Genetically Modified Wheat, Wheat Intolerance, and Food Safety Concerns

Authors: Atul Munish Chander,1,2 Sanjay Kumar Bhadada,2 *Devinder Kumar Dhawan1

1. Department of Biophysics, Panjab University, Chandigarh, India 2. Department of Endocrinology, Postgraduate Institute of Medical Education and Research, Chandigarh, India *Correspondence to [email protected]

Disclosure: The authors have declared no conflicts of interest.

Acknowledgements: Mr Chander is thankful to the Indian Council of Medical Research (ICMR), New Delhi, India, for providing fellowship.

Received: 22.05.17

Accepted: 20.02.18

Keywords: Food safety, , metabolomics, RNA interference (RNAi), transgenic wheat.

Citation: EMJ Allergy Immunol. 2018;3[1]:114-122.

Abstract Wheat intolerance is a common problem for certain individuals. A gluten-free diet is the only option for people with wheat-associated disorders (WAD) to manage their condition. The elimination of immunogenic from the wheat is the most appropriate approach to ameliorate the symptoms of affected individuals, while also meeting their nutritional requirements. RNA interference technology can be exploited to silence the expression of to produce a wheat variety lacking the immunogenic proteins associated with WAD, but there are challenges before implementation of transgenic varieties in the market will occur. This review is focussed on RNA interference approaches acquired to produce wheat that patients with different WAD can tolerate. The authors also discuss the advantages and disadvantages of current omics approaches that are being used to validate the food safety issues related to the applicability and clinical relevance of genetically modified wheat.

INTRODUCTION was measured between 1.2 and 75.3 per 100,000 people for dermatitis herpetiformis.2-8 Research Wheat is the causal factor for a number of conducted in the USA reported the prevalence 9 diseases, including coeliac disease (CD), of NCGS to be 6.0% in a hospital-based study, wheat-dependent exercise-induced anaphylaxis but another study reported a 0.55% prevalence (WDEIA), wheat allergy, dermatitis herpetiformis, of NCGS in the general population of the USA.10 and non-coeliac gluten sensitivity (NCGS). CD is the most studied and prevalent among all The literature is scarce about the prevalence of wheat-related diseases. CD was first reported to most wheat-associated disorders (WAD) except have a prevalence of 1:184 in an Italian school- for CD. Recently, the prevalence of wheat aged paediatric population.11 Prevalence of allergy in a birth cohort (10 and 11 year olds) was CD varies from 0.006% to 5.600% in different reported as 0.48% in the UK,1 whereas prevalence populations across the world.12-20 The Saharawi

114 ALLERGY & IMMUNOLOGY • July 2018 EMJ EUROPEAN MEDICAL JOURNAL population of Africa accounts for 5.6% of CD have the potential of elucidating better safety cases. CD is considered the most prevalent measures from a health point of view. wheat-related disease across the world.21 In addition, the prevalence of CD is around 11% in IMMUNOGENIC GLUTEN PEPTIDES: 22 the Type 1 diabetes mellitus population in India. THE CAUSAL FACTOR FOR CD is also the cause of other diverse health COELIAC DISEASE problems23,24 because CD is an autoimmune disorder of the small intestine that leads to Gluten is the component of wheat destruction of intestinal villi as a consequence of that contains α-gliadins, γ-gliadins, and inflammation.25,26 Though there are some genetic glutenins as immunogenic components, and and environmental factors associated with ω5-gliadins as the allergen component. Human disease,27-31 wheat gluten (wheat protein) remains proteolytic enzymes cannot effectively digest the antigenic trigger of CD, and its withdrawal gluten proteins,35 and these proteins have been from the diet improves the clinical conditions reported to possess differential immune targets of CD patients. The extent of gluten intake is that make them immunotoxic by nature. strongly associated with the severity and During proteolytic digestion in the intestine, 20 prevalence of CD. immunogenic proline and glutamine-rich gluten Even after decades of research, there is no polypeptides are produced that can stimulate 36 successful treatment available as an alternative T cells. These peptides are resistant to further to a gluten-free diet (GFD). Strict lifelong hydrolysis due to enrichment of proline residues in the amino acid sequences.37 The peptides adherence to a GFD always remains a challenge 33-mer and 26-mer, derived from α- and for CD patients and even occasional ingestion of γ-gliadin, respectively, subsequently trigger gluten-containing food facilitates reoccurrence immunological responses in the intestines of of gluten-induced inflammation. Moreover, some CD patients.25,38-40 Peptide 31−49 of α-gliadin CD patients never recover from symptoms, was reported to be a potent activator of innate even after remaining on a GFD for a long time, immune processes in the mucosa of CD patients due to intestinal dysbiosis.32 Although a GFD is when tested on T cell lines established from the beneficial for patients, some clinical reports have CD intestinal mucosa.41 Another similar peptide shown that its effectiveness is compromised in of α-gliadin was tested in a Caco-2 cell line malnourished patients.33 Therefore, exclusion model and was reported to be resistant of wheat from the diet may lead to secondary to digestive enzymes, with the potential problems that may persist, along with an altered to penetrate across a Caco-2 monolayer.42 34 intestinal microbiota. The immunogenic potential of some peptides, Currently, trials of ongoing therapies in CD such as α-9 (57–68) and α-2 (62–75), was patients are still evoking different opinions confirmed when they were recognised by a 43 with regard to eliciting a beneficial response. T cell line derived from Norwegian CD patients. Thus, a wheat variety lacking the immunogenic Gliadin peptide 31–43 promotes an endoplasmic 2+ components of gluten may prove to be reticulum-stress pathway by inducing Ca mobilisation from the endoplasmic reticulum, beneficial for patients with this condition. whereas peptides 31–43 and 57–68 can induce Similarly, RNA interference (RNAi) is being immune dysfunction.44 In addition, peptides used by the scientific community to produce 31–43 and 57–68 can alter immune regulators a wheat variety devoid of immunogenic and induce deamidation of gluten peptides proteins. Other studies have used metabolomics and gliadin–tissue transglutaminase crosslinking techniques as markers of biosafety for crops, in enterocytes.36,43 As most of the peptides with an aim to elucidate the effect of fertilisers are immunogenic, some of them (p10-mer, on the metabolome of plants. Although QQPQDAVQPF) have protective effects metabolomics in this field is in its infancy, this that prevent gliadin-dependent dendritic cell review elucidates the use of metabolomics in maturation (Table 1).45 the development and validation of safety measures with regard to genetically modified (GM) plants. Hence, metabolomic approaches

Creative Commons Attribution-Non Commercial 4.0 July 2018 • ALLERGY & IMMUNOLOGY 115 Table 1: Immunotoxic gluten peptides and their diverse effects.

Immunotoxic peptides Protein Study model Key findings 31–43 α2-gliadin In vitro study on Protein induces anti-endomysial duodenal samples36 antibody production. 31–49 Prolamins of In vivo study on CD patients46 Patients with CD display α-gliadin variable sensitivity. 56–75 α-gliadin In vivo study on CD patients47 Coeliac-specific intestinal morphology and intraepithelial lymphocyte count increased significantly after treatment with both gliadin and the test peptide in all subjects. 57–89, 33-mer epitope α2-gliadin CD patients40 These peptides induced gut-derived human T cell lines derived from 14 CD patients. α-9(57–68) and α-gliadin T cell line derived from CD These are the common α-gliadin α-2(62–75) patients43 epitopes recognised by T cells in Norwegian CD patients. 31−49 α-gliadin T cell lines established from Potent activator of the innate CD intestinal mucosa41 immune activation in CD mucosa. 31−55 α-gliadin Caco-2 cell line model42 The peptide is resistant to digestive enzymes and can penetrate across a Caco-2 monolayer. 31–43 and 57–68 α-gliadin Caco-2 cell line model44 Promote an ER-stress pathway that may be relevant in CD pathogenesis and involved in inflammation. Decapeptides: Gliadin peptides Human monocyte-derived DC45 These peptides significantly QQPQRPQQPF (pRPQ) prevent the gliadin-induced and its homologue maturation of DC. QQPQDAVQPF (pDAV), and from human thyroid peroxidase (hTPO) (LDPLIRGILLARPAKLQV)

CD: coeliac disease; DC: dendritic cell; ER: endoplasmic reticulum.

RNA INTERFERENCE AND WHEAT endosperm of wheat and have the potential to downregulate gliadin proteins in A conserved biological response to double- the transgenic lines. Three transgenic wheat stranded RNA, known as RNAi or post- lines did not elicit T cell responses during transcriptional gene silencing, mediates in vitro treatment with T cell clones derived resistance to both endogenous parasitic and from intestinal lesions of CD patients. Another exogenous pathogenic nucleic acids, and study by the same group found that the regulates the expression of protein-coding downregulation of γ-gliadins proved beneficial genes. RNAi has been cultivated as a means to for enhancing the quality of the dough.50 manipulate gene expression experimentally and The subsequent transgenic clones resulted in to probe gene function on a whole-genome stronger dough quality that had tolerance to scale.48 RNAi technology has been used to over-mixing, from an industry perspective. In a produce GM plants, and to provide benefits recent study,51 all of the gliadin fraction of wheat to CD patients. Gil-Humanes et al.49 designed was successfully downregulated using RNAi hairpin constructs that are expressed in the so that the new wheat line exhibited stability

116 ALLERGY & IMMUNOLOGY • July 2018 EMJ EUROPEAN MEDICAL JOURNAL and tolerance to over-mixing, thereby showing with WDEIA. To the best of the authors’ better bread-making qualities. Continuing their knowledge, transgenic wheat varieties knocked work on previous wheat lines, they evaluated down for α-gliadins have not yet entered the physical properties and contents of clinical trials for CD in humans.56 However, the gliadins, thus further predicting the amount present trial of ω5-gliadins revealed that its of safe bread intake possible for CD patients. administration in the WDEIA population could Hence, Gil-Humanes et al.52 claimed their wheat reduce the incidence of this food allergy.55 line possessed similar baking and sensory The current clinical trial has also raised questions properties to normal flour but lacked 97% of on the applicability of transgenic lines, thereby the gliadin content. The wheat they developed pointing towards the complexity of WAD; also had better nutritional properties because i.e., WDEIA, CD, and wheat allergy, and the of higher content of lysine, an essential amino complex status of overall wheat proteins. acid. Although, no clinical trial was performed As the wide range of complex immunogenic in this study, a safe consumption of 67 g of proteins of wheat is still a challenge in developing their bread per day by the CD patients was immune-tolerant wheat lines, wheat proteins claimed as per descriptions of per day maximum as a whole should be targeted while trying to safe limits of gluten intake by Catassi et al.53 give an alternative to the GFD. This study also The ω5-gliadins are the major sensitising raised questions on the use of such silenced GM allergens in WDEIA, a disorder in which a patient plants even after a number of quality checks. experiences an allergic response during exercise. The effect of gene knockdown on other proteins To contain such a response, Altenbach et al.54 and metabolites that remain untraceable generated transgenic lines of wheat that were by current molecular techniques is another knocked down for ω5-gliadins. Furthermore, challenge regarding GM plants. Furthermore, in a set of additional experiments, it was what makes other proteins immunogenic for revealed that the protein content of flour was the WDEIA population is still not evident, and determined by the fertiliser regime in both whether it is because of a changed proportion transgenic samples as well as normal wheat of a targeted protein component, a consequence samples. Subsequently, ω5-gliadin was also of the gene silencing on the plant’s metabolic indicated to have a negative effect on flour control, or an imbalanced proportion of quality; thereby, suggesting that transgenic proteins is a subject of further investigations. wheat lines produce better flour quality than Gil-Humanes et al.57 reported that silencing wild-type wheat. induced a strong reduction in all the gliadins but caused a compensatory effect on the Altenbach et al.55 further observed that the synthesis of non-gluten proteins by upregulation. allergenic response of ω5-gliadins knock- down wheat lines was reduced in patients, Furthermore, the effect of gliadin knockdown as determined by serum immunoglobulin E on nutritional values of wheat remained reactivity in a clinical trial. Two transgenic wheat contradictory; however, Barro et al.58 attempted lines were assessed for their allergenic potential, to solve this by validating that silencing WAD- in which ω5-gliadin genes were silenced by associated proteins did not affect the total RNAi. Sera from 7 of 11 WDEIA patients showed protein and starch content of wheat. The authors greatly reduced levels of immunoglobulin E designed a combination of seven plasmids reactivity to ω5-gliadins for both transgenic containing RNAi fragments to mask all major lines. However, the sera also showed low levels components of WAD; i.e., α, γ, ω-gliadins, and of reactivity to other gluten proteins, but sera low molecular weight glutenin subunits. Out from three patients showed the greatest of these combinations, two provided a >90% reactivity to proteins other than ω5-gliadins, reduction in gluten content in comparison with which included either high molecular the wild-type when measured by anti-gliadin weight glutenin subunits (HMW-GS), α-gliadins, 33-mer monoclonal antibody. However, total or non-gluten proteins. The complexity of protein and starch contents remained unaffected immunological responses among these patients in all types of combinations. Gel electrophoresis, suggests that flour from the transgenic lines reversed-phase high-performance liquid would not be suitable for individuals diagnosed chromatography, and liquid chromatography–

Creative Commons Attribution-Non Commercial 4.0 July 2018 • ALLERGY & IMMUNOLOGY 117 mass spectrometry were used to measure the of risk proteins (immunogenic proteins i.e., extent of silencing. Though promising results gliadins).63,64 These adverse effects due to were also observed in experiments by Altenbach genomic level changes need validations using et al.,54 questions will remain unanswered metabolomics and other omics approaches until preclinical and clinical trials likewise integrated with clinical practice or trials in yield convincing results. Table 2 represents suitable models. Furthermore, the dysregulations information about the different RNAi approaches at the genetic level that modulate the metabolic profiles of plants is a food safety concern.65,66 used to produce safe wheat lines, and their characteristic features. Altenbach et al.63 revealed that environmental factors, including fertilisers, affected the FERTILISERS AS PREDOMINATING composition of specific flour proteins and their regulation. While conducting experiments, FACTORS FOR ACCUMULATION Triticum aestivum was grown with and without OF RISK PROTEINS IN WHEAT post-anthesis fertilisation (PAF), followed by quantitative two-dimensional gel electrophoresis Currently, both the prevalence and incidence of of the flour. Subsequently, proteomic profiling 59-62 food intolerances are increasing, but the clarified that the proportions of 54 unique reason for widespread food intolerance is proteins were altered in the treatment group; unknown. Studies have reported the adverse PAF treatment resulted in an increased effects of sulfur fertilisers on gene regulation of proportion of most ω-gliadins, HMW-GS, serpins, wheat plants, thereby increasing the content and some α-gliadins.

Table 2: RNA interference approaches to mask the expression of immunogenic proteins and their clinical relevance.

Wheat variety Silencing technique Targeted peptides Preclinical Effect on quality of wheat studies ‘Gazul’, ‘Podenco’, RNAi-mediated γ-gliadins No Downregulation of targeted and ‘Arpain’ silencing proteins resulted in improved quality and strength of gluten.50 Triticum aestivum RNAi-mediated α, γ, and ω-gliadin No All targeted fractions of gluten cv ‘Bobwhite 208’ silencing were downregulated and produced (BW208) and wheat lines with better tolerance T. aestivum cv to over-mixing, showing bread ‘Bobwhite 2003’ making qualities.51 (BW2003) T. aestivum cv. RNAi-mediated ω-5 gliadins No Knocked-down wheat lines show Butte 86 silencing improvement in quality and dough mixing properties. Response to fertilisers was also observed.54 T. aestivum RNAi-mediated ω-5 gliadins Trial on Complex immunological responses silencing WDEIA in patients were observed in WDEIA patients patients when administered with transgenic wheat lines, thus unsafe for WDEIA patients.55 T. aestivum RNAi-mediated ω-gliadins and In vitro Gluten content of six wheat lines cv. Bobwhite silencing γ-gliadins, and antibody was observed as highly reduced 208 (BW208) three of these also test when antigliadin 33-mer monoclonal silenced α-gliadins antibody. LC-MS/MS confirmed that wheat lines with three plasmid combinations was totally devoid of immunogenic peptides.58

LC: liquid chromatography; MS: mass spectrometry; RNAi: RNA interference; WDEIA: wheat-dependent exercise- induced anaphylaxis.

118 ALLERGY & IMMUNOLOGY • July 2018 EMJ EUROPEAN MEDICAL JOURNAL Hurkman et al.,64 while conducting studies to by proteomic analysis of different parts of observe the differential effects of temperature the plant (grains, leaves, straw, and husk).39,40 and fertiliser treatment on the development and Comparison of proteomic profiles of transgenic yield of grain, reported that both the treatments lines with that of normal lines gives an elicited the accumulation of gluten proteins in overview of the disturbed gene function or wheat flour. Under high temperature conditions, a description of knocked-down genes.63,64 gluten protein accumulation was not observed RNAi-induced silencing of 75 α-gliadin genes by PAF treatment, but the majority of HMW- that completely eliminated all gliadin proteins, GS, ω-gliadins, and some α-gliadins were as confirmed by proteome analysis,71 raises found to be elevated, thereby affirming that the safety issues70,72 related to such varieties; environmental stimuli do influence the these should be considered a priority, especially accumulation of risk proteins of CD. The studies those that are caused by a disturbance in a presented provide evidence that the man-made single plant metabolite or whole metabolome,73 means for increasing productivity of crops with and without treatment with fertilisers. can be a prominent cause of food intolerances Recently, metabolomics has helped in better because of a lack of food safety measures. The understanding the effects of any external aforementioned studies highlight the adverse stimuli (fertiliser) or genetic modifications effects of sulfur in terms of food safety and (gene knockdown) on plant metabolism and that they need to be validated by targeting the associated issues of biosafety. studies to evaluate the effect of sulfur-induced metabolome change and its effect on food Zörb et al.69 performed gas chromatography safety in relation to human health. Although mass spectrometry-based metabolite profiling there is lack of literature in this regard, there of flour of wheat line silenced for 75 α-gliadin is some evidence in support of the side effects genes and reported that, in comparison to wild- of sulfur fertiliser in GM wheat, as described in type plants, no appreciable difference in 109 the next section. metabolites was seen when plants were grown without sulfur. No unintended side effects of CROSS TALK ON RNA RNAi-induced gene silencing were observed. Conversely, the effect of fertilisation or single INTERFERENCE, FOOD SAFETY, nutrient (sulfur) availability in disturbing AND METABOLOMICS IN WHEAT metabolomic status was much higher than RNAi-induced silencing. The concentration It is worthwhile to elucidate the unwarranted of metabolites was also found to increase side effects of knocked-down wheat lines of with increasing sulfur supply. Zörb et al.69 also α-gliadins on plant metabolites since α-gliadins revealed that variable amounts of sulfur contain sulfur-rich amino acids, including supply influenced the yield aspects of grain cysteine and methionine. Furthermore, in wheat metabolome in both the wild-type and lines knocked down for α-gliadin genes, sulfur transgenic wheat line. Plants grown with higher amino acids used in the synthesis of α-gliadin sulfur content showed elevated grain metabolite might remain accumulated in the plant.67,68 concentration in comparison to the plants The accumulation of sulfur is reported to induce grown without sulfur treatment. Moreover, its incorporation into other plant metabolites principal component analysis showed that the that may become a food safety concern, but levels of β-amino isobutyric acid were affected this was not observed in metabolomic studies the most by variation in sulfur supply in wild- in transgenic versus wild lines.69,70 Therefore, type wheat. Furthermore, when α-gliadins with the support of metabolomics, the recent were knocked down, alanine, glycine, serine, studies advocated that transgenic varieties are homoserine, and tyrosine were found to be safe, because no change in the metabolomic associated with sulfur-induced differences in signature in transgenic verses wild wheat lines the grains. These studies confirm the effects was observed. of sulfur fertilisers in GM wheat but, noting RNAi-produced wheat lines lack antigenic the concerns discussed above it is pertinent components but result in diverse side effects to elucidate the effects of sulfur alone on on the plants. Initially, evaluation was performed gene regulation and also the associated

Creative Commons Attribution-Non Commercial 4.0 July 2018 • ALLERGY & IMMUNOLOGY 119 metabolomics in order to make the food safety sulfur fertilisers induce the accumulation of concerns relevant. gliadins in wheat, which makes the flour more immunogenic. Thus, the techniques adopted Collectively, it can be concluded that sulfur are useful for validating that fertilisers induce was not only potent in inducing genetic metabolic disturbances in plants. However, 63 dysregulations in wheat, but it might have clinical trials of wheat lines knocked down produced metabolic changes that were reflected for ω-gliadin showed reduction in disease- 69 in metabolomics studies. specific markers in WDEIA patients, but reactivity to other wheat protein components CONCLUSION could not be ruled out. This raises controversy on the gene expression status of other gluten The results of various experiments related to proteins that may be dysregulated as a proteomics and metabolomics are in consequence of interference exerted by RNAi. accordance with each other and reveal that Therefore, though omics techniques are useful fertilisers affect the proteome and metabolome to validate the global metabolic control or gene of wheat flour; thereby, raising issues related expression status, initial in vitro or in vivo to food safety. Although RNAi-induced gene preclinical studies are needed to guide better silencing does not affect the metabolomic future clinical output. Future research in this signature of wheat flour, fertilisers do have an field should be attributed to validation of effect. Sulfur fertilisers remain the determining food safety issues through improved platforms factor for disturbance in plant metabolism, of studying the transcriptome, proteome, followed by dysregulation in the proteome. metabolome, and immunome. Moreover, proteomic studies demonstrate that

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