Why is Fam83h critical for enamel formation?

为什么 Fam83h 对于牙釉质形成至关重要？

• 批准号: 8246309
• 负责人: JAN Ching Chun HU
• 金额: $ 35.96万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-04-01 至 2014-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8246309
• 关键词: 8q24.3; Ameloblasts; Amelogenesis; Amelogenesis Imperfecta; Bacteria; Biochemical; Biological; Candidate Disease Gene; Cells; Characteristics; Chromosomes; Chromosomes, Human, Pair 8; Co-Immunoprecipitations; Code; Complex; Defect; Dental Enamel; Development; Disease; Dominant-Negative Mutation; Enamel Formation; Etiology; Exons; Extracellular Matrix Proteins; Family; Genes; Genetic; Goals; Histologic; Immunohistochemistry; In Situ Hybridization; In Vitro; Inferior; Inherited; Lead; Learning; Link; MMP-20; Mass Spectrum Analysis; Monoclonal Antibodies; Mutate; Mutation; Nonsense Mutation; Odontogenesis; Oral health; Pain; Patients; Pattern; Peptides; Phenotype; Phosphoserine; Phosphothreonine; Phosphotyrosine; Play; Post-Translational Protein Processing; Principal Investigator; Proteins; Quality of life; Recombinants; Research; Role; Scanning Electron Microscopy; Secondary to; Structural Protein; Structure; System; Testing; Time Study; Tissues; Tooth structure; Transgenes; Transgenic Mice; Translations; Western Blotting; amelogenin; base; enamelin; genome-wide; glycosylation; in vivo; light microscopy; malformation; member; mouse model; novel; polyclonal antibody; programs; self esteem; yeast two hybrid system

Inherited diseases of dental enamel formation are grouped under the designation of amelogenesis imperfecta (AI). About 25% of all amelogenesis imperfecta (AI) cases are caused by the genes encoding four enamel extracellular matrix proteins, AMELX, ENAM, MMP20, and KLK4. Recently we identified mutations in FAM83H that cause AI in six different families suffering from amelogenesis imperfecta. This demonstrates that FAM83H is necessary for proper enamel formation. Virtually nothing is known about the Fam83h protein. Our objective is to learn the roles played by Fam83h in normal and defective enamel formation. Hypotheses: Because the phenotype in people with FAM83H mutations is limited to developing teeth, we hypothesize that Fam83H is expressed during odontogenesis. Because all of the AI- causing FAM83H mutations are dominant nonsense mutations that terminate translation in the last coding exon (exon 5), we further hypothesize that the Fam83h interacts with other cellular proteins to form functional complexes. We propose four Specific Aims: SA 1: To characterize the temporal and spatial pattern of Fam83h expression during odontogenesis and to determine its subcellular localization. SA 2: To isolate Fam83H protein for structural and functional characterization. SA 3: To identify Fam83H interacting proteins. SA 4: To determine if the expression of truncated Fam83h interferes with amelogenesis. Approach: The temporal and spatial pattern of Fam83h expression during odontogenesis is determined by in situ hybridization and immunohistochemistry. Recombinant and native Fam83h are used in structural and functional studies. Fam83h interacting proteins are identified using the yeast two-hybrid system and validated using a mammalian two-hybrid system, co- immunoprecipitation, and Far Western analyses. Normal and mutated Fam83h are expressed in ameloblasts as transgenes and their effects on enamel formation characterized. Heath Relatedness: Patients with inherited enamel defects (AI) have painful, disfigured teeth, lower self-esteem, and perceive themselves as having an inferior quality of life. The discovery that FAM83H is critical for dental enamel formation is a significant advance in our understanding of the etiology of AI. The proposed research may lead to a breakthrough in our understanding of important activities within ameloblasts and the discovery of new candidate gene(s) for AI.

牙釉质形成的遗传性疾病被归类为 釉质形成不全（AI）。约 25% 的釉质形成不全 (AI) 病例是由 由编码四种牙釉质细胞外基质蛋白 AMELX、ENAM、MMP20 和 KLK4。最近我们发现了 FAM83H 的突变导致六个不同家族的 AI 患有釉质发育不全。这表明 FAM83H 对于 适当的牙釉质形成。事实上，人们对 Fam83h 蛋白一无所知。我们的目标是 了解 Fam83h 在正常和有缺陷的牙釉质形成中所扮演的角色。 假设：因为 FAM83H 突变人群的表型仅限于发育 牙齿，我们假设 Fam83H 在牙发育过程中表达。因为所有的人工智能—— 引起 FAM83H 突变的是显性无义突变，其终止翻译 最后一个编码外显子（外显子 5），我们进一步假设 Fam83h 与其他细胞相互作用 蛋白质形成功能复合物。我们提出四个具体目标： SA 1：表征 Fam83h 表达的时间和空间模式 牙发生并确定其亚细胞定位。 SA 2：分离 Fam83H 蛋白以进行结构和功能表征。 SA 3：识别 Fam83H 相互作用蛋白。 SA 4：确定截短的 Fam83h 的表达是否干扰釉质形成。 方法：牙发生过程中 Fam83h 表达的时空模式是 通过原位杂交和免疫组织化学测定。重组和天然 Fam83h 用于结构和功能研究。 Fam83h 相互作用蛋白被鉴定 使用酵母双杂交系统并使用哺乳动物双杂交系统进行验证，共同 免疫沉淀和远西分析。正常和突变的 Fam83h 表达于 成釉细胞作为转基因及其对牙釉质形成的影响。 健康相关性：患有遗传性牙釉质缺陷 (AI) 的患者牙齿疼痛、畸形， 自尊心较低，认为自己的生活质量较差。发现 FAM83H 对于牙釉质形成至关重要，这是我们认识上的重大进步 AI 的病因学。拟议的研究可能会给我们的理解带来突破 成釉细胞内的重要活动以及人工智能新候选基因的发现。