Cellular and Molecular Genetics of Keratoconus

摘要 我们的长期目标是确定和表征参与发病机制的基因突变， 圆锥角膜（KC）。KC是一种双侧、不对称的角膜变性，其特征是局部变薄， 变薄的角膜突出。KC导致高度近视、不规则散光和角膜瘢痕形成。 尽管遗传因素有助于KC的发病机制，但其遗传原因仍有待确定。只 已知VSX 1和MIR 184基因的突变会导致KC，但它们占<10%的KC病例。KC是 通常通过常染色体显性模式遗传，尽管常染色体隐性模式已被 报道它对男性和女性的影响相似，在世界各地的所有族裔群体中都发现了这种疾病。社会生产 与其他人合作，从600多名KC个人及其家人那里收集DNA样本， 导致在三个基因中鉴定出KC分离突变，包括PPIP 5 K2（二磷酸肌醇 五磷酸激酶2）。PPIP 5 K2编码双功能激酶和磷酸酶，对 InsP 5/ 5-InsP 7和PtdIns（3，4，5）P3。在两个多代家庭中， 个体，我们已经确定了两个杂合突变，影响位于 PPIP 5 K2的多聚磷酸肌醇结合结构域（PBD）。PPIP 5 K2在人和小鼠中高度表达 角膜组织PPIP 5 K2相关的磷酸肌醇-3-激酶（PI-3激酶）途径，当被抑制时，影响 KC中AKT磷酸化和角膜基质细胞存活，导致角膜基质细胞凋亡 患者我们假设PPIP 5 K2突变改变TGFβ/AKT通路，导致TGFβ/AKT的变性。 角膜上皮和基质细胞，随后受影响的角膜变薄/隆起。我们将确定 使用原代人上皮细胞，在体外鉴定的突变如何影响PPIP 5 K2的细胞功能， Aim 1中的基质细胞。我们将使用以下方法确定Ppip 5 k2敲除小鼠中KC相关的角膜表型： 包括光学相干断层扫描、功能性视力筛查和裂隙灯在内的综合方法 目标2中的形态学检查。另一方面，我们将筛选PPIP 5 K2 使用全外显子组测序在另外的家族性和散发性患者中检测突变。PPIP 5 K2阴性 将用全外显子组测序进一步检查多重家族，以鉴定Aim 3中的新突变。 成功完成后，我们将拥有一个经过验证的KC动物模型和未来潜在的新靶点 KC诊断和临床治疗的研究。

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.