Mechanisms of Revertant Mosaicism in Ichthyosis with Confetti

五彩纸屑鱼鳞病中回复性嵌合的机制

• 批准号: 10335133
• 负责人: KEITH A CHOATE
• 金额: $ 51.17万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-19 至 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10335133
• 关键词: Affect; Alleles; Apoptosis; Area; Birth; CRISPR screen; Candidate Disease Gene; Cell Cycle; Cell Cycle Progression; Cell Cycle Stage; Cell Nucleolus; Cell Nucleus; Cells; Childhood; Clinical; Clonal Expansion; Clustered Regularly Interspaced Short Palindromic Repeats; Comet Assay; Cytosol; DNA Damage; DNA Double Strand Break; DNA Repair; DNA lesion; Development; Disease; Dominant Genetic Conditions; Epidermis; Event; Frameshift Mutation; Frequencies; Funding; Genes; Genetic; Genetic Diseases; Genetic Recombination; Growth; Haplotypes; Histologic; Human; Ichthyosis en confetti; Image; In Vitro; Individual; Inherited; Intermediate Filaments; Island; Ker10 protein; Keratin; Kinetics; Knock-in; Knock-out; Lead; Libraries; Loss of Heterozygosity; Mediating; Mediator of activation protein; Mitosis; Modeling; Molecular; Mosaicism; Mus; Mutation; Normal Cell; Normal tissue morphology; Pathogenicity; Pathway interactions; Patients; Process; Proteins; Reporter; Resolution; Role; Self-Correction; Site; Skin; Spottings; System; Tail; Therapeutic; Time; Tissues; cell behavior; cell type; experimental study; genetic approach; homologous recombination; human disease; in vivo; intravital imaging; keratinocyte; knock-down; mouse model; mutant; p53-binding protein 1; pressure; recruit; repaired; response; single-cell RNA sequencing; skin disorder; tool; two-photon

PROJECT SUMMARY Revertant mosaicism (RM) occurs from spontaneous, somatic correction of pathogenic mutation, giving rise to areas of normal tissue. Ichthyosis with confetti (IWC), an autosomal dominant skin disorder caused by mutations affecting the tail domains of keratin 10 (K10) and keratin 1 (K1), is remarkable for its high frequency of RM, as patients develop hundreds to thousands of revertant islands of normal skin beginning in childhood. Interestingly, each revertant clone arises from independent copy-neutral loss-of-heterozygosity (CN-LOH), likely via homologous recombination (HR) of the mutant haplotype. Furthermore, the revertant macules are observed to grow in size and number over time, suggesting intercellular competition favoring the selection and expansion of revertant clones over their mutant neighbors. We have successfully demonstrated that expression of IWC mutant keratins uniquely increase the rate of HR, while inducing the formation of DNA double- strand breaks (DSBs). Furthermore, we have developed a conditional knock-in model of IWC, which clinically and histologically recapitulates disease including revertant mosaicism via CN-LOH, and demonstrates expansion of revertant, wild type clones. We now propose to systematically identify and interrogate the mechanisms and mediators governing IWC keratin-induced HR and cellular competition. We will investigate which DNA damage response (DDR) components are recruited to sites of keratin-induced DSBs, characterize the kinetics of DSB formation and resolution, and pinpoint the stage in the cell cycle at which damage occurs. We propose to utilize intravital live-imaging to explore, at the cellular level, whether altered rates of mitosis, apoptosis, or differentiation/delamination underlie the intercellular competition in IWC. Finally, we will perform single-cell RNA sequencing and CRISPR knockout screens, to identify mediators of intercellular competition and determinants or modifiers of HR in IWC. We will further examine compelling candidates in IWC patient and murine tissue and cells. Elucidating the previse mechanisms of genetic reversion and intercellular competition in IWC has the potential to identify pathways which may enable therapeutic recombination to treat inherited and acquired dominant genetic disorders.

项目摘要 恢复镶嵌（RM）来自自发的，躯体校正致病性突变， 引起正常组织的区域。带五彩纸屑（IWC）的鱼质病，一种常染色体显性皮肤 由影响角蛋白10（K10）和角蛋白1（K1）的突变引起的障碍，为 随着患者产生数百至数千个逆转的频率，它以其高频的频率而引人注目 从童年开始的正常皮肤岛。有趣的是，每个恢复克隆都来自 独立的拷贝性损失损失损失（CN-LOH），可能是通过同源重组的 突变单倍型的（HR）。此外，观察到恢复斑块的大小增长，并且 随着时间的流逝，数字表明细胞间竞争有利于选择和扩展 在其突变邻居上恢复克隆。我们已经成功证明了 IWC突变角素独特地提高了HR的速率，同时诱导DNA双重形成 链断裂（DSB）。此外，我们已经开发了一个有条件的IWC敲门模型 临床和组织学从临床和组织学概括疾病，包括通过CN-LOH恢复镶嵌物， 展示了恢复野生型克隆的扩展。 现在，我们建议系统地识别和询问管理机制和调解员 IWC角蛋白引起的HR和细胞竞争。我们将研究哪些DNA损伤响应 （DDR）组件被招募到角蛋白诱导的DSB的位置，以DSB的动力学为特征 形成和分辨率，并查明发生损害的细胞周期中的阶段。我们 提议利用插入的现场现场模仿，以在细胞水平上探索 有丝分裂，凋亡或分化/分层是IWC的细胞间竞争。最后， 我们将执行单细胞RNA测序和CRIS敲除屏幕，以识别 IWC中人力资源的细胞间竞争和决定因素或修饰符。我们将进一步研究 IWC患者，鼠组织和细胞中令人信服的候选人。阐明预期 IWC中遗传回归和细胞间竞争的机制有潜力识别 可以使治疗重组能够治疗遗传和获得的途径 遗传疾病。