Dental malformations associated with biallelic MMP20 mutations

Abstract

Background: Matrix metallopeptidase 20 (MMP20) is an evolutionarily conserved protease that is essential for processing enamel matrix proteins during dental enamel formation. MMP20 mutations cause human autosomal recessive pigmented hypomaturation-type amelogenesis imperfecta (AI2A2; OMIM #612529). MMP20 is expressed in both odontoblasts and ameloblasts, but its function during dentinogenesis is unclear.

Methods: We characterized 10 AI kindreds with MMP20 defects, characterized human third molars and/or Mmp20^-/- mice by histology, Backscattered Scanning Electron Microscopy (bSEM), µCT, and nanohardness testing.

Results: We identified six novel MMP20 disease-causing mutations. Four pathogenic variants were associated with exons encoding the MMP20 hemopexin-like (PEX) domain, suggesting a necessary regulatory function. Mutant human enamel hardness was softest (13% of normal) midway between the dentinoenamel junction (DEJ) and the enamel surface. bSEM and µCT analyses of the third molars revealed reduced mineral density in both enamel and dentin. Dentin close to the DEJ showed an average hardness number 62%-69% of control. Characterization of Mmp20^-/- mouse dentin revealed a significant reduction in dentin thickness and mineral density and a transient increase in predentin thickness, indicating disturbances in dentin matrix secretion and mineralization.

Conclusion: These results expand the spectrum of MMP20 disease-causing mutations and provide the first evidence for MMP20 function during dentin formation.

Keywords: MMP20 mutations; amelogenesis imperfecta; dentin defects; enamel hardness; hypomineralization.

FIGURE 1

Family 1 (Turkey). (a) Pedigree showing an autosomal recessive pattern of inheritance with four persons recruited (asterisks). Two were affected. (b) Both affected individuals (III:1 and III:2) were homozygous for an MMP20 splice junction mutation at the 3′ end of Intron 9 (NG_012151.1: g.52906G > A; NM_004771.3: c.1352‐1G > A)¹. Both parents (II:4 and II:5) were heterozygous for this mutation. (c) The proband (III:1) at age 11 showed thin (hypoplastic), brown‐stained hypomineralized enamel that was susceptible to rapid attrition and did not contrast well with dentin on radiographs. (d) The proband's younger sister (III:2) at age 6 was in the mixed dentition stage. The primary teeth showed significant attrition and the newly erupted permanent first molars had a rough, irregular crown form. The thinness of the enamel was apparent on the newly erupted mandibular central incisors

FIGURE 2

Family 2 (Turkey). (a) Pedigree showing an autosomal recessive pattern of inheritance with no reported consanguinity. Three persons were recruited (asterisks); one was affected. (b) The proband (III:1) was homozygous for an MMP20 splice junction mutation at the 3′ end of Intron 6 (NG_012151.1: g.35574A > G; NM_004771.3: c.954‐2A > G. p.?)¹. Both parents (II:3 and II:4) were heterozygous for this mutation. (c) The dentition of the proband (III:1; at age 10) showed light brown staining. (d) The panoramic radiograph of the proband showed stainless‐steel crowns covering the four first molars that had undergone significant attrition. Enamel thickness and contrast with dentin was variable

FIGURE 3

Family 6 (Taiwan). (a) Pedigree showing an autosomal recessive pattern of inheritance with no consanguinity. Three individuals were recruited (asterisks); one was affected. (b) The proband (III:1) was heterozygous for two MMP20 missense mutations, with a novel Exon 6 mutation (NG_012151.1: g.23756C > G; NM_004771.3: c.911C > G, p.Ala304Gly)¹ from her mother (II:5) and a novel Exon 7 mutation (g.35668C > T; c.1046C > T, p.Ala349Val)¹ from her father (II:4). (c) The proband (III:1) presented with an anterior open bite and attrition of her posterior occlusal enamel. (d) The panoramic radiograph showed variable enamel thickness and contrast with dentin among teeth

FIGURE 4

Nanohardness of Enamel and Dentin. Top: Sagittally cut third molars, one from an unaffected (MMP20 ^+/+) individual and two from the proband in Family 6 (MMP20^mut), were subjected to nanohardness testing. Indents (blue dots) in enamel (a–e) were made at five positions (a: 100 µm from the enamel surface; b: midway between the DEJ and enamel surface; c: 100 µm from the DEJ; d: 50 µm from DEJ; and e: 10 µm from DEJ), at two locations: under the cusp tip and near the cervical margin. The MMP20^mut enamel varied in hardness at different locations, but was significantly softer at all locations. The enamel was hardest (Point A, 55% of normal) nearest the enamel surface, and softest (Point B, 13% of normal) half‐way between the DEJ and the enamel surface. Indents in dentin (f‐l) were made at seven positions (Point F, 10 µm from DEJ; g, 30 µm from DEJ; h, 50 µm from DEJ; i, 100 µm from DEJ; j, 500 µm from DEJ; k, midway between DEJ and pulp surface; and l, 100 µm away from the pulp surface)

FIGURE 5

Backscattered SEM Analysis of human molars. The same sagittally cut third molars from an unaffected (MMP20 ^+/+) individual, and the proband of Family 6 (MMP20^mut) that were prepared for nanohardness testing were imaged by bSEM (Top) and analyzed using IMAGEJ (Bottom). Mineral in a sample increases the whiteness of the bSEM image. The grayscale images at the top show the enamel layer is more highly mineralized than dentin and that the MMP20^mut enamel below a relatively hard surface layer is severely hypomineralized. Differences in the degree of mineralization were highlighted by ImageJ analysis where grayscale ranges were assigned different colors: 0–66 black; 67–85 white; 86–123 blue; 124–148 orange; and 149–255 red. The MMP20 ^+/+ enamel is red throughout, whereas the MMP20^mut enamel shows large areas of orange sometimes mixed with blue. The MMP20^mut dentin shows some increase in whiteness relative to the MMP20 ^+/+ dentin, but the difference in apparent mineral density is not striking

FIGURE 6

MicroCT and histological analyses of mouse mandibular first molars. (a‐d) microCT images and heat maps of mineral density of WT (a,b) and Mmp20 ^−/− (c,d) molars from 30 dpn mice. (M1, mandibular first molar; AB, alveolar bone; EN, enamel; DE, dentin) (e‐h) measurements of thickness and mineral density of crown (e,f) and root (g,h) dentin. Dentin thickness was measured in the crown dentin 150 µm above the cementoenamel junction (CEJ) and in the mesial root dentin 150 µm below CEJ (*p < .05; **p < .01; ***p < .01–0.001; ****p < .0001). (i‐l) Histology of WT (i,j) and Mmp20 ^−/− (k,l) molars. (PD, predentin; PDL, periodontal ligament). (m‐o) Histomorphometry of dentin structures. Thicknesses of dentin (m) and predentin (n) were measured at the level of CEJ, and the predentin/dentin ratio (o) was calculated from primary measurements