Evolutionary Analysis Predicts Sensitive Positions of MMP20 and Validates Newly- and Previously-Identified MMP20 Mutations Causing Amelogenesis Imperfecta

Abstract

Amelogenesis imperfecta (AI) designates a group of genetic diseases characterized by a large range of enamel disorders causing important social and health problems. These defects can result from mutations in enamel matrix proteins or protease encoding genes. A range of mutations in the enamel cleavage enzyme matrix metalloproteinase-20 gene (MMP20) produce enamel defects of varying severity. To address how various alterations produce a range of AI phenotypes, we performed a targeted analysis to find MMP20 mutations in French patients diagnosed with non-syndromic AI. Genomic DNA was isolated from saliva and MMP20 exons and exon-intron boundaries sequenced. We identified several homozygous or heterozygous mutations, putatively involved in the AI phenotypes. To validate missense mutations and predict sensitive positions in the MMP20 sequence, we evolutionarily compared 75 sequences extracted from the public databases using the Datamonkey webserver. These sequences were representative of mammalian lineages, covering more than 150 million years of evolution. This analysis allowed us to find 324 sensitive positions (out of the 483 MMP20 residues), pinpoint functionally important domains, and build an evolutionary chart of important conserved MMP20 regions. This is an efficient tool to identify new- and previously-identified mutations. We thus identified six functional MMP20 mutations in unrelated families, finding two novel mutated sites. The genotypes and phenotypes of these six mutations are described and compared. To date, 13 MMP20 mutations causing AI have been reported, making these genotypes and associated hypomature enamel phenotypes the most frequent in AI.

Keywords: MMP20; amelogenesis imperfecta; evolution; mutations; phenotype.

Figures

Figure 1

SLAC analysis. The dN-dS value was analyzed along the MMP20 codon sequence. dN, non-synonymous substitution rate; dS, synonymous substitution rate. When dN-dS 0 (i.e., dN > dS), the codon is considered positively selected.

Figure 2

Evolutionary chart of MMP20. This chart was obtained from the alignment of 75 sequences representative of the main mammalian lineages (approx. 180 million years of evolution) and was deduced from the results obtained when dN/dS was calculated at each codon of MMP20 by Consurf (see Supplementary Figures 1, 2). Human sequence is used as a reference. Positions subjected to purifying selecton are on black (conserved positions) and gray (conservative positions) background. Variable positions on white background.

Figure 3

Mutational analysis of the two new MMP20 mutations. (A,C) Pedigree of the AI kindred; (B,D) DNA sequencing chromatograms of control (+/+) and of heterozygous (+/−) mutations. Arrows point to the mutation sites. See Figure 2, Supplementary Figure 1, Supplementary Table 2 for the validation of these mutations by means of evolutionary analyses. (A,B) Patient 1 with the homozygous mutation (c.323 A>G; p.Y108C). (C,D) Patient 2 with the compound heterozygous mutation (c.567 T>C; p.L189P / c.910 G>A; p.A304T). The second mutation was already reported as homozygous MMP20 mutation (Lee et al., 2010).

Figure 4

Hypomature amelogenesis imperfecta encountered in patients with diverse MMP20 mutations. (A–D): Patient 1 (c. 323 A>G). Intraoral clinical views and panoramic radiograph of primary, mixed (A: 6 years old; B: 7 years old) and permanent (C,D: 11 year old) dentitions. Note the limited contrast between enamel and dentine on X-rays. (E,F): Patient 2 (c.567 T>C + c.910 G>A). Primary dentition of a 5 year old girl. No enamel or very thin enamel was visible on X-rays. (G–J): Patient 3 (c.126+6 t>g + c.954-2 a>t). A young boy at 5 (G,I: primary teeth) and then at 8 years (H,J: permanent teeth). Limited radio-opaque enamel, if none, was seen on X-rays. (K, L): Patient 4 (c.389 C>T + c.954-2 a>t). Permanent teeth of a 20-year old man. Hypomature enamel is clearly visible on X-rays. (M,N): Patient 5 and patient 6 (c.954-2 a>t). Two girls displaying the same mutation leading to similar phenotypes with hypomature amelogenesis imperfecta.

Figure 5

MMP20 mutations known to date. Schematical representation of the human MMP20 gene structure with indication of the previously reported mutations and the two new mutations identified in this study (in red).