THE FUNCTIONAL CHARACTERISATION OF TREM2 RISK VARIANTS USING BAC-BASED EXPRESSION VECTORS

Abstract

Background:A rare coding variant in the TREM2 gene has been associated with the common late-onset form of Alzheimer’s disease (AD). This variant, which results in an arginine (R) to histidine (H) substitution at residue 47 of the TREM2 protein, is predicted to triple disease risk. Recent studies led in part by the AD team at Cardiff University have identified a second risk variant within TREM2 (data not yet published). This variant is not in linkage disequilibrium with R47H and thus appears to exert independent effects on AD risk. TREM2 is a single-pass transmembrane protein which is expressed on the surface of microglia, macrophages and dendritic cells. It forms a receptor signalling complex with DAP12 and triggers activation of immune responses. The mechanisms by which TREM2 variants contribute to AD risk are unknown. Methods: To investigate the putative pathogenic effects of TREM2 risk variants we have created BAC-based expression vectors and transfected them into human immortalised cell lines. Risk variants were generated by site-directed mutagenesis and incorporated into the BAC vectors using recombineering technology. A peptide tag was incorporated within the TREM2 open reading frame to enable us to distinguish between endogenously expressed TREM2 and the TREM2 expressed from our vectors. Vectors were transfected into microglia cells via lipofection and the levels of transgene protein determined by western blotting. Results: We have validated our TREM2 vectors by detecting robust transgene expression in HEK cells. We are currently transfecting our TREM2 expression vectors into human microglia cells. Preliminary data will be presented in this poster. Conclusions: We have created and validated novel expression vectors which can be used to determine the pathogenic consequences of putative AD risk variants.