FCGR Locus Research Articles

Genetic variations in low-to-medium-affinity Fcγ receptors and autoimmune neutropenia in early childhood in a Danish cohort.

Autoimmune neutropenia (AIN) in early childhood is caused by autoantibodies directed against antigens on the neutrophil membrane and is a frequent cause of neutropenia in children. Association of AIN with Fcγ receptor (FCGR) 3B variants is well described. In this study, we investigate genetic variations in the FCGR locus and copy number variation of FCGR3B. A total of 130 antibody-positive AIN patients, 64 with specific anti-HNA-1a antibodies and 66 with broad-reacting anti-FcγRIIIb antibodies, were genotyped with a multiplex ligation probe assay and compared with healthy controls. Positive findings were confirmed with real-time q-PCR. We determined copy numbers of the FCGR2 and FCGR3 genes and the following SNPs: FCGR2A Q62W (rs201218628), FCGR2A H166R (rs1801274), FCGR2B I232T (rs1050501), FCGR3A V176F (rs396991), haplotypes for FCGR2B/C promoters (rs3219018/rs780467580), FCGR2C STOP/ORF and HNA-1 genotypes in FCGR3B (rs447536, rs448740, rs52820103, rs428888 and rs2290834). Generally, associations were antibody specific, with all associations being representative of the anti-HNA-1a-positive group, while the only association found in the anti-FcγRIIIb group was with the HNA-1 genotype. An increased risk of AIN was observed for patients with one copy of FCGR3B; the HNA genotypes HNA-1a, HNA-1aa or HNA-1aac; the FCGR2A 166H and FCGR2B 232I variations; and no copies of FCGR2B 2B.4. A decreased risk was observed for HNA genotype HNA-1bb; FCGR2A 166R; FCGR2B 232T; and one copy of FCGR2B promoter 2B.4. We conclude that in our Danish cohort, there was a strong association between variation in the FCGR locus and AIN. The findings of different genetic associations between autoantibody groups could indicate the presence of two different disease entities and disease heterogeneity.

OA13 Comprehensive genetic and functional analyses of Fc gamma receptors explain response to rituximab therapy for autoimmune rheumatic diseases

Abstract Background/Aims Rituximab is widely used to treat rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) but clinical response varies. Efficacy is determined by the efficiency of depletion, which may depend on a variety of Fc gamma receptor (FcγR)-dependent mechanisms. Previous research was limited by complexity of the FCGR locus, not integrating copy number variation with functional SNP, and small sample size. Here, we aimed to assess the effect of the full range of FcγRs variants on depletion, clinical response and functional effect on NK-cell-mediated killing in two rheumatic diseases with a view to personalised B-cell depleting therapies. Methods A prospective longitudinal study was conducted in 873 patients (RA = 611; SLE=262) from four cohorts (BSRBR-RA, BILAG-BR, Leeds RA and Leeds SLE Biologics). For RA, the outcome measures were 3C-DAS28CRP and 2C-DAS28CRP at 6 (+/-3) months post-rituximab (adjusted for baseline DAS28). For SLE, major clinical response (MCR) was defined as improvement of active BILAG-2004 domains to grade C/better at 6 months. B-cell depletion was evaluated by highly-sensitive flow cytometry. Qualitative and quantitative polymorphisms for five major FcγRs were measured using a commercial multiplex ligation-dependent probe amplification. Median NK cell FcγRIIIa expression (CD3-CD56+CD16+) and NK-cell degranulation (CD107a) in the presence of rituximab-coated Daudi/Raji B-cell lines were assessed using flow cytometry. Results In RA, for FCGR3A, carriage of V allele (coefficient -0.25 [SE 0.11]; p = 0.02) and increased copies of V allele (-0.20 [0.09]; p = 0.02) were associated with better 2C-DAS28 response. Irrespective of FCGR3A genotype, increased gene copies were associated with a better response. In SLE, 177/262 (67.6%) achieved BILAG response (MCR=34.4%; Partial=33.2%). MCR was associated with increased copies of FCGR3A-158V allele, OR 1.64 (95% CI 1.12-2.41) and FCGR2C-ORF allele 1.93 (1.09-3.40). Of patients with B-cells data in the combined cohort, 236/413 (57%) achieved complete depletion post-rituximab. Only homozygosity for V allele and higher copies of FCGR3A V allele were associated with increased odds of depletion. Patients with complete depletion had higher NK cell FcγRIIIa expression at rituximab initiation than those with incomplete depletion (p = 0.04) and this higher expression was associated with improved EULAR response in RA. Moreover, for FCGR3A, degranulation activity was increased in V allele carriers vs FF genotype in the combined cohort; p = 0.02. Conclusion FcγRIIIa is the major low affinity FcγR and increased copies of the FCGR3A-158V allele, encoding the allotype with a higher affinity for IgG1, was associated with clinical and biological responses to rituximab in two autoimmune diseases. This was supported by functional data on NK cell-mediated cytotoxicity. In SLE, increased copies of the FCGR2C-ORF allele was also associated with improved response. Our findings indicate that enhancing FcγR-effector functions could improve the next generation of CD20-depleting therapies and genotyping could stratify patients for optimal treatment protocols. Disclosure M. Md Yusof: None. J. Robinson: None. V. Davies: None. D. Wild: None. M. Morgan: None. J. Taylor: None. Y. El-Sherbiny: None. D. Morris: None. L. Liu: None. A. Rawstron: None. M. Buch: None. D. Plant: None. H. Cordell: None. J. Isaacs: None. I. Bruce: None. P. Emery: Grants/research support; PE has received consultancy fees and funding for research from Roche within the last 3 years. A. Barton: None. T. Vyse: None. J. Barrett: None. E. Vital: Grants/research support; EMV has received consultancy fees and funding for research from Roche within the last 3 years. A. Morgan: None.

FCGR Genetic Variation in Two Populations From Ecuador Highlands-Extensive Copy-Number Variation, Distinctive Distribution of Functional Polymorphisms, and a Novel, Locally Common, Chimeric FCGR3B/A (CD16B/A) Gene.

Fcγ receptors (FcγR), cell-surface glycoproteins that bind antigen-IgG complexes, control both humoral and cellular immune responses. The FCGR locus on chromosome 1q23.3 comprises five homologous genes encoding low-affinity FcγRII and FcγRIII, and displays functionally relevant polymorphism that impacts on human health. Recurrent events of non-allelic homologous recombination across the FCGR locus result in copy-number variation of ~82.5 kbp-long fragments known as copy-number regions (CNR). Here, we characterize a recently described deletion that we name CNR5, which results in loss of FCGR3A, FCGR3B, and FCGR2C, and generation of a recombinant FCGR3B/A gene. We show that the CNR5 recombination spot lies at the beginning of the third FCGR3 intron. Although the FCGR3B/A-encoded hybrid protein CD16B/A reaches the plasma membrane in transfected cells, its possible natural expression, predictably restricted to neutrophils, could not be demonstrated in resting or interferon γ-stimulated cells. As the CNR5-deletion was originally described in an Ecuadorian family from Llano Grande (an indigenous community in North-Eastern Quito), we characterized the FCGR genetic variation in two populations from the highlands of Ecuador. Our results reveal that CNR5-deletion is relatively frequent in Llano Grande (5 carriers out of 36 donors). Furthermore, we found a high frequency of two strong-phagocytosis variants: the FCGR3B-NA1 haplotype and the CNR1 duplication, which translates into an increased FCGR3B and FCGR2C copy-number. CNR1 duplication was particularly increased in Llano Grande, 77.8% of the studied sample carrying at least one such duplication. In contrast, an extended haplotype CD16A-176V – CD32C-ORF+2B.2 – CD32B-2B.4 including strong activating and inhibitory FcγR variants was absent in Llano Grande and found at a low frequency (8.6%) in Ecuador highlands. This particular distribution of FCGR polymorphism, possibly a result of selective pressures, further confirms the importance of a comprehensive, joint analysis of all genetic variations in the locus and warrants additional studies on their putative clinical impact. In conclusion, our study confirms important ethnic variation at the FCGR locus; it shows a distinctive FCGR polymorphism distribution in Ecuador highlands; provides a molecular characterization of a novel CNR5-deletion associated with CD16A and CD16B deficiency; and confirms its presence in that population.

Variability at the FCGR locus: characterization in Black South Africans and evidence for ethnic variation in and out of Africa.

This study set out to comprehensively investigate all known functional FcγR variants in South African Black and Caucasian individuals. Population diversity was further assessed using data from the 1000 Genomes Project. In our cohort, Black South Africans neither possessed the haplotypes previously associated with increased surface densities of FcγRIIb and FcγRIIIa nor the FCGR2C haplotype recently associated with increased vaccine efficacy in the RV144 HIV-1 vaccine trial (despite 48.7% bearing the c.134-96T tag allele). Moreover, Africans (South Africans, Luhya Kenyans and Yoruba Nigerians) lack the FCGR2C c.798+1G splice-site allele required for the expression of functional FcγRIIc. Although the presence or absence of surface FcγRIIc did not affect natural killer cell-mediated antibody-dependent cellular cytotoxicity capability, this may be significant for other FcγRIIc-mediated functions. Overall, allele distribution and linkage disequilibrium in Africans and Caucasians differed in a manner that would suggest a differentially maintained balance of FcγR-mediated cell activation in these populations. Finally, significant variation observed among different African populations precludes the use of any one African population as a proxy for FcγR diversity in Africans. In conclusion, the findings of this study highlight further ethnic variation at the FCGR gene locus, in particular for FCGR2C, a gene with increasingly recognized clinical significance.

Association analysis of copy numbers of FC-gamma receptor genes for rheumatoid arthritis and other immune-mediated phenotypes.

Segmental duplications (SDs) comprise about 5% of the human genome and are enriched for immune genes. SD loci often show copy numbers variations (CNV), which are difficult to tag with genotyping methods. CNV in the Fcγ receptor region (FCGR) has been suggested to be associated with rheumatic diseases. The objective of this study was to delineate association of FCGR-CNV with rheumatoid arthritis (RA), coeliac disease and Inflammatory bowel disease incidence. We developed a method to accurately quantify CNV in SD loci based on the intensity values from the Immunochip platform and applied it to the FCGR locus. We determined the method's validity using three independent assays: segregation analysis in families, arrayCGH, and whole genome sequencing. Our data showed the presence of two separate CNVs in the FCGR locus. The first region encodes FCGR2A, FCGR3A and part of FCGR2C gene, the second encodes another part of FCGR2C, FCGR3B and FCGR2B. Analysis of CNV status in 4578 individuals with RA and 5457 controls indicated association of duplications in the FCGR3B gene in antibody-negative RA (P=0.002, OR=1.43). Deletion in FCGR3B was associated with increased risk of antibody-positive RA, consistently with previous reports (P=0.023, OR=1.23). A clear genotype-phenotype relationship was observed: CNV polymorphisms of the FCGR3A gene correlated to CD16A expression (encoded by FCGR3A) on CD8 T-cells. In conclusion, our method allows determining the CNV status of the FCGR locus, we identified association of CNV in FCGR3B to RA and showed a functional relationship between CNV in the FCGR3A gene and CD16A expression.

RhD Immunization Despite Adequate Immunoprophylaxis: Role of Fc Gamma Receptor Gene Polymorphisms

▪Introduction: One of the most effective immunological interventions in clinical medicine is the prevention of hemolytic disease of the newborn by prophylactic Rh immune globulin (Rh-Ig) therapy. The administration of ante- and postnatal Rh-Ig has reduced the risk of RhD immunization in the Netherlands from 17% to a mere 0.31%, yet its mechanism of action is still unknown. To gain more insight into the possible working mechanism of the Rh-Ig prophylaxis we analyzed potential risk factors and genotyped all known IgG-Fc receptor (protein FcγR, gene FCGR) variants known to date, on a cohort of Dutch women who failed Rh-Ig prophylaxis and developed anti-D antibodies. Adequate Rh-Ig immunoprophylaxis was defined as an antenatal and postnatal prophylaxis of 1,000 IU (200 µg) in both current and previous pregnancies, according to the Dutch guidelines.Material and Methods: Between 1999 and 2013 we identified 274 women who produced anti-D antibodies. Through a structured questionnaire we collected information about Rh-Ig prophylaxis and additional clinical data for potential risk factors. In 122 cases, adequate Rh-Ig prophylaxis was given, and clinical risk factors for fetal maternal hemorrhage (FMH) could be collected. Their clinical circumstances were compared to a control group of 339 randomly selected pregnant women. The Rh-Ig therapy failure of 57 of those women could not be explained through our risk factor analysis. From these 57 cases, DNA was obtained, and used for the FcγR-specific multiplex ligation-dependent probe amplification (MLPA) assay, identifying both single nucleotide polymorphisms and copy number variations in the FCGR locus. The results were compared to a control group of 200 healthy donors.Results: A history of red blood cell transfusion (p=0.05) and caesarean section (p<0.0001) were identified to be independent risk factors for RhD immunization. All other described risk factors for FMH such as miscarriage, termination of pregnancy, or invasive diagnostic procedures, requiring an additional Rh-Ig dose according to the guidelines, were not found to increase the risk of immunoprophylaxis failure. RhD-immunization due to caesarian section or red blood cell transfusion accounted for 53% of our cohort, suggesting an alternative explanation for the production of Rh-Ig alloantibodies in the remaining 47% of the cases - despite adequate amount of prophylaxis given in current and previous pregnancies. We therefore postulate the existence of a genetic variation that puts women at increased risk for RhD immunization during pregnancy. To test this hypothesis we analyzed the genetic variation in the FCGR locus and found a significantly (p=0.02) increased prevalence of the FCGR2 -ORF, expressing a functional copy of the activating FcγRIIc, which is otherwise a pseudogene. Strikingly, the prevalence of the 2B.4-promotor haplotype of the FCGR2B gene, associated with a 1.5 fold increase of the inhibitory FcγRIIb, was strongly (p=0.0001) increased.Conclusion: Caesarian section and red blood cell transfusion are risk factors that increase RhD immunization during pregnancies, accounting for about half failed Rh-Ig prophylactic cases. Genetic variation in the FCGR-gene might be a possible explanation for increased immunization risk. In our cohort we encountered a significantly increased frequency of individuals expressing FcγRIIc, along with a polymorphism encoding for a higher expression of the inhibitory receptor FcγRIIb, suggesting these genes to influence immune responses to RBC in a manner previously unrecognized. DisclosuresNo relevant conflicts of interest to declare.

Genomic Dissection of the Fcγ Receptor Region in the Context of Monoclonal Antibody Therapy

Development of the anti-CD20 antibody, rituximab, heralded the start of monoclonal antibody (mAb) therapy as an effective means of treating cancer. Despite its undoubted impact, clinical responses remain variable and cures are rarely achieved. Evidence from pre-clinical models and human trials indicates that mAbs primarily act through engaging low-affinity Fc gamma receptor (FcγR)-expressing effector immune cells. The low-affinity FcγR genes, FCGR2A, FCGR2B, FCGR2C, FCGR3A and FCGR3B, are located within a highly homologous 200 kb region at 1q23, which is the result of an ancestral segmental duplication event (Figure 1). The locus also contains numerous single nucleotide polymorphisms (SNPs), many of which can affect receptor affinity and/or function and are associated with differential responses following mAb immunotherapy. Moreover, the region contains extensive copy number variation (CNV) that also can affect the expression and function of these receptors, but its impact on mAb immunotherapy remains unknown.To investigate the full impact of SNPs and CNV in the FcγR locus, we have optimised a number of sensitive and specific assays which are amenable to formalin fixed paraffin embedded (FFPE) material in order to apply them to clinical trial samples in a high-throughput manner. Initially we assessed the accuracy of established TaqMan and novel allele-specific (KASP) genotyping assays for FCGR2A-131H/R (rs1801274), FCGR3A-158F/V (rs396991) and FCGR2B-232I/T (rs1050501) SNPs by analysing 2085 DNA samples derived from peripheral blood lymphocytes (PBL) from a large, multi-centre cohort. Our data showed that although clear discrimination was possible at the FCGR2A-131H/R SNP, we needed additional selective Sanger sequencing to discriminate the FF/FV and IT/TT genotypes for the FCGR3A-158F/V and FCGR2B-232I/T SNPs, respectively. This difficulty in genotype discrimination in the cases of FCGR3A and FCGR2B is likely due to sequence homology with other genes and CNV in the gene regions which complicate assay design and interpretation of certain genotypes. Secondly, we applied a combined KASP genotyping and Sanger sequencing approach to matched PBL DNA and FFPE-extracted DNA from follicular lymphoma (FL) patients [n=14] and showed that while FFPE material was more likely to fail genotyping, successfully genotyped cases were concordant with the matched genomic DNA samples. FFPE samples which failed to amplify PCR products of at least 100 bp using the BIOMED-2 multiplex PCR protocol were more likely to fail genotyping assays.Finally, we assessed the ability of a multiplex ligation-dependent probe amplification (MLPA) assay to concurrently determine SNP genotype and CNV in the low-affinity FCGR locus in a cohort of 155 normal donors and DNA from seven matched PBL-/FFPE-derived FL cases. We employed a paralog ratio test (PRT) assay for FCGR3A and FCGR3B CNV confirmation. In our normal donors, MLPA and PRT results were concordant. 16% of normal donors harboured a deletion [n=15] or duplication [n=10] affecting the FCGR2C locus (A summary of regions of CNV is shown in Figure 1). CNV of FCGR3B was associated with variation at FCGR2C and no CNV was observed in FCGR2A and FCGR2B. CNV affecting FCGR3A was observed in 5% of donors with deletions and duplications in 4 and 5 donors, respectively. In the FFPE-derived DNA samples, we observed elevated variability in data quality that was most noticeable in probes targeting HSPA6, FCGR2C exon 4 and HSPA7. Poor quality data correlated with samples that failed to amplify at least the 100 bp PCR product using the BIOMED-2 multiplex PCR protocol. As such, the preclusion of HSPA6, FCGR2C exon 4 and HSPA7 probes and FFPE samples that failed to amplify any BIOMED-2 PCR product from the analysis permitted the production of high-quality MLPA data. Finally, we designed, and are currently optimising, a targeted re-sequencing platform (Haloplex, Agilent) to interrogate informative regions of the FcγR region, which includes those with unique sequence identify for CNV analysis, and those that include known SNPs.In conclusion, we have evaluated a suite of assays for the genomic analysis of the FcγR locus that are scalable for application in large clinical trials of antibody therapy. This work will ultimately provide a detailed architecture of the region and establish the importance of FcγR genetics in predicting response to antibody therapeutics. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

CNVrd, a Read-Depth Algorithm for Assigning Copy-Number at the FCGR Locus: Population-Specific Tagging of Copy Number Variation at FCGR3B

The extent of contribution from common gene copy number (CN) variants in human disease is currently unresolved. Part of the reason for this is the technical difficulty in directly measuring CN variation (CNV) using molecular methods, and the lack of single nucleotide polymorphisms (SNPs) that can tag complex CNV that has arisen multiple times on different SNP haplotypes. One CNV locus implicated in human disease is FCGR. Here we aimed to use next-generation sequencing (NGS) data from the 1000 Genomes Project to assign CN at FCGR3A and FCGR3B and to comprehensively assess the ability of SNPs to tag specific CN variants. A read-depth algorithm was developed (CNVrd) and validated on a subset of HapMap samples using CN assignments that had previously been determined using molecular and microarray methods. At 7 out of 9 other complex loci there was >90% concordance with microarray data. However, given that some prior knowledge of CN is required, the generalizability of CNVrd is limited and should be applied to other complex CNV loci with caution. Subsequently, CN was assigned et FCGR3B using CNVrd in a total of 952 samples from the 1000 Genomes Project, using three classes and SNPs that correlated with duplication were identified. The best tag SNP was observed in the Mexican-American sample set for duplication at FCGR3B. This SNP (rs117435514, r2 = 0.79) also tagged similar duplication in Chinese and Japanese (r2 = 0.35–0.60), but not in Caucasian or African. No tag SNP for duplication at FCGR3A or deletion at FCGR3B was identified in any population. We conclude that it is possible to tag CNV at the FCGR locus, but CN and SNPs have to be characterized and correlated on a population-specific basis.

Lack of support for association between the copy number variants in the FCGR locus and schizophrenia: A case control study

The missing heritability of polygenic schizophrenia after genome-wide association studies (GWAS) can be potentially accounted for by the fact that most dynamic multiallelic copy number variants (CNVs) overlap segmental duplications (SDs). The FCGR locus covers this category of complex CNVs and it has long been postulated to harbor variants conferring the risk of schizophrenia. However, such association remains unproven. We used a case-control design to investigate CNV-based association with the disease. Data were obtained from 598 unrelated schizophrenia patients and 959 normal controls of Han ancestry from Shanghai. A total of four copy number (CN) probes in the FCGR locus were detected using TaqMan® Copy Number Assay. SPSS version 16.0 was used for the statistical analyses. And the frequency distributions of target CN in FCGR locus were very similar between controls and cases, whereas the CNV frequency differed markedly among different target CN analyzed in the two cohorts. When compared with the predominant two copies per diploid genome, a distinct non-protein-coding CN deletion region containing regulatory sequences was detected by probe Hs04194069_cn. Taken together, we found no evidence of association of target CNVs in the FCGR locus with schizophrenia. However, our negative findings suggest that more detailed next generation sequencing-based association studies are needed to fully evaluate the contribution of this category of complex CNVs to the disease.

Confirmation of association ofFCGR3Bbut notFCGR3Acopy number with susceptibility to autoantibody positive rheumatoid arthritis

The FCGR locus encoding the low-affinity Fcγ receptors (FcγR) for immunoglobulin G has largely been missed by genome-wide association studies due to complications with structural variation and segmental duplication. Recently identified copy number variants (CNVs) affecting FCGR3A and FCGR3B have been linked to a number of autoimmune disorders. We have developed and validated a novel quantitative sequence variant assay in combination with an adapted paralogue ratio test to examine independent CNVs carrying FCGR3A and FCGR3B in rheumatoid arthritis (RA) compared with healthy volunteers (n = 1,115 and 654, respectively). Implementation of a robust statistical analysis framework (CNVtools) allowed for systematic batch effects and for the inherent uncertainty of copy number assignment, thus avoiding two major sources of false positive results. Evidence for association with neither duplications nor deletions of FCGR3A was found; however, in line with previous studies, there was evidence of overrepresentation of FCGR3B deletions in RA (odds ratio [OR] 1.50, P = 0.028), which was more apparent in rheumatoid factor positive disease (OR 1.61, P = 0.011). The level of FcγRIIIb, encoded by FCGR3B, expression on neutrophils was shown to correlate with gene copy number. Thus, our results may highlight an important role for neutrophils in the pathogenesis of RA, potentially through reduced FcγRIIIb-mediated immune complex clearance.

Copy number, linkage disequilibrium and disease association in the FCGR locus

The response of a leukocyte to immune complexes (ICs) is modulated by receptors for the Fc region of IgG (FcγRs), and alterations in their affinity or function have been associated with risk of autoimmune diseases, including systemic lupus erythematosus (SLE). The low-affinity FcγR genomic locus is complex, containing regions of copy number variation (CNV) which can alter receptor expression and leukocyte responses to IgG. Combined paralogue ratio tests (PRTs) were used to distinguish three intervals within the FCGR locus which undergo CNV, and to determine FCGR gene copy number (CN). There were significant differences in FCGR3B and FCGR3A CNV profiles between Caucasian, East Asian and Kenyan populations. A previously noted association of low FCGR3B CN with SLE in Caucasians was supported [OR = 1.57 (1.08–2.27), P = 0.018], and replicated in Chinese [OR = 1.65 (1.25–2.18), P = 4 × 10−4]. There was no association of FCGR3B CNV with vasculitis, nor with malarial or bacterial infection. Linkage disequilibrium (LD) between multi-allelic FCGR3B CNV and SLE-associated SNPs in the FCGR locus was defined for the first time. Despite LD between FCGR3B CNV and a variant in FcγRIIB (I232T) which abolishes inhibitory function, both reduced CN of FCGR3B and homozygosity of the FcγRIIB-232T allele were individually strongly associated with SLE risk. Thus CN of FCGR3B, which controls IC responses and uptake by neutrophils, and variations in FCGR2B, which controls factors such as antibody production and macrophage activation, are important in SLE pathogenesis. Further interpretations of contributions to pathogenesis by FcγRs must be made in the context of LD involving CNV regions.

Copy number variation at theFCGRlocus includesFCGR3A, FCGR2CandFCGR3Bbut notFCGR2AandFCGR2B

Human Fcgamma receptors (FcgammaRs) are glycoproteins that bind the Fc region of IgG. The genes encoding the low-affinity FcgammaRs are located on chromosome 1q23-24. Beside single nucleotide polymorphisms (SNPs), gene copy number variation (CNV) is now being recognized as an important indicator for inter-individual differences. Recent studies on identifying CNV in the human genome suggest large areas at chromosome 1q23-24 to be involved, and CNV in this region has been associated with manifestations of systemic autoimmune disease. To study both SNPs and CNV of the low-affinity FcgammaRs in one assay, we have developed a Multiplex Ligation-dependent Probe Amplification (MLPA) assay. A novel CNV for FCGR3A was observed. Similar to FCGR3B and FCGR2C, a gene-dosage effect of FCGR3A was found, that seemed to correlate nicely with the FcgammaRIIIa expression on NK cells. Next, we delineated the approximate boundaries of CNV at the FCGR locus. Variation in co-segregation of neighboring FCGR genes was limited to four variants, with patterns of Mendelian inheritance. No CNV of the FCGR2A and FCGR2B genes was observed in over 600 individuals. In conclusion, we report a novel CNV of the FCGR3A gene that correlates with FcgammaRIIIa expression and function on NK cells. Only FCGR3A, FCGR2C and FCGR3B show CNV, in contrast to FCGR2A and FCGR2B.

Association of FCGR2A and FCGR2A-FCGR3A haplotypes with susceptibility to giant cell arteritis

The Fc gamma receptors have been shown to play important roles in the initiation and regulation of many immunological and inflammatory processes and to amplify and refine the immune response to an infection. We have investigated the hypothesis that polymorphism within the FCGR genetic locus is associated with giant cell arteritis (GCA). Biallelic polymorphisms in FCGR2A, FCGR3A, FCGR3B and FCGR2B were examined for association with biopsy-proven GCA (n = 85) and healthy ethnically matched controls (n = 132) in a well-characterised cohort from Lugo, Spain. Haplotype frequencies and linkage disequilibrium (D') were estimated across the FCGR locus and a model-free analysis performed to determine association with GCA. There was a significant association between FCGR2A-131RR homozygosity (odds ratio (OR) 2.10, 95% confidence interval (CI) 1.12 to 3.77, P = 0.02, compared with all others) and carriage of FCGR3A-158F (OR 3.09, 95% CI 1.10 to 8.64, P = 0.03, compared with non-carriers) with susceptibility to GCA. FCGR haplotypes were examined to refine the extent of the association. The haplotype showing the strongest association with GCA susceptibility was the FCGR2A-FCGR3A 131R-158F haplotype (OR 2.84, P = 0.01 for homozygotes compared with all others). There was evidence of a multiplicative joint effect between homozygosity for FCGR2A-131R and HLA-DRB1*04 positivity, consistent with both of these two genetic factors contributing to the risk of disease. The risk of GCA in HLA-DRB1*04 positive individuals homozygous for the FCGR2A-131R allele is increased almost six-fold compared with those with other FCGR2A genotypes who are HLA-DRB1*04 negative. We have demonstrated that FCGR2A may contribute to the 'susceptibility' of GCA in this Spanish population. The increased association observed with a FCGR2A-FCGR3A haplotype suggests the presence of additional genetic polymorphisms in linkage disequilibrium with this haplotype that may contribute to disease susceptibility. These findings may ultimately provide new insights into disease pathogenesis.

Analysis of Fcγ receptor haplotypes in rheumatoid arthritis: FCGR3A remains a major susceptibility gene at this locus, with an additional contribution from FCGR3B

The Fcγ receptors play important roles in the initiation and regulation of many immunological and inflammatory processes, and genetic variants (FCGR) have been associated with numerous autoimmune and infectious diseases. The data in rheumatoid arthritis (RA) are conflicting and we previously demonstrated an association between FCGR3A and RA. In view of the close molecular proximity with FCGR2A, FCGR2B and FCGR3B, additional polymorphisms within these genes and FCGR haplotypes were examined to refine the extent of association with RA. Biallelic polymorphisms in FCGR2A, FCGR2B and FCGR3B were examined for association with RA in two well characterized UK Caucasian and North Indian/Pakistani cohorts, in which FCGR3A genotyping had previously been undertaken. Haplotype frequencies and linkage disequilibrium were estimated across the FCGR locus and a model-free analysis was performed to determine association with RA. This was followed by regression analysis, allowing for phase uncertainty, to identify the particular haplotype(s) that influences disease risk. Our results reveal that FCGR2A, FCGR2B and FCGR3B were not associated with RA. The haplotype with the strongest association with RA susceptibility was the FCGR3A–FCGR3B 158V-NA2 haplotype (odds ratio 3.18, 95% confidence interval 1.13–8.92 [P = 0.03] for homozygotes compared with all genotypes). The association was stronger in the presence of nodules (odds ratio 5.03, 95% confidence interval 1.44–17.56; P = 0.01). This haplotype was also more common in North Indian/Pakistani RA patients than in control individuals, but not significantly so. Logistic regression analyses suggested that FCGR3A remained the most significant gene at this locus. The increased association with an FCGR3A–FCGR3B haplotype suggests that other polymorphic variants within FCGR3A or FCGR3B, or in linkage disequilibrium with this haplotype, may additionally contribute to disease pathogenesis.