Allergens of wild house dust mites: Environmental Der p 1 and Der p 2 sequence polymorphisms

Background: Sequence diversity is a common feature of mite allergens, Previous studies, using predominantly commercial mite clones, have described several polymorphic residues for Der p 1 and Her p 2. Objective: This study aimed at determining the occurrence of sequence diversity in environmental mite isolates. Methods: Mites were isolated from houses in Perth and Sydney, Australia. Total RNA was extracted from 1 to 30 Perth mites, and cDNA was synthesized by reverse transcriptase PCR, Der p 1 and Der p 2 cDNAs were PCR amplified and sequenced, Genomic Her p 1 DNA was amplified from whole Sydney mites directly by PCR and then sequenced, Results: Twelve Der p 1 and 9 Der p 2 cDNA clones and 3 Der p 1 genomic DNA were analyzed and showed a high frequency of amino acid polymorphisms. Der p 2 displayed a clear pattern of divergence toward 2 alleles that differed by 4 amino acids and had characteristic silent nucleotide changes. The pattern for Der p 1 was different and unusual, with almost no silent nucleotide substitutions but frequent sporadic missense changes. Proliferative responses of peripheral blood mononuclear cells to peptides containing polymorphic residues of Der p 1 were detected in 8 of 19 subjects, with stimulation being found only for either one of the variant forms of the peptides, However, the responses to variants of whole recombinant allergens were similar, as shown for 4 variants of Der p 2, Conclusion: Two clones for each of the allergens were identified as containing sequences that were largely representative of environmental isolates, A small-scale reverse transcriptase PCR used to produce cDNA from individual mites isolated From house dust will have wide application for studies on mite genetics and the production of recombinant mite allergens. Differences in T-cell responses to peptides representing variant epitopes were found, but responses to variants of whole recombinant allergens were similar. The GenBank and Swiss Prot database entries for Her p 1 (U11695) and Her p 2 (P49278) have been updated with the inclusion of the sequence polymorphisms described in this study.