FCGR3B Research Articles

Fc gamma receptor IIIb polymorphism and systemic lupus erythematosus: association with disease susceptibility and identification of a novel FCGR3B*01 variant.

The objective of this study was to evaluate the association between Fc gamma receptor IIIb polymorphism and susceptibility to systemic lupus erythematosus and clinical traits of the disease. Genomic DNA was obtained from 303 consecutive systemic lupus erythematosus patients and 300 healthy blood donors from the southeastern region of Brazil. The polymorphic region of the FCGR3B gene was sequenced and the alleles FCGR3B*01, FCGR3B*02 and FCGR3B*03 were analyzed. The FCGR3B*01 allele was more frequent in systemic lupus erythematosus patients (43.1%) while the FCGR3B*02 allele prevailed among controls (63.7%) (P = 0.001). The FCGR3B*03 allele was found equally in both groups. The FCGR3B*01/*01 (20.7%) and FCGR3B*01/*02 (41.1%) genotypes were more frequent among systemic lupus erythematosus patients (P = 0.028 and P = 0.012, respectively) while the FCGR3B*02/*02 genotype was more frequent in controls (45.5%) (P < 0.001). One variant of the FCGR3B*01 allele previously described in Germany was found in only one control. A new variant of the FCGR3B*01 allele with two substitutions (A227G/G277A) was found in one control. Three variants of the FCGR3B*02 allele previously described in African-Americans, Brazilians, Chinese and Japanese were found in ten 10 patients and two controls. In addition, several single nucleotide polymorphisms at non-polymorphic positions were identified in both patients and controls. Susceptibility to systemic lupus erythematosus was associated with the FCGR3B*01 allele, as well as with the FCGR3B*01/*01 and FCGR3B*01/*02 genotypes. No association was found between FCGR3B genotypes and clinical manifestations, disease severity or the presence of autoantibodies.

FCGR3A and FCGR3B copy number variations are risk factors for sarcoidosis.

Sarcoidosis is a multisystem granulomatous disorder that causes significant morbidity. Genetic factors contribute to sarcoidosis risks. In this study, we investigated whether copy number variations (CNVs) of FCGR3A (coding for FcγRIIIA) and FCGR3B (coding for FcγRIIIB) genes are associated with sarcoidosis susceptibility and whether the expressions of FcγRIIIA on NK cells and FcγRIIIB on neutrophils are altered in sarcoidosis patients. TaqMan real-time PCR assays were used to analyze the CNV of FCGR3A and FCGR3B genes. FCGR3A and FCGR3B CNV genotypes were compared between 671 biopsy-proven sarcoidosis patients and the same number of healthy controls matched with age, sex, race, and geographic area from the ACCESS (A Case Control Etiologic Study of Sarcoidosis) cohort. Flow cytometry analyses were used to determine expressions of FcγRIIIA on NK cells and FcγRIIIB on neutrophils in phenotype analyses. We found that FCGR3A CNVs were significantly associated with sarcoidosis in females (CN=1 vs. CN=2 logistic regression adjusted for sex and race, OR 4.0156, SE=2.2784, P=0.0143; CN=3 vs. CN=2 logistic regression adjusted for sex and race, OR 2.8044, SE=1.1065, P=0.0090), suggesting that FCGR3A gene abnormality influences sarcoidosis development in a gender-specific manner. Furthermore, FcγRIIIA expressions were significantly decreased on NK cells from sarcoidosis patients compared to those from healthy controls (P=0.0007). Additionally, low FCGR3B CN was associated with sarcoidosis (CN <2 vs. CN=2 logistic regression adjusted for sex and race, OR 1.5025, SE=0.2682, P=0.0226), indicating that the functions of FCGR3B gene may also contribute to the pathogenesis of sarcoidosis. We conclude that FCGR3A CNVs are a major risk factor for female sarcoidosis and FCGR3B CNVs may also affect the development of sarcoidosis.

Response to Intravenous Cyclophosphamide Treatment for Lupus Nephritis Associated with Polymorphisms in the FCGR2B-FCRLA Locus.

Cyclophosphamide (CYC) is an immunosuppressant drug widely used to treat various diseases including lupus nephritis, but its efficacy highly varies from individual to individual. This pharmacogenomics association study searched for genetic variations associated with CYC efficacy. Genome-wide association scan was performed for 109 Korean patients with systemic lupus erythematosus with lupus nephritis (classes III-V) who received intravenous CYC induction therapy. Genetic differences between responders and nonresponders were examined using Cochran-Armitage trend tests, and genotype imputation was used for defining the association locus. Genetic polymorphisms in the Fcγ receptor gene (FCGR) cluster at human chromosome 1q23, previously associated with lupus nephritis susceptibility, were associated with the response to CYC treatment for lupus nephritis. Significant response association was found for 3 perfectly correlated (r(2) = 1) single-nucleotide polymorphisms (SNP): rs6697139, rs10917686, and rs10917688, located between the FCGR2B and FCRLA genes (p = 3.4 × 10(-8)). Carriage of the minor alleles in these SNP was found only in nonresponders (31%) and none in responders (0%). This first genome-wide association approach for CYC response yielded a robust profile of genetic associations including large-effect SNP in the FCGR2B-FCRLA locus, which may provide better insights to CYC metabolism and efficacy.

Genetic Variants That Are Associated with Neuropsychiatric Systemic Lupus Erythematosus.

While genetic risks have been implicated in systemic lupus erythematosus (SLE), the involvement of various genotypes in neuropsychiatric SLE (NPSLE) remains uncertain. The present metaanalysis aimed to combine data from different studies and evaluate the association between each genotype and the risk of developing NPSLE. Studies were searched and retrieved from online databases (PubMed, EMBASE, BIOSIS, and ScienceDirect). Case-control studies were chosen if they reported genotype frequencies of the γ Fc region (FCγR) receptors II-A, III-A, and III-B; tumor necrosis factor-α (TNF-α); mannan-binding lectin (MBL); integrin alpha M (ITGAM); interleukin (IL) 1, IL-1β, and IL-6; IL-10 promoter; and vitamin D genes. The OR were used to assess the strength of this association between patients with NPSLE and SLE. A total of 33 studies were considered in this metaanalysis. The results suggest that these genotypes demonstrated a significant association with NPSLE: the homozygous FCγR IIIa 158 FF genotype (OR 1.89, p = 0.03 for FF vs VV + FV), heterozygous FCγR IIIb NA1/2 genotype (OR 2.14, p = 0.03 for NA1/2 vs NA1/1; OR 1.81, p = 0.04 for NA1/2 vs NA1/1 + NA2/2), and homozygous ITGAM rs1143679 HH genotype (OR 3.39, p = 0.04 for HH vs RH; OR 3.11, p = 0.048 for HH vs RR + RH). Polymorphisms of the TNF-α, MBL2, IL-1, IL-1β, IL-6, IL-10 promoter, and vitamin D receptor genes did not show a statistically significant association with the risk of developing NPSLE (p > 0.05). This metaanalysis indicates that polymorphisms in the pathways of immune complex clearance, such as the FcγRIIIa, FcγRIIIb, and ITGAM genotypes, are potential susceptibility genes for NPSLE.

Association study of copy number variants in FCGR3A and FCGR3B gene with risk of ankylosing spondylitis in a Chinese population.

Ankylosing spondylitis (AS) is a common inherited autoimmune disease. Copy number variation (CNV) of DNA segments has been found to be an important part of genetic variation, and the FCGR3A and FCGR3B gene CNVs have been associated with various autoimmune disorders. The aim of the study was to determine whether CNVs of FCGR3A and FCGR3B were also associated with the susceptibility of AS. A total of 801 individuals including 402 AS patients and 399 healthy controls were enrolled in this study. The copy numbers of FCGR3 gene (two fragments, included FCGR3A and FCGR3B) were measured by AccuCopy™ methods. Chi-square test and logistic regression model were used to evaluate association between FCGR3 gene CNVs and AS susceptibility. P values, odds ratio, and 95% confidence intervals (CIs) were used to estimate the effects of risk. Significantly, difference in the frequencies of FCGR3A and FCGR3B gene CNVs was founded between the patients with AS and controls. For the FCGR3A gene, a low (≤3) copy number was significantly associated with AS [for ≤3 copies versus 4 copies, (OR 2.17, 95% CI (1.41, 3.34), P < 0.001, adjusted OR 2.22, 95% CI (1.44, 3.43), P < 0.001)]. A low FCGR3B copy number was also significantly associated with increasing risk of AS [for ≤3 copies versus 4 copies, (OR 1.87, 95% CI (1.25, 2.79), P = 0.002, adjusted OR 1.94, 95% CI (1.29, 2.91), P = 0.001)]; however, both the high FCGR3A and FCGR3B copy numbers (≥5) were not significantly associated with the risk of AS (≥5 copies versus 4 copies). The lower copy numbers (≤3) of FCGR3A and FCGR3B genes confer a risk factor for AS susceptibility.

Accurate interrogation of FCGR3A rs396991 in European and Asian populations using a widely available TaqMan genotyping method.

A polymorphism in the receptor for the Fc region of IgG, Fc γ-receptor IIIa (FcγRIIIa, FCGR3A rs396991), has been inconsistently shown in the literature to have an effect on response to monoclonal antibody therapy in several indications. The rs396991 (T/G) polymorphism leads to an F176V substitution and increased affinity for IgG. This variant has proven difficult to genotype accurately, primarily because of extensive homology between the FCGR3A and FCGR3B genes. We have shown that rs396991 can be genotyped by PCR amplification, followed by direct Sanger sequencing of the product, without coamplification of FCGR3B, and that the rs396991 TaqMan assay (C__25815666_10) agrees with Sanger sequencing results in 100% of European and Asian samples tested, but it has a small error rate in African and American populations. C__25815666_10 is therefore suitable to interrogate rs396991 in studies involving Europeans and Asians; however for other populations, the default genotyping method should be PCR followed by Sanger sequencing.

Nonallelic homologous recombination of the FCGR2/3 locus results in copy number variation and novel chimeric FCGR2 genes with aberrant functional expression.

The human FCGR2/3 locus, containing five highly homologous genes encoding the major IgG receptors, shows extensive copy number variation (CNV) associated with susceptibility to autoimmune diseases. Having genotyped >4000 individuals, we show that all CNV at this locus can be explained by nonallelic homologous recombination (NAHR) of the two paralogous repeats that constitute the majority of the locus, and describe four distinct CNV regions (CNRs) with a highly variable prevalence in the population. Apart from CNV, NAHR events also created several hitherto unidentified chimeric FCGR2 genes. These include an FCGR2A/2C chimeric gene that causes a decreased expression of FcγRIIa on phagocytes, resulting in a decreased production of reactive oxygen species in response to immune complexes, compared with wild-type FCGR2A. Conversely, FCGR2C/2A chimeric genes were identified to lead to an increased expression of FCGR2C. Finally, a rare FCGR2B null-variant allele was found, in which a polymorphic stop codon of FCGR2C is introduced into one FCGR2B gene, resulting in a 50% reduction in protein expression. Our study on CNRs and the chimeric genes is essential for the correct interpretation of association studies on FCGR genes as a determinant for disease susceptibility, and may explain some as yet unidentified extreme phenotypes of immune-mediated disease.

Abstract CT129: Association of the FCGR2A and FCGR3A genotypes with trastuzumab benefit in NSABP B-31

Abstract Background: The FCGR3A-158V and FCGR2A-131H alleles are referred to as favorable alleles with regards to trastuzumab response because the FCGR3A-V/V and FCGR2A-H/H genotypes have been associated with higher trastuzumab induced antibody-dependent cell-mediated cytotoxicity (ADCC) response in vitro. The objective of this study was to assess the interaction between these genotypes and the degree of benefit from trastuzumab in a phase III trial which demonstrated the benefit of adding trastuzumab to the standard adjuvant chemotherapy in the treatment of HER2-positive breast cancer (NSABP B-31). Methods: Genotypes for FCGR3A-158V/F and FCGR2A-131H/R alleles were determined in a representative cohort of NSABP clinical trial B-31 which included all available pre-treatment blood samples (N = 1251). Genotypes were determined by Typeplex chemistry and mass spectrometry on the Seqeunom platform. A custom nested PCR design was required to enable specific amplification of the FCGR3A gene due to the extensive homology between the FCGR3A and FCGR3B genes. Cox regression analyses were used to test for genotype-treatment interactions. Endpoint was disease free survival (DFS). Results: The genotyped cohort resembled the entire B-31 cohort based on clinical variables and the degree of benefit from trastuzumab. Results are summarized in the table. While there were trends for interaction between polymorphisms and trastuzumab for both genes, only FCGR3A-158 polymorphism reached statistical significance for interaction (p = 0.005). Conclusion: In NSABP B-31 there was a trend for benefit in the favorable genotypes of the FCGR2A-131 SNP, but only the favorable FCGR3A-158 genotypes showed a significant association with trastuzumab benefit with genotype-treatment interaction. However, polymorphism alone could not identify a subgroup with no benefit from trastuzumab, since even the patients with less favorable genotypes received significant benefit. Support: NCI U10-CA-12027, -69651, -37377, -69974. PA DOH disclaims responsibility for analysis, interpretations, or conclusions. Citation Format: Patrick G. Gavin, Nan Song, Nicole L. Johnson, Corey Lipchik, Seong-Rim Kim, Melanie Finnigan, Hanna Bandos, Jong-Hyeon Jeong, Joseph P. Costantino, Priya Rastogi, Edward H. Romond, Louis Fehrenbacher, Eleftherios P. Mamounas, Sandra M. Swain, D. Lawrence Wickerham, Charles E. Geyer, Jr., Norman Wolmark, Soonmyung Paik, Katherine L. Pogue-Geile. Association of the FCGR2A and FCGR3A genotypes with trastuzumab benefit in NSABP B-31. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr CT129. doi:10.1158/1538-7445.AM2015-CT129

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.

FCGR3B gene frequencies among ethnic Thai blood donors from a regional hospital in Eastern Thailand.

The FCGR3B gene encodes three human neutrophil antigens which consist of HNA-1a, HNA-1b, and HNA-1c. These antigens are encoded by three alleles in the FCGR3B locus: FCGR3B*01, FCGR3B*02, and FCGR3B*03 alleles, respectively. The frequencies of FCGR3B alleles have been reported in different ethnic populations. This study compared the FCGR3B gene frequencies among 230 unrelated healthy Eastern Thai blood donors in Rayong hospital with the previously published studies. The polymerase chain reaction-sequence-specific primers method was performed to determine FCGR3B genotypes. The results showed that the allele frequencies of FCGR3B*01, FCGR3B*02, and FCGR3B*03 were 0.722, 0.274, and 0.009, respectively. The FCGR3B*01 and FCGR3B*02 frequencies found in the Eastern Thais were similar to the previous reports investigating in Northern Thais, Chinese Han, Taiwanese, and Japanese populations. Interestingly, our data showed statistically significant difference (P < 0.05) to Central Thais, Korean, Indian, Turkish, Australian, Tunisian, American, German, and Italian populations. In addition, one FCGR3Bnull , which represents a gene deletion, was also found in this study. This information is important not only for the assessment of neutrophil antibody-mediated clinical conditions and for disease association studies but also for anthropological studies.

Polymorphism of FCGR2A, FCGR2C, and FCGR3B Genes in the Pathogenesis of Sarcoidosis.

We have previously presented evidence that the polymorphism of the FCGR3A gene, encoding the receptor for Fc fragment of immunoglobulin G IIIa (FcγRIIIa) plays a role in the enhancement of circulating immune complexes (CIs) with the occurrence of Mycobacterium tuberculosis heat shock proteins in patients with sarcoidosis (SA). The immunocomplexemia might be caused by decreased affinity of CIs to Fcγ receptors, with the subsequently decreased receptor clearance by immune cells. In the present study we examined whether the polymorphisms of other related genes (FCGR2A, FCGR2C, FCGR3B) encoding other activatory Fcγ receptors, could have a similar effect. To this end, we genotyped 124 patients with sarcoidosis and 148 healthy volunteers using polymerase chain reaction with sequence-specific primers. We revealed a significant decrease in the percentage of the FCGR2A and FCGR2C variants that ensure effective CIs clearance, with a concomitant increase of less functional variants of these genes in Stages I/II, compared with Stages III/IV of SA. There was no aberration in FCGR3B allele/genotype frequencies. We conclude that the FCGR2A and FCGR2C polymorphisms may also contribute to immunocomplexemia present in SA. The assessment of FCGR genes could become a tool in presaging a clinical course of sarcoidosis and in its personalized therapy.

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.

De-functioning polymorphism in the inhibitory receptor FcγRIIB does not impact upon kidney allograft survival.

De-functioning polymorphism in the inhibitory receptor FcγRIIB does not impact upon kidney allograft survival.

Abstract 4698: Gene expression profiling distinguishes between endometrial stromal tumors subtypes

Abstract Introduction Endometrial stromal tumors (EST) are the second most prevalent uterine sarcomas. EST are divided into three histosubtypes: benign endometrial stromal nodules (ESN), low-grade endometrial stromal sarcomas (ESS) and the most malignant undifferentiated stromal sarcomas (UES). Moreover, due to clinicopathological features intermediate between classical low-grade ESS and UES, some tumors have been described in the literature as “high-grade ESS”. The histological distinction between these three subtypes has important prognostic implications. The molecular biology of these tumors remains poorly understood because of their low incidence. Patients and Methods In the present study, we performed gene expression profiling of 22 EST using microarray, RNA-Seq and qRT-PCR techniques. Our cohort included 10 classical low-grade ESS (8 with ESS-associated gene fusions), 8 UES and 4 tumors which were classified as high-grade ESS based on the presence of YWHAE-FAM22A/B fusion and histological features. Results Using gene expression microrarrays, we have identified 81 entities differentially expressed in analyzed samples (fold change &amp;gt; 2, p &amp;lt; 0.005). Hierarchical clustering analysis revealed that high-grade ESS formed a separate sub-cluster with a distinct molecular profile as compared with the low-grade ESS and UES cases. Low-grade ESS cases formed 2 distinct sub-clusters, each containing 5 cases, one of which presented gene expression profile partially corresponding to high-grade ESS cases. GO analysis of genes strongly up-regulated in UES specimens showed over-representation of immune response (GO:0006955) and immune system process (GO:0002376) pathways (MARCO, MYO1F, FCN1, GPR183, HLA-DPB1, FCGR2B, NCF1, CCL18, CD28, PTAFR, CCL13, STK4, CD14, NCF4 and CTLA4 genes) (p &amp;lt; 0.05). UES cases were also characterized by significant down-regulation of WT1 tumor suppressor gene as compared to the low- and high-grade ESS. Moreover, both high-grade ESS and UES cases were characterized by significantly lower expression level of PEG3 gene, which has been reported as a tumor suppressor gene. All low-grade ESS cases presented significant over-expression of estrogen receptor ESR1 gene. Microarray results were validated by RNA-Seq and qRT-PCR experiments. Conclusions This study provides gene expression profiles that distinguish between EST subtypes and sheds light on the biology of these tumors. Our results point to immune system involvement in the pathogenesis of UES and support the notion that the current EST classification should discriminate high-grade ESS as a distinct subtype. Citation Format: Joanna Przybyl, Magdalena Kowalewska, Anna Quattrone, Barbara Dewaele, Vanessa Vanspauwen, Julio Finalet-Ferreiro, Michal Swierniak, Elwira Bakula-Zalewska, Janusz A. Siedlecki, Mariusz Bidzinski, Jan Cools, Maria Debiec-Rychter. Gene expression profiling distinguishes between endometrial stromal tumors subtypes. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4698. doi:10.1158/1538-7445.AM2014-4698

HLA-DQA1 and PLA2R1 Polymorphisms and Risk of Idiopathic Membranous Nephropathy

Single nucleotide polymorphisms (SNPs) within HLA complex class II HLA-DQ α-chain 1 (HLA-DQA1) and M-type phospholipase A2 receptor (PLA2R1) genes were identified as strong risk factors for idiopathic membranous nephropathy (IMN) development in a recent genome-wide association study. Copy number variants (CNVs) within the Fc gamma receptor III (FCGR3) locus have been associated with several autoimmune diseases, but their role in IMN has not been studied. This study aimed to validate the association of HLA-DQA1 and PLA2R1 risk alleles with IMN in a Spanish cohort, test the putative association of FCGR3A and FCGR3B CNVs with IMN, and assess the use of these genetic factors to predict the clinical outcome of the disease. A Spanish cohort of 89 IMN patients and 286 matched controls without nephropathy was recruited between October of 2009 and July of 2012. Case-control studies for SNPs within HLA-DQA1 (rs2187668) and PLA2R1 (rs4664308) genes and CNVs for FCGR3A and FCGR3B genes were performed. The contribution of these polymorphisms to predict clinical outcome and renal function decline was analyzed. This study validated the association of these HLA-DQA1 and PLA2R1 SNPs with IMN in a Spanish cohort and its increased risk when combining both risk genotypes. No significant association was found between FCGR3 CNVs and IMN. These results revealed that HLA-DQA1 and PLA2R1 genotype combination adjusted for baseline proteinuria strongly predicted response to immunosuppressive therapy. HLA-DQA1 genotype adjusted for proteinuria was also linked with renal function decline. This study confirms that HLA-DQA1 and PLA2R1 genotypes are risk factors for IMN, whereas no association was identified for FCGR3 CNVs. This study provides, for the first time, evidence of the contribution of these HLA-DQA1 and PLA2R1 polymorphisms in predicting IMN response to immunosuppressors and disease progression. Future studies are needed to validate and identify prognostic markers.

FCGR3B Gene Variants In Brazilians: New Alleles Should Be Considered?

Background FCGR3B gene encoding the receptor FcγRIIIb has three polymorphic forms (FCGR3B*01, FCGR3B*02 and FCGR3B*03) which represent the human neutrophil antigens HNA-1a, -1b and -1c, respectively. It has been postulated that variants of these alleles might represent new FCGR3B alleles, however there are few studies assessing the frequency of such variants in different populations. In this study we performed an analysis of the frequency of the FCGR3B alleles and its variant forms examining the polymorphic region of the FCGR3B gene in a large Brazilian population sample including healthy blood donors and patients with systemic lupus erythematosus (SLE). Study Design and Methods A segment containing the polymorphic nucleotide positions 141, 147, 227, 266 and 277 in exon 3 was sequenced from genomic DNA of 615 individuals including 310 blood donors and 305 patients with SLE. Additionally, nucleotide databases were screened for variant FCGR3B sequences. Results The allele frequencies for FCGR3B*01, FCGR3B*02 and FCGR3B*03 were 0.37, 0.59 and 0.04, respectively. In two patients with SLE (0.3%) there was no gene amplification characterizing the FCGR3B-null phenotype. Considering the 5 FCGR3B polymorphic sites, 4 variant forms were observed. One variant was linked to the FCGR3B*01 allele with SNPs occurring simultaneously in two positions: A227G and G277A (n=2; 0.3%). The other three variants, linked to the FCGR3B*02 allele were: A277G (n=8; 1.3%); C141G (n=3; 0.5%); and T147C (n=1; 0.2%). All mutations observed were missenses. The results are summarized in the Table. In 103/613 (16.8%) sequenced individuals we found 19 SNPs on 14 polymorphic sites distinct from those which characterize the FCGR3B alleles. Among the most frequent, T230A was observed in 36 cases (5.9%), T230G in 20 cases (3.3%), G249A in 6 cases (1%) and C337A in 6 cases (1%), all shown as heterozygous. The comparative analysis between the two groups of individuals revealed an association between the presence of the allele FCGR3B*01 and the HNA-1a/a and HNA-1a/b genotypes with increased susceptibility to SLE (p &lt;0.001; p = 0.028; and p = 0.012, respectively), however there was no significant correlation between these genotypes and disease severity. Discussion The observed frequencies of the FCGR3B*01, FCGR3B*02, and FCGR3B*03 alleles were similar to those reported by genotyping studies which included Europeans or North Americans individuals. The frequency of the null phenotype (0.3%) was similar to that reported by Europeans (0.2-0.8%). The variant FCGR3B*02A277G showed a frequency (1.3%) sufficiently high to be considered a new allele as proposed by the last Granulocyte Working Party/ISBT 2013. Interestingly, this variant was significantly more prevalent in patients with SLE than in blood donors (p=0.03). All the variants described in this study have been observed in different populations however we emphasize the presence of SNPs (T230A, T230G, G249A and C337A) in polymorphic sites different from those that characterize the FCGR3B gene alleles. We conclude that the FCGR3B gene is highly polymorphic and that the occurrence of new alleles must be considered, however the functional consequences of such changes has not yet been elucidated. Disclosures: No relevant conflicts of interest to declare.

A novel specific pyrosequencing method for genotyping FCGR3A rs396991 without coamplification of homologous gene FCGR3B

Monoclonal antibodies, such as rituximab, trastuzumab, and cetuximab, mediate immune response by binding to Fcγ receptors. The frequently occurring Phe158Val variant of the FCGR3A gene has increased binding affinity and consequently may affect immune response. Several pharmacogenetic association studies have genotyped this variant (FCGR3A rs396991), but with disconcordant results. In addition, in some of these studies genotype distribution was not in Hardy-Weinberg equilibrium, and samples were excluded from analysis because of genotype inconsistency. Genotyping problems of FCGR3A rs396991 are most likely due to sequence homology with the FCGR3B gene. For that reason, we developed a novel pyrosequencing method specifically for genotyping FCGR3A rs396991 and confirmed that the FCGR3B gene is not coamplified.

Impact of Allele Copy Number of Polymorphisms in FCGR3A and FCGR3B Genes on Susceptibility to Ulcerative Colitis

Polymorphisms in the Fcγ receptor genes have been implicated in several autoimmune diseases, including ulcerative colitis (UC). However, most of these reports had not taken into account the effect of copy number variation at this region. We investigated the combined effect of allele and gene copy number of FCGR3A-158F/V and FCGR3B-NA1/NA2 on susceptibility to UC. Study subjects were composed of a total of 752 Japanese patients with UC and 2062 Japanese control subjects. To estimate allele copy number of the 2 polymorphisms, we integrated the results of PCR-based real-time Invader assay (PCR-RETINA) that measures the allelic ratio and Taqman assay that detects the total copy number. We analyzed the associations of allele copy number with UC using logistic regression model. Gene and allele copy numbers of FCGR3A and FCGR3B were successfully determined in more than 99.5% of the study subjects. Allele copy number of FCGR3A-158F/V demonstrated significant association with susceptibility to UC (P = 0.02), although each single-nucleotide polymorphism and copy number variation alone did not show significant association. Although allele copy number of FCGR3B-NA1/NA2 (P = 0.002) also showed significant association with UC susceptibility, this association seemed to reflect the effect of FCGR3B gene copy number. Subsequent haplotype analyses revealed a strong association of a haplotype FCGR2A-131H/R and copy number of FCGR3B gene (P = 6.5 × 10). Allele copy number of FCGR3A-158F/V and FCGR3B gene copy number were associated with UC susceptibility. Our results suggest that organizing handling of immune complex by FCGR3A, FCGR3B, and FCGR2A may play a crucial role in the pathogenesis of UC.

No Association between FC γ R3B Copy Number Variation and Susceptibility to Biopsy-Proven Giant Cell Arteritis.

Objective. To determine the relationship between FCGR3B gene copy number variation (CNV) and biopsy proven giant cell arteritis (GCA). Methods. FCGR3B CNV was determined in 139 Australian biopsy proven GCA patients and 162 population matched controls, using a duplex qPCR assay and RNase P as the reference gene. Copy number was determined using Copy Caller software (v.1.0, Applied Biosystems, USA). CNV genotypes were classified into 3 groups (<2, 2, 3+) for analysis purposes, and analysis was performed using logistic regression. Results. All GCA patients had a positive temporal artery biopsy, and the most common presenting symptoms were visual disturbance and temporal headache. The mean age of patients at biopsy was 74 years (range 51–94) and 88/139 (63%) were female. The frequency of low (<2) FCGR3B copy number was comparable between GCA patients (9/139 = 6.5%) and controls (10/162 = 6.2%), as was the frequency of high (3+) FCGR3B copy number (15/130 (10.8%) in GCA patients versus 13/162 (8.0%) in controls). Overall there was no evidence that FCGR3B CNV frequencies differed between GCA patients and controls (χ 2 = 0.75, df = 2, P = 0.69). Conclusion. FCGR3B CNV is not associated with GCA; however, replicate studies are required.

AB0005 Association of FCGR2A with response to infliximab in rheumatoid arthritis patients

BackgroundReceptors for the Fc fragment of IgG play an important role in homeostasis and function of antibodies. In addition of their functions related with endogenous antibodies, they also bind biologic...