Mechanisms of Genetic Reversion in Ichthyosis With Confetti

五彩纸屑鱼鳞病的遗传逆转机制

基本信息

批准号：
8332886
负责人：
KEITH A CHOATE
金额：
$ 37.37万
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 2016-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8332886
关键词：
17q Affect Alleles Area Arginine Biological Assay Blood Cells Bloom Syndrome Breeding C-terminal Cell Line Cell Nucleolus Cell Nucleus Cells Characteristics Chemicals Chromosomes Chromosomes, Human, Pair 17 Contracts Cutaneous DNA Damage DNA Double Strand Break DNA Repair DNA Repair Pathway Defect Detection Development Disadvantaged Disease Distal Epidermis Event Exposure to Family Study Fanconi&apos s Anemia Frameshift Mutation Frequencies Gene Expression Profile Gene Mutation Generations Genes Genetic Genetic Recombination Genetic Skin Diseases Genotype Goals Growth Hereditary Disease Human Human Cell Line Ichthyoses Ichthyosis en confetti Imagery Immunohistochemistry Inborn Genetic Diseases Individual Inherited Intermediate Filament Proteins Investigation Ker10 protein Keratin Lead Loss of Heterozygosity Manuals Measures Mechanics Mediating Methods Microscopic Mitotic Mitotic Recombination Modality Modeling Mus Mutation Natural Selections Normal Cell Orthologous Gene Pathway interactions Patients Pattern Peptides Phenotype Play Polymorphic Microsatellite Marker Proteins Rare Diseases Reading Frames Relative (related person)Reporting Resistance Role SNP genotyping Science Severities Shapes Sister Chromatid Exchange Site Skin Skin Transplantation Skin graft Spottings Stem cells TK1 gene Therapeutic Thymidine Kinase Time Tissues Transgenes Transgenic Mice Transgenic Model Trifluridine Tumor Suppressor Proteins Water assault carcinogenesis cell type gene replacement homologous recombination interest irradiation keratinocyte mouse model mutant preference retinal rods skin disorder tool trait

项目摘要

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.

描述（由申请人提供）：将遗传疾病的广泛回归至未受影响的状态极为罕见。因此，在患有五彩纸屑的人（IWC）的个体中观察到数百至数千个正常皮肤区域，这是一种严重的常染色体显性皮肤疾病，具有两种不同类型，是一种了不起的现象。我们已经发现，影响中间细丝蛋白角蛋白角蛋白1和角蛋白10（KRT1，KRT10）的羧基末端非螺旋结构域的突变导致它们错误地定位到细胞核上，并引起独特的IWC表型，并带有一个统一特征：超大型皮肤的发展，这是正常皮肤的发展，这会增加数量和大小的尺寸，这是杂种的尺寸和杂种的复制范围，这是杂种的杂模。（LOH）事件。值得注意的是，高度保守的杆域KRT1和KRT10的突变导致了独特的显性疾病，表皮溶虫病（EI），其中未观察到回归事件。尽管在其他疾病中已经观察到了逆转，但在遗传性皮肤病中很少见，通常在分布中受到限制。 IWC个体中看到的大量回归事件表明，由于遗传回归的速率增加，或者部分或完全或全部是由于选择性增长或生存优势，因此IWC中出现了正常的皮肤克隆。该项目的总体目标是确定IWC恢复的机理，并且有三种研究途径可以探索这一点：1。分析野生型和突变体KRT1和KRT10对同源重组的影响。 IWC中的恢复事件仅通过明显的有丝分裂重组而出现，而这些重组是同源重组途径（HR）的产物。 WT和突变体KRT1和KRT10在人力资源中的功能将通过在人体细胞系中的稳定表达进行检查，该细胞系在胸苷激酶（TK）基因座的功能等位基因中是杂合的，从而赋予三氟甲氨基胺（TFT）的敏感性，并允许检测tfte loh loh loh loh loh loh loh loh loh loh loh loh tfft tfft。我们将通过在存在和不存在X-Irradiation（DSB的有效诱导剂）的情况下，通过DNA双链断裂（DSB）速率以及LOH速率和机制来检查HR。 2。分析来自KRT1和KRT10突变个体的细胞和组织中的反转事件和选择性优势。 IWC重新转换事件必须发生在角质形成细胞的水平上，而恢复克隆需要选择性优势才能在皮肤内膨胀。鉴于表皮液化性鱼质病（EI）是由相同基因突变导致的，但没有临床上明显的回归，如果IWC突变不影响回归速率，EI应证明回归速率等效速率，而逆转克隆的选择性优势较小。我们将检查在EI患者组织的大部分中的重新转换事件，并将在人类皮肤等效的移植模型中进行竞争测定，其中将不同比例的恢复或正常角质形成细胞与突变体IWC或EI细胞混合，并通过每种细胞类型的每种细胞类型分析表演表皮以分析表皮以进行分析。 3。确定在IWC鼠标模型中增加回归事件频率的方法。我们已经生成了IWC的转基因小鼠模型，该模型能够皮肤遗传回归事件。我们将通过免疫组织化学和免疫定位在物理和化学DNA损害剂的设置中检查自发和诱导的恢复的速率和特征，从而确定了IWC中诱导治疗性重组的潜在方式，以及其他主要遗传的遗传疾病。