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

Niederer HA, Willcocks LC, Rayner TF, Yang W, Lau YL, Williams TN, Scott JA, Urban BC, Peshu N, Dunstan SJ, Hien TT, Phu NH, Padyukov L, Gunnarsson I, Svenungsson E, Savage CO, Watts RA, Lyons PA, Clayton DG, Smith KG - Hum. Mol. Genet. (2010)

Bottom Line: 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 FcgammaRIIB (I232T) which abolishes inhibitory function, both reduced CN of FCGR3B and homozygosity of the FcgammaRIIB-232T allele were individually strongly associated with SLE risk.Further interpretations of contributions to pathogenesis by FcgammaRs must be made in the context of LD involving CNV regions.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Cambridge Institute for Medical Research, University of Cambridge School of Clinical Medicine, Addenbrooke's Hospital, Hills Road, Cambridge CB2 0XY, UK.

ABSTRACT
The response of a leukocyte to immune complexes (ICs) is modulated by receptors for the Fc region of IgG (FcgammaRs), and alterations in their affinity or function have been associated with risk of autoimmune diseases, including systemic lupus erythematosus (SLE). The low-affinity FcgammaR 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 x 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 FcgammaRIIB (I232T) which abolishes inhibitory function, both reduced CN of FCGR3B and homozygosity of the FcgammaRIIB-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 FcgammaRs must be made in the context of LD involving CNV regions.

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CNV and paralogue ratio test (PRT) in the FCGR l ocus. A segmental duplication resulted in the production of FCGR3B, as well as FCGR2C which combines the 5′ end of FCGR2B and the 3′ end for FCGR2A. Repeated black, grey and white regions reflect areas of homology within the genes. The PRT primer pairs used in this study to determine copy number variation (CNV) are PRT-2C3′/2A3′ (circle) and PRT-3B/3A (square), which each bind at and amplify two locations in the genome on Chromosome 1; and PRT-3(A+B)/Chr18 (triangle) which binds at three locations—two on chromosome 1 and one on chromosome 18. Relative gene locations are derived from the Database of Genomic Variants, human genome build 36 (hg18). Three CNV regions are identified by variation at PRT primer binding sites (marked): CNR1 (blue) includes CNV at the PRT primer sites in FCGR2C and FCGR3B; CNR2 (red) includes CNV at the PRT primer sites in FCGR2A 3′-UTR and FCGR3A, but not in FCGR2C 3′-UTR; and CNR3 (green) includes CNV at the PRT primer sites in FCGR3A and FCGR2C 3′-UTR. Solid colour bars indicate areas suggested to be included in the CNV interval and thin grey lines indicate maximum plausible extent. Diploid copy number variations of each region observed in this study are noted, with other probable variations in brackets. For further details of the relationship between relative PRT product, underlying gene CN and CNV regions, see Supplementary data online.
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DDQ216F1: CNV and paralogue ratio test (PRT) in the FCGR l ocus. A segmental duplication resulted in the production of FCGR3B, as well as FCGR2C which combines the 5′ end of FCGR2B and the 3′ end for FCGR2A. Repeated black, grey and white regions reflect areas of homology within the genes. The PRT primer pairs used in this study to determine copy number variation (CNV) are PRT-2C3′/2A3′ (circle) and PRT-3B/3A (square), which each bind at and amplify two locations in the genome on Chromosome 1; and PRT-3(A+B)/Chr18 (triangle) which binds at three locations—two on chromosome 1 and one on chromosome 18. Relative gene locations are derived from the Database of Genomic Variants, human genome build 36 (hg18). Three CNV regions are identified by variation at PRT primer binding sites (marked): CNR1 (blue) includes CNV at the PRT primer sites in FCGR2C and FCGR3B; CNR2 (red) includes CNV at the PRT primer sites in FCGR2A 3′-UTR and FCGR3A, but not in FCGR2C 3′-UTR; and CNR3 (green) includes CNV at the PRT primer sites in FCGR3A and FCGR2C 3′-UTR. Solid colour bars indicate areas suggested to be included in the CNV interval and thin grey lines indicate maximum plausible extent. Diploid copy number variations of each region observed in this study are noted, with other probable variations in brackets. For further details of the relationship between relative PRT product, underlying gene CN and CNV regions, see Supplementary data online.

Mentions: The qPCR-based CNV assays previously used to interrogate the FCGR locus (21–25) have been shown to be limited by the requirement to make discrete CN calls from continuous data distributions (23,26). Therefore, we developed an assay involving a combination of three PRTs to better analyse CNV in the FCGR locus. Both PRT and qPCR compare the signal generated at a variable locus to that at a reference locus. The ratio between amplified products, and thus relative gene CN, can then be determined. The key advantage to the PRT assay is that a single pair of primers amplifies two genomic loci, producing products which differ in size by a few nucleotides, in the same tube. In contrast, in qPCR, the separate reference and CNV reactions introduce variability, with significant plate-to-plate variation (23). Two PRT assays developed in this study compare the CN of a region in FCGR3B with one in FCGR3A, and CN of a region in the FCGR2C 3′-untranslated region (3′-UTR) with one in the FCGR2A 3′-UTR (Fig. 1, Supplementary Material, Fig. S1A and B). These were used in conjunction with a third previously described PRT assay which compared total CN in both FCGR3B and FCGR3A with a region on Chromosome 18 (27). Comparison of these three PRT assays allowed CN of FCGR3A, FCGR3B and the 3′-UTR of FCGR2A and FCGR2C to be determined, without the requirement of a restriction enzyme digest as used previously (27) (full description of PRT in Supplementary Material; Figs S1 and S2). FCGR2B was previously shown not to undergo CNV (6,20). The CN variation over these loci suggested at least three CNV regions: CNR1, CNR2 and CNR3 (Fig. 1, Supplementary Material, Fig. S1C and D). The more common CNR1 and CNR2 show similarity to, and extend, previously identified CNV regions (6,20), and a novel CNR3 is seen primarily in East Asians. Both duplication and deletion were observed at CNR1 and CNR2 (and thus the CNV at these sites is multi-allelic), whereas only a duplication of CNR3 was able to be identified (Supplementary Material, Fig. S2E and Table S1).Figure 1.


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

Niederer HA, Willcocks LC, Rayner TF, Yang W, Lau YL, Williams TN, Scott JA, Urban BC, Peshu N, Dunstan SJ, Hien TT, Phu NH, Padyukov L, Gunnarsson I, Svenungsson E, Savage CO, Watts RA, Lyons PA, Clayton DG, Smith KG - Hum. Mol. Genet. (2010)

CNV and paralogue ratio test (PRT) in the FCGR l ocus. A segmental duplication resulted in the production of FCGR3B, as well as FCGR2C which combines the 5′ end of FCGR2B and the 3′ end for FCGR2A. Repeated black, grey and white regions reflect areas of homology within the genes. The PRT primer pairs used in this study to determine copy number variation (CNV) are PRT-2C3′/2A3′ (circle) and PRT-3B/3A (square), which each bind at and amplify two locations in the genome on Chromosome 1; and PRT-3(A+B)/Chr18 (triangle) which binds at three locations—two on chromosome 1 and one on chromosome 18. Relative gene locations are derived from the Database of Genomic Variants, human genome build 36 (hg18). Three CNV regions are identified by variation at PRT primer binding sites (marked): CNR1 (blue) includes CNV at the PRT primer sites in FCGR2C and FCGR3B; CNR2 (red) includes CNV at the PRT primer sites in FCGR2A 3′-UTR and FCGR3A, but not in FCGR2C 3′-UTR; and CNR3 (green) includes CNV at the PRT primer sites in FCGR3A and FCGR2C 3′-UTR. Solid colour bars indicate areas suggested to be included in the CNV interval and thin grey lines indicate maximum plausible extent. Diploid copy number variations of each region observed in this study are noted, with other probable variations in brackets. For further details of the relationship between relative PRT product, underlying gene CN and CNV regions, see Supplementary data online.
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DDQ216F1: CNV and paralogue ratio test (PRT) in the FCGR l ocus. A segmental duplication resulted in the production of FCGR3B, as well as FCGR2C which combines the 5′ end of FCGR2B and the 3′ end for FCGR2A. Repeated black, grey and white regions reflect areas of homology within the genes. The PRT primer pairs used in this study to determine copy number variation (CNV) are PRT-2C3′/2A3′ (circle) and PRT-3B/3A (square), which each bind at and amplify two locations in the genome on Chromosome 1; and PRT-3(A+B)/Chr18 (triangle) which binds at three locations—two on chromosome 1 and one on chromosome 18. Relative gene locations are derived from the Database of Genomic Variants, human genome build 36 (hg18). Three CNV regions are identified by variation at PRT primer binding sites (marked): CNR1 (blue) includes CNV at the PRT primer sites in FCGR2C and FCGR3B; CNR2 (red) includes CNV at the PRT primer sites in FCGR2A 3′-UTR and FCGR3A, but not in FCGR2C 3′-UTR; and CNR3 (green) includes CNV at the PRT primer sites in FCGR3A and FCGR2C 3′-UTR. Solid colour bars indicate areas suggested to be included in the CNV interval and thin grey lines indicate maximum plausible extent. Diploid copy number variations of each region observed in this study are noted, with other probable variations in brackets. For further details of the relationship between relative PRT product, underlying gene CN and CNV regions, see Supplementary data online.
Mentions: The qPCR-based CNV assays previously used to interrogate the FCGR locus (21–25) have been shown to be limited by the requirement to make discrete CN calls from continuous data distributions (23,26). Therefore, we developed an assay involving a combination of three PRTs to better analyse CNV in the FCGR locus. Both PRT and qPCR compare the signal generated at a variable locus to that at a reference locus. The ratio between amplified products, and thus relative gene CN, can then be determined. The key advantage to the PRT assay is that a single pair of primers amplifies two genomic loci, producing products which differ in size by a few nucleotides, in the same tube. In contrast, in qPCR, the separate reference and CNV reactions introduce variability, with significant plate-to-plate variation (23). Two PRT assays developed in this study compare the CN of a region in FCGR3B with one in FCGR3A, and CN of a region in the FCGR2C 3′-untranslated region (3′-UTR) with one in the FCGR2A 3′-UTR (Fig. 1, Supplementary Material, Fig. S1A and B). These were used in conjunction with a third previously described PRT assay which compared total CN in both FCGR3B and FCGR3A with a region on Chromosome 18 (27). Comparison of these three PRT assays allowed CN of FCGR3A, FCGR3B and the 3′-UTR of FCGR2A and FCGR2C to be determined, without the requirement of a restriction enzyme digest as used previously (27) (full description of PRT in Supplementary Material; Figs S1 and S2). FCGR2B was previously shown not to undergo CNV (6,20). The CN variation over these loci suggested at least three CNV regions: CNR1, CNR2 and CNR3 (Fig. 1, Supplementary Material, Fig. S1C and D). The more common CNR1 and CNR2 show similarity to, and extend, previously identified CNV regions (6,20), and a novel CNR3 is seen primarily in East Asians. Both duplication and deletion were observed at CNR1 and CNR2 (and thus the CNV at these sites is multi-allelic), whereas only a duplication of CNR3 was able to be identified (Supplementary Material, Fig. S2E and Table S1).Figure 1.

Bottom Line: 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 FcgammaRIIB (I232T) which abolishes inhibitory function, both reduced CN of FCGR3B and homozygosity of the FcgammaRIIB-232T allele were individually strongly associated with SLE risk.Further interpretations of contributions to pathogenesis by FcgammaRs must be made in the context of LD involving CNV regions.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Cambridge Institute for Medical Research, University of Cambridge School of Clinical Medicine, Addenbrooke's Hospital, Hills Road, Cambridge CB2 0XY, UK.

ABSTRACT
The response of a leukocyte to immune complexes (ICs) is modulated by receptors for the Fc region of IgG (FcgammaRs), and alterations in their affinity or function have been associated with risk of autoimmune diseases, including systemic lupus erythematosus (SLE). The low-affinity FcgammaR 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 x 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 FcgammaRIIB (I232T) which abolishes inhibitory function, both reduced CN of FCGR3B and homozygosity of the FcgammaRIIB-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 FcgammaRs must be made in the context of LD involving CNV regions.

Show MeSH
Related in: MedlinePlus