Dental characteristics of patients with four different types of skeletal dysplasias.

OBJECTIVE: Skeletal dysplasia (SD) comprises more than 450 separate disorders. We hypothesized that their dental features would be distinctive and investigated the tooth characteristics of four patients with different SDs. MATERIAL AND METHODS: Four SD patients with molecularly confirmed diagnoses, Pt-1 acromicric dysplasia, Pt-2 hypophosphatasia and hypochondroplasia, Pt-3 cleidocranial dysplasia, and Pt-4 achondroplasia, were recruited. A tooth from each patient was evaluated for mineral density (micro-computerized tomography), surface roughness (surface profilometer), microhardness, mineral contents (energy-dispersive X-ray), and ultrastructure (scanning electron microscopy and histology), and compared with three tooth-type matched controls. RESULTS: Pt-1 and Pt-3 had several unerupted teeth. Pt-2 had an intact-root-exfoliated tooth at 2 years old. The lingual surfaces of the patients' teeth were significantly smoother, while their buccal surfaces were rougher, than controls, except for Pt-1's buccal surface. The patients' teeth exhibited deep grooves around the enamel prisms and rough intertubular dentin. Pt-3 demonstrated a flat dentinoenamel junction and Pt-2 had an enlarged pulp, barely detectable cementum layer, and ill-defined cemento-dentinal junction. Reduced microhardnesses in enamel, dentin, and both layers were observed in Pt-3, Pt-4, and Pt-1, respectively. Pt-1 showed reduced Ca/P ratio in dentin, while both enamel and dentin of Pt-2 and Pt-3 showed reduced Ca/P ratio. CONCLUSION: Each SD has distinctive dental characteristics with changes in surface roughness, ultrastructure, and mineral composition of dental hard tissues. CLINICAL RELEVANCE: In this era of precision dentistry, identifying the specific potential dental problems for each patient with SD would help personalize dental management guidelines.

Functional consequences of C-terminal mutations in RUNX2.

Cleidocranial dysplasia (CCD) is a genetic disorder caused by mutations in the RUNX2 gene, affecting bone and teeth development. Previous studies focused on mutations in the RUNX2 RHD domain, with limited investigation of mutations in the C-terminal domain. This study aimed to investigate the functional consequences of C-terminal mutations in RUNX2. Eight mutations were analyzed, and their effects on transactivation activity, protein expression, subcellular localization, and osteogenic potential were studied. Truncating mutations in the PST region and a missense mutation in the NMTS region resulted in increased transactivation activity, while missense mutations in the PST showed activity comparable to the control. Truncating mutations produced truncated proteins, while missense mutations produced normal-sized proteins. Mutant proteins were mislocalized, with six mutant proteins detected in both the nucleus and cytoplasm. CCD patient bone cells exhibited mislocalization of RUNX2, similar to the generated mutant. Mislocalization of RUNX2 and reduced expression of downstream genes were observed in MSCs from a CCD patient with the p.Ser247Valfs*3 mutation, leading to compromised osteogenic potential. This study provides insight into the functional consequences of C-terminal mutations in RUNX2, including reduced expression, mislocalization, and aberrant transactivation of downstream genes, contributing to the compromised osteogenic potential observed in CCD.

Cleidocranial dysplasia and novel RUNX2 variants: dental, craniofacial, and osseous manifestations.

OBJECTIVES: Cleidocranial dysplasia (CCD) is a skeletal disorder affecting cranial sutures, teeth, and clavicles, and is associated with the RUNX2 mutations. Although numerous patients have been described, a direct genotype-phenotype correlation for RUNX2 has been difficult to establish. Further cases must be studied to understand the clinical and genetic spectra of CCD. To characterize detailed phenotypes and identify variants causing CCD in five unrelated patients and their family members. METHODOLOGY: Clinical and radiographic examinations were performed. Genetic variants were identified by exome and Sanger sequencing, data were analyzed by bioinformatics tools. RESULTS: Three cases were sporadic and two were familial. Exome sequencing successfully detected the heterozygous pathogenic RUNX2 variants in all affected individuals. Three were novel, comprising a frameshift c.739delA (p.(Ser247Valfs*)) in exon 6 (Patient-1), a nonsense c.901C>T (p.(Gln301*)) in exon 7 (Patient-2 and affected mother), and a nonsense c.1081C>T (p.(Gln361*)) in exon 8 (Patient-3). Two previously reported variants were missense: the c.673C>T (p.(Arg225Trp)) (Patient-4) and c.674G>A (p.(Arg225Gln)) (Patient-5) in exon 5 within the Runt homology domain. Patient-1, Patient-2, and Patient-4 with permanent dentition had thirty, nineteen, and twenty unerupted teeth, respectively; whereas Patient-3 and Patient-5, with deciduous dentition, had normally developed teeth. All patients exhibited typical CCD features, but the following uncommon/unreported phenotypes were observed: left fourth ray brachymetatarsia (Patient-1), normal clavicles (Patient-2 and affected mother), phalangeal malformations (Patient-3), and normal primary dentition (Patient-3, Patient-5). CONCLUSIONS: The study shows that exome sequencing is effective to detect mutation across ethnics. The two p.Arg225 variants confirm that the Runt homology domain is vital for RUNX2 function. Here, we report a new CCD feature, unilateral brachymetatarsia, and three novel truncating variants, expanding the phenotypic and genotypic spectra of RUNX2 , as well as show that the CCD patients can have normal deciduous teeth, but must be monitored for permanent teeth anomalies.

The Prostacyclin Analog Iloprost Promotes Cementum Formation and Collagen Reattachment of Replanted Molars and Up-regulates Mineralization by Human Periodontal Ligament Cells.

INTRODUCTION: This study evaluated the use of the prostacyclin analog iloprost as a root surface treatment agent in promoting acellular cementum (AC) formation and collagen reattachment after tooth replantation in vivo. In addition, its effect on human periodontal ligament cell (hPDLC) mineralization was assessed in vitro. METHODS: First molars of 8-week-old Wistar rats were extracted. In 1 group, the root surfaces were treated with Hank's balanced salt solution (HBSS), and the other group's root surfaces were treated with 10-6 mol/L iloprost before replantation. At day 30, maxillae were prepared for micro-computed tomographic imaging and histomorphometric analysis. The effect of iloprost on mineralization by hPDLCs was analyzed by mineralized nodule formation and quantitative polymerase chain reaction at 7 and 14 days. RESULTS: Micro-computed tomographic imaging demonstrated a significant higher bone volume in the iloprost groups, whereas the HBSS groups had extensive bone and root resorption. Histologic analysis revealed deposition of a thick AC layer along the root in the iloprost group with well-organized periodontal ligament fibers inserted into the cementum. The HBSS group demonstrated more osteoclasts than the iloprost group. In vitro, iloprost-treated hPDLCs had a significantly increased RUNX2, OSX, BSP, and ALP gene expression that coincided with an increased deposition of mineralized nodules. These effects were abrogated by a PGI2 receptor inhibitor. CONCLUSIONS: Our results revealed that iloprost promoted PDL regeneration in replanted molars. Furthermore, resorption of the roots was decreased, whereas AC deposition was stimulated. Iloprost-treatment increased hPDLC mineralization and was mediated by PGI2 receptor activation. These observations indicate that iloprost may be a promising root surface treatment agent.

Age-related dental phenotypes and tooth characteristics of FAM83H-associated hypocalcified amelogenesis imperfecta.

OBJECTIVES: Autosomal-dominant hypocalcified amelogenesis imperfecta (ADHCAI) shows phenotypic heterogeneity. Our aim was to characterise the ADHCAI phenotypes, tooth properties and genotypes. METHODS: Three unrelated ADHCAI probands and seven additional affected members of the three families were recruited. Mutations were identified by exome and Sanger sequencing, and haplotypes by SNP array. Tooth colour, roughness, density, nanohardness, minerals and ultrastructure were investigated. RESULTS: Ten participants were heterozygous for the FAM83H mutation c.1387C>T (p.Gln463*). All shared a 3.43 Mbp region on chromosome 8q24.3 encompassing the FAM83H variant, indicating a common ancestry. The c.1387C>T was estimated to be 23.8 generations or 600 years. The FAM83H enamel had higher roughness and lower lightness, density, nanohardness, and calcium and phosphorus levels than controls. Blunted enamel rods, wide interrod spaces and disorganised dentinoenamel junctions were observed. Evaluating the patients with the same mutation and reviewing others with different mutations in FAM83H revealed that the FAM83H heterogeneous phenotypes are age-influenced. Tooth colour and surface texture change with ageing. CONCLUSIONS: FAM83H enamel demonstrated decreased lightness, density, hardness, calcium, phosphorus and defective ultrastructure. We have identified that the phenotypic variation in FAM83H-associated ADHCAI is age-related. Awareness of the correlation between age and clinical features of FAM83H-ADHCAI can help dentists make an accurate diagnosis.

Cleidocranial dysplasia and novel RUNX2 variants: dental, craniofacial, and osseous manifestations

Abstract Cleidocranial dysplasia (CCD) is a skeletal disorder affecting cranial sutures, teeth, and clavicles, and is associated with the RUNX2 mutations. Although numerous patients have been described, a direct genotype-phenotype correlation for RUNX2 has been difficult to establish. Further cases must be studied to understand the clinical and genetic spectra of CCD. Objectives To characterize detailed phenotypes and identify variants causing CCD in five unrelated patients and their family members. Methodology Clinical and radiographic examinations were performed. Genetic variants were identified by exome and Sanger sequencing, data were analyzed by bioinformatics tools. Results Three cases were sporadic and two were familial. Exome sequencing successfully detected the heterozygous pathogenic RUNX2 variants in all affected individuals. Three were novel, comprising a frameshift c.739delA (p.(Ser247Valfs*)) in exon 6 (Patient-1), a nonsense c.901C>T (p.(Gln301*)) in exon 7 (Patient-2 and affected mother), and a nonsense c.1081C>T (p.(Gln361*)) in exon 8 (Patient-3). Two previously reported variants were missense: the c.673C>T (p.(Arg225Trp)) (Patient-4) and c.674G>A (p.(Arg225Gln)) (Patient-5) in exon 5 within the Runt homology domain. Patient-1, Patient-2, and Patient-4 with permanent dentition had thirty, nineteen, and twenty unerupted teeth, respectively; whereas Patient-3 and Patient-5, with deciduous dentition, had normally developed teeth. All patients exhibited typical CCD features, but the following uncommon/unreported phenotypes were observed: left fourth ray brachymetatarsia (Patient-1), normal clavicles (Patient-2 and affected mother), phalangeal malformations (Patient-3), and normal primary dentition (Patient-3, Patient-5). Conclusions The study shows that exome sequencing is effective to detect mutation across ethnics. The two p.Arg225 variants confirm that the Runt homology domain is vital for RUNX2 function. Here, we report a new CCD feature, unilateral brachymetatarsia, and three novel truncating variants, expanding the phenotypic and genotypic spectra of RUNX2 , as well as show that the CCD patients can have normal deciduous teeth, but must be monitored for permanent teeth anomalies.

Phenotypic features of dentinogenesis imperfecta associated with osteogenesis imperfecta and COL1A2 mutations.

OBJECTIVE: Dentinogenesis imperfecta (DI) requires dental treatment. This study investigated the characteristics of DI teeth associated with osteogenesis imperfecta (OI) and COL1A2 mutations. STUDY DESIGN: Whole exome and Sanger sequencing were performed. Three primary teeth (called "OIDI teeth") obtained from 3 unrelated COL1A2 patients were investigated and compared with 9 control teeth from age-matched healthy individuals using colorimetry, micro-computed tomography, Knoop microhardness, energy dispersive X-ray spectroscopy, scanning electron microscopy, and histology. RESULTS: All patients were identified with heterozygous glycine substitutions in COL1A2. The COL1A2 mutations, c.1531G>T and c.2027G>T, were de novo, whereas c.3106G>C was inherited. OIDI1, 2, and 3 teeth had a substantial decrease in dentin microhardness and lightness. OIDI2 enamel microhardness was significantly reduced, whereas OIDI1 and 3 had enamel microhardness comparable to that of control individuals. The OIDI1 pulp cavity was large; OIDI2 was narrow; and OIDI3 was obliterated. OIDI1 and 3 had significantly higher carbon levels than those in control individuals. Numerous ectopic calcified masses, sparse and obstructed dentinal tubules, dentin holes, and collagen disorientation were observed. CONCLUSIONS: OIDI teeth had reduced lightness and variable pulp morphology. Weak dentin, mineral disproportion, and abnormal ultrastructure could contribute to the brittleness of OIDI teeth and adhesive restoration failure. Here, we expand the phenotypic spectrum of COL1A2 mutations and raise awareness among dentists seeing patients with OI.

Phenotypic and Genotypic Features of Thai Patients With Nonsyndromic Tooth Agenesis and WNT10A Variants.

Tooth agenesis is one of the most common orodental anomalies that demonstrate phenotypic and genotypic heterogeneity with a prevalence of 2.5%-7%. Mutations in WNT10A have been proposed to be the most common cause of nonsyndromic tooth agenesis (NSTA). The aim of this study was to characterize the dental features and genetic variants of NSTA in a Thai population. We recruited 13 unrelated patients with NSTA who attended the Faculty of Dentistry, Chulalongkorn University, Thailand, from 2017 to 2019. All 13 underwent whole exome sequencing that identified likely pathogenic genetic variants, all in WNT10A, in five patients. All five patients had second premolar agenesis, while three also had absent or peg-shaped upper lateral incisors. Patient 1 possessed a novel heterozygous duplication, c.916_918dupAAC (p.Asn306dup) in WNT10A. Patients 2 and 3 harbored a heterozygous and homozygous c.637G > A (p.Gly213Ser) in WNT10A, respectively. Patients 4 possessed a heterozygous c.511C > T (p.Arg171Cys) in WNT10A. Patient 5 harbored a homozygous c.511C > T (p.Arg171Cys) in WNT10A and a novel heterozygous c.413A > T (p.Asn138Ile) in EDARADD, suggesting digenic inheritance. We recruited another 18 family members of these five patients. Out of 23 participants, homozygous WNT10A variants were identified in 2 patients and heterozygous variants in 17 individuals. Both homozygous patients had NSTA. Eight out of 17 heterozygous individuals (8/17) had NSTA or a peg-shaped lateral incisor, indicating a 47% penetrance of the heterozygous variants or 53% (10/19) penetrance of either homozygous or heterozygous variants in WNT10A. The frequencies of the c.511C > T in our in-house 1,876 Thai exome database, Asian populations, and non-Asian populations were 0.016, 0.005-0.033, and 0.001, respectively; while those of the c.637G > A were 0.016, 0.004-0.029, and 0.000, respectively. In conclusion, our study reports two novel variants with one each in WNT10A and EDARADD, expanding the genotypic spectra of NSTA. Second premolar agenesis is a common phenotype in affected individuals with variants in WNT10A; however, its penetrance is incomplete. Lastly, the different frequencies of WNT10A variants, c.511C > T and c.637G > A, in diverse populations might contribute to the prevalence range of NSTA between continents.

Human serum enhances the proliferative capacity and immunomodulatory property of MSCs derived from human placenta and umbilical cord.

BACKGROUND: Mesenchymal stromal cells (MSCs) are considered potential candidates that hold great promise in the treatment of immune-related diseases. For therapeutic applications, it is necessary to isolate and expand MSCs with procedures complying with good manufacturing practice (GMP). Recent studies reported the use of human serum (HS) instead of fetal bovine serum (FBS) for the expansion of bone marrow-derived MSCs. Nevertheless, there are only limited data on HS as an alternative to FBS for the isolation and expansion of umbilical (UC-MSCs) and placenta-derived MSCs (PL-MSCs). In this study, we evaluate the effect of HS compared to FBS on the proliferative and immunosuppressive capacities of these MSCs. METHODS: PL-MSCs and UC-MSCs were isolated and cultured in HS- or FBS-supplemented media. The MSC characteristics, including morphology, immunophenotype, and differentiation ability, were verified. The proliferative and immunosuppressive capacities were also examined. In addition, the proliferative-enhancing factors in both sera were explored using proteomic analysis. RESULTS: PL-MSCs and UC-MSCs proliferated faster in HS-supplemented medium than in equivalent levels of FBS-supplemented medium. Adipogenic and osteogenic differentiations occurred at nearly identical levels in HS- and FBS-supplemented media. Interestingly, MSCs cultured in HS-supplemented medium had a similar immunosuppressive effect as MSCs cultured in FBS-supplemented medium. Proteomic analysis revealed that Con-A binding glycoproteins with a molecular weight > 100 kDa in FBS could significantly enhance MSC proliferation. In contrast, the proliferative enhancing factors in HS were found in the Con-A non-binding fraction and WGA binding fraction with a molecular weight > 100 kDa. CONCLUSIONS: Taken together, our results suggest applications for the use of HS instead of FBS for the isolation and expansion of PL-MSCs and UC-MSCs for cell therapy in the future. Furthermore, this study identifies factors in HS that are responsible for its proliferative and immunosuppressive effects and might thus lead to the establishment of GMPs for the therapeutic use of MSCs.

Genotype-phenotype correlation and expansion of orodental anomalies in LTBP3-related disorders.

The latent transforming growth factor-beta-binding protein 3 (LTBP3), encoding extracellular matrix proteins, plays a role in skeletal formation. Mutations in LTBP3 have been associated with various types of skeletal dysplasia. We aimed to characterize clinical and molecular features of more patients with mutations in the gene, which may help suggest genotype-phenotype correlation. The first two East Asian patients with short stature, heart defects, and orodental anomalies having LTBP3 mutations were identified. Whole exome and Sanger sequencing revealed that the one with a novel heterozygous missense (c.2017G>T, p.Gly673Cys) mutation in LTBP3 had clinical features consistent with acromicric dysplasia (ACMICD). The variant was located in the highly conserved EGF-like calcium-binding domain adjacent to the single reported LTBP3 variant associated with ACMICD. This finding supports that LTBP3 is a disease gene for ACMICD. Another patient with a novel homozygous splice site acceptor (c.1721-2A>G) mutation in LTBP3 was affected with dental anomalies and short stature (DASS). Previously undescribed orodental features included multiple unerupted teeth, high-arched palate, and microstomia found in our patient with ACMICD, and extensive dental infection, condensing osteitis, and deviated alveolar bone formation in our patient with DASS. Our results and comprehensive reviews suggest a genotype-phenotype correlation: biallelic loss-of-function mutations cause DASS, monoallelic missense gain-of-function mutations in the EGF-like domain cause ACMICD, and monoallelic missense gain-of-function mutations with more drastic effects on the protein functions cause geleophysic dysplasia (GPHYSD3). In summary, we expand the phenotypic and genotypic spectra of LTBP3-related disorders, support that LTBP3 is a disease gene for ACMICD, and propose the genotype-phenotype correlation of LTBP3 mutations.

The Biological Characteristics of Placenta Derived Mesenchymal Stem Cells Cultured in Human Serum.

Objective: Evidence exists indicating that mesenchymal stem cells (MSCs) are promising candidate for therapeutic applications. One major obstacle for their clinical use is the biosafety of fetal bovine serum (FBS), which is a crucial part of all media currently used for culture of MSCs. Although some recent studies recommended substituting FBS with human serum (HS) for the expansion of MSCs for clinical use, the characteristics and functional capacity of the expanded cells has only been partially explored. In addition, limited experience indicates that HS may replace FBS in some but not all culture systems. Currently, relatively little is known about using HS instead of FBS for isolation and expansion of placenta derived MSCs. Therefore, this study aimed to compare the exploit of HS and FBS as a supplement in terms of their impact on biological characteristics of MSCs. Material and Method: MSCs derived from placenta were cultured in Dulbecco's Modified Eagle's Medium supplemented with 10% fetal bovine serum or 10% human serum. The morphology, the expression of MSC markers, the differentiation ability and the proliferation characteristics were examined. Results: The results demonstrated that MSCs cultured in DMEM supplemented with 10% HS had similar characteristics to MSCs cultured in DMEM supplemented with 10% FBS. Interestingly, MSCs cultured in DMEM supplemented with 10% HS had greater expansion potential than that of MSCs cultured in DMEM supplemented with 10% FBS. Conclusion: The results obtained from this study imply some application in the use of HS instead of FBS for expansion of placenta derived MSCs. The HS-expanded MSCs might be useful and safe for use as a therapeutic tool in regenerative medicine.