Sensory Functions Section, Research Institute, National Rehabilitation Center for Persons with Disabilities, 4-1 Namiki, Tokorozawa, 359-8555, Japan.
Department of Ophthalmology, Hospital, National Rehabilitation Center for Persons with Disabilities, 4-1 Namiki, Tokorozawa, 359-8555, Japan.
Stem Cell Res Ther. 2018 Oct 25;9(1):279. doi: 10.1186/s13287-018-1016-9.
Generation of induced photoreceptors holds promise for in vitro modeling of intractable retinal diseases. Retinitis pigmentosa is an inherited retinal dystrophy that leads to visual impairment. The EYS gene was reported to be the most common gene responsible for autosomal recessive retinitis pigmentosa (arRP). arRP with defects in the EYS gene is denoted by "EYS-RP". We previously established a "redirect differentiation" method to generate photosensitive photoreceptor-like cells from commercially available human dermal fibroblasts. In this study, we produced photoreceptor-like cells from dermal fibroblasts of EYS-RP patients as a replacement for the degenerative retinas using "redirect differentiation". We analyzed defective transcripts of the EYS gene in these cells to elucidate phenotypes of EYS-RP patients because decay of transcripts was previously suggested to be involved in phenotypic variation associated with diseases.
Using "redirect differentiation" by CRX, RAX, NeuroD and OTX2, we made photoreceptor-directed fibroblasts derived from three normal volunteers and three EYS-RP patients with homozygous or heterozygous mutations. We tested inducible expression of the photoreceptor-specific genes (blue opsin, rhodopsin, recoverin, S-antigen, PDE6C) in these cells. We then analyzed transcripts derived from three different types of the defective EYS gene, c.1211dupA, c.4957dupA and c.8805C > A, expressed in these cells by RT-PCR and sequencing.
Photoreceptor-specific genes including the EYS gene were up-regulated in all the photoreceptor-directed fibroblasts tested. However, expression levels of defective transcripts were markedly different depending on the type of mutation. Transcripts derived from these three defective genes were scarcely detected, expressed at a lower level, and expressed at almost the same level as in normal volunteers, respectively.
Expression levels of genetically defective EYS gene transcripts in photoreceptor-directed fibroblasts of EYS-RP patients vary depending on the type of mutation. Variation in expression levels in transcripts having c.1211dupA, c.4957dupA and c.8805C > A suggests that almost complete nonsense-mediated mRNA decay (NMD), partial NMD and escape from NMD occurred for these transcripts, respectively. To determine the relationship with phenotypic variations in EYS-RP patients, more samples are needed. The present study also suggests that the redirect differentiation method could be a valuable tool for disease modeling despite some limitations.
诱导光感受器的产生有望用于体外模拟难治性视网膜疾病。色素性视网膜炎是一种遗传性视网膜营养不良，可导致视力损害。EYS 基因被报道是导致常染色体隐性遗传色素性视网膜炎（arRP）的最常见基因。EYS 基因缺陷的 arRP 被称为“EYS-RP”。我们之前建立了一种“重定向分化”方法，可从市售的人真皮成纤维细胞中产生光敏光感受器样细胞。在这项研究中，我们使用“重定向分化”方法，从 EYS-RP 患者的成纤维细胞中产生光感受器样细胞，以替代退行性视网膜。我们分析了这些细胞中 EYS 基因的缺陷转录本，以阐明 EYS-RP 患者的表型，因为先前有人提出转录本的衰减可能与疾病相关的表型变异有关。
使用 CRX、RAX、NeuroD 和 OTX2 的“重定向分化”，我们从三个正常志愿者和三个带有纯合或杂合突变的 EYS-RP 患者的成纤维细胞中制造了光感受器定向成纤维细胞。我们测试了这些细胞中光感受器特异性基因（蓝色视蛋白、视紫红质、恢复蛋白、S-抗原、PDE6C）的诱导表达。然后，我们通过 RT-PCR 和测序分析了这些细胞中表达的三种不同类型的缺陷 EYS 基因（c.1211dupA、c.4957dupA 和 c.8805C> A）的转录本。
所有测试的光感受器定向成纤维细胞中均上调了光感受器特异性基因，包括 EYS 基因。然而，缺陷转录本的表达水平因突变类型而异。这些三种缺陷基因的转录本几乎检测不到，表达水平较低，且与正常志愿者的表达水平几乎相同。
EYS-RP 患者的光感受器定向成纤维细胞中遗传缺陷 EYS 基因转录本的表达水平因突变类型而异。c.1211dupA、c.4957dupA 和 c.8805C> A 转录本的表达水平存在差异，提示这些转录本分别发生了近乎完全的无义介导的 mRNA 衰减（NMD）、部分 NMD 和逃逸 NMD。为了确定与 EYS-RP 患者表型变异的关系，还需要更多的样本。本研究还表明，尽管存在一些局限性，但重定向分化方法可能是一种有价值的疾病建模工具。
