Cellular and Molecular Genetics of Keratoconus

圆锥角膜的细胞和分子遗传学

基本信息

批准号：
9920145
负责人：
Yutao Liu
金额：
$ 43.54万
依托单位：
AUGUSTA UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-05-01 至 2024-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9920145
关键词：
1-Phosphatidylinositol 3-Kinase Affect Affinity American Amino Acids Animal Model Apoptosis Astigmatism Bilateral Binding Blindness Cell Culture Techniques Cell Proliferation Cell physiology Cells Clinical Code Collaborations Complementary DNA Cornea Corneal Diseases Corneal Topography DNA DNA Sequence Alteration Data Data Set Diagnosis Dideoxy Chain Termination DNA Sequencing Disease Epithelial Cells Ethnic group Exons Family Female Fibroblasts Functional disorder Generations Genes Genetic Genetic Research Genetic Transcription Goals Grant Human In Vitro Individual Inherited InsP5 Investigation Keratoconus Knockout Mice Location Messenger RNA Molecular Molecular Genetics Morphology Mus Mutation Myopia Optical Coherence Tomography Outcome Pathogenesis Pathogenicity Pathologic Pathology Pathway interactions Patients Pattern Phenotype Phosphatidylinositol Phosphates Phosphatidylinositols Phosphoric Monoester Hydrolases Phosphorylation Phosphotransferases Prevalence Proteins Proto-Oncogene Proteins c-akt Reporting Role Sampling Site-Directed Mutagenesis Stromal Cells Testing Thinness Time Tissues Transforming Growth Factor beta Validation Variant Vision Screening base cell motility corneal epithelium corneal scar diphosphoinositol pentakisphosphate kinase exome sequencing improved in vivo in vivo Model inositol heptakisphosphate lymphoblast male mouse model novel overexpression phosphatidylinositol 3,4,5-triphosphate prevent protein function response therapy development transcriptome sequencing translational impact

项目摘要

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.

抽象的我们的长期目标是识别和表征与疾病发病机制相关的基因突变圆锥角膜 (KC) 是一种双侧不对称角膜变性，其特征是局部变薄和变薄。变薄的角膜突出会导致高度近视、不规则散光和角膜疤痕。尽管遗传因素导致 KC 发病，但其遗传原因仍有待确定。已知 VSX1 和 MIR184 基因突变会导致 KC，但它们占 KC 病例的比例低于 10%。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证实据报道，它对男性和女性的影响相似，并且在世界各地的所有种族群体中都有发现。与其他人合作收集了 600 多名 KC 个人及其家人的 DNA 样本导致三个基因中 KC 分离突变的鉴定，包括 PPIP5K2（二磷酸肌醇）五磷酸激酶 2) 编码具有高亲和力的双功能激酶和磷酸酶。 InsP5 / 5-InsP7 和 PtdIns(3,4,5)P3 分别存在于具有多个受影响的两个多代家庭中。在个体中，我们发现了两个影响氨基酸氨基酸的杂合突变 PPIP5K2 的多磷酸肌醇结合域 (PBD) 在人和小鼠中高度表达。当 PPIP5K2 相关的磷酸肌醇 3 激酶（PI-3 激酶）途径受到抑制时，会影响角膜组织。 AKT 磷酸化和角膜细胞存活，导致 KC 中角膜基质细胞凋亡我们认为 PPIP5K2 突变会改变 TGFβ/AKT 通路，导致退化。我们将确定角膜上皮和基质细胞随后受影响的角膜变薄/凸出。使用原代人上皮和体外实验，确定的突变如何影响 PPIP5K2 的细胞功能目标 1 中的基质细胞。我们将使用以下方法确定 Ppip5k2 敲除小鼠中 KC 相关的角膜表型：综合方法，包括光学相干断层扫描、功能性视力筛查和裂隙灯目标 2 中进行形态学检查。另一方面，我们将筛选 PPIP5K2 使用全外显子组测序检测其他家族性和散发性患者的突变。将通过全外显子组测序进一步检查多重家族，以识别 Aim 3 中的新突变。成功完成后，我们将拥有经过验证的 KC 动物模型和未来潜在的新靶点研究方向为KC诊断和临床治疗。