Novel Mechanisms of Congenital Dyserythropoietic Anemia

摘要 先天性红细胞生成性贫血(CDA)是一组异质性的红血球遗传病 细胞产生以再生能力低下的贫血为特征，伴有多核红细胞 骨髓中的前体。许多基因座与CDA有关，最常见的是 Codanin-1(CDAI)和SEC23B(CDA II)。虽然CDA是世界范围内的共同事业，但它的作用 Codanin-1在很大程度上是未知的。我们已经证明了Codanin-1是一种染色质结合蛋白，其 在红系分化过程中保持表达。此应用程序的目标是标识 CDA相关蛋白在红细胞生成中的作用，尤其是Codanin-1和一种新的 鉴定出CDA蛋白MACF1。这将通过审查三个具体目标来探讨：(1)研究 Codanin-1在红细胞生成过程中调节基因表达的机制；(2)小鼠的特征 Codanin-1 CDA相关点突变的CRISPR/Cas9敲击模型；以及(3) MACF1在红细胞生成和CDA的病理生理学中的作用，包括敲门和 基因敲除突变MACF1连锁的CDA小鼠模型。中心假设是Codanin-1和MACF1 参与调节红系发育和分化。该试验计划的重点是 使用细胞系、人和小鼠原代红系细胞系统和小鼠来理解这一调节 模特们。初步数据支持假设和拟议的研究。Codanin-1在血管内皮细胞中的表达减弱 由siRNA产生的CDA I和患者来源的点突变Codanin-1的过度表达均可导致红系 前体多核化和血红素化减少。遗传分析有力地支持了 CDA中的MACF1。研究人员为拟议的研究创造了许多重要的工具。这个 这项建议的理论基础是，通过了解像CDA这样罕见疾病的病理生理学，我们将获得 对控制红细胞生成的机制有广泛的了解，从而获得适用于开发 遗传性和获得性红细胞生成障碍的治疗策略。这个项目需要 利用使用多PI格式的丰富专业知识，加入2名经验丰富的血液学研究人员 耶鲁大学的儿科。加拉格尔博士有研究红细胞生成机制及其扰动的经验 在遗传病以及红系发育和分化的基因组学方面。库普费尔博士研究了 以范可尼贫血为模型的造血及其扰动的基本问题。把这些放在一起 研究人员是唯一有资格研究与红细胞生成功能障碍相关的疾病的人。共同个人投资指数 积极参与耶鲁大学的分子血液学研究；两人都是耶鲁合作中心的合作PI 血液学卓越中心(YCCEH)，为该提案中的一些研究提供支持。一起， 这些研究将为遗传性和获得性疾病中的红细胞生成及其扰动提供重要的线索。 红血球的产生。

Abstract The congenital dyserythropoietic anemias (CDAs) are a heterogeneous group of genetic diseases of red blood cell production marked by hyporegenerative anemia with the presence of multinucleated red blood cell precursors in the bone marrow. Numerous gene loci have been associated with CDA, the most common being codanin-1 (CDAI) and SEC23B (CDA II). Although a common cause of CDA worldwide, the function of codanin-1 is largely unknown. We have demonstrated that codanin-1 is a chromatin binding protein whose expression is maintained during erythroid differentiation. The objective of this application is to identify the function of CDA-associated proteins in erythropoiesis, with a particular focus on codanin-1 and a newly identified CDA protein, MACF1. This will be explored by examining three Specific Aims: (1) study of mechanisms of codanin-1 regulation of gene expression during erythropoiesis; (2) characterization of a murine CRISPR/Cas9 knockin model of a codanin-1 CDA- associated point mutations; and (3) characterization of the role of MACF1 in erythropoiesis and in the pathophysiology of CDA, including development of a knockin and knockout mutant MACF1-linked murine model of CDA. The central hypothesis is that codanin-1 and MACF1 are involved in regulation of erythroid development and differentiation. The experimental plan focuses on understanding this regulation using cell lines, human and murine primary erythroid cell systems, and mouse models. Preliminary data support the hypotheses and proposed studies. Diminished expression of codanin-1 in CDA I by siRNA and overexpression of a patient-derived point mutant codanin-1 both result in erythroid precursor multinuclearity and decreased hemoglobinization. Genetic analyses strongly support the role of MACF1 in CDA. The investigators have created numerous important tools for the proposed studies. The rationale for this proposal is that by understanding the pathophysiology of a rare disease like CDA, we will gain broad knowledge of mechanisms controlling erythropoiesis, resulting in insights applicable to development of therapeutic strategies for inherited and acquired disorders of red blood cell production. This project takes advantage of a wealth of expertise using the multiple PI format, joining 2 experienced hematologic researchers in Pediatrics at Yale. Dr. Gallagher has experience studying mechanisms of erythropoiesis and its perturbation in genetic disease as well as genomics of erythroid development and differentiation. Dr. Kupfer has studied fundamental aspects of hematopoiesis and its perturbation using Fanconi anemia as a model. Together these investigators are uniquely qualified to study diseases associated with dysfunctional erythropoiesis. The co-PIs are actively involved in molecular hematology research at Yale; both are co-PIs of the Yale Cooperative Center of Excellence in Hematology (YCCEH), providing support for some of the studies in this proposal. Together, these studies will shed important light on erythropoiesis and its perturbation in inherited and acquired disorders of erythrocyte production.