90: 2075-2083.PubMed CentralView ArticlePubMedGoogle Scholar
- Sullivan KE, Wooten C, Goldman D, Petri M: Mannose-binding protein genetic polymorphisms in black patients with systemic lupus erythematosus. Arthritis Rheum. 1996, 39: 2046-2051.View ArticlePubMedGoogle Scholar
- Madsen HO, Garred P, Thiel S, Kurtzhals JA, Lamm LU, Ryder LP, Svejgaard A: Interplay between promoter and structural gene variants control basal serum level of mannan-binding protein. J Immunol. 1995, 155: 3013-3020.PubMedGoogle Scholar
- Ip WK, Chan SY, Lau CS, Lau YL: Association of systemic lupus erythematosus with promoter polymorphisms of the mannose-binding lectin gene. Arthritis Rheum. 1998, 41: 1663-1668.PubMedGoogle Scholar
- Minchinton RM, Dean MM, Clark TR, Heatley S, Mullighan CG: Analysis of the relationship between mannose-binding lectin (MBL) genotype, MBL levels and function in an Australian blood donor population. Scand J Immunol. 2002, 56: 630-641. 10.1046/j.1365-3083.2002.01167.x.View ArticlePubMedGoogle Scholar
- Davies EJ, Snowden N, Hillarby MC, Carthy D, Grennan DM, Thomson W, Ollier WE: Mannose-binding protein gene polymorphism in systemic lupus erythematosus. Arthritis Rheum. 1995, 38: 110-114.View ArticlePubMedGoogle Scholar
- Blakemore AI, Tarlow JK, Cork MJ, Gordon C, Emery P, Duff GW: Interleukin-1 receptor antagonist gene polymorphism as a disease severity factor in systemic lupus erythematosus. Arthritis Rheum. 1994, 37: 1380-1385.View ArticlePubMedGoogle Scholar
- Suzuki H, Matsui Y, Kashiwagi H: Interleukin-1 receptor antagonist gene polymorphism in Japanese patients with systemic lupus erythematosus. Arthritis Rheum. 1997, 40: 389-390.View ArticlePubMedGoogle Scholar
- Tan EM, Cohen AS, Fries JF, Masi AT, McShane DJ, Rothfield NF, Schaller JG, Talal N, Winchester RJ: The 1982 revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum. 1982, 25: 1271-1277.View ArticlePubMedGoogle Scholar
- Jonsson H, Nived O, Sturfelt G: Outcome in systemic lupus erythematosus: a prospective study of patients from a defined population. Medicine (Baltimore). 1989, 68: 141-150.View ArticleGoogle Scholar
- Gladman D, Ginzler E, Goldsmith C, Fortin P, Liang M, Urowitz M, Bacon P, Bombardieri S, Hanly J, Hay E, et al: The development and initial validation of the Systemic Lupus International Collaborating Clinics/American College of Rheumatology Damage Index for systemic lupus erythematosus. Arthritis Rheum. 1996, 39: 363-369.View ArticlePubMedGoogle Scholar
- Miller SA, Dykes DD, Polesky HF: A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 1988, 16: 1215-PubMed CentralView ArticlePubMedGoogle Scholar
- Grant SF, Kristjansdottir H, Steinsson K, Blondal T, Yuryev A, Stefansson K, Gulcher JR: Long PCR detection of the C4A null allele in B8-C4AQ0-C4B1-DR3. J Immunol Methods. 2000, 244: 41-47. 10.1016/S0022-1759(00)00251-9.View ArticlePubMedGoogle Scholar
- Flesch BK, Bauer F, Neppert J: Rapid typing of the human Fc gamma receptor IIA polymorphism by polymerase chain reaction amplification with allele-specific primers. Transfusion. 1998, 38: 174-176. 10.1046/j.1537-2995.1998.38298193100.x.View ArticlePubMedGoogle Scholar
- Leppers-van de Straat FG, van der Pol WL, Jansen MD, Sugita N, Yoshie H, Kobayashi T, van de Winkel JG: A novel PCR-based method for direct Fc gamma receptor IIIA (CD16) allotyping. J Immunol Methods. 2000, 242: 127-132. 10.1016/S0022-1759(00)00240-4.View ArticlePubMedGoogle Scholar
- Tjernstrom F, Hellmer G, Nived O, Truedsson L, Sturfelt G: Synergetic effect between interleukin-1 receptor antagonist allele (IL1RN*2) and MHC class II (DR17, DQ2) in determining susceptibility to systemic lupus erythematosus. Lupus. 1999, 8: 103-108. 10.1191/096120399678847560.View ArticlePubMedGoogle Scholar
- Hosmer DW, Lemeshow S: Confidence interval estimation of interaction. Epidemiology. 1992, 3: 452-456.View ArticlePubMedGoogle Scholar
- Manger K, Repp R, Spriewald BM, Rascu A, Geiger A, Wassmuth R, Westerdaal NA, Wentz B, Manger B, Kalden JR, et al: Fcgamma receptor IIa polymorphism in Caucasian patients with systemic lupus erythematosus: association with clinical symptoms. Arthritis Rheum. 1998, 41: 1181-1189. 10.1002/1529-0131(199807)41:7<1181::AID-ART6>3.0.CO;2-C.View ArticlePubMedGoogle Scholar
- Sturfelt G, Hellmer G, Truedsson L: TNF microsatellites in systemic lupus erythematosus-a high frequency of the TNFabc 2-3-1 haplotype in multicase SLE families. Lupus. 1996, 5: 618-622.View ArticlePubMedGoogle Scholar
- Koene HR, Kleijer M, Swaak AJ, Sullivan KE, Bijl M, Petri MA, Kallenberg CG, Roos D, dem Borne AE, de Haas M: The Fc gammaRIIIa-158F allele is a risk factor for systemic lupus erythematosus. Arthritis Rheum. 1998, 41: 1813-1818. 10.1002/1529-0131(199810)41:10<1813::AID-ART13>3.3.CO;2-Y.View ArticlePubMedGoogle Scholar
- Garred P, Voss A, Madsen HO, Junker P: Association of mannose-binding lectin gene variation with disease severity and infections in a population-based cohort of systemic lupus erythematosus patients. Genes Immun. 2001, 2: 442-450. 10.1038/sj.gene.6363804.View ArticlePubMedGoogle Scholar
- Duits AJ, Bootsma H, Derksen RH, Spronk PE, Kater L, Kallenberg CG, Capel PJ, Westerdaal NA, Spierenburg GT, Gmelig-Meyling FH: Skewed distribution of IgG Fc receptor IIa (CD32) polymorphism is associated with renal disease in systemic lupus erythematosus patients. Arthritis Rheum. 1995, 38: 1832-1836.View ArticlePubMedGoogle Scholar
- Norsworthy P, Theodoridis E, Botto M, Athanassiou P, Beynon H, Gordon C, Isenberg D, Walport MJ, Davies KA: Overrepresentation of the Fcgamma receptor type IIA R131/R131 genotype in caucasoid systemic lupus erythematosus patients with autoantibodies to c1q and glomerulonephritis. Arthritis Rheum. 1999, 42: 1828-1832. 10.1002/1529-0131(199909)42:9<1828::AID-ANR6>3.0.CO;2-F.View ArticlePubMedGoogle Scholar
- Dijstelbloem HM, Bijl M, Fijnheer R, Scheepers RH, Oost WW, Jansen MD, Sluiter WJ, Limburg PC, Derksen RH, van de Winkel JG, et al: Fcgamma receptor polymorphisms in systemic lupus erythematosus: association with disease and in vivo clearance of immune complexes. Arthritis Rheum. 2000, 43: 2793-2800. 10.1002/1529-0131(200012)43:12<2793::AID-ANR20>3.0.CO;2-6.View ArticlePubMedGoogle Scholar
- Sehouli J, Mustea A, Konsgen D, Katsares I, Lichtenegger W: Polymorphism of IL-1 receptor antagonist gene: role in cancer. Anticancer Res. 2002, 22: 3421-3424.PubMedGoogle Scholar
- Santtila S, Savinainen K, Hurme M: Presence of the IL-1RA allele 2 (IL1RN*2) is associated with enhanced IL-1beta production in vitro. Scand J Immunol. 1998, 47: 195-198. 10.1046/j.1365-3083.1998.00300.x.View ArticlePubMedGoogle Scholar
- Suzuki H, Takemura H, Kashiwagi H: Interleukin-1 receptor antagonist in patients with active systemic lupus erythematosus. Enhanced production by monocytes and correlation with disease activity. Arthritis Rheum. 1995, 38: 1055-1059.View ArticlePubMedGoogle Scholar
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