Alpha-tryptase gene variation is associated with levels of circulating IgE and lung function in asthma

Abstract

Background: Tryptase, a major secretory product of human mast cells has been implicated as a key mediator of allergic inflammation. Genetic variation in the tryptases is extensive, and α-tryptase, an allelic variant of the more extensively studied β-tryptase, is absent in substantial numbers of the general population. The degree to which α-tryptase expression may be associated with asthma has not been studied. We have investigated the α-tryptase gene copy number variation and its potential associations with phenotypes of asthma.

Objectives: Caucasian families (n = 341) with at least two asthmatic siblings (n = 1350) were genotyped for the α-tryptase alleles, using high-resolution melting assays. Standards for the possible α-/β-tryptase ratios were constructed by cloning α-and β-tryptase PCR products to generate artificial templates. Association analysis of asthma affection status and related phenotypes [total and allergen-specific serum IgE, bronchial hyperresponsiveness to methacholine, forced expiratory volume in 1s (FEV1 ) and atopy and asthma severity scores] was undertaken using family-based association tests (FBAT).

Results: Four consistent melting patterns for the α-tryptase genotype were identified with alleles carrying null, one or two copies of the α-tryptase allele. Possessing one copy of α-tryptase was significantly associated with lower serum levels of total and dust mite-specific IgE levels and higher FEV1 measurements, while two copies were related to higher serum concentrations of total and dust mite-specific IgE and greater atopy severity scores.

Conclusions and clinical relevance: Associations of α-tryptase copy number with serum IgE levels, atopy scores and bronchial function may reflect roles for tryptases in regulating IgE production and other processes in asthma.

Keywords: IgE; asthma; gene copy number variation; mast cell; tryptase.

Figure 1

Genotyping assay development. (a) Comparison of α- and β- tryptase nucleotide sequences for the qPCR melting assay. Nucleotide numbers and GenBank accession numbers are indicated. MF, melting forward, MR, melting reverse primers. Six-base pair differences are shown. (b) Melt curve of DNA from the cell lines HMC-1 (β-tryptase alone), KU812 and LAD2 cells (both with α- and β- tryptases).

Figure 2

Genotyping of asthma family cohort for α-tryptase copy number. High-resolution melting analysis of possible α-tryptase ratios using (a) cloned PCR DNA fragments, and (b) DNA from the asthmatic family cohort. Difference plots are normalized to the β-tryptase only genotype, and four consistent plots were identified in DNA samples.