Dental defects in the primary dentition associated with hypophosphatasia from biallelic ALPL mutations

Abstract

ALPL encodes tissue-nonspecific alkaline phosphatase (TNAP), an enzyme expressed in bone, teeth, liver, and kidney. ALPL loss-of-function mutations cause hypophosphatasia (HPP), an inborn error-of-metabolism that produces skeletal and dental mineralization defects. Case reports describe widely varying dental phenotypes, making it unclear how HPP comparatively affects the three unique dental mineralized tissues: enamel, dentin, and cementum. We hypothesized that HPP affected all dental mineralized tissues and aimed to establish quantitative measurements of dental tissues in a subject with HPP. The female proband was diagnosed with HPP during childhood based on reduced alkaline phosphatase activity (ALP), mild rachitic skeletal effects, and premature primary tooth loss. The diagnosis was subsequently confirmed genetically by the presence of compound heterozygous ALPL mutations (exon 5: c.346G>A, p.A116T; exon 10: c.1077C>G, p.I359M). Dental defects in 8 prematurely exfoliated primary teeth were analyzed by high resolution micro-computed tomography (micro-CT) and histology. Similarities to the Alpl^−/− mouse model of HPP were identified by additional analyses of murine dentoalveolar tissues. Primary teeth from the proband exhibited substantial remaining root structure compared to healthy control teeth. Enamel and dentin densities were not adversely affected in HPP vs. control teeth. However, analysis of discrete dentin regions revealed an approximate 10% reduction in the density of outer mantle dentin of HPP vs. control teeth. All 4 incisors and the molar lacked acellular cementum by micro-CT and histology, but surprisingly, 2 of 3 prematurely exfoliated canines exhibited apparently normal acellular cementum. Based on dentin findings in the proband’s teeth, we examined dentoalveolar tissues in a mouse model of HPP, revealing that the delayed initiation of mineralization in the incisor mantle dentin was associated with a broader lack of circumpulpal dentin mineralization. This study describes a quantitative approach to measure effects of HPP on dental tissues. This approach has uncovered a previously unrecognized novel mantle dentin defect in HPP, as well as a surprising and variable cementum phenotype within the teeth from the same HPP subject.

1. Introduction

Hypophosphatasia (HPP) is an inborn error-of-metabolism caused by loss-of-function mutations in the ALPL gene (OMIM #241500, #241510, #146300) that encodes tissue-nonspecific alkaline phosphatase (TNAP/TNALP/TNSALP) [1, 2]. TNAP is an enzyme expressed in bones, teeth, liver, and kidneys [3]. Reduced TNAP function in HPP results in the accumulation of substrates, including inorganic pyrophosphate (PP_i), a potent inhibitor of mineralization, leading to skeletal defects that may include rickets (in children) and osteomalacia (in children and adults) that result in fractures and bone pain. HPP manifests with a wide range of severity, from life-threatening perinatal and infantile forms to milder forms that manifest in childhood or adulthood or affect primarily the dentition. Prevalence estimates in Europe report severe forms at 1:300,000 live births and milder forms at about 1:6,370; although these estimates will presumably be revised given the increased awareness of mild HPP forms and significantly increased genetic testing. HPP has been linked to more than 400 pathogenic variants found across all 12 coding exons of ALPL (http://www.sesep.uvsq.fr/03_hypo_mutations.php), with severe forms associated with autosomal recessive inheritance, moderate forms with autosomal dominant or recessive inheritance, and mild forms with autosomal dominant patterns [4–6].

Orodental pathology has been described across all clinical forms of HPP [7, 8], prompting speculation that dentoalveolar mineralized tissues are particularly sensitive to ALPL mutations. Murine studies demonstrate that ameloblasts, odontoblasts, cementoblasts, osteoblasts, and periodontal ligament (PDL) cells express TNAP [1, 9, 10], suggesting the enzyme functions in all aspects of dentoalveolar mineralization. Dental defects reported in association with HPP include lack of acellular cementum, premature loss of fully rooted primary teeth, loss of secondary teeth, delayed eruption, periodontal disease, enamel alterations, thin and hypomineralized dentin, widened pulp chambers, and tooth root malformations [7, 8, 11]. However, dental case reports to date that use varying criteria and largely observational and qualitative descriptions have reported widely variable effects of HPP on enamel, dentin, and cementum. As a result, it remains unclear how HPP specifically affects each of the dentoalveolar tissues. In addition, it remains unclear how reported HPP dental defects correlate to known genetic, biochemical, and musculoskeletal findings. The goal of this study was to identify the genetic variants and dental effects of HPP in the female proband and apply a quantitative approach to defining dental defects in exfoliated primary teeth. We hypothesized that all dental tissues would be affected by HPP. Analyses of dental tissues in a mouse model HPP accompany human findings.

2. Methods

2.1. Human Subjects

Retrospective dental and medical findings are reported from a child with HPP (“not human subject research” as regulated and defined by 45 CRR 46, the “Common Rule,” as well as institutional guidelines). Blood and genetic analyses were performed as part of the routine clinical diagnosis and provided with the consent of the family. Eight primary teeth (4 incisors, 3 canines, 1 molar) from the proband were freely donated and subsequently analyzed. Healthy primary control teeth (n=10 including incisors, canines, and a molar) from 4 male and female individuals (ages 6–11 years) were donated through an IRB exempted protocol and analyzed for comparison.

2.2. Mice

Animal studies were approved by the Ohio State University Institutional Animal Care and Use Committee (Columbus, OH). Characterization and genotyping of Alpl knockout (Alpl^−/−) mice has been described [9]. Littermate wild-type (WT) control and Alpl^−/− mice were on a mixed 129 genetic background [12]. Dietary supplementation with vitamin B6 briefly suppresses seizures and extends lifespan, therefore, mice were given free access to modified laboratory rodent diet with increased levels (325 ppm) of pyridoxine. Alpl^−/− mice typically die within 2–4 weeks after birth. Mice were euthanized and tissues harvested at 23–24 days postnatal (dpn) with n=3–4 mice/group for experiments.

2.3. Micro-computed Tomography (Micro-CT)

For human subjects, exfoliated HPP and control teeth were scanned by micro-CT 50 (Scanco Medical, Bruttisellen, Switzerland) with parameters: 70 kVp, 76 μA, 0.5 mm Al Filter, 900 ms integration time, and 5 or 10 μm voxel size), as previously described [13]. Mouse mandibles were scanned with the same parameters with 6 μm voxel size. DICOM images were analyzed with AnalyzePro 1.0 (AnalyzeDirect, Overland Park, KS) and calibrated to five known densities of hydroxyapatite (mg/cm³ HA). Images were oriented anatomically using the midsagittal slice, where the mesial and distal cementum-enamel junctions (CEJs) were used to identify an axis that was made perpendicular to the length of the root. Dentin was segmented at 650–1600 mg/cm³ HA, and enamel was segmented above 1600 mg/cm³ HA. Acellular cementum was segmented as previously described [13]. Briefly, a median filter with a kernel size of 11 was applied. Acellular cementum was then segmented between 450–1050 mg/cm³ HA with manual corrections to exclude softer dentin that was highlighted adjacent to the pulp. The segmentation map was then loaded back onto the original calibrated image and used as a mask to highlight any cementum under this mask with a density over 650 mg/cm³ HA. Quantitative analysis was then performed on the original calibrated image. Average density was determined from these volumes. Thickness was determined using cortical bone thickness algorithms from the most apical 25–50 slices that formed a hollow cylinder of acellular cementum (depending on whether sufficient root structure remained).

Primary teeth are challenging to analyze because of different degrees of enamel attrition and root resorption, therefore regions of interest (ROI) were selected near the cementum-enamel junction (CEJ). For crown dentin and enamel thickness, as well as interglobular dentin area, we defined the ROI by identifying the CEJ in the axial plane (in this case, the first slice where enamel made a complete ring), moved coronally 0.5 mm, and outlined an ROI consisting of 0.5 mm. For root dentin thickness, we identified the most apical extension of enamel in the axial orientation and moved apically 150 µm to outline an ROI consisting of 0.5 mm. Enamel and dentin thicknesses were measured using a cortical bone thickness algorithm for the defined ROIs that was adapted from Bouxsein and colleagues, where average thickness of the cylinder over the z-stack is calculated [14]. Subdivision of dentin mineral density was performed in the root dentin ROI by designating mantle dentin (outermost 150 µm, excluding cementum), proximal pulpal dentin (innermost 150 µm), and circumpulpal dentin (all dentin between mantle and proximal pulpal regions). ROIs are illustrated in Supplemental Fig. 1. There is not a consensus on mantle dentin thickness in humans. Some texts and publications report a layer on the scale of tens of µm, while Goldberg and colleagues report a less mineralized outer dentin layer of about 200 µm [15]. In our analyses, HPP teeth exhibited alterations in the outer 150 µm of dentin, roughly corresponding to mantle dentin in location and size. Proximal pulpal dentin is not a previously recognized anatomical form of dentin but is rather a term we used in order to interrogate the most recently formed dentin.

For mouse mandibles, images were oriented anatomically as described previously [13] and a region of interest (ROI) was defined from 240 µm mesial to the first mandibular molar mesial root to 240 µm distal to the distal root. Bone and dentin were segmented at 450–1600 mg/cm³ HA, and enamel was segmented above 1600 mg/cm³ HA. Because of the young age, cementum accumulation was minimal, and the mineralization defect in Alpl^−/− mice prevented segmentation of cementum from dentin, therefore dentin and cementum were jointly analyzed. Analyses of enamel and dentin in the mouse ever erupting incisor were performed on the segment defined by the ROI above.

2.4. Histology

Human teeth were fixed in 10% neutral buffered formalin and decalcified in a formic acid and formaldehyde-based solution (Polysciences, Inc., Warrington, PA) after micro-CT scanning. Mouse tissues for standard histology were decalcified in an acetic acid/formalin/sodium chloride solution [13, 16]. Both human and mouse decalcified tissues were paraffin embedded for 6 µm sections. [17]. Hematoxylin and eosin (H&E) and picrosirius red staining were previously described [17].

Mouse tissues for undecalcified histology were fixed in paraformaldehyde, embedded in methylmethacrylate, sectioned, and stained by Goldner’s trichrome stain, as described previously [9, 18, 19].

2.5. Genetic and Bioinformatic Analyses

Effects of the observed missense mutations and amino acid substitutions on TNAP were evaluated using a variety of bioinformatic tools. Multiple sequence alignment of TNAP sequences from different species (sequences obtained through the protein database, PDB; https://www.rcsb.org) was performed using Clustal Omega (http://clustal.org/omega/) for the following species: Homo sapiens (P05186), Pan troglodytes (K7B4Y6), Macaca mulatta (A0A1D5R5B1), Rattus norvegicus (P08289), Mus musculus (P09242), Canis lupus familiaris (F1PF95), Bos taurus (P09487), Ovis aries (W5PFB8), Sus scrofa (A0A287BSC3), Gallus gallus (Q92058), and Xenopus laevis (Q7ZYJ4). The 3D structure of TNAP has not been elucidated, therefore we and others use the 3D model of a human placental alkaline phosphatase (PLAP) dimer (record 1zefA), a closely related phosphatase thought to have a highly similar 3D conformation to TNAP. Effects of A116T and I359M amino acid substitutions on TNAP secondary structure were modeled by Phyre2 protein fold recognition server (http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index)[20] using records c1ew2a (crystal structure of human phosphatase) to model 90% of the sequence (474 residues) with 100% confidence. PDB files generated for normal, A116T, and I359M versions of TNAP were loaded into the RCSB PDB protein comparison tool (http://www.rcsb.org/pdb/workbench/workbench.do) to generate superimposed 3D structure alignments [21].

2.6. Statistical Analysis

Data are expressed as mean ± standard deviation (SD) in graphs. Using measurements from healthy control teeth, 95% confidence intervals (CI) were calculated for comparison with measurements from HPP teeth from the single proband (Prism version 8.02; GraphPad Software, La Jolla, CA). Significance was determined by P<0.05 and is reported as such.

3. Results

3.1. Clinical Findings and Genetic Analysis

The female proband was 2.5 years old at the time medical and dental records were assessed, and the HPP diagnosis made. Premature primary tooth loss and skin dimples (characteristic of rickets) prompted medical evaluation. Radiographs of femur, tibia, and fibula exhibited bowing of the first segments, flared metaphyses, and beaking (Fig. 1A). No evidence of fracture was noted, and no fractures were reported at that time. Oral examination and occlusal dental radiographs of the anterior maxilla (Fig. 1B) and mandible (Fig. 1C) revealed thin dentin and enlarged pulp chambers in several teeth. The proband experienced premature loss of 4 primary incisors (D, N, O, and P), 3 primary canines (H, M, and R), and 1 primary molar (A) by age 2.5 years (Fig. 1D). Prematurely exfoliated teeth exhibited substantial remaining root structure. Reduced circulating alkaline phosphatase activity (ALP) in the proband of 29 U/L (Normal range for age and sex: 150–440 U/L) contributed to the diagnosis of HPP (Fig. 1E) that was confirmed by similar low circulating ALP activity at age 5 (data not shown).

Figure 1. Diagnosis of Hypophosphatasia in the Proband.

(A) Radiograph of right leg featuring bowing of first segments, flared metaphyses, and beaking consistent with rachitic effects of HPP. (B) Occlusal radiograph of anterior maxilla of proband showing absence of primary upper central incisors and left lateral incisor due to premature exfoliation. Right primary lateral incisor and both primary canines can be seen with developing secondary dentition in the intraosseous stage within the alveolar bone. (C) Occlusal radiograph of anterior mandible showing absence of primary central incisors and lower right lateral incisor due to premature exfoliation. Lower left primary lateral incisor and both primary canines are visible, as well as developing secondary central and lateral incisors in the intraosseous stage within the alveolar bone. (D) Prematurely exfoliated primary teeth of the proband, including 4 incisors (D, N, O, and P), 3 canines (H, R, and M), and 1 molar (A), that all exhibit excessive remaining root structure for exfoliated primary teeth. (E) Normal range (pink shading) and mean levels (red line) of circulating alkaline phosphatase (ALP) in females. The proband’s ALP measured 29 U/L at age 2.5 years. (F) Pedigree indicating ALPL variants inherited from father (blue; c.1077C>G, p.I359M) and mother (red; c.346G>A, p.A116T) in the proband (black arrowhead).

The proband’s family reported no prior skeletal or dental abnormalities within the pedigree (Fig. 1F). Genetic analysis revealed biallelic ALPL mutations in the proband, confirming the diagnosis of HPP: c.346G>A/p. Ala116Thr (A116T) in exon 5 inherited from mother and c.1077C>G/p.Iso359Met (I359M) in exon 10 inherited from father (indicated by red and blue shading, respectively, in Fig. 1F). This combination of variants has not been previously reported among the 411 ALPL mutations reported to date (http://www.sesep.uvsq.fr/03_hypo_mutations.php), although each individual mutation was found previously in separate individuals with HPP. The A116T substitution was reported in a large cohort with skeletal and dental manifestations [22]. The I359M substitution was identified in a homozygous individual with premature tooth loss and a relatively mild form of childhood HPP [23], as well as associated with infantile HPP in conjunction with an additional A378V substitution [24].

3.2. Bioinformatic Analysis of ALPL Mutations

In the simplified 2D schematic representation of human TNAP primary protein structure, the p.A116T substitution in exon 5 falls near the enzyme active site and a Zn²⁺ binding site (Fig. 2A). The p.I359M substitution in exon 10 lies near the crown domain and a Zn²⁺ binding site. Phylogenetic analysis revealed that the identity of the A116 residue is perfectly conserved across all mammals assessed, as well as in chicken and frog TNAP sequences (Fig. 2B). The identity of I359 residue is nearly perfectly conserved across all species, with chicken showing a conservative substitution with valine (V), an amino acid with similar chemistry and side chain (Fig. 2C).

Figure 2. Bioinformatic Analysis and Predicted Structures of TNAP Variants.

(A) Protein model of TNAP showing functional regions including active site, ion binding, crown domain, and disulfide bonds. Nucleotide mutations (c.346G>A and c.1077C>G) and amino acids substitutions (p.A116T and p.I359M) are indicated in exon locations. (B) Phylogenetic analysis reveals that A116 is highly conserved across mammals, chicken, and frog TNAP sequences. (C) The identity of I359 is conserved across all species analyzed with the exception of a conservative substitution in chicken (Gallus gallus). In panels B and C, the asterisk (*) represents an exact match in all sequences; a colon (:) represents a conserved substitution; a period (.) represents a semi-conserved substitution; no symbol represents no conservation at that site. (D, E) 3D model of native TNAP dimer (monomer 1 shown in green, monomer 2 shown in yellow) showing the locations of A116 (shaded red) and I359 (shaded blue) in monomer 1. Both residues reside in predicted alpha helical domains. (F) Superimposed 3D structure alignments of native TNAP (gray) vs. A116T mutant (red) predict structural changes in the crown domain, the dimerization region, and within a limited number of alpha helices. (G) Superimposed 3D structure alignments of native TNAP (gray) vs. I359M mutant (blue) predict structural changes in the crown domain, the dimerization region, and in multiple alpha helices. For F and G, structural differences are indicated when the red/blue structure deviates from the native gray structure.

Considering the 3D structure of the TNAP dimer, the substitution at position 116 of alanine, a small amino acid with a hydrophobic side chain, for threonine, a larger amino acid with a polar side chain, could potentially cause deleterious chemical or structural changes to the TNAP protein. Likewise, replacement of isoleucine at position 359 with methionine, a somewhat larger residue with similar chemistry, might affect protein folding and/or function domains. While the 3D structure of TNAP has not been determined, the X-ray crystal structure of placental alkaline phosphatase (PLAP) has been routinely used to model TNAP structure and mutations [25, 26]. Both identities and positions of A116 and I359 are conserved between TNAP and PLAP. In the 3D structure of the normal TNAP/PLAP homodimer, A116 is localized within an alpha helical region that is somewhat concealed (Fig. 2D). I359 is localized within another alpha helical region nearer to the surface and on the opposite aspect of the TNAP protein (Fig. 2E). Computer modeling of both substitutions was performed to predict whether 3D structural differences would likely result from these specific substitutions. The superimposed 3D structure alignments of native (gray) vs. each substituted form (red/blue) predicted specific structural changes in the altered forms (Fig. 2F). The T116 mutation model revealed limited changes both up- and downstream of residue 116, including in the crown domain and dimerization regions. The M359 mutation model revealed even greater divergence from the native form, with several altered domains including the crown domain, dimerization region, and within multiple alpha helical regions (Fig. 2G).

3.3. Mantle Dentin Defects Associated with HPP

Eight prematurely exfoliated and donated primary teeth (incisors D, N, O, and P; canines H, M, and R; and molar A) from the proband and 10 healthy control teeth (including incisors, canines, and a molar) were analyzed by high-resolution micro-CT (Fig. 3A–D). Enamel appeared grossly normal in teeth from the proband. Acellular cementum, which in healthy primary teeth can be digitally separated from dentin by application of threshold and filters to find the cementum-dentin junction (CDJ), could not be reliably traced in any of the HPP incisors, one canine, and the molar. However, a cementum layer was detected in canines H and R (Supplemental Fig. 2). Radiolucency in the outer dentin of HPP teeth (yellow arrow in Fig. 3D) prompted analysis of teeth using 2D heat maps of hydroxyapatite (HA) mineral density where highly mineralized regions (yellow-red) could be more easily differentiated from areas with low mineral density (shades of blue) (Fig. 3E–F). Heat maps confirmed the consistent presence of a hypomineralized region in dentin, roughly corresponding to the outermost mantle dentin layer that forms during primary dentinogenesis.

Figure 3. Mantle Dentin Defects in HPP Teeth.

(A, C) 3D micro-CT renderings of representative control and HPP incisors where white indicates enamel (EN), gray indicates dentin (DE), and yellow indicates acellular cementum (AC), which was undetectable on HPP incisors. Additional HPP teeth are illustrated in Supplemental Fig. 2. (B, D) 2D images in frontal and sagittal plane. HPP teeth exhibited excessive remaining root structure and radiolucent regions in outer dentin (yellow arrow). Green dotted lines indicate regions shown in panels E and F. (E, F) Heat maps of hydroxyapatite (HA) mineral density (see color coded key on left margin) in DE from control and HPP incisors. The HPP tooth features a hypomineralized (blue) zone of outer DE (indicated by yellow arrow) furthest from the dental pulp (DP). (G) Quantitative measurements of lengths, thicknesses, and mineral densities of control and HPP teeth. Individual measurements are shown and group means ± standard deviation are indicated by bars and brackets. HPP incisors are indicated by red dots, HPP canines by orange dots, and the molar by green dots. A 95% confidence interval calculated from control values is shown as a gray box for all parameters.

Next, quantitative analyses of tissue linear measurements and densities were conducted. Average remaining tooth root length for nearly all HPP teeth fell above the 95% CI generated from measurements of control teeth, confirming substantial remaining root structure associated with premature exfoliation of these teeth and lack of physiological resorption (Fig. 3G). Enamel thickness was not diminished by HPP, but was increased above the 95% CI for several teeth. Likewise, enamel density was not reduced in HPP vs. control teeth. Crown and root dentin thicknesses and densities did not appear to be altered in HPP vs. control teeth. Based on heat maps indicating hypomineralized outer dentin, root dentin was subdivided into 3 regions: mantle (outermost 150 µm, excluding cementum when present), circumpulpal (bulk of the central dentin), and proximal pulpal dentin (newest dentin, 150 µm nearest the pulp chamber). In control teeth, circumpulpal dentin exhibited higher mean density than mantle dentin, and the more recently formed proximal pulpal dentin was least mineralized, as expected. While neither circumpulpal nor proximal pulpal zones of dentin showed mean differences in HPP vs. control teeth, mantle dentin of HPP teeth exhibited a greater than 10% reduction in mean mineral density (Fig. 3G), identifying mantle dentin as a specific focal point for HPP mineralization defects. HPP teeth are marked by tooth type (incisors in red, canines in orange, and the molar in green), and interestingly, two canines and the molar featured mantle dentin density within the normal range, while the other HPP teeth fell at the lower border or below the 95% CI. Though bulk mineralization of enamel and dentin was not perturbed with HPP, variation in mineral densities of both enamel and crown dentin of some teeth was higher in HPP vs. control teeth, suggesting an altered distribution of mineral in those tissues (Supplemental Fig. 3).

3.4. Variable Cementum Defects in HPP Teeth

Histology was performed on decalcified HPP and control teeth to further investigate structural details. In control teeth, acellular cementum was present on all incisor, canine, and molar root surfaces by H&E staining, and embedded Sharpey’s fibers (primarily green staining indicative of small fibers) were evident by picrosirius red staining observed under polarized light microscopy (Fig. 4A–F). All 4 HPP incisors showed a complete lack of acellular cementum structure on root surfaces, and substantial accumulation of plaque was often noted along the root surfaces, though was not present on control root surfaces (Fig. 4G–J). Two of the HPP canines (H, R) featured apparently normal acellular cementum, while the third canine (M) exhibited cementum hypoplasia like the incisors (respectively labeled as “unaffected” and “affected” in Fig. 4). Molar A featured no acellular cementum, though some cellular cementum was present in the furcation. Picrosirius red staining of HPP teeth confirmed observations by H&E, where Sharpey’s fibers were only visible in teeth with a recognizable cementum layer (Fig. 4K–N). By picrosirius red staining, HPP incisors and the affected canine featured an interglobular pattern of staining in the outer region of mantle dentin that indicated disrupted structures (Fig. 4K and M). Histological measurements revealed that canines H and R had normal acellular cementum thickness (Fig. 4O). Similarly, micro-CT measurements demonstrated undiminished cementum density in these two teeth (Fig. 4P).

Figure 4. Lack of Acellular Cementum in HPP Teeth.

Representative H&E staining for control (A) incisor, (B) canine, and (C) molar exhibiting acellular cementum (AC) on the surfaces of root dentin (DE). Periodontal ligament (PDL) is present around some teeth. (D-F) Picrosirius red (PR) staining imaged under polarized light microscopy reveals embedded Sharpey’s fibers (green) in the AC layer. (G-J) H&E staining indicates a lack of AC in HPP incisors, affected canine, and molar accompanied by plaque accumulation (*), while unaffected HPP canines exhibit grossly normal AC. (K-N) PR staining of HPP teeth confirms absence of Sharpey’s fibers on incisor, affected canine, and molar root surfaces, as well as an unusual “interglobular” patterns (white #) in underlying mantle dentin of affected teeth. Sharpey’s fibers are present on unaffected canine root surfaces. (O) AC thickness measured from histomorphometry shows normal dimensions for unaffected canines and no measurable cementum in the remaining affected teeth. (P) AC mineral density measured from micro-CT shows undiminished density in unaffected canines, while the remaining HPP teeth have no AC for which to quantify density. HPP incisors are indicated by red dots, HPP canines by orange dots, and the HPP molar by green dots. A 95% confidence interval calculated from control values is shown as a gray box for all parameters.

3.5. Dentin Mineralization Defects in a Mouse Model of HPP

Based on the dentin findings in the proband’s teeth, dentoalveolar tissues in a mouse model of HPP were examined. We and others have described dentoalveolar defects in the Alpl^−/− mouse model of severe infantile HPP [9, 10, 27–32]. While dental tissues have been previously assessed in these mice, multimodal analyses were applied to dentin, including qualitative and quantitative micro-CT, mineral density heat maps, and decalcified and undecalcified histology.

Compared to WT littermates, Alpl^−/− mice had dentin hypomineralization defects so severe that substantial portions of these tissues were below the threshold for micro-CT to detect, making molar roots appear abnormally short or lacking root structures (Fig. 5A–D). Mineralization defects resulted in all crown and root dentin volumes being significantly reduced in Alpl^−/− vs. WT molars and incisors (Fig. 5E, F). Interestingly, for dentin that was detected by micro-CT, crown and root dentin mineral densities were not different between Alpl^−/− vs. WT. Because of the thin dentin resulting from the phenotype and shortened lifespan of Alpl^−/− mice, it was not possible to reliably subdivide dentin into multiple regions as described for the human teeth. A different strategy was used to further explore dentin defects in Alpl^−/− mouse incisors, as described below. Alpl^−/− vs. WT alveolar bone showed significant defects in both volume and density (Fig. 5G).

Figure 5. Dentoalveolar Defects in Alpl^−/− Mice.

(A-D) 3D and 2D micro-CT renderings of first molars (M1) reveal severe dentin (DE) defects (yellow arrows) and alveolar bone (AB) defects (yellow *) in Alpl^−/− vs. WT mice. Both (E) first molar and (F) incisor teeth show significant defects in Alpl^−/− vs. WT mice, including reduced crown and root dentin volumes (n=3–4). (G) Alveolar bone shows decreased volume and density in Alpl^−/− vs. WT mice (n=3–4). Statistical analysis by independent samples t-test where * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. EN = enamel.

We focused on identifying patterns of dentin mineralization in the continually erupting incisors to simultaneously observe multiple stages of dentinogenesis from apical (early stages of development) to incisal locations (late stages of development) along the incisor length. Apical to incisal locations are labeled I-IV in 3D micro-CT mandible renderings in Fig. 6A, B, and these specific locations are shown as coronal sections in panels D and F. Heat maps confirmed that regions of Alpl^−/− mouse molar and incisor dentin were extremely hypomineralized (Fig. 6C–F; red arrows indicate defective regions in panels E and F). In these regions, mineral content was entirely beneath detection limits (black in the color spectrum). Dentin defects were more severe on the root analogue (lingual aspect) of the incisor. Lack of mantle dentin mineralization was always associated with a complete lack of mineralization of the adjacent circumpulpal dentin (Fig. 6F locations I and II). However, as development advanced from the apical end towards the incisal tip, hypomineralized regions progressively exhibited “breakthrough” mineralization. In breakthrough areas, low density mantle dentin first appeared (red-yellow dentin indicated by white arrowheads in I-III) and then adjacent circumpulpal dentin subsequently mineralized. The dentin that overcame inhibition of mineralization showed increased mineral density that reached the normal range and was similar to WT dentin by incisal location IV.

Figure 6. Delayed Dentin Mineralization in Alpl^−/− Mouse Molars and Incisors.

(A, B) 3D micro-CT renderings of WT and Alpl^−/− mandibles in sagittal orientation. Regions of the incisor shown by heat map analysis in panels D and F are indicated by roman numerals I, II, III, and IV (apical to incisal locations). Images in panels C-F depict heat maps of molars (sagittal plane) and incisors (coronal plane) where mineral density is indicated by color spectrum shown below. (C-F) Alpl^−/− molars and incisors show regions of extreme dentin hypomineralization below density 450 mg/cm³, the threshold of detection (red arrows), as well as hypomineralized bone (red * in E). In the continually erupting incisor, Alpl^−/− defects in mantle dentin are associated with lack of mineralization of the adjacent circumpulpal dentin. When mantle dentin shows “breakthrough” mineralization (white arrowheads), the initial dentin matrix is hypomineralized (red-yellow), but adjacent circumpulpal dentin is then able to progressively mineralize (compare I to II) until it is similar in structure and mineral content to WT incisor in the same region (compare III to IV, and Alpl^−/− vs. WT in region IV).

Histology confirmed that short molar roots in Alpl^−/− vs. WT mouse molars resulted from lack of mineralization of secreted dentin matrix. H&E stained sections of molars revealed substantial regions of abnormal root dentin in Alpl^−/− vs. WT mice (Fig. 7A, C; red arrows in panel C). Hypomineralization of large regions of Alpl^−/− root dentin accounts for these structures being undetectable by micro-CT (e.g. in Fig. 5A–D and Fig. 6C, E). Goldner’s trichrome staining of undecalcified sections confirmed accumulation of unmineralized dentin matrix in Alpl^−/− molars (Fig. 7B, D; red arrow in panel D). Serial coronal sections of incisors of WT and Alpl^−/− mice were examined by H&E stain in the regions indicated by yellow boxes in Fig. 7E, F. Compared to normal dentin mineralization in WT incisors, Alpl^−/− mice show severely hypomineralized root analogue dentin matrix (Fig. 7G, H; red arrows in panel H). Over 100 µm increments from apical to incisal locations, close observation of the mineralization front (indicated by black arrowheads in higher magnification images in Fig. 7I; regions indicated by red dotted boxes in panel H) shows that outer mantle dentin first mineralizes, followed by adjacent circumpulpal dentin mineralization. While delayed compared to WT incisors, this process progressively mineralizes Alpl^−/− mouse incisors until they are grossly similar in structure and mineralization to controls (as in Fig. 6D, F).

Figure 7. Dentin Defects in Alpl^−/− Mouse Molars and Incisors.

(A, C) H&E stained sections show defective dentin (DE) regions (red arrows in C) in Alpl^−/− first molar (M1) roots, as well as accumulation of osteoid (red * in C) in alveolar bone (AB). (B, D) Goldner trichrome stained undecalcified sections confirm hypomineralized DE matrix (red arrow in D; red stained outer dentin). DP = dental pulp; PDL = periodontal ligament. (E, F) 3D micro-CT renderings indicate the locations (yellow dotted boxes) of incisor sections in panels G and H. (G, H) Location matched serial coronal sections of mandibular incisors from WT and Alpl^−/− mice are shown in approximatey 100 µm increments from apical to incisal locations. Hypomineralized DE in Alpl^−/−incisors is indicated by red arrows. Red dotted boxes in Alpl^−/− images indicate areas of higher magnification in lower panel (I) where the location of the mineralization front (black arrowhead) is initiated in outermost mantle DE and migrates around the lingual aspect of the incisor to mineralize circumpulpal DE.

4. Discussion

A novel combination of biallelic ALPL mutations causing substitutions in exons 5 (p.Ala116Thr; A116T) and 10 (p.Iso359Met; I359M) in a female child diagnosed with HPP based on premature primary tooth loss, skeletal findings, low ALP, and genetic results were identified. We report for the first time high resolution micro-CT analysis of primary teeth from a child with HPP, identifying increased remaining root structure associated with premature exfoliation. While some exfoliated primary teeth featured the expected lack of functional cementum (incisors and one molar and canine), two canines unexpectedly featured normal acellular cementum structure. Mantle dentin defects were found in HPP teeth. Examination of the Alpl^−/− mouse model of HPP confirmed in the continually erupting incisor delayed mantle and circumpulpal dentin mineralization.

4.1. Genetic Variants and Their Effects on TNAP Function

Genetic analysis of the pedigree described in this study revealed biallelic ALPL mutations in the proband resulting in two amino acid substitutions in the TNAP protein: p. Ala116Thr (A116T) inherited from mother and p.Iso359Met (I359M) inherited from father. From the changes in the predicted 3D structure of the TNAP homodimer, these mutations have the potential to adversely impact enzyme activity, stability, and allosteric properties, based on genetic analyses and functional studies of mutations that span the exons of the ALPL gene [6, 33, 34]. Such molecular changes likely contribute to reduced enzyme activity, lower than normal circulating ALP enzyme levels, and thus the HPP diagnosis of this proband. This specific combination of variants has not been reported among the individuals carrying the 411 identified ALPL mutations reported to date (http://www.sesep.uvsq.fr/03_hypo_mutations.php). However, both individual mutations have been separately identified in different individuals with HPP [22–24].

The A116T substitution was reported in a large multigenerational cohort with HPP exhibiting skeletal manifestations in some individuals (variable, but including short stature, rickets, scoliosis, Wormian bones in the skull, and bone fractures) and dental manifestations (enamel hypoplasia, widened pulp chambers, lack of acellular cementum, and premature tooth loss) in most individuals [22]. This variant was shown to exert a dominant negative effect in functional assays [33–35]. A recent report describing a genetic-based nosology for HPP found that the A116T mutation is a common variant in the U.S. and Europe, and is found in dominant genotypes (moderate severity) and recessive genotypes (moderate or severe)[6]. A genetically engineered knock-in mouse model heterozygous for the same A116T substitution, exhibited a 50% reduction of ALP, no detectable postcranial skeletal alterations, and very mild craniofacial and dentoalveolar effects [36]. Species-specific differences in manifestations of the A116T substitution may be attributed to differences in physiology or sensitivity or may reflect other genetic modifiers of PP_i metabolism or biomineralization. The modifier hypothesis is supported by genetic testing for candidate modifying factors in individuals heterozygous for ALPL mutations, which to date have identified variants in the COL1A2 gene [37].

The I359M variant is a relatively rare mutation based on reports to date, and was not found among 424 European HPP patients genetically analyzed [6]. The I359M substitution was previously found in a homozygous individual with a history of premature tooth loss and a relatively mild form of childhood HPP that included skeletal effects such as radiolucent lesions and bone pain [23]. The I359M substitution was also previously associated with infantile HPP [24], though in that individual it was present in conjunction with an A378V substitution, a residue that has been linked in combination with other ALPL variants to infantile, childhood, adult, and odonto-HPP forms [34, 38–41]. Compound heterozygous ALPL mutations associated with HPP have been reported numerous times and make it challenging to parse out genotype-phenotype correlations. In addition, a sheep knock-in model with the I359M substitution that we developed resulted in an approximate 30% decrease in ALP, with signs of rickets, measurable reduction in bone mineral density, and significant alveolar bone loss [42].

The clinical scope of HPP in this proband to date falls toward the milder end of the spectrum described in the original large A116T cohort, where most individuals had dental effects and some had moderate skeletal effects in childhood and/or adulthood [22]. However, the mild skeletal effects and premature tooth loss in the proband are entirely consistent with the individual with homozygous I359M substitutions, supporting similar contributions from both variants to the proband phenotype. It should be noted that HPP manifestations can be progressive in some cases, with mildly affected or undiagnosed individuals, or even “carriers” of variants, developing more debilitating clinical burden of the disease later in life [43–45]. This may be an important consideration when clinicians determine whether or not to treat children with the recombinant enzyme replacement therapy, asfotase alfa (Strensiq™) [1, 46].

4.2. Insights into Dental Effects of HPP

The proband demonstrated several hallmarks of HPP-associated dental disease. At age 2.5 years, she would be expected to have her complete and erupted primary dentition with a full complement of 20 teeth. Maxillary and mandibular primary incisors typically exfoliate at 6–8 years, primary canines at age 9–10 years, and primary molars at 9–12 years. The proband exhibited premature loss of several primary incisors and canines, and a primary molar by 2.5 years. All showed substantial remaining root structure, in contrast to healthy teeth undergoing normal physiological root resorption and exfoliation. This scenario is entirely consistent with numerous case reports in the literature and familiar to clinicians involved in treatment of individuals with HPP [as summarized in [1, 8, 11, 47]]. The proband did not exhibit other dental signs of HPP sometimes reported, featuring no obvious enamel abnormalities (e.g. hypoplasia, hypomineralization, or discoloration), no dramatic dentin-pulp alterations or root morphology defects, and no alveolar bone loss (which would be uncommon for a child with mixed dentition).

Application of high-resolution micro-CT analysis allowed for a systematic and quantitative approach to define dental mineralization defects in teeth from the proband compared to healthy controls. Most bulk properties of enamel and dentin appeared largely unaffected in HPP vs. control teeth, though when dentin was digitally subdivided for finer analysis, substantial mantle dentin defects in HPP teeth became apparent. Mantle dentin refers to the outermost, initial layer of dentin secreted at the onset of dentinogenesis. While the circumpulpal dentin houses odontoblast processes surrounded by peritubular dentin and intertubular dentin, mantle dentin has been described as predominantly atubular [15]. Greater sensitivity of mantle dentin to HPP suggests different mechanisms for mantle vs. circumpulpal dentin mineralization. We further explored this mantle dentin effect in molars and incisors of Alpl^−/− mice, noting by micro-CT, decalcified histology, and Goldner’s trichrome staining of undecalcified sections, regions where both mantle and circumpulpal dentin were severely hypomineralized. Interestingly in Alpl^−/−mouse incisors, only once there was breakthrough mineralization in mantle dentin matrix would circumpulpal dentin mineralization become established. This suggests a sequential relationship where mantle dentin mineralization may stimulate or be required for correct circumpulpal dentin formation and mineralization. In contrast, in human HPP teeth, mantle dentin defects existed in the absence of apparent circumpulpal defects. This may be a species-specific difference, or possibly mantle dentin defects in the proband were not severe enough to disrupt circumpulpal dentinogenesis. This is the first time a mantle dentin defect of this nature has been associated with HPP in human teeth, suggesting a mechanism that we are exploring in additional individuals with HPP, as well as in other mineralization disorders and anatomical contexts [48].

Despite the resolution of the micro-CT analysis, we were unable to differentiate cementum from dentin in most of the HPP teeth, and histological analysis was employed. As expected, the 4 incisors (D, N, O, and P), 1 canine (M), and molar (A) exhibited no recognizable acellular cementum, exactly as described in numerous case reports spanning back to the original report on cementum defects in HPP by Bruckner and colleagues [49]. In the absence of cementum, these affected teeth exhibited accumulation of dental plaque deep on root surfaces, an observation also noted previously [50]. Surprisingly, 2 canines (H and R) from the proband exhibited apparently normal acellular cementum structure. The potential mechanism underlying differential effects of HPP on acellular cementum of incisors vs. canines vs. molars remains unclear. The tooth types have slightly different times of initiation and mineralization in utero, as well eruption at postnatal ages. However, these processes are overlapping in time and employ largely identical developmental processes between tooth types. Patterning of tooth types is not fully understood, though differential roles for patterning genes (e.g. MSX1 and 2, BARX1, and DLX1 and 2) in directing dental morphogenesis along the arch could also potentially lead to differential expression of downstream effectors including ALPL/TNAP and other mineralization-associated genes [51]. This concept is borne out in other disorders that differentially affect specific tooth types, e.g. forms of tooth agenesis and molar-incisor hypomineralization [52, 53].

Anterior teeth are most frequently reported to exfoliate in children with HPP. The hypothesis has been suggested that incisors exfoliate most frequently because their simple conical roots rely almost entirely on acellular cementum for attachment. Similarly, canines fall out less frequently because of their relatively larger and longer roots and molars fall out least frequently because of multiple, complex roots that are mechanically more difficult to dislodge. Many HPP case reports that microscopically examine prematurely lost teeth have described only incisors [49, 54–59], defining HPP dental effects based predominantly on this single tooth type. However, there are some exceptions. Van den Bos and colleagues observed 19 primary incisors and 12 primary canines from 7 subjects with HPP, and reported uniform absence of acellular cementum, with no differences noted in incisors vs. canines, and no qualitative effects on dentin by histology [60]. Luder also described an HPP canine tooth where acellular cementum was absent but dentin was normal [50]. Wei and coauthors examined one primary canine tooth and found absence of cementum and PDL attachment directly to dentin [61]. Our results suggest that in some subjects, status of acellular cementum may vary by tooth type. It is unclear why canines H and R exfoliated prematurely considering the grossly normal cementum. Perhaps attachment was compromised in a way that cannot be appreciated by examining tooth structures post-exfoliation. It is possible that alveolar bone alterations, that cannot be analyzed within the limits of the study design, play some part. This idea is supported by the reduced alveolar bone volume and density as well as osteoid accumulation observed here and in previous analyses of Alpl^−/− mice [28, 62]. Perhaps the most surprising observation was that all HPP incisors and canines that exhibited mantle dentin defects also featured absence of acellular cementum; these two traits were linked among all the teeth except for molar A. During root formation in human teeth, the mantle dentin remains unmineralized when the initial cementum collagen fibers are secreted, allowing intermingling of fibers at the dentin-cementum junction [63]. Is it possible that mineralization of the mantle dentin and cementum are in some way linked? While we are unaware of this hypothesis being suggested previously, a similar concept is accepted for crown mineralization, where dentin mineralization always precedes and prompts initiation of enamel mineralization. This potential link between mantle dentin and acellular cementum is a provocative hypothesis that deserves further study.

In summary, from this first high-resolution quantitative study of exfoliated primary teeth from an individual with HPP, a novel mantle dentin mineralization defect and surprising differential effects of HPP on acellular cementum of different tooth types, were identified. This study is limited to a single subject, therefore further studies are necessary to determine if these patterns are identified in additional individuals with HPP, particularly in association with different ALPL mutations, clinical forms, and musculoskeletal effects. This work is currently underway to better understand the pathologic effects of HPP on dental mineralization in a large cohort of subjects with different types and severities of HPP. We posit that an accurate definition of the dental effects of HPP will assist in not only defining the pathology and its mechanisms, but in guiding clinical decisions on therapy to maximally benefit dental tissues.

Highlights.

• A novel combination of biallelic ALPL substitutions resulting in hypophosphatasia were identified in the proband

• Prematurely exfoliated primary teeth from the proband exhibited the expected lack of acellular cementum, however two teeth featured normal cementum thickness and density

• Primary teeth featured novel mantle dentin mineralization defects

• A mouse model of severe infantile hypophosphatasia exhibited delayed mantle dentin mineralization in association with more severe dentin hypomineralization in incisors