Alopecia areata is an immune-mediated disorder, occurring with the highest observed frequency in the rare recessive autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) syndrome caused by mutations of the autoimmune regulator (AIRE) gene on chromosome 21q22.3. We have previously detected association between alopecia areata and a single nucleotide polymorphism (SNP) in the AIRE gene in patients without APECED, and we now report the findings of an extended examination of the association of alopecia areata with haplotype analysis including six SNPs in the AIRE gene: C-103T, C4144G, T5238C, G6528A, T7215C and T11787C. In Caucasian groups of 295 patients and 363 controls, we found strong association between the AIRE 7215C allele and AA [P = 3.8 x 10(-8), OR (95% CI): 2.69 (1.8-4.0)]. The previously reported association between AA and the AIRE 4144G allele was no longer significant on correction for multiple testing. The AIRE haplotypes CCTGCT and CGTGCC showed a highly significant association with AA [P = 6.05 x 10(-6), 9.47 (2.91-30.8) and P = 0.001, 3.51 (1.55-7.95), respectively]. To select the haplotypes most informative for analysis, we tagged the polymorphisms using SNPTag software. Employing AIRE C-103T, G6528A, T7215C and T11787C as tag SNPs, two haplotypes were associated with AA; AIRE CGCT and AIRE CGCC [P = 3.84 x 10(-7), 11.40 (3.53-36.9) and P = 3.94 x 10(-4), 2.13 (1.39-3.24) respectively]. The AIRE risk haplotypes identified in this study potentially account for a major component of the genetic risk of developing alopecia areata.
The link between vitiligo and AIRE raises the possibility that defective skin peripheral antigen selection in the thymus is involved in the changes that result in melanocyte destruction in this disorder.
BackgroundThe autoimmune regulator (AIRE) is expressed in the thymus, particularly in thymic medullary epithelial cells (mTECs), and is required for the ectopic expression of a diverse range of peripheral tissue antigens by mTECs, facilitating their ability to perform negative selection of auto-reactive immature T-cells. The expression profile of peripheral tissue antigens is affected not only by AIRE deficiency but also with variation of AIRE activity in the thymus.Method and ResultsTherefore we screened 591bp upstream of the AIRE transcription start site including AIRE minimal promoter for single nucleotide polymorphism (SNPs) and identified two SNPs -655R (rs117557896) and -230Y (rs751032) respectively. To study the effect of these variations on AIRE promoter activity we generated a Flp-In host cell line which was stably transfected with a single copy of the reporter vector. Relative promoter activity was estimated by comparing the luciferase specific activity for lysates of the different reporter AIRE promoter-reporter gene constructs including AIRE-655G AIRE-230C, AIRE-655G AIRE-230T and AIRE-655A AIRE-230C. The analysis showed that the commonest haplotype AIRE-655G AIRE-230C has the highest luciferase specific activity (p<0.001). Whereas AIRE-655G AIRE-230T has a luciferase specific activity value that approaches null. Both AIRE promoter polymorphic sites have one allele that forms a CpG methylation site which we determined can be methylated in methylation assays using the M.SssI CpG methyltransferase.Conclusion AIRE-230Y is in a conserved region of the promoter and is adjacent to a predicted WT1 transcription factor binding site, suggesting that AIRE-230Y affects AIRE expression by influencing the binding of biochemical factors to this region. Our findings show that AIRE-655GAIRE-230T haplotype could dramatically alter AIRE transcription and so have an effect on the process of negative selection and affect susceptibility to autoimmune conditions.
Autoimmune regulator (AIRE) regulates promiscuous expression of tissue-restricted antigens in medullary epithelial cells (mTEC) of the thymus. To understand the diverse effects of AIRE, it is crucial to elucidate the molecular mechanisms underlying the process of AIRE-regulated gene expression. In this study, we generated a recombinant AIRE expression variant of the TEC 1A3 human cell line, TEC 1A3 AIREhi, to determine genes targeted by AIRE, and using microarray analysis, we identified 482 genes showing significant differential expression (P < 0.05; false discovery rate <5%), with 353 upregulated and 129 downregulated by AIRE expression. Microarray data were validated by quantitative PCR, confirming the differential expression of 12 known AIRE-regulated genes. Comparison of AIRE-dependent differential expression in our cell line model with murine datasets identified 447 conserved genes with a number of transcription regulatory interactions, forming several key nodes, including STAT1, which had over 30 interactions with other AIRE-regulated genes. As STAT1 mutations cause dominant chronic mucocutaneous candidiasis and decreased STAT1 levels in monocytes of autoimmune polyglandular syndrome 1 (APS-1) patients, it was important to further characterize AIRE–STAT1 interactions. TEC 1A3AIREhi were treated with the STAT1 phosphorylation inhibitors fludarabine and LLL3 showed that phosphorylated STAT1 (p-STAT1) was not responsible for any of the observed differential expression. Moreover, treatment of TEC 1A3 AIREhi with STAT1 shRNA did not induce any significant variation in the expression of unphosphorylated STAT1 (U-STAT1) downstream genes, suggesting that these genes were directly regulated by AIRE but not via U-STAT1. The novel model system we have developed provides potential opportunities for further analysis of the pathogenesis of (APS-1) and the wider roles of the AIRE gene.
Autoimmune polyglandular syndrome type 1 (APS-1) is an inherited autosomal disorder. The most common clinical features of the disease include adrenocortical failure, hypoparathyroidism (HP), and chronic mucocutaneous candidiasis (CMC). APS-1 is caused by mutations in the autoimmune regulator (AIRE) gene. AIRE is a transcriptional factor involved in the regulation of thousands of genes in the thymus. It facilitates central tolerance by promoting the ectopic expression of tissue-specific antigens (TSAs) in medullary thymic epithelial cells (mTECs), leading to the deletion of self-reactive thymocytes. Several Aire-deficient mice were developed separately, on different backgrounds; seven published Aire knockout mice show a variety of phenotypes depending on the strain used to generate the experimental model. The first Aire-deficient mice were generated on a “black 6” background almost 20 years ago. The model showed mild phenotype with relatively modest penetrance compared to models generated on BALBc or NOD backgrounds. The generation of all these experimental models is crucial for development and testing new therapeutics as well as reading the response to treatments.
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