Transcriptomic profile of cystic fibrosis patients identifies type I interferon response and ribosomal stalk proteins as potential modifiers of disease severity

Abstract

Cystic Fibrosis (CF) is the most common monogenic disease among people of Western European descent and caused by mutations in the CFTR gene. However, the disease severity is immensely variable even among patients with similar CFTR mutations due to the possible effect of 'modifier genes'. To identify genetic modifiers, we applied RNA-seq based transcriptomic analyses in CF patients with a mild and severe lung phenotype. Global gene expression and enrichment analyses revealed that genes of the type I interferon response and ribosomal stalk proteins are potential modifiers of CF related lung dysfunction. The results provide a new set of CF modifier genes with possible implications as new therapeutic targets for the treatment of CF.

Fig 1. RNA-seq analyses of Mild CF and severe CF patients.

A) Heat-map of differentially expressed genes. Each column represents a separate patient, and each horizontal line represents a separate gene. Dendrogram of clustered samples and genes, in which mild CF and severe CF samples cluster with respect to their expression similarity. Expression profiles are measured by counts per million reads (CPM- model). B) Volcano plot of RNA-seq data, in which the -Log₁₀ of the false discovery rate is plotted against Log₂ fold change.

Fig 2. qPCR validation of RNA-seq findings.

A) Expression profile of RNA-seq data and qPCR data for selected genes were compared using the same samples. Severe CF samples were used as control group and the expression level set to 1 or -1. Expression was normalized using 18s as a reference gene. Results represent mean values and are expressed as Log₂ values of the fold change. Significant difference observed in qPCR results between mild CF and severe CF patients (**P value < 0.05; ***P value < 0.001). The level of significance was set to a P-value of < 0.0001 for RNA-seq data. B) Correlation analysis of RNA-seq and qPCR. (Log₂ values of the fold change; Spearman’s rho, ρ = 0.53, P value = 0.04).

Fig 3. IL-8 expression by qPCR and RNA seq.

A) Dot-plot shows the expression of IL-8 in severe and mild CF patients. Expression was normalized using 18s as a reference gene. Results represent mean values and are expressed as Log₂ values of the fold change. B) Using the same data of Fig 2A for IL-8 to understand different results between platforms.

Fig 4. Functional enrichment analysis of RNA-seq data.

A) Representative Gene Ontology (GO) terms enrichment among differentially expressed genes for biological processes. Genes involved in Type I interferon response, and ribosomal proteins responsible for endoplasmic reticulum transport and protein synthesis were over represented. B) ISMARA analysis predicts significant difference in IRF1,2,8 and STAT2 activity between mild CF and severe CF patients (*P value < 0.05).