CRISPR/Cas9-mediated multiplex gene editing of gamma and omega gliadins, paving the way for gliadin-free wheat.

Wheat is a staple cereal in the human diet. Despite its significance, an increasing percentage of the population suffers adverse reactions to wheat, which are triggered by wheat gluten, particularly the gliadin fractions. In this study, we employed CRISPR/Cas multiplexing to introduce targeted mutations into γ- and ω-gliadin genes of wheat, to produce lines deficient in one or both immunogenic gliadin fractions simultaneously. For this work, eight single guide RNAs (sgRNAs) were designed and combined into four plasmids to produce 59 modified wheat lines, of which 20 exhibited mutations in the target genes. Characterization of these lines through Sanger or NGS sequencing revealed a complex pattern of InDels, including deletions spanning multiple sgRNAs. The mutations were transmitted to the offspring, and the analysis of homozygous derived lines by RP-HPLC and monoclonal antibodies showed a 97.7% reduction in gluten content. Crossing these lines with other CRISPR/Cas lines deficient in the α-gliadins allowed multiple mutations to be combined. This work represents an important step forward in the use of CRISPR/Cas to develop gluten-free wheat.

Rapid analysis of wheat gluten composition using a triple ELISA.

BACKGROUND: Gluten composition is an important quality parameter of wheat flour. Reversed-phase high-performance liquid chromatography (RP-HPLC) is a state-of-the-art method for its analysis. As this is a very labour-intensive and time-consuming procedure, alternative faster methods are desirable. Enzyme-linked immunosorbent assay (ELISA) is a high-throughput method often used for the analysis of gluten traces in gluten-free products. In this proof-of-principle study, we introduce an experimental triple ELISA for the relative quantitation of gliadins, high-molecular-weight glutenin subunits (HMW-GS) and low-molecular-weight glutenin subunits (LMW-GS) of one wheat flour extract. RESULTS: The results of 80 common wheat flour samples obtained from the triple ELISA and RP-HPLC were correlated. The results for gliadins (r = 0.69) and HMW-GS (r = 0.81) showed a medium and high correlation, respectively. Only a very weak correlation of ELISA and RP-HPLC results was observed for LMW-GS (r = 0.49). Results for glutenins (r = 0.69) and gluten (r = 0.72) had a medium correlation. The gliadin/glutenin ratio (r = 0.47) and LMW-GS/HMW-GS ratio (r = 0.40) showed a weak or no correlation. The gliadin, LMW-GS and gluten contents were lower and the HMW-GS content was higher in the ELISA measurement compared to RP-HPLC. CONCLUSION: The quantitation of gliadins and HMW-GS by the experimental triple ELISA showed comparable results to RP-HPLC, whereas no strong correlation between the results from the two methods was found for LMW-GS. Overall, the experimental triple ELISA is suitable for relative gluten quantitation, especially for the analysis of large sample sets. Further work will focus on improving the experimental procedure of the ELISA. © 2024 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Agro-Industrial Protein Waste and Co-Products Valorization for the Development of Bioplastics: Thermoprocessing and Characterization of Feather Keratin/Gliadin Blends.

Biopolymers based on plant and animal proteins are interesting alternatives in the development of films with future prospects as food packaging. Considering that in recent years there has been an increasing interest in the valorization of agro-industrial residues and by-products and that the blending of polymers can lead to materials with improved properties, in this work, keratin-rich feather fibers and gliadins were blended at different ratios in order to develop sustainable and biodegradable films. Control gliadin G100, feather F100 films, and their blends at 3:1 (G75F25), 2:2 (G50F50), and 1:3 (G25F75) ratios were successfully developed through thermoprocessing. The physical properties were differentiated as a function of the concentration of both polymeric matrices. Although gliadins showed higher hydrophilicity as confirmed by their highest swelling degree, films with high gliadin ratios exhibited lower water vapor permeability values at low and medium relative humidities. On the other hand, the feather fiber-based films displayed the highest Young's modulus values and provided an oxygen barrier to the blends, principally at the highest relative humidity. In conclusion, the blend of these protein-based polymers at different ratio resulted in interesting composites whose physical properties could be adjusted.

Recombinant Cathepsin L of Tribolium castaneum and Its Potential in the Hydrolysis of Immunogenic Gliadin Peptides.

Wheat gliadins contain a large amount of glutamine- and proline-rich peptides which are not hydrolyzed by human digestive peptidases and can cause autoimmune celiac disease and other forms of gluten intolerance in predisposed people. Peptidases that efficiently cleave such immunogenic peptides can be used in enzyme therapy. The stored product insect pest Tribolium castaneum efficiently hydrolyzes gliadins. The main digestive peptidase of T. castaneum is cathepsin L, which is from the papain C1 family with post-glutamine cleavage activity. We describe the isolation and characterization of T. castaneum recombinant procathepsin L (rpTcCathL1, NP_001164001), which was expressed in Pichia pastoris cells. The activation of the proenzyme was conducted by autocatalytic processing. The effects of pH and proenzyme concentration in the reaction mixture on the processing were studied. The mature enzyme retained high activity in the pH range from 5.0 to 9.0 and displayed high pH-stability from 4.0 to 8.0 at 20 °C. The enzyme was characterized according to electrophoretic mobility under native conditions, activity and stability at various pH values, a sensitivity to various inhibitors, and substrate specificity, and its hydrolytic effect on 8-, 10-, 26-, and 33-mer immunogenic gliadins peptides was demonstrated. Our results show that rTcCathL1 is an effective peptidase that can be used to develop a drug for the enzyme therapy of various types of gluten intolerance.

Does Nitrogen Fertilization Affect the Secondary Structures of Gliadin Proteins in Hypoallergenic Wheat?

One of the macronutrients indispensable for plant growth and development is nitrogen (N). It is responsible for starch and storage protein (gliadins and glutenins) biosynthesis and, in consequence, influences kernels' quality and yields. However, applying N-fertilizers increases gluten content in wheat, and it may intensify the risk of developing allergy symptoms in gluten-sensitive individuals. The purpose of our research was to analyse whether and how the elimination of N-fertilizers during the cultivation of wasko.gl- wheat (modified genotype lacking ω-gliadins) changes the secondary structures of gliadin proteins. To this aim, using the FT-Raman technique, we examined flour and gliadin protein extracts obtained from kernels of two winter wheat lines: wasko.gl+ (with a full set of gliadin proteins) and wasko.gl- (without ω-gliadin fraction) cultivated on two different N-fertilization levels-0 and 120 kg N·ha-1. On the basis of the obtained results, we proved that nitrogen fertilization does not have a major impact on the stability of the secondary structures of gliadin proteins for wasko.gl- wheat line with reduced allergenic properties. Furthermore, the results presented herein suggest the possibility of increasing the stability of glutenin structures as a result of the N-fertilization of wasko.gl- wheat line, which gives hope for its use in the production of wheat articles devoted to people suffering from diseases related to gluten sensitivity.

A Bioinformatic Workflow for InDel Analysis in the Wheat Multi-Copy α-Gliadin Gene Family Engineered with CRISPR/Cas9.

The α-gliadins of wheat, along with other gluten components, are responsible for bread viscoelastic properties. However, they are also related to human pathologies as celiac disease or non-celiac wheat sensitivity. CRISPR/Cas was successfully used to knockout α-gliadin genes in bread and durum wheat, therefore, obtaining low gluten wheat lines. Nevertheless, the mutation analysis of these genes is complex as they present multiple and high homology copies arranged in tandem in A, B, and D subgenomes. In this work, we present a bioinformatic pipeline based on NGS amplicon sequencing for the analysis of insertions and deletions (InDels) in α-gliadin genes targeted with two single guides RNA (sgRNA). This approach allows the identification of mutated amplicons and the analysis of InDels through comparison to the most similar wild type parental sequence. TMM normalization was performed for inter-sample comparisons; being able to study the abundance of each InDel throughout generations and observe the effects of the segregation of Cas9 coding sequence in different lines. The usefulness of the workflow is relevant to identify possible genomic rearrangements such as large deletions due to Cas9 cleavage activity. This pipeline enables a fast characterization of mutations in multiple samples for a multi-copy gene family.

FT-Raman Spectroscopy as a Tool to Study the Secondary Structures of Wheat Gliadin Proteins.

Raman spectroscopy is a useful method in biological, biomedical, food, and agricultural studies, allowing the simultaneous examination of various chemical compounds and evaluation of molecular changes occurring in tested objects. The purpose of our research was to explain how the elimination of ω-fractions from the wheat gliadin complex influences the secondary structures of the remaining αßγ-gliadins. To this aim, we analyzed the endosperm of wheat kernels as well as gliadin proteins extracted from two winter wheat genotypes: wasko.gl+ (control genotype containing the full set of gliadins) and wasko.gl- (modified genotype lacking all ω-gliadins). Based on the decomposition of the amide I band, we observed a moderate increase in ß-forms (sheets and turns) at the expense of α-helical and random coil structures for gliadins isolated from the flour of the wasko.gl- line. Since ω-gliadins contain no cysteine residues, they do not participate in the formation of the disulfide bridges that stabilize the protein structure. However, they can interact with other proteins via weak, low-energetic hydrogen bonds. We conclude that the elimination of ω-fractions from the gliadin complex causes minor modifications in secondary structures of the remaining gliadin proteins. In our opinion, these small, structural changes of proteins may lead to alterations in gliadin allergenicity.

Effects of Physical and Chemical Factors on the Structure of Gluten, Gliadins and Glutenins as Studied with Spectroscopic Methods.

This review presents applications of spectroscopic methods, infrared and Raman spectroscopies in the studies of the structure of gluten network and gluten proteins (gliadins and glutenins). Both methods provide complimentary information on the secondary and tertiary structure of the proteins including analysis of amide I and III bands, conformation of disulphide bridges, behaviour of tyrosine and tryptophan residues, and water populations. Changes in the gluten structure can be studied as an effect of dough mixing in different conditions (e.g., hydration level, temperature), dough freezing and frozen storage as well as addition of different compounds to the dough (e.g., dough improvers, dietary fibre preparations, polysaccharides and polyphenols). Additionally, effect of above mentioned factors can be determined in a common wheat dough, model dough (prepared from reconstituted flour containing only wheat starch and wheat gluten), gluten dough (lack of starch), and in gliadins and glutenins. The samples were studied in the hydrated state, in the form of powder, film or in solution. Analysis of the studies presented in this review indicates that an adequate amount of water is a critical factor affecting gluten structure.

Exploiting the reference genome sequence of hexaploid wheat: a proteomic study of flour proteins from the cultivar Chinese Spring.

Although the economic value of wheat flour is determined by the complement of gluten proteins, these proteins have been challenging to study because of the complexity of the major protein groups and the tremendous sequence diversity among wheat cultivars. The completion of a high-quality wheat genome sequence from the reference wheat Chinese Spring recently facilitated the assembly and annotation of a complete set of gluten protein genes from a single cultivar, making it possible to link individual proteins in the flour to specific gene sequences. In a proteomic analysis of total wheat flour protein from Chinese Spring using quantitative two-dimensional gel electrophoresis combined with tandem mass spectrometry, gliadins or low-molecular-weight glutenin subunits were identified as the predominant proteins in 72 protein spots. Individual spots were associated with 40 of 56 Chinese Spring gene sequences, including 16 of 26 alpha gliadins, 10 of 11 gamma gliadins, six of seven omega gliadins, one of two delta gliadins, and nine of ten LMW-GS. Most genes that were not associated with protein spots were either expressed at low levels in endosperm or encoded proteins with high similarity to other proteins. A wide range of protein accumulation levels were observed and discrepancies between transcript levels and protein levels were noted. This work together with similar studies using other commercial cultivars should provide new insight into the molecular basis of wheat flour quality and allergenic potential.

Towards reducing the immunogenic potential of wheat flour: omega gliadins encoded by the D genome of hexaploid wheat may also harbor epitopes for the serious food allergy WDEIA.

BACKGROUND: Omega-5 gliadins are a group of highly repetitive gluten proteins in wheat flour encoded on the 1B chromosome of hexaploid wheat. These proteins are the major sensitizing allergens in a severe form of food allergy called wheat-dependent exercise-induced anaphylaxis (WDEIA). The elimination of omega-5 gliadins from wheat flour through biotechnology or breeding approaches could reduce the immunogenic potential and adverse health effects of the flour. RESULTS: A mutant line missing low-molecular weight glutenin subunits encoded at the Glu-B3 locus was selected previously from a doubled haploid population generated from two Korean wheat cultivars. Analysis of flour from the mutant line by 2-dimensional gel electrophoresis coupled with tandem mass spectrometry revealed that the omega-5 gliadins and several gamma gliadins encoded by the closely linked Gli-B1 locus were also missing as a result of a deletion of at least 5.8 Mb of chromosome 1B. Two-dimensional immunoblot analysis of flour proteins using sera from WDEIA patients showed reduced IgE reactivity in the mutant relative to the parental lines due to the absence of the major omega-5 gliadins. However, two minor proteins showed strong reactivity to patient sera in both the parental and the mutant lines and also reacted with a monoclonal antibody against omega-5 gliadin. Analysis of the two minor reactive proteins by mass spectrometry revealed that both proteins correspond to omega-5 gliadin genes encoded on chromosome 1D that were thought previously to be pseudogenes. CONCLUSIONS: While breeding approaches can be used to reduce the levels of the highly immunogenic omega-5 gliadins in wheat flour, these approaches are complicated by the genetic linkage of different classes of gluten protein genes and the finding that omega-5 gliadins may be encoded on more than one chromosome. The work illustrates the importance of detailed knowledge about the genomic regions harboring the major gluten protein genes in individual wheat cultivars for future efforts aimed at reducing the immunogenic potential of wheat flour.

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 α-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 α-gliadin genes. Twenty-one mutant lines were generated, all showing strong reduction in α-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.

Effects of water deficit on breadmaking quality and storage protein compositions in bread wheat (Triticum aestivum L.).

BACKGROUND: Water deficiency affects grain proteome dynamics and storage protein compositions, resulting in changes in gluten viscoelasticity. In this study, the effects of field water deficit on wheat breadmaking quality and grain storage proteins were investigated. RESULTS: Water deficiency produced a shorter grain-filling period, a decrease in grain number, grain weight and grain yield, a reduced starch granule size and increased protein content and glutenin macropolymer contents, resulting in superior dough properties and breadmaking quality. Reverse phase ultra-performance liquid chromatography analysis showed that the total gliadin and glutenin content and the accumulation of individual components were significantly increased by water deficiency. Two-dimensional gel electrophoresis detected 144 individual storage protein spots with significant accumulation changes in developing grains under water deficit. Comparative proteomic analysis revealed that water deficiency resulted in significant upregulation of 12 gliadins, 12 high-molecular-weight glutenin subunits and 46 low-molecular-weight glutenin subunits. Quantitative real-time polymerase chain reaction analysis revealed that the expression of storage protein biosynthesis-related transcription factors Dof and Spa was upregulated by water deficiency. CONCLUSION: The present results illustrated that water deficiency leads to increased accumulation of storage protein components and upregulated expression of Dof and Spa, resulting in an improvement in glutenin strength and breadmaking quality. © 2018 Society of Chemical Industry.

Variations in yield and gluten proteins in durum wheat varieties under late-season foliar versus soil application of nitrogen fertilizer in a northern Mediterranean environment.

BACKGROUND: With the increasing demand for high-quality foodstuffs and concern for environmental sustainability, late-season nitrogen (N) foliar fertilization of common wheat is now an important and widespread practice. This study investigated the effects of late-season foliar versus soil N fertilization on yield and protein content of four varieties of durum wheat, Aureo, Ariosto, Biensur and Liberdur, in a three-year field trial in northern Italy. RESULTS: Variations in low-molecular-weight glutenins (LMW-GS), high-molecular-weight glutenins (HMW-GS) and gliadins were assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). It was found that N applied to the canopy did not improve protein rate compared with N application to the soil (general mean 138 mg g-1 ), but moderately increased productivity in the high-yielding varieties Liberdur and Biensur (three-year means 7.23 vs 7.13 and 7.53 vs 7.09 t ha-1 respectively). Technological quality was mainly related to variety choice, Aureo and Ariosto having higher protein rates and glutenin/gliadin ratios. Also found was a strong 'variety × N application method' interaction in the proportions of protein subunits within each class, particularly LMW-GS and gliadins. A promising result was the higher N uptake efficiency, although as apparent balance, combined with higher HMW/LMW-GS ratio in var. Biensur. CONCLUSION: Late-season foliar N fertilization allows N fertilizer saving, potentially providing environmental benefits in the rainy climate of the northern Mediterranean area, and also leads to variety-dependent up-regulation of essential LMW-GS and gliadins. Variety choice is a key factor in obtaining high technological quality, although it is currently associated with modest grain yield. This study provides evidence of high quality in the specific high-yielding variety Biensur, suggesting its potential as a mono-varietal semolina for pasta production. © 2017 Society of Chemical Industry.

Production and molecular characterization of bread wheat lines with reduced amount of α-type gliadins.

BACKGROUND: Among wheat gluten proteins, the α-type gliadins are the major responsible for celiac disease, an autoimmune disorder that affects about 1% of the world population. In fact, these proteins contain several toxic and immunogenic epitopes that trigger the onset of the disease. The α-type gliadins are a multigene family, encoded by genes located at the complex Gli-2 loci. RESULTS: Here, three bread wheat deletion lines (Gli-A2, Gli-D2 and Gli-A2/Gli-D2) at the Gli-2 loci were generated by the introgression in the bread wheat cultivar Pegaso of natural mutations, detected in different bread wheat cultivars. The molecular characterization of these lines allowed the isolation of 49 unique expressed genes coding α-type gliadins, that were assigned to each of the three Gli-2 loci. The number and the amount of α-type gliadin transcripts were drastically reduced in the deletion lines. In particular, the line Gli-A2/Gli-D2 contained only 12 active α-type gliadin genes (-75.6% respect to the cv. Pegaso) and a minor level of transcripts (-80% compared to cv. Pegaso). Compensatory pleiotropic effects were observed in the two other classes of gliadins (ω- and γ-gliadins) either at gene expression or protein levels. Although the comparative analysis of the deduced amino acid sequences highlighted the typical structural features of α-type gliadin proteins, substantial differences were displayed among the 49 proteins for the presence of toxic and immunogenic epitopes. CONCLUSION: The deletion line Gli-A2/Gli-D2 did not contain the 33-mer peptide, one of the major epitopes triggering the celiac disease, representing an interesting material to develop less "toxic" wheat varieties.

Prolidase is a critical enzyme for complete gliadin digestion in Tenebrio molitor larvae.

Prolidase is a proline-specific metallopeptidase that cleaves imidodipeptides with C-terminal Pro residue. Prolidase was purified and characterized from the Tenebrio molitor larval midgut. The enzyme was localized in the soluble fraction of posterior midgut tissues, corresponding to a predicted cytoplasmic localization of prolidase according to the structure of the mRNA transcript. Expression of genes encoding prolidase and the major digestive proline-specific peptidase (PSP)-dipeptidyl peptidase 4-were similar. The pH optimum of T. molitor prolidase was 7.5, and the enzyme was inhibited by Z-Pro, indicating that it belongs to type I prolidases. In mammals, prolidase is particularly important in the catabolism of a proline-rich protein-collagen. We propose that T. molitor larval prolidase is a critical enzyme for the final stages of digestion of dietary proline-rich gliadins, providing hydrolysis of imidodipeptides in the cytoplasm of midgut epithelial cells. We propose that the products of hydrolysis are absorbed from the luminal contents by peptide transporters, which we have annotated in the T. molitor larval gut transcriptome. The origin of prolidase substrates in the insect midgut is discussed in the context of overall success of grain feeding insects.

Evaluation of molecular weight distribution of unreduced wheat gluten proteins associated with noodle quality.

Unreduced gluten proteins of Indian wheat varieties viz.C306, DBW16, HI977 and HW2004 were separated using size-exclusion chromatography (SEC). Statistical correlation of area % of eluted peaks with properties of flour, dough and noodles was elucidated. Chromatograms of gluten proteins were classified primarily into five peaks in decreasing molecular size range and relative proportion were expressed in terms of area % of individual peaks which depicts the quantitative variation in protein eluted at different retention times. Cooking time and cooked weight of noodles depicted positive correlation with peak I and negative correlation with peak II which predominantly composed of glutenins and gliadins, respectively. Oil uptake and cooking loss were negatively association with peak I and positively with peak II. Noodle hardness, springiness, cohesiveness and chewiness were positively correlated with peak I and negatively to peak II, though adhesiveness was unaffected by SEC eluted peaks statistically.

Biochemical and functional properties of wheat gliadins: a review.

Gliadins account for 40-50% of the total storage proteins of wheat and are classified into four subcategories, α-, ß-, γ-, and ω-gliadins. They have also been classified as ω5-, ω1, 2-, α/ß-, and γ-gliadins on the basis of their primary structure and molecular weight. Cysteine residues of gliadins mainly form intramolecular disulfide bonds, although α-gliadins with odd numbers of cysteine residues have also been reported. Gliadins are generally regarded to possess globular protein structure, though recent studies report that the α/ß-gliadins have compact globular structures and γ- and ω-gliadins have extended rod-like structures. Newer techniques such as Mass Spectrometry with the development of matrix-assisted laser desorption/ionization (MALDI) in combination with time-of-flight mass spectrometry (TOFMS) have been employed to determine the molecular weight of purified ω- gliadins and to carry out the direct analysis of bread and durum wheat gliadins. Few gliadin alleles and components, such as Gli-B1b, Gli-B2c and Gli-A2b in bread wheat cultivars, γ-45 in pasta, γ-gliadins in cookies, lower gliadin content for chapatti and alteration in Gli 2 loci in tortillas have been reported to improve the product quality, respectively. Further studies are needed in order to elucidate the precise role of gliadin subgroups in dough strength and product quality.

Biochemical modifications of gliadins induced by microbial transglutaminase on wheat flour.

BACKGROUND: Celiac disease (CD) is an immune-mediated disorder caused by the ingestion of wheat gluten. A lifelong, gluten-free diet is required to normalize the intestinal mucosa. We previously found that transamidation by microbial transglutaminase (mTGase) suppressed the gliadin-specific immune response in intestinal T-cell lines from CD patients and in models of gluten sensitivity. METHODS: SDS-PAGE, Western blot, ELISA, tissue transglutaminase (tTGase) assay and nano-HPLC-ESI-MS/MS experiments were used to analyze prolamins isolated from treated wheat flour. RESULTS: Gliadin and glutenin yields decreased to 7.6±0.5% and 7.5±0.3%, respectively, after a two-step transamidation reaction that produced a water-soluble protein fraction (spf). SDS-PAGE, Western blot and ELISA analyses confirmed the loss of immune cross-reactivity with anti-native gliadin antibodies in residual transamidated gliadins (K-gliadins) and spf as well as the occurrence of neo-epitopes. Nano-HPLC-ESI-MS/MS experiments identified some native and transamidated forms of celiacogenic peptides including p31-49 and confirmed that mTGase had similar stereo-specificity of tTGase. Those peptides resulted to be 100% and 57% modified in spf and K-gliadins, respectively. In particular, following transamidation p31-49 lost its ability to increase tTGase activity in Caco-2 cells. Finally, bread manufactured with transamidated flour had only minor changes in baking characteristics. CONCLUSIONS: The two-step transamidation reaction modified the analyzed gliadin peptides, which are known to trigger CD, without influencing main technological properties. GENERAL SIGNIFICANCE: Our data shed further light on a detoxification strategy alternative to the gluten free diet and may have important implications for the management of CD patients.

Wheat allergy in children - new tools for diagnostics.

BACKGROUND: The detection of wheat-specific IgE in children often leads to a suspicion of wheat allergy, but little information is available on the most reliable wheat allergens for predicting clinical reactivity. OBJECTIVE: To evaluate the role of allergenic components of wheat in wheat allergy diagnostics. METHODS: One hundred and eight children (median age 1.5 years; range 0.6-17.3 years) with suspected wheat allergy underwent open or double-blinded, placebo-controlled oral wheat challenges. Responsiveness to different allergenic components of wheat was studied by skin prick tests and by determination of serum IgE antibodies using a semi-quantitative microarray assay. RESULTS: Thirty (28%) children reacted with immediate symptoms, and 27 (25%) with delayed symptoms to ingested wheat, whereas 51 (47%) children exhibited no reactions in oral wheat challenges. Positive IgE responses to any of the 12 allergenic components of wheat was seen in 93%, 41%, and 43% of those with immediate, delayed or no reactions to ingested wheat, respectively (P < 0.001 to P < 0.05 in every comparisons between those with immediate reactions and those with no reactions). Positive IgE responses to ≥5 different allergenic components improved significantly the diagnostic accuracy (with a positive likelihood ratio (LR+) of 5.10). Alpha-amylase inhibitors (AAI), in particular dimeric AAI 0.19 (LR+ 6.12), alpha-, beta-, and gamma-gliadins (LR+ from 3.57 to 4.53), and high-molecular-weight (HMW) glutenin subunits (LR+ 4.37) were the single allergenic components of wheat differentiating most effectively those with immediate symptoms from those who did not exhibit any reactions. CONCLUSIONS AND CLINICAL RELEVANCE: Wheat allergy diagnostics is difficult, even using sophisticated component methods. Our results confirm earlier findings about gliadins and identify the dimeric AAI 0.19, as a relevant allergen in clinically reactive patients when compared to non-reactive subjects. The accuracy of wheat allergy diagnosis may be improved by measuring IgE responses to several components of wheat.

Unraveling the celiac disease-related immunogenic complexes in a set of wheat and tritordeum genotypes: implications for low-gluten precision breeding in cereal crops.

The development of low-gluten immunogenic cereal varieties is a suitable way to fight the increment of pathologies associated with the consumption of cereals. Although RNAi and CRISPR/Cas technologies were effective in providing low-gluten wheat, the regulatory framework, particularly in the European Union, is an obstacle to the short- or medium-term implementation of such lines. In the present work, we carried out a high throughput amplicon sequencing of two highly immunogenic complexes of wheat gliadins in a set of bread and durum wheat, and tritordeum genotypes. Bread wheat genotypes harboring the 1BL/1RS translocation were included in the analysis and their amplicons successfully identified. The number of CD epitopes and their abundances were determined in the alpha- and gamma-gliadin amplicons, including 40k-γ-secalin ones. Bread wheat genotypes not containing the 1BL/1RS translocation showed a higher average number of both alpha- and gamma-gliadin epitopes than those containing such translocation. Interestingly, alpha-gliadin amplicons not containing CD epitopes accounted for the highest abundance (around 53%), and the alpha- and gamma-gliadin amplicons with the highest number of epitopes were present in the D-subgenome. The durum wheat and tritordeum genotypes showed the lowest number of alpha- and gamma-gliadin CD epitopes. Our results allow progress in unraveling the immunogenic complexes of alpha- and gamma-gliadins and can contribute to the development of low-immunogenic varieties within precision breeding programs, by crossing or by CRISPR/Cas gene editing.