22.    A study on the effect of genetic variants of the autoimmune regulator (AIRE) promoter, on AIRE activity and downstream AIRE-regulated genes. 
Thomas Lovewell(1), Mike Cork(1&2), David Wengraf(1), Andrew Messenger(2), Andrew McDonagh(2), Rachid Tazi-Ahnini(1&2). 1.Division of Genomic Medicine, University of Sheffield, Sheffield S10 2RX, 2.Dermatology Immunogenetics Group, University of Sheffield, Royal Hallamshire Hospital, Sheffield, UK.

Alopecia areata has been found to occur ~40 times more commonly and with greater severity amongst Autoimmune Polyglandular Syndrome-1 (APS-1) patient populations. APS-1 is a monogenic disease, caused by loss of function mutations in the Autoimmune Regulator (AIRE) gene. The AIRE gene is primarily expressed in the thymus and is believed to modulate promiscuous gene expression, which in turn, seems to be involved in T-cell negative selection. It has been shown that the function of Aire is dose dependent, thus we intend to investigate whether natural polymorphisms in the AIRE promoter region affect the level of AIRE transcription. We used WAVE denaturing HPLC to screen the first 591bp up-stream of the AIRE transcription start site from 32 alopecia areata patients and 32 control samples. Using the MatInspector 2.1 and the rVista 2.0 programmes we have investigated the predicted binding sites of Aire protein to the promoters of the human and mouse homologues of casein alpha S1, keratin 10 and keratin 12. We identified the -103C/T SNP, found within the minimal promoter region, and the -528G/A SNP, found upstream of the minimal promoter region. We cloned the -528G/-103C, -528G/-103T and -528A/-103C promoter haplotypes up-stream of a firefly luciferase reporter gene. We are currently performing reporter gene assays in thymic cells TEC 1A3 in order to measure the efficiency of the different promoter variants. Binding sites were identified in the analysed promoters and were conserved between mouse and human. We have cloned these promoters into the pCR2.1 vector, and are currently using DNase I protection assays and electrophoretic mobility shift assays to investigate the physical binding of Aire protein to these promoters. Our main aim is on one hand to analyse the effect of SNPs on the AIRE promoter activity and on the other hand to dissect the mechanisms by which Aire regulate gene expression.