Deficiency in FAM20A leads to skeletal and dental defects – a study in FAM20A knockout mice
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Family with sequence similarity 20 (FAM20) consists of three members: FAM20A, FAM20B and FAM20C. Mutations in FAM20 family have been linked to developmental disorders involving bones, cartilage and teeth. FAM20A mutations in humans are associated with amelogenesis imperfecta with gingival fibromatosis and enamel renal syndrome. Fam20a knockout (KO) mouse showed growth retardation. The aim of this study was to characterize the skeletal and dental phenotypes using Fam20a KO mouse. Our results showed that body size and bone length of KO mice were smaller than those of WT. The microcomputed tomography (μCT) analyses of trabecular and cortical bones in KO displayed lower bone volume, thinner trabeculae and thinner bone cortex as compared to WT. Histological examination of KO growth plate demonstrated disorganized chondrocyte zones and extended hypertrophic zone. qRT-PCR results showed downregulation of several osteoblast differentiation markers in KO long bone. Immunohistochemical examination demonstrated reduced chondrocyte proliferation, apoptosis and increased collagen X expression in KO growth plate. Our data showed a lower number of osteoblasts and osteoclasts in KO as compared to WT. In vitro study, Fam20a KO showed a lower number of bone marrow stromal cells and osteoprogenitors. In vitro mineralization was impaired in KO osteoblasts. Fam20a KO had hypoplastic enamel, delayed tooth eruption and gingival overgrowth. The µCT results demonstrated that enamel in Fam20a KO was not fully mineralized and enamel matrix was detached from dentin. Scanning electron microscopy displayed absence of decussation patterns in Fam20a KO enamel. Histological examination of maxillary first molar at differentiation stage showed no difference between WT and KO. At the secretory stage, Fam20a KO ameloblasts were short and non-polarized as compared to WT. Immunohistochemical analysis showed diffuse staining pattern of amelogenin in Fam20a KO first molar compared to WT. Western blot analysis demonstrated that amelogenin proteolytic process was impaired in KO and showed slower migration pattern of MMP20. In conclusion, endochondral ossification defects and reduced number of osteoblasts and their precursors led to the bone phenotype in Fam20a KO. Amelogenin processing defects caused amelogenesis imperfecta phenotype in KO. Our study indicated that Fam20a plays a role in skeletal development and amelogenesis.