RNA-amplicon sequencing method

“Gli attuali metodi commerciali di rilevamento del glutine non sono in grado di distinguere tra varianti di epitopi CD immunogeniche e non immunogeniche e quindi di quantificare con precisione il carico complessivo di epitopi CD di una data varietà di frumento. Il metodo di sequenziamento dell’RNA-amplicon è stato sviluppato per le trascrizioni di alfa-gliadina che comprendono i tre principali epitopi di CD e le loro varianti. Tale quantificazione è indispensabile per la corretta selezione delle varietà di frumento con basso potenziale in grado di causare la celiachia. “Quantitative and qualitative differences in celiac disease epitopes among durum wheat varieties identified through deep RNA-amplicon sequencing
Elma MJ Salentijn, Danny G Esselink, Svetlana V Goryunova, Ingrid M van der Meer, Luud JWJ Gilisse and Marinus JM Smulders Salentijn et al. BMC Genomics 2013, 14:905 http://www.biomedcentral.com/1471-2164/14/905

Abstract
“Background: Wheat gluten is important for the industrial quality of bread wheat (Triticum aestivum L.) and durum wheat (T. turgidum L.). Gluten proteins are also the source of immunogenic peptides that can trigger a T cell reaction in celiac disease (CD) patients, leading to inflammatory responses in the small intestine. Various peptides with three major T cell epitopes involved in CD are derived from alpha-gliadin fraction of gluten. Alpha-gliadins are encoded by a large multigene family and amino acid variation in the CD epitopes is known to influence the immunogenicity of individual gene family members. Current commercial methods of gluten detection are unable to distinguish between immunogenic and non-immunogenic CD epitope variants and thus to accurately quantify the overall CD epitope load of a given wheat variety. Such quantification is indispensable for correct selection of wheat varieties with low potential to cause CD.

Results: A 454 RNA-amplicon sequencing method was developed for alpha-gliadin transcripts encompassing the three major CD epitopes and their variants. The method was used to screen developing grains on plants of 61 different durum wheat cultivars and accessions. A dedicated sequence analysis pipeline returned a total of 304 unique alpha-gliadin transcripts, corresponding to a total of 171 ‘unique deduced protein fragments’ of alpha-gliadins. The numbers of these fragments obtained in each plant were used to calculate quantitative and quantitative differences between the CD epitopes expressed in the endosperm of these wheat plants. A few plants showed a lower fraction of CD epitope-encoding alpha-gliadin transcripts, but none were free of CD epitopes.

Conclusions: The dedicated 454 RNA-amplicon sequencing method enables 1) the grouping of wheat plants according to the genetic variation in alpha-gliadin transcripts, and 2) the screening for plants which are potentially less CD-immunogenic. The resulting alpha-gliadin sequence database will be useful as a reference in proteomics analysis regarding the immunogenic potential of mature wheat grains.

……omissis Limiting the abundance of CD epitopes in food products may reduce the risk of sensitization of the immune system of the group of people that are genetically susceptible for CD. In order to breed and select for wheat varieties with significantly reduced immunogenic potential to cause CD it is necessary to accurately estimate the quantity and quality of the CD epitope load in gluten. Up to now, the ability for high throughput quantification of CD epitopes by presently available assays based on T cell clones and on monoclonal antibodies is very limited, mainly because of the high complexity of the wheat material on the one hand, and the laboriousness of in vitro T cell assays and the promiscuity of the monoclonal antibodies on the other hand [22,23]. In addition, most commercial kits with monoclonal antibodies detect gluten, not CD epitopes.

A custom 454 sequence analysis pipeline was used to quantify CD epitopes and their variants in the alpha-gliadin transcriptomes of a set of 77 individual plants from 61 different durum wheat accessions, by determining the normalised transcript abundances for the respective CD epitopes and variants thereof.”

More information: https://www.yourgenome.org/facts/what-is-the-454-method-of-dna-sequencing

Nota:

Il sequenziamento del DNA è la determinazione dell’ordine dei diversi nucleotidi (quindi delle quattro basi azotate che li differenziano, cioè adenina, citosina, guanina e timina) che costituiscono l’acido nucleico.

La sequenza del DNA contiene tutte le informazioni genetiche ereditarie del nucleo, plasmidi, mitocondri e cloroplasti che sono alla base per lo sviluppo di tutti gli organismi viventi. All’interno di questa sequenza sono codificati i geni di ogni organismo vivente, nonché le istruzioni per esprimerli nel tempo e nello spazio (regolazione dell’espressione genica). Determinare la sequenza è dunque utile nella ricerca del perché e come gli organismi vivono.

La conoscenza del genoma risulta quindi utile in ogni campo della biologia e l’avvento di metodi per il sequenziamento del DNA ha accelerato significativamente la ricerca.