Zhang Xiao, Thompson Jennifer A, Zhang Dan, Charng Jason, Arunachalam Sukanya, McLaren Terri L, Lamey Tina M, De Roach John N, Jennings Luke, McLenachan Samuel, Chen Fred K
Centre for Ophthalmology and Visual Science, The University of Western Australia, Nedlands, WA, Australia.
Lions Eye Institute, Nedlands, WA, Australia.
Mol Genet Genomic Med. 2020 Nov;8(11):e1489. doi: 10.1002/mgg3.1489. Epub 2020 Sep 15.
Mutations in the human crumbs homologue 1 (CRB1) gene are associated with a spectrum of inherited retinal diseases. However, functional studies demonstrating the impact of individual CRB1 mutations on gene expression are lacking for most variants. Here, we investigated the effect of two CRB1 variants on pre-mRNA splicing using neural retinal organoids (NRO) derived from a patient with recessive rod-cone dystrophy caused by compound heterozygous mutations in CRB1 (c.1892A>G and c.2548G>A).
The patient received ophthalmological examinations including multimodal imaging. NRO were differentiated from induced pluripotent stem cells (iPSCs) derived from the patient and a control subject. CRB1 transcripts were characterized by RT-PCR and Sanger sequencing.
The Patient displayed retinal thickening with disorganization of retinal layers and preservation of para-arteriolar retinal pigment epithelium. Both patient and control iPSC produced NRO containing photoreceptor progenitor cells expressing CRB1 mRNA. Patient NRO expressed a novel CRB1 transcript displaying skipping of exon 6. CRB1 transcripts containing the c.2548G>A substitution in exon 7 were expressed in patient NRO.
Together, these results confirm the pathogenicity of the c.1892A>G and c.2548G>A CRB1 variants in a family with recessive adult-onset rod-cone dystrophy and further demonstrate the effects of these variants on pre-mRNA splicing. This data provide important insights into the pathogenic mechanisms associated with these variants.
人类crumbs同源物1（CRB1）基因突变与一系列遗传性视网膜疾病相关。然而，对于大多数变异体而言，缺乏能证明单个CRB1突变对基因表达影响的功能研究。在此，我们利用源自一名患有由CRB1复合杂合突变（c.1892A>G和c.2548G>A）导致的隐性视杆-视锥营养不良患者的神经视网膜类器官（NRO），研究了两种CRB1变异体对前体mRNA剪接的影响。
该患者接受了包括多模态成像在内的眼科检查。NRO由源自该患者和一名对照受试者的诱导多能干细胞（iPSC）分化而来。通过逆转录聚合酶链反应（RT-PCR）和桑格测序对CRB1转录本进行表征。
该患者表现出视网膜增厚，视网膜层结构紊乱，动脉旁视网膜色素上皮得以保留。患者和对照的iPSC均产生了含有表达CRB1 mRNA的光感受器祖细胞的NRO。患者的NRO表达了一种新的CRB1转录本，显示外显子6跳跃。在患者的NRO中表达了含有外显子7中c.2548G>A替换的CRB1转录本。
总之，这些结果证实了c.1892A>G和c.2548G>A这两种CRB1变异体在一个患有隐性成人发病视杆-视锥营养不良的家族中的致病性，并进一步证明了这些变异体对前体mRNA剪接的影响。这些数据为与这些变异体相关的致病机制提供了重要见解。
