AMELX Mutations and Genotype-Phenotype Correlation in X-Linked Amelogenesis Imperfecta.

AMELX mutations cause X-linked amelogenesis imperfecta (AI), known as AI types IE, IIB, and IIC in Witkop's classification, characterized by hypoplastic (reduced thickness) and/or hypomaturation (reduced hardness) enamel defects. In this study, we conducted whole exome analyses to unravel the disease-causing mutations for six AI families. Splicing assays, immunoblotting, and quantitative RT-PCR were conducted to investigate the molecular and cellular effects of the mutations. Four AMELX pathogenic variants (NM_182680.1:c.2T>C; c.29T>C; c.77del; c.145-1G>A) and a whole gene deletion (NG_012494.2:g.307534_403773del) were identified. The affected individuals exhibited enamel malformations, ranging from thin, poorly mineralized enamel with a "snow-capped" appearance to severe hypoplastic defects with minimal enamel. The c.145-1G>A mutation caused a -1 frameshift (NP_001133.1:p.Val35Cysfs*5). Overexpression of c.2T>C and c.29T>C AMELX demonstrated that mutant amelogenin proteins failed to be secreted, causing elevated endoplasmic reticulum stress and potential cell apoptosis. This study reveals a genotype-phenotype relationship for AMELX-associated AI: While amorphic mutations, including large deletions and 5' truncations, of AMELX cause hypoplastic-hypomaturation enamel with snow-capped teeth (AI types IIB and IIC) due to a complete loss of gene function, neomorphic variants, including signal peptide defects and 3' truncations, lead to severe hypoplastic/aplastic enamel (AI type IE) probably caused by "toxic" cellular effects of the mutant proteins.

Phenotypic variability in LAMA3-associated amelogenesis imperfecta.

OBJECTIVE: Amelogenesis imperfecta (AI) is defined as inherited enamel malformations. LAMA3 (laminin alpha-3) encodes a critical protein component of the basement membrane (laminin-332). Individuals carrying heterozygous LAMA3 mutations have previously been shown to have localized enamel defects. This study aimed to define clinical phenotypes and to discern the genetic etiology for four AI kindreds. MATERIALS AND METHODS: Whole-exome analyses were conducted to search for sequence variants associated with the disorder, and micro-computed tomography (µCT) to characterize the enamel defects. RESULTS: The predominant enamel phenotype was generalized thin enamel with defective pits and grooves. Horizonal bands of hypoplastic enamel with chalky-white discoloration and enamel hypomineralization were also observed and demonstrated by µCT analyses of affected teeth. Four disease-causing LAMA3 mutations (NM_198129.4:c.3712dup; c.5891dup; c.7367del; c.9400G > C) were identified. Compound heterozygous MMP20 mutations (NM_004771.4:c.539A > G; c.692C > T) were also found in one proband with more severe enamel defects, suggesting a mutational synergism on disease phenotypes. Further analyses of the AI-causing mutations suggested that both α3A (short) and α3B (long) isoforms of LAMA3 are essential for enamel formation. CONCLUSIONS: Heterozygous LAMA3 mutations can cause generalized enamel defects (AI1A) with variable expressivity. Laminin-332 is critical not only for appositional growth but also enamel maturation.

FAM20A mutations and transcriptome analyses of dental pulp tissues of enamel renal syndrome.

AIM: Biallelic loss-of-function FAM20A mutations cause amelogenesis imperfecta (AI) type IG, better known as enamel renal syndrome (ERS), characterized by severe enamel hypoplasia, delayed/failed tooth eruption, intrapulpal calcifications, gingival hyperplasia and nephrocalcinosis. FAM20A binds to FAM20C, the Golgi casein kinase (GCK) and potentiates its function to phosphorylate secreted proteins critical for biomineralization. While many FAM20A pathogenic mutations have been reported, the pathogeneses of orodental anomalies in ERS remain to be elucidated. This study aimed to identify disease-causing mutations for patients with ERS phenotypes and to discern the molecular mechanism underlying ERS intrapulpal calcifications. METHODOLOGY: Phenotypic characterization and whole exome analyses were conducted for 8 families and 2 sporadic cases with hypoplastic AI. A minigene assay was performed to investigate the molecular consequences of a FAM20A splice-site variant. RNA sequencing followed by transcription profiling and gene ontology (GO) analyses were carried out for dental pulp tissues of ERS and the control. RESULTS: Biallelic FAM20A mutations were demonstrated for each affected individual, including 7 novel pathogenic variants: c.590-5T>A, c.625T>A (p.Cys209Ser), c.771del (p.Gln258Argfs*28), c.832_835delinsTGTCCGACGGTGTCCGACGGTGTC CA (p.Val278Cysfs*29), c.1232G>A (p.Arg411Gln), c.1297A>G (p.Arg433Gly) and c.1351del (p.Gln451Serfs*4). The c.590-5T>A splice-site mutation caused Exon 3 skipping, which resulted in an in-frame deletion of a unique region of the FAM20A protein, p.(Asp197_Ile214delinsVal). Analyses of differentially expressed genes in ERS pulp tissues demonstrated that genes involved in biomineralization, particularly dentinogenesis, were significantly upregulated, such as DSPP, MMP9, MMP20 and WNT10A. Enrichment analyses indicated overrepresentation of gene sets associated with BMP and SMAD signalling pathways. In contrast, GO terms related to inflammation and axon development were underrepresented. Among BMP signalling genes, BMP agonists GDF7, GDF15, BMP3, BMP8A, BMP8B, BMP4 and BMP6 were upregulated, while BMP antagonists GREM1, BMPER and VWC2 showed decreased expression in ERS dental pulp tissues. CONCLUSIONS: Upregulation of BMP signalling underlies intrapulpal calcifications in ERS. FAM20A plays an essential role in pulp tissue homeostasis and prevention of ectopic mineralization in soft tissues. This critical function probably depends upon MGP (matrix Gla protein), a potent mineralization inhibitor that must be properly phosphorylated by FAM20A-FAM20C kinase complex.

A genetic model for the secretory stage of dental enamel formation.

The revolution in genetics has rapidly increased our knowledge of human and mouse genes that are critical for the formation of dental enamel and helps us understand how enamel evolved. In this graphical review we focus on the roles of 41 genes that are essential for the secretory stage of amelogenesis when characteristic enamel mineral ribbons initiate on dentin and elongate to expand the enamel layer to the future surface of the tooth. Based upon ultrastructural analyses of genetically modified mice, we propose a molecular model explaining how a cell attachment apparatus including collagen 17, α6ß4 and αvß6 integrins, laminin 332, and secreted enamel proteins could attach to individual enamel mineral ribbons and mold their cross-sectional dimensions as they simultaneously elongate and orient them in the direction of the retrograde movement of the ameloblast membrane.

The spatial distribution of focal stacks within the inner enamel layer of mandibular mouse incisors.

Focal stacks are an alternative spatial arrangement of enamel rods within the inner enamel of mandibular mouse incisors where short rows comprised of 2-45 enamel rods are nestled at the side of much longer rows, both sharing the same rod tilt directed mesially or laterally. The significance of focal stacks to enamel function is unknown, but their high frequency in transverse sections (30% of all rows) suggests that they serve some purpose beyond representing an oddity of enamel development. In this study, we characterized the spatial distribution of focal stacks in random transverse sections relative to different regions of the inner enamel and to different locations across enamel thickness. The curving dentinoenamel junction (DEJ) in transverse sections complicated spatial distribution analyses, and a technique was developed to "unbend" the curving DEJ allowing for more linear quantitative analyses to be carried out. The data indicated that on average there were 36 ± 7 focal stacks located variably within the inner enamel in any given transverse section. Consistent with area distributions, focal stacks were four times more frequent in the lateral region (53%) and twice as frequent in the mesial region (33%) compared to the central region (14%). Focal stacks were equally split by tilt (52% mesial vs. 48% lateral, not significant), but those having a mesial tilt were more frequently encountered in the lateral and central regions (2:1) and those having a lateral tilt were more numerous in the mesial region (1:3). Focal stacks having a mesial tilt were longer on average compared to those having a lateral tilt (7.5 ± 5.6 vs. 5.9 ± 4.0 rods per row, p < 0.01). There was no relationship between the length of a focal stack and its location within the inner enamel. All results were consistent with the notion that focal stacks travel from the DEJ to the outer enamel the same as the longer and decussating companion rows to which they are paired. The spatial distribution of focal stacks within the inner enamel was not spatially random but best fit a null model based on a heterogenous Poisson point process dependent on regional location within the transverse plane of the enamel layer.

Recessive Mutations in ACPT, Encoding Testicular Acid Phosphatase, Cause Hypoplastic Amelogenesis Imperfecta.

Amelogenesis imperfecta (AI) is a heterogeneous group of genetic disorders affecting tooth enamel. The affected enamel can be hypoplastic and/or hypomineralized. In this study, we identified ACPT (testicular acid phosphatase) biallelic mutations causing non-syndromic, generalized hypoplastic autosomal-recessive amelogenesis imperfecta (AI) in individuals from six apparently unrelated Turkish families. Families 1, 4, and 5 were affected by the homozygous ACPT mutation c.713C>T (p.Ser238Leu), family 2 by the homozygous ACPT mutation c.331C>T (p.Arg111Cys), family 3 by the homozygous ACPT mutation c.226C>T (p.Arg76Cys), and family 6 by the compound heterozygous ACPT mutations c.382G>C (p.Ala128Pro) and 397G>A (p.Glu133Lys). Analysis of the ACPT crystal structure suggests that these mutations damaged the activity of ACPT by altering the sizes and charges of key amino acid side chains, limiting accessibility of the catalytic core, and interfering with homodimerization. Immunohistochemical analysis confirmed localization of ACPT in secretory-stage ameloblasts. The study results provide evidence for the crucial function of ACPT during amelogenesis.

Mutations in the pH-Sensing G-protein-Coupled Receptor GPR68 Cause Amelogenesis Imperfecta.

Amelogenesis is the process of dental enamel formation, leading to the deposition of the hardest tissue in the human body. This process requires the intricate regulation of ion transport and controlled changes to the pH of the developing enamel matrix. The means by which the enamel organ regulates pH during amelogenesis is largely unknown. We identified rare homozygous variants in GPR68 in three families with amelogenesis imperfecta, a genetically and phenotypically heterogeneous group of inherited conditions associated with abnormal enamel formation. Each of these homozygous variants (a large in-frame deletion, a frameshift deletion, and a missense variant) were predicted to result in loss of function. GPR68 encodes a proton-sensing G-protein-coupled receptor with sensitivity in the pH range that occurs in the developing enamel matrix during amelogenesis. Immunohistochemistry of rat mandibles confirmed localization of GPR68 in the enamel organ at all stages of amelogenesis. Our data identify a role for GPR68 as a proton sensor that is required for proper enamel formation.

Mutations in RELT cause autosomal recessive amelogenesis imperfecta.

Amelogenesis imperfecta (AI) is a collection of isolated (non-syndromic) inherited diseases affecting dental enamel formation or a clinical phenotype in syndromic conditions. We characterized three consanguineous AI families with generalized irregular hypoplastic enamel with rapid attrition that perfectly segregated with homozygous defects in a novel gene: RELT that is a member of the tumor necrosis factor receptor superfamily (TNFRSF). RNAscope in situ hybridization of wild-type mouse molars and incisors showed specific Relt mRNA expression by secretory stage ameloblasts and by odontoblasts. Relt-/- mice generated by CRISPR/Cas9 exhibited incisor and molar enamel malformations. Relt-/- enamel had a rough surface and underwent rapid attrition. Normally unmineralized spaces in the deep enamel near the dentino-enamel junction (DEJ) were as highly mineralized as the adjacent enamel, which likely altered the mechanical properties of the DEJ. Phylogenetic analyses showed the existence of selective pressure on RELT gene outside of tooth development, indicating that the human condition may be syndromic, which possibly explains the history of small stature and severe childhood infections in two of the probands. Knowing a TNFRSF member is critical during the secretory stage of enamel formation advances our understanding of amelogenesis and improves our ability to diagnose human conditions featuring enamel malformations.

Characteristics of the transverse 2D uniserial arrangement of rows of decussating enamel rods in the inner enamel layer of mouse mandibular incisors.

The 2D arrangement of rows of enamel rods with alternating (decussating) tilt angles across the thickness of the inner layer in rat and mouse incisor enamel is well known and assumed to occur in a uniform and repetitive pattern. Some irregularities in the arrangement of rows have been reported, but no detailed investigation of row structure across the entire inner enamel layer currently exists. This investigation was undertaken to determine if the global row pattern in mouse mandibular incisor enamel is predominately regular in nature with only occasional anomalies or if rows of enamel rods have more spatial complexity than previously suspected. The data from this investigation indicate that rows of enamel rods are highly variable in length and have complex transverse arrangements across the width and thickness of the inner enamel layer. The majority of rows are short or medium in length, with 87% having < 100 rods per row. The remaining 13% are long rows (with 100-233 rods per row) that contain 46% of all enamel rods seen in transverse sections. Variable numbers of rows were associated with the lateral, central and mesial regions of the enamel layer. Each region contained different ratios of short, medium and long rows. A variety of relationships was found along the transverse length of rows in each region, including uniform associations of alternating rod tilts between neighboring rows, and instances where two rows having the same rod tilt were paired for variable distances then moved apart to accommodate rows of opposite tilt. Sometimes a row appeared to branch into two rows with the same tilt, or conversely where two rows merged into one row depending upon the mesial-to-lateral direction in which the row was viewed. Some rows showed both pairing and branching/merging along their length. These tended to be among the longest rows identified, and they often crossed the central region with extensions into the lateral and mesial regions. The most frequent row arrangement was a row of petite length nestled at the side of another row having the same rod tilt (30% of all rows). These were termed 'focal stacks' and may relate to the evolution of uniserial rat and mouse incisor enamel from a multilayered ancestor. The mesial and lateral endpoints of rows also showed complex arrangements with the dentinoenamel junction (DEJ), the inner enamel layer itself, and the boundary area to the outer enamel layer. It was concluded that the diversity in row lengths and various spatial arrangements both within and between rows across the transverse plane provides a method to interlock the enamel layer across each region and keep the enamel layer compact relative to the curving DEJ surface. The uniserial pattern for rows in mouse mandibular incisors is not uniform, but diverse and very complex.

Quantitative analysis of the core 2D arrangement and distribution of enamel rods in cross-sections of mandibular mouse incisors.

Considerable descriptive information about the overall organization of mouse mandibular incisor enamel is available but almost nothing is known about the quantitative characteristics of enamel rod arrangement and distribution in these teeth. This has important implications concerning cell movement during the secretory stage because each ameloblast makes one enamel rod. Knowing how many enamel rods are cut open in a cross-section of the enamel layer could provide insights into understanding the dynamics of how groups of ameloblasts form the enamel layer. In this study, cross-sections of fully mineralized enamel were cut on 24 mandibular mouse incisors, polished and etched, and imaged by scanning electron microscopy in backscatter mode. Montaged maps of the entire enamel layer were made at high magnification and the enamel rod profiles in each map were color-coded based upon rod category. Quantitative analyses of each color layer in the maps were then performed using standard routines available in imagej. The data indicated that that there were on average 7233 ± 575 enamel rod profiles per cross-section in mandibular incisors of 7-week-old mice, with 70% located in the inner enamel layer, 27% located in the outer enamel layer, and 3% positioned near the mesial and lateral cementoenamel junctions. All enamel rod profiles showed progressive increases in tilt angles, some very large in magnitude, from the lateral to mesial sides of the enamel layer, whereas only minor variations in tilt angle were found relative to enamel thickness at given locations across the enamel layer. The decussation angle between alternating rows of rod profiles within the inner enamel layer was fairly constant from the lateral to central labial sides of the enamel layer, but it increased dramatically in the mesial region of the enamel layer. The packing density of all rod profiles decreased from lateral to central labial regions of the enamel layer and then in progressing mesially, decreased slightly (inner enamel, mesial tilt), increased slightly (outer enamel layer) or almost doubled in magnitude (inner enamel, lateral tilt). It was concluded that these variations in rod tilt angle and packing densities are adaptations that allow the tooth to maintain a sharp incisal edge and shovel-shape as renewing segments formed by around 7200 ameloblasts are brought onto the occluding surface of the tooth by continuous renewal.

Mineral Trioxide Aggregate and Diluted Formocresol Pulpotomy: Prospective and Retrospective Study Outcomes.

Mineral Trioxide Aggregate (MTA) has been used in many endodontic procedures with success. We hypothesized that MTA as a pulpotomy medicament elicits outcomes no different than that of the diluted formocresol (DFC). PURPOSE: The purpose of this study was to compare the outcomes of grey MTA and DFC in primary molar pulpotomies at a teaching institution and a pediatric dental practice. METHODS: At the teaching institution, 206 primary molars of 122 children were enrolled. At 48-months, 20 teeth treated with MTA and 25 teeth treated with DFC, were available for evaluation. At the private practice, dental records of 245 primary molars of 68 patients were available for evaluation. RESULTS: At 48 months, the results from both sites showed a radiographic success rate of 80 percent for DFC and 95 percent for MTA. The odds of radiographic failure were not affected by study sites. The Cox-regression analysis revealed that patient's age at the time of pulpotomy impacted the "hazard of exfoliation." Each year following the completion of DFC or MTA pulpotomy, there is a 4.6-times-more-likely chance for early exfoliation of the pulpotomized tooth. CONCLUSION: Grey MTA is an acceptable alternative for primary molar pulpotomies.

ITGB6 loss-of-function mutations cause autosomal recessive amelogenesis imperfecta.

Integrins are cell-surface adhesion receptors that bind to extracellular matrices (ECM) and mediate cell-ECM interactions. Some integrins are known to play critical roles in dental enamel formation. We recruited two Hispanic families with generalized hypoplastic amelogenesis imperfecta (AI). Analysis of whole-exome sequences identified three integrin beta 6 (ITGB6) mutations responsible for their enamel malformations. The female proband of Family 1 was a compound heterozygote with an ITGB6 transition mutation in Exon 4 (g.4545G > A c.427G > A p.Ala143Thr) and an ITGB6 transversion mutation in Exon 6 (g.27415T > A c.825T > A p.His275Gln). The male proband of Family 2 was homozygous for an ITGB6 transition mutation in Exon 11 (g.73664C > T c.1846C > T p.Arg616*) and hemizygous for a transition mutation in Exon 6 of Nance-Horan Syndrome (NHS Xp22.13; g.355444T > C c.1697T > C p.Met566Thr). These are the first disease-causing ITGB6 mutations to be reported. Immunohistochemistry of mouse mandibular incisors localized ITGB6 to the distal membrane of differentiating ameloblasts and pre-ameloblasts, and then ITGB6 appeared to be internalized by secretory stage ameloblasts. ITGB6 expression was strongest in the maturation stage and its localization was associated with ameloblast modulation. Our findings demonstrate that early and late amelogenesis depend upon cell-matrix interactions. Our approach (from knockout mouse phenotype to human disease) demonstrates the power of mouse reverse genetics in mutational analysis of human genetic disorders and attests to the need for a careful dental phenotyping in large-scale knockout mouse projects.

FAM20A mutations can cause enamel-renal syndrome (ERS).

Enamel-renal syndrome (ERS) is an autosomal recessive disorder characterized by severe enamel hypoplasia, failed tooth eruption, intrapulpal calcifications, enlarged gingiva, and nephrocalcinosis. Recently, mutations in FAM20A were reported to cause amelogenesis imperfecta and gingival fibromatosis syndrome (AIGFS), which closely resembles ERS except for the renal calcifications. We characterized three families with AIGFS and identified, in each case, recessive FAM20A mutations: family 1 (c.992G>A; g.63853G>A; p.Gly331Asp), family 2 (c.720-2A>G; g.62232A>G; p.Gln241_Arg271del), and family 3 (c.406C>T; g.50213C>T; p.Arg136* and c.1432C>T; g.68284C>T; p.Arg478*). Significantly, a kidney ultrasound of the family 2 proband revealed nephrocalcinosis, revising the diagnosis from AIGFS to ERS. By characterizing teeth extracted from the family 3 proband, we demonstrated that FAM20A(-/-) molars lacked true enamel, showed extensive crown and root resorption, hypercementosis, and partial replacement of resorbed mineral with bone or coalesced mineral spheres. Supported by the observation of severe ectopic calcifications in the kidneys of Fam20a null mice, we conclude that FAM20A, which has a kinase homology domain and localizes to the Golgi, is a putative Golgi kinase that plays a significant role in the regulation of biomineralization processes, and that mutations in FAM20A cause both AIGFS and ERS.

Evolution of Klk4 and enamel maturation in eutherians.

Kallikrein-related peptidase 4 (KLK4) is a secreted serine protease that degrades residual enamel proteins to facilitate their removal by ameloblasts, which increases mineralization and hardens the enamel. Mutations in human KLK4 cause hypomaturation amelogenesis imperfecta. Enamel formed by Klk4 null mice is normal in thickness and prism structure, but the enamel layer retains proteins, is hypomineralized, and undergoes rapid attrition following tooth eruption. We searched multiple databases, retrieved Klk4 and Klk5 from various mammalian genomes, and identified Klk4 in 46 boreoeutherian genomes. In non-Boreoeutheria, Klk4 was detected in only one afrotherian genome (as a pseudogene), and not in the other six afrotherian, two xenarthran, or three marsupial genomes. In contrast, Klk5 was detected in both marsupial and eutherian mammals. Our phylogenetic and mutation rate analyses support the hypothesis that Klk4 arose from Klk5 by gene duplication near the divergence of Afrotheria, Xenarthra and Boreoeutheria, and that functionally-differentiated Klk4 survived only in Boreoeutheria. Afrotherian mammals share the feature of delayed dental eruption relative to boreoeutherian mammals. KLK4 shortens the time required for enamel maturation and could have alleviated negative selection following mutations that resulted in thicker enamel or earlier tooth eruption, without reducing enamel hardness or causing dental attrition.

Ameloblast transcriptome changes from secretory to maturation stages.

The purpose of this study was to identify the major molecular components in the secretory and maturation stages of amelogenesis through transcriptome analyses. Ameloblasts (40 sections per age group) were laser micro-dissected from Day 5 (secretory stage) and Days 11-12 (maturation stage) first molars. PolyA+ RNA was isolated from the lysed cells, converted to cDNA, and amplified to generate a cDNA library. DNA sequences were obtained using next generation sequencing and analyzed to identify genes whose expression had increased or decreased at least 1.5-fold in maturation stage relative to secretory stage ameloblasts. Among the 9198 genes that surpassed the quality threshold, 373 showed higher expression in secretory stage, while 614 genes increased in maturation stage ameloblasts. The results were cross-checked against a previously published transcriptome generated from tissues overlying secretory and maturation stage mouse incisor enamel and 34 increasing and 26 decreasing expressers common to the two studies were identified. Expression of F2r, which encodes protease activated receptor 1 (PAR1) that showed 10-fold higher expression during the secretory stage in our transcriptome analysis, was characterized in mouse incisors by immunohistochemistry. PAR1 was detected in secretory, but not maturation stage ameloblasts. We conclude that transcriptome analyses are a good starting point for identifying genes/proteins that are critical for proper dental enamel formation and that PAR1 is specifically expressed by secretory stage ameloblasts.

Novel WDR72 Mutations Causing Hypomaturation Amelogenesis Imperfecta.

Amelogenesis imperfecta (AI) is a heterogeneous collection of hereditary enamel defects. The affected enamel can be classified as hypoplastic, hypomaturation, or hypocalcified in form. A better understanding of normal amelogenesis and improvements in our ability to diagnose AI through genetic testing can be realized through more complete knowledge of the genes and disease-causing variants that cause AI. In this study, mutational analysis was performed with whole exome sequencing (WES) to identify genetic etiology underlying the hypomaturation AI condition in affected families. Mutational analyses identified biallelic WDR72 mutations in four hypomaturation AI families. Novel mutations include a homozygous deletion and insertion mutation (NM_182758.4: c.2680_2699delinsACTATAGTT, p.(Ser894Thrfs*15)), compound heterozygous mutations (paternal c.2332dupA, p.(Met778Asnfs*4)) and (maternal c.1287_1289del, p.(Ile430del)) and a homozygous 3694 bp deletion that includes exon 14 (NG_017034.2:g.96472_100165del). A homozygous recurrent mutation variant (c.1467_1468delAT, p.(Val491Aspfs*8)) was also identified. Current ideas on WDR72 structure and function are discussed. These cases expand the mutational spectrum of WDR72 mutations causing hypomaturation AI and improve the possibility of genetic testing to accurately diagnose AI caused by WDR72 defects.

Dentin defects caused by a Dspp-1 frameshift mutation are associated with the activation of autophagy.

Dentin sialophosphoprotein (DSPP) is primarily expressed by differentiated odontoblasts (dentin-forming cells), and transiently expressed by presecretory ameloblasts (enamel-forming cells). Disease-causing DSPP mutations predominantly fall into two categories: 5' mutations affecting targeting and trafficking, and 3' - 1 frameshift mutations converting the repetitive, hydrophilic, acidic C-terminal domain into a hydrophobic one. We characterized the dental phenotypes and investigated the pathological mechanisms of DsppP19L and Dspp-1fs mice that replicate the two categories of human DSPP mutations. In DsppP19L mice, dentin is less mineralized but contains dentinal tubules. Enamel mineral density is reduced. Intracellular accumulation and ER retention of DSPP is observed in odontoblasts and ameloblasts. In Dspp-1fs mice, a thin layer of reparative dentin lacking dentinal tubules is deposited. Odontoblasts show severe pathosis, including intracellular accumulation and ER retention of DSPP, strong ubiquitin and autophagy activity, ER-phagy, and sporadic apoptosis. Ultrastructurally, odontoblasts show extensive autophagic vacuoles, some of which contain fragmented ER. Enamel formation is comparable to wild type. These findings distinguish molecular mechanisms underlying the dental phenotypes of DsppP19L and Dspp-1fs mice and support the recently revised Shields classification of dentinogenesis imperfecta caused by DSPP mutations in humans. The Dspp-1fs mice may be valuable for the study of autophagy and ER-phagy.

PAX9 mutations and genetic synergism in familial tooth agenesis.

Familial tooth agenesis (FTA) is one of the most common craniofacial anomalies in humans. Loss-of-function mutations in PAX9 and WNT10A have been known to cause FTA with various expressivity. In this study, we identified five FTA kindreds with novel PAX9 disease-causing mutations: p.(Glu7Lys), p.(Val83Leu), p.(Pro118Ser), p.(Ser197Argfs*23), and c.771+4A>G. Concomitant PAX9 and WNT10A pathogenic variants found in two probands with severe phenotypes suggested an effect of mutational synergism. All overexpressed PAX9s showed proper nuclear localization, excepting the p.(Pro118Ser) mutant. Various missense mutations caused differential loss of PAX9 transcriptional ability. PAX9 overexpression in dental pulp cells upregulated LEF1 and AXIN2 expression, indicating a positive regulatory role for PAX9 in canonical Wnt signaling. Analyzing 176 cases with 63 different mutations, we observed a distinct pattern of tooth agenesis for PAX9-associated FTA: Maxillary teeth are in general more frequently affected than mandibular ones. Along with all second molars, maxillary bicuspids and first molars are mostly involved, while maxillary lateral incisors and mandibular bicuspids are relatively less affected. Genotypically, missense mutations are associated with fewer missing teeth than frameshift and nonsense variants. This study significantly expands the phenotypic and genotypic spectrums of PAX9-associated disorders and reveals a molecular mechanism of genetic synergism underlying FTA variable expressivity.

Effect of kallikrein 4 loss on enamel mineralization: comparison with mice lacking matrix metalloproteinase 20.

Enamel formation depends on a triad of tissue-specific matrix proteins (amelogenin, ameloblastin, and enamelin) to help initiate and stabilize progressively elongating, thin mineral ribbons of hydroxyapatite formed during an appositional growth phase. Subsequently, these proteins are eradicated to facilitate lateral expansion of the hydroxyapatite crystallites. The purpose of this study was to investigate changes in enamel mineralization occurring in mice unable to produce kallikrein 4 (Klk4), a proteinase associated with terminal extracellular degradation of matrix proteins during the maturation stage. Mice lacking functional matrix metalloproteinase 20 (Mmp20), a proteinase associated with early cleavage of matrix proteins during the secretory stage, were also analyzed as a frame of reference. The results indicated that mice lacking Klk4 produce enamel that is normal in thickness and overall organization in terms of layers and rod/inter-rod structure, but there is a developmental defect in enamel rods where they first form near the dentinoenamel junction. Mineralization is normal up to early maturation after which the enamel both retains and gains additional proteins and is unable to mature beyond 85% mineral by weight. The outmost enamel is hard, but inner regions are soft and contain much more protein than normal. The rate of mineral acquisition overall is lower by 25%. Mice lacking functional Mmp20 produce enamel that is thin and structurally abnormal. Relatively high amounts of protein remain throughout maturation, but the enamel is able to change from 67 to 75% mineral by weight during maturation. These findings reaffirm the importance of secreted proteinases to enamel mineral acquisition.