PPIP5K2 mutations & Keratoconus in Human Patients and Mouse Models

Abstract

Purpose : Genetic factors contribute to the pathogenesis of Keratoconus (KC). A linkage locus (Chr5q14.3-q21.1) was reported in a four-generation KC pedigree with an autosomal dominant mode; however, no mutations have been identified. We aimed to identify and characterize the underlying mutation and the related molecular mechanism.

Methods : We selected 10 subjects for whole exome sequencing (WES) and 5 subjects for whole genome sequencing. WES was also performed in other 20 multiplex families. We filtered and focused on exonic, non-synonymous variants with good quality and read depth, segregated with the disease phenotype, and with MAF≤1% in public databases. We validated the variants using Sanger sequencing. The biochemical impact of the identified PPIP5K2 mutations was evaluated using an established HPLC-based in vitro assay with purified overexpressed proteins carrying these mutations. We assessed the gene expression in human primary corneal epithelial and stromal cells using droplet digital PCR and the protein expression in mouse and human corneas using immunofluorescence. We examined a Ppip5k2 gene trap mouse model with no phosphatase activity, but elevated kinase activity (Ppip5k2^{K^}) using slit lamp, OCT (Optical Coherence Tomography) scanning, and histology staining for KC-related corneal phenotypes.

Results : We identified a missense variant in PPIP5K2 (c.1255T>G, p.Ser419Ala) in the linkage family and another variant (c.2528A>G, p.Asn843Ser) in a second family. PPIP5K2 encodes a bifunctional kinase/phosphatase that controls metabolism and signaling of inositol phosphates. In vitro biochemical assays indicate reduced phosphatase activity of PPIP5K2 carrying these two mutations. Both human corneal epithelial and stromal cells showed higher expression of PPIP5K2 mRNA. Protein staining indicated significantly higher expression of PPIP5K2 than PPIP5K1 in human and mouse cornea. Mouse corneal OCT-derived tomography and histology demonstrated the presence of KC-related corneal surface irregularities in the Ppip5k2^{K^}/Ppip5k2^{K^} mice compared to littermate controls at 3-4 months of age. Slit lamp data indicated varying degrees of corneal opacity and shallow anterior chamber compared to littermate controls at 3 months.

Conclusions : We identified two potential mutations in the phosphatase domain of PPIP5K2 gene in multiplex KC families. Our in vitro and in vivo data strongly suggest an important role of PPIP5K2 in KC pathogenesis.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.