Mechanisms of Revertant Mosaicism in Ichthyosis with Confetti

项目摘要 回复突变嵌合体（RM）发生于致病突变的自发体细胞校正， 产生正常组织区域。鱼鳞病与五彩纸屑（IWC），一种常染色体显性皮肤 由影响角蛋白10（K10）和角蛋白1（K1）的尾部结构域的突变引起的病症， 值得注意的是其高频率的RM，因为患者发展数百至数千个回复突变体， 正常皮肤的岛从童年开始。有趣的是，每个回复突变体克隆来自 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，观察到回复突变斑的尺寸增大， 随着时间的推移，这表明细胞间的竞争有利于选择和扩大 回复突变体克隆超过它们的突变体邻居。我们已经成功地证明了 IWC突变角蛋白独特地增加HR的速率，同时诱导DNA双链的形成。 链断裂（DSB）。此外，我们还建立了IWC的条件敲入模型， 临床上和组织学上通过CN-LOH概括疾病，包括回复突变嵌合体， 证实了回复突变体、野生型克隆的扩增。 我们现在建议系统地确定和询问管理的机制和调解人， IWC角蛋白诱导的HR和细胞竞争。我们将研究哪种DNA损伤反应 (DDR)组分被募集到角蛋白诱导的DSB的位点，表征DSB的动力学 形成和解决，并精确定位细胞周期中发生损伤的阶段。我们 建议利用活体成像来探索，在细胞水平上，是否改变了 有丝分裂、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 IWC中细胞间竞争和HR的决定因素或修饰因素。我们将进一步审查 在IWC患者和鼠组织和细胞中的令人信服的候选物。解释previse IWC的遗传逆转和细胞间竞争机制有可能确定 可能使治疗性重组能够治疗遗传性和获得性显性遗传的途径 遗传疾病

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.