Genes and Immunity (2006) 7, 250–263 & 2006 Nature Publishing Group All rights reserved 1466-4879/06 $30.00 www.nature.com/gene ORIGINAL ARTICLE Overlapping BXSB congenic intervals, in combination with microarray gene expression, reveal novel lupus candidate genes MEK Haywood1, SJ Rose1, S Horswell2, MJ Lees1,GFu3, MJ Walport4 and BJ Morley1 1Rheumatology Section, Division of Medicine, Imperial College Faculty of Medicine, London, UK; 2Medical Research Council, Clinical Sciences Centre, Hammersmith Hospital, London, UK; 3Oxitec Limited, Oxford, UK and 4The Wellcome Trust, London, UK The BXSB mouse strain is an important model of glomerulonephritis observed in systemic lupus erythematosus (SLE). Linkage studies have successfully identified disease-susceptibility intervals; however, extracting the identity of the susceptibility gene(s) in such regions is the crucial next step. Congenic mouse strains present a defined genetic resource that is highly amenable to microarray analysis. We have performed microarray analysis using a series of chromosome 1 BXSB congenic mice with partially overlapping disease-susceptibility intervals. Simultaneous comparison of the four congenic lines allowed the identification of expression differences associated with both the initiation and progression of disease. Thus, we have identified a number of novel SLE disease gene candidates and have confirmed the identity of Ifi202 as a disease candidate in the BXSB strain. Sequencing of the promoter regions of Gas5 has revealed polymorphisms in the BXSB strain, which may account for the differential expression profile. Furthermore, the combination of the microarray results with the different phenotypes of these mice has allowed the identification of a number of expression differences that do not necessarily map to the congenic interval, but may be implicated in disease pathways. Genes and Immunity (2006) 7, 250–263. doi:10.1038/sj.gene.6364294; published online 16 March 2006 Keywords: autoimmunity; systemic lupus erythematosus; rodent; microarray; candidate genes Introduction consistently linked to SLE in different models, particu- larly distal chromosome 1.5–7 However, it is also clear The BXSB mouse is a recombinant inbred strain1 that that several other intervals, unique to each mouse model, provides a model of the proliferative glomerulonephritis play crucial roles in the development of disease. For observed in human systemic lupus erythematosus (SLE). example, in BXSB mice, Bxs1 on chromosome 1 is now This strain develops a range of autoantibodies directed at known to be an important locus for the development of nuclear components, particularly anti-DNA and anti- nephritis,8,9 but this interval is not linked to disease in 2 10,11 chromatin. Alongside this spectrum of autoantibodies, either the (NZB Â NZW)F1 or MRL models. Identifi- more general indicators of immune system dysregulation cation of the genetic basis of these disease-susceptibility can be observed, specifically splenomegaly and lympha- intervals is fundamental to developing our understand- denopathy.1 The culmination of disease occurs in the ing of this prototypic autoimmune disease. kidneys, where mesangial matrix increase and cellular In BXSB mice, in addition to the autosomal loci, the Y- infiltration are observed as characteristic features of the chromosome carries an autoimmune accelerator gene glomerulonephritis in the BXSB strain.2 (Yaa).12 Though the identity of this gene is still unclear, it Genetic studies in both mouse models and human has been demonstrated to accelerate disease observed in populations have indicated that SLE is a complex genetic BXSB male mice in comparison to their female counter- disease; thus, multiple loci contribute to disease suscept- parts.12 Transfer of the BXSB Y-chromosome by breeding ibility, with no absolute requirement for any single onto other autoimmune prone strains of mice, such as locus.3 Comparison of the many linkage studies per- NZB and NZW, has been shown to accelerate disease formed in the lupus prone mouse strains, MRL, BXSB progression.13 It has only recently become clear that Yaa and (NZB Â NZW)F1, amply illustrates the complexity of is capable of breaking tolerance in a non-autoimmune this disease.4 Some chromosomal intervals have been prone strain such as C57BL/10 (B10).8 Whereas linkage studies are invaluable for identifying chromosomal intervals in which disease-susceptibility Correspondence: Dr B Morley, Rheumatology Section, Division of loci are located, they are limited in their resolution. The Medicine, Imperial College, Hammersmith Campus, Du Cane Road, resultant disease-associated interval may contain several London W12 0NN, UK. E-mail: [email protected] hundred positional candidate genes, of which several Received 9 September 2005; revised 2 February 2006; accepted 2 may present as reasonable functional candidates. In February 2006; published online 16 March 2006 order to confirm our linkage data, we generated congenic Microarray analysis of BXSB congenic mice MEK Haywood et al 251 mice, in which the disease-associated intervals of potential candidates, even within the congenic intervals, chromosome 1 were bred onto a non-autoimmune strain microarrays offer a rapid way of screening for expression (B10) containing the Yaa gene.8 Thus, the individual and level differences. Although expression profiling may not isolated contribution of each linkage region to the overall identify functional differences underlying the disease, it phenotype could be interrogated. These novel mouse may detect perturbations in downstream expression strains present a restricted and defined genetic resource, levels secondary to the primary genetic defects on with minimal genetic variation from the non-affected B10 chromosome 1. Such a ‘disease pathway’ may be parental strain, and each interval contributes different identified by examining gene expression in those strains aspects of the overall phenotype8 (Figure 1). with related phenotypes and determining any common The next stage to dissecting the genetic basis of SLE is differentially expressed genes. the identification of candidate genes. One approach is to A major problem in the expression profiling approach select genes that are differentially expressed between is the large number of candidates that may be identified. congenic and control strains. Given the large number of These congenic strains provide an ideal resource to Figure 1 Schematic representation of chromosome 1 BXSB congenic mouse strains. The microsatellite markers used to direct the breeding for each strain are shown on the left-hand side (scale in Mb). Disease-susceptibility linkage intervals are indicated by grey boxes and named according to Hogarth et al.7 and Haywood et al.9 The congenic interval is indicated by the dark grey bar, and the pale grey bars indicate regions in which the location of genetic recombination remains to be determined. Thus, the B10.Yaa.Bxs1 strain contains the BXSB-derived interval from 46.3 to 89.2 Mb, B10.Yaa.Bxs1/4 spans 20.0–65.9 Mb, B10.Yaa.Bxs1/2 encompasses 64.4–159.0 Mb and B10.Yaa.Bxs2/3, 105.4– 189.0 Mb. The phenotypes associated with each interval,8 or overlapping interval, are indicated on the right-hand side. Genes and Immunity Microarray analysis of BXSB congenic mice MEK Haywood et al 252 reduce background in such an analysis.14,15 Each con- tosis in B10.Yaa.Bxs2/3.8 However, the samples used for genic interval is relatively small in comparison to the the gene expression analysis presented here were whole genome, so the confounding influence of natural collected prior to this change and to the development variations between strains can be minimised. In addition, of disease. each congenic interval partially overlaps with at least one of the other strains. Thus, the validity of candidate genes Overlapping congenic intervals aid identification of novel can, in part, be determined by the expression profile candidate genes across the strains. By definition, a differentially ex- Over- and underexpressed probes for each congenic pressed candidate gene should be expressed at a similar strain were identified using the arbitrary level of twofold level in all strains containing the BXSB allele of that gene, change in expression levels with respect to B10.16,17 The and must be differentially expressed in all strains results for each congenic strain were overlapped with carrying the B10 allele. To this end, we have carried those for both BXSB and B10.Yaa mice. This allowed us to out an extensive microarray analysis on all four congenic identify the primary genetic causes of lupus attributable strains together with B10.Yaa, B10 and BXSB as controls, to the specific chromosome 1 congenic interval rather to direct our future studies. than effects associated with Yaa. Our results therefore B10.Yaa.Bxs2/3 mice develop a phenotype that is show those genes with altered expression levels in both closest to the parental BXSB strain, though with later the congenic line and BXSB, but not in B10.Yaa. All onset, 12 months for 50% mortality compared to 6 differentially expressed probe sequences were BLAST months for BXSB.8 This congenic strain displays acceler- searched using NCBI Genome Blast (http://www.ncbi.- ated mortality, a broad spectrum of autoantibodies and nih.gov/entrez/) to identify chromosomal locations for age-dependent glomerulonephritis with focal mesangial each sequence, and checked against the Ensembl hypercellularity accompanied by a matrix increase that database. As many Affymetrix probe sequences origi- was qualitatively similar to the lesions observed in BXSB nated from complementary DNA (cDNA) sequences, mice.8 B10.Yaa.Bxs1/4 mice also developed similar ne- NCBI Unigene was used to identify the genes repre- phritic lesions, along with a broad spectrum of auto- sented by some probes. Differentially expressed se- antibodies, but the onset was later still (12 months quences that were located on chromosome 1 were mortality 25%).8 B10.Yaa.Bxs1 mice developed glomer- plotted with respect to the congenic intervals on ulonephritis in the absence of autoantibodies, whereas chromosome 1 (Figures 2 and 3). B10.Yaa.Bxs1/2 mice developed autoantibodies without concomitant nephritis.