MMP20 hemopexin domain mutation in amelogenesis imperfecta

Abstract

Proteolytic enzymes serve important functions during dental enamel formation, and mutations in the kallikrein 4 (KLK4) and enamelysin (MMP20) genes cause autosomal-recessive amelogenesis imperfecta (ARAI). So far, only 1 KLK4 and 3 MMP20 mutations have been reported in ARAI kindreds. To determine whether ARAI in a family with a hypomaturation-type enamel defect is caused by mutations in the genes encoding enamel proteolytic enzymes, we performed mutational analysis on candidate genes. Mutational and haplotype analyses revealed an ARAI-causing point mutation (c.910G>A, p.A304T) in exon 6 of MMP20 that results in a single amino acid substitution in the hemopexin domain. Western blot analysis showed decreased expression of the mutant protein, but zymogram analysis demonstrated that this mutant was a functional protein. The proband and an affected brother were homozygous for the mutation, and both unaffected parents were carriers. The enamel of newly erupted teeth had normal thickness, but was chalky white and became darker with age.

Figure 1.

Pedigrees, mutational analyses of the AI kindred, and alignment of amino acid sequences. (A) Pedigree and haplotype of the kindred with the (g.18,742G>A) MMP20 mutation. (B) DNA sequencing chromatogram of the normal individual IV:2. (C) DNA sequencing chromatogram of the carrier individual IV:1. (D) DNA sequencing chromatogram of the proband IV:4. (E) MMP20 amino acid sequence alignment of various species. Note the high conservation of the affected alanine (A) among the species.

Figure 2.

Clinical photographs and radiographs of proband (IV:4) at age 10 yrs. (A) Frontal view. Overjet and overbite are within normal limits. Anterior teeth have dull, chalky hypomatured enamel. (B) Maxillary occlusal view. Enamel breakdown can be seen in the permanent first molars and deciduous teeth. (C) Mandibular occlusal view. Anterior teeth have slight staining on the labial cervical portion. (D) Full-mouth intra-oral radiographs. Enamel has reduced radiopacity, but normal thickness can be seen in the unerupted teeth.

Figure 3.

Clinical photographs and radiographs of the affected brother of the proband (IV:3) at age 12 yrs. (A) Frontal view. Overjet and overbite are minimal, showing the edge-to-edge bite. Anterior teeth have dark brown pigmented areas. (B) Maxillary occlusal view. Enamel breakdown can be seen in the permanent first molars and deciduous teeth. Newly erupting premolars do not have pigmentation. (C) Mandibular occlusal view. Anterior teeth have dark staining. (D) Full-mouth intra-oral radiographs. Enamel has reduced radiopacity, but normal thickness can be seen in the newly erupted premolars.

Figure 4.

Zymography and Western blot analysis. The zymogram revealed that mutant MMP20 was catalytically active; however, Western blot analysis showed that the amount of mutant MMP20 in the culture media was much less than that of normal MMP20. Lane Ev indicates empty expression vector only; Wt indicates normal MMP20; M1 and M2 indicate mutant MMP20.