Genomic pathology of SLE-associated copy-number variation at the FCGR2C/FCGR3B/FCGR2B locus.

Reduced FCGR3B copy number is associated with increased risk of systemic lupus erythematosus (SLE). The five FCGR2/FCGR3 genes are arranged across two highly paralogous genomic segments on chromosome 1q23. Previous studies have suggested mechanisms for structural rearrangements at the FCGR2/FCGR3 locus and have proposed mechanisms whereby altered FCGR3B copy number predisposes to autoimmunity, but the high degree of sequence similarity between paralogous segments has prevented precise definition of the molecular events and their functional consequences. To pursue the genomic pathology associated with FCGR3B copy-number variation, we integrated sequencing data from fosmid and bacterial artificial chromosome clones and sequence-captured DNA from FCGR3B-deleted genomes to establish a detailed map of allelic and paralogous sequence variation across the FCGR2/FCGR3 locus. This analysis identified two highly paralogous 24.5 kb blocks within the FCGR2C/FCGR3B/FCGR2B locus that are devoid of nonpolymorphic paralogous sequence variations and that define the limits of the genomic regions in which nonallelic homologous recombination leads to FCGR2C/FCGR3B copy-number variation. Further, the data showed evidence of swapping of haplotype blocks between these highly paralogous blocks that most likely arose from sequential ancestral recombination events across the region. Functionally, we found by flow cytometry, immunoblotting and cDNA sequencing that individuals with FCGR3B-deleted alleles show ectopic presence of FcγRIIb on natural killer (NK) cells. We conclude that FCGR3B deletion juxtaposes the 5'-regulatory sequences of FCGR2C with the coding sequence of FCGR2B, creating a chimeric gene that results in an ectopic accumulation of FcγRIIb on NK cells and provides an explanation for SLE risk associated with reduced FCGR3B gene copy number.

Interstitial fibrosis is associated with increased COL1A2 transcription in AA-injured renal tubular epithelial cells in vivo.

Accumulation of type I collagen is a key event in renal interstitial fibrosis. As there is no effective treatment, understanding the site where collagen is transcribed and the factors driving it in response to disease in vivo is critical for designing future therapies. The present research investigated the transcriptional activity of the COL1A2 gene in a mouse model of progressive fibrosis induced by aristolochic acid (aristolochic acid nephropathy, AAN). To achieve this we genetically modified mice to express a reporter gene (LacZ) and CCN2 (connective tissue growth factor) under the transcriptional control of the COL1A2 promoter /enhancer sequences. Using these mice we asked where is collagen actively transcribed and secondly, what is the role of CCN2 in AAN. Here, we report that de-novo transcription of the COL1A2 gene occurred predominantly in damaged tubular epithelial cells during progressive interstitial fibrosis in vivo. The activation of COL1A2 was studied by detection of the reporter gene LacZ and COL1A2 mRNA in interstitial, glomerular, vascular, and tubular epithelial tissue from laser capture microscopy. We also demonstrated that LacZ-positive cells co-express E-Cadherin a marker of epithelial origin which is consistent with an epithelial phenotype which is capable of collagen expression during injury. There was no evidence of detachment of these cells from tubules to become myofibroblasts. Moreover, we showed that the transgenic mice show a modest enhancement of CCN2 expression; however fibrosis induced by AA is the same in transgenics and controls suggesting that CCN2, at this level of expression, is not sufficient to enhance fibrogenesis. Overall our study provides a better understanding into the expression patterns and roles of two major extracellular matrix proteins: type I collagen and CCN2.

Evidence for both copy number and allelic (NA1/NA2) risk at the FCGR3B locus in systemic lupus erythematosus.

The Fcgamma-receptor locus on chromosome 1q23 shows copy-number variation (CNV), and it has previously been shown that individuals with reduced numbers of copies of the Fcgamma-receptor-IIIB gene (FCGR3B) have an increased risk of developing systemic lupus erythematosus (SLE). It is not understood whether the association arises from FCGR3B (CD16b) itself, is observed because of linkage disequilibrium with actual causal alleles and/or is an effect of CNV on flanking FCGR genes. Thus, we extended this previous work by genotyping the FCGR3B alleles NA1/NA2 and re-assaying CNV using a paralogue ratio test assay in a family study (365 families). We have developed a novel case/pseudo-control approach to analyse family data, as the phase of copy number (CN) is not known in parents and cannot always be inferred in offspring. The results, obtained by fitting logistic regression models, confirm the association of low CN of FCGR3B with SLE (P=0.04). The risk conferred by low copies (<2) was contingent on FCGR3B allotype, being greater for deletion of NA1 than the for lower-affinity NA2. The simpler model with just CN was rejected in favour of the biallelic-CN model (P=0.03). We observed a correlation (R(2)=0.75, P<0.0001) between FCGR3B CNV and neutrophil expression in both healthy controls and patients with SLE. Our results suggest that one mechanism by which CNV at this locus confers disease risk is directly as a result of reduced FcgammaRIIIb function, either because of reduced expression (related to CNV) or because of reduced affinity for its ligand (NA1/NA2 allotype).