CCDC78: unveiling the function of a novel gene associated with hereditary myopathy

Congenital myopathies are a large group of inherited muscle disease affecting the skeletal muscle tissue caused by genetically determined muscle protein defects. The coiled-coil domain-containing protein 78 gene (CCDC78) has been suggested in 2012 by Majczenko et al. as novel candidate gene for the autosomal dominant centronuclear myopathy-4 (CNM4; OMIM#614807). However, to date, only one family has been described and CCDC78 role in muscle function remains unclear. Here we deeply analyze for the first time a family harbouring a nonsense mutation in CCDC78 gene. We performed optic and electron microscopy study and multiple immunofluorescent assays on muscle biopsy. Moreover, we analyzed CCDC78 transcripts and protein in different tissues from healthy and mutated subjects, and we performed transcriptome profiling by RNA-seq and RT-qPCR from muscle tissues. Lastly, coimmunoprecipitation (Co-Ip) assay and mass spectroscopy (LC-MS/MS) studies were carried out on extracted muscle proteins from healthy and mutated subjects. Histopathological features encompassed fibre size variation, nuclear centralizations and as novel histological hallmark, a peculiar, dilated terminal sarcoplasmic reticulum (SR), whirls of redundant membranes and areas of dilated and swollen SR. We provided evidence of nonsense mediated decay, suggesting an haploinsufficiency mechanism; we found novel CCDC78 transcripts selectively expressed in muscle, and we detected 1035 muscular differentially expressed genes (DEGs). The identified transcripts clustered in specific pathways: in the discovery set we observed a statistically significant enrichment of functionally interesting biological processes including muscle contraction and development. Among the identified DEGs, we found upregulation of a series of genes involved in SR: TMOD1, JPH1, CACNA2D1, MTM1, ASPH, CASQ1 and ATP2A1. Lastly, though Co-Ip assay and LC-MS/MS, we discovered for the first time that CCDC78 interacts with two pivotal SR proteins: SERCA1 and CASQ1. We also found an interaction with other proteins outside the SR: MYH1, ACTN2 and ACTA1. Our findings shed light for the first time on interactors and possible role of CCDC78 in skeletal muscle, thus allowing us to definitely locate the protein in SR. Moreover, our data expand the phenotype previously associated with CCDC78 mutations, indicating new possible histopathologic hallmarks of disease in humans; according to our data we can consider CCDC78 as CNM4 causative gene.