Characterization of novel MSX1 mutations identified in Japanese patients with nonsyndromic tooth agenesis.

[oligodontia]

Since MSX 1 and PAX 9 are linked to the pathogenesis of nonsyndromic tooth agenesis , we performed detailed mutational analysis of these two genes sampled from Japanese patients . We identified two novel MSX 1 variants with an amino acid substitution within the homeodomain ; Thr 174 I le (T 174 I ) from a sporadic hypodontia case and Leu 205 A rg (L 205 R ) from a familial oligodontia case . Both the Thr 174 and Leu 205 residues in the MSX 1 homeodomain are highly conserved among different species . To define possible roles of mutations at these amino acids in the pathogenesis of nonsyndromic tooth agenesis , we performed several functional analyses . It has been demonstrated that MSX 1 plays a pivotal role in hard tissue development as a suppressor for mesenchymal cell differentiation . To evaluate the suppression activity of the variants in mesenchymal cells , we used the myoD-promoter , which is one of convenient reporter assay system for MSX 1 . Although the gene products of these MSX 1 variants are stable and capable of normal nuclear localization , they do not suppress myoD-promoter activity in differentiated C 2 C 12 cells . To clarify the molecular mechanisms underlying our results , we performed further analyses including electrophoretic mobility shift assays , and co -immunoprecipitation assays to survey the molecular interactions between the mutant MSX 1 proteins and the oligonucleotide DNA with MSX 1 consensus binding motif or EZH 2 methyltransferase . Since EZH 2 is reported to interact with MSX 1 and regulate MSX 1 mediated gene suppression , we hypothesized that the T 174 I and L 205 R substitutions would impair this interaction . We conclude from the results of our experiments that the DNA binding ability of MSX 1 is abolished by these two amino acid substitutions . This illustrates a causative role of the T 174 I and L 205 R MSX 1 homeodomain mutations in tooth agenesis , and suggests that they may influence cell proliferation and differentiation resulting in lesser tooth germ formation in vivo .