There is increasing evidence that gene copy number (CN) variation influences clinical phenotype. The low-affinity Fc receptor 3B (FCGR3B) located in the FCGR gene cluster is a CN polymorphic gene involved in the recruitment of polymorphonuclear neutrophils to sites of inflammation and their activation. Given the genetic overlap between systemic lupus erythematosus and systemic sclerosis (SSc) and the strong evidence for FCGR3B CN in the pathology of SLE, we hypothesised that FCGR3B gene dosage influences susceptibility to SSc. We obtained FCGR3B deletion status in 777 European Caucasian cases and 1000 controls. There was an inverse relationship between FCGR3B CN and disease susceptibility. CN of ⩽1 was a significant risk factor for SSc (OR=1.55 (1.13–2.14), P=0.007) relative to CN⩾2. Although requiring replication, these results suggest that impaired immune complex clearance arising from FCGR3B deficiency contributes to the pathology of SSc, and FCGR3B CN variation is a common risk factor for systemic autoimmunity.
Systemic sclerosis (SSc) is an uncommon autoimmune disease, in which abnormalities in the immune and vascular system lead to a variety of dermatological and systemic abnormalities. Although it differs from systemic autoimmune diseases such as systemic lupus erythematosus (SLE) in several respects (the inflammatory infiltrates are milder, and neither immunoglobulin deposits nor glomerulonephritis is a feature of SSc), a number of loci associated with SSc appear to be common risk factors for several autoimmune connective tissue and rheumatic disorders, including PTPN22 rs2476601, STAT4 rs7574864 and IRF5 rs2004640.1
Fc gamma receptor 3B (FcγR3B) is the most abundant neutrophil binding site for polymeric IgG and immune complexes (IC), and is involved in both the recruitment of polymorphonuclear neutrophils (PMN) to sites of inflammation and the clearance of circulating IC. Copy number (CN) variation in FCGR3B has been associated with a variety of autoimmune disorders, including rheumatoid arthritis,2, 3, 4 ulcerative colitis5 and glomerular disease.6, 7 In particular, CN<2 has been reported to be a risk factor for SLE in a number of studies.7, 8, 9, 10, 11, 12, 13 Given the evidence for shared genetic susceptibility between SSc and SLE, we hypothesised that copy-number variation in FCGR3B has a role in the pathology of SSc. In order to evaluate this hypothesis, we tested for association between FCGR3B gene dosage and SSc in a Caucasian case–control sample set under the specific hypothesis that deletion of FCGR3B is a risk factor in comparison with diploid and increased CN.
CN assignment, aided by neutrophil antigen typing on a subset of 56 samples selected because they were positioned near the CN 1–2 boundary on histograms (Supplementary Figure 1a), showed that 11.3% of the SSc sample set carried a single-copy deletion of FCGR3B compared with 7.6% of controls, with one SSc patient having a null FCGR3B genotype. (This patient also has Sjögren’s syndrome with arthritis, which is possibly of clinical significance given a report of association of FCGR3B deletion with Sjögren’s syndrome.14) The FCGR3B CN allele distribution among the controls (CN<2=7.6%, CN 2=82.6% and CN>2=9.8%) is similar to that observed in other healthy Caucasian populations as determined by multiplex ligand-dependent probe amplification or paralog ratio testing (CN<2=6.7–7.8%, CN 2=81.1–82.2% and CN>2=9.4–11.8%).8, 12, 13, 15, 16
The influence of possessing <2 copies of FCGR3B on SSc risk was tested under the hypothesis that any association of FCGR3B with disease would be similar to that evident in SLE, where CN<2 is a risk factor.5, 6, 7, 8, 9, 10 There was significant evidence that possessing fewer than two copies of FCGR3B is a risk factor for SSc (odds ratio (OR)=1.55 (1.13–2.14), P=0.007) (Table 1). This risk was not diminished after accounting for country of origin in the association analysis (Table 1).
We tested for association between FCGR3B CN<2 and limited cutaneous or diffuse cutaneous SSc, and the presence or absence of anti-topoisomerase I and anti-centromere antibodies. There was no significant association of CN<2 with any of these stratifications (Supplementary Table 1).
Our results provide evidence that deletion of FCGR3B is associated with SSc (OR=1.55, P=0.007). This is the first report in SSc and is consistent with the association reported for SLE, where published ORs range from 1.47 to 1.65.7, 8, 9, 10, 11, 12, 13 Although the P value is modest (0.007), this likely represents genuine association given the prior evidence supporting a role for FCGR3B deletion in SSc. Membrane-bound FcγR3B is involved in the adherence of neutrophils to endothelial cells at sites of infection as well as in IC uptake. The soluble form of the receptor (sFcγR3B) released from activated PMN also binds IC, preventing these complexes being taken up by other, activating Fc receptors. FcγR3B expression, PMN adhesion, IC formation and sFcγR3B levels are all correlated with FCGR3B CN.12 FCGR3B deletion is therefore likely to decrease recruitment of PMN to sites of inflammation as well as reduce IC sequestration and clearance. It has been hypothesised that the primary effect of deletion in the FCGR3B gene is a reduced uptake of IgG complexes, leading to tissue deposition of IC.5, 6, 12, 15 Our results, however, suggest that this may not be the only explanation for the observed associations. Although SLE and SSc are characterised by the presence of autoantibodies, the absence of IgG deposits in SSc is one of the major differences between SLE and SSc.17 This suggests that decreased FcγR3B expression leading to the reduced clearance of circulating IC containing autoantibodies may contribute to systemic inflammation, with tissue-specific effects if IC deposition occurs. Although the role of autoantibodies in the pathogenesis of SSc is a subject of debate, it is known that toll-like receptor-mediated production of type 1 interferons by plasmacytoid dendritic cells is a characteristic feature of SLE and a similar interferon signature has been reported in SSc patients.17 This observation is consistent with a model whereby FCGR3B deficiency decreases IC uptake by PMN, leading to higher levels of circulating IC and enhanced autoantibody stimulation of plasmacytoid dendritic cells.
Although it is possible that the association is an artefact of experimental methodology, we are confident that this is not the case. The quantitative PCR assay method we have used has been validated previously.2 The use of an internal reference in the PCR removes variations due to amplification efficiency and experimental conditions that can occur when test and reference genes are measured in separate wells. Cases and controls were also assayed concurrently, and a-posteriori assignment of CN rather than arbitrary rounding of non-integer values was done to minimise batch effects and systematic errors. Furthermore, all plates where the cutoff between CN=1 and CN=2 was unclear were excluded from the analysis. NA typing was also used as a partial validation of cluster analysis, as presence of heterozygosity in individuals assigned as CN=1 would indicate misidentification.
Another potential source of error is the presence of SNPs within the amplification primers; the forward primer was originally designed so that the two bases at the 3′ end of the forward primer differ between FCGR3B and the closely related FCGR3A.2 The 1000 Genomes project has recently identified two SNPs at these 3′ positions (rs115802971 and rs115130696) in individuals of Yoruban descent in which the minor allele corresponds to the FCGR3A sequence. Although an individual carrying these minor alleles would be falsely determined to have a deletion, the allele frequencies in Caucasian populations are very low, with the minor allele of these SNPs being observed in only one of the first 380 Caucasians sequenced in the 1000 Genomes project.
Given the complexity of the FCGR locus and the fact that deletion of FCGR3B can extend to FCGR2C and/or FCGR2B,11, 15 it is also possible that the apparent association with FCGR3B is in fact caused by variations in one of these other genes. Although FCGR2C has been associated with idiopathic thrombocytopenic purpura,18 CN variation is unlikely to have a major biological effect; in most individuals the gene contains a premature stop codon. It is the presence of a SNP (found in ∼18% of the population) within this stop codon that creates an open reading frame and results in the expression of this inhibitory receptor that appears to be the aetiological variant. Despite the presence of linkage disequilibrium between FCGR3B CN and a functional polymorphism within FCGR2B that abolishes its inhibitory capacity, the effects of FCGR3B deletion and homozygosity for the inactivated FcγR2B allele are independently associated with SLE.11 This suggests that FCGR3B CN is also likely to contribute to disease risk independently of an accompanying deletion in FCGR2B.
In conclusion, the results presented here suggest that FCGR3B CN influences susceptibility to SSc. Future work should focus on development of more efficient approaches for measuring CN at FCGR3B, replication of this association and investigation of association in clinically-defined subgroups. Given the role of this receptor in the uptake of IC and the fact that impaired clearance of autoantibodies is strongly implicated in the pathogenesis of SLE,19 it is possible that a similar deficiency contributes to the development of SSc. This association also adds to the list of diseases in which FCGR3B CN has been implicated, suggesting that FCGR3B may be a common autoimmune-related locus.
Materials and methods
All the patients fulfilled the 1980 American College of Rheumatology (ACR) classification criteria for SSc.20 The local ethical committee from each centre approved the study. Both patients and controls were included in the study after obtaining written informed consent. All patients included in this study were classified as having limited cutaneous (LcSSc) or diffuse cutaneous SSc (DcSSc) using the criteria postulated by LeRoy et al.21 Patients with scleroderma changes limited to the skin distal to the elbows and/or knees, regardless of facial involvement, fulfil the definition for LcSSc. Those SSc patients with more proximal scleroderma skin changes were classified as having DcSSc. The presence of pulmonary fibrosis was investigated by a high-resolution computed tomography scan. Restrictive syndrome and diffusion capacity of the lungs was defined as a forced vital capacity <75% of the predicted value, and the diffusion capacity of the lung for carbon monoxide was defined as <75% of that predicted. Controls were derived from the same country and region as the patients originated from and included 723 previously genotyped samples from the Netherlands that exhibited good clustering.2 Clinical details of the SSc patients are given in Supplementary Table 2. Collection of blood samples and clinical information from case and control subjects was undertaken with informed consent and relevant ethical review board approval from each contributing centre in accordance with the tenets of the Declaration of Helsinki.
Determination of FCGR3B copy number
FCGR3B CN was measured using Taqman Real-Time PCR as described previously, with detection probe 5′-CCTCAGCTTTCCC-3′.2 To minimise DNA degradation (which can increase the risk of false-positive calls22), lysophilised samples were first assayed within 24 h of resuspension and stored at −20 °C between experiments. All samples were assayed on two separate occasions in duplicate on each occasion and the raw CN values averaged, with an overall average difference of 0.21 copies between duplicates. A maximum of 183 samples were assayed simultaneously in 384-well plates. Genotyping was successful for 90.1% (1284/1425) of cases and 89.4% (1218/1362) of controls. Two approaches were used to convert continuous CN estimates to integers. In the first approach, ΔΔCT analysis was carried out using Applied Biosystems CopyCaller V2.0 software with the assumption that the most common CN estimate was 2, and by discarding samples where bin assignment was done with <50% confidence. The second approach was based on the standard curve method described previously,2 whereby the distribution of calculated CN for each experiment was examined by displaying them histographically, with an average of 80 samples assayed in the same batch. Each histogram was drawn in a manner blinded to the origin of the samples and the cutoff between CN=1 and CN>1 independently determined by two people based on the clustering of values and the location of the first-identified NA heterozygote (Supplementary Figure 1a). Samples exhibiting discrepancy with confident (>50%) CopyCaller assignments were excluded. Ten plates (containing 507 cases and 218 controls) were excluded from the analysis because of unclear clustering between CN=1 and CN=2 (Supplementary Figure 1b).
When sufficient DNA was available for standard PCR to be performed, the functional NA1 and NA2 alleles of FCGR3B were genotyped over samples spanning the boundary between CN=1 and CN=2 using conventional PCR and gel visualisation using an established assay.23
Subjects were grouped as those with FCGR3B deletion and were compared with the reference group of those having two or more copies of FCGR3B. Chi-square analysis was used to compare the distribution of FCGR3B CN between patients and controls after CN assignment. Logistic regression analysis was used to measure the SSc-associated risks of low FCGR3B CN as the OR relative to CN⩾2 using the STATA 8.0 statistical package.
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The Health Research Council of NZ and NZ Lottery Health are thanked for financial support. JM was funded by grant SAF2009-11110 from the Spanish Ministry of Science, CTS-4977 from Junta de Andalucía, Spain, by Redes Temáticas de Investigación Cooperativa Sanitaria Programme, RD08/0075 (RIER) from Instituto de Salud Carlos III (ISCIII), Spain, and by Fondo Europeo de Desarrollo Regional (FEDER).
The authors declare no conflict of interest.
Supplementary Information accompanies the paper on Genes and Immunity website
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McKinney, C., Broen, J., Vonk, M. et al. Evidence that deletion at FCGR3B is a risk factor for systemic sclerosis. Genes Immun 13, 458–460 (2012). https://doi.org/10.1038/gene.2012.15
- systemic sclerosis
- copy number
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