Analysis of polymorphisms affecting immune complex handling in systemic lupus erythematosus
K. E. Sullivan,
A. F. Jawad,
L. M. Piliero1,
N. Kim1,
X. Luan1,
D. Goldman2 and
M. Petri2
University of Pennsylvania School of Medicine, Philadelphia,
1 Children's Hospital of Philadelphia, Philadelphia, PA and
2 Johns Hopkins University School of Medicine, Baltimore, MD, USA
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Abstract
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Objectives. Systemic lupus erythematosus (SLE) is a polygenic disorder of dysregulated inflammation. Numerous specific candidate genes have been identified and most relate to the handling of immune complexes or antigen presentation. This is consistent with the classic finding of immune complex deposition in affected end organs. We wished to examine combinatorial effects of polymorphic variants of genes involved in immune complex clearance in susceptibility to lupus.
Methods. This study examined the occurrence of polymorphisms in genes which encode proteins known to be involved in immune complex handling and clearance. Each polymorphic variant of a complement protein (C2, mannose binding protein and C4), complement receptor (CR1) or Fc receptor (Fc
RIIA and Fc
RIIIA) gene is known to affect function adversely. One hundred and sixty SLE patients and 212 control subjects were genotyped using polymerase chain reaction methods.
Results. We found an increasing association of SLE with increasing numbers of gene defects. Combinations of severe defects in Fc
RIIA and Fc
RIIIA were particularly deleterious for both African American and Caucasian patients, even though only one defective variant was individually statistically significantly associated with SLE.
Conclusions. The results of the study suggest that genes may interact in ways that either synergize or modify the effect of a single genetic effect and imply that association studies must be interpreted within the genetic background of the populations.
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Introduction
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Systemic lupus erythematosus (SLE) is a disorder characterized by the production of autoantibodies and the deposition of immune complexes in affected end organs. There is an implied loss of both B-cell and T-cell tolerance. However, most genes that have been implicated in human SLE have a role in immune complex handling. To date, the strongest evidence support roles for HLA, Fc
receptors, complement and tumour necrosis factor polymorphisms in SLE [110].
Fc receptors and complement receptors play a critical role in the handling of immune complexes. Under normal circumstances, immune complexes are tagged with complement products (C1, C4 and C3). CR1 receptors on the surface of erythrocytes bind the C3b-tagged immune complexes and transport them to the liver and spleen, where fixed cells of the reticuloendothelial system take them up [11, 12]. Binding to erythrocytes and subsequent fixation of complement is dependent on the size and nature of the immune complexes [13]. For those complexes that are successfully transported by erythrocytes, uptake in the liver and spleen is mediated by both Fc receptors and complement receptors [1417]. In general, SLE patients have rapid clearance of immune complexes due to early clearance by the liver. Subsequently, these immune complexes are released. Uptake by the spleen is both delayed and inefficient [18, 19]. The cause of this phenomenon in SLE patients is presumably hypocomplementaemia and a decreased concentration of CR1 on the surface of erythrocytes. Most of this is presumably secondary to the disease process, but there is evidence that inherited polymorphisms in Fc receptors, complement genes and complement receptor genes could play a role in the inherited predisposition to SLE. Aberrant handling of immune complexes may be important in both immune complex-mediated inflammation and in the aberrant B-cell tolerance which is thought to occur in SLE.
Fc receptors constitute a large family of genes belonging to the family of multichain immune recognition receptors (with the exception of Fc
RIIIB) [20]. Fc
RI is not known to be polymorphic in humans, although rare individuals lack Fc
RI without consequence [21]. Fc
RII (CD32) is a low-affinity receptor present on phagocytic cells, B cells and dendritic cells. It exhibits significant population polymorphism, the 131-Arg (R131) allele binding IgG2 much less avidly than the 131-His (H131) allele [22]. RR131 homozygosity is not thought to be a risk factor for SLE but has been associated with worse disease, renal disease, and earlier onset in some studies [4, 6, 10, 23, 24] but not others [2527]. Fc
RIIIA (CD16) encodes a single polymorphic protein which is expressed on NK cells and monocytes. The wild-type sequence at position 176 encodes a phenylalanine (176-F) whereas the polymorphic variant is 176-valine (176-V). This change results in increased binding of IgG1 and IgG3 [7, 28, 29]. The F allele has been associated with SLE in some studies [7, 2830] and not others [27]. Fc
RIIIB (CD16) encodes a single GPI-linked polymorphic protein expressed on neutrophils. The NA1 and NA2 polymorphisms reflect a number of amino acid changes. These polymorphic variants have not been consistently shown to be implicated in autoimmune disorders.
Complement defects have long been known to be a risk factor for the development of SLE. The strongest genetic risk factor for SLE that is known is complete deficiency of one of the early complement components [3136]. Nevertheless, in most SLE cohorts the frequency of complete complement deficiencies is only 12% [37], implying that for most SLE patients there are other genetic risk factors. Partial complement deficiencies, such as C4A deficiency, also predispose to SLE [3, 3843]. Although C4A deficiency is in strong linkage disequilibrium with DR3 in European populations, the independent contribution of C4A to disease susceptibility has been confirmed [42]. In addition, complement receptor defects have been associated with SLE. A CR3 mutation was found in one SLE patient [44] and quantitative defects in CR1 expression have been documented in some studies. There is an inherited polymorphism in CR1 expression levels on erythrocytes, but most of the variation in CR1 expression is thought to be due to removal by the reticuloendothelial system [4547].
This study examined whether combinations of polymorphisms known to affect function were over-represented in SLE patients compared with controls. We hypothesized that there might be a threshold effect or a particular combination of genetic polymorphisms which is particularly deleterious.
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Materials and methods
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Patients
SLE patients were recruited from the Johns Hopkins Lupus Cohort. Controls were recruited from Philadelphia and Baltimore. The Johns Hopkins Lupus Cohort, established in 1987, consists of nearly 1000 patients diagnosed with SLE and followed by members of the Division of Molecular and Clinical Rheumatology at The Johns Hopkins Hospital. Patient inclusion in the cohort is based on a clinical diagnosis of SLE. A cumulative SLE database was constructed for each patient during 1989 that included nine demographic items, onset and duration of SLE, symptoms and signs of SLE in each organ system, laboratory features and hospitalizations. Patients are seen prospectively every 3 months after cohort entry. The cumulative database is updated yearly. We studied 160 SLE patients (91% female, 53% African American) and 212 controls (52% female, 27% African American). Patients were recruited from consecutive clinic visits during three separate recruitment phases. Controls were recruited from the Baltimore and Philadelphia areas. Exclusion criteria were autoimmune diseases in the donor or SLE in a first-degree relative. Ethnicity was self-reported by the donors. Approximately one-third of the African American patients in this study had been included in previous studies of mannose binding protein (MBP), Fc
RII and C4A [27, 48, 49]. Approximately one-quarter of the Caucasians had had previous C4A analyses reported. All of the controls were new. Institutional review board approval was granted for this study and informed consent was obtained from the patients and controls.
Polymerase chain reaction analysis
DNA was prepared by either GenePure (Gentra Systems, Minneapolis, MN, USA) or phenol extraction of lysed cells. MBP polymorphisms were identified according to methods published previously [48]. Fc
RII H/R alleles and Fc
RIII F/V alleles were assigned according to methods described previously [27, 50]. The 2 base-pair (bp) insertion in C4A was identified by direct polymerase chain reaction (PCR) analysis [49]. The 28 bp deletion in C2 was also detected by direct PCR analysis [9]. CR1 alleles were detected by HindIII digestion of PCR amplified products, as described previously [51].
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Statistical analyses
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The
2 test, Fisher's exact test or the odds ratios (OR) and their associated 95% confidence intervals (CI) were used to compare gene frequencies between different populations. The MantelHaenszel test was used to determine the association of individual polymorphisms with SLE using race to stratify. Three genotypes known to have a particularly deleterious effect on function were designated as severe genotypes: Fc
RIIA RR-131, Fc
RIIIA FF-176 and the MBP O/O type. The MBP summary nomenclature is used here, where O/O indicates nearly absent functional MBP levels due to any one of several combinations of structural or promoter polymorphisms (O=MBP 54A, MBP 57A, LX). A/O indicates a partial deficiency and A/A indicates a normal level of MBP, as predicted by the codon 54, codon 57 and MBP promoter haplotypes [52]. We used
2 analysis to determine whether increasing numbers of these severe genotypes were seen in SLE patients. There is no linkage disequilibrium between these genes [20, 53]. We also analysed whether combinations of deleterious genotypes contributed independently to susceptibility to SLE by the use of step-wise variable selection, multiple logistic regression modelling.
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Results
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Differences in allele frequencies
Six genes whose protein products are involved in immune complex handling were examined initially. The polymorphic variants examined have all been shown to have functional significance. The frequencies of each of the alleles in the SLE and control populations and the appropriate statistical tests for proportions and their P values are shown in Table 1
. Codon 54A and the low-producing promoter haplotype (LX) were over-represented in African American patients compared with controls. In contrast, codon 57A was over-represented in Caucasian SLE patients compared with controls, while the promoter haplotypes and codon 54A were found with similar frequencies in Caucasian SLE patients and controls. The low-binding Fc
RIIA R131 allele (R) was over-represented in Caucasian SLE patients only and the low-binding Fc
RIIIA F176 (F) allele in the African American SLE patients only.
Two defects in early classical complement components were examined. The second most common cause of a C4 null allele in Caucasian populations is a 2 bp insertion which results in premature termination [54]. Neither the C4A 2 bp insertion nor the C2 28 bp deletion were found with increased frequency in the SLE populations compared with controls. However, the number of people bearing either of these polymorphisms was small compared with other studies which found an association.
A polymorphic variant of the CR1 receptor results in decreased expression of this molecule on erythrocytes. There was no difference in the frequency of the different CR1 alleles.
The number of comparisons performed on each population increases the likelihood of a false-positive result. The Bonferroni correction compensates for this. In these experiments, the Bonferroni correction would require a P value of <0.006 for significance. Therefore, the association of the LX MBP promoter haplotype in African Americans and the association of MBP codon 57A and Fc
RIIA R131 with SLE in Caucasians are the most firmly established.
Combinatorial analysis
The original intent of this study was to determine whether combinations of functionally relevant polymorphisms were particularly deleterious in SLE. We hypothesized that there might be an increasing association of SLE with increasing numbers of deleterious polymorphic variants. For this purpose we identified three homozygous polymorphisms which are known to adversely affect handling of immune complexes [22, 29, 55]. The frequencies of the C4 2 bp insertion and the C2 28 bp deletion were too small to be included in this analysis. Table 2
shows the odds ratio and P values for the entire SLE population for two homozygous polymorphisms [Fc
RIIA R131/R131 (RR), Fc
RIIIA F176/F176 (FF)] and the very low-producing MBP combinations denoted by MBP O/O. In addition, we compared the number of patients who had any one of these three genotypes, two of the genotypes or all three of the genotypes (Table 3
). There is a significant association of SLE with increasing numbers of deleterious genotypes.
Multiple logistic regression analyses were used to determine whether MBP O/O, Fc
RIIA RR, Fc
RIIIA FF or combinations thereof were associated with SLE susceptibility (Table 4
). The distribution of genotypes is race-dependent and therefore race was included in the final logistic model as a covariate. The contribution of each genotype combination to the risk of developing lupus is shown. The results of the analyses identified MBP O/O as the most potent single-gene effect (adjusted OR, 2.74; 95% CI, 1.355.55). The combination of Fc
RIIA RR and Fc
RIIIA FF was particularly deleterious. This is notable because neither was independently significantly associated with SLE in this model. Table 5
demonstrates the predicted probabilities associated with each genotype combination in African American and Caucasian populations separately. For African Americans, MBP O/O increased the probability of SLE by 24% while Fc
RIIA RR and Fc
RIIIA FF increased the risk by only 1216% individually. The strongest probability was conferred by all three deleterious genotypes, which increased the risk by 30% in both populations. In both populations, the duplex combination of Fc
RIIA RR and Fc
RIIIA FF increased the probability by 1821%. For both populations, the probability associated with the Fc
RIIA RR and Fc
RIIIA FF combination is higher than each individually. This is significant because the association studies in Table 1
demonstrated that only a single allele studied in isolation was statistically significantly associated with SLE. For Caucasians, only Fc
RIIA R was associated with SLE, while in African Americans only Fc
RIIIA F was associated with SLE.
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TABLE 4. Odds ratios and 95% CIs from the multiple logistic model using maximum likelihood estimates predicting SLE
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TABLE 5. Predicted probabilities associated with deleterious genotypes based on the final logistic regression model
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Clinical phenotypes
We hypothesized that the clinical phenotype of patients carrying more of the severe genotypes might be different from that of patients carrying fewer of the severe genotypes. Specifically, increased renal disease, vasculitis or photosensitivity was predicted. Therefore, we evaluated the frequency of 138 different clinical variables in patients with 0, 1, 2 or 3 of the severe genotypes. Because this represents a large number of variables, it is difficult to interpret simple P values. A clear result would be indicated if there was a gradient of an effect with increasing numbers of gene defects, or an effect of such magnitude that it would be significant even if the large number of variables was corrected for (i.e. P < 0.00036). This was not observed for any variable.
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Discussion
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Several previous studies have investigated the possibility of interactions among SLE candidate genes [1, 3, 5659]. Most of these have focused on the relative contributions of HLA and complement loci on chromosome 6. Our approach relied on data suggesting that multiple genes involved in immune complex handling and clearance are implicated in SLE [36, 10, 22, 23, 25, 38, 40, 46, 48, 60]. We hypothesized that the accumulation of functional defects might demonstrate either a threshold effect for the susceptibility to SLE or a progressive association with increasing numbers of dysfunctional alleles. Therefore, we examined genes which were known to participate in immune complex handling and we specifically evaluated polymorphic variants which had been shown by others to have functional consequences. This was not an exhaustive analysis of all genes implicated in SLE, but rather a selection of those with known functional consequences in which the analysis could be performed using high-throughput techniques on stored samples. A small subset of patients in this study had been studied previously. The present study largely confirms previous findings, although there are two differences. We have reported previously the MBP genotypes from a small subset of the African American patients [48]. The previous study found that codon 54A, codon 57A and the LX promoter haplotype were over-represented in African American SLE patients compared with controls. This study confirmed over-representation of codon 54A and the LX promoter haplotype in African American SLE patients compared with controls. There was no significant difference in the frequencies of the codon 57 alleles in African American SLE patients compared with controls. This discrepancy is probably due to the larger sample size of the previous study. This study was not powered to evaluate C2 and C4A in the same fashion as the previous studies and thus could not be expected to confirm the associations identified previously.
This study clearly demonstrated an increasing association of SLE with increasing numbers of dysfunctional alleles. This was true for both the African American population and the Caucasian population. This finding confirms the longstanding hypothesis that the inherited susceptibility to SLE is the accumulation of multiple independent genetic effects. However, this is the first study to examine more than two-fold combinations. Population differences in the frequency of the individual polymorphic variants make it likely that specific genetic combinations will differ in different ethnic groups and in different individuals.
This study also sought to determine whether specific combinations of individual genes were particularly deleterious. Two homozygous Fc
R polymorphisms and the very low-producing MBP variants were evaluated in this analysis. Thus, our analysis of epistatic interactions included only people with substantial functional deficits. The Fc
RIIA RR genotype and the Fc
RIIIA FF genotype are known to be associated with significant alterations in IgG binding [22, 29, 61]. MBP O/O genotypes are associated with nearly absent serum MBP [52, 55, 62]. All three deleterious genotypes were statistically associated with SLE in at least one population. Interestingly, although in some cases the individual genes had no statistically significant association on their own, they were still capable of participating in an additive effect (the Fc
RII RR+Fc
RIIIA FF combination in Caucasians and the MBP O/O+Fc
RII RR+Fc
RIIIA FF in African Americans)
Monozygotic twin concordance rates are estimated at 2964% [6365], suggesting that environmental or stochastic processes are at least as important as genetic susceptibility. Understanding the genetic contribution to SLE is important because it is likely to reveal disease mechanisms which could be amenable to therapeutic intervention. The main conclusion from this study is that genetic contributions within a functional class (immune complex clearance) may be additive or synergistic even with genes that by themselves have a minimal effect. Simple association studies may therefore exclude genes which could be important in the context of a specific genetic background. New types of analyses and larger studies will be required to define genetic interactions. Nevertheless, this study and other studies provide provocative evidence that combinatorial analysis may provide unique insights into the genetic susceptibility to SLE.
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Acknowledgments
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This work was supported by the Wallace Chair of Pediatrics and the Lupus Foundation of Delaware Valley (KES) and by NIH grant R01-AR-43727 and The Johns Hopkins Outpatient Clinical Research Center grant RR-00052 (MP). We would like to acknowledge the contributions of Saurabh Moonat, Bradley Weinberger and Young Hwangbo, who performed certain PCR analyses.
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Notes
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Correspondence to: K. E. Sullivan, Division of Immunologic and Infectious Diseases, The Children's Hospital of Philadelphia, 34th Street and Civic Ctr. Blvd., Philadelphia, PA, USA. E-mail: sullivak{at}mail.med.upenn.edu. 
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Submitted 23 May 2003;
Accepted 9 October 2002