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Copy number polymorphism in Fcgr3 predisposes to glomerulonephritis in rats and humans
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"� 2006 Nature Publishing Group Copy number polymorphism in Fcgr3 predisposes to glomerulonephritis in rats and humans Timothy J. Aitman 1 , Rong Dong 1 *, Timothy J. Vyse 2 *, Penny J. Norsworthy 1 *, Michelle D. Johnson 1 , Jennifer Smith 3 , Jonathan Mangion 1 , Cheri Roberton-Lowe 1,2 , Amy J. Marshall 1 , Enrico Petretto 1 , Matthew D. Hodges 1 , Gurjeet Bhangal 3 , Sheetal G. Patel 1 , Kelly Sheehan-Rooney 1 , Mark Duda 1,3 , Paul R. Cook 1,3 , David J. Evans 3 , Jan Domin 3 , Jonathan Flint 4 , Joseph J. Boyle 5 , Charles D. Pusey 3 & H. Terence Cook 5 Identification of the genes underlying complex phenotypes and the definition of the evolutionary forces that have shaped eukary- otic genomes are among the current challenges in molecular genetics 1?3 . Variation in gene copy number is increasingly recog- nized as a source of inter-individual differences in genome sequence and has been proposed as a driving force for genome evolution and phenotypic variation 3?5 . Here we show that copy number variation of the orthologous rat and human Fcgr3 genes is a determinant of susceptibility to immunologically mediated glomerulonephritis. Positional cloning identified loss of the newly described, rat-specific Fcgr3 paralogue, Fcgr3-related sequence (Fcgr3-rs), as a determinant of macrophage overactivity and glo- merulonephritisinWistarKyotorats.Inhumans,lowcopynumber of FCGR3B,anorthologueofratFcgr3, was associated with glomerulonephritis in the autoimmune disease systemic lupus erythematosus. The finding that gene copy number polymorphism predisposes to immunologically mediated renal disease in two mammalian species provides direct evidence for the importance of genome plasticity in the evolution of genetically complex phenotypes, including susceptibility to common human disease. Glomerulonephritis is a major cause of kidney failure in humans, and is one of the most serious complications of autoimmune disorders such as systemic lupus erythematosus (SLE). In its most severeform,necrosisofmesangialandendothelialcellswithaccumu- lation of inflammatory and epithelial cells in Bowman?s space give rise to the morphological appearance of glomerular ?crescents? or crescentic glomerulonephritis. Several rodent models of crescentic glomerulonephritis have been developed. The Wistar Kyoto (WKY) rat strain is uniquely susceptible to crescentic glomerulonephritis among rat strains tested, as demonstrated by susceptibility to experimentally induced nephrotoxic nephritis (NTN) (ref. 6 and H. Rennke, personal communication) and experimental auto- immune glomerulonephritis 7 . Glomerular injury and crescent for- mation in rat glomerulonephritis are macrophage-dependent 8 , with morphological changes closely resembling those seen in human focal and segmental necrotizing glomerulonephritis 9 . The genetic com- plexity of glomerulonephritis in humans led us to study the genetics of glomerulonephritis in WKY rats. Ten days after injection of nephrotoxic serum, WKY rats consist- entlydevelopedNTN,whereasLewis,BrownNorwayandWistarrats showed no crescent formation, minimal glomerular macrophage infiltration and no proteinuria (Supplementary Fig. 1 and data not shown). F 1 rats showed intermediate phenotypes for crescent for- mation, proteinuria and macrophage infiltration, and phenotypes in F 2 rats spanned the range of the parental strains (Supplementary Fig. 1). Crescent formation in F 2 rats was highly correlated with proteinuria (r 2 � 0.66; P , 0.0001) and with macrophage infiltra- tion (r 2 � 0.30; P , 0.0001). Heritability was 0.96 for crescent formation, 0.80for proteinuria and0.52for macrophageinfiltration. AgenomescreenforNTNsusceptibilitylociinF 2 ratsrevealedtwo major quantitative trait loci (QTLs) on chromosomes 13 and 16 (designated crescentic glomerulonephritis 1 (Crgn1) and 2 (Crgn2)), both of which were linked to crescent formation and proteinuria (logarithm of the odds (LOD) scores 7.4?9.1; Fig. 1a). Infiltration of macrophages was also strongly linked only to Crgn1 (LOD 6.1; Fig. 1b, c). Several additional linkages (LOD scores 3?4) to crescent formation and proteinuria were detected on other chromosomes and weredesignatedCrgn3?7.Crgn1andCrgn2accounted,respectively,for 21.8% and 16.8% of the genetic variance in crescent formation and 16.7% and 17.7% of the genetic variance in proteinuria. Crgn1 accountedfor12.9%ofthegeneticvarianceinglomerularmacrophage infiltration. Several biological candidates were found in the Crgn1 region of linkage including the genes encoding the activatory Fc receptor for IgG, Fcgr3 (also known as FcgRIII), the inhibitory Fc receptor Fcgr2 (FcgRII), and the common g-subunit Fcer1g (FcRg). We sequenced the coding region of Fcer1g and found no sequence variants. In Fcgr2 we found a single nucleotide substitution, G388T, which changed codon 103 from arginine in Lewis rats to leucine in WKY rats. Sequence analysis of Fcgr3 revealed evidence of a genomic rearrange- mentinvolvingtheFcgr3locusandwasthereforeinvestigatedfurther. Polymerase chain reaction (PCR) amplification of Fcgr3 exons from genomic DNA revealed single bands for exons 1?4 in Lewis and WKY rats, and a single band from WKYexon 5 but two discrete PCR products from Lewisexon 5 (Fig. 1d), suggesting duplication ofexon 5 in Lewis genomic DNA, with loss of the shorter exon 5 from WKY genomic DNA. Direct sequence analysis of gel-purified products of exon 5 genomic DNA indicated the presence of a 226-base pair (bp) sequence in the longer PCR product (designated exon5_226�) that was absent in the shorter product (exon5_2262). The 226-bp sequence was situated in the 3 0 -untranslated region and contained a 153-bp short interspersed repetitive element (SINE). PCR analysis of 27 divergent rat strains showed an identical pattern to Lewis, whereas spontaneously hypertensive rats (SHR) and stroke-prone SHR (SHRSP) showed the same pattern as WKY. Because WKY, SHR and SHRSP rats were derived in the mid- twentieth century from the same outbred Kyoto colony of Wistar rats 10 , we determined the genotype of five additional strains derived LETTERS 1 Physiological Genomics and Medicine Group, MRC Clinical Sciences Centre, and Sections of 2 Rheumatology and 3 Renal Medicine, Imperial College, London W12 0NN, UK. 4 Wellcome Trust Centre for Human Genetics, Oxford OX3 7BN, UK. 5 Department of Histopathology, Imperial College, London W12 0NN, UK. *These authors contributed equally to this work. Vol 439|16 February 2006|doi:10.1038/nature04489 851 � 2006 Nature Publishing Group from this colony. As with WKY, four of these also show loss of exon5_2262 (Fig. 1e). The loss of exon5_2262 from seven strains, all descended from the Kyoto Wistar colony, suggests that these strains have inherited this chromosomal segment identical- by-descent. Because mesangial cell damage and glomerular necrosis are key features of NTN and are macrophage-dependent, we developed an in vitro assay of macrophage-mediated killing of antibody-coated glomerular mesangial cells as an intermediate phenotype of glome- rular pathology in NTN. Macrophages from WKY rats showed markedly enhanced antibody-dependent cellular cytotoxicity (ADCC) compared with macrophages from Lewis rats (Fig. 2a). We further phenotyped Lewis, WKY and five of the other Kyoto- derived strains using a semi-automated, fluorescence-based assay of ADCC andfound that five strains (WKY, SHR, WTC, WKYO, DON) hadincreasedmacrophagekillingcomparedwithLewis,whereasone (IS/KYO)hadmacrophageactivitycomparabletoLewis(Fig.2b).All of the strains with increased macrophage activity have lost exon5_2262 from their genome. Haplotype analysis on chromo- some 13 defined a 27-kilobase (kb) region, containing only one annotated gene (Fcgr3), that co-segregated with increased macro- phageactivityacrosstheseratstrains(Fig.2c;seealsoSupplementary Information), excluding other genes in this region, including Fcgr2, as a cause of increased macrophage activity in WKY rats. In addition to identification of the exon5_226�/2 variant, sequence analysis of Fcgr3 exon 5 revealed a single nucleotide deletion in the coding sequence at position 129 (DG129), found only in the shorter exon 5 (exon5_2262) (Fig. 3a). Reverse tran- scriptase PCR (RT?PCR) showed that the DG129-containing exon 5 is transcribed (Fig. 3b), and we designate the gene from which this transcription product is derived as Fcgr3-rs. No copies of the DG129 variant were found in 65 separate clone inserts amplified from WKY complementary DNA or genomic DNA, confirming loss of Fcgr3-rs from the WKY genome. The DG129 variant results in a frameshift in the Fcgr3-rs coding sequence that predicts a novel cytoplasmic domain six amino acids longer than that encoded by Fcgr3. Sequence comparison showed thatalthoughFcgr3-rsishighlysimilartotheotherisoformsofFcgr3 across the extracellular and transmembrane domains, the Fcgr3-rs cytoplasmic domain has no homology to other known proteins. Figure 1 | Genome screen for NTN susceptibility loci, and duplication and loss of Fcgr3 exon 5. a?c, Multi-point linkage plots showing location of susceptibility genes for crescent formation, proteinuria and macrophage infiltration on a whole genome plot (a), chromosome 13 (b) and chromosome 16 (c). cM, centimorgan. d, e, PCR amplification of Fcgr3 exon 5 from Lewis and WKY rats with Brown Norway strain as reference, showing absence of exon5_2262 in WKY (d), and in six out of seven other Kyoto-derived rat strains (e). 2, negative control PCR; M, FX174 HaeIII size marker. Figure 2 | Macrophage activity and haplotype analysis. a, b, Macrophage- mediated killing of antibody-coated mesangial cells by Lewis and WKY macrophages determined by visual assessment (n � 5 rats per strain; asterisk indicates P , 0.001) (a), and in different rat strains determined by release of fluorescence, normalized to Lewis macrophages (n � 4 rats per strain; asterisk indicates P , 0.01 compared to Lewis) (b). Data are mean ^ s.e.m. c, Chromosome 13 haplotype. The vertical bar indicates the interval shared between rat strains with macrophage overactivity. No other marker genotypes co-segregated with macrophage overactivity within the Crgn1 linkage region. High and low macrophage activity denote, respectively, strains with macrophage activity that is significantly increased, or not, compared to Lewis. Numbers denote classification of alleles: 1, Lewis; 2, WKY; 3, 4, other alleles. The Fcgr3-rs DG129 genotype is indicated in bold. LETTERS NATURE|Vol 439|16 February 2006 852 � 2006 Nature Publishing Group Southern analysisconfirmedthepresenceofatleastthreecopiesof Fcgr3-like exon 5 sequences in Lewis and WKY genomic DNA and reaffirmed loss of Fcgr3-rs exon 5 from the WKY genome (Fig. 3c); clonotype analysis (Supplementary Fig. 2) indicated the presence in bothLewisandWKYratsofatleasttwodistincttranscribedcopiesof exon5_ins226�. Additional Southern analysis of two Fcgr3-contain- ing bacterial artificial chromosomes from the rat genome project (data not shown) showed identically sized restriction fragments to those shown in Fig. 3c, indicating that these genes reside in contiguous genomic DNA on chromosome 13. We then carried out western analysis of macrophage lysates from Lewis and WKY rats using anti-Fcgr3-rs-specific antiserum. This detected a 66-kDa band in Lewis but not in WKY rats (Fig. 3d), confirming expressionofFcgr3-rs protein inLewis macrophages and its deficiency in WKY. To compare function of Fcgr3 and Fcgr3-rs, we stably transfected COS-1 cells with the common g-subunit (Fcer1g) together with either the Fcgr3 or Fcgr3-rs a-subunits. We also transfected COS-1 cells with the Fcgr3 construct after modification by site-directed mutagenesis to delete the single nucleotide G129 from exon 5, yielding a chimaeric construct, Fcgr3-DG, that contains Fcgr3 extra- cellular and transmembrane domains with an Fcgr3-rs cytoplasmic domain.WetestedthevariousstablytransfectedCOS-1cellsfortheir potential to phagocytose opsonized sheep red blood cells (SRBCs). Similar expression of all Fcgr3 constructs was confirmed by western analysis using an antibody to the Myc tag (data not shown), and cell surfaceexpressionwasconfirmedbyflowcytometry(Supplementary Fig. 3). Cells transfected with both Fcer1g and Fcgr3 showed levels of phagocytosisovertenfoldgreaterthancellstransfectedwithFcer1gand either Fcgr3-rs or the Fcgr3-DG constructs (Fig. 3e). Co-transfection of Fcgr3 with Fcgr3-rs showed a 70% inhibition of Fcgr3-mediated phagocytosis (Fig. 3f), indicating that loss of Fcgr3-rs-mediated inhibition of Fcgr3 is the likely mechanism for macrophage over- activity in WKY compared to Lewis rats. Our previous studies of Fc receptor polymorphisms in northern European nuclear families with SLE showed unexpected mendelian errors for two polymorphisms in the FCGR3B gene in 14% of these families 11 . The association of Fcgr3 copy number variation with immunologically mediated glomerulonephritis in the rat led us to test the hypothesis that copy number variation in human FCGR3B might explain these mendelian errors and that this could contribute to glomerulonephritis susceptibility. We developed a quantitative PCR assay to measure FCGR3B gene copynumberandappliedthisassayto30individualsfromasubsetof 8 of the nuclear families shown previously to have non-mendelian inheritanceatFCGR3B.ThisshowedsignificantvariationinFCGR3B copy number that was consistent with an estimate by Southern analysis (Fig. 4a). FCGR3B copy number differed significantly in this sample from that expected for a single copy gene in a diploid genome (P � 0.0004; Supplementary Fig. 4). We tested for an association between FCGR3B copy number and disease in SLE patients and in the subset of SLE patients with glomerulonephritis, referred to as lupus nephritis. Discordant sib pairanalysis(onesibpairperfamily)showednoassociation between FCGR3BcopynumberandSLE(n � 187sibpairs,Wilcoxonsign-rank test,P 2-tailed � 0.083,95%confidenceinterval(CI)0.082?0.085),but Figure 3 | Identification and functional characterization of Fcgr3-rs. a, Sequence analysis of exon 5 in Lewis and WKY rats showing a guanidine deletion at position 129 (DG129) in Lewis exon5_2262, denoted with an asterisk. The new reading frame is underlined in red. b, RT?PCR of Lewis and WKY RNA indicates expression of Fcgr3-rs exon5_2262 in Lewis but not in WKY rats. M, FX174 HaeIII size marker. c, Southern blot of the Fcgr3 locus with genomic DNA from WKY (W) and Lewis (L) rats. All of the restriction enzymes cut within the exon 5 SINE-containing insertion, and do not cut within the probe sequence. XmnI, EarI and Bsu36I digests were loaded after the NcoI and BlpI digests and have separate size markers (kb). d, Western blot of peritoneal macrophage lysate using a custom antibody to the Fcgr3-rs cytoplasmic tail, showing presence of Fcgr3-rs in Lewis but not in WKY macrophages on replicate loadings. e, Phagocytosis of antibody- coated erythrocytes by COS-1 cells transfected with Fcgr3 constructs. Asterisk, P , 0.01 compared to all other constructs. f, Co-transfection of Fcgr3 with Fcgr3-rs showing inhibition of Fcgr3-mediated phagocytosis by Fcgr3-rs (n � 8 paired transfections). Double asterisk, P, 0.002. Data are mean ^ s.e.m. NATURE|Vol 439|16 February 2006 LETTERS 853 � 2006 Nature Publishing Group showed a weak association with lupus nephritis (n � 61 pairs, Wilcoxon sign-rank test, P 2-tailed � 0.038, 95% CI 0.037?0.039). Separate association analysis, carried out between all available lupus nephritis patients (n � 63) and unrelated, seronegative con- trols (n � 141) from the lupus cohort also showed association with lupus nephritis (Mann?Whitney U-test, P � 0.001, 95% CI 0.001? 0.002; Fig. 4b). Logistic regression analysis, using age and gender as covariates,confirmedtheassociationbetweenFCGR3Bcopynumber andlupusnephritisinthissample (x 2 (3degreesoffreedom) � 13.5, P � 0.004; Supplementary Table 1A). Because previous studies have shown an association between SLE and the FCGR2A and FCGR3A genes within the Fc receptor cluster on chromosome 1q23 (refs 12, 13), we carried out a sequential logistic regression analysis to test whether the effect of FCGR3B copy number on lupus nephritis was independent of FCGR2A and FCGR3A. Using data from 60 lupus nephritis patients and 109 unrelated, seronegative controls in this cohort for whom complete genotypedatawasavailable,thesequentiallogisticregressionanalysis model predicted FCGR3B copy number as being significantly and independently associated with lupus nephritis (x 2 (3 degrees of freedom) � 13.4, P � 0.004), whereas the inclusion of FCGR2A- G548A and FCGR3A-T559G produced poorer models, indicating that the effects of FCGR3A and FCGR2A are very small compared with that observed for FCGR3B (Supplementary Table 1B). An analysis of FCGR2A-G548A and FCGR3A-T559G haplotypes in this group did not show any association with copy number at FCGR3B (Fisher?s exact test, P 2-tailed . 0.5), providing further evidence that reduced copy number at FCGR3B is an independent risk factor for lupus nephritis. Fc receptors, the genes of which are located in clusters across mammalian genomes, functionally link the humoral and cellular branches of the immune system and have a key role in activation and modulation of the immune response 14,15 . Our findings in rats that loss of Fcgr3-rs results in macrophage overactivity and glomerulo- nephritis susceptibility, and that Fcgr3-rs inhibits Fcgr3-mediated phagocytosis, suggest that Fcgr3-rs may be a potential therapeutic agent for autoimmune disease. Human FCGR3B is expressed mainly in neutrophils and is necessary for neutrophil tethering to immune complexes 15?17 . It is therefore plausible that reduced neutrophil expression of FCGR3B in patients with low FCGR3B copy number maylead to reduced glomerularclearance of immune complexes and susceptibility to autoimmune renal disease in patients with SLE, and possibly to other autoimmune disorders. So far, there is little evidence that common, complex disease phenotypes can be caused by stably transmitted gene duplication/ deletion or copy number polymorphisms 4 . Two examples that have been reported are Cd36 deletion in insulin resistance in rats and CCL3L1 copy number polymorphism in HIV-1/AIDS susceptibility inhumans 18?20 .AlthoughmultiplegenomiccopiesofFcgr3havebeen suggested in the rat 21 , and gene duplication and deletion in human FCGR3B havebeen reported in isolated cases or infamilies identified because of FCGR3B deficiency, their association with immune phenotypes has not been clear 22?24 . Our studies have demonstrated naturally occurring gene copy number variation within the syntenic Fcgr3 clusters in rats and humans, and showedthat loss ofFcgr3-rs in the rat andlowFCGR3B copynumberin humans are associated with susceptibility to immunologically mediated forms of glomerulo- nephritis in these two mammalian species. To our knowledge, this is the first demonstration in any species that gene copy number polymorphism predisposes toautoimmune disease. Ourfindingthat copy number polymorphism at orthologous regions of diverse genomes is associated with immunologically related disease suggests that genome plasticity, manifested by gene duplication/deletion and copy number polymorphism, is a more common cause of genetically complex phenotypes than has hitherto been observed. METHODS See Supplementary Information for detailed Methods. Inbred strains and linkage studies. WKY/NCrlBR (abbreviated to WKY) and SHR/NCrlBR (SHR) rats were obtained from Charles River and Lew/HanHsd (Lewis) rats from Harlan. WTC, WKYO, DON and IS/KYO rats were obtained from T. Serikawa. Reciprocal crosses between WKYand Lewis (both Rt1-l) rats were used to generate 177 F 2 rats that were phenotyped for NTN and used in a genome screen for susceptibility genes with 128 polymorphic microsatellite markers. No phenotypic differences were found between the reciprocal crosses, which were combined for linkage studies. NTN phenotypes were assessed in 200?220g male rats 10days after intravenous injection of 0.1ml of a rabbit antiserum to rat glomerular basement membrane 25 . Crescent formation, 24h urinary protein and glomerular macrophage infiltration were assessed as described 26 . Direct sequencing and sequencing of cloned PCR products. RNA extraction fromfrozentissues,cDNApreparationandPCRwereundertakenasdescribed 18 , and clonotype analysis was performed using the high-fidelity polymerases Pfu (Promega) or KOD1 (Novagen). Southernandwesternanalyses.Southernanalyseswerecarriedoutasdescribed 18 . For western analysis, 10mg denatured protein from lysed thioglycollate-elicited macrophages or COS-1 cells was resolved by 4?12% gradient SDS?PAGE. Proteins were electro-blotted onto PVDF membranes (Invitrogen) and stained with antibodies against the Fcgr3-rs cytoplasmic domain (macrophages) or against Myc tag (COS-1 cells). Antibody-directed cellular cytotoxicity. ADCC assays were carried out by visual assessment of cell death of antibody-coated mesangial cells and by a semi-automated dye release assay, as described 27 . Mesangial cells were derived from WKY kidneys. Similar results were found with Lewis mesangial cells. For the dye release assay, thioglycollate-elicited peritoneal macrophages were added to cultured mesangial cellsloaded with calcein fluorescent dye in the presence of 10mgml 21 anti-Thy1.1 antibody. Calcein release into the supernatant was measured in a microplate fluorimeter. Differences between inbred strains were assessed by Student?s t-test (1-tailed). Studies of Fc receptor function in COS-1 cells. Constructs of Fcer1g, Fcgr3, Fcgr3-rs and Fcgr3-DG were created from PCR-amplified cDNA. A Myc tag was introduced into the amino terminus of all three Fcgr3 a-subunit constructs to allow confirmation of expression by western blot. COS-1 cells stably transfected with Fcer1g were electroporated with Fcgr3, Fcgr3-rs or Fcgr3-DG and selected with G418 and hygromycin B. Fc-receptor-mediated function was assessed by measuring internalization into COS-1 cells of SRBCs coated with anti-SRBC antibody. Phagocytic index (RBCs per COS-1 cell) was calculated as described 28 . The effect of Fcgr3-rs on Fcgr3-mediated phagocytosis was determined by transfection of COS-1 cells that had been transfected with, and were expressing, Fcer1g and Fcgr3. Differences between groups were compared using Student?s t-test (1-tailed). Nucleotide and protein sequence analysis. Homologous sequences to rat Fcgr3 and Fcgr3-rs exon 5 sequences were sought by searching the NCBI non- redundant and high-throughput genomic sequence databases using BLAST. Quantification of FCGR3B copy number. Quantitative PCR was carried out Figure 4 | Copy number polymorphism in human FCGR3 and association withlupusnephritis. a, Southern blot of genomic DNA from four unrelated individuals, selected according to the quantitative PCR estimate of FCGR3B copy number, shown above the blot. The Southern probe was designed to cross-hybridize to FCGR3A and FCGR3B restriction fragments. FCGR3B band intensity, measured by densitometry, is normalized to FCGR3A band intensity, and shown below the plot as the ratio of FCGR3B:FCGR3A. Genomic DNA was digested with the restriction enzyme HpaI. b, Histogram showing frequency distribution of FCGR3B copy number, per diploid genome, in lupus nephritis cases (filled columns) and unaffected controls (open columns). LETTERS NATURE|Vol 439|16 February 2006 854 � 2006 Nature Publishing Group using SYBR Green Jumpstart Taq Readymix (Sigma) and analysed by the standard curve method. Oligonucleotides were designed to amplify specifically FCGR3Bandtoavoidparalogousorallelicsequencevariants.CD36wasusedasa single-copycontrol.DatafromFCGR3BandCD36werenormalizedto forkhead box P2 (FOXP2) to give an estimate of copy number. Sequence analysis was undertaken to confirm specificity of the FCGR3B PCR product. Human genetics. DNAwas available from 256 SLE nuclear families (consisting of parents and progeny) of northern European origin in which genotypes for FCGR3B had been previously determined 29 . SLE and lupus nephritis were definedaccordingtoACRcriteria 30 .ThiscohortconsistedofasingleSLEpatient per family. Of the 256 SLE patients studied, 63 had nephritis and were available for analysis. Discordant sib pair analyses were performed for all families with available sib pairs (one sib pair per family) using the nonparametric Wilcoxon sign-rank test. We also compared the 63 lupus nephritis cases with unrelated controls (n � 141) (one per family) from within the SLE cohort. All controls used were seronegative for antinuclear antibody. The study was ethically approved under MREC 98/2/6. A one-sample sign test of the median was used to determine whether gene copy number differed significantly from 2. To test for the significance of the differencebetween the distributions of the FCGR3B gene copy numbers we used the nonparametric Mann?Whitney U-test (2-tailed). For both the Wilcoxon sign-rank test and the Mann?Whitney U-test, 100,000 Monte Carlo simulations wereperformedtoestimateexactPvaluesand95%confidenceintervals.Logistic regressionwas performed using SPSS 12.0 with FCGR3B gene copy number and genotypes of FCGR2A and FCGR3A as predictor variables. Haplotypes for FCGR2A-G548A and FCGR3A-T559G were constructed as described 11 . Received 3 August; accepted 22 November 2005. 1. Ohno, S., Wolf, U. & Atkin, N. B. Evolution from fish to mammals by gene duplication. Hereditas 59, 169?-187 (1968). 2. Glazier, A. M., Nadeau, J. H. & Aitman, T. J. 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Supplementary Information is linked to the online version of the paper at www.nature.com/nature. Acknowledgements We acknowledge intramural funding from the CSC, and support from the Wellcome Trust Cardiovascular Functional Genomics award, the British Heart Foundation and the Medical Research Council. P.R.C. is a Medical Research Council Clinical Fellow. We thank E. Sodergren and G. Weinstock for BAC clone DNA; H. Hedrich, A. Dominiczak and J. Rapp for rat genomic DNA; T. Serikawa and the National Bio Resource Project in Japan for rat strains and genomic DNA; B. Foxwell for bicistronic vector; and M. Botto, B. Morley, P. Froguel, C. Shoulders and S. Cook for constructive criticism of the manuscript. We acknowledge the CSC Genomics Laboratory for DNA sequencing, and bioinformatics support from M. Mu�ller, N. J. Dickens and the Imperial College Bioinformatics Support Service. Author Contributions The study was conceived and funded by T.J.A., H.T.C. and C.D.P. H.T.C., J.S., P.R.C. and D.J.E. carried out the rodent phenotyping. T.J.A., M.D., P.J.N., P.R.C. and J.F. carried out the rodent linkage studies. T.J.A., R.D., M.D.J., J.M., A.J.M., M.D.H., S.G.P. and K.S.-R. carried out the genomic analysis of rat and human Fcgr3. Cellular immunology studies were carried out by R.D., J.J.B., M.D.J., G.B., M.D., J.D., C.D.P. and H.T.C. Human genetics was carried out by P.J.N., A.J.M., C.R.-L., T.J.V., E.P. and T.J.A., and the manuscript was written by T.J.A., H.T.C., T.J.V. and J.M. Author Information The sequence of Fcgr3-rs exon 5 has been deposited in GenBank under accession number AY561710. Reprints and permissions information is available at npg.nature.com/reprintsandpermissions. The authors declare no competing financial interests. Correspondence and requests for materials should be addressed to T.J.A. (t.aitman@csc.mrc.ac.uk) or H.T.C. (t.h.cook@imperial.ac.uk). NATURE|Vol 439|16 February 2006 LETTERS 855 "
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