Fc{gamma}RIIIA-158V and rheumatoid arthritis: a confirmation study

A. W. Morgan1,2,, V. H. Keyte2, S. J. Babbage2, J. I. Robinson2, F. Ponchel2, J. H. Barrett3, B. B. Bhakta1, S. J. Bingham1, M. H. Buch1, P. G. Conaghan1, A. Gough1, M. Green1, C. A. Lawson1,2, C. T. Pease1, A. F. Markham2, W. E. R. Ollier4, P. Emery1, J. Worthington4 and J. D. Isaacs1,2

1 Rheumatology and Rehabilitation Research Unit,
2 Molecular Medicine Unit and
3 Cancer Research UK, Genetic Epidemiology Division, University of Leeds and
4 ARC Epidemiology Unit, University of Manchester, UK


    Abstract
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Objectives. To develop a robust assay for genotyping the Fc{gamma}RIIIA-158V/F polymorphism and to confirm the putative association between the Fc{gamma}RIIIA-158V allele and rheumatoid arthritis (RA).

Methods. This allelic association study examined the Fc{gamma}RIIIA-158V/F polymorphism for association with RA. A novel single-stranded conformational polymorphism assay was used to genotype 828 RA patients and 581 controls from the UK.

Results. The Fc{gamma}RIIIA-158V allele was associated with both RA (P=0.02) and nodules (P=0.04). Individuals homozygous for this higher affinity allele had a significantly increased risk of RA (OR 1.53, 95% CI 1.08–2.18) and the development of nodules (OR 2.20, 95% CI 1.20–4.01). There was no evidence of an interaction with the shared epitope.

Conclusions. We have developed a novel assay to genotype the Fc{gamma}RIIIA-158F/V polymorphism and confirmed that homozygosity for the Fc{gamma}RIIIA-158V allele is associated with UK Caucasian RA, particularly in those individuals with nodules, suggesting Fc{gamma}RIIIA may play a role in determining disease severity or in the development of nodules per se.

KEY WORDS: Fc gamma receptor, Rheumatoid arthritis, HLA-DRB1, Polymorphisms.


    Introduction
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
We have previously demonstrated an association between the Fc{gamma}RIIIA-158V allele and rheumatoid arthritis (RA), in both UK Caucasian and North Indian/Pakistani ethnic groups. In the Caucasian cohort the effect was stronger in a subgroup of individuals with nodules [1]. Fc{gamma}RIIIA-158F/V genotyping is problematical and a loss of assay specificity for Fc{gamma}RIIIA over Fc{gamma}RIIIB results in an over-representation of the Fc{gamma}RIIIA-158V allele, due to sequence homology. The association between the Fc{gamma}RIIIA-158V allele and RA has not been confirmed in all studies [2, 3], with the latter group using two different genotyping methods owing to technical difficulties with published genotyping assays [3]. These conflicting results may therefore be secondary to differences in the methods of genotyping, analytical approaches [4], the method of recruitment of both patients and controls, or a genuine difference.

The difficulties inherent in confirming the results of genetic association studies in complex human diseases have been highlighted previously [57]. Even the precise role of the MHC genes, where an association with RA was initially discovered more than 20 yr ago, remains to be fully defined. RA is likely to mirror other autoimmune diseases where many different disease susceptibility genes and environmental factors each contribute a small amount to the overall risk, possibly at different stages in the disease process and varying between different populations. In addition, RA is characterized by phenotypic heterogeneity and could be considered a clinical syndrome composed of many different clinical subtypes, each of which may have different genetic and environmental triggers. Stochastic epigenetic and physiological events that remain undefined in RA may additionally contribute to low penetrance. Consequently, large sample sizes are necessary to confirm modest associations in different populations and many confirmatory studies do not have adequate power to confirm or refute initial associations. We have already highlighted the methodological difficulties involved in genotyping the Fc{gamma}RIIIA-158F/V polymorphism, a frequently overlooked fact when comparing association studies from different laboratories [4].

The Fc gamma receptors (Fc{gamma}Rs) are a family of receptors expressed on the cell surface of leucocytes, which bind IgG antibodies and immune complexes. The activating and inhibitory Fc{gamma}Rs are frequently co-expressed on the same cell, thus providing a means for regulating activation thresholds [8]. The biological plausibility of a role of Fc{gamma}Rs, specifically the activating Fc{gamma}RIIIa and inhibitory Fc{gamma}RIIb, in the pathogenesis of RA has been established in animal models of both spontaneous and induced autoimmunity [8]. Deletion of the common signalling component ({gamma}-chain) of Fc{gamma}RI and Fc{gamma}RIII pathways results in the loss of susceptibility to collagen-induced arthritis [9]. In contrast, mice lacking the inhibitory Fc{gamma}RIIb receptor suffer accentuated arthritis [9, 10]. The potential for therapeutically manipulating Fc{gamma}R pathways has also been highlighted in a murine model of idiopathic thrombocytopenic purpura [11]. Autoantibody-mediated inflammation could be abolished by either blocking Fc{gamma}RIIIa or by increasing the level of inhibitory Fc{gamma}RIIb expression.

Genetic polymorphisms within the Fc{gamma}R genes have been widely investigated as potential susceptibility and severity factors in several human autoimmune diseases [12, 13]. Functional polymorphisms in Fc{gamma}RIIA, Fc{gamma}RIIIA and Fc{gamma}RIIIB have been shown to influence the biological activity of phagocytes directly [12, 13]. Additionally, tumour necrosis factor-{alpha} (TNF{alpha}) [14] and monocyte chemoattractant protein-1 (MCP-1) release [15] have been demonstrated following cross-linking of Fc{gamma}RIIIa, which can thus be directly implicated in RA pathogenesis. Fc{gamma}RIIIa is predominantly expressed on natural killer (NK) cells and macrophages, although differential expression during B- and T-cell development [16] and following activation of {gamma}{delta} T cells has been shown [17]. The Fc{gamma}RIIIA-158V allele has been demonstrated to have an increased affinity for IgG1, IgG3 [18, 19] and possibly IgG4 immune complexes [Morgan AW, unpublished observation], which may have important implications for autoimmunity, antibody-mediated immune surveillance and defence against pathogens [12].

In this paper we present a novel and robust genotyping assay for the Fc{gamma}RIIIA-158F/V polymorphism and use this to confirm our original finding of a genetic association between the Fc{gamma}RIIIA-158V allele and RA in a large cohort of UK Caucasian RA patients.


    Patients and methods
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Study design
This was an allelic association study where the role of the Fc{gamma}RIIIA-158F/V polymorphism was examined in a new cohort of Caucasian RA patients and controls, resident in the UK. RA patients were recruited from rheumatology out-patient clinics. They were all 16 yr of age or older at disease onset and fulfilled the 1987 American Rheumatism Association criteria for RA [20]. All patients were assessed clinically and serologically, and the available demographic data included age at onset, sex and IgM rheumatoid factor (RF) status. Additional data regarding radiographic evidence of erosions and the presence of nodules were available on 500 and 414 RA patients, respectively. Caucasian controls were obtained from two sources: healthy blood donors from the same geographical area and healthy laboratory staff. Power calculations (power 95%, significance 5%) indicated that 575 patients and the same number of controls were required to confirm an association between Fc{gamma}RIIIA-158V and RA with an odds ratio of 2.0, assuming a recessive model. Ethical approval was obtained from the Local Research Ethics Committee.

HLA-DRB1 typing
DNA was extracted from EDTA anti-coagulated blood using a salt precipitation technique. HLA-DRB1*01–16 types were determined using commercially available semi-automated PCR-SSOP typing techniques (Dynal RELITM SSO HLA DRB test, Merseyside, UK or Inno-LiPa, Abbott laboratories Ltd, Maidenhead, UK). The ‘shared epitope’ was defined as HLA-DRB1*01, *0401, *0404, *0405, *0408, *0409, *0410, *0413, *0416, *0419, *0421 or *10.

Genotyping
The Fc{gamma}RIIIA-158-F/V polymorphism was genotyped by a novel single-stranded conformational polymorphism (SSCP) assay. A 199-base pair polymerase chain reaction (PCR) product that contained the polymorphic site was amplified using our previously published PCR conditions [1]. The PCR product was denatured at 95°C for 5 min, placed on ice to prevent re-annealing and was electrophoresed at 14°C, 600 V, 25 mA and 15 W for 90 min using a pre-prepared polyacrylamide gel (Amersham, Little Chalfont, UK). PCR products were visualized by silver staining (Amersham, Little Chalfont, UK). A single upper band denoted Fc{gamma}RIIIA-158FF individuals, a single lower band denoted Fc{gamma}RIIIA-158VV individuals and multiple bands indicated Fc{gamma}RIIIA-158VF individuals (Fig. 1Go). Confirmatory fluorescent automated-cycle sequencing was performed on 60 PCR products as previously described [1]. This analysis provided identical results to SSCP in every case.



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FIG. 1. Single-stranded conformational polymorphism assay for the Fc{gamma}RIIIA-158F/V polymorphism. Lane 1 contains molecular weight markers, lane 2 DNA from a Fc{gamma}RIIIA-158F homozygote, lane 3 DNA from a Fc{gamma}RIIIA-158FV heterozygote and lane 4 DNA from a Fc{gamma}RIIIA-158V homozygote.

 

Statistical analysis
Statistical analyses were performed using the SPSS 9.0 statistical package for Windows, (Chicago, Illinois, USA). Allele and genotype frequencies were compared using 2x2 and 3x2 contingency tables, respectively, and the {chi}2-test was used for statistical comparisons. Two-sided P values below 0.05 were considered statistically significant throughout. The data were initially stratified according to the presence of nodules in order to confirm our previous finding. Further stratifications based on the presence or absence of erosions and the age of onset of RA were performed, as they were felt to be indicators of disease severity or different aetiopathological groups of RA.

Odds ratios (OR) and their 95% confidence intervals (CI) were calculated for Fc{gamma}RIIIA-158VV and Fc{gamma}RIIIA-158VF individuals to quantify the magnitude of the association between the Fc{gamma}RIIIA-158V allele and RA, as an approximation of the relative risk. The Fc{gamma}RIIIA-158FF individuals served as the reference group for these analyses. Under a dominant model the ORs for homozygotes and heterozygotes would be equal, under a co-dominant model (in which each allele contributes to disease risk) a trend in ORs would be expected, and under a recessive model the OR for heterozygotes would be 1.0 [21]. When the data supported more than one model, the mode of inheritance was tested using a likelihood ratio test, for example to examine whether a simpler model (dominant or recessive) fitted the data as well as a more complex co-dominant model. The possibility of an interaction between Fc{gamma}RIIIA and the shared epitope was formally tested, within a logistic regression framework, by performing a test for interaction using the Stata statistical software (Stata Corporation 1999, Stata Statistical Software: Release 6.0. College Station Texas, USA).


    Results
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Characteristics of the RA patients
The study comprised 828 RA patients and 581 controls (511 blood donors and 70 laboratory staff). The HLA-DRB1 status was available on 798 RA patients and 458 controls. The RA and control groups had shared epitope frequencies of 79 and 42%, respectively. The shared epitope was associated with RA in this cohort of patients (OR 4.89, 95% CI 3.81–6.28). In addition, 73% of the RA patients were female and 76% seropositive for IgM RF. The median age at onset was 47 yr and in those for whom data were available 74% had erosive disease and 24% had documented nodules.

Fc{gamma}RIIIA-158V/F allele and genotype distributions and association with RA
There was no departure from Hardy–Weinberg equilibrium in the control group. There was an increase in the frequency of the Fc{gamma}RIIIA-158V allele in RA patients compared with controls (Table 1Go), which was statistically significant for both the total RA cohort (P=0.02) and the subgroup with nodules (P=0.04). There was borderline significance for the 2x3 genotype distributions in the total RA cohort (P=0.05), but this was statistically significant in individuals with nodules (P=0.03). The ORs and 95% CI for the risk of RA and nodular RA for both Fc{gamma}RIIIA-158FV and Fc{gamma}RIIIA-158VV individuals are shown in Table 2Go. In the total RA cohort, there was a significant association with individuals homozygous for the Fc{gamma}RIIIA-158V allele (OR 1.53, 95% CI 1.08–2.18) with a weak trend towards significance in heterozygotes (OR 1.13, 95% CI 0.90–1.41). The data were therefore in keeping with either a co-dominant or recessive mode of inheritance. A likelihood ratio test demonstrated that the data fitted a recessive model equally as well as the more complex co-dominant model (P=0.32). Similarly, homozygosity for the Fc{gamma}RIIIA-158V allele was significantly associated with nodule formation (OR 2.20, 1.20–4.01) and the data were again consistent with a recessive model (Table 2Go).


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TABLE 1. Comparison of Fc{gamma}RIIIA-158F/V genotype and allele frequencies in UK Caucasian RA patients and matched healthy controls

 

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TABLE 2. Odds ratios for the association of the Fc{gamma}RIIIA-158V allele with RA

 
Additionally, there was a trend towards an association between Fc{gamma}RIIIA and the presence of erosions with each Fc{gamma}RIIIA-158V allele appearing to contribute to the overall risk (Fc{gamma}RIIIA-158FV: OR 1.43, 95% CI 0.93–2.19; Fc{gamma}RIIIA-158VV: OR 1.48, 95% CI 0.80–2.74) (Table 3Go). There was no statistically significant association with the age at onset of RA.


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TABLE 3. Comparison of Fc{gamma}RIIIA-158F/V genotype frequencies in RA patients in the presence and absence of radiographic erosions

 

Interaction of the Fc{gamma}RIIIA-158V/F polymorphism and the HLA-DRB1 shared epitope in determining the risk of RA
Our previous work had highlighted a possible interaction between Fc{gamma}RIIIA and the shared epitope in determining susceptibility to RA [1]. Therefore, we calculated the risk of developing RA conferred by either homozygosity for the Fc{gamma}RIIIA-158V allele or the shared epitope allele in isolation, and then formally tested for an interaction within a logistic regression model (Table 4Go). The simpler recessive model (see above) was used for these analyses. These data were consistent with homozygosity for the Fc{gamma}RIIIA-158V allele providing a contribution to the genetic susceptibility of RA (OR 1.56, 95% CI 0.88–2.78), although this was weaker than the contribution of the HLA-DRB1 alleles comprising the shared epitope (OR 4.42, 95% CI 3.06–6.38) (Table 4Go). There was no evidence of an interaction between these two genetic loci (P=0.41). The increased risk in individuals with both Fc{gamma}RIIIA-158VV and shared epitope (OR 5.71, 95% CI 3.44–9.48) was consistent with a multiplicative effect of these two risk factors.


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TABLE 4. Odds ratios demonstrating an association between the Fc{gamma}RIIIA-158V allele and the ‘shared epitope’ (SE) in predicting the development of RA

 


    Discussion
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
We have developed a robust SSCP assay for genotyping the Fc{gamma}RIIIA-158F/V polymorphism, the more widespread use of which may reduce genotyping errors at this locus. We have confirmed our initial association between the Fc{gamma}RIIIA-158V allele and RA in a large cohort of patients and controls recruited from the UK. This association was most marked in the presence of nodules, suggesting Fc{gamma}RIIIA may play a role in determining disease severity or in the development of nodules per se.

Although there was a slight difference in the allele frequencies compared with our previous UK Caucasian control and RA populations, this was not significant and may reflect the relatively small sample sizes or genuine differences in the genetic background between individuals from different parts of the UK. Utilization of family controls, such as parents or unaffected siblings, would have removed any potential bias from population stratification, but they were not available to us. The two UK Caucasian RA populations each had a similar sex ratio, median age of disease onset and RF status. The observed shared epitope frequencies were also comparable with those previously reported in both UK Caucasian RA and control groups [22, 23]. One notable difference between our two UK Caucasian cohorts was the decreased duration of RA. Indeed, where known, 40% of the current cohort had disease of less than 2 yr standing, compared with 15% of the Birmingham UK Caucasian and 25% of the North Indian/Pakistani cohort, from our original publication [1]. This difference is the most likely explanation for the decreased frequency of erosions (74 vs 100%) and nodules (24 vs 31%) in the current population.

The Fc{gamma}Rs play important roles in the initiation and regulation of many immunological and inflammatory processes [13] and it is therefore relatively easy to construct hypotheses to create potential roles for them in the pathogenesis of RA. In humans, Fc{gamma}RIIIa is expressed on NK cells, macrophages, {gamma}{delta} T cells, a subset of monocytes, cultured mast cells [24, 25] and possibly on specific dendritic cells subsets, as seen in mice [26]. These are potentially important effector cells in RA, and Fc{gamma}RIIIa may play a key role in the perpetuation of inflammatory responses to IgG immune complexes. The relative balance of opposing signals from activating and inhibitory Fc{gamma}Rs is likely to determine the overall response to an IgG immune complex [27]. Furthermore, the absolute level of receptor expression is modulated by pro- and anti-inflammatory cytokines [28, 29]. Polymorphic variants that increase the expression or affinity of activating receptors or enhance their ability to bind specific IgG isotypes, such as the Fc{gamma}RIIIA-158V allelic variant, may thus play an important role in determining the severity and persistence of inflammation to IgG (auto)antibodies and immune complexes in RA.

IgG autoantibodies will also bind Fc{gamma}Rs and in RA increased titres of IgG RFs correlate with articular disease severity and with the extra-articular manifestations of RA [30]. Other IgG autoantibodies are found in RA, most notably type II collagen antibodies [31, 32]. These latter may exist fixed to the tissues and the subsequent cross-linking of Fc{gamma}Rs on immune effector cells may induce cytokine release by a mechanism akin to antibody-dependent cellular cytotoxicity. Strong candidates for mediating this are the intermediate-affinity Fc{gamma}RIIIa expressed on NK cells and macrophages and the high-affinity Fc{gamma}RI expressed on macrophages and neutrophils. However, Fc{gamma}RI is believed to be fully saturated with monomeric IgG in vivo, thus reducing its potential role [24].

The genetic effect was relatively weak, however, and the possibility of an additive effect with the rheumatoid shared epitope in RA susceptibility was again raised. One likely mechanism whereby Fc{gamma}RIIIa may influence RA susceptibility is via antigen presentation. The Fc{gamma}Rs play important roles in delivering (auto)antigens, activation and maturation signals to dendritic cells [26, 33]. These events are likely to be important in the initiation of autoimmune responses and the continued presentation of ‘arthritogenic’ peptides, by both professional and non-professional antigen-presenting cells, resulting in disease persistence. Continued activation of antigen-presenting cells and subsequent T-cell responses, by a variety of Fc{gamma}Rs, may thus contribute to episodic flares of inflammation in response to non-specific synovial stimuli [34]. The roles that functional polymorphic variants play in modulating these events and thus initiating or perpetuating RA remain to be clarified. Thus, the Fc{gamma}RIIIA-158V allele may enhance capture of IgG opsonized pathogens or IgG immune complexes, feeding them directly into the antigen-processing pathway and resulting in more efficient presentation of ‘arthritogenic’ peptides.

In summary, our results support an association between the Fc{gamma}RIIIA locus and RA. Thus, the presence of the Fc{gamma}RIIIA-158V allele resulted in increased susceptibility to RA in all three populations we have studied. Individuals homozygous for this allele had a 1.5- to 2- fold increased risk of RA and therefore it is unsurprising that the chromosomal region containing Fc{gamma}RIIIA has not been highlighted as an independent genetic susceptibility locus for RA in genome-wide screens [3537]. The power of the latter studies was only sufficient to detect genetic loci with an approximate genotype-related risk of 4.0 [38]. Further studies focusing on the remaining Fc{gamma}R genes located in this gene cluster on chromosome 1q22 and potentially in linkage disequilibrium with Fc{gamma}RIIIA, are underway.


    Acknowledgments
 
Supported by the Arthritis Research Campaign and the Medical Research Council, UK.


    Notes
 
Correspondence to: A. W. Morgan, Molecular Medicine Unit, Clinical Sciences Building, St. James's University Hospital, Leeds, LS9 7TF, UK. E-mail: a.w.morgan{at}leeds.ac.uk Back


    References
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 

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Submitted 25 January 2002; Accepted 6 November 2002