Methylation Status of Differentially-Expressed Genes in Equine Common Variable Immunodeficiency

Common Variable Immunodeficiency (CVID) is a late-onset primary immunodeficiency that occurs in horses and humans.  Circulating B lymphocytes become depleted in CVID-affected horses, resulting in decreased serum immunoglobulin concentrations and recurrent bacterial infections.  No shared familial history has been identified in the 30 equine CVID cases diagnosed to date.  Our laboratory has demonstrated that B lymphocyte differentiation is blocked at the pro-B cell stage in the bone marrow of affected horses.  In particular, PAX5 mRNA expression is markedly decreased in the bone marrow of CVID-affected horses (p < 0.01).  PAX5 is critical for successful B lymphocyte differentiation, in that it promotes B cell-specific gene expression (i.e., CD19) and blocks genes specific for other lineages (i.e., NOTCH1 for T lymphocytes).  We hypothesized that loss of PAX5 gene expression would be associated with increased PAX5 gene methylation in the bone marrow of CVID-affected horses.  To this end, regulatory elements in the PAX5 gene were amplified from bisulfite-treated bone marrow genomic DNA of CVID-affected and healthy horses.  PAX5 regulatory elements had significantly more methylation in 8 of 9 CVID-affected horses (p=0.0000).  Preliminary analysis of the CD19 promoter region indicates that CVID-affected horses also harbor differential methylation upstream of the CD19 gene (p=0.0042).  The acquisition of aberrant methylation in a B lineage progenitor cell is consistent with the late-onset and progressive nature of CVID, as well as the lack of shared familial history between affected horses.  Future studies will investigate the upstream factors responsible for maintaining the B lymphocyte lineage epigenome.