Elucidating molecular basis of ICF Syndrome with human pluripotent stem cells

描述（由申请人提供）：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病。最近的人类遗传学研究发现，大多数ICF综合征患者携带编码新DNA甲基转移酶的DNMT3B基因突变。因此，来自受影响患者的细胞表现出甲基转移酶活性显著降低，中心粒周围卫星、亚端粒和X染色体区域的CpG位点异常低甲基化。反映人类ICF综合征基因突变的转基因小鼠模型并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理解ICF综合征分子基础的人类干细胞模型。在Specific Aim 1中，我们将生成并表征携带ICF突变的人类iPSCs，并确定DNMT3B缺陷是否会影响细胞增殖和基因组稳定性。ICF-iPSCs的特点是表达多能性标记物和通过胚胎样体形成和畸胎瘤形成的细胞分化的多种潜力。特异性Aim 2旨在比较对照和ICF突变iPSCs向造血祖细胞（HPCs）和神经嵴谱系细胞（nc）的定向分化，以确定DNMT3B缺乏对谱系特异性细胞分化的影响。在特异性目标3中，我们将尝试通过在ICF突变iPSCs、HPCs和nc中引入野生型DNMT3B表达来挽救ICF表型。我们将重点了解DNMT3B表达是否可以挽救细胞分化过程中特异性DNA低甲基化对基因组稳定性和基因表达的影响。我们的研究将为了解ICF综合征的发病机制提供新的途径，从而有可能通过干细胞治疗ICF综合征开辟新的途径。

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.