Characterization of a COPD-Associated NPNT Functional Splicing Genetic Variant in Human Lung Tissue via Long-Read Sequencing

Abstract

Chronic obstructive pulmonary disease (COPD) is a leading cause of death worldwide. Genome-wide association studies (GWAS) have identified over 80 loci that are associated with COPD and emphysema, however for most of these loci the causal variant and gene are unknown. Here, we utilize lung splice quantitative trait loci (sQTL) data from the Genotype-Tissue Expression project (GTEx) and short read sequencing data from the Lung Tissue Research Consortium (LTRC) to characterize a locus in nephronectin (NPNT) associated with COPD case-control status and lung function. We found that the rs34712979 variant is associated with alternative splice junction use in NPNT , specifically for the junction connecting the 2nd and 4th exons (chr4:105898001-105927336) (p=4.02x10-38). This association colocalized with GWAS data for COPD and lung spirometry measures with a posterior probability of 94%, indicating that the same causal genetic variants in NPNT underlie the associations with COPD risk, spirometric measures of lung function, and splicing. Investigation of NPNT short read sequencing revealed that rs34712979 creates a cryptic splice acceptor site which results in the inclusion of a 3 nucleotide exon extension, coding for a serine residue near the N-terminus of the protein. Using Oxford Nanopore Technologies (ONT) long read sequencing we identified 13 NPNT isoforms, 6 of which are predicted to be protein coding. Two of these are full length isoforms which differ only in the 3 nucleotide exon extension whose occurrence differs by genotype. Overall, our data indicate that rs34712979 modulates COPD risk and lung function by creating a novel splice acceptor which results in the inclusion of a 3 nucelotide sequence coding for a serine in the nephronectin protein sequence. Our findings implicate NPNT splicing in contributing to COPD risk, and identify a novel serine insertion in the nephronectin protein that warrants further study.

Proteome-wide Mendelian randomization implicates nephronectin as an actionable mediator of the effect of obesity on COVID-19 severity

“…Thus, we evaluated whether the cis-pQTL influenced either total RNA expression level or a specific isoform level of NPNT. Given that the lung is a primary target organ in the context of COVID-19 severity, NPNT is highly expressed in the lung, NPNT splice isoforms have previously been implicated as a risk factor for COPD and other lung outcomes 38,39,40 , and SNPs influencing a specific isoform level would be a splice QTLs (sQTLs), we performed colocalization analysis of the cis-pQTL with eQTLs and sQTLs for NPNT in lung tissue from GTEx 41 .…”

“…Notably, the lead cis-pQTL from the deCODE study, i.e., rs34712979, was also identified by the FENLAND study 27 and has been reported to create a cryptic splice acceptor site, which inserts a three-nucleotide sequence coding a serine residue at the 5'-splice site of exon 2, resulting in perturbations of the alpha-helix motif 38 . Further, this lead cis-pQTL (rs34712979) and another cis-pQTL (rs78213340) from the AGES Reykjavik study-that was not in high LD with rs34712979 (r 2 = 0.234)-were both associated with the same exon-skipping splicing in lung in GTEx (Supplementary Table 14).…”

Nephronectin as a Matrix Effector in Cancer

“…A recent preprint indicates that a splice variant of NPNT includes an additional serine residue in the splice site, and is associated with COPD. The serine residue is predicted to be located between the signal peptide and the first EGF-like repeat [ 57 ]. The structure and function of this newly discovered variant of NPNT is still to be resolved.…”

Nephronectin-integrin α8 signaling is required for proper migration of periocular neural crest cells during chick corneal development

“…Other studies have also demonstrated that Npnt plays a potential role in diseases such as chronic obstructive pulmonary disease (COPD) (Saferali et al, 2020), diabetic glomerulosclerosis (Nakatani et al, 2012), Fraser syndrome (Kiyozumi et al, 2012), and in various cancers (Magnussen et al, 2021). Despite the pleiotropic functions of Npnt in development and disease, its expression and function have not yet been described in the cornea.…”