An immunological and genomic study of canine hypoadrenocorticism (Addison's disease)

BACKGROUND Canine hypoadrenocorticism is a well-established spontaneous animal model of human Addison's disease. The disease is characterised by a loss of adrenocortical secretory capacity, leading to glucocorticoid and/or mineralocorticoid deficiency. Iatrogenic hypoadrenocorticism can also occur as an adverse effect of drug treatment for hyperadrenocorticism. In primary hypoadrenocorticism the pathogenesis of disease is believed to result from immune-mediated destruction of adrenocortical tissue. Of all the autoimmune disorders of the dog, hypoadrenocorticism is perhaps the least heterogeneous in terms of the clinical phenotype, making this disease ideal for studying immune response genes associated with susceptibility to autoimmunity, which are also likely to be involved in other, more common, but more heterogeneous canine autoimmune diseases. A genetic predisposition for hypoadrenocorticism has been demonstrated in a number of dog breeds, including Standard poodles, Bearded collies and Portuguese water dogs. A recent microsatellite-based genome-wide association study has indicated two disease-associated loci, which are syntenic with human HLA-DRB1 (MHC Class II) and CTLA4 (Chase et al., 2006). In the current project we will characterise the autoimmune response in dogs suffering from hypoadrenocorticism and take a comparative genetic and functional genomic approach to better understand the relationship between MHC Class II and CTLA4 genotypes and this autoimmune phenotype. AIMS AND OBJECTIVES The aims of the current project are to characterise the autoimmune response in dogs suffering from hypoadrenocorticism and to investigate the molecular basis for breed-susceptibility to the disease. We will test the hypotheses that autoantibodies to adrenal antigens can be identified in the serum of dogs suffering from hypoadrenocorticism and that disease-susceptibility is associated with polymorphisms in Dog Leukocyte Antigen (DLA) and CTLA4 genes. Furthermore, we will investigate whether the CTLA4 genotype influences gene expression at the transcriptome level, potentially leading to aberrant production of soluble CTLA-4 protein, an important molecule for immune regulation. We will test these hypotheses by pursuing the following objectives: 1) To clone and express canine adrenal autoantigens (including 21-hydroxylase, 17alpha-hydroxylase and P450scc) and to develop a radioimmunoassay for detection of serum autoantibodies. (Year 1-2) 2) To screen sera from dogs with spontaneous and iatrogenic hypoadrenocorticism to determine the prevalence of anti-adrenal antibodies and to compare autoantibody profiles in dogs with glucocorticoid deficiency to those dogs with combined glucocorticoid and mineralocorticoid deficiency. (Year 2-3) 3) To perform a case:control association study of polymorphisms in DLA and CTLA4 candidate genes to identify susceptibility alleles (Year 3) 4) To investigate how variation in CTLA4 genotype influences CTLA-4 mRNA and protein expression (Year 3-4) PROJECT EVOLUTION As well as providing core milestones and deliverables, the project will allow the student flexibility to develop the research into new areas. In particular, they will be responsible for facilitating the established collaboration between the RVC and Universities of Manchester and Newcastle, where funding is in place to perform a genome-wide (SNP-based) association study in canine hypoadrenocorticism. Thus, as novel susceptibility genes are identified, the student will be ideally placed to exploit this information (e.g. for development of genetic tests). REFERENCE Chase K, Sargan D, Miller K, Ostrander EA, Lark KG. Understanding the genetics of autoimmune disease: two loci that regulate late onset Addison's disease in Portuguese Water Dogs. Int J Immunogenet. 2006; 33:179-84.