Cellular and Molecular Genetics of Keratoconus

圆锥角膜的细胞和分子遗传学

• 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
• 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

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.

摘要 我们的长期目标是识别和表征与糖尿病发病机制有关的基因突变。 圆锥角膜(KC)。KC是一种双眼不对称的角膜变性，其特征是局限性变薄和 变薄的角膜突出。KC会导致高度近视、不规则散光和角膜瘢痕形成。 虽然遗传因素在KC的发病机制中起作用，但其遗传原因仍有待确定。仅限 VSX1和MIR184基因的突变是已知的引起KC的原因，但它们占KC病例的10%。KC是 通常通过常染色体显性遗传，尽管常染色体隐性遗传模式 据报道。它对男性和女性的影响相似，在世界各地的所有种族中都发现了这种疾病。我们的生产力 与其他人合作，收集了600多名KC个人及其家人的DNA样本 导致在包括PPIP5K2(二磷酸肌醇)在内的三个基因中鉴定出KC分离突变 五磷酸激酶2)。PPIP5K2编码高亲和力的双功能激酶和磷酸酶 InsP5/5-InsP7和PtdIns(3，4，5)P3。在两个受多个影响的多代家庭中 对于个体，我们发现了两个杂合突变，它们影响位于 PPIP5K2的多磷肌醇结合区(PBD)。PPIP5K2在人和小鼠中高表达 角膜组织。PPIP5K2相关的磷脂酰肌醇-3-激酶(PI-3)通路被抑制时，会影响 AKT磷酸化与角膜基质细胞存活导致KC中角膜基质细胞的凋亡 病人。我们推测，PPIP5K2突变改变了转化生长因子β/akt通路，导致肝细胞退变。 角膜上皮细胞和基质细胞，随后受影响的角膜变薄/膨胀。我们将决定 已识别的突变如何影响PPIP5K2的细胞功能 Aim 1中的基质细胞。我们将使用以下方法确定Ppi5k2基因敲除小鼠的KC相关角膜表型 包括光学相干断层扫描、功能视力筛选和裂隙灯在内的综合方法 在AIM 2中，我们将对PPIP5K2进行筛查 使用完整外显子组测序在额外的家族性和散发性患者中发现突变。PPIP5K2-阴性 多重家族将通过整个外显子组测序进一步检查，以确定AIM 3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目标 针对KC的诊断和临床治疗的研究。