Identification of Peptidyl-Prolyl Cis-Trans Isomerase-Like 4 as a Disease Causing Gene in Intracranial Aneurysms and its Role in Vertebrate CNS Specific Angiogenesis

Abstract

Abstract INTRODUCTION Intracranial aneurysm (IA) is a significant health burden affecting nearly 6 million people in the United States with an estimated prevalence of 3.2% worldwide. Using whole exome sequencing (WES) of 728 European IA cases, we identified damaging mutations in and RNA binding protein peptidyl-prolyl cis-trans isomerase-like 4 (PPIL4) in both familial and sporadic IA cases. METHODS We performed WES in 728 European IA cases followed by a mutational burden analysis between IA cases and a European (Finnish + Non-Finnish) control group of 1443 unaffected individuals, and European subjects (non-Finnish + Finnish) in the ExAC and gnomAD databases. Fisher exact test was used to determine the significance of the enrichment of rare (ExAC MAF <0.0001) and deleterious variants consisting of loss of function and deleterious missense mutations (MetSVM-D). Mutant ppil4 lines were generated using Crispr-CAS9 in zebrafish and X.tropicalis. RNA-seq and Slam-Seq were performed to identify transcriptome-wide changes in RNA half-life mediated by PPIL4. RESULTS Burden analysis revealed a significant enrichment of rare and deleterious PPIL4 variants in European IA cases when compared to 1443 controls (OR = INF., Fisher P = 3.17 × 10–4), ExAC (OR = 12.26, Fisher P = 5 × 10–4) or gnomAD (OR = 13.14, Fisher P = 3.4 × 10–4). PPIL4 mutants both in zebrafish and X.tropicalis models displayed cerebral hemorrhage and cerebrovascular simplification. Slam-seq analysis revealed that PPIL4 promotes mRNA stability of the genes implicated in VEGF signaling (FDR = 3.75E-07) and Focal adhesion pathways (FDR = 2.04E-06). CONCLUSION In this study, we identified PPIL4 as a candidate gene in IA pathogenesis and demonstrated morphological and hemodynamic impairment in vivo upon abrogating PPIL4 in 2 vertebrate models. Furthermore, we demonstrated that PPIL4 plays a major role in CNS angiogenesis and IA pathogenesis through promoting mRNA stability of key angiogenesis related genes, emphasizing the role of post-transcriptional RNA modification in cerebrovascular development and pathologies.