A Promoter Haplotype of the Immunoreceptor Tyrosine-Based Inhibitory Motif-Bearing FcγRIIb Alters Receptor Expression and Associates with This information is current as Autoimmunity. I. Regulatory FCGR2B of September 27, 2021. Polymorphisms and Their Association with Systemic Erythematosus Kaihong Su, Jianming Wu, Jeffrey C. Edberg, Xiaoli Li, Polly Ferguson, Glinda S. Cooper, Carl D. Langefeld and Downloaded from Robert P. Kimberly J Immunol 2004; 172:7186-7191; ; doi: 10.4049/jimmunol.172.11.7186 http://www.jimmunol.org/content/172/11/7186 http://www.jimmunol.org/

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

A Promoter Haplotype of the Immunoreceptor Tyrosine-Based Inhibitory Motif-Bearing Fc␥RIIb Alters Receptor Expression and Associates with Autoimmunity. I. Regulatory FCGR2B Polymorphisms and Their Association with Systemic Lupus Erythematosus1,2

Kaihong Su,* Jianming Wu,* Jeffrey C. Edberg,* Xiaoli Li,* Polly Ferguson,3* Glinda S. Cooper,† Carl D. Langefeld,‡ and Robert P. Kimberly4*

Fc␥RIIb, the immunoreceptor tyrosine-based inhibitory motif-containing receptor for IgG (Mendelian Inheritance in Man no. Downloaded from 604590), plays an important role in maintaining the homeostasis of immune responses. We have identified 10 novel single- nucleotide polymorphisms in the promoter region of human FCGR2B gene and characterized two functionally distinct haplotypes in its proximal promoter. In luciferase reporter assays, the less frequent promoter haplotype leads to increased expression of the reporter gene in both B lymphoid and myeloid cell lines under constitutive and stimulated conditions. Four independent genome- wide scans support linkage of the human Fc␥R region to the systemic lupus erythematosus (SLE; Online Mendelian Inheritance

in Man no. 152700) phenotype. Our case-control study in 600 Caucasians indicates a significant association of the less frequent http://www.jimmunol.org/ The FCGR2B haplotype has no linkage .(0.0054 ؍ p ;1.65 ؍ FCGR2B promoter haplotype with the SLE phenotype (odds ratio disequilibrium with previously identified FCGR2A and FCGR3A polymorphisms, and after adjustment for FCGR2A and These results suggest that an .(0.0083 ؍ p ;1.72 ؍ FCGR3A, FCGR2B showed a persistent association with SLE (odds ratio expression variant of FCGR2B is a risk factor for human lupus and implicate FCGR2B in disease pathogenesis. The Journal of Immunology, 2004, 172: 7186–7191.

ithin the classical IgG Fc-binding receptor family, and proliferation (2–6). Coligation of Fc␥RIIb also provides a Fc␥RIIb (CD32B) is the only receptor that bears an negative-feedback mechanism for Ig production by B cells. On immunoreceptor tyrosine-based inhibitory motif myeloid lineage cells, Fc␥RIIb coclustering with the activating by guest on September 27, 2021 W5 (ITIM) in its cytoplasmic domain (1). Fc␥RIIb is expressed on B Fc␥Rs, such as Fc␥RIa (CD64), Fc␥RIIa (CD32A), and Fc␥RIIIa lymphocytes, myeloid cell lineages, and dendritic and mast cells. (CD16A), down-modulates their function (2). Ab-mediated phago- On B lymphocytes, coligation of Fc␥RIIb with the Ag re- cytosis by macrophages is decreased by exaggerated Fc␥RIIb co- ceptor by IgG immune complexes down-regulates B cell Ag re- clustering and is enhanced by disruption of Fc␥RIIb (7–9). On ceptor signaling and modulates the threshold for B cell activation follicular dendritic cells (FDC), Fc␥RIIb mediates the retention and conversion of immune complexes to a highly immunogenic form, which facilitate B cell recall responses (10–13). Thus, *Division of Clinical Immunology and Rheumatology, Departments of Medicine and Fc␥RIIb plays multiple roles in modulating immune function and Microbiology, University of Alabama, Birmingham, AL 35294; †Epidemiology thus maintaining immune homeostasis. Indeed, studies in mouse Branch, National Institute on Environmental Health Sciences, Research Triangle Park, NC 27709; and ‡Department of Public Health Sciences, Wake Forest Univer- models have highlighted the role of FCGR2B in the development sity, Winston-Salem, NC 27157 of autoimmune diseases (14–19). For example, targeted disruption Received for publication November 26, 2003. Accepted for publication March of FCGR2B in the mouse leads to elevated serum Ig levels and, on 26, 2004. the susceptible C57BL/6 background, leads to the development of The costs of publication of this article were defrayed in part by the payment of page lupus-like phenotypes (20, 21). charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Human systemic lupus erythematosus (SLE) is a prototypic au- toimmune disease characterized by production of antinuclear au- 1 This work was supported by Grants R01 AR42476, P50 AR45231, P01 AR49084, and R01 33062 from the National Institutes of Health. The University of Alabama at toantibodies and tissue deposition of immune complexes (22–25). Birmingham (UAB) Pittman General Clinical Research Center is supported by Grant This complex polygenic disease has strong genetic components MO1 RR00032 from the National Center for Research Resources at the National ␭ Ϸ Institutes of Health. The FACS Core Facility of the UAB Arthritis and Musculoskel- ( s 20) (26, 27). In humans, outside of MHC class II, genetic etal Center was supported by Rheumatic Diseases Core Center (P30 AR48311). polymorphisms or defects in genes involved in Ag uptake and/or 2 The authors declare that they have no competing financial interests. process and in clearance such as complement, 3 Current address: Department of Pediatrics, University of Iowa College of Medicine, FCGR2A and FCGR3A have been identified to contribute to SLE Iowa City, IA 52242. susceptibility (26, 28–33). Recently, programmed cell death gene 4 Address correspondence and reprint requests to Dr. Robert P. Kimberly, Tinsley 1(PDCD1) which regulates B cell activation has been identified as Harrison Tower 429, 1900 University Boulevard, University of Alabama, Birming- an autoimmunity candidate gene in the mouse (34, 35), and a sin- ham, AL 35294-0006. E-mail address: [email protected] gle-nucleotide polymorphism (SNP) in a putative RUNX1 binding 5 Abbreviations used in this paper: ITIM, immunoreceptor tyrosine-based inhibitory motif; FDC, follicular dendritic cell; SLE, systemic lupus erythematosus; SNP, sin- site in the promoter of human PDCD1 gene has been implicated as gle-nucleotide polymorphism; RLA, relative luciferase activity. a risk allele for SLE (34, 35). However, potential variations in the

Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00 The Journal of Immunology 7187 regulatory regions of human FCGR2B as a disease susceptibility cals, Indianapolis, IN). The cells were recovered overnight and treated with gene have not yet been characterized. 0.5 mM dibutyryl cAMP, or 400 U/ml IFN-␥, or nonstimulated for addi- Unlike the SNPs in human FCGR2A and FCGR3A that affect the tional 24 h. The cells were then lysed and measured for luciferase activities using the Dual Luciferase Reporter Assay System (Promega, Madison, ligand-binding properties of the receptors (29, 36), we could not WI). The firefly luciferase activity was normalized by Renilla luciferase find any nonsynonymous SNPs encoding the extracellular domains activity to yield the relative luciferase activity (RLA). of Fc␥RIIb in Ͼ120 donors. However, we identified 10 polymor- phic sites in the 2-kb promoter region of human FCGR2B and Statistical analysis defined two SNP haplotypes in its proximal promoter. In luciferase Data for comparison of mean values among samples were analyzed by reporter assays, the less frequent variant FCGR2B haplotype in- Student’s t test or Kruskal-Wallis test. To test for an association between creases the promoter activity both constitutively and under induc- FCGR2B and human SLE, we computed four separate logistic regression ible conditions. In our case-control study of 600 Caucasians, the models. The four models contained only the FCGR2B haplotype and were sequentially partitioned into two degree of freedom tests for general asso- variant FCGR2B haplotype is significantly associated with the ciation and three a priori genetic models (i.e., dominant, additive, and SLE phenotype. This association is not due to the effects of pre- recessive). We estimated the degree of linkage disequilibrium among viously identified FCGR2A or FCGR3A polymorphisms. Our ob- FCGR loci using the D and DЈ statistics (38). In the joint analysis and the servation not only provides evidence for the genetic association of conditional tests of association, to adjust for the effects of FCGR2A and FCGR2B with human lupus but also is the first study to charac- FCGR3A on our FCGR2B tests of association, we computed a parallel set of logistic regression models that contain the effects of all three genes with terize the functionally important promoter polymorphisms in tests of FCGR2B conditional on the FCGR2A/3A genotypes viewed as a FCGR2B, one of the key regulators in immune responses. priori tests. Downloaded from Materials and Methods Results Donors Identification of SNPs in the human FCGR2B promoter Caucasian SLE patients and controls were recruited as part of the Univer- To identify functional SNPs in the human FCGR2B gene, cDNA sity of Alabama at Birmingham-based DISCOVERY cohort and as part of Ͼ the Carolina Lupus Study (37), a population-based case-control study. The from 120 donors was amplified and sequenced. We did not find any nonsynonymous SNPs in the IgG-binding extracellular do-

studies were reviewed and approved by the Institution Review Board, and http://www.jimmunol.org/ all donors provided written informed consent. mains of Fc␥RIIb (39). However, we found that the expression levels of Fc␥RIIb is variable among individuals (data not shown). FCGR2B genotyping Therefore, we searched for SNPs in the regulatory region of Long-range PCR was performed to specifically amplify FCGR2B from FCGR2B gene. Study of polymorphisms in the noncoding regions genomic DNA using Failsafe PCR system (Epicenter Technologies, Mad- of FCGR2B is complicated by the extremely high homology be- ison, WI). The sense primer (5Ј-CTCCACAGGTTACTCGTTTCTACCT TATCTTAC-3Ј) anneals at both FCGR2B/C Ϫ2-kb promoters, and the tween the FCGR2B and FCGR2C genes, which reflects gene du- antisense primer (5Ј-GCTTGCGTGGCCCCTGGTTCTCA-3Ј) anneals at plication and crossover events during evolution of FcR cluster the FCGR2B-specific sequence in intron 6 between exons 6 and 7. The (40–42). To characterize the promoter region of FCGR2B,we PCR conditions were 94°C for 2 min, 14 cycles of 98°C for 20 s and 68°C screened a bacterial artificial library, identified the by guest on September 27, 2021 for 17 min, followed by 10 more cycles with the extension time increasing by 15 s each cycle, and a 7-min extension at 68°C. The resultant 15-kb FCGR2B and FCGR2C genes, and sequenced a 12-kb region of the PCR product was gel-purified and used as the template for the nested PCR 5Ј portion of each gene (42). FCGR2B and FCGR2C are nearly to amplify the 2-kb promoter of FCGR2B with the sense primer (5Ј-GT 100% identical within the first 3.4 kb of the 5Ј flanking region and TACTCGTTTCTACCTTATCTTAC-3Ј) and the antisense primer (5Ј-TT regions through exon 3. However, a stretch of 31 nt in the intron Ј GCAGTCAGCCCAGTCACTCTC-3 ). The PCR conditions were 95°C 6 (between exons 6 and 7) of FCGR2B is unique to the FCGR2B for 5 min, 35 cycles of 94°C for 30 s, 56°C for 30 s, and 72°C for 2 min, and followed by a 7-min extension at 72°C. The nested-PCR product was gene (41, 42). Based on this information, we developed a long- then gel-purified and sequenced with BigDye terminator cycle sequencing range PCR to specifically amplify the 15 kb of FCGR2B from Ϫ2 on an ABI 377 (Applied Biosystems, Foster City, CA). The sequencing kb to intron 7 from genomic DNA and a subsequent nested PCR Ј Ј primer was 5 -ATTTCAAGAAGCATCCAGATTC-3 . The rare alleles using the long PCR product as a template to amplify the FCGR2B were confirmed by sequencing from both directions. For genotyping the FCGR2B promoter SNPs, pan-PCR was performed promoter for genotyping. to amplify both FCGR2B/C promoters containing Ϫ120 or Ϫ386 SNP. For Among 66 non-SLE controls and 66 SLE patients, we found 10 the PCR amplicon of 114 bp containing Ϫ120 SNP, the sense primer is SNPs in the first 2-kb promoter of FCGR2B (Fig. 1). We did not 5Ј-AAAGAGGGTGGAAAGGGAGGAG-3Ј and the antisense primer is identify any deletions or insertions in this FCGR2B promoter re- Ј Ј 5 -biotin-CTCTCAAAGCTTGGCGGATTCTAC-3 . For the PCR ampli- gion. The gene specificity of the nested-PCR strategy was verified con containing Ϫ386 SNP, the sense primer is 5Ј-TCAAGAAGCATCCA GATTCCAG-3Ј and the antisense primer is 5Ј-biotin-AAACTCAGCTCA by gene-specific SNPs in the FCGR2C exon 3 (42). GAACCTCCTGTT-3Ј. The PCR conditions were 95°C for 5 min, 40 cycles of 95°C for 30 s, 56°C for 30 s, and 72°C for 45 s, followed by a Haplotypes in the FCGR2B proximal promoter alter promoter 7-min extension at 72°C. The PCR product was genotyped by pyrose- activity quencing on a PSQ 96 system following the manufacturer’s instructions (Biotage, Westborough, MA). The pyrosequencing primers for Ϫ120 and In the mouse, key elements regulating FCGR2B expression are Ϫ386 SNPs were 5Ј-CCTGTGATAAAACAGAACAT-3Ј and 5Ј-TGCT located within the first several hundred base pairs of the 5Ј pro- GGTGCACGCTGTCCT-3Ј, respectively. For the donors who have the Ϫ Ϫ moter. To focus on potential functionally important polymor- uncommon A or C allele at nt 120 or 386, FCGR2B-specific long PCR Ј was then performed to assign the origin of these uncommon alleles by phisms in the human FCGR2B promoter, we made a series of 5 pyrosequencing. deletion promoter-reporter constructs and transfected them into BJAB cells, a B-lymphoid cell line. Luciferase reporter assays Transient transfection and luciferase reporter assays showed that 1.0-kb promoter of FCGR2B retains Ϸ100% activity For BJAB cells, the FCGR2B-promoter reporter plasmid (40 ␮g) was co- as compared with 4.3-, 2.0-, and 1.4-kb promoter (Fig. 2). There 6 transfected with 300 ng of the reference plasmid pRL-SV40 into 10 ϫ 10 may be a repressor element between Ϫ0.6 and Ϫ1.0 kb, because cells by electroporation at 200 V and 960 ␮F. For U937 cells, the FCGR2B-promoter reporter plasmid (1 ␮g) was cotransfected with 100 ng deletion of this promoter fragment leads to a 1.9-fold increase of of the reference plasmid into 5 ϫ 105 cells using 3 ␮l of FuGENE 6 reagent the luciferase activity (Fig. 2). We obtained similar results using according to the manufacturer’s instructions (Roche Molecular Biochemi- the same 5Ј-promoter constructs in U937 cells, a myeloid cell line. 7188 FCGR2B PROMOTER POLYMORPHISMS ASSOCIATE WITH LUPUS

FIGURE 1. SNPs in the 2-kb FCGR2B promoter region. The polymorphic alleles are indicated in parentheses with the common allele in the upper left and the uncommon allele in the lower right. The nucleotide position is relative to the translation start site.

Therefore, we focused our further study on the three SNPs in the U937 cells were transfected with the reporter plasmid containing proximal 1.0-kb promoter of human FCGR2B. Ϫ893C-386G-120T or Ϫ893C-386C-120A haplotype for 16 h and Among the 132 individuals, we found that the variant “C” allele then stimulated with dibutyryl cAMP or IFN-␥ for 24 h. In BJAB at nt Ϫ386C always accompanied the variant “A” allele at nt cells, cAMP up-regulated FCGR2B promoter activity by 1.5-fold, Ϫ120. The possibility that the Ϫ386C and Ϫ120A alleles might and IFN-␥ slightly down-regulated FCGR2B promoter in the con- Downloaded from form a haplotype was confirmed by cloning and sequencing the text of both Ϫ386G-120T and Ϫ386C-120A haplotypes (Fig. 3C). 1.0-kb FCGR2B promoter from doubly heterozygous donors, In U937 cells, for both the FCGR2B promoter haplotypes, cAMP which identified only two haplotypes, Ϫ386G-120T and Ϫ386C- slightly up-regulated the promoter activity, and IFN-␥ down-reg- 120A. The variant G allele at nt Ϫ893 (allele frequency is Ϸ7%) ulated the promoter by 50% (Fig. 3D). These data indicate that the was about equally represented in both the Ϫ386G-120T and

Ϫ386C-120A haplotypes. http://www.jimmunol.org/ To determine whether the variant alleles affect the promoter activity, we performed luciferase reporter assays using constructs containing the 1.0-kb FCGR2B promoter incorporating different alleles in front of the luciferase gene. The Ϫ893 “C/G” alleles did not influence promoter activity in the context of both Ϫ386G-120T and Ϫ386C-120A haplotypes in BJAB and U937 cells (Fig. 3, A and B). However, the FCGR2B promoter with the Ϫ386C-120A haplotype showed a 1.8-fold greater expression of the luciferase by guest on September 27, 2021 reporter, compared with the Ϫ386G-120T haplotype, in both BJAB and U937 cells (Fig. 3, A and B). This difference is apparent in the context of either common “C” or uncommon “G” allele at nt Ϫ893. This result showed clearly that the two proximal FCGR2B promoter haplotypes differentially affect constitutive promoter activity. Recently, several studies have shown that the expression of Fc␥RIIb is regulated by cytokines and hormones (7, 8, 43–45). We next examined whether the two FCGR2B promoter haplotypes have differential activity under stimulated conditions. BJAB and

FIGURE 3. The variant Ϫ386C-120A haplotype of FCGR2B promoter drives higher luciferase reporter expression than the Ϫ386G-120T haplo- type. The reporter constructs incorporating the four haplotypes (“CGT,” “GGT,”“CCA,” and “GCA” are shortened haplotype names and represent alleles at nt Ϫ893, Ϫ386, and Ϫ120, respectively) in the context of 1.0 kb FIGURE 2. 5Ј-Deletion analysis of the FCGR2B promoter. A series of of the FCGR2B promoter were transiently transfected into BJAB (A) and 5Ј-deletion FCGR2B promoter fragments was placed in front of the firefly U937 (B) cells. For C and D, the reporter constructs with the CGT or CCA luciferase report gene, and the plasmid was cotransfected with the refer- haplotype in the context of the 1-kb FCGR2B promoter were transfected ence plasmid pRL-SV40 (SV40 promoter drives Renilla luciferase gene) into BJAB (C) or U937 (D) cells for 16 h and then either unstimulated (Ⅺ), into BJAB cells. Dual Luciferase assay was performed 24–40 h after trans- or stimulated with 0.5 mM dibutyryl cAMP (o) or 400 U/ml IFN-␥ (2) for fection. The firefly luciferase activity was normalized by Renilla luciferase an additional 24 h. The firefly luciferase activity was measured and nor- levels, and the ratio is designated as RLA. The results represent the malized by Renilla luciferase levels to yield RLA. The results represent the mean Ϯ SEM from three independent experiments. mean Ϯ SEM from three independent experiments. The Journal of Immunology 7189 less frequent variant Ϫ386C-120A haplotype has greater promoter Table I. Distribution of the FCGR2B promoter haplotypes in SLE activity than the more common Ϫ386G-120T haplotype under patients and controlsa both constitutive and stimulated conditions. We have examined the Fc␥RIIb expression levels on primary Caucasian Controls Caucasian SLE ϭ ϭ cells, and in agreement with our in vitro luciferase assay, the do- (n 366) Patients (n 243) nors with the Ϫ386C-120A haplotype express more receptor on B Genotype (no. of subjects (% of group)) lymphocytes and than donors homozygous for the 2B.1/2B.1 300 (82.0%) 180 (74.1%) Ϫ386G-120T haplotype (46). The differential promoter activity of 2B.1/2B.4 63 (17.2%) 56 (23.0%) 2B.4/2B.4 3 (0.8%) 7 (2.9%) the two FCGR2B haplotype is due to their differential binding capacity for transcription factors GATA4 and YY1 (46). Haplotype frequency 2B.1 (Ϫ386G-120T) 90.6% 85.6% The association of FCGR2B haplotype with SLE 2B.4 (Ϫ386C-120A) 9.4% 14.4% To investigate the relationship of the two functionally important a Haplotype frequency in SLE patients vs controls (2 ϫ 2 ␹2 test, odds ratio ϭ FCGR2B promoter haplotypes to an autoimmune phenotype, we 1.62, p ϭ 0.007; logistic regression analyses, additive model, odds ratio ϭ 1.65, p ϭ 0.0054, 95% confidence interval ϭ 1.16–2.36). developed a strategy to genotype the two SNPs at nt Ϫ386 and Ϫ120 in a larger collection of samples. PCR was performed to amplify 114-bp promoter regions containing the Ϫ120 SNP of both FCGR2B and FCGR2C genes from genomic DNA. The pan- 2.36). Unlike FCGR2B, there was no association of FCGR2C al-

ϭ Downloaded from PCR products were applied to quantitative pyrosequencing in a leles or haplotypes with SLE ( p 0.975). 96-well format which gave 100, 75, 50, or 25% allele distributions The association of FCGR2B promoter haplotype with SLE was reflecting the four from both FCGR2B and FCGR2C not due to the presence of an extended haplotype containing the genes that were amplified. For the donors with the variant Ϫ120A recently reported nonsynonymous exon 5 SNP that encodes a allele, FCGR2B-specific long PCR, followed by nested PCR, was transmembrane polymorphism and that associates with the SLE performed and applied to pyrosequencing to determine the allele phenotype in a Japanese population (47). The uncommon trans- 3 1873 187 frequency in the FCGR2B gene. By this method, the frequency of membrane allele, 775T C encoding Ile Thr , was found in http://www.jimmunol.org/ the variant Ϫ120A allele in the FCGR2C gene was also deter- only several donors with the 2B.4 promoter haplotype, and the 187 mined. A similar strategy was also used for Ϫ386 G/C SNPs. For variant Thr is not associated with SLE in a Caucasian the FCGR2B gene in our Caucasian population, we have found that population (39). the frequency of the common haplotype Ϫ386G-120T (named Combined analysis of FCGR2B, FCGR2A, FCGR3A 2B.1 haplotype) is 91%, the uncommon Ϫ386C-120A (2B.4) hap- polymorphisms lotype is ϳ9% (Fig. 4). The Ϫ386C-120T (2B.2) haplotype is very rare, and the frequency is ϳ0.41%. We have not observed the Functional polymorphisms in the extracellular domains of Fc␥RIIa Ϫ386G-120A (2B.3) haplotype in the FCGR2B gene from our and Fc␥RIIIa have been shown to associate with SLE in a number populations. For the FCGR2C gene, however, the haplotype fre- of studies (26). Because FCGR2B is located ϳ200 kb telomeric to by guest on September 27, 2021 quencies are distinct from FCGR2B. The Ϫ386C-120T (2B.2) FCGR2A/3A within the classical FcR cluster, we next examined haplotype occurs much more frequently than in the FCGR2B gene the potential linkage disequilibrium among the polymorphisms of (12 vs 0.4% haplotype frequency), and the Ϫ386C-120A (2B.4) these three genes in our collection of Caucasians. Analyses of the haplotype is much more rare than in the FCGR2B gene (1 vs 9%) SNP genotyping data show that there is no linkage disequilibrium (Fig. 4). As with FCGR2B, we have not observed the Ϫ386G- of FCGR2B promoter haplotypes with FCGR2A and FCGR3A 120A (2B.3) haplotype in the FCGR2C gene. Having established polymorphisms, because the calculated DЈ was low (DЈϭ0.221 these haplotype frequencies, we focused our further association and DЈϭ0.486, respectively), and there is no statistical interaction studies on the common Ϫ386G-120T and variant Ϫ386C-120A between FCGR2B and FCGR3A loci under a dominant-additive haplotype. genetic model ( p ϭ 0.6629). We also computed the three condi- In our collection of 243 Caucasian SLE patients and 366 tional association tests for FCGR2A, FCGR3A, and FCGR2B poly- matched controls, the less frequent variant 2B.4 (Ϫ386C-120A) morphisms. Logistic regression, adjusted for FCGR2A and haplotype in the FCGR2B gene promoter was significantly asso- FCGR3A, showed a persistent effect for FCGR2B (Table II; p ϭ ciated with the autoimmune SLE phenotype (Table I; single locus 0.0083; odds ratio ϭ 1.72; 95% confidence interval ϭ 1.15–2.58). association test using logistic regression analyses, additive model; After adjusting for FCGR2B polymorphisms, FCGR3A is also sig- p ϭ 0.0054; odds ratio ϭ 1.65; 95% confidence interval ϭ 1.16– nificantly associated with SLE (Table II; p ϭ 0.0288; odds ratio ϭ 0.65; 95% confidence interval ϭ 0.44–0.96). This lack of sub- stantial linkage disequilibrium from FCGR2B to FCGR2A/3A is consistent with the physical distance of ϳ250 kb across this clus- ter, which is larger than the median haplotype block in Caucasians (42, 48). Therefore, FCGR2B and FCGR3A may contribute to SLE independently and, perhaps, synergistically.

Discussion The ITIM-bearing IgG receptor Fc␥RIIb is widely expressed on immune cells and plays an important role in maintaining immune FIGURE 4. Four proximal promoter haplotypes and their frequencies in response homeostasis. FCGR2B-deficient mice have elevated Ig the FCGR2B and FCGR2C genes. The four haplotypes (2B.1–4) have levels in response to both thymus-dependent and -independent different allele combination at nt Ϫ386 and Ϫ120 but the same “C” allele Ags, and, on a susceptible genetic background, FCGR2B-deficient at nt Ϫ893. The FCGR2B and FCGR2C genes have distinct haplotype mice develop a lupus-like autoimmune disease (20, 21). Polymor- frequencies. phisms in the mouse homolog of the human FCGR2B gene have 7190 FCGR2B PROMOTER POLYMORPHISMS ASSOCIATE WITH LUPUS

Table II. Joint analysis and the conditional tests of associationa

General Test of Association Best Genetic Model Test of OR (95% CI) under Best (p Value) Association (p Value) Genetic Model

FCGR2A 0.9929 Dominant, 0.9720 1.01 (0.61–1.67) FCGR3A 0.0949 Dominant, 0.0288 0.65 (0.44–0.96) FCGR2B 0.0204 Additive, 0.0083 1.72 (1.15–2.58)

a OR, Odds ratio; CI, confidence interval. been identified in several autoimmune-prone strains (14, 16). extended haplotype including both promoter and the nonsynony- Taken together, these observations have focused attention on mous exon 5 (transmembrane) SNPs will explain the proposed Fc␥RIIb both as a disease susceptibility gene and as a potential association of the FCGR2B transmembrane polymorphism with therapeutic target for autoimmunity. SLE in Japanese patients. Such studies will need to take care to To assess the role of Fc␥RIIb in human autoimmunity, we iden- differentiate the promoters of human FCGR2B and FCGR2C, tified the functional genetic variations in the FCGR2B gene and which have exceptionally high homology, and to consider the con- assessed their association with the SLE phenotype. We character- tribution of other genes as suggested in the mouse (21). As with ized the two FCGR2B proximal promoter haplotypes which were susceptibility genes in other complex diseases, one can anticipate found in Ͼ99% of all 600 donors studied. The two FCGR2B hap- that the contribution of each gene will have a modest odds ratio Downloaded from lotypes have differential promoter activity in cell lines of lymphoid (54). Nonetheless, identification of single-gene variants will help and myeloid lineages under both constitutive and stimulated con- characterize pathologic pathways for these complex autoimmune ditions. The less frequent, variant gain-of-function promoter hap- disorders, and combined multigene analysis will be important for lotype of FCGR2B is significantly enriched in SLE patients in our the ultimate understanding of the disease pathogenesis. case-control study of Caucasians with an odds ratio of 1.65. This Most importantly, our data demonstrate the occurrence of two

disease association is not due to linkage disequilibrium with other functionally distinct FCGR2B promoter haplotypes that affect pro- http://www.jimmunol.org/ FcR family genes (FCGR2A or FCGR3A). moter activity in both lymphoid and myeloid cell lines. The two The association of the gain-of-function promoter variant of FCGR2B promoter haplotypes have differential binding capacity FCGR2B with human SLE might be considered a surprise. How- for transcription factors GATA4 and YY1 and lead to differential ever, the effect of the homozygous FCGR2B knockout mouse is expression levels of the endogenous Fc␥RIIb on primary cells background dependent, and the repertoire of FcRs in mouse is (46). Identification of the FCGR2B promoter variants as a disease different from that in humans. Mouse has a single CD32 gene (the risk factor also supports the notion that duplicated regions within ITIM-containing FCGR2B), whereas humans have two additional the genome are likely the hot spots of genomic instability and are CD32 genes, the immunoreceptor tyrosine-based activation motif- associated with genetic diseases (55). Furthermore, apart from au- containing activation receptors, FCGR2A and FCGR2C.Fc␥RIIb toimmunity, FCGR2B promoter genotypes may also play an im- by guest on September 27, 2021 is expressed on multiple cell types and may have distinct func- portant role in the variations of human Ab responses to vaccines as tion(s) depending on its cell context. For example, expressed on predicted by its function on B cells and studies in the mouse (56). mononuclear phagocytes, Fc␥RIIb can decrease the of immune complexes, a process important for the in vivo clear- Acknowledgments ance of immune complex. On FDC, Fc␥RIIb promotes the matu- We thank S. McKenzie for discussion regarding genomic structure of the ration of FDC reticulum and mediates the uptake and conversion FcR cluster, and Drs. G. S. Alarco´n, H. Bastian, and B. Fessler for assis- of immune complexes on FDCs to potentially more highly immu- tance in patient recruitment. nogenic forms (10–13, 49). In contrast, on B cells, Fc␥RIIb down- modulates B cell activation and Ab production. Thus, Fc␥RIIb References may play distinct roles according to the disease stage and the cell 1. Daeron, M. 1997. Fc receptor biology. Annu. Rev. Immunol. 15:203. 2. Ravetch, J. V., and S. Bolland. 2001. IgG Fc receptors. Annu. Rev. Immunol. types involved in the development of autoimmunity. In consider- 19:275. ing modulation of Fc␥RIIb as a therapeutic target, it may be im- 3. Takai, T. 2002. Roles of Fc receptors in autoimmunity. Nat. Rev. Immunol. 2:580. 4. Coggeshall, K. M. 2002. 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