Effects of Fam83h truncation mutation on enamel developmental defects in male C57/BL6J mice.

Truncation mutations in family with sequence similarity, member H (FAM83H) gene are considered the main cause of autosomal dominant hypocalcified amelogenesis imperfecta (ADHCAI); however, its pathogenic mechanism in amelogenesis remains poorly characterized. This study aimed to investigate the effects of truncated FAM83H on developmental defects in enamel. CRISPR/Cas9 technology was used to develop a novel Fam83h c.1186C > T (p.Q396*) knock-in mouse strain, homologous to the human FAM83H c.1192C > T mutation in ADHCAI. The Fam83h mice showed poor growth, a sparse and scruffy coat, scaly skin and early mortality compared to control mice. Moreover, the forelimbs of homozygous mice were swollen, exhibiting a significant inflammatory response. Incisors of Fam83h mice appeared chalky white, shorter, and less sharp than those of control mice, and energy dispersive X-ray spectroscopy (EDS) analysis and Prussian blue staining helped identify decreased iron and increased calcium (Ca) and phosphorus (P) levels, with an unchanged Ca/P ratio. The expression of iron transportation proteins, transferrin receptor (TFRC) and solute carrier family 40 member 1 (SLC40A1), was decreased in Fam83h-mutated ameloblasts. Micro-computed tomography revealed enamel defects in Fam83h mice. Fam83h enamel showed decreased Vickers hardness and distorted enamel rod structure and ameloblast arrangement. mRNA sequencing showed that the cell adhesion pathway was most notably clustered in LS8-Fam83h-mutated cells. Immunofluorescence analysis further revealed decreased protein expression of desmoglein 3, a component of desmosomes, in Fam83h-mutated ameloblasts. The FAM83H-casein kinase 1α (CK1α)-keratin 14 (K14)-amelogenin (AMELX) interaction was detected in ameloblasts. And K14 and AMELX were disintegrated from the tetramer in Fam83h-mutated ameloblasts in vitro and in vivo. In secretory stage ameloblasts of Fam83h mice, AMELX secretion exhibited obvious retention in the cytoplasm. In conclusion, truncated FAM83H exerted dominant-negative effects on gross development, amelogenesis, and enamel biomineralization by disturbing iron transportation, influencing the transportation and secretion of AMELX, and interfering with cell-cell adhesion in ameloblasts.