ABSTRACT

Background: To identify and investigate the effects of a novel splicing variant, c.1444–2A>C of OPA1, on its transcript, translation, and mitochondrial function, which was found in an 8-year-old patient with dominantly inherited optic atrophy (DOA).

Materials and Methods: The clinical evaluations were performed at the Eye Center. Lymphoblast cell lines were generated from the patient, mother, and a normal control with the same haplotype of mitochondrial genome. The novel variant was confirmed by Sanger sequencing. The splicing alteration of cDNA was checked by both Sanger sequencing and agarose gel. OPA1 expression was carried out by RT-PCR and Western blotting. Transmission electron microscopy was used for mitochondrial morphology. Mitochondrial functions, including the rates of oxygen consumption, ATP generation, ROS product and membrane potential were assayed in lymphoblast cells.

Results: The novel OPA1 splicing variant, c.1444–2A>C, led to a deletion of the 15th exon in mRNA transcript. Approximately 50% reduction of mRNA and protein expression was present in mutant cells as compared with controls. No marked depletion of mtDNA nor mitochondrial mass was caused by the splicing variant. However, defects that the impaired capacity of OXPHOS, reduced ATP generation, increased ROS and decreased membrane potential were observed in the mutant cells, which promoted a ubiquitin-binding mitophagy instead of apoptosis.

Conclusions: The novel splicing variant, c.1444–2A>C resulted in OPA1 haploinsufficiency effect on its expression and mitochondrial function without mtDNA depletion. Our findings may provide new insights into the understanding of pathophysiology of DOA.

摘要

背景：为了鉴定和研究OPA1 的c.1444-2A>C 新剪接变异体对其转录、翻译和线粒体功能的影响，该变异体在一名 8 岁显性遗传性视神经萎缩患者中发现(DOA)。

材料和方法：临床评估在眼科中心进行。从患者、母亲和具有相同线粒体基因组单倍型的正常对照产生淋巴母细胞系。通过 Sanger 测序证实了新变体。通过桑格测序和琼脂糖凝胶检查 cDNA 的剪接变化。OPA1 表达通过 RT-PCR 和蛋白质印迹进行。透射电子显微镜用于线粒体形态。在淋巴母细胞中测定了线粒体功能，包括耗氧率、ATP 生成、ROS 产物和膜电位。

结果：新的OPA1剪接变体 c.1444-2A>C 导致 mRNA 转录物中第 15 个外显子的缺失。与对照相比，突变细胞中存在大约 50% 的 mRNA 和蛋白质表达减少。剪接变体没有引起 mtDNA 和线粒体质量的明显消耗。然而，在突变细胞中观察到 OXPHOS 能力受损、ATP 生成减少、ROS 增加和膜电位降低等缺陷，这促进了泛素结合线粒体自噬而不是细胞凋亡。

结论：新的剪接变体 c.1444-2A>C 导致 OPA1 单倍体不足对其表达和线粒体功能的影响，而没有 mtDNA 耗竭。我们的发现可能为理解 DOA 的病理生理学提供新的见解。