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Hypophosphatasia (HPP) is a group of inherited disorders characterised by the impaired mineralisation of bones and/or teeth and low serum alkaline phosphatase (ALP) activity. It is caused by a mutation in the ALPL gene encoding the tissue-non-specific isoenzyme of ALP (TNSALP) resulting in a loss of function. The disease is highly heterogenous in its clinical expression ranging from stillbirth without mineralised bone to the mild form of late adult onset with symptoms and signs such as musculoskeletal pain, arthropathy, lower-extremity fractures, premature loss of teeth or an incidental finding of reduced serum ALP activity. A classification based on the age at diagnosis and the presence or absence of bone symptoms was historically used: perinatal, prenatal benign, infantile, childhood, adult and odontohypophosphatasia. These subtypes are known to have overlapping signs and complications. Three forms of HPP distinguishable by their genetic characteristics have been described: severe, moderate and mild. Severe forms of HPP (perinatal and infantile severe) are recessively inherited, whereas moderate HPP may be dominantly or recessively inherited. The biochemical hallmark of HPP is persistently low serum ALP for age and increase in natural substrates of TNSALP, pyridoxal 5'-phosphate and phosphoethanolamine supported by radiological findings. The diagnosis is confirmed by ALPL sequencing. A multidisciplinary team of experts is essential for the effective management. Calcium restriction is recommended in infants/children to manage hypercalcaemia. A targeted enzyme replacement therapy for HPP has become available and correct diagnosis is crucial to allow early treatment., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. No commercial re-use. See rights and permissions. Published by BMJ.)

Objectives: The purpose of this study was to determine if shear bond resistance of orthodontic brackets bonded to enamel is associated with genes implicated in the enamel mineralization process., Methods: Ninety-two permanent, caries-free premolars extracted for orthodontic purposes and their associated saliva samples were obtained. Eighteen single nucleotide polymorphisms (SNPs) were studied for association with shear bond resistance. The genes of interest in this study were those previously associated with dental caries by our group. All tooth samples were bonded on the buccal surface with metallic lower lateral brackets, and then subjected to physical debonding. The force required to debond the bracket was recorded in Newtons (N) and converted to a shear bond resistance value in Megapascals (N/mm 2 ). The data were analyzed for statistical significance as compared with the mean shear bond resistance value via PLINK whole genome analysis software., Results: Associations were found between the SNPs for tuftelin (rs7526319, P = 0.004) and amelogenin (rs17878486, P = 0.04) and a higher shear bond resistance., Conclusion: The collected data support the proposed hypothesis that genes involved in the mineralization process affect the bonding of orthodontic brackets, and such an association is of value for the field of orthodontics, particularly in evaluating the efficacy of enamel-resin bond strength for patients receiving treatment., (Copyright © 2020 World Federation of Orthodontists. Published by Elsevier Inc. All rights reserved.)

Alagille syndrome (AGS) is a multisystem disorder classically involving liver and heart failure, characteristic vertebral and facial features and ocular abnormalities. AGS is caused by heterozygous mutations in JAG1 or NOTCH2, with variable phenotype penetrance. We report two cases of AGS in children with tooth defects characterised by green discolouration and hypomineralisation. The role of hyperbilirubinaemia (HB) in this atypical colour, a classical feature of AGS, has been well described. However, it does not totally explain the dental phenotype. As JAG1 and NOTCH2 mutations can affect bone development and considering common physiological pathways between bone and tooth mineralisation, both mutations could participate in this unusual dental phenotype. The role of HB and genetics in the development of the dental phenotype of AGS is discussed in two prototypical cases. Future research should focus on the underlying genetic component of tooth abnormalities., Competing Interests: Competing interests: None declared., (© BMJ Publishing Group Limited 2020. No commercial re-use. See rights and permissions. Published by BMJ.)

Amelogenesis imperfecta (AI) is a heterogeneous group of genetic diseases characterised by dental enamel malformation. Pathogenic variants in at least 33 genes cause syndromic or non-syndromic AI. Recently variants in RELT, encoding an orphan receptor in the tumour necrosis factor (TNF) superfamily, were found to cause recessive AI, as part of a syndrome encompassing small stature and severe childhood infections. Here we describe four additional families with autosomal recessive hypomineralised AI due to previously unreported homozygous mutations in RELT. Three families carried a homozygous missense variant in the fourth exon (c.164C>T, p.(T55I)) and a fourth family carried a homozygous missense variant in the 11th exon (c.1264C>T, p.(R422W)). We found no evidence of additional syndromic symptoms in affected individuals. Analyses of tooth microstructure with computerised tomography and scanning electron microscopy suggest a role for RELT in ameloblasts' coordination and interaction with the enamel matrix. Microsatellite genotyping in families segregating the T55I variant reveals a shared founder haplotype. These findings extend the RELT pathogenic variant spectrum, reveal a founder mutation in the UK Pakistani population and provide detailed analysis of human teeth affected by this hypomineralised phenotype, but do not support a possible syndromic presentation in all those with RELT-variant associated AI., (© 2020 The Authors. Clinical Genetics published by John Wiley & Sons Ltd.)

Hypophosphatasia (HPP) is a rare and intractable metabolic bone disease caused by mutations in the ALPL gene. Here, we undertook a nationwide survey of HPP in Japan, specifically regarding the prominent genetic and dental manifestations of odonto (n = 16 cases) and other (termed "non-odonto") (n = 36 cases) types. Mean serum alkaline phosphatase (ALP) values in odonto-type patients were significantly greater than those of non-odonto-type patients (P<0.05). Autosomal dominant and autosomal recessive inheritance patterns were detected, respectively, in 89% of odonto-type and 96% of non-odonto-type patients. The ALPL "c.1559delT" mutation, associated with extremely low ALP activity, was found in approximately 70% of cases. Regarding dental manifestations, all patients classified as odonto-type showed early exfoliation of the primary teeth significantly more frequently than patients classified as non-odonto-type (100% vs. 56%; P<0.05). Tooth hypomineralisation was detected in 42% of non-odonto-type patients, but not in any odonto-type patients (0%; P<0.05). Collectively, these results suggest that genetic and dental manifestations of patients with odonto-type and non-odonto-type HPP are significantly different, and these differences should be considered during clinical treatment of patients with HPP., Competing Interests: The authors have declared that no competing interests exist.

X-linked hypophosphatemia (XLH) is a skeletal disease caused by inactivating mutations in the PHEX gene. Mutated or absent PHEX protein/enzyme leads to a decreased serum phosphate level, which cause mineralization defects in the skeleton and teeth (osteomalacia/odontomalacia). It is not yet altogether clear whether these manifestations are caused solely by insufficient circulating phosphate availability for mineralization or also by a direct, local intrinsic effect caused by impaired PHEX activity. Here, we evaluated the local role of PHEX in a 3-dimensional model of extracellular matrix (ECM) mineralization. Dense collagen hydrogels were seeded either with human dental pulp cells from patients with characterized PHEX mutations or with sex- and age-matched healthy controls and cultured up to 24 d using osteogenic medium with standard phosphate concentration. Calcium quantification, micro-computed tomography, and histology with von Kossa staining for mineral showed significantly lower mineralization in XLH cell-seeded scaffolds, using nonparametric statistical tests. While apatitic mineralization was observed along collagen fibrils by electron microscopy in both groups, Raman microspectrometry indicated that XLH cells harboring the PHEX mutation produced less mineralized scaffolds having impaired mineral quality with less carbonate substitution and lower crystallinity. In the XLH cultures, immunoblotting revealed more abundant osteopontin (OPN), dentin matrix protein 1 (DMP1), and matrix extracellular phosphoglycoprotein (MEPE) than controls, as well as the presence of fragments of these proteins not found in controls, suggesting a role for PHEX in SIBLING protein degradation. Immunohistochemistry revealed altered OPN and DMP1 associated with an increased alkaline phosphatase staining in the XLH cultures. These results are consistent with impaired PHEX activity having local ECM effects in XLH. Future treatments for XLH should target both systemic and local manifestations.

Dentin sialophosphoprotein (DSPP) is one of the major non-collagenous proteins present in dentin, cementum and alveolar bone; it is also transiently expressed by ameloblasts. In humans many mutations have been found in DSPP and are associated with two autosomal-dominant genetic diseases - dentinogenesis imperfecta II (DGI-II) and dentin dysplasia (DD). Both disorders result in the development of hypomineralized and mechanically compromised teeth. The erupted mature molars of Dspp(-/-) mice have a severe hypomineralized dentin phenotype. Since dentin and enamel formations are interdependent, we decided to investigate the process of enamel onset mineralization in young Dspp(-/-) animals. We focused our analysis on the constantly erupting mouse incisor, to capture all of the stages of odontogenesis in one tooth, and the unerupted first molars. Using high-resolution microCT, we revealed that the onset of enamel matrix deposition occurs closer to the cervical loop and both secretion and maturation of enamel are accelerated in Dspp(-/-) incisors compared to the Dspp(+/-) control. Importantly, these differences did not translate into major phenotypic differences in mature enamel in terms of the structural organization, mineral density or hardness. The only observable difference was the reduction in thickness of the outer enamel layer, while the total enamel thickness remained unchanged. We also observed a compromised dentin-enamel junction, leading to delamination between the dentin and enamel layers. The odontoblast processes were widened and lacked branching near the DEJ. Finally, for the first time we demonstrate expression of Dspp mRNA in secretory ameloblasts. In summary, our data show that DSPP is important for normal mineralization of both dentin and enamel., (Copyright © 2016 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.)

Hypophosphatasia (HPP) is a genetic disease characterized by defective calcification of hard tissues such as bone and teeth accompanying deficiency of serum alkaline phosphatase (ALP) activity. Its development results from various mutations in the ALPL gene encoding tissue-nonspecific ALP (TNSALP). HPP is known to be transmitted in an autosomal recessive or autosomal dominant manner. A point mutation (c.323C>T) in the ALPL gene leading to a proline to leucine substitution at position 108 of TNSALP was first reported in a patient diagnosed with odonto-HPP (M Herasse et al., J Med Genet 2003;40:605-609), although the effects of this mutation on the TNSALP molecule have not been elucidated. To understand the molecular basis of this dominantly transmitted HPP, we first characterized TNSALP (P108L) by expressing it in COS-1 cells transiently. In contrast to wild-type TNSALP (WT), TNSALP (P108L) showed virtually no ALP activity. When coexpressed with TNSALP (WT), TNSALP (P108L) significantly inhibited the enzyme activity of TNSALP (WT), confirming that this mutant TNSALP exerts a dominant negative effect on TNSALP (WT). Using immunofluorescence and digestion with phosphatidylinositol-specific phospholipase C, we demonstrated that TNSALP (P108L) was anchored to the cell surface via glycosylphosphatidylinositol-like TNSALP (WT) in a Tet-On CHO cell expression system. Consistent with this, TNSALP (P108L) acquired endo-β-N-acetylglucosaminidase H resistance and sialic acids, as evidenced by glycosidase treatments. Importantly, TNSALP (WT) largely formed a functional dimeric structure, while TNSALP (P108L) was found to be present as a monomer in the cell. This indicates that the molecular structure of TNSALP is affected by a missense mutation at position 108, which is in contact with the active site, such that it no longer assembles into the functional dimeric form. Collectively, these results may explain why TNSALP (P108L) loses its ALP activity, even though it is able to gain access to the cell surface., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Mutations in ALPL result in hypophosphatasia (HPP), a disease causing defective skeletal mineralization. ALPL encodes tissue nonspecific alkaline phosphatase (ALP), an enzyme that promotes mineralization by reducing inorganic pyrophosphate, a mineralization inhibitor. In addition to skeletal defects, HPP causes dental defects, and a mild clinical form of HPP, odontohypophosphatasia, features only a dental phenotype. The Alpl knockout (Alpl (-/-)) mouse phenocopies severe infantile HPP, including profound skeletal and dental defects. However, the severity of disease in Alpl (-/-) mice prevents analysis at advanced ages, including studies to target rescue of dental tissues. We aimed to generate a knock-in mouse model of odontohypophosphatasia with a primarily dental phenotype, based on a mutation (c.346G>A) identified in a human kindred with autosomal dominant odontohypophosphatasia. Biochemical, skeletal, and dental analyses were performed on the resulting Alpl(+/A116T) mice to validate this model. Alpl(+/A116T) mice featured 50% reduction in plasma ALP activity compared with wild-type controls. No differences in litter size, survival, or body weight were observed in Alpl(+/A116T) versus wild-type mice. The postcranial skeleton of Alpl(+/A116T) mice was normal by radiography, with no differences in femur length, cortical/trabecular structure or mineral density, or mechanical properties. Parietal bone trabecular compartment was mildly altered. Alpl(+/A116T) mice featured alterations in the alveolar bone, including radiolucencies and resorptive lesions, osteoid accumulation on the alveolar bone crest, and significant differences in several bone properties measured by micro-computed tomography. Nonsignificant changes in acellular cementum did not appear to affect periodontal attachment or function, although circulating ALP activity was correlated significantly with incisor cementum thickness. The Alpl(+/A116T) mouse is the first model of odontohypophosphatasia, providing insights on dentoalveolar development and function under reduced ALP, bringing attention to direct effects of HPP on alveolar bone, and offering a new model for testing potential dental-targeted therapies in future studies., (© International & American Associations for Dental Research 2015.)

Amelogenesis Imperfecta (AI) is a clinical diagnosis that encompasses a group of genetic mutations, each affecting processes involved in tooth enamel formation and thus, result in various enamel defects. The hypomaturation enamel phenotype has been described for mutations involved in the later stage of enamel formation, including Klk4, Mmp20, C4orf26, and Wdr72. Using a candidate gene approach we discovered a novel Wdr72 human mutation in association with AI to be a 5-base pair deletion (c.806&#95;810delGGCAG; p.G255VfsX294). To gain insight into the function of WDR72, we used computer modeling of the full-length human WDR72 protein structure and found that the predicted N-terminal sequence forms two beta-propeller folds with an alpha-solenoid tail at the C-terminus. This domain iteration is characteristic of vesicle coat proteins, such as beta'-COP, suggesting a role for WDR72 in the formation of membrane deformation complexes to regulate intracellular trafficking. Our Wdr72 knockout mouse model (Wdr72(-/-)), containing a LacZ reporter knock-in, exhibited hypomineralized enamel similar to the AI phenotype observed in humans with Wdr72 mutations. MicroCT scans of Wdr72(-/-) mandibles affirmed the hypomineralized enamel phenotype occurring at the onset of the maturation stage. H&E staining revealed a shortened height phenotype in the Wdr72(-/-) ameloblasts with retained proteins in the enamel matrix during maturation stage. H(+)/Cl(-) exchange transporter 5 (CLC5), an early endosome acidifier, was co-localized with WDR72 in maturation-stage ameloblasts and decreased in Wdr72(-/-) maturation-stage ameloblasts. There were no obvious differences in RAB4A and LAMP1 immunostaining of Wdr72(-/-) mice as compared to wildtype controls. Moreover, Wdr72(-/-) ameloblasts had reduced amelogenin immunoreactivity, suggesting defects in amelogenin fragment resorption from the matrix. These data demonstrate that WDR72 has a major role in enamel mineralization, most notably during the maturation stage, and suggest a function involving endocytic vesicle trafficking, possibly in the removal of amelogenin proteins., (Copyright © 2014. Published by Elsevier B.V.)

Background: Congenital forms of hearing impairment can be caused by mutations in the estrogen related receptor beta (ESRRB) gene. Our initial linkage studies suggested the ESRRB locus is linked to high caries experience in humans., Methods: We tested for association between the ESRRB locus and dental caries in 1,731 subjects, if ESRRB was expressed in whole saliva, if ESRRB was associated with the microhardness of the dental enamel, and if ESRRB was expressed during enamel development of mice., Results: Two families with recessive ESRRB mutations and DFNB35 hearing impairment showed more extensive dental destruction by caries. Expression levels of ESRRB in whole saliva samples showed differences depending on sex and dental caries experience., Conclusions: The common etiology of dental caries and hearing impairment provides a venue to assist in the identification of individuals at risk to either condition and provides options for the development of new caries prevention strategies, if the associated ESRRB genetic variants are correlated with efficacy.

Hypophosphatasia (HPP) is an inherited disorder of mineral metabolism caused by mutations in ALPL, encoding tissue non-specific alkaline phosphatase (TNAP). Here, we report the molecular findings from monozygotic twins, clinically diagnosed with tooth-specific odontohypophosphatasia (odonto-HPP). Sequencing of ALPL identified two genetic alterations in the probands, including a heterozygous missense mutation c.454C>T, leading to change of arginine 152 to cysteine (p.R152C), and a novel heterozygous gene deletion c.1318&#95;1320delAAC, leading to the loss of an asparagine residue at codon 440 (p.N440del). Clinical identification of low serum TNAP activity, dental abnormalities, and pedigree data strongly suggests a genotype-phenotype correlation between p.N440del and odonto-HPP in this family. Computational analysis of the p.N440del protein structure revealed an alteration in the tertiary structure affecting the collagen-binding site (loop 422-452), which could potentially impair the mineralization process. Nevertheless, the probands (compound heterozygous: p.[N440del];[R152C]) feature early-onset and severe odonto-HPP phenotype, whereas the father (p.[N440del];[=]) has only moderate symptoms, suggesting p.R152C may contribute or predispose to a more severe dental phenotype in combination with the deletion. These results assist in defining the genotype-phenotype associations for odonto-HPP, and further identify the collagen-binding site as a region of potential structural importance for TNAP function in the biomineralization., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

Objective: The aims of the study were to examine tooth and enamel disturbances in individuals with 22q11.2 deletion syndrome and to analyze associations with medical conditions, birth characteristics and blood values of calcium and PTH., Materials and Methods: Fifty individuals participated in the study (27 females, median age 10 years, range 1.5-44). Congenital absence of teeth was studied on orthopantomograms; 1148 teeth were examined, both clinically and radiologically, and enamel hypomineralizations and hypoplasias were recorded. Medical history and findings were recorded as part of a larger study on the manifestations of 22q11.2-deletion syndrome in Norway., Results: Tooth agenesis was observed in 15% of study participants. Sixty-six percent of the participants and 26.0% of teeth presented with enamel disturbances. Of these, 12 individuals (24.0%) and 215 teeth (18.7%) had hypomineralizations and four individuals (8.0%) and 86 teeth (7.5%) had hypoplasias. Seventeen participants (34.0%) presented with both types of disturbance, but rarely in the same tooth. Only two teeth (0.17%) had both types of disturbance. Hypomineralizations were twice as frequent in permanent as in primary teeth. No correlations were found to medical conditions, except that participants with congenital cardiac anomalies presented with fewer total enamel disturbances and hypomineralizations in permanent teeth than those without., Conclusions: Enamel disturbances were frequently seen. There were more hypomineralizations than hypoplasias. Hypoparathyroidism and/or hypocalcemia are not clear etiological factors for enamel disturbances and there were no major correlations between medical conditions and enamel disturbances.

Defects in FAM83H on human chromosome 8q24.3 cause autosomal-dominant hypocalcified amelogenesis imperfecta (ADHCAI). FAM83H does not encode a recognizable signal peptide, so we predicted that the Fam83h protein functions within the cell. We tested this hypothesis by constitutively expressing mouse Fam83h with green fluorescent protein (GFP) fused to its C-terminus in HEK293 and HeLa cell lines. Green fluorescent signal from the Fam83h-GFP fusion protein was associated with perinuclear vesicles, usually in the vicinity of the Golgi apparatus. No signal was observed within the nucleus. In addition, we identified FAM83H nonsense mutations in Hispanic (C1330C>T; p.Q444X) and Caucasian (c.1192C>T; p.Q398X) families with ADHCAI. We conclude that Fam83h localizes in the intracellular environment, is associated with vesicles, and plays an important role in dental enamel formation. FAM83H is the first gene involved in the etiology of amelogenesis imperfecta (AI) that does not encode a secreted protein.