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Premature Stop Codon in MMP20 Causing Amelogenesis Imperfecta
1 Department of Biologic and Materials Sciences, Correspondence: * corresponding author, University of Michigan Dental Research Lab, 1210 Eisenhower Place, Ann Arbor, MI 48108, USA; janhu{at}umich.edu
Proteolytic enzymes are necessary for the mineralization of dental enamel during development, and mutations in the kallikrein 4 (KLK4) and enamelysin (MMP20) genes cause autosomal-recessive amelogenesis imperfecta (ARAI). So far, only one KLK4 and two MMP20 mutations have been reported. We have identified an ARAI-causing point mutation (c.102G>A, g.102G>A, and p.W34X) in exon 1 of MMP20 in a proband with autosomal-recessive hypoplastic-hypomaturation amelogenesis imperfecta. The G to A transition changes the tryptophan (W) codon (TGG) at amino acid position 34 into a translation termination (X) codon (TGA). No disease-causing sequence variations were detected in KLK4. The affected enamel is thin, with mild spacing in the anterior dentition. The enamel layer is hypomineralized, does not contrast with dentin on radiographs, and tends to chip away from the underlying dentin. An intrinsic yellowish pigmentation is evident, even during eruption. The phenotype supports current ideas concerning the function of enamelysin.
Key Words: enamelysin • MMP20 • enamel • tooth • amelogenesis imperfecta
Dental enamel forms in stages. During the secretory stage, amelogenin enamelin, and ameloblastin are secreted by ameloblasts. These proteins have been isolated from developing enamel and accumulate during the secretory stage, mainly as proteolytic cleavage products of the originally secreted proteins (Fincham et al., 1999). Early in the maturation stage, the accumulated cleavage products are degraded and re-absorbed into the overlying ameloblasts, and the rate of mineral deposition accelerates as the enamel crystallites thicken (Smith, 1998). Protease inhibition studies of secretory and maturation-stage enamel extracts have demonstrated that matrix metalloproteinase (MMP) activity predominates during the secretory stage, while serine protease activity is mainly observed during the maturation stage (Overall and Limeback, 1988). Cloning of mRNA extracted from porcine developing teeth revealed 2 novel proteases: enamelysin (MMP-20) (Bartlett et al., 1996) and kallikrein 4 (KLK4) (Simmer et al., 1998). Enamelysin appears as a doublet at 45 and 41 kDa on casein zymograms (Fukae et al., 1998). Both bands represent the active protease, containing the catalytic and hemopexin domains, but lack the propeptide (Yamada et al., 2003). KLK4 appears as 2 smeared bands between 30 and 36 kDa on gelatin zymograms (Tanabe, 1984). The expression of MMP-20 and KLK4 during tooth formation has been characterized by in situ hybridization (Hu et al., 2002; Simmer et al., 2004). Both enzymes are expressed by ameloblasts and odontoblasts. Ameloblasts express MMP-20 prior to the onset of mineralization (Bègue-Kirn et al., 1998), and expression continues into early maturation. MMP-20 catalyzes the partial degradation, or processing, of enamel proteins during the secretory stage. Recombinant enamelysin cleaves recombinant amelogenin (Ryu et al., 1999) and recombinant ameloblastin (Iwata et al., 2007) at the same sites in vitro as are cleaved in vivo, to generate the cleavage products that accumulate in the secretory-stage matrix. Ameloblasts first express KLK4 in the transition between the secretory and maturation stages, and KLK4 expression continues throughout the maturation stage (Hu et al., 2000). KLK4 degrades recombinant amelogenin in vitro, generating cleavage products that are not the same as those found in secretory-stage enamel (Ryu et al., 2002). The distinctive, but partially overlapping, patterns of MMP-20 and KLK4 expression, as well as the sharp differences in the way they individually cleave enamel proteins, suggest that these enzymes play similar but separate roles in enamel formation. It is a general principle that disturbances of tooth development are manifested as dental malformations that are characteristic of the timing of the disturbance (Hu and Simmer, 2007). For instance, defects in genes expressed early in tooth development, such as PAX9, MSX1, and AXIN2, are associated with oligodontia or familial tooth agenesis (Vastardis et al., 1996; Stockton et al., 2000; Lammi et al., 2004). This principle also applies to the stages of amelogenesis. Amelogenin, enamelin, and ameloblastin null mice all exhibit thin or absent enamel layers, which correlate with their critical expression during the secretory stage (Gibson et al., 2001; Fukumoto et al., 2004; Masuya et al., 2005). Humans with AMELX mutations in males (Kida et al., 2007) or ENAM mutations affecting both alleles (Ozdemir et al., 2005) have severely hypoplastic (thin) enamel. The relationship between the normal timing of a gene’s expression and the characteristics of the enamel malformations that occur in its absence appears to be more complex in the case of enamelysin. The enamelysin null mice deposit an enamel layer that is thinner than normal, tends to chip off from the underlying dentin, has disorganized prisms, is less highly mineralized, and retains more protein than normal enamel (Caterina et al., 2002; Bartlett et al., 2004). The phenotype is indicative of disturbances occurring in both the secretory and maturation stages of amelogenesis. The enamel phenotype in two autosomal-recessive amelogenesis imperfecta (ARAI) kindreds caused by MMP20 mutations has been described (Kim et al., 2005; Ozdemir et al., 2005). In both cases, the enamel was pigmented, typically yellowish to brownish, but appeared to be normal in thickness. The enamel layer was less highly mineralized and did not contrast with dentin on radiographs. This phenotype is classic hypomaturation amelogenesis imperfecta, and suggests that, in humans, MMP20 functions primarily in enamel maturation (Witkop and Sauk, 1977). A classic hypomaturation phenotype was also observed in a kindred with ARAI caused by defects in KLK4 (Hart et al., 2004). In this report, we ask the experimental questions: What is the range of phenotypes exhibited by kindreds with MMP20 defects? and How can these phenotypes be rationalized in terms of MMP-20 function? We describe the third MMP20 mutation in a kindred with ARAI, and the first MMP20 phenotype displaying hypoplastic and hypomaturation enamel.
Protocol Approval The study protocol and participant consent forms were reviewed and approved by the Institutional Review Boards at the University of Michigan and at Taipei Medical University Hospital.
Individual Recruitment and Examination
Mutation Analysis
DNA Sequence Analysis
Evaluation of Study Participants Clinically, the enamel layer of the proband was characterized by a generalized surface roughness and non-homogenous yellowish-brown stain (Fig. 1  ). The erupting second permanent molars were severely discolored, suggesting that the discoloration was intrinsic in nature. Vertical shear fractures of the enamel on functional cusps exposed the underlying dentin on many teeth. Radiographically, tooth number and size were within normal limits, but the enamel layer was thin and showed little contrast with dentin, so the position of the DEJ could not be discerned (Fig. 2). No additional oral pathology was noted. Mild bone loss was observed around the first molars, and was associated with stainless steel crown restorations and inadequate dental hygiene. Following a thorough clinical and radiographic evaluation of the proband’s father, mother, and older brother, it was concluded that no other members of the family were affected. Taken together, the clinical findings are consistent with a diagnosis of autosomal-recessive hypoplastic-hypomaturation AI.
Mutational analysis DNA amplification of the KLK4 coding region was performed first. Single-nucleotide changes were identified, but all of the variations are recognized single-nucleotide polymorphisms in the databases and are considered not likely to be disease-causing. Mutational analysis of MMP20 identified a nonsense mutation in the coding region of exon 1 (Fig. 1  ). This single-nucleotide (G to A) substitution converts the tryptophan (W) codon (TGG) at amino acid position 34 into a translation termination (X) codon (TGA). The standard designations for this mutation in the cDNA, gene, and protein are c.102G>A, g.102G>A, and p.W34X, respectively. The distribution of this mutation in the four participating members of the kindred was determined. The proband is the only person with both MMP20 alleles affected and the only one displaying an enamel phenotype.
The G>A transition in codon 34 of MMP20 was observed in both MMP alleles in the proband with autosomal-recessive hypoplastic-hypomaturation amelogenesis imperfecta. This single-nucleotide substitution introduces a premature translation termination codon into exon 1 and would likely cause the defective MMP20 transcripts to be degraded by the nonsense-mediated decay system (Wagner and Lykke-Andersen, 2002). If translated, the defective MMP20 mRNA transcripts could have directed the synthesis of only a 33-amino-acid peptide (MKVLPASGLAVFLIMALKFSTAAP SLVAASPRT) containing the 22-residue signal peptide and 11 amino acids from the beginning of the propeptide domain (Llano et al., 1997). It is certain, then, that no functional MMP-20 was expressed during tooth formation in the proband. Based upon the known importance of enamelysin in dental enamel formation, it is highly probable that the p.W34X mutation caused the enamel phenotype in the proband. It also seems safe to conclude that the enamel defects observed in the proband resulted from the MMP20 null condition, although a toxic effect from translation of the truncated protein cannot be ruled out. There are now 3 defined MMP20 mutations reported in families with ARAI. The affected enamel phenotype in these families all showed stained, hypomineralized enamel that did not sharply contrast with dentin on radiographs and tended to chip away from the underlying dentin. The pigmentation is evident during eruption, suggesting that the stain is intrinsic and not acquired, and is presumed to be due to the retention of enamel proteins. The enamel layer is clearly thin in our proband and caused a mild spacing in the anterior dentition, in contrast to the tight contacts observed in her older brother. Although thin enamel was not evident in the two previously reported MMP20 phenotypes, a reduction in enamel thickness is hard to quantify, because the DEJ is not plainly delineated on radiographs, and because the formed enamel layer is altered by chipping and accelerated wear. Given the difficulties in the assessment of hypoplasia when associated with enamel hypomaturation, the enamel phenotypes observed in the three MMP20 kindreds are remarkably consistent, and the minor variations can be attributed to differences in genetic background. The enamel phenotype displayed in the MMP20 null condition provides insights into the function of enamelysin, although it must be kept in mind that the phenotype might reflect secondary events resulting from ameloblasts reacting pathologically to disturbances in the matrix caused by the absence of MMP-20 activity. The cleavage of enamel proteins by enamelysin may not be critical for crystal elongation, since the enamel crystals must lengthen significantly to achieve the observed thickness of hypoplastic enamel. The absence of secretory-stage proteolytic activity resulting in enamel hypomaturation recalls the results of earlier studies showing that significant amounts of enamel maturation occur during the secretory stage, where enamel crystals increase in both width and thickness while going from the enamel surface to the DEJ (Daculsi and Kerebel, 1978). The accumulation of enamel matrix cleavage products during the secretory stage is a steady-state process. Enamel proteins and MMP-20 are continually secreted by ameloblasts at the enamel surface. Enamelysin cleaves the secreted proteins into progressively smaller products. The abundance of any particular cleavage product is determined by its rate of generation (by proteolysis or secretion) and its rate of elimination (by further proteolysis) (Simmer and Hu, 2002). With increasing distance from the secretory front at the enamel surface, there is a decline in the amount of organic matrix, and the crystallites are able to increase in width and thickness. Failure to process and slowly degrade enamel proteins during the secretory stage may interfere with the progressive thickening of enamel crystals with depth. The resulting structural weaknesses may account for the observed chipping of enamel formed without enamelysin. The intrinsic staining suggests that KLK4 activity during the maturation stage is insufficient to overcome the increased enamel protein content and reduced crystal thickness in the deeper enamel that occurs in the absence of MMP-20 activity. It may also be that MMP-20 activity is necessary to activate KLK4. It is not known how the propeptide is removed from the KLK4 zymogen in vivo, but enamelysin is able to catalyze removal of the KLK4 propeptide in vitro (Ryu et al., 2002). We conclude that MMP-20 is necessary for the gradual degradation of enamel proteins during the secretory stage, to remove the organic matrix between enamel crystallites and allow them to thicken progressively with depth. Without MMP-20, there may be insufficient maturation of the crystallites in the deeper enamel, which manifests itself clinically as a tendency for the enamel to shear.
We thank the family members for their participation and contributions. This investigation was supported by USPHS Research Grants DE15846 and DE016276 from the National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 29892, USA. Received for publication June 27, 2007. Revision received September 25, 2007. Accepted for publication October 17, 2007.
Journal of Dental Research, Vol. 87, No. 1,
56-59 (2008) This article has been cited by other articles:
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