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.

MSX1 mutations and associated disease phenotypes: genotype-phenotype relations

“…25 MSX1 and non-syndromic tooth agenesis Non-syndromic tooth agenesis (ns TA) is another common developmental anomaly that can be caused by MSX1 variants. 14 To date, nearly 20 MSX1 mutations have been related to ns TA 8,13,[26][27][28][29][30][31][32][33][34][35][36][37] (Table 2). Functional analyses of the mutant proteins suggest that haploinsufficiency of MSX1 underlies this phenotype.…”

“…As a result, the function of MSX1 as a transcriptional repressor can be greatly impaired. 27,39,40 This could also be the result of epigenetic silencing of MSX1 by DNA methylation giving rise to either or combinations of its phenotypes or increase risk for tumoral growth Syndromes caused by MSX1 mutations Mutations in or including the MSX1 gene can also cause syndromic forms of tooth agenesis, including Wolf-Hirschhorn syndrome, Witkop syndrome, and tooth agenesis combined with orofacial clefting (Table 3). The Wolf-Hirschhorn syndrome is caused by a deletion of the WHS locus -including MSX1 -on chromosome 4p.…”

Novel human mutation and CRISPR/Cas genome-edited mice reveal the importance of C-terminal domain of MSX1 in tooth and palate development

“…It has been demonstrated that transcriptional repression by MSX1 occurs in the absence of DNA-binding sites; the repressor function is attributed to multiple domains in the N- and C-terminal regions of MSX136. In silico analysis studies have indicated that missense mutations affecting MH4 in MSX1 alter the encoded 3D structures of the proteins, specifically by destabilizing the helix-turn-helix motif or altering its protein-DNA interactions3738. Moreover in vitro studies have demonstrated that nuclear localization of MSX1 protein is mediated by the homeodomain37.…”

Risk factors for teeth aplasia and hypoplasia in cleft lip and palate children

“…Традиционно считается, что аномалии зубов входят в состав генетической парадигмы, определяющей и развитие самой расщелины [17]. Действительно, доказано, что мутации генов MSX1 и PAX9, приводящие к селективной аплазии зубов [4,13,[18][19][20], также отвечают за развитие некоторых расщелин лица [11,15]. Кроме этих генов, однако, идентифицировано более 100, также сопряженных с РГН, но не влияющих на одонтогенез.…”