Mechanisms of Genetic Reversion in Ichthyosis With Confetti

五彩纸屑鱼鳞病的遗传逆转机制

• 批准号: 8703506
• 负责人: KEITH A CHOATE
• 金额: $ 36.71万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 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/8703506
• 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' 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 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

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）：遗传性疾病广泛逆转为未受影响的状态是极其罕见的。出于这个原因，在患有五彩纸屑鱼鳞病（IWC）（一种具有两种不同类型的严重常染色体显性皮肤病）的个体中观察到的数百至数千个正常皮肤区域的发育是一个显著的现象。我们已经发现，影响中间丝蛋白角蛋白1和角蛋白10（KRT 1，KRT 10）的羧基末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的功能：正常皮肤斑块的发育，其数量和大小随时间增加，并且是拷贝中性杂合性丢失（洛）事件的结果。值得注意的是，高度保守的杆结构域KRT 1和KRT 10的突变导致一种独特的显性疾病，表皮增生性鱼鳞病（EI），其中没有看到逆转事件。虽然在其他疾病中观察到逆转，但在遗传性皮肤病中很少见，并且通常分布有限。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增加，要么部分或全部是由于选择性生长或生存优势。本研究的主要目的是探讨IWC的回复机制，主要从三个方面进行研究：1。分析野生型和突变型KRT 1和KRT 10对同源重组的影响。IWC中回复突变体的发生率非常高，完全是通过同源重组途径（HR）的产物--表观有丝分裂重组。将通过在人细胞系中稳定表达来检查WT和突变型KRT 1和KRT 10在HR中的功能，所述人细胞系对于胸苷激酶（TK）基因座的功能等位基因是杂合的，赋予对三氟胸苷（TFT）的敏感性，并允许通过选择TFT抗性来检测自发和诱导的洛事件。我们将通过检测DNA双链断裂（DSB）率、姐妹染色单体交换和洛率以及在存在和不存在X射线照射（DSB的一种有效诱导剂）的情况下的机制来检查HR。2.检测KRT 1和KRT 10突变个体细胞和组织中的回复突变事件和选择优势。IWC回复事件必须发生在角质形成细胞干细胞的水平上，并且回复突变克隆需要选择性优势以在皮肤内扩增。鉴于表皮癣性鱼鳞病（EI）由相同基因的突变引起，但没有临床上明显的回复，如果IWC突变不影响回复率，EI应表现出等效的回复率，但回复突变克隆的选择性优势较小。我们将检查EI患者组织的大切片中的回复突变事件，并将在人皮肤等效移植模型中进行竞争测定，其中将不同比例的回复突变或正常角质形成细胞与突变IWC或EI细胞混合，并测定所得表皮随时间推移的每种细胞类型的组成。3.确定增加IWC小鼠模型中回复突变频率的方法。我们已经建立了一个转基因小鼠模型的IWC，这是能够皮肤遗传逆转事件。我们将通过免疫组化和免疫定位研究在物理和化学DNA损伤剂的环境中自发和诱导逆转的速率和特征，确定诱导IWC和其他显性遗传性遗传性皮肤病治疗重组的潜在方式。