Functional characterisation and pharmacogenetic relevance of a novel gene associated with poor lung function

Abstract

Introduction: The single nucleotide polymorphism (SNP) rs6889822 has been reported to be sentinel SNP in a leading genome wide association study (GWAS) which strongly associated the HTR4 gene with the phenotype for altered lung function and COPD. It has also been identified as an expression quantitative trait locus (eQTL) of FBXO38 gene expression in humans and although to date literature supports no direct role for the FBXO38 gene in lung diseases, the eQTL association between rs6889822 and FBXO38 provides a basis for this. Aim: To study the influence of experimentally altered knockdown and overexpression of the FBXO38 gene on downstream pathways, using an in vitro airway cell culture model. Methodology: Knockdown of the FBXO38 gene in H460 airway lung model and HEK-293 experimental model was carried out through transfection of FBXO38 specific siRNA duplexes. An overexpression plasmid vector for delivery of wild type FBXO38 into the H460 lung model and HEK-293 experimental model was designed. Delivery of both the siRNA and recombinant FBXO38 plasmid was carried out by magnetofection. A comparative transcriptome carried out on duplicate samples between normal and altered FBXO38 overexpression and knockdown was obtained through RNA sequencing. The resulting sequencing data was bioinformatically analysed using a differential expressed genes (DEG) and gene set enrichment analysis (GSEA) approaches for patterns of differential gene expression and pathways associated with poor lung function. Results: Following analysis of RNAseq knockdown and overexpression data, 11 significantly differentially expressed genes were isolated through DEG analysis, and 3 significantly activated pathways were identified using GSEA analysis. A combination of bioinformatics and literature review particularly identified three differentially expressed genes, JUN, MAP2 and ITCH to have important airway regulatory roles tied to FBXO38 knockdown. The ubiquitination and proteasome degradation pathway was identified as being significantly activated with respect to FBXO38 knockdown. No differential expression was observed in overexpression samples. Conclusion: The results propose a role for FBXO38 in deregulation of the airway proteasome through its action as an E3 ubiquitin ligase capable of exerting an SCF dependant regulatory effect characteristic of its F-box protein class. In addition to this the genes JUN, MAP2 and ITCH were identified to have important airway regulatory roles tied to FBXO38 knockdown. The combined strengths of these observations propose FBXO38 to be a strong and promising potential candidate for further study in relation to altered lung function and the pathophysiology of chronic airway disease.