Characterisation of a COPD-associated nephronectin (NPNT) functional splicing genetic variant in human lung tissuevialong-read sequencing

Background Identification of COPD disease-causing genes is an important tool for understanding why COPD develops, who is at highest COPD risk and how new COPD treatments can be developed. Previous COPD genetic studies have identified a highly significant genetic association near NPNT (nephronectin), a gene involved in tissue repair, but the biological mechanisms underlying this association are unknown. Methods Splicing quantitative trait locus (sQTL) analysis was performed to identify common genetic variants that alter RNA splicing in lung tissues. These lung sQTL signals were compared to COPD genetic association results near the NPNT gene using colocalisation analysis to determine whether genetic risk for COPD in this region may act through altered splicing. Long-read sequencing characterised COPD-associated splicing events at isoform-level resolution and in silico protein structural analysis identified likely functional effects of this alternative splicing. Results An established COPD genetic risk variant, rs34712979-A, creates a cryptic splice acceptor site that causes four separate splicing changes in NPNT . The only one of these splicing changes that was associated with COPD phenotypes involved a cassette exon (exon 3). Long-read RNA sequencing demonstrated that the COPD risk allele causes a shift in isoform usage away from the dominant NPNT isoform B precursor, which excludes exon 3, to the isoform A precursor, which splices-in exon 3. AlphaFold protein structural analysis reveals that inclusion of this exon disrupts an epidermal growth factor-like functional domain in NPNT. Conclusion Genetic variants in the NPNT gene increase COPD risk by changing RNA splicing of NPNT in the lung.