Date of Award

January 2024

Document Type

Thesis

Degree Name

Medical Doctor (MD)

Department

Medicine

First Advisor

Keith A. Choate

Abstract

Revertant mosaicism Revertant mosaicism (RM) is a rare but naturally occurring phenomenon wherein cells carrying disease-causing mutations coexist with cells in which the mutation has been spontaneously replaced with a normal allele. Ichthyosis with confetti (IWC) is a rare skin disorder that commonly displays cutaneous revertant mosaicism. Patients with IWC are born with generalized erythematous scaly skin, and during childhood they start to develop confetti-like patches of histologically normal appearing skin, in which the mutation has been lost. These normal skin patches increase in size and number over time, eventually becoming thousands, each of which is an independent event of reversion of the disease-causing mutation. The expansion of these clones suggests the existence of opposing selective pressures on the revertant clones and mutant cells, which is suggestive of cellular competition. Our lab originally discovered that IWC is caused by dominant negative mutations in the tail domains of the keratin 1 (KRT1) or 10 (KRT10) intermediate filament genes which normally contribute to the stability of the cytoskeleton and reside in the cytoplasm. These mutations result in nuclear mislocalization of the affected keratins and copy-neutral loss of heterozygosity. The factors regulating reversion and the steps leading to recombination are largely unknown. In addition, the relationship between mutant and revertant cells is not well understood. To investigate the development and growth of revertant spots in IWC, we employed intravital live imaging (IVLI). To achieve visualization of wild type (WT) or K10 IWC keratinocytes and their nuclei, we crossed K10IWC/K14-CreERT/mTmG with K14CreERT/H2BmCherry mouse (mCherry nucleus) to create K10-WT (mTomato) or K10IWC (mGFP) keratinocytes with readily observable mCherry nuclei. To interrogate the mechanism underlying intercellular competition, we used this novel triple-fluorescence IWC mouse to perform IVLI of mosaic K10IWC epidermis to study the cellular behaviors that result in clonal advantage of wild type keratinocytes over mutant keratinocytes, and determine whether differential rates in proliferation of wild type cells, apoptosis or differentiation of mutant cells, or a combination of all, underlie the robust competition of revertant clones in IWC; and to perform single cell RNA sequencing (scRNAseq) of mutant and wild type keratinocytes from K10IWC mice, which helped us identify candidates affecting competition and reversion via doublet analysis. Also, we performed spatial transcriptomics analysis through Visium Spatial Gene Expression (Visium), a next-generation molecular profiling method for examining gene expression and its relation to tissue organization, which identified candidate mediators/effectors of IWC keratins involved in reversion and cellular competition at the affected-revertant junction in human skin. Lastly, to determine the role of IWC mutant keratins in the induction of revertant mosaicism we performed in vitro experiments and found that IWC-expressing cells showed increased levels of DNA damage via comet assay. Deciphering the molecular mechanisms that govern this form of natural gene therapy could be used to correct mutations that cause a wide array of diseases, including oncogenic mutations in cancer.

Comments

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