In search of genetic mutations for familial keratoconus

Abstract

Keratoconus (KC) is the most common corneal degenerative disorder and a leading cause of corneal transplantation in developed countries. KC is a multi-factorial disease with involvement of genetic, environmental, and hormonal factors. Although KC has been widely studied, the main cause of the disease and the molecular mechanism remain unknown. We aimed to study the molecular genetics of KC via utilizing next-generation sequencing technology including RNA-Seq, whole exome sequencing, and whole genome sequencing. We used RNA-Seq to study the KC-affected corneal transcriptome. We identified 436 coding RNAs and 584 lncRNAs with differential expression in the KC-affected corneas with a |fold change| ≥ 2 and a false discovery rate ≤ 0.05. Pathway analysis, using WebGestalt, indicated the enrichment of the genes involved in the extracellular matrix, protein binding, glycosaminoglycan binding, and cell migration. Co-expression analysis revealed 296 pairs of genes with significant KC-specific correlations. The RNA-Seq data analysis highlighted the potential roles of several genes (CTGF, SFRP1, AQP5, lnc-WNT4-2:1, and lnc-ALDH3A2-2:1) and pathways (TGF-β, WNT signaling, and PI3K/AKT pathways) in KC pathogenesis. Next, we used whole genome and exome sequencing to figure out the causal mutation(s) in a four-generation KC family with a linkage locus on Chr5q14.3-q21.1. We found a missense mutation in the phosphatase domain of PPIP5K2 (c.1255T>G, p.Ser419Ala). We found another missense mutation in the same domain of PPIP5K2 (c.2528A>G, p.Asn843Ser) in a second KC family. PPIP5K2 is a bifunctional enzyme involved in the inositol phosphate metabolic pathway. In vitro functional assays indicated the impact of the identified mutations on the enzymatic activity of PPIP5K2. PPIP5K2 expresses at a higher level than its homolog PPIP5K1 in both human and mouse corneas. A transgenic mouse model with the loss of phosphatase activity and elevated kinase activity of Ppip5k2 exhibited corneal structural abnormalities emphasizing the important role of PPIP5K2 in the homeostasis of corneal integrity. This study advances our knowledge of KC genetic etiology and helps in identifying a potential therapeutic target for KC.