title: Characterization of novel MSX1 mutations identified in Japanese patients with nonsyndromic tooth agenesis.
abstract: Since MSX1 and PAX9 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 MSX1 variants with an amino acid substitution within the homeodomain; Thr174Ile (T174I) from a sporadic hypodontia case and Leu205Arg (L205R) from a familial oligodontia case. Both the Thr174 and Leu205 residues in the MSX1 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 MSX1 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 MSX1. Although the gene products of these MSX1 variants are stable and capable of normal nuclear localization, they do not suppress myoD-promoter activity in differentiated C2C12 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 MSX1 proteins and the oligonucleotide DNA with MSX1 consensus binding motif or EZH2 methyltransferase. Since EZH2 is reported to interact with MSX1 and regulate MSX1 mediated gene suppression, we hypothesized that the T174I and L205R substitutions would impair this interaction. We conclude from the results of our experiments that the DNA binding ability of MSX1 is abolished by these two amino acid substitutions. This illustrates a causative role of the T174I and L205R MSX1 homeodomain mutations in tooth agenesis, and suggests that they may influence cell proliferation and differentiation resulting in lesser tooth germ formation in vivo.
authors.affiliation: Department of Maxillofacial Surgery, School of Dentistry, Aichi-Gakuin University, Nagoya, Aichi, Japan; Department of Dentistry and Oral Surgery, Aichi Children's Health and Medical Center, Obu, Aichi, Japan; Department of Perinatology, Institute for Developmental Research, Aichi Human Service Center, Kasugai, Aichi, Japan.
authors.forename: Seishi
authors.initials: S
authors.lastname: Yamaguchi
authors.affiliation: Department of Maxillofacial Surgery, School of Dentistry, Aichi-Gakuin University, Nagoya, Aichi, Japan; Department of Oral and Maxillofacial Surgery, Toyota Memorial Hospital, Toyota, Aichi, Japan.
authors.forename: Junichiro
authors.initials: J
authors.lastname: Machida
authors.affiliation: Department of Maxillofacial Surgery, School of Dentistry, Aichi-Gakuin University, Nagoya, Aichi, Japan; Department of Oral and Maxillofacial Surgery, Japanese Red Cross Society Himeji Hospital, Himeji, Hyogo, Japan.
authors.forename: Munefumi
authors.initials: M
authors.lastname: Kamamoto
authors.affiliation: Department of Maxillofacial Surgery, School of Dentistry, Aichi-Gakuin University, Nagoya, Aichi, Japan; Department of Perinatology, Institute for Developmental Research, Aichi Human Service Center, Kasugai, Aichi, Japan.
authors.forename: Masashi
authors.initials: M
authors.lastname: Kimura
authors.affiliation: Department of Maxillofacial Surgery, School of Dentistry, Aichi-Gakuin University, Nagoya, Aichi, Japan; Department of Perinatology, Institute for Developmental Research, Aichi Human Service Center, Kasugai, Aichi, Japan.
authors.forename: Akio
authors.initials: A
authors.lastname: Shibata
authors.affiliation: Department of Maxillofacial Surgery, School of Dentistry, Aichi-Gakuin University, Nagoya, Aichi, Japan; Department of Perinatology, Institute for Developmental Research, Aichi Human Service Center, Kasugai, Aichi, Japan.
authors.forename: Tadashi
authors.initials: T
authors.lastname: Tatematsu
authors.affiliation: Department of Maxillofacial Surgery, School of Dentistry, Aichi-Gakuin University, Nagoya, Aichi, Japan.
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Miyachi
authors.affiliation: Department of Perinatology, Institute for Developmental Research, Aichi Human Service Center, Kasugai, Aichi, Japan.
authors.forename: Yujiro
authors.initials: Y
authors.lastname: Higashi
authors.affiliation: Department of Periodontology, University of Alabama at Birmingham School of Dentistry, Institute of Oral Health Research, Birmingham, Alabama, United States of America.
authors.forename: Peter
authors.initials: P
authors.lastname: Jezewski
authors.affiliation: Department of Embryology, Institute for Developmental Research, Aichi Human Service Center, Kasugai, Aichi, Japan.
authors.forename: Atsuo
authors.initials: A
authors.lastname: Nakayama
authors.affiliation: Department of Maxillofacial Surgery, School of Dentistry, Aichi-Gakuin University, Nagoya, Aichi, Japan.
authors.forename: Kazuo
authors.initials: K
authors.lastname: Shimozato
authors.affiliation: Department of Maxillofacial Surgery, School of Dentistry, Aichi-Gakuin University, Nagoya, Aichi, Japan; Department of Perinatology, Institute for Developmental Research, Aichi Human Service Center, Kasugai, Aichi, Japan.
authors.forename: Yoshihito
authors.initials: Y
authors.lastname: Tokita
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000848:Anodontia / Q000235:genetics*; D001665:Binding Sites; D017124:Conserved Sequence; D004252:DNA Mutational Analysis; D024202:Electrophoretic Mobility Shift Assay; D000071221:Enhancer of Zeste Homolog 2 Protein; D005260:Female; D005786:Gene Expression Regulation; D006801:Humans; D007564:Japan; D051957:MSX1 Transcription Factor / Q000235:genetics*; D008297:Male; D008969:Molecular Sequence Data; D010375:Pedigree; D063151:Polycomb Repressive Complex 2 / Q000378:metabolism; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein
chemical_list: D051957:MSX1 Transcription Factor; C484083:MSX1 protein, human; C498409:EZH2 protein, human; D000071221:Enhancer of Zeste Homolog 2 Protein; D063151:Polycomb Repressive Complex 2
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