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：
10398841
负责人：
Yutao Liu
金额：
$ 42.3万
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/10398841
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参与的遗传突变角膜结构（KC）。 KC是一种双边，不对称的角膜变性，其特征是局部变薄和稀薄的角膜的突出。 KC导致近视，不规则的散光和角膜疤痕。尽管遗传因素有助于KC发病机理，但其遗传原因仍有待鉴定。仅有的已知VSX1和MIR184基因中的突变会引起KC，但它们占KC病例<10％。 KC是尽管常染色体隐性模式已经是报告。它类似地影响男性和女性，并在全球所有种族中被发现。我们的生产力与其他人合作，积累了来自600多个公里个人及其家人的DNA样本导致在三个基因中鉴定KC分离突变，包括pPIP5K2（二磷酸肌醇五磷酸磷酸酯激酶2）。 PPIP5K2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。在两个有多个受影响的多代家族中个体，我们已经确定了两个影响位于氨基酸的杂合突变 PPIP5K2的聚磷酸结合结构域（PBD）。 PPIP5K2在人和小鼠中高度表达角膜组织。 PPIP5K2相关的磷酸肌醇3-激酶（PI-3激酶）途径在抑制时会影响 Akt磷酸化和角膜细胞的存活，导致角膜基质角膜细胞凋亡患者。我们假设PPIP5K2突变改变了TGFβ/AKT途径，导致变性角膜上皮和基质细胞，随后的受影响的角膜的变薄/膨胀。我们将确定鉴定的突变如何使用原发性人类上皮和 AIM 1中的基质细胞。我们将使用PPIP5K2敲除小鼠中KC相关的角膜表型全面的方法，包括光学相干断层扫描，功能视觉筛选和缝隙灯考试随后在AIM 2中进行形态学检查。另一方面，我们将筛选PPIP5K2 使用整个外显子组测序的其他家庭和零星患者的突变。 PPIP5K2阴性将通过整个外显子组测序进一步检查多重家族，以鉴定目标3中的新突变。成功完成后，我们将建立一个验证的KC动物模型，并为未来的潜在新颖目标针对KC诊断和临床疗法的研究。