First successful reduction of clinical allergenicity of food by genetic modification: Mal d 1-silenced apples cause fewer allergy symptoms than the wild-type cultivar.

BACKGROUND: Genetic modification of allergenic foods such as apple has the potential to reduce their clinical allergenicity, but this has never been studied by oral challenges in allergic individuals. METHODS: We performed oral food challenges in 21 apple-allergic individuals with Elstar apples which had undergone gene silencing of the major allergen of apple, Mal d 1, by RNA interference. Downregulation of Mal d 1 gene expression in the apples was verified by qRT-PCR. Clinical responses to the genetically modified apples were compared to those seen with the wild-type Elstar using a visual analogue scale (VAS). RESULTS: Gene silencing produced two genetically modified apple lines expressing Mal d 1.02 and other Mal d 1 gene mRNA levels which were extensively downregulated, that is only 0.1-16.4% (e-DR1) and 0.2-9.9% (e-DR2) of those of the wild-type Elstar, respectively. Challenges with these downregulated apple lines produced significantly less intense maximal symptoms to the first dose (Vmax1) than with Elstar (Vmax1 Elstar 3.0 mm vs 0.0 mm for e-DR1, P = 0.017 and 0.0 mm for e-DR2, P = 0.043), as well as significantly less intense mean symptoms per dose (meanV/d) than with Elstar (meanV/d Elstar 2.2 mm vs 0.2 mm for e-DR1, P = 0.017 and 0.0 mm for e-DR2, P = 0.043). Only one subject (5%) remained symptom-free when challenged with the Elstar apple, whereas 43% did so with e-DR1 and 63% with e-DR2. CONCLUSION: These data show that mRNA silencing of Mal d 1 results in a marked reduction of Mal d 1 gene expression in the fruit and reduction of symptoms when these apples are ingested by allergic subjects. Approximately half of the subjects developed no symptoms whatsoever, and virtually all subjects wished to consume the apple again in the future.

Commentary: "A systems view on the future of medicine: inspiration from Chinese medicine?".

Chinese medicine could serve as a source of inspiration for drug development. Using systems biology in combination with reverse pharmacology is a novel way for the discovery of novel biological active compounds and targets as well as for proving the occurrence of synergy and prodrugs. A key factor for coming to evidence-based Chinese medicine will be the quality control. Metabolomics is a very promising tool for this purpose.

Detailed analysis of the expression of an alpha-gliadin promoter and the deposition of alpha-gliadin protein during wheat grain development.

BACKGROUND AND AIMS: Alpha-gliadin proteins are important for the industrial quality of bread wheat flour, but they also contain many epitopes that can trigger celiac (coeliac) disease (CD). The B-genome-encoded alpha-gliadin genes, however, contain very few epitopes. Controlling alpha-gliadin gene expression in wheat requires knowledge on the processes of expression and deposition of alpha-gliadin protein during wheat grain development. METHODS: A 592-bp fragment of the promotor of a B-genome-encoded alpha-gliadin gene driving the expression of a GUS reporter gene was transformed into wheat. A large number of transgenic lines were used for data collection. GUS staining was used to determine GUS expression during wheat kernel development, and immunogold labelling and tissue printing followed by staining with an alpha-gliadin-specific antibody was used to detect alpha-gliadin protein deposited in developing wheat kernels. The promoter sequence was screened for regulatory motifs and compared to other available alpha-gliadin promoter sequences. KEY RESULTS: GUS expression was detected primarily in the cells of the starchy endosperm, notably in the subaleurone layer but also in the aleurone layer. The alpha-gliadin promoter was active from 11 days after anthesis (DAA) until maturity, with an expression similar to that of a 326-bp low molecular weight (LMW) subunit gene promoter reported previously. An alpha-gliadin-specific antibody detected alpha-gliadin protein in protein bodies in the starchy endosperm and in the subaleurone layer but, in contrast to the promoter activity, no alpha-gliadin was detected in the aleurone cell layer. Sequence comparison showed differences in regulatory elements between the promoters of alpha-gliadin genes originating from different genomes (A and B) of bread wheat both in the region used here and upstream. CONCLUSIONS: The results suggest that additional regulator elements upstream of the promoter region used may specifically repress expression in the aleurone cell layer. Observed differences in expression regulator motifs between the alpha-gliadin genes on the different genomes (A and B) of bread wheat leads to a better understanding how alpha-gliadin expression can be controlled.

The role of profilin and lipid transfer protein in strawberry allergy in the Mediterranean area.

BACKGROUND: In contrast to other Rosaceae fruit, only few cases of patients with adverse reactions to strawberry are listed in literature. OBJECTIVE To identify allergenic proteins in strawberry and to express and characterize recombinant strawberry lipid transfer protein (LTP; rFra a 3). METHODS: Established apple-allergic patients were recruited on the basis of a reported allergic reaction to strawberry (n=28, confirmed by double-blind placebo-controlled food challenge in four patients) or on the basis of IgE reactivity to LTP (n=34). Sensitization to purified natural and recombinant allergens was assessed by RAST, immunoblot (inhibition) and basophil histamine release (BHR). A strawberry cDNA library was screened for genes homologous to known fruit allergens. Fra a 3 was cloned and expressed in the yeast Pichia pastoris and compared with peach and apple LTP by RAST, immunoblot-inhibition and BHR tests. RESULTS: Genes homologous to Bet v 1, Bet v 6, profilin and LTP were identified in a strawberry cDNA library. In BHR the rFra a 3 induced histamine release at a 100-fold higher concentration than peach LTP. RAST inhibition showed high cross-reactivity to peach and apple LTP, although IgE reactivity was lower by a factor 5. On strawberry immunoblot, patients' IgE showed reactivity to a Bet v 1 homologue, profilin, LTP and high-molecular weight bands. CONCLUSION: In addition to a Bet v 1 homologue, strawberry also contains IgE-binding profilin and LTP. The rFra a 3 has less allergenic potency than peach and apple LTP, and therefore is an interesting tool for future immunotherapy. Fra a 3 does not seem to be clinically relevant.

Genomic characterization and linkage mapping of the apple allergen genes Mal d 2 (thaumatin-like protein) and Mal d 4 (profilin).

Four classes of apple allergens (Mal d 1, -2, -3 and -4) have been reported. By using PCR cloning and sequencing approaches, we obtained genomic sequences of Mal d 2 (thaumatin-like protein) and Mal d 4 (profilin) from the cvs Prima and Fiesta, the two parents of a European reference mapping population. Two copies of the Mal d 2 gene (Mal d 2.01 A and Mal d 2.01 B) were identified, which primarily differed in the length of a single intron (378 or 380 nt) and in one amino acid in the signal peptide. Both Mal d 2.01 A and Mal d 2.01 B were mapped at identical position on linkage group 9. Genomic characterization of four Mal d 4 genes (Mal d 4.01 A and B, Mal d 4.02 A and Mal d 4.03 A) revealed their complete gDNA sequences which varied among genes in length from 862 to 2,017 nt. They all contained three exons of conserved length: 123, 138, and 135 nt. Mal d 4.01 appeared to be duplicated in two copies and located on linkage group 9. Mal d 4.02 A and Mal d 4.03 A were single copy genes located on linkage group 2 and 8, respectively.

Genomic cloning and linkage mapping of the Mal d 1 (PR-10) gene family in apple (Malus domestica).

Fresh apples can cause birch pollen-related food allergy in northern and central European populations, primarily because of the presence of Mal d 1, the major apple allergen that is cross-reactive to the homologous and sensitizing allergen Bet v 1 from birch. Apple cultivars differ significantly in their allergenicity. Knowledge of the genetic basis of these differences would direct breeding for hypoallergenic cultivars. The PCR genomic cloning and sequencing were performed on two cultivars, Prima and Fiesta, which resulted in 37 different Mal d 1 gDNA sequences. Based on the mapping of sequence-specific molecular markers, these sequences appeared to represent 18 Mal d 1 genes. Sixteen genes were located in two clusters, one cluster with seven genes on linkage group (LG) 13, and the other cluster with nine genes on the homoeologous LG 16. One gene was mapped on LG 6, and one remained unmapped. According to sequence identity, these 18 genes could be subdivided into four subfamilies. Subfamilies I-III had an intron of different size that was subfamily and gene-specific. Subfamily IV consisted of 11 intronless genes. The deduced amino acid sequence identity varied from 65% to 81% among subfamilies, from 82% to 100% among genes within a subfamily, and from 97.5% to 100% among alleles of one gene. This study provides a better understanding of the genetics of Mal d 1 and the basis for further research on the occurrence of allelic diversity among cultivars in relation to allergenicity and their biological functions.

Linkage map positions and allelic diversity of two Mal d 3 (non-specific lipid transfer protein) genes in the cultivated apple (Malus domestica).

Non-specific lipid transfer proteins (nsLTPs) of Rosaceae fruits, such as peach, apricot, cherry, plum and apple, represent major allergens for Mediterranean atopic populations. As a first step in elucidating the genetics of nsLTPs, we directed the research reported here towards identifying the number and location of nsLTP (Mal d 3) genes in the apple genome and determining their allelic diversity. PCR cloning was initially performed on two cultivars, Prima and Fiesta, parents of a core apple mapping progeny in Europe, based on two Mal d 3 sequences (AF221502 and AJ277164) in the GenBank. This resulted in the identification of two distinct sequences (representing two genes) encoding the mature nsLTP proteins. One is identical to accession AF221502 and has been named Mal d 3.01, and the other is new and has been named Mal d 3.02. Subsequent genome walking in the upstream direction and DNA polymorphism analysis revealed that these two genes are intronless and that they could be mapped on two homoeologous segments of linkage groups 12 and 4, respectively. Further cloning and sequencing of the coding and upstream region of both Mal d 3 genes in eight cultivars was performed to identify allelic variation. Assessment of the deduced nsLTP amino acid sequences gave a total of two variants at the protein level for Mal d 3.01 and three for Mal d 3.02. The consequences of our results for allergen nomenclature and the breeding of low allergenic apple cultivars are discussed.