L18.    Genetic analysis of autoimmune regulator (AIRE) haplotypes in alopecia areata.
David Wengraf, Thomas Lovewell, Mike Cork, Andrew Messenger, Andrew McDonagh, Rachid Tazi-Ahnini; Division of Genomic Medicine, University of Sheffield, Sheffield, UK.

Alopecia areata (AA) is a disorder primarily affecting the hair and nails associated with autoimmune diseases. Autoimmune Polyglandular Syndrome-1 (APS-1) is caused by loss-of-function mutations in the AIRE gene on chromosome 21. There are several associated autoimmune diseases including hypoparathyroidism, thyroid disorders, vitiligo, and diabetes mellitus. In particular, AA has ~40 times increased prevalence amongst APS-1 patients, suggesting that AIRE is a strong candidate gene for AA. We identified six different SNP’s in the AIRE gene to be used in genetic analysis. These were at positions C-103T, C4144G, T5238C, G6528A, T7215C and T11787C. SNP’s were genotyped in 295 AA patients and 363 healthy controls using RFLP analysis. The PMPLUS program was used to analyse the different haplotypes of these six polymorphisms. The haplotype analysis was repeated using tagged SNPs (C-103T, G6528A, T7215C and T11787C). The online program, Mfold (Version 3.2) was used to predict secondary mRNA structure for these four SNP haplotypes. All SNPs were in Hardy-Weinberg equilibrium in the control group. Genetic analysis of individual SNP revealed that the only significant association with AA was with the 7215C allele (p=2x10-10). Standardised coefficients of disequilibrium were calculated between all pairs of these SNPs and showed that T7215C is in complete linkage disequilibrium (LD) with T-103C and T11787C. Haplotype analysis has shown a highly significant association between AA and the haplotypes CCTGCT and CGTGCC (p= 6.05x10-6 and p=0.001 respectively), whereas CCTGTC was shown to have a protective effect (p=0.004). The haplotype anaylsis using the tagged SNPs showed two haplotypes having significant associations with AA; these were CGCT and CGCC (p=3.84x10-7 and p=3.94x10-4, respectively). The hapolotype CGTC was also shown to have a protective effect (p=2.10x10-4). The predicted mRNA structures for the CGTC, CGCC and CGCT haplotypes are different. The haplotype CGCT has the lowest free energy (deltaG-975.75 kcal/mol) suggesting a more stable mRNA structure than for the mRNA of haplotypes CGCC and CGTC (deltaG-964.42 kcal/mol and deltaG-964.77 respectively). These findings suggest that the AIRE susceptibility haplotypes may affect the stability of AIRE mRNA which could play a central role in the pathogenesis of AA.