Association Alopecia Areata | Genetics variants of the autoimmune regulator (aire) promoter : analysis of their effect on aire gene expression, activity and possible role in the pathogenesis of alopecia areata

Genetics variants of the autoimmune regulator (aire) promoter : analysis of their effect on aire gene expression, activity and 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 2004) à hauteur de 3 800 €.

Subvention débloquée le 08 janvier 2005.

Compte rendu fourni le 24 janvier 2006.

COMPTE RENDU DETAILLE (texte intégral)

Scientific Report


Alopecia areata is an organ specific autoimmune disease (Messenger & Simpson. 1997) characterised by patches of non-scarring hair loss from any hairy part of the body, and sometimes involves pitting of an individuals nails as well (Sinclair et al. 1999).

Alopecia areata has been estimated to affect about 0.15% of the UK population, with the majority of people affected only developing the mild form of the disease, where hair is lost in patches. However, approximately 30% of the population develop more severe forms of the disease, which can include loss of all hair from the scalp, or even complete loss of all hair from the body (Sinclair et al. 1999).

Human lesional skin has been shown to regrow hair when grafted onto SCID (severe combined immunodeficiency) mice. However, when T-lympocytes extracted from alopecia areata patients were injected into these mice, the hair loss recurred, indicating a role of T-lymphocytes in the pathogenesis of alopecia areata (Gilhar et al. 1998). This is supported by the observation that dystrophic anagen hair follicles have been shown to have peri- and intra-follicular inflammation caused by T-lymphocytes infiltration (Van Scott. 1958, Perret et al. 1984, and Ranki et al. 1984)

Alopecia areata has been found to affect approximately 30% of patients suffering from autoimmune polyglandular syndrome 1 (APS-1, a.k.a. APECED), with approximately 80-90% of the alopecia areata/APS-1 patient population suffering from the more severe forms of alopecia areata (Betterle. 1998 and personal communication from P. Peterson). This strongly suggests that there is an association between APS-1 and alopecia areata. APS-1 is an autosomal recessive disease caused by loss of function mutations in the AutoImmune REgulator (AIRE) gene (Scott et al. 1998, Nagamine et al. 1997, and The Finnish-German APECED consortium, 1997).

Mature Aire protein is about 57.7 kDa, is expressed in a tissue specific manner, particularly thymic medullary epithelial cells (TMEC’s), and is localised to the nucleus (Nagamine et al. 1997, The Finnish-German APECED consortium. 1997, Heino et al. 1999, Heino et al. 2000, Pitkänen et al. 2000, and Adamson et al. 2004). Aire is thought to be a transcription factor, partly based on its homology with other transcription factors and its DNA binding ability (Kumar et al. 2001). This is further backed up by AIRE knockout mice study by Anderson et al. (2002), which indicated that Aire was involved in regulating the expression of a selection of peripheral specific genes in the thymus. Liston et al. (2004) also demonstrated that Aire regulated gene expression in a dose dependent manner.

TMEC’s appear to play a role in the process of clonal deletion or inactivation of T-lymphocytes that are self-reactive (Naquet et al. 1999, and Kishimoto et al. 2000), and AIRE gene deficient transgenic mice have been shown to a defect in this process (Liston et al. 2003).

It is our hypothesis that a reduction in the overall activity of Aire in the thymus, due to either reduced levels of Aire or from a reduced efficacy as a transcription factor could lead to a reduction in the expression of Aire-regulated genes in the thymus. This could lead to a knock-on effect of self-reactive immature T-lymphocytes escaping the clonal deletion process in the thymus, which in turn could lead to an autoimmune response.

We looked at whether polymorphisms in the AIRE promoter could lead to reduced expression of Aire, and whether these could be susceptibility factors for alopecia areata.


The first 600bp upstream of the AIRE transcription start site (TSS), which includes the minimal promoter region, was screened for predicted transcription factor binding sites using the Match 1.0 programme (available at Predicted sites for some transcription factors were excluded based on their expression profile, whereas other sites have previously been confirmed by Murumägi et al. (2003).


We used WAVE denaturing HPLC to screen 64 individual DNA samples (34 controls and 30 cases) for the presence of SNPs. Currently three SNP’s have been screening the first 600bp upstream of the AIRE TSS for SNPs using dHPLC. Two SNPs have been identified in this region. One at position –103 which is within the minimum promoter region of the AIRE gene and could affect the activity of AIRE promoter. The other SNP was located in the proximal region of the AIRE promoter at position –528. The AIRE –528 has been identified in two of the samples (fig.1), which was confirmed and characterised by DNA sequencing. A PCR-RFLP assay were designed to determine the frequency of –103 and –528 in both control and diseases groups.


Figure 1. Wave dHPLC trace showing samples containing novel SNP, -528A (pink and purple traces), along with samples lacking this SNP (other traces).

In preparation for reporter gene assays, different promoter haplotypes have been cloned and then sub-cloned into the pGL3-basic reporter vector. We are only considering SNP’s that have been validated for the different haplotypes, and at present have constructs with the following haplotypes; -528G/-103C, -528G/-103T and -528A/-103C. A negative control construct has also been produced. It contains the -528G allele, but will have the region -315 to -77 removed, as this is most of the minimal promoter region, but the construct stills contain the TATA box so that a basal level of transcription can still occur. These different promoter haplotypes have been sub-cloned with the firefly luciferase gene into a pcDNA5/FRT expression vector that had its Human Cytomegalovirus (CMV) immediate early promoter removed.

Work in progress

We have started screening our collection of alopecia areata patient DNA samples and control DNA samples using our PCR-RFLP assay for both AIRE polymorphisms at positions –528 and –103, in order to determine the frequency of this allele, and to investigate whether the SNP is associated with alopecia areata.

To investigate potential effects of SNPs in the AIRE promoter, we intend to investigate the expression of the firefly luciferase gene in thymic cells and COS-7 cells, when under the control of promoters with different haplotypes. The different haplotypes will be -528G/-103C, -528G/-103T, -528A/-103C, -528A/-103T and the negative control construct. We will use the Invitrogen “Flp-In” system to insert a single copy of the AIRE promoter/firefly luciferase construct into the genome of the host eukaryotic cell. Thus, any differences between luciferase expression levels in a cell line will be due to changes in the promoter, as there will only be one copy of promoter/luciferase construct per cell, and they will all be in the same genomic environment.


  1. Messenger, R. and Simpson, N. 1997.   Alopecia areata. In: Diseases of the Hair and Scalp, 3rd edition, pp 338–369. Edited by R. Dawber. Oxford: Blackwell Science Ltd.
  2. Sinclair, R.D., Banfield, C.C. and Dawber R.P.R. 1999. Alopecia areata. In: Handbook of diseases of the hair and scalp, pp. 75-84. Blackwell Science.
  3. Gilhar, A., Ullmann, Y, Berkutzki, T., Assy, B., and Kalish, R.S. 1998. Autoimmune hair loss (alopecia areata) transferred by T lymphocytes to human scalp explants on SCID mice. J Clin Invest, 101:62-67.
  4. Van Scott, E.J. 1958. Morphologic changes in pilosebaceous units and anagen hairs in alopecia areata. J Invest Dermat. 31, 35-43.
  5. Perrett, C., Wiesner-Menzel, L., and Happle, R. 1984. Immunohistochemical analysis of T-cell subsets in the peribulbar and intrabulbar infiltrates of alopecia areata. Acta Derm-Venereol, 64:26-30.
  6. Ranki, A, Kianto, U, Kanerva, L, Tolvanen, E and Johansson, E, 1984. Immunohistochemical and electron microscopic characterisation of the cellular infiltrate in alopecia (areata, totalis and universalis). J Invest Dermat, 83:7-11.
  7. Betterle, C., Greggio, N. A. and Volpato, M. 1998. Clinical review 93: Autoimmune polyglandular syndrome type 1. J Clin EndocMetab 83, 1049-1055.
  8. Scott, HS, Heino, M, Peterson, P, Mittaz, L, Lalioti, MD, Betterle, C, Cohen, A, Seri, M, Lerone, M., Romeo, G., Collin, P., Salo, M., Metcalfe, R., Weetman, A., Papasavvas, M-P., Rossier, C., Ngamine, K., Kudoh, J., Shimizu, N., Krohn, K.E.J., and Antonarakis, S.E. 1998. Common mutations in autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy patients of different origins. Mol Endoc, 12:1112-1119.
  9. Nagamine, K, Peterson, P, Scott, HS, Kudoh, J, Minoshima, S, Heino, M, Krohn, KJE, Lalioti, MD, Mullis, PE, Antonarakis, SE, Kawasaki, K, Asakawa, S, Ito, F, and Shimizu, N, 1997. Positional cloning of the APECED gene. Nat Genet, 17:393-398.
  10. The Finnish-German APECED consortium, 1997. An autoimmune disease, APECED, caused by mutations in a novel gene featuring two PHD-type zinc finger domains. Nat Genet, 17:399-403.
  11. Heino, M., Peterson, P., Kudoh, J., Nagamine, K., Lagerstedt, A., Ovod, V., Ranki, A., Rantala, I., Nieminen, M., Tuukkanen, J., Scott, H.S., Antonarakis, S.E., Shimizu, N., and Krohn, K. Autoimmune regulator is expressed in the cells regulating immune tolerance in thymus medulla. Bioch Biophy Res Comm. 257, 821-825.
  12. Heino, M., Peterson, P., Sillanpaa, N., Guerin, S., Wu, L., Anderson, L., Scott, H. S., Antonarakis, S.E., Kudoh, J., Shimizu, N., Jenkinson, E.J., Naquet, P., and Krohn, K.J.R. RNA and protein expression of the murine autoimmune regulator gene (Aire) in normal, RelB-deficient and in NOD mouse. Eur J Immunol. 30, 1884-1893.
  13. Pitkanen, J., Doucas, V., Sternsdorf, T., Nakajima, T., Aratani, S., Jensen, K., Will, H., Vahamurto, P., Ollila, J., Vihinen, M., Scott, H.S. Antonarakis, S.E., Kudoh,J., Shimizu, N., Krohn, K., and Peterson, P. 2000. The autoimmune regulator protein has transcriptional transactivating properties and interacts with the common coactivator CREB-binding protein. J Biol Chem. 275:16802-16809.
  14. Adamson, K.A., Pearce, S.H.S., Lamb, J.R., Seckl, J.R., and Howie, S.E.M., 2004. A comparative study of mRNA and protein expression of the autoimmune regulator gene (Aire) in embryonic and adult murine tissues. J Pathol, 202:180-187.
  15. Kumar, P.G., 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. J Biol Chem, 276:41357-41364.
  16. 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.
  17. Liston, A., Gray, D.H., Lesage, S., Fletcher, A.L., Wilson, J., Webster, K.E., Scott, H.S., Boyd, R.L., Peltonen, L., and Goodnow, C.C. 2004. Gene dosage-limiting role of Aire in thymic expression, clonal deletion and organ-specific autoimmunity. J Exp Med. 200(8):1015-26
  18. Naquet, P., Naspetti, N. and Boyd, R. 1999. Development, organisation and function of the thymic medulla in normal, immunodeficient or autoimmune mice. Sem Immunol. 11, 47 – 55.
  19. Kishimoto, H. and Sprent, J. 2000. The thymus and central tolerance. Clin Immunol. 95, S3-7.
  20. Liston, A., Lesage, S., Wilson, J., Peltonen, L., and Goodnow, C.C., 2003. Aire regulates negative selection of organ-specific T cells. Nat Immunol, 4:350-354.
  21. Murumägi, A., Vähämurto, P. and Peterson, P. 2003. Characterisation of regulatory elements and methylation pattern of AIRE (autoimmune regulator) promoter. J Biol Chem. 278, 19784 – 19790.