Conclusions
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 CCDC78 mutations, indicating potential histopathological hallmarks of the disease in human muscle. Based on our data, we can consider CCDC78 as the causative gene for CNM4.
Methods
We conducted a comprehensive histopathological analysis on muscle biopsies, including immunofluorescent assays to detect multiple sarcoplasmic proteins. We examined CCDC78 transcripts and protein using WB in CCDC78-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.
Results
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 CCDC78 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. Conclusions: 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 CCDC78 mutations, indicating potential histopathological hallmarks of the disease in human muscle. Based on our data, we can consider CCDC78 as the causative gene for CNM4.