Assessing the role of SEC16B in bone fragility

In the current study, we have identified a single de novo homozygous mutation in SEC16B in exon 4 (NM_033127.3: c.424CàT; p.Arg142àTry) in a ten-year-old boy with typical clinical, radiological and bone ultrastructural features of OI using whole exome sequencing. Growing these patients’ fibroblasts in our lab, we found that these cells accumulate collagen type I in the ER when compared to healthy control cells with no mutation. This is supported by comparing the immunofluorescence (IF) staining of patient and control cells using antibodies to identify the location of collagen type I within the ER and Golgi in the cells. Pulse chase experiment indicated that patient cells with homozygous SEC16B mutation had accelerated cellular collagen type I secretion compared to healthy control cells with intact SEC16B. This may be explained by the fact that collagen proteins do not retain in the Golgi but instead get transported directly to the extracellular matrix. Interestingly, using transmission electron microscopy and western blotting, we found that SEC16B mutant fibroblasts exihibit enhanced autophagosome formation combined with increased apoptosis. Most interestingly, mass spectrometry demonstrated that the quality of COL1A1 in mutated SEC16B cells is different than the healthy controls. Particularly; two regions of COL1A1 are not detected in patient fibroblasts when compared to healthy fibroblasts. These defects in type I collagen can be explained by altered post translational modification (PTMs) in SEC16B mutant cells.