Functional Fcgamma receptor polymorphisms are associated with human allergy.

Objective IgG Fc receptors (FcγRs) play important roles in immune responses. It is not clear whether FcγR receptors play a role in human asthma and allergy. The aim of current study was to investigate whether functional single nucleotide polymorphisms (SNPs) of FcγR genes (FCGR) are associated with human asthma and allergy. Methods Functional SNPs of FCGR2A (FcγRIIA-131His>Arg, rs1801274), FCGR2B (FcγRIIB-187Ile>Thr, rs1050501), FCGR2C (FcγRIIC-13Gln>Stop, rs10917661), FCGR3A (FcγRIIIA-158Val>Phe, rs396991), and FCGR3B variants (FcγRIIIB NA1 and NA2) were genotyped in an asthma family cohort including 370 atopy positive, 239 atopy negative, and 169 asthma positive subjects. The genotype and phenotype data (asthma, bronchial hyper-responsiveness, and atopy) of subjects were analyzed using family-based association tests (FBAT) and logistic regression adjusted for age and sex. Result The FcγRIIA-131His>Arg SNP is significantly associated with atopy in a family-based association test (P = 0.00287) and in a logistic regression analysis (P = 0.0269, OR 0.732, 95% CI: 0.555–0.965). The FcγRIIA-131His (or rs1801274-A) allele capable of binding human IgG2 has a protective role against atopy. In addition, the rare FcγRIIB-187Thr (or rs1050501-C) allele defective for the receptor-mediated inhibitory signals is a risk factor for atopy (P = 0.0031, OR 1.758, 95% CI: 1.209–2.556) and IgE production (P<0.001). However, variants of activating FcγRIIIA (rs396991), and FcγRIIIB (NA1 and NA2), and FcγRIIC (rs10917661) are not associated with asthma, BHR, and atopy (P>0.05). Conclusions FcγRIIA and FcγRIIB functional polymorphisms may have a role in the pathogenesis of allergy.


Introduction
Asthma is a complex syndrome characterized by airflow obstruction, bronchial hyper-responsiveness (BHR), and airway inflammation. Both genetic and environmental factors contribute to the development of asthma. Evidence for a genetic component in asthma includes familial clustering and higher concordance rates in monozygotic twins than in dizygotic twins [1,2]. Approximately 48-79% of asthma risk is attributable to genetic factors [1,2]. According to the American Academy of Allergy, Asthma and Immunology, half of the 20 million Americans with asthma have allergic asthma. Thus, allergic reactions to foreign antigens are considered as the most common causes for asthma. To date, no genes have been definitely shown to influence asthma/allergy development. It is well-known that IgE and its cognate receptor (FceRI) are important mediators in allergic reactions [3]. However, the role of human IgG Fc receptors (FccRs) in asthma and allergy remains unknown.
A recent meta-analysis of human genome-wide association study (GWAS) revealed a significant asthma susceptibility locus on chromosome 1q23, where FccR (FCGR) genes are located [4]. Human FccRs are glycoproteins that bind the Fc region of immunoglobulin G (IgG). FccRs mediate a variety of immune functions such as antigen presentation, immune complex clearance, phagocytosis of pathogens, degranulations, ADCC, and cytokine production [5]. In humans, five genes (FCGR2A, FCGR2B, FCGR2C, FCGR3A, and FCGR3B) in the 1q23 chromosome region code for five classical low affinity Fcc receptors (FccRIIA, FccRIIB, FccRIIC, FccRIIIA, and FccRIIIB). Coordination between the activating FccRs (FccRIIA, FccRIIC, FccRIIIA, and FccRIIIB) and the inhibitory FccR (FccRIIB) is crucial in balancing immune responses and determining the outcomes of local and systemic inflammations [6]. FccRs have important roles in the pathogenesis of a variety of human inflammatory diseases [7]. Not surprisingly, functional polymorphisms of FccR have robust effects on susceptibility and severity of inflammatory diseases as demonstrated in genetic association studies by our group and others [8,9,10,11]. However, comprehensive genetic analysis of human FCGR genes in asthma/allergy patients has yet been performed. It remains unknown whether human FccRs play a role in the development of allergy.

Study Subjects
Genomic DNA was isolated from anti-coagulated peripheral blood of human subjects from 27 multigenerational families with multiple asthmatic members, which were originally recruited as part of the Collaborative Study on the Genetics of Asthma (CSGA) [12]. For the CSGA, asthma families were ascertained through two asthmatic siblings. Additional relatives in the families were then recruited either by extending the families through asthmatic relatives or by including no more than one unaffected relative to permit a lineage to incorporate other relatives with asthma. The inclusion criteria for each family consisted of each of the two asthmatic siblings having met the following criteria for the proband: (1) being at least 6 years of age; (2) having either bronchial hyper-responsiveness (BHR), defined as a fall from baseline FEV1 greater than 20% in one second after inhalation of 25 mg/ml or less of methacholine, or reversibility, defined as a 15% or greater increase from baseline FEV1 after inhaled bronchodilator (albuterol) for those with reduced baseline FEV1; (3) having the presence of two or more of the symptoms of coughing, wheezing and shortness of breath; (4) having less than three pack/years of cigarette smoking; and (5) having a physician's diagnosis of asthma with no conflicting pulmonary disease. All family members went through a standardized protocol consisting of an interviewer administered questionnaire, pulmonary function studies including a methacholine challenge and/or reversibility studies, blood drawing for serum IgE levels at a single time not during an acute exacerbation and skin prick testing using standardized allergens [12]. Additional details of the study design can be found in an earlier publication [12]. The 27 multigenerational Caucasian families were recruited in Minnesota as previously described [13]. These families had 169 asthmatic members, 347 who were not asthmatic and 129 for whom the diagnosis was unavailable [13]. Pulmonary function data were available on 619 individuals. The study (Title: Genetics of Asthma. Study Number: 920M05150) was approved by The Institutional Review Board of Human Study at the University Of Minnesota. The informed written consent was obtained from all participants recruited in this study. The written consents containing participants' signatures were kept in locked file cabinets for record. The traits of asthma, BHR, atopy, and IgE levels were analyzed in the current genetic study.

Genotyping of FCGR SNPs
FCGR family member genes were generated through duplication and divergence during evolution [14]. SNPs in five FCGR genes are not suitable for direct TaqMan assays due to near 100% sequence identity surrounding the functional SNPs between homologous genes. Consequently, we used a modified FCGR SNP TaqMan assay in which FCGR gene-specific PCR fragments were used as templates instead of genomic DNA for TaqMan assays. The genomic DNA fragments containing functional SNPs of FCGR2A and FCGR3A were amplified using the gene specific primers as described previously [9]. For the FCGR2B SNP, a genomic DNA fragment containing FccRIIB-187Ile.Thr was amplified using the gene specific primers as described [8,15]. To genotype FccRIIC-13Gln.STP, a long FCGR2C genomic fragment (6227 bps) containing the SNP was amplified using Platinum Taq DNA Polymerase High Fidelity (Invitrogen) with a sense primer (59-CTG CAT ATG TTG TCC CCC TGT GTT GCT AAA T-39) annealing to the FCGR2C intron 2 and an antisense primer (59-AAC ATG AGA GAG AAA AAG AGA GGC AGG GAG GGA GCT TA-39) annealing to the FCGR2C intron 6. The TaqMan assays for FCGR2A SNP (FccRIIA-131His.Arg), FCGR2B SNP (FccRIIB-187Ile.Thr), FCGR2C SNP (FccRIIC-13Gln.STP), and FCGR3A SNP (FccRIIIA-158Val.Phe) were designed using the Software Primer Express v3.0 (Applied Biosystems Inc.). TaqMan genotyping assays were carried out according to the standard protocol on an ABI 7500 Real-Time PCR System using Genotyping Master Mix (Applied Biosystems). The primers and probes used in FCGR TaqMan genotyping assays are listed in Table 1. Genotyping of the respective SNPs of FCGR2A, FCGR2B, FCGR2C, and FCGR3A was carried out with four independent TaqMan allele discrimination assays that were developed and validated in the lab. The specificity and accuracy of individual TaqMan assays were validated by the perfect match (100%) with at least 300 genotyped human subjects published previously [8,9,15]. For FCGR3B allele determination, a primer pair that specifically amplifies the FCGR3B fragment containing FCGR3B coding SNPs (cSNPs) was used. The 1.6 kb FCGR3B PCR fragment was treated with ExoSAP-IT PCR Product Clean-UP reagent (Affymetrix) before being sequenced on an ABI 3730xl DNA Analyzer with BigDye Terminator kit (Applied Biosystems) with the sequencing primer (59-TCC TCA CCC CAC ATT ATC TTG-39). The FCGR3B alleles and genotypes were determined based on the published reference [16,17].

Statistical Analysis
The IgE levels were log-transformed to correct for skewed distribution. Family-based association tests (FBAT) [18] were used to examine whether individual FCGR SNPs are associated with phenotypes of human subjects in the asthma family cohort. Alternatively, we used conditional logistic regression to estimate odds ratios of FCGR SNPs for their association with asthma, BHR, and atopy, adjusting for age and sex. The association between logtransformed IgE levels and FCGR genotypes were analyzed using one-way analysis of variance (ANOVA) in addition to the nonparametric t-test (Mann-Whitney test). In both FBAT and regression analysis, an additive model was assumed for SNP genotypes. To correct for multiple hypothesis tests, the Bonferroni method was used and the null hypothesis was reject at 0.05/ number of tests.

Results
The FccRIIA SNP is Associated with Atopy As shown in Table 2, the FCGR2A SNP (FccRIIA-131His.Arg, rs1801274) is significantly associated with atopy in the familybased association test (FBAT) (P = 0.003). The FCGR2A SNP is also associated with asthma and BHR in FBAT (P,0.05). Conditional logistic regression analysis estimated an OR of 0.732 (P = 0.027, 95% CI: 0.555-0.965) for FCGR2A SNP with atopy, suggesting a protective role against atopy for carriers of the FccRIIA-131His allele (population allele frequency = 0.488). Although the FCGR2A SNP is significantly associated with asthma and BHR in FBAT (P,0.05), the association were not significant in logistic regression analyses adjusted for age and sex. Further validation may be needed to confirm our findings. Furthermore, the functional SNPs of the other three activating FccRs (FccRIIIA, FccRIIIB, and FccRIIC) were not associated with asthma, BHR, and atopy (P.0.05) ( Table 3).

The Inhibitory FccRIIB SNP is Associated with Atopy and IgE Production
Although the FCGR2B SNP (FccRIIB-187Ile.Thr, rs1050501) is not associated with asthma, BHR, and atopy in FBAT analyses, conditional logistic regression analyses showed that FCGR2B SNP is significantly associated with atopy and that the FccRIIB-187Thr (allele frequency = 0.088) is a risk allele for atopy (P = 0.003, OR 1.758, 95% CI: 1.209-2.556) ( Table 2). Because immunoglobulin E (IgE) play an important role in allergic diseases and elevated total IgE is frequently considered as a diagnostic criterion for allergic diseases [3], we subsequently analyzed whether the FccRIIB SNP is associated with IgE levels in human subjects. As shown in Figure 1A, FccRIIB genotypes are significantly associated with the serum IgE levels. The human subjects carrying rare FccRIIB-Thr allele produced significantly more IgE (P = 0.0002 for 187Ile/Thr heterozygous subjects and P = 0.0004 for 187Thr/Thr homozygous subjects) than those homozygous (187-Ile/Ile) subjects carrying the common allele, suggesting that the functional FccRIIB SNP may have a role in allergy through IgE production. On the other hand, FccRIIA SNP is not associated with IgE production in humans ( Figure 1B).

Discussion
This study shows the association of two functional SNPs (the activating FccRIIA-131His.Arg and the inhibitory FccRIIB-187Ile.Thr) with human atopy. Furthermore, we demonstrated an association between FccRIIB SNP and IgE production. Our   [19,20]. The FccRIIA-131His. Arg SNP significantly affects receptor binding affinity and specificity for IgG subclasses [21,22]. Although both FccRIIA-131His and 131Arg alleles bind IgG1 and IgG3, the FccRIIA-131His allele displays a higher binding affinity for IgG3 and is capable of binding IgG2 most effectively as compared to the FccRIIA-131Arg allele [21,22]. The functional FccRIIA-131His. Arg SNP affects the receptor binding affinity for IgG and thus influences the clinical phenotype in response to infectious diseases and inflammation [21]. The FccRIIA-131His.Arg SNP affects the functions of bacterial phagocytosis [23,24] and immune complex handling [22,25,26]. The FccRIIA-131His.Arg SNP has been reported to be associated with ulcerative colitis [27], Kawasaki diseases [28], systemic lupus erythematosus [29], and chronic inflammatory disorders such as periodontitis [30,31] and Guillain-Barré syndrome [32]. In addition, FccRIIA-131His.Arg polymorphism is associated with infections including recurrent bacterial respiratory tract infections [33], bacteremic pneumococcal pneumonia [34], severe acute respiratory syndrome [35], severe sepsis [36], HIV [37], and EB virus infection [38]. IgG2 is produced primarily in response to polysaccharide/carbohydrate antigens commonly found in allergens. The protective effect of the FccRIIA-131His allele on atopy is possibly due to the increased capacity of this allele to efficiently internalize and destroy allergen-IgG2 immune complexes. The role of FccRIIA in allergy was also demonstrated in transgenic mouse models [39]. Therefore, FccRIIA likely contributes to allergy development in humans. Although the FccRIIA-131His.Arg SNP is associated with atopy, the SNP is not associated with IgE production ( Figure 1B), suggesting that FccRIIA likely affects allergy through pathways of immune complex clearance and receptor-mediated cell activation. Future studies are required to reveal whether IgG2 levels are associated with the asthma or atopy in the context of FccRIIA SNP and whether FccRIIA-mediated functions (immune complex clearance and phagocytosis of allergens) are different between asthmatic and non-asthmatic human subjects.
FccRIIB, mainly expressed on B cells and myeloid cells, is a classical inhibitory IgG Fc receptor [40,41,42]. Cross-linking of FccRIIB by immune complexes leads to the down-regulation of B cell activation and antibody production, which is an important feedback mechanism to maintain the homeostasis of immune responses [5,40,43]. Therefore, FccRIIB overexpression (or enhanced FccRIIB functions) reduces the immunoglobulin production in T-dependent immune responses [44]. In humanized mouse models of immunoglobulin production, co-engagement of IgE B-cell receptor with FccRIIB drastically inhibited human IgE production [45]. FccRIIB-187Ile.Thr SNP (rs1050501) is located within the receptor transmembrane segment and the FccRIIB-187Thr allele is less efficient in mediating inhibitory signals than the FccRIIB-187Ile allele [46,47,48]. We observed that the low function FccRIIB-187Thr allele is significantly associated with elevated IgE levels (Figure 1), suggesting that the reduced FccRIIB function may promote IgE antibody production by B cells in humans. Interestingly, the low function FccRIIB-187Thr allele is also associated with protection against malaria [49], signifying FccRIIB functions play important roles in controlling the immune response to parasites [50]. Nevertheless, FccRIIB-187Ile.Thr SNP may also be in linkage equilibrium with SNPs of the FCER1A gene encoding for the alpha chain of the high affinity receptor for IgE (FceRIA) because a GWAS identified the FCER1A functional variants strongly associated with total IgE levels [51].
FccRIIB on immune cells also inhibits cellular functions including phagocytosis, ADCC, degranulation, and cytokine release [40]. Mast cells from FccRIIB 2/2 mice are highly sensitive to IgG-triggered degranulation compared to those from the wildtype mice. FccRIIB-deficient mice have an enhanced passive cutaneous analphylaxis reaction, as a result of the decreased threshold for mast-cell activation through activating Fc receptors Thr were significantly associated with IgE levels in ANOVA (P,0.0001). The rare FCGR2B-187Thr carriers (187Ile/Thr and 187Thr/Thr genotypes) produced significantly more IgE than the homozygous subjects for common allele (187Ile) in Mann-Whitney tests. B. The genotypes of FCGR2B-131His.Arg were not associated with IgE levels in ANOVA (P = 0.817). No significant differences were found between FCGR2A-131Arg carriers (131his/Arg and 131Arg/Arg genotypes) and the 131His homozygous subjects in Mann-Whitney tests (P.0.05). doi:10.1371/journal.pone.0089196.g001 [52]. FccRIIB negatively regulates cell activation triggered by high-affinity IgE receptors (FceRI) [53]. FccRIIB binds to the Fc domains of IgE and IgG with similar low affinity [54,55]. Mast cells and basophils could be regulated by immune complexes of allergen-IgG or allergen-IgE. FccRIIB-deficient mice developed more severe eosinophilia compared to wild-type mice, suggesting an important regulatory role for FccRIIB in the onset of allergic diseases [56]. FccRIIB-knockout mice developed the exacerbated lung inflammation [57]. Taken together, FccRIIB seems to play a critical role in allergic inflammations. In the current study, the dysfunctional FccRIIB-187Thr allele was found to be a risk factor for atopy. A decreased activation threshold for immune cells carrying FccRIIB-187Thr allele may be responsible for the increased sensitivity to allergens that trigger the allergic responses, which may explain the association between the defective FccRIIB allele and atopy.
On the other hand, the functional SNPs of the other three activating FccRs (FccRIIIA, FccRIIIB, and FccRIIC) were not associated with asthma, BHR, and atopy, suggesting that functions of the restrictively expressed activating FccRs (FccRIIIA, FccRIIIB, and FccRIIC) may not play prevailing roles in the development of allergy. Our current study had more than 80% power to detect an association between a FCGR SNP and atopy with an OR of 1.75.
In summary, the functional SNPs of FccRIIA and FccRIIB are associated with atopy, signifying that FccRIIA and FccRIIB may serve as important modifiers in the development of allergy. Therefore, targeting FccRIIA and FccRIIB for enhanced receptor expressions and functions may be an important avenue for therapeutic discovery in allergy and asthma treatment.