Birch pollen allergy: molecular characterization and hypoallergenic products

M.F. Schenk

Research output: Thesisinternal PhD, WU

Abstract

Allergic diseases, such as hay fever and food allergy, affect a substantial part of the population in westernized countries. Pollen of the European white birch (Betula pendula) is a considerable cause of hay fever (seasonal allergic rhinitis) in northern and central Europe. The major birch pollen allergen is Bet ν 1, which is the conventional allergen name for the birch pollen proteins of a large group of proteins otherwise known as PR-10 proteins. Individuals that suffer from birch pollen allergy are particularly prone to develop Oral Allergy Syndrome (OAS) due to the occurrence of an IgE-mediated cross-reaction between Bet ν 1 and PR-10 proteins in various plant foods. The high prevalence of apple allergy among hay fever sufferers is a good example of OAS. Allergic diseases can be severe for patients and have considerable medical and economical costs as well. Consequently, prevention of birch pollen allergy and OAS would contribute to an improvement of the quality of life of many patients. This thesis examines the feasibility of strategies that are directed towards development of hypoallergenic (= having a reduced allergenicity) birch trees or plant foods in order to alleviate Bet ν 1 [PR-10]- associated complaints.
When the development of hypoallergenic products involves application of genetic modification (GM), societal concerns about this technology should be taken into account. The attitude towards hypoallergenic products developed by GM was examined in the survey that is described in Chapter 2. This study differentiated between patients and non-patients. Attitude towards GM was measured for two applications directed against hay fever (hypoallergenic birch and grass) and one application directed against food allergy (hypoallergenic apple). Attitude was described in terms of two constructs that were labelled as 'benefits' and 'rejection factors'. Hay fever sufferers perceived greater 'benefits' associated with application of GM to develop hypoallergenic birch trees as compared to non-sufferers. The perceived 'benefits' increased with a higher self-reported impact of hay fever on quality of life. No attitudinal differences were observed between sufferers and non-sufferers for the attitudinal construct 'rejection factors'. Furthermore, the impact of perceived 'benefits' on acceptance of GM was larger than the impact of 'rejection factors'.
The perceptions of 'benefits' were further explored in Chapter 3. Here, the attitude towards hypoallergenic apples was examined in a survey in which consumers rate a set of apple profiles that varied in the breeding method that was applied during development (GM vs. traditional breeding), in pesticide usage, and in the degree of allergenicity. Acceptance of hypoallergenic apples was high among all consumers, also when GM was involved in their development. Acceptance of hypoallergenic products was higher among consumers with an apple allergy, presumably because they find a personal 'benefit' associated with these products. Novel GM products that are recognised as beneficial by some consumers may consequently experience an increased acceptance. However, both Chapter 2 and 3 indicated a clear consumer preference for
traditional breeding over breeding by GM for the development of hypoallergenic birch trees or food products. Approaches which focus on traditional breeding should thus be explored first.
Chapter 3 also evaluated appreciation of the hypoallergenic apple cultivar Santana, which was introduced in shops in a large-scale sales pilot labelled as 'suitable for individuals suffering from mild apple allergy' in 2006. A survey among consumers that bought the Santana measured the self-reported response to this apple. Almost half (42%) of the apple allergic consumers had no allergic reaction at all after eating the Santana. Most (96%) consumers who did experience an allergic reaction reported the symptoms as minor. The self-reported severity of the apple allergy, the occurrence of other fruit allergies, and age were associated with the occurrence of an allergic reaction to the Santana. Overall, the Santana was valued positively by the majority of apple allergic consumers, regardless of whether these consumers could eat the apple without experiencing an allergic reaction. Development of hypoallergenic foods such as Santana may therefore contribute to food allergy management, although variation among individual consumers in the allergic response to hypoallergenic products should be taken into account when developing and marketing such products.
A prerequisite for designing strategies for selection and breeding of hypoallergenic birch trees is knowledge on diversity of Bet ν 1 genes and on allergenicity of the protein variants (=isoforms) that are encoded by these genes. Chapter 4 describes a study on the variation of Bet ν 1 isoforms in .the most common birch species in Europe, Betula pendula. PR-10 sequences from three B. pendula cultivars were amplified, cloned and sequenced. Forty-four unique PR-10 sequences were recovered from B. pendula and these were assigned to thirteen putative genes based on sequence identity and intron length. Information on gene transcription was inferred from a comparison with existing mRNA sequences and suggested that seven of these genes are transcribed in pollen. Bet ν 1 isoforms are known to vary in IgE-reactivity. The study in Chapter 4 showed that isoforms with high and low IgE-reactivity are encoded by different genes. Thus, one birch pollen grain has the genetic background to produce a mixture of isoforms with varying IgE-reactivity. The sequence of one of the isoforms with a high IgE-reactivity was present in all examined B. pendula trees. The search for hypoallergenic birch trees was, therefore, expanded to other Betula species.
The selection of a representative set of species which cover variation among Betula species requires knowledge on phylogenetic relationships within the genus Betula. Chapter 5 describes a study on the reconstruction of the phylogeny of this genus using multilocus data from AFLP markers. The taxonomy of the genus Betula is complicated by the occurrence of parallel evolution of morphological traits, of polyploidisation events, and of extensive hybridisation among species. A large number of polymorphic AFLP markers (321 variable bands) were produced in 107 Betula accessions from 23 species and 11 hybrids. The analysis identified four distinct groups within the
genus. These groups are partly in disagreement with the traditional, but disputed, division of the genus. The majority of the species and all hybrids fell within subgenus Betula and are thus closely related to B. pendula. Subgenus Chamaebetula and part of the Neurobetula species should be merged with subgenus Betula. Apart from subgenus Betula, the subgenera Betulenta, Betulaster, and the remaining part of Neurobetula formed distinct and well-supported groups and should thus be maintained. The results from the AFLP study are to a large extent congruent with results from previous studies that made use of molecular (sequence) data.
The allergenic potency of Betula species other than B. pendula is described in Chapter 6 in which the PR-10 genes from eight birch species are cloned and sequenced. These species represent the various groups that were previously identified in the genus Betula. In total, 134 unique PR-10 sequences were recovered, including both sequences with a full open reading frame and pseudogenes. Sequences were attributed to putative genes, which could, in turn, be subdivided into seven subfamilies. Five subfamilies were common to all birch species. Q-TOF LC-MSE was applied to detect peptide fragments of Bet ν 1 that are unique for particular isoforms, in order to identify which PR-10 genes are expressed in pollen. The relative abundance of individual isoforms in the pollen proteome was also determined by Q-TOF LC-MSE. Each of the five examined birch species expressed a mixture of isoforms with at least 4-5 different isoforms. Both isoforms with a high and low IgE-reactivity were abundant in the Bet ν 1 mixture of B. pendula. The other birch species lacked Bet ν 1 isoforms that are similar to known isoforms with a low IgE-reactivity, but isoforms that are similar to known isoforms with a high IgE-reactivity were abundant in all species except B. lenta.
In Chapter 7, the antigenic and allergenic profiles of pollen extracts from twenty-four different birch trees were determined by SDS-PAGE and Western blotting. Fifteen different Betula species were examined, covering all previously identified subgenera/groups in the genus Betula. The major birch allergen Bet ν 1 was an abundant protein in all examined pollen extracts. Immune-reactivity of the extracts was tested using a pool of sera that were obtained from birch pollen allergic patients. A strong 17 kDa band, representing Bet ν 1, was recognized by the serum pool in all pollen extracts. The degree of immune-reactivity correlated well with the total amount of Bet ν 1 in the extract, which varied from 44% to 61% of the total protein content. Pollen extracts from different birch trees varied in the total protein content, presumably the result of variation in pollen quality due to pollen rupture and varying hydration during the extraction. Bet ν 1 isoforms in the pollen of eleven Betula species were subsequently digested with trypsin and the resulting fragments were analyzed and quantified by Q-TOF LC-MSE. Peptides that contained amino acid residues that are associated with high IgE-reactivity were detected in all examined species, and were abundant as well. Differences between Betula species in the relative presence of these amino acid residues were small. As a consequence, differences in allergenicity between birch trees
are probably far too small to have clinical relevance, implying that all examined Betula species will be highly allergenic.
The research presented here did not identify any birch trees in which Bet ν 1 variants with a high IgE-reactivity are reduced in abundance or are absent. Development of hypoallergenic birch trees thus must rely on other approaches, such as selection or introduction of male sterility (trees that do not produce male catkins were observed during the study), or the application of RNAi to silence the Bet ν 1 genes in pollen. Also, the conducted research showed that acceptance of hypoallergenic GM products is quite high, particularly for allergic sufferers who were allergic to the product being modified. This supports the hypothesis that consumer attitude towards GM is partly driven by recognition of specific and personally relevant benefits. The hypoallergenic apple cultivar Santana, which was developed by selection and not by GM, was well received by allergic consumers, indicating that there is a market for hypoallergenic products.

Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Wageningen University
Supervisors/Advisors
  • Jacobsen, Evert, Promotor
  • Frewer, Lynn, Promotor
  • Smulders, Rene, Co-promotor
  • Gilissen, Luud, Co-promotor
Award date19 Feb 2008
Place of Publication[S.l.]
Print ISBNs9789085048732
Publication statusPublished - 2008

Keywords

  • betula
  • allergies
  • allergens
  • apples
  • food allergies
  • diets
  • transgenic plants
  • consumer attitudes
  • cultivars
  • plant breeding
  • selection
  • ige
  • dna sequencing
  • genetic variation
  • pollen allergy
  • molecular breeding

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