Mechanisms of Revertant Mosaicism in Ichthyosis with Confetti

五彩纸屑鱼鳞病中回复性嵌合的机制

基本信息

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
项目类别：
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

项目摘要

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.

项目概要回复嵌合体 (RM) 是由致病性突变的自发体细胞校正而产生的，产生正常组织区域。伴有五彩纸屑的鱼鳞病 (IWC)，一种常染色体显性皮肤病由影响角蛋白 10 (K10) 和角蛋白 1 (K1) 尾部结构域的突变引起的疾病是因其高频率的 RM 而引人注目，因为患者会出现数百至数千个回复突变正常皮肤的岛屿从童年开始。有趣的是，每个回复克隆都来自独立拷贝中性杂合性丢失（CN-LOH），可能通过同源重组 (HR) 突变单倍型。此外，观察到回复斑疹尺寸增大并且随着时间的推移，数量不断增加，表明细胞间竞争有利于细胞的选择和扩张回复性克隆优于其突变邻居。我们已经成功地证明了 IWC 突变角蛋白独特地提高 HR 速率，同时诱导 DNA 双链的形成链断裂（DSB）。此外，我们还开发了IWC的条件敲入模型，通过 CN-LOH 在临床和组织学上重现疾病，包括回复嵌合体，以及展示回复野生型克隆的扩增。我们现在建议系统地识别和询问管理机制和调解员 IWC 角蛋白诱导 HR 和细胞竞争。我们将研究哪种 DNA 损伤反应 (DDR) 成分被招募到角蛋白诱导的 DSB 位点，表征 DSB 的动力学形成和分解，并查明损伤发生的细胞周期阶段。我们提议利用活体实时成像在细胞水平上探索是否改变了有丝分裂、细胞凋亡或分化/分层是 IWC 中细胞间竞争的基础。最后，我们将进行单细胞 RNA 测序和 CRISPR 敲除筛选，以确定介导的 IWC 中细胞间竞争和 HR 的决定因素或调节因素。我们将进一步审查 IWC 患者和小鼠组织和细胞中引人注目的候选者。阐明前提 IWC 中的遗传逆转和细胞间竞争机制有可能确定可能使治疗重组能够治疗遗传性和获得性显性遗传的途径遗传性疾病。