The manner of decay of genetically defective EYS gene transcripts in photoreceptor-directed fibroblasts derived from retinitis pigmentosa patients depends on the type of mutation.
The manner of decay of genetically defective EYS gene transcripts in photoreceptor-directed fibroblasts derived from retinitis pigmentosa patients depends on the type of mutation.
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DOI：
10.1186/s13287-018-1016-9
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发表时间：
2018-10-25
影响因子：
7.5
通讯作者：
Kato S
中科院分区：
文献类型：
--
作者：
Seko Y;Iwanami M;Miyamoto-Matsui K;Takita S;Aoi N;Umezawa A;Kato S
诱导光感受器的产生为难治性视网膜疾病的体外建模带来了希望。视网膜色素变性是一种遗传性视网膜营养不良，会导致视力障碍。EYS基因是常染色体隐性遗传性视网膜色素变性(Arrp)最常见的致病基因。EYS基因缺陷的ARRP称为“EYS-RP”。我们之前建立了一种“重定向分化”的方法，从商业上可获得的人皮肤成纤维细胞中产生光敏的光感受器样细胞。在这项研究中，我们从EYS-RP患者的真皮成纤维细胞中培养出光感受器样细胞，作为变性视网膜的替代细胞，使用“重定向分化”。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，因为转录本的衰退以前被认为与疾病相关的表型变异有关。我们利用CRX、RAX、NeuroD和OTX2的“重定向分化”技术，从三名正常志愿者和三名EYS-RP患者的纯合子或杂合子突变的EYS-RP患者那里获得了光感受器定向的成纤维细胞。我们测试了光感受器特异性基因(蓝视蛋白、视紫红质、恢复素、S抗原、PDE6C)在这些细胞中的诱导表达。然后，我们通过RTPCR和测序分析了三种不同类型的缺陷EYS基因c.1211dupA、c.4957dupA和c.8805C >； A在这些细胞中表达的转录产物。包括EYS基因在内的光感受器特异性基因在所有被测试的光感受器导向的成纤维细胞中上调。然而，缺陷转录本的表达水平因突变类型的不同而明显不同。来自这三个缺陷基因的转录本很少被检测到，表达水平较低，与正常志愿者的表达水平几乎相同。在EYS-RP患者的光感受器导向的成纤维细胞中，基因缺陷的EYS基因转录本的表达水平因突变类型而异。在具有c.1211dupA、c.4957dupA和c.8805C >； A的转录本中，表达水平的差异表明这些转录本分别发生了几乎完全的无意义介导的mR NA衰变、部分NMD和逃避NMD。要确定与EYS-RP患者表型变异的关系，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。本文的在线版本(10.1186/s13287-0181016-9)包含补充材料，可供授权用户使用。
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. The online version of this article (10.1186/s13287-018-1016-9) contains supplementary material, which is available to authorized users.
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