Discriminatory Molecular Biomarkers of Allergic and Nonallergic Asthma and Its Severity

Selene Baos, David Calzada, Lucía Cremades-Jimeno, MªÁngeles de Pedro, Joaquín Sastre, César Picado, Joaquín Quiralte, Fernando Florido, Carlos Lahoz, Blanca Cárdaba, Selene Baos, David Calzada, Lucía Cremades-Jimeno, MªÁngeles de Pedro, Joaquín Sastre, César Picado, Joaquín Quiralte, Fernando Florido, Carlos Lahoz, Blanca Cárdaba

Abstract

Asthma is a complex disease comprising various phenotypes and endotypes, all of which still need solid biomarkers for accurate classification. In a previous study, we defined specific genes related to asthma and respiratory allergy by studying the expression of 94 genes in a population composed of 4 groups of subjects: healthy control, nonallergic asthmatic, asthmatic allergic, and nonasthmatic allergic patients. An analysis of differential gene expression between controls and patients revealed a set of statistically relevant genes mainly associated with disease severity, i.e., CHI3L1, IL-8, IL-10, MSR1, PHLDA1, PI3, and SERPINB2. Here, we analyzed whether these genes and their proteins could be potential asthma biomarkers to distinguish between nonallergic asthmatic and asthmatic allergic subjects. Protein quantification was determined by ELISA (in serum) or Western blot (in protein extracted from peripheral blood mononuclear cells or PBMCs). Statistical analyses were performed by unpaired t-test using the Graph-Pad program. The sensitivity and specificity of the gene and protein expression of several candidate biomarkers in differentiating the two groups (and the severity subgroups) was performed by receiver operating characteristic (ROC) curve analysis using the R program. The ROC curve analysis determined single genes with good sensitivity and specificity for discriminating some of the phenotypes. However, interesting combinations of two or three protein biomarkers were found to distinguish the asthma disease and disease severity between the different phenotypes of this pathology using reproducible techniques in easy-to-obtain samples. Gene and protein panels formed by single biomarkers and biomarker combinations have been defined in easily obtainable samples and by standardized techniques. These panels could be useful for characterizing phenotypes of asthma, specifically when differentiating asthma severity.

Keywords: allergy; asthma; biomarkers; gene expression; protein expression.

Figures

Figure 1
Figure 1
Mean levels of protein expression. (A) Mean levels of PHLDA1. (B) Mean levels of SERPINB2. (C) Mean levels of CHI3L1. (D) Mean levels of IL-8. (E) Mean levels of IL-10. (F) Mean levels of PI3. (G) Mean levels of POSTN. *Statistically significant comparison (P < 0.05) between the indicated groups. Protein levels of PHLDA1 and SERPINB2 were measured by Western blot in 5 NA and 6 AA subjects, and 11 NA and 11 AA, respectively. Densitometric analysis was done in individual blots (see “Materials and Methods”) using the β-actin protein for normalization. CHI3L1, IL-8, IL-10, PI3, and POSTN were quantified by ELISA in all patients included in the study population. The levels and relative expression of the proteins studied were compared among groups by unpaired t-test, using the Graph-Pad InStat 3 program. The error bars indicate the standard deviation. NA, total nonallergic asthma group; AA, total allergic asthma group.
Figure 2
Figure 2
Mean levels of protein expression by asthma severity subgroup. (A) Mean levels of PHLDA1. (B) Mean levels of SERPINB2. (C) Mean levels of CHI3L1. (D) Mean levels of IL-8. (E) Mean levels of IL-10. (F) Mean levels of PI3. (G) Mean levels of POSTN. *Statistically significant comparison (p < 0.05) between the indicated groups. **Statistically significant comparison (p < 0.005) between the indicated groups. Protein levels of PHLDA1 were measured by Western blot in 3 severe NA and 2 moderate-mild NA patients, 3 patients with severe AA, and 3 with moderate-mild AA. Protein levels of SERPINB2 were measured by Western blot in 6 severe NA and AA subjects and in 5 moderate-mild NA and AA patients. Densitometric analysis was done in individual blots (see “Materials and Methods”) using the β-actin protein for normalization. CHI3L1, IL-8, IL-10, PI3, and POSTN were quantified by ELISA in all patients studied. The levels and relative expression of the proteins studied were compared among groups by unpaired t-test, using the Graph-Pad InStat 3 program. The error bars indicate the standard deviation. S, group of subjects with severe asthma; MM, group of subjects with moderate-mild asthma.

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