A novel DSPP frameshift mutation causing dentin dysplasia type 2 and disease management strategies.

The present study aims to investigate the mutation in a Chinese family with dentin dysplasia type II (DD-II) and to summarize mutation hotspots, clinical manifestations, and disease management strategies. Phenotype analysis, clinical intervention, mutation screening, and cosegregation analysis within the enrolled family were performed. A summary of the reported mutations in the dentin phosphoprotein (DPP) region of dentin sialophosphoprotein (DSPP) was analyzed. Pathogenicity prediction analysis of the physical properties and function of DSPP variants was performed by bioinformatic processing. Clinical management strategies are discussed. A novel pathogenic mutation (c.2035delA) in the DPP region of DSPP was identified, which was cosegregated in the family. The immature permanent teeth of patients with DD-II presented with X-shaped root canal phenotypes. Most of the identified mutations for DD-II were clustered in the DPP region between nucleotides 1686-2134. Points of differential diagnosis, clinical interventions, and management strategies are proposed. This study revealed a novel DSPP frameshift mutation and presented new clinical features of DD-II. The locus involving nucleotides 1686-2134 of DSPP may represent a mutational hotspot for the disease. Appropriate management of DD-II at different stages is important to avoid the development of secondary dental lesions.

Fam83h mutation causes mandible underdevelopment via CK1α-mediated Wnt/ß-catenin signaling in male C57/BL6J mice.

Truncation mutations in FAM83H are the major cause of autosomal dominant hypocalcified amelogenesis imperfecta. Some studies also indicated that FAM83H could be involved in osteogenic differentiation; however, the function of FAM83H in bone formation was rarely explored. This study aimed to explore the effect of Fam83h mutation on skeletal development. We generated Fam83h c.1186C>T (p.Q396*) knockin C57/BL6J mice by CRISPR/Cas9 technology and found that the Fam83hQ396⁎/Q396⁎ male mice presented skeletal development retardation that was inconspicuous at birth but progressively worsened as they grew up. Alcian and Alizarin Red staining of the whole-mount skeleton showed Fam83hQ396⁎/Q396⁎ mice presented obvious skeletal development retardation. Moreover, Micro-computed tomography (Micro-CT) analysis and H&E staining showed that the mandible of Fam83hQ396⁎/Q396⁎ mice exhibited decreased bone trabecula and slight bone rarefaction compared with wild-type mice. Calcium and phosphorus content of serum and bone, and serum ALP activity analysis showed that the serum ALP activity and value of bone calcium were decreased in Fam83hQ396⁎/Q396⁎ mice. The reduced expression of mineralization markers of RUNX2, OSX, OCN, and COL1, the reduced ALP activity and the weakened ARS staining exhibited in osteoblasts isolated from 3-day-old Fam83hQ396⁎/Q396⁎ mice. The increased protein expression of casein kinase 1α (CK1α) in the cytoplasm and the decreased expression of ß-CATENIN in the nucleus indicated the inhibiting Wnt/ß-catenin signaling in osteoblasts from Fam83hQ396⁎/Q396⁎ mice. Furthermore, agonists of Wnt/ß-catenin signaling and Ck1α siRNA partially reversed the mineralization inhibition and the decreased expression of key signaling molecules in osteoblasts of Fam83hQ396⁎/Q396⁎ mice. In conclusion, Fam83h mutation caused the increase of cytoplasmic CK1α (as one of the components of the degradation complex), which in turn promoted degradation of ß-CATENIN in the cytoplasm and reduced ß-CATENIN translocation into the nucleus, subsequently inhibited Wnt/ß-catenin signaling in osteoblast differentiation, and thus resulted in the mandible underdevelopment in Fam83hQ396⁎/Q396⁎ male mice.

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 Fam83hQ396⁎/Q396⁎ 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 Fam83hQ396⁎/Q396⁎ 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 Fam83hQ396⁎/Q396⁎ mice. Fam83hQ396⁎/Q396⁎ 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 Fam83hQ396⁎/Q396⁎ 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.

Mettl3 regulates hypertrophic differentiation of chondrocytes through modulating Dmp1 mRNA via Ythdf1-mediated m6A modification.

As the main cells in endochondral osteogenesis, chondrocytes have limited self-repair ability due to weak proliferation activity, low density, and dedifferentiation tendency. Here, a thorough inquiry about the effect and underlying mechanisms of methyltransferase like-3 (Mettl3) on chondrocytes was made. Functionally, it was indicated that Mettl3 promoted the proliferation and hypertrophic differentiation of chondrocytes. Mechanically, Dmp1 (dentin matrix protein 1) was proved to be the downstream direct target of Mettl3 for m6A modification to regulate the differentiation of chondrocytes through bioinformatics analysis and correlated experiments. The Reader protein Ythdf1 mediated Dmp1 mRNA catalyzed by Mettl3. In vivo, the formation of subcutaneous ectopic cartilage-like tissue further supported the in vitro results. In conclusion, the gene regulation of Mettl3/m6A/Ythdf1/Dmp1 axis in hypertrophic differentiation of chondrocytes for the development of endochondral osteogenesis may supply a promising treatment strategy for the repair and regeneration of bone defects.