Identification and Rescue of Splice Defects Caused by Two Neighboring Deep-Intronic ABCA4 Mutations Underlying Stargardt Disease
Identification and Rescue of Splice Defects Caused by Two Neighboring Deep-Intronic ABCA4 Mutations Underlying Stargardt Disease
复制标题
DOI：
10.1016/j.ajhg.2018.02.008
复制
发表时间：
2018-04-05
影响因子：
9.8
通讯作者：
Cremers, Frans P. M.
中科院分区：
文献类型：
--
作者：
Albert, Silvia;Garanto, Alejandro;Cremers, Frans P. M.
遗传性视网膜疾病的编码区和剪接位点序列的序列分析无法发现40%的致病变异。全基因组测序可以识别大多数非编码变体，但它们的解释仍然非常具有挑战性，特别是当相关基因以组织特异性方式表达时。ABCA 4的深内含子变异与常染色体隐性遗传性Stargardt病（STGD 1）有关，但确切的致病机制尚不清楚。通过从患有STGD 1的个体获得的成纤维细胞中产生光感受器前体细胞（PPC），我们证明了两个相邻的深内含子ABCA 4变体（c.4539thorn2001G>A和c.4539thorn2028C>T）导致视网膜特异性345-nt假外显子插入（预测的蛋白质变化：p.Arg1514Leufs*36），可能是由于外显子增强子的产生。靶向345-nt假外显子的反义寡核苷酸（AON）的管理可以显着挽救在具有这些突变的两个个体的PPC中观察到的剪接缺陷。有趣的是，与c.4539thorn2001G>A互补的AON仅在来自具有STGD 1的个体的PPC中挽救了剪接缺陷，而没有其他突变，证明了AON的高特异性。此外，单个AON分子挽救了与不同相邻突变相关的剪接缺陷，从而为STGD 1患者的治疗提供了新的策略。由于许多与人类遗传条件相关的基因在特定组织中表达，并且前mRNA剪接也可能依赖于器官特异性因素，因此我们使用来自STGD 1个体的分化细胞研究和治疗剪接变体的方法可以应用于任何感兴趣的组织。
Sequence analysis of the coding regions and splice site sequences in inherited retinal diseases is not able to uncover similar to 40% of the causal variants. Whole-genome sequencing can identify most of the non-coding variants, but their interpretation is still very challenging, in particular when the relevant gene is expressed in a tissue-specific manner. Deep-intronic variants in ABCA4 have been associated with autosomal-recessive Stargardt disease (STGD1), but the exact pathogenic mechanism is unknown. By generating photoreceptor precursor cells (PPCs) from fibroblasts obtained from individuals with STGD1, we demonstrated that two neighboring deep-intronic ABCA4 variants (c.4539thorn2001G>A and c.4539thorn2028C>T) result in a retina-specific 345-nt pseudoexon insertion (predicted protein change: p.Arg1514Leufs*36), likely due to the creation of exonic enhancers. Administration of antisense oligonucleotides (AONs) targeting the 345-nt pseudoexon can significantly rescue the splicing defect observed in PPCs of two individuals with these mutations. Intriguingly, an AON that is complementary to c.4539thorn2001G>A rescued the splicing defect only in PPCs derived from an individual with STGD1 with this but not the other mutation, demonstrating the high specificity of AONs. In addition, a single AON molecule rescued splicing defects associated with different neighboring mutations, thereby providing new strategies for the treatment of persons with STGD1. As many genes associated with human genetic conditions are expressed in specific tissues and pre-mRNA splicing may also rely on organ-specific factors, our approach to investigate and treat splicing variants using differentiated cells derived from individuals with STGD1 can be applied to any tissue of interest.