Department of Medicine, Surgery and Neurosciences, University of Siena, 53100 Siena, Italy.
UOC Neurologia, Azienda Ospedaliero-Universitaria Senese, 53100 Siena, Italy.
Cells. 2024 Sep 8;13(17):1504. doi: 10.3390/cells13171504.
was identified as a novel candidate gene for autosomal dominant centronuclear myopathy-4 (CNM4) approximately ten years ago. However, to date, only one family has been described, and the function of CCDC78 remains unclear. Here, we analyze for the first time a family harboring a nonsense mutation to better understand the role of CCDC78 in muscle.
We conducted a comprehensive histopathological analysis on muscle biopsies, including immunofluorescent assays to detect multiple sarcoplasmic proteins. We examined transcripts and protein using WB in -mutated muscle tissue; these analyses were also performed on muscle, lymphocytes, and fibroblasts from healthy subjects. Subsequently, we conducted RT-qPCR and transcriptome profiling through RNA-seq to evaluate changes in gene expression associated with CCDC78 dysfunction in muscle. Lastly, coimmunoprecipitation (Co-Ip) assays and mass spectrometry (LC-MS/MS) studies were carried out on extracted muscle proteins from both healthy and mutated subjects.
The histopathological features in muscle showed novel histological hallmarks, which included areas of dilated and swollen sarcoplasmic reticulum (SR). We provided evidence of nonsense-mediated mRNA decay (NMD), identified the presence of novel transcripts in muscle and lymphocytes, and identified 1035 muscular differentially expressed genes, including several involved in the SR. Through the Co-Ip assays and LC-MS/MS studies, we demonstrated that CCDC78 interacts with two key SR proteins: SERCA1 and CASQ1. We also observed interactions with MYH1, ACTN2, and ACTA1.
Our findings provide insight, for the first time, into the interactors and possible role of CCDC78 in skeletal muscle, locating the protein in the SR. Furthermore, our data expand on the phenotype previously associated with mutations, indicating potential histopathological hallmarks of the disease in human muscle. Based on our data, we can consider as the causative gene for CNM4.
大约十年前，被确定为常染色体显性中核肌病-4（CNM4）的一个新候选基因。然而，迄今为止，仅描述了一个家系，CCDC78 的功能仍不清楚。在这里，我们首次分析了一个携带无义突变的家系，以更好地了解 CCDC78 在肌肉中的作用。
我们对肌肉活检进行了全面的组织病理学分析，包括免疫荧光检测多种肌浆蛋白。我们使用 WB 在突变肌肉组织中检查了 转录本和蛋白质；还对来自健康受试者的肌肉、淋巴细胞和成纤维细胞进行了这些分析。随后，我们通过 RNA-seq 进行 RT-qPCR 和转录组谱分析，以评估与 CCDC78 功能障碍相关的基因表达变化。最后，我们对来自健康和突变受试者的提取肌肉蛋白进行了免疫共沉淀（Co-Ip）测定和质谱（LC-MS/MS）研究。
肌肉的组织病理学特征显示出新颖的组织学特征，包括扩张和肿胀的肌浆网（SR）区域。我们提供了证据表明存在无意义介导的 mRNA 降解（NMD），在肌肉和淋巴细胞中发现了新型 转录本的存在，并鉴定了 1035 个肌肉差异表达基因，包括几个与 SR 相关的基因。通过 Co-Ip 测定和 LC-MS/MS 研究，我们证明 CCDC78 与两个关键的 SR 蛋白：SERCA1 和 CASQ1 相互作用。我们还观察到与 MYH1、ACTN2 和 ACTA1 的相互作用。
我们的研究结果首次提供了有关 CCDC78 在骨骼肌中的相互作用体和可能作用的见解，将该蛋白定位在 SR 中。此外，我们的数据扩展了先前与 突变相关的表型，表明人类肌肉疾病的潜在组织病理学特征。基于我们的数据，我们可以将 视为 CNM4 的致病基因。
