Centre for Ophthalmology and Visual Science, The University of Western Australia, Perth, WA, Australia.
Ocular Tissue Engineering Laboratory, Lions Eye Institute, Perth, WA, Australia.
Mol Genet Genomic Med. 2021 Mar;9(3):e1601. doi: 10.1002/mgg3.1601. Epub 2021 Jan 26.
Mutations in CLN3 cause Batten disease, however non-syndromic CLN3 disease, characterized by retinal-specific degeneration, has been also described. Here, we characterized an induced pluripotent stem cell (iPSC)-derived disease model derived from a patient with non-syndromic CLN3-associated retinopathy.
Patient-iPSC, carrying the 1 kb-deletion and c.175G>A variants in CLN3, coisogenic iPSC, in which the c.175G>A variant was corrected, and control iPSC were differentiated into neural retinal organoids (NRO) and cardiomyocytes. CLN3 transcripts were analyzed by Sanger sequencing. Gene expression was characterized by qPCR and western blotting. NRO were characterized by immunostaining and electron microscopy.
Novel CLN3 transcripts were detected in adult human retina and control-NRO. The major transcript detected in patient-NRO displayed skipping of exons 2 and 4-9. Accumulation of subunit-C of mitochondrial ATPase (SCMAS) protein was demonstrated in patient-derived cells. Photoreceptor progenitor cells in patient-NRO displayed accumulation of peroxisomes and vacuolization of inner segments. Correction of the c.175G>A variant restored CLN3 mRNA and protein expression and prevented SCMAS and inner segment vacuolization.
Our results demonstrate the expression of novel CLN3 transcripts in human retinal tissues. The c.175G>A variant alters splicing of the CLN3 pre-mRNA, leading to features consistent with CLN3 deficiency, which were prevented by gene correction.
CLN3 基因突变会导致巴滕病，但也有报道称存在非综合征型 CLN3 疾病，其特征为视网膜特异性退行性变。在此，我们对一位非综合征型 CLN3 相关性视网膜病变患者来源的诱导多能干细胞（iPSC）衍生疾病模型进行了特征描述。
患者 iPSC 携带 CLN3 中的 1kb 缺失和 c.175G>A 变异，与患者 iPSC 携带的 c.175G>A 变异相匹配的同基因 iPSC 以及对照 iPSC 分化为神经视网膜类器官（NRO）和心肌细胞。通过 Sanger 测序分析 CLN3 转录本。通过 qPCR 和 Western blot 分析基因表达。通过免疫染色和电子显微镜对 NRO 进行特征描述。
在成人视网膜和对照 NRO 中检测到新的 CLN3 转录本。在患者 NRO 中检测到的主要转录本显示外显子 2 和 4-9 的跳跃。在患者来源的细胞中证实了线粒体 ATP 酶亚基-C（SCMAS）蛋白的积累。患者 NRO 中的光感受器祖细胞显示过氧化物酶体的积累和内节空泡化。c.175G>A 变异的校正恢复了 CLN3 mRNA 和蛋白的表达，并防止了 SCMAS 和内节空泡化。
我们的研究结果表明，新型 CLN3 转录本在人视网膜组织中表达。c.175G>A 变异改变了 CLN3 前体 mRNA 的剪接，导致与 CLN3 缺乏一致的特征，而基因校正可预防这些特征的出现。
