Hypercementosis Associated with ENPP1 Mutations and GACI

Abstract

Mineralization of bones and teeth is tightly regulated by levels of extracellular inorganic phosphate (P_i) and pyrophosphate (PP_i). Three regulators that control pericellular concentrations of P_i and PP_i include tissue-nonspecific alkaline phosphatase (TNAP), progressive ankylosis protein (ANK), and ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1). Inactivation of these factors results in mineralization disorders affecting teeth and their supporting structures. This study for the first time analyzed the effect of decreased PP_i on dental development in individuals with generalized arterial calcification of infancy (GACI) due to loss-of-function mutations in the ENPP1 gene. Four of the 5 subjects reported a history of infraocclusion, overretained primary teeth, ankylosis, and/or slow orthodontic tooth movement, suggesting altered mineral metabolism contributing to disrupted tooth movement and exfoliation. All subjects had radiographic evidence of unusually protruding cervical root morphology in primary and/or secondary dentitions. High-resolution micro-computed tomography (micro-CT) analyses of extracted primary teeth from 3 GACI subjects revealed 4-fold increased cervical cementum thickness ( P = 0.00007) and a 23% increase in cementum density ( P = 0.009) compared to age-matched healthy control teeth. There were no differences in enamel and dentin densities between GACI and control teeth. Histology revealed dramatically expanded cervical cementum in GACI teeth, including cementocyte-like cells and unusual patterns of cementum resorption and repair. Micro-CT analysis of Enpp1 mutant mouse molars revealed 4-fold increased acellular cementum thickness ( P = 0.002) and 5-fold increased cementum volume ( P = 0.002), with no changes in enamel or dentin. Immunohistochemistry identified elevated ENPP1 expression in cementoblasts of human and mouse control teeth. Collectively, these findings reveal a novel dental phenotype in GACI and identify ENPP1 genetic mutations associated with hypercementosis. The sensitivity of cementum to reduced PP_i levels in both human and mouse teeth establishes this as a well-conserved and fundamental biological process directing cementogenesis across species (ClinicalTrials.gov NCT00369421).

Keywords: cementum; enamel; generalized arterial calcification of infancy; mineralized tissue/development; periodontal tissues/development; tooth development.

Figure 1.

Clinical presentations of generalized arterial calcification of infancy (GACI) subjects 1 to 3. (A) Schematic of ENPP1 protein indicating locations of p.P305T (exon 8) and p.H500P (exon 15) amino acid substitutions identified in subject 1. (B) Pedigree of proband subject 1 (black arrowhead) showing unaffected parents, 2 unaffected older siblings (unknown genetic composition indicated by gray shading), and an older brother harboring the same biallelic ENPP1 mutations who died of cardiac arrest. (C, D) Clinical photographs at 6.7 y of age reveal infraoccluded primary maxillary lateral incisors (yellow arrows). (E) Periapical radiograph at 7.8 y shows eruption of permanent maxillary incisors (8, 9) while primary lateral incisors remain infraoccluded (yellow arrows) and secondary lateral incisors are unerupted. (F) Bitewing radiograph reveals a protruding cervical root morphology (yellow arrowheads) in primary and secondary molars, where cementum sometimes appears to overlap enamel at the cementum-enamel junction. (G) Schematic of ENPP1 protein indicating the location of the homozygous p.Y513C amino acid substitution in exon 15 identified in subject 2. (H) Pedigree of proband subject 2 (black arrowhead) showing unaffected consanguineous parents. (I) Clinical photograph of subject 2 at 7.11 y reveals a mixed dentition with gingival hyperplasia and mild discoloration in enamel of central incisors (inset). (J, K) Dental radiographs indicate overretained maxillary central and lateral incisors. (L) Schematic of ENPP1 protein indicating locations of p.C480R (exon 15) and p.G805V (exon 23) amino acid substitutions identified in GACI subject 3. (M) Pedigree of proband subject 3 (black arrowhead) showing unaffected parents and younger sister with GACI. (N) Oral photograph at 6.11 y and (O) panoramic radiograph at 6.9 y reveal a healthy mixed dentition. (P) Dental bitewing radiograph shows protruding cervical root morphology on primary molar K.

Figure 2.

Clinical presentations of generalized arterial calcification of infancy (GACI) in subjects 4 and 5. (A) Schematic of ENPP1 protein indicating locations of p.P250L (exon 7) and p.P305T (exon 8) amino acid substitutions, both within the catalytic domain, identified in GACI subject 4. (B) Pedigree of proband subject 4 (black arrowhead) showing unaffected parents, twin brother who died of GACI in infancy, and unaffected younger sister (unknown genetic composition indicated by gray shading). (C) Oral photograph at 26.5 y reveals a healthy dentition and absence of caries, with generalized gingival recession. Dental bitewing radiographs at (D) 22.2 y of age and (E) 26.0 y show consistent protruding cervical root morphology (yellow arrowheads) in molars. (F) Schematic of ENPP1 protein indicating locations of p.Y471C (exon 14) and p.R481Q (exon 15) amino acid substitutions, both within the catalytic domain, identified in GACI subject 5. (G) Pedigree of proband subject 5 (black arrowhead) showing unaffected parents and stepfather and unaffected brother and stepsister (unknown genetic composition indicated by gray shading). (H) Oral photograph at 25.7 y reveals a relatively healthy dentition with unilateral crossbite and localized areas of gingival recession. Mild gingival hyperplasia is noted between maxillary central incisors. Dental bitewing radiographs at (I) 22.11 and (J) 25.3 y show consistent protruding cervical root morphology (yellow arrowheads) in posterior teeth and slightly narrow root canals in incisors.

Figure 3.

Increased thickness and density of cervical cementum in teeth from generalized arterial calcification of infancy (GACI) subjects. (A) Three-dimensional micro–computed tomography (CT) reconstructions of representative incisors from 1 healthy control and 3 GACI subjects. Cementum layer is highlighted in yellow. Scale bars in panels A to C represent 2.5 mm. (B) Two-dimensional micro-CT cut plane showing hypercementosis in GACI teeth. Cementum layer is highlighted in yellow. Tooth E from GACI subject 2 features calcified pulp stone-like material (labeled in red). (C) Segmented cervical cementum layer used for thickness measurement in control and GACI teeth. Quantitative analyses of control teeth (6 teeth from 4 subjects) and 10 GACI teeth indicate (D) 4-fold significantly increased cementum thickness in GACI vs. control teeth (****adjusted P = 0.00007), (E) 23 % increase in cementum density in GACI vs. control teeth (**adjusted P = 0.009), and no differences in (F) dentin density or (G) enamel density (adjusted P > 0.05 for both). In graphs shown in panels D to G, each individual tooth measurement is displayed with color coding to indicate subject of origin, with mean ± standard deviation shown for 6 teeth from n = 4 controls and 10 teeth from n = 3 GACI subjects. For independent samples t test, multiple teeth from the same individual were first averaged to compare n = 4 control and n = 3 GACI measurements, followed by Benjamini-Hochberg false discovery rate correction for Q = 0.05.

Figure 4.

Histological analysis of hypercementosis in teeth from generalized arterial calcification of infancy (GACI) subjects. Histology sections of (A, B) healthy control incisor compared to (C, D) incisor and (E, F) canine from GACI subject 1 reveal dramatically increased acellular cementum (AC) in GACI teeth. Both GACI teeth exhibit embedded nucleated cementocyte-like cells and empty lacunae (D and F) unusual for cervical cementum. (G–I) GACI teeth exhibit patterns of adjacent resorption and reparative cellular cementum (black arrows), unusual for primary teeth undergoing exfoliation. Immunohistochemistry for ENPP1 protein in control (J, K) human and (L, M) mouse molar sections reveals selective elevated ENPP1 in cementoblasts (Cb) lining AC surfaces, with fewer positive Cb on the cellular cementum (CC) surfaces. AB, alveolar bone; DE, dentin; PDL, periodontal ligament.

Figure 5.

Enpp1 mutant mice feature increased acellular and cellular cementum dimensions. High-resolution micro–computed tomography analysis reveals hypercementosis in Enpp1^asj/asj versus wild-type (WT) mouse first mandibular molars (n = 4 to 5) in (A, B) 3-dimensional reconstructions and (C, D) 2-dimensional cut planes in sagittal, frontal, and transverse orientations. Cementum layer is highlighted in yellow. Quantitative analysis of molars shows significantly increased (E) acellular cementum (AC) thickness (**adjusted P = 0.002) and (F) volume (**adjusted P = 0.002), and (G) a nonsignificant increase in mean AC density in Enpp1^asj/asj versus WT mice. (H–L) Significant increases are observed in cellular cementum (CC) volume (*adjusted P = 0.02) and length on mesial (M) root (*adjusted P = 0.01), although differences are not found in thickness, density, or distal (D) root CC length (adjusted P > 0.05 for all). For independent samples t test, calculated P values were adjusted and statistical significance determined by the Benjamini-Hochberg procedure for Q = 0.05.