Seven different genes encode a diverse mixture of isoforms of Bet v 1, the major birch pollen allergen

Abstract

Background: Pollen of the European white birch (Betula pendula, syn. B. verrucosa) is an important cause of hay fever. The main allergen is Bet v 1, member of the pathogenesis-related class 10 (PR-10) multigene family. To establish the number of PR-10/Bet v 1 genes and the isoform diversity within a single tree, PCR amplification, cloning and sequencing of PR-10 genes was performed on two diploid B. pendula cultivars and one interspecific tetraploid Betula hybrid. Sequences were attributed to putative genes based on sequence identity and intron length. Information on transcription was derived by comparison with homologous cDNA sequences available in GenBank/EMBL/DDJB. PCR-cloning of multigene families is accompanied by a high risk for the occurrence of PCR recombination artifacts. We screened for and excluded these artifacts, and also detected putative artifact sequences among database sequences.

Results: Forty-four different PR-10 sequences were recovered from B. pendula and assigned to thirteen putative genes. Sequence homology suggests that three genes were transcribed in somatic tissue and seven genes in pollen. The transcription of three other genes remains unknown. In total, fourteen different Bet v 1-type isoforms were identified in the three cultivars, of which nine isoforms were entirely new. Isoforms with high and low IgE-reactivity are encoded by different genes and one birch pollen grain has the genetic background to produce a mixture of isoforms with varying IgE-reactivity. Allergen diversity is even higher in the interspecific tetraploid hybrid, consistent with the presence of two genomes.

Conclusion: Isoforms of the major birch allergen Bet v 1 are encoded by multiple genes, and we propose to name them accordingly. The present characterization of the Bet v 1 genes provides a framework for the screening of specific Bet v 1 genes among other B. pendula cultivars or Betula species, and for future breeding for trees with a reduced allergenicity. Investigations towards sensitization and immunotherapy should anticipate that patients are exposed to a mixture of Bet v 1 isoforms of different IgE-reactivity, even if pollen originates from a single birch tree.

Figure 1

Phylogenetic profiles for detection of recombination. Phylogenetic profile of the sequences from B. pendula 'Long Trunk' obtained after a PCR of (a) 30 cycles (n = 72 sequences) and (b) 20 cycles (n = 53). (c) Phylogenetic profile of the GenBank PR-10 sequences from B. pendula (n = 66). The x-axis represents the sequence position (5'-3' including only informative positions). The y-axis indicates the phylogenetic correlation. Low values are indicative for recombination [27]. Low values at the edges are artifacts of the employed method.

Figure 2

Bayesian phylogenetic tree of the PR-10 sequences from B. pendula. Bayesian phylogenetic tree of the PR-10 sequences from B. pendula 'Schneverdinger Goldbirke' (Sv), 'Tristis' (Tr), and the B. pendula alleles from 'Long Trunk' (Lt). The 'Long Trunk' alleles that belong to the unknown parental species are not included in this figure. Numbers on the branches represent posterior probabilities after running a Markov chain Monte Carlo search for 1,000,000 generations. Sequences of PR-10 genes from Malus domestica (apple, X83672, Z72425, Z72427), Prunus armeniaca (apricot, AF020784), P. avium (cherry, U66076), and Pyrus communis (pear, AF057030) were used as outgroup. Each cluster that is identified as a putative gene has maximally two alleles per cultivar. Genes are classified into five major groups. The intron length is indicated on the right. If multiple introns of the same length exist within one group, the different types are shown between brackets. ^*1 PR-10.03B02.01 from 'Tristis' was an in vivo recombination of the PR-10.03D gene and the original PR-10.03B gene.

Figure 3

Amino acid sequences, amino acids that affect IgE-reactivity, and T-cell epitopes of the PR-10 proteins. Amino acid sequences of the PR-10 proteins from B. pendula 'Tristis' (Tr), 'Schneverdinger Goldbirke' (Sv), and the B. pendula alleles from 'Long Trunk' (Lt). Amino acids associated with high allergenicity are marked with grey boxes and those associated with low IgE-reactivity (located within B-cell epitopes) are marked with black boxes [12, 14]. The locations of the two major T-cell activating regions are indicated above the consensus [22].