Auteur : Dr. Rachid TAZI-AHNINI (Biomedical Genetics Division of Genomic Medicine University of Sheffield Medical School Royal Hallamshire Hospital SHEFFIELD ENGLAND).
Travail subventionné par l’AAA (subvention 2000-2001) à hauteur de 77 000 F.
Subvention débloquée le 24 juin 2001.
Compte rendu fourni le 2 avril 2002.
COMPTE RENDU DETAILLE (texte intégral)
Role of the Autoimmune Regulator (AIRE) Gene
in Alopecia Areata
Final Report to Association Alopecia Areata 2001-2002
There is a high frequency of alopecia areata (approximately 40 %) in patients with the autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy syndrome (APECED), a recessive disorder caused by mutations of the autoimmune regulator (AIRE) gene located on chromosome 21. We believe that the AIRE gene is a strong candidate for alopecia areata. The aim is to show whether or not variants within the AIRE gene are associated with alopecia areata in index cases and families.
First we screened the 14 exons of the AIRE gene for single nucleotide polymorphisms (SNPs). Now we using assays for these variants to perform association studies using both case-control and transmission disequilibrium test (TDT) methods.
Primers we designed within the introns flanking each exon from the published AIRE gene sequence (GeneBank accession number : AB00684). PCR conditions were optimised using a range of magnesium concentrations at different alignment temperatures. DNA from 9 samples including 5 different ethnic origins were amplified. PCR products were then purified from agarose gel and automatically sequenced using an ABI 377 machine.
First six months report
Sequence alignments using GeneJockey II software allowed us to identify 23 SNPs, 11 of which are within the introns and 12 within the exons. The variants at positions G8723C ant T9827C gave rise to an amino acid change S278R and V301A respectively (Table 1). Our results confirmed the findings of Scott et al. in relation to the SNPs at positions 7094, 8385, 8723, 8924 and 16366. We have also identified a number of previously unreported variants (table 1).
Two variants within the coding sequence producing amino acid changes (S278R and V301A) have been confirmed in many samples. These variants are particulary interesting because of their potential functional relevance. Interestingly, valine at position 301 is located within the first PHD finger domain of Aire protein and this variant may have an effect on Aire protein in the chromatin-mediated regulation of transcriptional activity.
Table 1 : Positions of SNPs within the AIRE gene
|2||No SNPs detected|
|12||No SNPs detected|
|13||No SNPs detected|
*SNPs need to be confirmed
In the second tranche of this project we have concentrated on the two confirmed nonsynonymous substitutions at positions G961C and T1029C because they might have functional relevance. We developed assays to genotype these variants in unrelated alopecia areata patients and controls. If association is confirmed we will test transmission of the variant(s) associated with disease in families.
1) AIRE Exon 7
Sequencing of exon 7 detected a polymorphism at genomic position 8723, cDNA position 961. This is a GC polymorphism which leads to the peptide change S278R. Exon 7 of the AIRE gene was amplified in 233 Caucasian samples, 107 of which were alopecia areata patients, the remaining 126 were healthy controls. Each PCR product was split into two samples, one of these samples was digested with Fnu4H1 restriction endonuclease, and the other with NlaIV restriction endonuclease.
Allelic distribution of the AIRE C961G variant in control and disease groups has been determined in 126 controls and 107 unrelated patients. The results are summarised in table 2. There is an increase in allelic frequency of allele 2 (961G) in patients compared to the control group 0.12 and 0.10 respectively. We then grouped individuals heterozygotes for the rare allele with those homozygotes for the same allele and calculated the Odds ration OR = 1.35 (95 % C.I. (0.71, 2.55)). The OR lower value at 95 % confidence interval wass less than 1, suggesting the result is not statistically significant. When we divided patients according to the disease severity we found an increase the AIRE variant 961G allele frequency in patients with alopecia universalis compared to controls (0.13 and 0.10 respectively).
Table 2 : Allelic distribution of AIRE C961G in 126 healthy controls and 107 alopecia areata patients.
2) AIRE Exon 8
Sequencing has detected a polymorphism in exon 8. The genomic position is 9817, the cDNA position 1029. This is a TC polymorphism that leads to the peptide change V301A. Several essays have been tried for this polymorphism. Digestion with Fnu4H1, Tha1, BstU1 and Aci1 did not produce results that could be used. Currently, PCR is being used to create a recognition site for Mlu1 that includes this polymorphism.
We found 21 SNPs in AIRE gene and two in the 3’region. Nine SNPs are located in the intron-exon junctions and 12 within the exons. Six of the exonic substitutions are non-synonymous and have potential functional relevance. We initially concentrated on two AIRE variants C961G and T1029C respectively because of their potential importance in Aire protein. The polymorphism at position 961 gave a change from a polar amino acid to a charges one (S278R). Moreover, the AIRE variant at position T1029C giving an amino acide change from valine at position 301 is located within the first PHD finger domain of Aire protein and this variant may have an effect on Aire protein in the chromatin-mediated regulation of transcriptional activity. There is a slight increase in the frequency of the rare allele (961 G) in patients. When we divided patients according the disease severity, we found an increase the AIRE variant 961G allele frequency in patients with alopecia universalis compared to controls (0.13 and 0.10 respectively). However, equivalent frequency to the control group was found for the AIRE variant 961G allele in the mild disease group including patchy alopecia areata and alopecia totalis (0.097).
In conclusion we found a suggestive association between the rare allele of AIRE polymorphism at position 961 in both the overall data set, and in alopecia universalis. Therefore, we believe that a larger sample is needed to determine wheter or not this polymorphism is actually associated with alopecia areata.
We have also developed an assay to genotype the AIRE T1029C polymorphism and are currently genotyping samples from control and patient groups for this polymorphism. The results will be hopefully presented at the 20 th World Congress of Dermatology 1st-5th of July 2002 in Paris.
Rachid Tazi-Ahnini, PhD
Biomedical Genetics Project, Dermatology group
Divison of Genomic Medicine
University of Sheffield Medical School D floor
Royal Hallamshire Hospital Beech Hill Road Sheffield S. Yorks S10 2RX UK