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: 7623768
• 负责人: JAN Ching Chun HU
• 金额: $ 33.02万
• 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/7623768
• 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; Electron Microscopy; 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; 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; public health relevance; self esteem; yeast two hybrid system

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: Our group has determined that defects in FAM83H (family with sequence similarity 83 member H) on chromosome 8 cause severe enamel malformations (autosomal dominant hypocalcification amelogenesis imperfecta). Our objectives are to discover the normal function of Fam83h and how defects in this protein result in enamel malformations. Our long term goal is to advance understanding of normal and pathological tooth formation to stimulate improvements in the public's oral health.

描述（由申请人提供）：牙釉质形成的遗传性疾病被归为釉质生成不全症（AI）。约 25% 的牙釉质生成不全 (AI) 病例是由编码四种牙釉质细胞外基质蛋白 AMELX、ENAM、MMP20 和 KLK4 的基因引起的。最近，我们发现了 FAM83H 的突变，该突变导致六个不同的患有牙釉质发育不全的家庭出现 AI。这表明 FAM83H 对于正确的牙釉质形成是必需的。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：由于 FAM83H 突变人群的表型仅限于牙齿发育，因此我们假设 Fam83H 在牙发育过程中表达。由于所有引起 AI 的 FAM83H 突变都是显性无义突变，会终止最后一个编码外显子（外显子 5）的翻译，因此我们进一步假设 Fam83h 与其他细胞蛋白相互作用形成功能复合物。我们提出了四个具体目标： SA 1：表征牙发育过程中 Fam83h 表达的时间和空间模式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：通过原位杂交和免疫组织化学测定成牙过程中 Fam83h 表达的时间和空间模式。重组和天然 Fam83h 用于结构和功能研究。 Fam83h 相互作用蛋白使用酵母双杂交系统进行鉴定，并使用哺乳动物双杂交系统、免疫共沉淀和 Far Western 分析进行验证。正常和突变的 Fam83h 作为转基因在成釉细胞中表达，并表征了它们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，并认为自己的生活质量较差。 FAM83H 对牙釉质形成至关重要的发现是我们对 AI 病因学理解的重大进步。拟议的研究可能会导致我们对成釉细胞内重要活动的理解取得突破，并发现人工智能的新候选基因。 公共健康相关性：我们的小组已确定，8 号染色体上的 FAM83H（具有序列相似性的家族 83 成员 H）缺陷会导致严重的牙釉质畸形（常染色体显性低钙化釉质发育不全）。我们的目标是发现 Fam83h 的正常功能以及该蛋白质的缺陷如何导致牙釉质畸形。我们的长期目标是增进对正常和病理性牙齿形成的了解，以促进公众口腔健康的改善。