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畸形(ICF)综合征是一种常染色体隐性遗传性疾病，影响多个器官系统。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从头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显著的甲基转移酶活性降低和着丝粒周围卫星区、亚端粒和X染色体区域CpG位点的异常低甲基化。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人类ICF综合征的发病机制。为此，我们建议开发一种更适合于理解ICF综合征的分子基础的人类干细胞模型。在特定的目标1中，我们将产生并鉴定携带ICF突变的人IPSCs，并确定Dnmt3b缺陷是否影响细胞增殖和基因组稳定性。ICF-IPSCs的特征是表达多潜能标志物和通过胚样形成和畸胎瘤形成向细胞分化的多潜能。目的2比较对照和ICF突变型IPSCs定向分化为造血祖细胞(HPC)和神经脊系细胞(NCS)，以确定Dnmt3b缺乏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突变型IPSCs、HPC和NCS中引入野生型Dnmt3b表达来挽救ICF表型。我们将重点了解Dnmt3b的表达是否可以挽救阶段特异性DNA低甲基化对基因组稳定性和细胞分化过程中基因表达的影响。我们的研究将为理解ICF综合征的发病机制提供一种新的途径，从而可能开发一种通过干细胞治疗治疗ICF综合征的新途径。

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.