Mechanisms of Revertant Mosaicism in Ichthyosis with Confetti

项目总结 逆转性嵌合体(RM)发生于病原突变的自发、体细胞纠正， 形成正常组织的区域。常染色体显性遗传性鱼鳞病(IWC) 由角蛋白10(K10)和角蛋白1(K1)的尾部结构域突变引起的疾病，是 值得注意的是它的高频率的RM，因为患者发展成数百到数千个回复突变 从童年开始的正常皮肤孤岛。有趣的是，每个回复克隆人都是从 独立拷贝中性杂合性缺失(CN-LOH)，可能通过同源重组 (HR)突变单倍型。此外，观察到突变的斑点在大小和 数量随着时间的推移，表明细胞间的竞争有利于选择和扩大 变异克隆人凌驾于它们的变异邻居之上。我们已经成功地证明了 IWC突变型角蛋白独特地提高了HR的比率，同时诱导DNA双... 链断裂(DSB)。此外，我们还开发了IWC的有条件敲入模型，该模型 临床和组织学上通过CN-LOH重现包括突变嵌合体在内的疾病，以及 演示了突变体、野生型克隆的扩展。 我们现在提议系统地确定和审问管理 IWC角蛋白诱导的HR和细胞竞争。我们将调查哪种DNA损伤反应 (DDR)组分被招募到角蛋白诱导的DSB的位置，表征了DSB的动力学 形成和分解，并准确指出细胞周期中发生损伤的阶段。我们 建议利用活体内成像在细胞水平上探索是否改变了 有丝分裂、凋亡或分化/分层是IWC细胞间竞争的基础。最后， 我们将进行单细胞RNA测序和CRISPR基因敲除筛选，以确定 IWC中的细胞间竞争和HR的决定因素或修饰因素。我们将进一步研究 在IWC患者和小鼠组织和细胞方面有吸引力的候选人。阐明预防措施 IWC的遗传逆转和细胞间竞争的机制有可能确定 可能使治疗性重组治疗遗传性和获得性显性遗传的途径 遗传性疾病。

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.