Background: Focal segmental glomerulosclerosis (FSGS) is a kidney disease that is commonly associated with proteinuria and the progressive loss of renal function, which is characterized by podocyte injury and the depletion and collapse of glomerular capillary segments. The pathogenesis of FSGS has not been completely elucidated; however, recent advances in molecular genetics have provided increasing evidence that podocyte structural and functional disruption is central to FSGS pathogenesis. Here, we identified a patient with FSGS and aimed to characterize the pathogenic gene and verify its mechanism.
Methods: Using next-generation sequencing and Sanger sequencing, we screened the causative gene that was linked to FSGS in this study. The patient's total blood RNA was extracted to validate the messenger RNA (mRNA) expression of coenzyme Q10 monooxygenase 6 (COQ6) and validated it by immunohistochemistry. COQ6 knockdown in podocytes was performed in vitro with small interfering RNA, and then, F-actin was determined using immunofluorescence staining. Cell apoptosis was evaluated by flow cytometry, the expression of active caspase-3 was determined by Western blot, and mitochondrial function was detected by MitoSOX.
Results: Using whole-exome sequencing and Sanger sequencing, we screened a new causative gene, COQ6, NM_182480: exon1: c.G41A: p.W14X. The mRNA expression of COQ6 in the proband showed decreased. Moreover, the expression of COQ6, which was validated by immunohistochemistry, also had the same change in the proband. Finally, we focused on the COQ6 gene to clarify the mechanism of podocyte injury. Flow cytometry showed significantly increased in apoptotic podocytes, and Western blotting showed increases in active caspase-3 in si-COQ6 podocytes. Meanwhile, reactive oxygen species (ROS) levels were increased and F-actin immunofluorescence was irregularly distributed in the si-COQ6 group.
Conclusions: This study reported a possible mechanism for FSGS and suggested that a new mutation in COQ6, which could cause respiratory chain defect, increase the generation of ROS, destroy the podocyte cytoskeleton, and induce apoptosis. It provides basic theoretical basis for the screening of FSGS in the future.
COQ6突变导致FSGS患者足细胞损伤的新突变 摘要 背景: 局灶节段性肾小球硬化(focal segmental glomerularsclerosis，FSGS)是一种常见的肾病，常与蛋白尿和渐进性肾功能衰竭有关，其特征为足细胞损伤，肾小球毛细血管袢的缺失和塌陷。然而，FSGS的发病机制尚未完全阐明，近年来分子遗传学的进展提供了越来越多的证据表明足细胞的结构和功能紊乱成为FSGS发病机制的关键。在本研究中，通过围绕一例FSGS患者旨在找出致病基因并验证其致病机制。 方法: 采用新一代测序技术(next-generation sequencing，NGS)和Sanger测序筛出与FSGS相关的致病基因。后提取先证者全血检测COQ6-mRNA表达水平，并通过免疫组织化学验证。体外实验中，利用siRNA敲除足细胞中的COQ6基因，并利用免疫荧光技术检测F-actin表达。通过流式细胞检测细胞凋亡率及检测凋亡蛋白caspase3的表达来验证细胞凋亡情况，并利用MitoSOX检测线粒体功能。 结果: 通过全外显子测序和Sanger测序筛选出COQ6一种新致病突变，NM_182480: exon1: c.G41A:p.W14X。先证者全血COQ6-mRNA检测结果及免疫组化结果均显示COQ6表达明显下降，最终明确COQ6为足细胞损伤的致病基因。通过siRNA抑制足细胞COQ6表达后，流式细胞仪检测到凋亡细胞数明显增多，同时活性Capase-3的表达明显增加，并且线粒体产生的ROS水平明显升高，免疫荧光示F-actin排列紊乱。 结论: 本研究为FSGS发病机制提供了一种新的思路，并且发现了COQ6的一种新突变位点，该突变会致线粒体呼吸链受损，从而ROS生成过多，破坏足细胞骨架，诱导足细胞凋亡。本研究为今后的FSGS的筛查提供了基本的理论依据。.
Keywords: Apoptosis; Coenzyme Q10; Focal Segmental Glomerulosclerosis; Monooxygenase 6 Mutation; Podocyte.