Mechanisms of Genetic Reversion in Ichthyosis With Confetti

描述(由申请人提供)：遗传病广泛逆转到未受影响的状态是极其罕见的。因此，在五彩纸屑鱼鳞病(IWC)患者身上观察到的数百至数千个正常皮肤面积的发展是一个显著的现象。IWC是一种严重的常染色体显性皮肤病，有两种不同的类型。我们发现，影响中间丝蛋白KRT1和KRT10(KRT1，KRT10)的羧基末端非螺旋结构域的突变导致它们错误定位到细胞核，并导致不同的IWC表型，具有一个统一的特征：正常皮肤斑块的发展随着时间的推移数量和大小增加，这是复制中性杂合性丢失(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的突变导致了一种独特的显性疾病，表皮松解性鱼鳞病(EI)，在这种疾病中看不到逆转事件。虽然在其他疾病中也观察到了逆转，但在遗传性皮肤病中很少见，而且通常分布有限。在IWC患者身上看到的大量逆转事件表明，IWC患者中正常皮肤克隆的出现要么是由于遗传逆转速度的增加，要么是部分或全部由于选择性生长或生存优势。本项目的总体目标是确定IWC的逆转机制，并从三个方面进行研究：1.检测野生型和突变型KRT1和KRT10对同源重组的影响。IWC中的逆转事件仅通过明显的有丝分裂重组以非常高的比率发生，这是同源重组途径(HR)的产物。WT和突变体KRT1和KRT10在HR中的功能将通过在人类细胞系中稳定表达来检测，该细胞系是胸苷激酶(TK)基因座上的功能等位基因的杂合子，使人对三氟胸苷(TFT)敏感，并通过选择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、姐妹染色单体互换和杂合性缺失(LOH)率的分析，以及在存在和不存在DSB的有效诱导剂X射线照射的情况下，来检测HR。2.检测KRT1和KRT10突变个体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，而突变克隆需要选择性优势才能在皮肤内扩展。考虑到表皮松解性鱼鳞病(EI)是由相同基因的突变引起的，但临床上没有明显的逆转，如果IWC突变不影响复发率，EI应该表现出同等的复原率，但对突变克隆的选择优势较小。我们将在EI患者的大片组织中检测逆转事件，并将在人类皮肤等效移植模型中进行竞争分析，在该模型中，将不同比例的回复或正常角质形成细胞与突变的IWC或EI细胞混合，并根据每种细胞类型随着时间的推移分析产生的表皮成分。3.确定在IWC小鼠模型中增加回复事件频率的方法。我们已经建立了一种能够发生皮肤遗传逆转事件的IWC转基因小鼠模型。我们将通过免疫组织化学和免疫定位来研究在物理和化学DNA损伤剂的背景下自发和诱导逆转的比率和特征，确定在IWC和其他主要遗传性皮肤病中诱导治疗性重组的潜在方式。

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.