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.

National Institute of Health (NIH)
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Research Project (R01)
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Study Section
Therapeutic Approaches to Genetic Diseases (TAG)
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Baker, Carl
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Yale University
Schools of Medicine
New Haven
United States
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Lim, Young H; Fisher, Jonathan M; Choate, Keith A (2017) Revertant mosaicism in genodermatoses. Cell Mol Life Sci 74:2229-2238
Lim, Young H; Moscato, Zoe; Choate, Keith A (2017) Mosaicism in Cutaneous Disorders. Annu Rev Genet 51:123-141
Lim, Young H; Choate, Keith A (2016) Expanding the Mutation Spectrum of Ichthyosis with Confetti. J Invest Dermatol 136:1941-1943
Lim, Young H; Qiu, Jingyao; Saraceni, Corey et al. (2016) Genetic Reversion via Mitotic Recombination in Ichthyosis with Confetti due to a KRT10 Polyalanine Frameshift Mutation. J Invest Dermatol 136:1725-1728
Choate, Keith A; Lu, Yin; Zhou, Jing et al. (2015) Frequent somatic reversion of KRT1 mutations in ichthyosis with confetti. J Clin Invest 125:1703-7
Choate, Keith A; Milstone, Leonard M (2015) Phenotypic expansion in ichthyosis with confetti. JAMA Dermatol 151:15-6