 determined using a quantitative real-time polymerase chain reaction (qPCR) method21. Briefly, a duplex Taqman® CN assay was performed, using FCGR3B-specific primers (Applied Biosystems, Hs04211858, FAM-MGB dual labeled probe) and RNase P (Applied Biosystems, product 4403326, VIC-TAMRA dual labeled probe) as the reference assay. The assay was performed according to the manufacturer’s instructions and PCR were run on an Applied Biosystems 7300 Real Time PCR machine. All samples were tested in triplicate, and fluorescence signals were normalized to ROX dye. CN was determined using Copy Caller software (v.1.0, Applied Biosystems), and results were accepted only when calling confidence for discrete CN assignment was > 80%, and the ΔCq SD between replicates was < 0.20; otherwise, samples were retested.
The Taqman qPCR FCGR3B CN assay was initially validated against an endpoint PCR paralog ratio assay, measuring FCGR2C/FCGR2A CN ratios20, for which the signal intensity (peak height) for both amplicons (274 and 279 bp) was quantified through a multiplexed fluorescence capillary electrophoresis detection system (Qiaxcel). The rationale for this validation is that several studies have reported complete agreement (i.e., linkage disequilibrium) between FCGR2C and FCGR3B CN12,20. Three reference samples (1,2,3 CN) validated by this assay were included on each qPCR assay plate to control for possible batch effects.
The association between FCGR3B CN and disease susceptibility, and other dichotomous variables, was analyzed by chi-square and logistic regression analysis, with results reported as OR. Testing for ordinal relationships between FCGR3B CN and other variables was performed using the nonparametric gamma correlation coefficient, and proportional OR as appropriate. All analyses were performed using Statistica v6 (Statsoft).
A random effects metaanalysis of this study and a previous publication, examining the relationship between low CN of FCGR3B and pSS, was performed using the R metafor library25,26 and the restricted maximum likelihood method.
P values < 0.05 were considered to indicate statistical significance.
The number of genomic FCGR3B copies observed in our study varied from 0 to 4; however, 1−3 copies were the most common. One pSS patient carried no FCGR3B (null), and 1 patient and 1 control each carried 4 FCGR3B copies. Accordingly, the data were grouped for analysis into < 2, 2, and > 2 copies, respectively.
The distribution of FCGR3B CN variants was significantly different between pSS patients and controls (chi-square = 6.10, df = 2, p = 0.047; Table 1). Expressed as OR relative to the normal diploid 2 CN, low (< 2) CN was significantly increased in pSS (OR 2.6, 95% CI 1.2, 5.6, p = 0.016), whereas there was no difference in the frequencies of high (> 2) CN between pSS patients and controls (OR 1.1, 95% CI 0.5, 2.4, p = 0.80). Although the sample sizes in this study were comparatively small, a retrospective power analysis indicated that there was sufficient power (79.6% 1-sided test, 69.5% 2-sided test) to detect a difference in low FCGR3B CN frequency between patients and controls.
Low FCGR3B genomic copy numbers (< 2 CN) is associated with primary Sjögren’s syndrome (pSS), but not specifically Ro + La autoantibody (seropositive) pSS. The distribution fo FCGR3B CN was significantly different between pSS patients and controls (p = 0.047). When expressed as OR relative to the normal diploid 2 CN, low (< 2) CN was significantly increased in pSS (p = 0.016), wheras there was no difference in the frequencies of high (> 2) CN between pSS patients and controls (p = 0.80). The frequency distribution was also different twhen comparing seropositive versus seronegative pSS patients (p = 0.042), which was primarily attributable to a higher proportion of high (> 2) CN in seronegative patients with pSS. Thee was, however, no suggestion that low (< 2) CN is preferentially associated with Ro + La seropositive pSS.
One previous study evaluated FCGR3B CN in pSS19. In a metaanalytic approach, the combined results from both studies (Figure 1) yielded OR of 2.3 (95% CI 1.3, 3.9, p = 0.003) for low (< 2) CN, and 1.6 (95% CI 0.8, 3.1, p = 0.20) for high (> 2) CN, both expressed relative to the normal diploid 2 CN. Therefore, the association between low FCGR3B CN is confirmed in 2 separate studies, but the additional putative association with high FCGR3B CN19 remains unconfirmed. Our observation that high (> 2) FCGR3B CN was more frequent in patients with seronegative pSS (Table 1) may be relevant in this context, although the number of seronegative patients was very small.
Random effects (RE) metaanalysis of the association between FCGR3B copy number variations and primary Sjögren’s syndrome. The 2 studies available were Mamtani 201019 and the current study. (A) < 2 versus 2 FCGR3B copies; p = 0.003. (B) > 2 versus 2 FCGR3B copies; p = 0.20. RE model indicates the OR (95% CI), estimated by restricted maximum likelihood, for the 2 studies combined.
Given that FCGR play an important role in the clearance of IC, it is relevant to consider whether low FCGR3B CN, as a risk factor for pSS, is specifically associated with seropositivity for Ro ± La autoantibodies within patients with pSS. The distribution of FCGR3B CN in Ro ± La-seronegative versus seropositive patients with pSS is also shown in Table 1. Although the number of patients with seronegative pSS in this study was limited, the frequency distribution was different between the 2 groups (p = 0.042), which was primarily attributable to a higher proportion of high (> 2) CN in patients with seronegative pSS. The relevance of this observation is uncertain, given the low numbers of patients with seronegative pSS. There was, however, no suggestion that low FCGR3B CN is preferentially associated with Ro ± La-seropositive pSS.
In terms of other clinical/serological features, low FCGR3B CN was associated with lower rheumatoid factor (RF) titers and lower serum IgG levels (Table 2, Figure 2). There was also a trend for an inverse ordinal relationship between FCGR3B CN and the proportion of patients with a history of persistent, presumed autoimmune neutropenia, although the number of such patients was low (26/254 = 17%) and this analysis did not achieve statistical significance (p = 0.12; Table 2, Figure 2). There was no observed relationship with age of onset, sex, Schirmer’s test results, sialometry results, or C4 or serum BAFF levels (data not shown).
Box plots of FCGR3B copy number (CN) by (A) rheumatoid factor titer (n = 140) and (B) serum IgG (n = 138). The width of the box plots reflects the number of observations. It is evident that the ordinal trends reported in Table 3 are primarily due to low (< 2) FCGR3B CN, which is associated with lower RF titers and lower serum IgG. (C) The prevalence of low FCGR3B CN is slightly higher in patients with a history of neutropenia, and the prevalence of high FCGR3B CN slightly lower, indicative of an inverse ordinal trend.
Within patients with primary Sjögren’s syndrome there is a positive ordinal relationship between FCGR3B copy number and rheumatoid factor (RF) titer and serum IgG levels, and an inverse ordinal trend with the risk of neutropenia.
We have confirmed that low FCGR3B CN is a genetic susceptibility factor for pSS. Our results for the association with low FCGR3B CN were very similar to the single previous study of patients with pSS19, and collectively, the OR for the association with pSS (relative to the normal diploid 2 CN) was estimated as 2.6 (95% CI 1.2, 5.6, p = 0.003). Therefore low FCGR3B CN may be a common genetic risk factor for systemic autoimmune diseases such as SLE18,19,20, RA21,22, and pSS. However, it is also clear that a relatively low proportion (< 20%) of patients with systemic autoimmune diseases appear to carry this genetic risk factor. This may be indicative of underlying disease heterogeneity, in which low FCGR3B CN is a risk factor in an as-yet unidentified subgroup of patients.
The exposure of autoantigens in pSS is thought to occur during apoptosis of salivary gland acinar and ductal epithelial cells, which is accompanied by translocation of autoantigens to apoptotic blebs. This local event may initiate an autoimmune reaction that becomes systemic through upregulation of type I interferon signature and abnormal expression of BAFF7, and lymphocytic invasion and/or IC-mediated inflammation in other organ systems may result. Circulating IC can be demonstrated in a majority of patients with pSS, and anti-Ro-containing IC are related to continuing systemic immune activation27