Skip to content


Allergy, Asthma & Clinical Immunology

Open Access

Blood biomarkers of the late phase asthmatic response using RNA-Seq

  • Amrit Singh1, 2, 6Email author,
  • Casey P Shannon2, 6,
  • Gail M Gauvreau3,
  • Paul M O'Byrne3,
  • J Mark FitzGerald4,
  • Louis-Philippe Boulet5 and
  • Scott J Tebbutt1, 2, 6
Allergy, Asthma & Clinical Immunology201410(Suppl 2):A61

Published: 18 December 2014


Classification PerformanceComplement PathwayAllergen InhalationAlternative Complement PathwayClathrin Coated Vesicle


Asthmatic individuals respond differently, but reproducibly, to allergen inhalation challenge. Some individuals develop an isolated early response (ER), while others develop a dual response (DR). Peripheral blood cell transcriptome signatures can discriminate isolated early responders from dual asthmatic responders undergoing allergen inhalation challenge.


35 individuals (17 ERs and 18 DRs) participated in the allergen inhalation challenge. Blood samples were obtained prior to and 2 hours post allergen inhalation challenge. HiSeq-Illumina paired-ends 100bp sequencing was performed. A UCSC transcriptome using both the UCSC gene and gene-isoform transcripts was created using the RSEM (RNA-Seq by Expectation Maximization) package. RSEM uses Bowtie to align read files to the reference transcripts and estimates the expected number of counts per transcript. The biomarker pipeline consisted of 20x5-fold deep cross-validation using limma voom (linear models for microarrays and RNA-Seq using variance modeling at the observational level) for differential expression and elastic net for classification. Gene set enrichment analysis was performed using GeneGo.


Classification performance of the pre-challenge classifier across the 100 panels included an AUC of 0.76±0.02, a sensitivity of 0.70±0.03 and a specificity of 0.72±0.02. There were 511 gene transcripts identified across the 100 panels. Pathway analysis of the 511 gene transcripts identified lectin induced complement pathway, integrin inside-out signaling, alternative complement pathway and function of MEF2 in T lymphocytes as the top ranked pathways. Classification performance of the post-challenge classifier across the 100 panels included an AUC of 0.62±0.002, a sensitivity of 0.67±0.002 and a specificity of 0.54±0.003. There were 301 gene transcripts identified across the 100 panels. Pathway analysis of the 301 gene transcripts identified clathrin coated vesicles formation, CDC42 in cellular processes and regulation of actin cytoskeleton by Rho GTPases as the top ranked pathways.


The pre-challenge classifier out-performed the post-challenge classifier in the internal deep cross-validation as depicted by the classification performance measures. The lower performance of the post-challenge classifier in discriminating ERs from DRs may be due to a dilution of signal, given that both responder groups are undergoing an immune response to allergen inhalation. Incorporating changes in cellular composition and gene isoform expression estimates may improve classification performance and also reveal new insights into the mechanisms of the late phase asthmatic response.

Authors’ Affiliations

James Hogg Research Centre, St. Paul’s Hospital, University of British Columbia, Vancouver, Canada
Prevention of Organ Failure (PROOF) Centre of Excellence, Vancouver, Canada
Department of Medicine, McMaster University, Hamilton, Canada
Vancouver Coastal Health Research Institute, Vancouver General Hospital, Vancouver, Canada
Centre de Pneumologie de L’Hopital, Université Laval, Sainte-Foy, Canada
Department of Medicine, McMaster University, Hamilton, Canada


© Singh et al; licensee BioMed Central Ltd. 2014

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated.