Phase 2 – Statistical analysis

Asthmatics and non-asthmatics have increased expression of STAT2 and OAS genes after HRV challenge. Asthmatics have higher DGE than non-asthmatics.

Table 1: Summary of Phase 2 statistical analysis results of gene expression in pairwise comparison sets.

• Foldchange was calculated as ratio between averages of each sample set.

• Statistical analysis of each gene’s expression data produced p-values.

• Orange highlights OAS1 in Set B because its p-value is almost <0.05.

• STAT1 and OASL included for context.

• See Appendix 3 for supplementary text-based tables.

Table 1: Summary of Phase 2 statistical analysis results of gene expression in pairwise comparison sets.

Key

• MWU = Mann-Whitney U-test

• W = Wilcoxon signed rank (matched pairs) test

• ast = asthmatic

• non = non-asthmatic

• hrv = HRV16 treatment

• veh = vehicle treatment

• ‘A’ = null hypothesis accepted

• ‘R’ = null rejected.

Sets A & D – Wilcoxon Matched Pairs test

Null hypotheses rejected.

All tests showed HRV16 significantly affected DGE of OAS1,2&3 and STAT2 in asthmatic compared to non-asthmatic lung tissue (W(0) = 0, p≤0.05, two-tailed).

Sets B & C – Mann–Whitney U-test

Set B (α:γ): null hypotheses rejected, barring OAS1.

Three sets showed HRV16 challenge significantly affected DGE in asthmatic tissues compared to non-asthmatic. OAS1 had almost-significant DGE (p=0.055).

Set C (β:δ): null hypotheses accepted.

All tests showed vehicle didn’t significantly affect DGE in asthmatic tissues compared to non-asthmatic.

Combining results

A correlation is indicated in Set B between genetic expression and tissues being asthmatic; the upregulation may be an intensification of the antiviral response.

Figure 2 presents the second-order comparison between the differential expression ratios. All genes are upregulated in both non-asthmatic (Set D, turquoise) and asthmatic (Set A, orange) HAEC immune response to HRV16. Set B (purple), compares the asthmatic and non-asthmatic HAEC responses to HRV16. Comparing the foldchange from each gene indicates a correlation between tissues being asthmatic and upregulated genetic expression: the ratio of each gene is similar, suggesting the asthmatic upregulation could be an intensification of the regular antiviral response.

Figure 2: Comparison between DGE ratios of STAT2 and OAS genes, relative to control sample δ.

• DGE ratios were recalibrated relative to control sample δ (vehicle treatment on non-asthmatic tissue) to compare the DGE of each gene directly.

• DGE pairs were grouped by gene and by coloured by set for easy visual comparison.

• Foldchange ratios between samples in each set were labelled (FC).

• Legend key: Left: Comparison sets (A,B,C,D) are colour-coded. Right: sample sets (α,β,γ,δ) are colour-coded by which comparison sets they appear in.

A chart showing the comparison between differential expression ratios of STAT2, OAS1, OAS2 & OAS3 relative to control sample δ (vehicle treatment on non-asthmatic tissue). Foldchange ratios included for clarity, especially comparison set B (HRV effect on non-asthmatic vs asthmatic tissue).

Legend key

• Left: Comparison sets (A,B,C,D) are colour-coded.

• Right: sample sets (α,β,γ,δ) are colour-coded by which comparison sets they appear in.

Baseline expressions (Set C, lime) of STAT2, OAS2 and STAT1 (not pictured) were lower in asthmatic tissue than non-asthmatic (0.98x, 0.93x, 0.87x respectively), despite greater DGE in HRV16 response; but the analysis results were statistically insignificant, so likely represent natural fluctuations.