Alternative splicing in lung influences COVID-19 severity and respiratory diseases

Abstract

Alternative splicing generates functional diversity in isoforms, impacting immune response to infection. Here, we evaluate the causal role of alternative splicing in COVID-19 severity and susceptibility by applying two-sample Mendelian randomization to cis-splicing quantitative trait loci and the results from COVID-19 Host Genetics Initiative. We identify that alternative splicing in lung, rather than total expression of OAS1, ATP11A, DPP9 and NPNT, is associated with COVID-19 severity. MUC1 and PMF1 splicing is associated with COVID-19 susceptibility. Colocalization analyses support a shared genetic mechanism between COVID-19 severity with idiopathic pulmonary fibrosis at the ATP11A and DPP9 loci, and with chronic obstructive lung diseases at the NPNT locus. Last, we show that ATP11A, DPP9, NPNT, and MUC1 are highly expressed in lung alveolar epithelial cells, both in COVID-19 uninfected and infected samples. These findings clarify the importance of alternative splicing in lung for COVID-19 and respiratory diseases, providing isoform-based targets for drug discovery.

Fig. 1. Flow Diagram of Study Design.

MR Mendelian randomization.

Fig. 2. The violin plots of normalized intron excision ratio stratified by sQTL genotypes.

a OAS1, b ATP11A, c DPP9 and d NPNT show the sQTL genotypes in dark orange that are associated with COVID-19 severity. e MUC1 and f PMF1 show the sQTL genotypes in dark blue that are associated with SARS-CoV-2 reported infection. Normalized intron excision ratios were obtained from GTEx sQTL phenotype matrices (https://www.gtexportal.org/home/datasets). The genotypes were obtained from whole exome sequence data. Lower edge of the whisker: the lowest value within 1.5 * IQR of the hinge, lower hinge: 25% quantile, horizontal line contained within the box: median value, upper hinge: 75% quantile, the upper edge of the whisker: the highest value that is within 1.5 * IQR of the hinge.

Fig. 3. MR estimates of the effect of RNA splicing at ATP11A, DPP9, NPNT, OAS1, MUC1, and PMF1 in lung with COVID-19 outcomes.

Forest plot showing odds ratio and 95% confidence interval from two sample Mendelian Randomization analyses (two-sided). All significant results listed in Fig. 3. were estimated by Wald ratio. P values are unadjusted. Unit of exposure: standard deviation of intron excision ratios as quantified by LeafCutter. Centre: Odds ratio, error bar: 95% confidence interval (CI).

Fig. 4. Gene expression in lung and peripheral blood mononuclear cells (PBMCs).

a The consensus transcript expression levels summarized per gene in 54 tissues in Human Protein Atlas (HPA), which was calculated as the maximum transcripts per million value (TPM) value for each gene in all sub-tissues categories of each tissue, based on transcriptomics data from HPA and GTEx. The consensus transcript expression level for PMF1 was not available in HPA. b RNA single cell type tissue cluster data (transcript expression levels summarized per gene and cluster) of lung (GSE130148) and peripheral blood mononuclear cell (PBMC) (GSE112845) were visualized using log₁₀(protein-transcripts per million [pTPM]) values. Each c-X annotation is taken from the clustering results performed in HPA. c Single-cell RNA expression profile of 23 lung COVID-19 autopsy donor tissue samples from GSE171668. The RNA expression of PMF1 was not detected in this dataset. The mean value of RNA expression of the cells annotated in the same subcategory was represented as a dot per each sample. The cell type annotation was manually performed in the original publication. AT1 alveolar type 1 epithelial cells, AT2 alveolar type 2 epithelial cells, EC endothelial cells, KRT8⁺ PATS/ADI/DATPs KRT8⁺ pre-alveolar type 1 transitional cell state, MAST mast cells, XXhi XX (gene expression) ^high cells, RBC red blood cells. Lower edge of the whisker: the lowest value within 1.5 * IQR of the hinge, lower hinge: 25% quantile, horizontal line contained within the box: median value, upper hinge: 75% quantile, the upper edge of the whisker: the highest value that is within 1.5 * IQR of the hinge.