PMC8260930_01
Clinical images (A-E) Intraoral views of the proband. The teeth of the proband were typically amber and translucent and showed significant attrition, especially the molar teeth. (F-J) Panoramic radiographs and radiovisiography images. The pulp chambers and root canals of the affected teeth were smaller than those in normal teeth or completely obliterated. Radiographs of the bones and knees revealed no significant osteopenia, bony destruction, periosteal reactions, or evidence of any acute fractures, dislocations or injuries.
fgene-12-699278-g0001.jpg
PMC8260930_01_fgene-12-699278-g0001.jpg
F1
The nanoindentation test showed the nanoindentation load-displacement curves of the enamel and dentine, which indicated that the dentine hardness values and elastic modulus of the DGI-I teeth were significantly reduced compared to those of the control (Supplementary Figure 1).
Teeth ultrastructural analyses. (A) 3D reconstruction of the teeth CT data. 3D reconstruction of the pulp chambers. (B) Typical CT sections of the teeth are presented using false colors calibrated based on the mineral density to generate mineral density maps. (C) SEM of representative exfoliated teeth. The SEM images of the control dentine showed regularly organized dentine tubes and an evenly calcified matrix, while the teeth of the patients with DGI-I presented very few dentine tubules and enlarged malformed dentine tubes. (D) Toluidine blue staining of teeth. The number and morphology of odontoblasts adjacent to the mineralized dentine layer were visibly different. d, dentine; od, odontoblast; pd, predentine.
fgene-12-699278-g0002.jpg
PMC8260930_01_fgene-12-699278-g0002.jpg
F2
Micro-CT analysis showed a bulbous shape and color change in the proband teeth (Figure 2A), whereas the 3D image of the pulp presented an irregularly obliterated pulp chamber and scattered pulp stones. Mineral density analysis showed that the DGI-I teeth had similar enamel mineral density, but lower dentine mineral density compared with the control teeth (Figure 2B). The SEM images of the control dentine showed the regularly organized dentine tubes and an evenly calcified matrix, while the DGI-I teeth presented very few dentine tubules and enlarged malformed dentine tubules (Figure 2C). However, in the teeth of the patient, the odontoblasts became flattened as a result of lost polarity, and the odontoblast layer was disorganized (Figure 2E).
Analysis of COL1A1 mutation. (A) Pedigree of the family. (B) Sequence chromatogram of the affected individual (heterozygous) and control (wild type). (C) Genomic structure of COL1A1. The rectangles represent exons.
fgene-12-699278-g0003.jpg
PMC8260930_01_fgene-12-699278-g0003.jpg
F3
Sanger sequencing showed that this mutation was not identified in any other members of the family (Figure 3B).
Effect of the mutation on COL1A1 function. (A) Conservation analysis of this abnormal variation using Polyphen-2. (B) The 3D structure of mutated COL1A1 was different from that of the wild-type (WT) predicted structure using I-TASSER. (C) Subcellular localization of COL1A1 in HEK293 cells. The mutant COL1A1 was localized in the cytoplasm similar to the WT protein. (D) The mRNA and protein expression levels of COL1A1 in HEK293 cells. Mutant COL1A1 mRNA expression was no different than that of the WT in HEK293 cells, but the mutant COL1A1 protein expression was higher than that of the WT (P < 0.05). (E,F) Analysis of the mRNA and protein expression of COL1A1 and odontogenic differentiation markers (DSPP and OCN) during the osteogenic differentiation of hDPSCs. Values are represented as means +- SD of three independent experiments (*P < 0.05 and **P < 0.01). (G) Representative images from the ARS staining of DPSCs-MUT and DPSCs-CON at the indicated time points after differentiation induction.
fgene-12-699278-g0004.jpg
PMC8260930_01_fgene-12-699278-g0004.jpg
F4
I-TASSER indicated that the COL1A1 c.1463G>C mutation changed the 3D dimensional structure of the protein, causing changes in the alpha-helix and random coil structures (Figure 4B). The Gly488 position is highly conserved in the other known COL1A1 proteins (Figure 4A), suggesting that it has an important function. As shown in Figure 4C, there were no differences in the subcellular localization of the MUT compared with the WT protein. However, western blot analysis revealed that the expression level of mutant COL1A1 protein was increased compared with the WT protein (Figure 4D). The expression levels of COL1A1, DSPP, and OCN in hDPSCs with the COL1A1 mutation were significantly higher than those in control hDPSCs (Figure 4E). Moreover, western blotting showed that the expression level of COL1A1, DSPP, and OCN in DGI-I hDPSCs were significantly upregulated compared with those in the control hDPSCs after odontoblastic differentiation (Figure 4F). The results demonstrated that the DGI-I hDPSCs had a higher odontogenic differentiation ability, and the ARS staining results also confirmed this (Figure 4G).