Applications of Next-Generation Sequencing to Rare Disease

Abstract

Since the discovery of the structure of DNA in 1953, researchers and clinicians have been painstakingly paving the way for the use of genetic information in the treatment of disease. In order for this to be possible, specific genetic targets must be identified. For this dissertation, I use next generation, single-cell, and third generation RNA-sequencing techniques to identify markers of genetic heterogeneity and potential therapeutic targets in the rare diseases recessive dystrophic epidermolysis bullosa (RDEB) and cerebral childhood adrenoleukodystrophy (ccALD). RDEB is a an inherited blistering disorder caused by mutations in the key structural skin protein, type VII collagen (C7). It can partially treated by hematopoietic stem cell transplant (HSCT), however, how blistered RDEB skin signals to donor cells is unknown. In Chapter 2, to identify potential signals, I performed single-cell RNA-seq (scRNA-seq) on patient fibroblasts and implemented a variance-driven multitask clustering (scVDMC), that utilizes multiple single-cell populations from biological replicates or different samples. scVDMC clusters single cells in multiple scRNA-seq experiments of similar cell types and markers but varying expression patterns such that the scRNA-seq data are better integrated than typical pooled analyses which only increase the sample size. By controlling the variance among the cell clusters within each dataset and across all the datasets, scVDMC detects cell sub-populations in each individual experiment with shared cell-type markers but varying cluster centers among all the experiments. scVDMC was then applied to two previously published scRNA-seq datasets with several replicates and one large-scale Drop-seq dataset on three patient samples. scVDMC more accurately detected cell populations and known cell markers than pooled clustering and other recently proposed scRNA-seq clustering methods. When applied to the scRNA-seq RDEB patient fibroblast data, scVDMC revealed several new cell types and unknown markers that I validated by flow cytometry. ccALD is caused by mutations in the \emph{ABCD1} gene and manifests in early childhood with neuropathological symptoms and hyper-pigmentation, culminating in massive breakdown of the blood-brain barrier (BBB) and death if HSCT is not performed at an early stage. It is difficult to model the BBB of this disease as primary cells do not recapitulate the barrier in culture and the mouse model shows incomplete penetrance. In Chapter 3, I model the blood-brain barrier of ccALD patients and wild-type (WT) controls using directed differentiation of induced pluripotent stem cells (iPSCs) into induced brain microvascular endothelial cells (iBMECs). Immunocytochemistry and PCR confirmed characteristic expression of brain microvascular endothelial cell (BMEC) markers. Barrier properties of iBMECs were measured via trans-endothelial electrical resistance (TEER), sodium fluorescein permeability, and frayed junction analysis. Electron microscopy and RNA-seq were used to further characterize disease-specific differences. Oil-Red-O staining was used to quantify differences in lipid accumulation. To evaluate whether treatment with block copolymers of poly(ethylene oxide) and poly(propylene oxide) (PEO-PPO) could mitigate defective properties, ccALD-iBMECs were treated with PEO-PPO block copolymers and their barrier properties and lipid accumulation levels were quantified. iBMECs from patients with ccALD had significantly decreased TEER (2592 ± 110 $\Omega \cdot cm^2$) compared to WT controls (5001 ± 172 $\Omega \cdot cm^2$). They also accumulated lipid droplets to a greater extent than WT-iBMECs. Upon treatment with a PEO-PPO diblock copolymer during the differentiation process, an increase in TEER and a reduction in lipid accumulation were observed for the polymer treated ccALD-iBMECs compared to untreated controls. The finding that BBB integrity is decreased in ccALD and can be rescued with block copolymers opens the door for the discovery of BBB-specific molecular markers that can indicate the onset of ccALD and has therapeutic implications for preventing the conversion to ccALD. Revertant mosaicism in RDEB patients is seen as patches of skin that have never blistered. At the molecular level, these patches of skin contain detectable amounts of C7, indicating that a reversion of the disease-causing mutation has occurred at the DNA level. One of the limited treatment options available for treating RDEB is the use of C7 expressing stem cells or differentiated skin cells to replace C7 at the dermal-epidermal junction and restore the overall integrity of the skin architecture. However, this typically requires the use of gene therapy or allogeneic cells, which can be costly and cause adverse reactions in the recipient. Mosaic cells could potentially be used for these purposes, however, isolating and purifying them has proven difficult. In Chapter 4, I describe a method utilizing synthetic micro RNA (miR) switches, whereby differences in endogenous miR activity are exploited to purify mosaic cells in culture, which may be useful in generating pure populations of mosaic cells that can then be used in future clinical applications. Chapter 5 of this dissertation uses third generation or long read sequencing to look more closely at the underlying genetic event resulting in mosaic expression in one particular RDEB patient. These studies identify genetic heterogeneity in cell types relevant to the respective rare diseases being examined and give support to developing precision medicine techniques to treat these rare diseases.