Specific Aims
Harlequin ichthyosis is a deadly disease resulting from the improper formation of the skin or epidermis. Patients develop hyperkeratosis, or thickening of the epidermis, and are susceptible to impaired barrier functions resulting in dehydration, infection, and heat stroke [1]. Harlequin ichthyosis is caused by a loss of function mutation in the gene ABCA12. The ABCA12 gene encodes a lipid transport protein localized to lamellar granules. Lamellar granules form lipid-containing bodies that release components required for proper keratin shedding in the epidermis [2]. Abnormal lamellar granules are only found in the harlequin ichthyosis phenotype, not in other less severe ichthyosis diseases. How ABCA12 mediates the regulation of formation of lamellar granules in the epidermis remains unclear.
The overall goal is to determine how ABCA12 mediates lipid transport and abnormal lamellar granule formation in the epidermis. The model organism Mus musculus provides a good model as they exhibit visibly hyperkeratotic skin, as well as measurably impaired barrier function and abnormal lamellar bodies. The long-term goal is to understand how the ABCA12 gene allows for proper skin function. Aim 1: Identify conserved amino acids in ABCA12 necessary for lamellar granule formation and lipid transport in the epidermis. Approach: Clutstal Omega will be used to determine specific amino acids in ABCA12 that are conserved in species with similar epidermal layers as humans. Then, CRISPR will be used to mutate these sequences in mice. I will then assay skin punches to determine which of these amino acids are important for proper formation of lamellar granules by measuring epidermal thickness and using skin permeability and lipid staining assays in both mutant and wild type (WT) mice. Hypothesis: Specific amino acid mutations in the ABC transport domain will result in impaired lipid transport, thus, abnormal lamellar granule formation. Rationale: Site specific knockouts in the ABC transport domain that result in hyperkeratosis, impaired barrier function, and abnormal lamellar bodies are critical amino acids necessary for the maintenance of the epidermis. Aim 2: Identify genes that are differentially expressed in ABCA12 mutants, important for lamellar granule formation and lipid transport. Approach: Tissue punches will be taken from the epidermis where ABCA12 RNA is highly expressed. RNA-sequencing will be used on the tissues samples and GO analysis will sort differentially expressed genes into categories like skin development and small molecule transport. Assays from aim 1 be performed to test the genes are involved in lamellar granule formation and lipid transport. Hypothesis: WT and mutant mice will differ in gene expression in tissues that highly express ABCA12. Rationale: Determining differentially expressed genes will identify gene interactions related to hyperkeratosis, impaired barrier function, and abnormal lamellar granules. Aim 3: Quantify protein expression important for lamellar granule formation and lipid transport in the epidermis Approach: Using BioID, protein abundance levels of all proteins will be gathered from epidermal punches of WT and ABCA12 mice. Proteins of interested will be sorted by GO terms, specifically, skin development and small molecule transport. Assays from aim 1 will be performed to determine how differential protein expression levels affect lamellar granule formation and lipid transport. Hypothesis: Proteins involved in lamellar granule formation and lipid transport will be found in lower levels in mutant versus WT mice, leading to the diseased skin phenotype. Rationale: Identifying protein levels in epidermal tissues between WT and mutant mice will help further elucidate the role of ABCA12 in abnormal lamellar granule formation and lipid transport in the epidermis. References [1] Harlequin ichthyosis - Genetics Home Reference - NIH. (n.d.). Retrieved March 3, 2020, from https://ghr.nlm.nih.gov/condition/harlequin-ichthyosis#genes [2] Lai, Y. (2013). Membrane transporters and the diseases corresponding to functional defects. Transporters in Drug Discovery and Development, 1–146. doi: 10.1533/9781908818287.1 |
This web page was produced as an assignment for Genetics 564, an undergraduate capstone course at UW-Madison
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