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: 8219845
• 负责人: KEITH A CHOATE
• 金额: $ 37.26万
• 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/8219845
• 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; Gene Mutation; Generations; Genes; Genetic; Genetic Recombination; Genetic Skin Diseases; Genotype; Goals; Growth; Hereditary Disease; Human; Human Cell Line; Ichthyoses; 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

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. PUBLIC HEALTH RELEVANCE: The skin functions as a critical defense to external mechanical, chemical, and bacterial assault, while serving as the primary barrier to water loss. It achieves this through a precisely orchestrated pattern of gene expression, defects in which cause genetic skin disorders of varying severity. Despite the fact that many of these disorders are rare, the genetic mutations underlying them have fundamentally shaped our understanding of how the skin functions. Ichthyosis with confetti (IWC) is a severe rare skin disorder with a remarkable feature: progressive genetic self-correction which occurs over time and results in thousands of expanding independent patches of normal skin. In other skin diseases, self-correction has been only rarely reported, and never to the extent seen in IWC. We have determined that this correction occurs by loss of a portion of the chromosome carrying the disease gene and replacement with the same segment from a paired normal chromosome, and we have identified mutations in two genes causing this disorder. This project investigates how these self-correction events occur - whether they are the direct result of the mutant proteins or if strong natural selection for the resulting normal skin clones plays a role. By defining mechanisms critical for the genetic self-correction events observed in IWC, it may become possible to develop tools to treat other skin or systemic disorders resulting from one copy of a mutant gene.

描述（由申请人提供）：遗传疾病广泛逆转至未受影响状态的情况极其罕见。因此，在患有五彩纸屑鱼鳞病 (IWC) 的个体中观察到数百至数千个正常皮肤区域的发育，这是一种严重的常染色体显性皮肤病，具有两种不同的类型，这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）羧基末端非螺旋结构域的突变会导致它们错误定位到细胞核，并导致具有一个统一特征的不同 IWC 表型：正常皮肤斑块的数量和大小随着时间的推移而增加，这是复制中性杂合性丢失 (LOH) 事件的结果。值得注意的是，高度保守的杆结构域 KRT1 和 KRT10 中的突变会导致一种独特的显性疾病，即表皮松解性鱼鳞病 (EI)，但未观察到逆转事件。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，而且分布范围通常有限。在 IWC 个体中观察到的大量回复事件表明，IWC 中出现正常皮肤克隆要么是由于遗传回复率增加，要么部分或完全由于选择性生长或生存优势。该项目的总体目标是确定 IWC 的回复机制，并通过三种研究途径来探索这一点： 1. 测定野生型和突变型 KRT1 和 KRT10 对同源重组的影响。 IWC 中的回复事件仅通过表观有丝分裂重组以非常高的速率发生，表观有丝分裂重组是同源重组途径 (HR) 的产物。 WT 和突变型 KRT1 和 KRT10 在 HR 中的功能将通过在人细胞系中的稳定表达来检查，该细胞系是胸苷激酶 (TK) 位点的功能等位基因杂合的，赋予对三氟胸苷 (TFT) 的敏感性并允许检测自发的并通过选择 TFT 电阻来诱导 LOH 事件。我们将通过检测 DNA 双链断裂 (DSB) 率、姐妹染色单体交换以及 LOH 率和存在或不存在 X 射线（DSB 的有效诱导剂）的情况下的机制来检查 HR。 2. 检测具有 KRT1 和 KRT10 突变的个体的细胞和组织中的逆转事件和选择优势。 IWC 回复事件必须发生在角质形成细胞干细胞水平，回复克隆需要选择性优势才能在皮肤内扩张。鉴于表皮松解性鱼鳞病 (EI) 是由相同基因突变引起的，但没有临床上明显的回复，如果 IWC 突变不影响回复率，EI 应表现出相同的回复率，但对回复克隆的选择性优势较小。我们将检查 EI 患者组织大切片中的回复事件，并将在人体皮肤等效移植模型中进行竞争测定，其中不同比例的回复或正常角质形成细胞与突变 IWC 或 EI 细胞混合，并分析所得表皮的成分随时间变化的细胞类型。 3. 确定提高 IWC 小鼠模型中回复事件频率的方法。我们已经建立了 IWC 转基因小鼠模型，能够进行皮肤遗传逆转事件。我们将通过免疫组织化学和免疫定位检查物理和化学 DNA 损伤剂环境中自发和诱导逆转的速率和特征，确定 IWC 和其他显性遗传性皮肤病中诱导治疗重组的潜在方式。 公共健康相关性：皮肤是抵御外部机械、化学和细菌攻击的重要屏障，同时也是防止水分流失的主要屏障。它通过精确协调的基因表达模式来实现这一目标，其中的缺陷会导致不同严重程度的遗传性皮肤病。尽管许多这些疾病都很罕见，但它们背后的基因突变从根本上影响了我们对皮肤功能的理解。伴有五彩纸屑的鱼鳞病 (IWC) 是一种严重的罕见皮肤病，具有一个显着特征：随着时间的推移，进行性基因自我修正，导致正常皮肤出现数千个独立的斑块。在其他皮肤病中，自我纠正的报道很少，而且从未达到IWC所见的程度。我们已经确定，这种校正是通过丢失携带疾病基因的染色体的一部分并用配对正常染色体的相同片段替换来实现的，并且我们已经确定了导致这种疾病的两个基因中的突变。该项目研究了这些自我校正事件是如何发生的——它们是否是突变蛋白的直接结果，或者对所产生的正常皮肤克隆的强烈自然选择是否发挥了作用。通过定义对 IWC 中观察到的基因自我校正事件至关重要的机制，开发出治疗由突变基因的一个拷贝引起的其他皮肤或系统性疾病的工具可能成为可能。