Modeling ciliogenesis in Leber congenital amaurosis using fibroblasts from patients with CEP290 mutations

摘要

Purpose : Leber congenital amaurosis (LCA) is a severe retinal dystrophy that presents shortly after birth. 20% of LCA is attributed to mutations in ciliary centrosomal protein CEP290 and is currently untreatable. CEP290 mutations disrupt development of the connecting cilium in photoreceptors. Here, we focus on ciliogenesis in CEP290-LCA patient fibroblasts with the aim to compare phenotypes caused by different gene mutations. Methods : The study included two families: one with one patient and unaffected mother and the second with two sibling patients and their unaffected mother. Skin biopsies from LCA patients and controls were obtained from University of Pennsylvania Scheie Eye Institute. Skin-derived fibroblasts were cultured in MEM?±+Glutamax media with 10% FBS for a maximum of 10 passages. Cells were seeded on collagen coated chamber slides and serum starved for 72 hours to promote cilia growth. After fixation, fibroblasts were immunostained with cilia marker acetylated tubulin, basal body marker ?3-tubulin, and DAPI. Slides were imaged by confocal microscopy and cilia length was measured using ImageJ. Percentage of cells with cilia and cilia length were compared between patients and controls. Ciliated fibroblasts were analyzed by electron microscopy (EM) to examine cilia morphology. Results : More than 90% of cells in all patient and control cultures developed cilia irrespective of the genotype. No significant difference in cilia length was observed between any CEP290 patients and controls (n=3, one-way ANOVA test). Mean cilia lengths (??m?±SEM) for controls were 3.96?±1.10 and 3.67?±1.04; and for patients were 4.10?±1.13, 4.13?±1.01, and 3.74?±1.10. Cilia structure in patient fibroblasts was comparable to control fibroblasts by EM. Conclusions : Extensive analysis of fibroblasts from three CEP290-LCA patients and their unaffected parent controls did not show any difference in number of ciliated cells, cilia length, or structure. Burnight et al. (2014) and Gerard et al. (2012) observed significantly reduced cilia expression and, when present, significantly shorter cilia in CEP290-LCA patients compared to control. Furthermore, Burnight et al. (2014) showed that the shorter cilia phenotype in fibroblasts of CEP290-LCA patients can be rescued using gene therapy strategies. Our results suggest caution in extending the application of gene therapy for CEP290-LCA based uniquely on ciliogenesis as a readout. This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.