Elucidating molecular basis of ICF Syndrome with human pluripotent stem cells

描述（申请人提供）：免疫缺陷、着丝粒不稳定和面部异常（ICF）综合征是一种影响多器官系统的常染色体隐性遗传疾病。最近的人类遗传学研究已经确定，大多数ICF综合征患者携带编码从头DNA甲基转移酶的DNMT 3B基因突变。因此，来自受影响患者的细胞表现出显着降低的甲基转移酶活性和异常低甲基化的CpG位点内的pericentromeric卫星，亚端粒，和X染色体区域。反映人类ICF综合征基因突变的转基因小鼠模型不能完全概括该综合征的人类发病机制。为此，我们建议开发一种更适合于了解ICF综合征分子基础的人类干细胞模型。在具体目标1中，我们将产生和表征携带ICF突变的人iPSC，并确定DNMT 3B缺陷是否影响细胞增殖和基因组稳定性。ICF-iPSC将表征多能性标志物的表达和通过胚状体形成和畸胎瘤形成的细胞分化的多潜能。具体目标2旨在比较对照和ICF突变iPSC向造血祖细胞（HPC）和神经嵴谱系细胞（NC）的定向分化，以确定DNMT 3B缺陷对谱系特异性细胞分化的影响。在具体目标3中，我们将尝试通过在ICF突变体iPSC、HPC和NC中引入野生型DNMT 3B表达来挽救ICF表型。我们将专注于了解DNMT 3B表达是否可以拯救阶段特异性DNA低甲基化对基因组稳定性和细胞分化过程中的基因表达。本研究为深入了解ICF综合征的发病机制提供了新的思路，并为干细胞治疗ICF综合征开辟了新的途径。

DESCRIPTION (provided by applicant): Immunodeficiency, Centromeric instability and Facial anomalies (ICF) syndrome is an autosomal recessive disorder affecting multiple organ systems. Recent human genetic studies have identified that most ICF syndrome patients carry genetic mutations in DNMT3B encoding a de novo DNA methyltransferase. Consequently, cells derived from affected patients exhibited significantly reduced methyltransferase activity and abnormal hypomethylation of CpG sites within pericentromeric satellite, subtelomeric, and X chromosome regions. Transgenic mouse models that mirror genetic mutations in human ICF syndrome do not fully recapitulate the human pathogenesis of the syndrome. For this purpose, we propose to develop a human stem cell model that is better suited to understand the molecular basis of ICF syndrome. In Specific Aim 1, we will generate and characterize human iPSCs carrying ICF mutations and determine whether DNMT3B deficiency affects cell proliferation and genome stability. ICF-iPSCs will be characterized for expression of pluripotency markers and multi-potentials of cell differentiation through embryoid formation and teratoma formation. Specific Aim 2 is designed to compare directed differentiation of control and ICF mutant iPSCs into hematopoietic progenitor cells (HPCs) and neural crest lineage cells (NCs) to determine the impact of DNMT3B deficiency on lineage-specific cell differentiation. In Specific Aim 3, we will attempt to rescue ICF phenotypes by introducing wild-type DNMT3B expression in ICF mutant iPSCs, HPCs, and NCs. We will focus on understanding whether DNMT3B expression can rescue stage-specific DNA hypomethylation on the genome stability and gene expression during cell differentiation. Our proposed research will provide a novel approach to understanding the pathogenesis of ICF syndrome, thus potentially develop a new approach to cure ICF syndrome through stem cell therapy.