Cellular and Molecular Genetics of Keratoconus

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ABSTRACT Our long term goal is to identify and characterize genetic mutations involved in the pathogenesis of keratoconus (KC). KC is a bilateral, asymmetric corneal degeneration characterized by localized thinning and protrusion of the thinned cornea. KC leads to high myopia, irregular astigmatism, and cornea scarring. Although genetic factors contribute to KC pathogenesis, its genetic causes remain to be identified. Only mutations in the VSX1 and MIR184 genes are known to cause KC, but they account for <10% KC cases. KC is typically inherited through autosomal dominant patterns, though autosomal recessive patterns have been reported. It affects males and females similarly and is detected in all ethnic groups worldwide. Our productive collaborations with others amassing DNA samples from more than 600 KC individuals and their families have led to the identification of KC-segregating mutations in three genes, including PPIP5K2 (diphosphoinositol pentakisphosphate kinase 2). PPIP5K2 encodes a bifunctional kinase and phosphatase with high affinity to InsP5 / 5-InsP7 and PtdIns(3,4,5)P3 respectively. In two multi-generation families with multiple affected individuals, we have identified two heterozygous mutations affecting amino acids located in the polyphosphoinositide binding domain (PBD) of PPIP5K2. PPIP5K2 is highly expressed in human and mouse cornea tissue. The PPIP5K2-related phosphoinositide-3-kinase (PI-3 kinase) pathway, when inhibited, affects AKT phosphorylation and keratocyte survival, leading to the apoptosis of corneal stromal keratocytes in KC patients. We hypothesize that PPIP5K2 mutations alter TGFβ/AKT pathways, leading to degeneration of corneal epithelial and stromal cells with subsequent thinning/bulging of the affected cornea. We will determine how the identified mutations affect the cellular function of PPIP5K2 in vitro using primary human epithelial and stromal cells in Aim 1. We will determine the KC-related corneal phenotypes in Ppip5k2-knockout mice using comprehensive approaches including optical coherence tomography, functional vision screening, and slit lamp exams followed by morphological examinations in Aim 2. On the other hand, we will screen PPIP5K2 mutations in additional familial and sporadic patients using whole exome sequencing. PPIP5K2-negative multiplex families will be further examined with whole exome sequencing to identify novel mutations in Aim 3. Upon successful completion, we will have a validated animal model of KC and potential novel targets for future research directed at KC diagnosis and clinical therapy.