Caracterización de nuevas variantes alélicas de prolaminas en Triticíneas: potencial para la selección de variedades no tóxicas para celíacos

El trigo harinero (Triticum aestivum) es parte de la alimentación básica y constituye una de las principales fuentes de proteínas de la dieta humana. Entre estas proteínas del trigo, el 80% constituyen las proteínas del gluten, estas son causantes de la enfermedad celiaca (EC) en el 0,7 al 2% en la población (Rewers, 2005). La EC es una patología crónica caracterizada por una lesión del intestino delgado (Catassi, 2005). El desarrollo de la EC implica la activación de células T CD4+ reactivas al gluten, que reaccionan ante los péptidos particulares del gluten reconocidos por los HLA-DQ2 o HLA-DQ8. De entre todos los péptidos relacionados con la EC, el 33-mer, presente en las -gliadinas de trigo, es el más inmunogénico ya que contiene seis epítopos superpuestos con altas propiedades estimulatorias (Shan et al. 2002). También existen otros tres péptidos más sobre las -gliadinas, que son el p31-43 que es capaz de desencadenar la respuesta inmune innata, el DQ2.5-gli-3 que solapa parcialmente con el 33-mer y el DQ8-glia-1 que se encuentra en la región C-terminal. Además hay otros péptidos inmunogénicos sobre las fracciones de las y -gliadinas. El único tratamiento efectivo para la EC es la exclusión completa y permanente del gluten de la dieta. En esta tesis se hará un estudio exhaustivo del fragmento que contiene los péptidos más inmunogénicos de las -gliadinas, también se analizará el contenido de gluten y como ha cambiado con la domesticación, para lo cual se dispone de colecciones de trigos, Aegilops, cebada y centeno. Finalmente estudiaremos la seguridad de una línea transgénica con bajo contenido en gliadinas

The Dietary Intervention of Transgenic Low-Gliadin Wheat Bread in Patients with Non-Celiac Gluten Sensitivity (NCGS) Showed No Differences with Gluten Free Diet (GFD) but Provides Better Gut Microbiota Profile

The study evaluated the symptoms, acceptance, and digestibility of bread made from transgenic low-gliadin wheat, in comparison with gluten free bread, in Non-coeliac gluten sensitivity (NCGS) patients, considering clinical/sensory parameters and gut microbiota composition. This study was performed in two phases of seven days each, comprising a basal phase with gluten free bread and an E82 phase with low-gliadin bread. Gastrointestinal clinical symptoms were evaluated using the Gastrointestinal Symptom Rating Scale (GSRS) questionnaire, and stool samples were collected for gluten immunogenic peptides (GIP) determination and the extraction of gut microbial DNA. For the basal and E82 phases, seven and five patients, respectively, showed undetectable GIPs content. The bacterial 16S rRNA gene V1-V2 hypervariable regions were sequenced using the Illumina MiSeq platform and downstream analysis was done using a Quantitative Insights into Microbial Ecology (QIIME) pipeline. No significant differences in the GSRS questionnaires were observed between the two phases. However, we observed a significantly lower abundance of some gut genera Oscillospira, Dorea, Blautia, Bacteroides, Coprococcus, and Collinsella, and a significantly higher abundance of Roseburia and Faecalibacterium genera during the E82 phase compared with the basal phase. The consumption of low-gliadin bread E82 by NCGS subjects induced potentially positive changes in the gut microbiota composition, increasing the butyrate-producing bacteria and favoring a microbial profile that is suggested to have a key role in the maintenance or improvement of gut permeability.España, MINECO Projects AGL2013-48946-C3-1-R, AGL2013-48946-C and AGL2016-80566-

Low-gluten, nontransgenic wheat engineered with CRISPR/Cas9

Coeliac disease is an autoimmune disorder triggered in genetically predisposed individuals by the ingestion of gluten proteins from wheat, barley and rye. The a-gliadin gene family of wheat contains four highly stimulatory peptides, of which the 33-mer is the main immunodominant peptide in patients with coeliac. We designed two sgRNAs to target a conserved region adjacent to the coding sequence for the 33-mer in the a-gliadin genes. Twenty-one mutant lines were generated, all showing strong reduction in a-gliadins. Up to 35 different genes were mutated in one of the lines of the 45 different genes identified in the wild type, while immunoreactivity was reduced by 85%. Transgene-free lines were identified, and no off-target mutations have been detected in any of the potential targets. The low-gluten, transgene-free wheat lines described here could be used to produce low-gluten foodstuff and serve as source material to introgress this trait into elite wheat varieties.Ministerio de Economía y Competitividad AGL2013-48946-C3-1-R ; AGL2016-80566-

Celiac Immunogenic Potential of α-Gliadin Epitope Variants from Triticum and Aegilops Species

The high global demand of wheat and its subsequent consumption arise from the physicochemical properties of bread dough and its contribution to the protein intake in the human diet. Gluten is the main structural complex of wheat proteins and subjects affected by celiac disease (CD) cannot tolerate gluten protein. Within gluten proteins, α-gliadins constitute the most immunogenic fraction since they contain the main T-cell stimulating epitopes (DQ2.5-glia-α1, DQ2.5-glia-α2, and DQ2.5-glia-α3). In this work, the celiac immunotoxic potential of α-gliadins was studied within Triticeae: diploid, tetraploid, and hexaploid species. The abundance and immunostimulatory capacity of CD canonical epitopes and variants (with one or two mismatches) in all α-gliadin sequences were determined. The results showed that the canonical epitopes DQ2.5-glia-α1 and DQ2.5-glia-α3 were more frequent than DQ2.5-glia-α2. A higher abundance of canonical DQ2.5-glia-α1 epitope was found to be associated with genomes of the BBAADD, AA, and DD types; however, the abundance of DQ2.5-glia-α3 epitope variants was very high in BBAADD and BBAA wheat despite their low abundance in the canonical epitope. The most abundant substitution was that of proline to serine, which was disposed mainly on the three canonical DQ2.5 domains on position 8. Interestingly, our results demonstrated that the natural introduction of Q to H at any position eliminates the toxicity of the three T-cell epitopes in the α-gliadins. The results provided a rational approach for the introduction of natural amino acid substitutions to eliminate the toxicity of three T-cell epitopes, while maintaining the technological properties of commercial wheats

Targeting of prolamins by RNAi in bread wheat: Effectiveness of seven silencing-fragment combinations for obtaining lines devoid of coeliac disease epitopes from highly immunogenic gliadins

Gluten proteins are responsible for the viscoelastic properties of wheat flour but also for triggering pathologies in susceptible individuals, of which coeliac disease (CD) and noncoeliac gluten sensitivity may affect up to 8% of the population. The only effective treatment for affected persons is a strict gluten-free diet. Here, we report the effectiveness of seven plasmid combinations, encompassing RNAi fragments from a-, c-, x-gliadins, and LMW glutenin subunits, for silencing the expression of different prolamin fractions. Silencing patterns of transgenic lines were analysed by gel electrophoresis, RP-HPLC and mass spectrometry (LC-MS/ MS), whereas gluten immunogenicity was assayed by an anti-gliadin 33-mer monoclonal antibody (moAb). Plasmid combinations 1 and 2 downregulated only c- and a-gliadins, respectively. Four plasmid combinations were highly effective in the silencing of x-gliadins and c-gliadins, and three of these also silenced a-gliadins. HMW glutenins were upregulated in all but one plasmid combination, while LMW glutenins were downregulated in three plasmid combinations. Total protein and starch contents were unaffected regardless of the plasmid combination used. Six plasmid combinations provided strong reduction in the gluten content as measured by moAb and for two combinations, this reduction was higher than 90% in comparison with the wild type. CD epitope analysis in peptides identified in LC-MS/MS showed that lines from three plasmid combinations were totally devoid of CD epitopes from the highly immunogenic a- and x-gliadins. Our findings raise the prospect of breeding wheat species with low levels of harmful gluten, and of achieving the important goal of developing nontoxic wheat cultivars