Title : Application of TaqMan probes to study the expression kinetics of celiac disease-related toxic epitopes and their presence in the gDNA of contrasted spelt accessions
Abstract:
Gluten is the water insoluble protein fraction found in the flour of several cereals such as wheat and spelt. Its ingestion is responsible of celiac disease (CD) in genetically predisposed individuals (1-2% of the human population). Alpha-gliadins are a class of proteins of the gluten fraction encoded by a multigene family which play a key role in this pathogenesis with 4 main toxic epitopes recognized by the immune system. These epitopes display a full toxicity in their canonical form but amino acid substitutions or deletions naturally occur in some epitope sequences which lower or suppress their toxicity. On this basis, TaqMan probes targeting only the canonical form of each epitope have previously been developed and epitope expression data in contrasted spelt accessions have been collected. In this study, these TaqMan probes have been applied to the genomic DNA (gDNA) of the same spelt accessions. The results are globally correlated to epitope expression levels previously measured on cDNA samples with the same probes, despite the high proportion of pseudogenes displayed in α-gliadin sequences. Moreover, a kinetic study of the epitope expression was carried out to evaluate the best moment to apply these probes. The analysis was performed with a European and an Asian spelt accession, given that genetic differences between spelts from these two origins have previously been underlined by several authors. The results highlighted interesting differences, the expression peak being observed sooner in the European than in the Asian spelt. The results obtained with gDNA samples and from the kinetic analysis provide useful information with a view to studying the toxic content of spelt or bread wheat accessions and to tracking it in accessions involved in breeding programs aiming at developing safer varieties for CD patients.
