Original Articles: Mechanisms Of Allergy
Allergens of wild house dust mites: Environmental Der p 1 and Der p 2 sequence polymorphisms,☆☆

https://doi.org/10.1067/mai.2001.114652Get rights and content

Abstract

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 Der 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 Der 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 Der p 1 (U11695) and Der p 2 (P49278) have been updated with the inclusion of the sequence polymorphisms described in this study. (J Allergy Clin Immunol 2001;107:985-92.)

Section snippets

Materials

D pteronyssinus mites were collected in Perth, Western Australia, and by Dr Euan Tovey from Sydney. They were collected by vacuuming carpet or soft furnishings for 2 to 3 minutes with filter paper in a filter apparatus attached to the head of a vacuum cleaner pipe. The dust collected in the filter from the Perth houses was placed in a mesh container secured at the top of a funnel; a 1.5-mL Eppendorf was placed at the base of the funnel and used to collect the mites after exposure of the dust to

Wild mite reverse transcriptase PCR

The first batch of mites to be amplified were the dried mite cultures from Sydney. For the remaining mites, a reverse transcriptase PCR method was used. RNA was successfully extracted from every sample of mites, even when only a single mite was used as starting material. The RNA extracted from a single mite was sufficient for the synthesis of good yields of first-strand cDNA with gene-specific primers.

Der p 2 wild mite cDNA sequences

Nine Der p 2 cDNAs were isolated from the Perth wild mites. The sequences revealed 16

Discussion

Der p 1 and Der p 2 are major allergic specificities and targets for new immunotherapeutic strategies such as the injection of peptides, modified allergens, or DNA vaccines. cDNA has provided the information to pursue these objectives but has also revealed that they exhibit a high frequency of allelic polymorphism. This study examined which variants found in the environment could best represent the spectrum of polymorphic proteins that induce sensitization. It was found that although the

Acknowledgements

We would like to thank Dr Euan Tovey, from the Institute of Respiratory Medicine, University of Sydney, Australia, for collecting and preparing the dried house dust mite samples from the Sydney homes.

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    Supported by the Australian National Health and Medical Research Council.

    ☆☆

    Reprint requests: Wayne R. Thomas, PhD, TVW Telethon Institute for Child Health Research, PO Box 855, West Perth, Western Australia 6872, Australia.

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