Mechanisms of Genetic Reversion in Ichthyosis With Confetti

描述（由申请人提供）：遗传疾病广泛地恢复到未受影响的状态是极其罕见的。因此，五彩鱼鳞病（IWC）是一种严重的常染色体显性皮肤病，有两种不同的类型，在患者中观察到数百至数千个正常皮肤区域的发育是一个值得注意的现象。我们发现，影响中间丝蛋白角蛋白1和角蛋白10 （KRT1、KRT10）的羧基端非螺旋结构域的突变导致它们错误定位于细胞核，并导致具有一个统一特征的不同IWC表型：正常皮肤斑块的发育随着时间的推移而增加数量和大小，这是复制中性杂合性丧失（LOH）事件的结果。值得注意的是，高度保守的杆状结构域KRT1和KRT10的突变导致一种明显的显性疾病，表皮松解性鱼鳞病（EI），其中未见逆转事件。虽然在其他疾病中也观察到这种逆转，但在遗传性皮肤病中很少见，而且通常分布有限。在IWC个体中观察到的大量逆转事件表明，IWC中出现的正常皮肤克隆要么是由于遗传逆转率的增加，要么部分或全部是由于选择性生长或生存优势。这个项目的总体目标是确定IWC的回归机制，有三个调查途径来探讨这一点：1。分析KRT1和KRT10野生型和突变型对同源重组的影响。在IWC中，仅通过同源重组途径（HR）产生的明显有丝分裂重组，就会出现非常高的逆转事件。WT和突变体KRT1和KRT10在HR中的功能将通过在胸腺嘧啶激酶（TK）位点的一个功能等位基因杂合的人类细胞系中的稳定表达来检测，该细胞系对三氟胸腺嘧啶（TFT）敏感，并允许通过选择TFT抗性来检测自发和诱导的LOH事件。我们将通过DNA双链断裂（DSB）率、姐妹染色单体交换、LOH率和存在和不存在x照射（DSB的有效诱导剂）的机制来检测HR。2. 分析KRT1和KRT10突变个体的细胞和组织中的逆转事件和选择优势。IWC逆转事件必须发生在角质形成细胞干细胞水平上，逆转克隆需要选择优势才能在皮肤内扩展。鉴于表皮松解性鱼鳞病（EI）是由相同基因的突变引起的，但在临床上没有明显的逆转，如果IWC突变不影响逆转率，EI应该显示出相同的逆转率，但对逆转克隆的选择优势较小。我们将在EI患者组织的大切片中检查逆转事件，并将在人类皮肤等效移植模型中进行竞争分析，其中将不同比例的逆转或正常角质形成细胞与突变IWC或EI细胞混合，并分析产生的表皮中每种细胞类型随时间的组成。3. 确定在IWC小鼠模型中增加逆转事件频率的方法。我们已经产生了一个转基因小鼠模型IWC能够皮肤遗传逆转事件。我们将通过免疫组织化学和免疫定位来研究物理和化学DNA损伤剂环境下自发和诱导逆转的速率和特征，确定IWC和其他主要遗传遗传性皮肤疾病诱导治疗性重组的潜在模式。

DESCRIPTION (provided by applicant): Widespread reversion of genetic disease to an unaffected state is exceedingly rare. For this reason, the development of hundreds to thousands of area of normal skin observed in individuals with ichthyosis with confetti (IWC), a severe autosomal dominant skin disease with two distinct types, is a remarkable phenomenon. We have found that mutations affecting the carboxy terminal non-helical domains of the intermediate filament proteins keratin 1 and keratin 10 (KRT1, KRT10) lead to their mis-localization to the nucleus and cause distinct IWC phenotypes with one unifying feature: the development of patches of normal skin which increase in number and size over time and which are the result of copy-neutral loss of heterozygosity (LOH) events. Remarkably, mutations in the highly conserved rod domains KRT1 and KRT10 result in a distinct dominant disorder, epidermolytic ichthyosis (EI) in which reversion events are not seen. While reversion has been observed in other diseases, it is rare in genetic skin disease and is typically limited in distribution. The large number of reversion events seen in individuals with IWC suggests that normal skin clones arise in IWC either due to an increased rate of genetic reversion or partly or wholly due to selective growth or survival advantage. The overall goal of this project is to determine the mechanism of reversion of IWC and there are three avenues of investigation to explore this: 1. To assay the effects of wild-type and mutant KRT1 and KRT10 on homologous recombination. Revertant events in IWC arise at a very high rate solely through apparent mitotic recombination which a product of homologous recombination pathways (HR). The function of WT and mutant KRT1 and KRT10 in HR will be examined via stable expression in a human cell line which is heterozygous for a functional allele at the thymidine kinase (TK) locus, conferring sensitivity to trifluorothymidine (TFT) and allowing detection of spontaneous and induced LOH events by selection for TFT resistance. We will examine HR via assays of DNA double strand break (DSB) rates, sister chromatid exchange, and LOH rates and mechanisms in the presence and absence of X-irradiation, a potent inducer of DSB. 2. To assay reversion events and selective advantage in cells and tissues from individuals with KRT1 and KRT10 mutations. IWC reversion events must occur at the level of the keratinocyte stem cell and revertant clones require selective advantage to expand within the skin. Given that epidermolytic ichthyosis (EI) results from mutations in the same genes but does not have clinically apparent reversion, if IWC mutations do not affect reversion rate, EI should demonstrate an equivalent rate of reversion but less selective advantage for revertant clones. We will examine reversion events in large sections of EI patient tissue and will perform competition assays in a human skin equivalent graft model in which varying proportions of revertant or normal keratinocytes are mixed with mutant IWC or EI cells and resulting epidermis is assayed for composition by each cell type over time. 3. To identify methods to increase the frequency of reversion events in an IWC mouse model. We have generated a transgenic mouse model of IWC which is capable of cutaneous genetic reversion events. We will examine the rate and characteristics of spontaneous and induced reversion in the setting of physical and chemical DNA damaging agents via immunohistochemistry and immunolocalization, identifying potential modalities for induction of therapeutic recombination in IWC and other dominantly inherited genetic skin disorders.