Novel Mechanisms of Congenital Dyserythropoietic Anemia

摘要 先天性红细胞生成异常性贫血（CDAs）是一组异质性红细胞遗传性疾病 以再生不足性贫血为标志的细胞生成，伴有多核红细胞的存在 骨髓中的前体细胞许多基因位点与CDA相关，最常见的是 codanin-1（CDAI）和SEC23B（CDA II）。虽然是一个共同的原因，CDA世界各地，功能 codanin-1在很大程度上未知。我们已经证明，codanin-1是一种染色质结合蛋白， 在红系分化期间维持表达。本申请的目的是确定 CDA相关蛋白在红细胞生成中的功能，特别关注codanin-1和新的 鉴定了CDA蛋白，MACF 1。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控基因表达的机制;（2）小鼠 codanin-1CDA相关点突变的CRISPR/Cas9敲入模型;和（3）codanin-1CDA相关点突变的CRISPR/Cas9敲入模型的表征。 MACF 1在红细胞生成和CDA的病理生理学中的作用，包括敲入和 CDA的敲除突变MACF 1连锁的鼠模型。中心假设是codanin-1和MACF 1 参与调节红细胞发育和分化。实验计划的重点是 使用细胞系、人和鼠原代红系细胞系统和小鼠红系细胞系统来理解这种调节， 模型初步数据支持的假设和拟议的研究。可达宁-1的表达减少， 通过siRNA的CDA I和患者来源的点突变型codanin-1的过表达都导致红系细胞凋亡。 前体多核和血红蛋白化减少。基因分析强烈支持 CDA中的MACF 1。研究人员为拟议的研究创造了许多重要工具。的 这一建议的基本原理是，通过了解像CDA这样的罕见疾病的病理生理学，我们将获得 控制红细胞生成的机制的广泛知识，导致适用于发展的见解， 用于遗传性和获得性红细胞生成障碍的治疗策略。本项目以 使用多PI格式的丰富专业知识的优势，加入2名经验丰富的血液学研究人员 在耶鲁大学的儿科Gallagher博士有研究红细胞生成机制及其扰动的经验 以及红细胞发育和分化的基因组学。库普弗博士研究了 造血的基本方面及其扰动使用范可尼贫血作为模型。综合这些 研究者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。合作伙伴 积极参与耶鲁大学的分子血液学研究;两人都是耶鲁大学合作中心的联合PI 卓越的血液学（YCCEH），为本提案中的一些研究提供支持。在一起， 这些研究将对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启示 红细胞的产生。

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.