Association Alopecia Areata | Genetic and functional analysis of the autoimmune regulator (aire) promoter and its possible role in the pathogenesis of alopecia areata

Genetic and functional analysis of the autoimmune regulator (aire) promoter and its possible role in the pathogenesis of alopecia areata

Auteur : Dr. Rachid TAZI-AHNINI (Biomedical Genetics – Division of Genomic Medicine – University of Sheffield Medical School – The Royal Hallamshire Hospital – Beech Hill Road – Sheffield S10 2RX ENGLAND).

Travail subventionné par l’AAA (subvention 2003) à hauteur de 12 000 €.

Subvention débloquée le 31 décembre 2003.

Compte rendu fourni le 12 novembre 2004.


COMPTE RENDU DETAILLE (texte intégral)

Report 2003


Alopecia areata is considered to be an autoimmune disease mediated by activated T lymphocytes. The lifetime risk of approximately 1.4% in the general population is increased to 40% or more in the autoimmune polyendocrinopathy-candidiasis ectodermal dystrophy (APECED) syndrome. APECED is caused by mutations in the autoimmune regulator (AIRE) gene on chromosome 21 resulting in autoimmunity directed against endocrine and other organs. We have recently demonstrated strong association between a potentially functional genetic variant in the AIRE gene and alopecia universalis outwith the context of APECED (Tazi-Ahnini et al. 2002). Evidence from a mouse knockout model suggests that the level of AIRE expression is pivotal in regulating a number of genes that could be directly involved in the pathogenesis of APECED associated diseases including alopecia areata.

Aim of the study

In this project the AIRE promoter region will be screened for genetic variants. AIRE promoter alleles will then be investigated in an in-vitro system to measure their expression activities. AIRE promoter alleles showing variation of promoter activity could have a direct effect on the level of expression of downstream genes. Variation in the expression of the AIRE promoter could be directly associated with AIRE transcriptional activity. Finally, we will perform a case-control study of the functionally variant AIRE promoter alleles to assess their possible role in determining disease status.

Results and current work

Part of the AIRE promoter region sequence (first 400 bp upstream of the transcription start site) was analysed using the Match 1.0 programme (available from http// This programme used the TRANSFAC 6.0 database to predict potential binding sites of transcription factors within the AIRE promoter. We narrowed down the number of hits by using a minimum core similarity of 0.9 and minimum matrix similarity of 0.85, and by removing hits from transcription factors that are not expressed in the thymus. The predicted sites were then combined with confirmed binding sites (Murumägi, et al. 2003) to produce a diagram of the promoter region (figure 1), indicating the position of the identified transcription factor binding sites within it. This analysis was performed in order to identify any potential effects of SNP’s discovered in the promoter region.

Figure 1.          Positions of predicted and confirmed transcription factor binding sites on the AIRE promoter

relative to the transcription start site (1)

PCR amplification, cloning and sequencing of the promoter region from a selection of alopecia areata patient samples and control samples was undertaken to search for novel SNP’s and to generate clones of promoter regions with different haplotypes for future work. Nine novel SNPs were identified during this investigation, but RFLP analysis to confirm the validity of these SNPs indicated that six were artefacts. The three SNPs which were validated by RFLP are –524 C/G, -523 G/A and –103 C/T. This later is located in a hot spot region which contains sequences for many potential transcription factor sites (Fig1).

As a result, we have altered our strategy to screen for SNP’s. We have recently developed a dHPLC assay to screen the promoter region in order to identify the presence of SNPs within this region. We have started to screen samples, and any that are identified as containing a SNP will be sequenced in order to determine the position and type of SNP, and then validated using RFLP. This method should reduce the number of false positives compared to our previous strategy.

Future work

Within the next few weeks, we should have completed our dHPLC screening of the AIRE promoter from at least 30 samples, the minimum size required to have 95% confidence that all SNP’s (with the frequency of rare allele >0.1) in the region will have been detected and any SNPs discovered will have been characterised. Our next aim is to clone the AIRE promoter from individuals with known genotypes in order to generate catalogue of promoters with different haplotypes. These promoters will then be inserted in front of a firefly luciferase gene, for use in reporter gene experiments.

Our next aim will be generating the required cell lines and vectors for reporter gene assays. We intend to use the Invitrogen Flp-In system to introduce a single copy of the AIRE promoter/firefly luciferase gene construct into a mammalian host cell-line. The advantages of this system, is that all different AIRE haplotypes will be in the same position in the genome, and same copy number, so that the only difference in firefly luciferase expression will be due to the different haplotypes. The preparation for this reporter gene assay will likely take several months due to some of the steps involved, and that each step needs confirmation that it was successful.

Some of the processes involved include:

  • Generating a stable thymus cell-line that contains a single FRT recombination site.
  • Generating insertion vectors for each AIRE promoter/firefly luciferase gene construct that has a different promoter haplotype.
  • Transforming thymus cells (generated above) with promoter/luciferase gene/insertion vector constructs, and then selecting for successful recombinants.

Once these products have been generated, the reporter assay can take place. As controls for these experiments, we intend to use COS-7 cells as a control cell-line, and AIRE promoter/luciferase gene constructs that either lack the promoter entirely, or has had part of the promoter selectively removed.

Finally, we will screen alopecia areata patient samples and control group samples for any SNPs that we identify as having an effect on the efficiency of the AIRE promoter, in order to see if there is an association between the SNP and alopecia areata.


  1. Anderson, MS, Venanzi, ES, Klein, L, Chen, Z, Berzins, SP, Turley, SJ, von Boehmer, H, Bronson, R, Dierich, A, Benoist, C, and Mathis, D, 2002. Projection of an immunological self shadow within the thymus by the Aire protein. Science, 298:1395-1401.
  2. Kumar, PG, Laloraya, M, Wang, C-Y, Ruan, Q-G, Davoodi-Semiromi, A, Kao, K-J, and She, J-X, 2001. The autoimmune regulator (AIRE) is a DNA-binding protein. Journal of Biological Chemistry, 276:41357-41364.
  3. Murumägi, A., Vähämurto, P. & Peterson, P. (2003). Characterisation of regulatory elements and methylation pattern of AIRE (autoimmune regulator) promoter. Journal of Biological Chemistry. 278, 19784 – 19790.
  4. Sillanpää, N., Magureanu, C.G., Murumägi, A., Reinikainen, A., West, A., Manninen, A., Lathi, M., Ranki, A., Saksela, K., Krohn, K., Lahesmaa, R. and Peterson, P., 2004. Autoimmune regulator induced changes in the gene expression profile of human monocyte-dendritic cell-lineage. Molecular Immunology. 41: 11185-1198.