Tadashi Tatematsu

Novel nonsense mutation in MSX1 in familial nonsyndromic oligodontia: subcellular localization and role of homeodomain/MH4

Nonsyndromic tooth agenesis is one of the most common anomalies in human development. Part of the malformation is inherited and is associated with paired box 9 (PAX9), msh homeobox 1 (MSX1), and axin 2 (AXIN2) mutations. To obtain a comprehensive understanding of the genetic and molecular mechanisms that underlie this genetic disease, we investigated six familial and seven sporadic Japanese cases of nonsyndromic tooth agenesis. Searches for mutations in these candidate genes detected a novel nonsense mutation (c.416G>A) in exon 1 of MSX1 from a family with oligodontia. This mutation co-segregated in the affected family members. Moreover, this mutation produced a termination codon in the first exon and therefore the gene product (W139X) was truncated at the C terminus, hence, the entire homeodomain/MH4, which has many functions, such as DNA binding, protein-protein interaction, and nuclear localization, was absent. We characterized the properties of this truncated MSX1 by investigating the subcellular localization of the mutant gene product in transfected cells. The wild-type MSX1 localized exclusively at the nuclear periphery of transfected cells, whereas the mutant MSX1 was stable but localized diffusely throughout the whole cell. These results indicate that W139X MSX1 is responsible for tooth agenesis.

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

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.

An aberrant splice acceptor site due to a novel intronic nucleotide substitution in MSX1 gene is the cause of congenital tooth agenesis in a Japanese family.

Congenital tooth agenesis is caused by mutations in the MSX1, PAX9, WNT10A, or AXIN2 genes. Here, we report a Japanese family with nonsyndromic tooth agenesis caused by a novel nucleotide substitution in the intronic region between exons 1 and 2 of the MSX1 gene. Because the mutation is located 9 bp before exon 2 (c.452-9G>A), we speculated that the nucleotide substitution would generate an abnormal splice site. Using cDNA analysis of an immortalized patient blood cell, we confirmed that an additional 7-nucleotide sequence was inserted at the splice junction between exons 1 and 2 (c.451_452insCCCTCAG). The consequent frameshift generated a homeodomain-truncated MSX1 (p.R151fsX20). We then studied the subcellular localization of truncated MSX1 protein in COS cells, and observed that it had a whole cell distribution more than a nuclear localization, compared to that of wild-type protein. This result suggests a deletion of the nuclear localization signal, which is mapped to the MSX1 homeodomain. These results indicate that this novel intronic nucleotide substitution is the cause of tooth agenesis in this family. To date, most MSX1 variants isolated from patients with tooth agenesis involve single amino acid substitutions in the highly conserved homeodomain or deletion mutants caused by frameshift or nonsense mutations. We here report a rare case of an intronic mutation of the MSX1 gene responsible for human tooth agenesis. In addition, the missing tooth patterns were slightly but significantly different between an affected monozygotic twin pair of this family, showing that epigenetic or environmental factors also affect the phenotypic variations of missing teeth among patients with nonsyndromic tooth agenesis caused by an MSX1 haploinsufficiency.

Characterisation of novel RUNX2 mutation with alanine tract expansion from Japanese cleidocranial dysplasia patient

Cleidocranial dysplasia (CCD; MIM 119600) is an autosomal dominant skeletal dysplasia characterised by hypopalstic and/or aplastic clavicles, midface hypoplasia, absent or delayed closure of cranial sutures, moderately short stature, delayed eruption of permanent dentition and supernumerary teeth. The molecular pathogenesis can be explained in about two-thirds of CCD patients by haploinsufficiency of the RUNX2 gene. In our current study, we identified a novel and rare variant of the RUNX2 gene (c.181_189dupGCGGCGGCT) in a Japanese patient with phenotypic features of CCD. The insertion led an alanine tripeptide expansion (+3Ala) in the polyalanine tract. To date, a RUNX2 variant with alanine decapeptide expansion (+10Ala) is the only example of a causative variant of RUNX2 with polyalanine tract expansion to be reported, whilst RUNX2 (+1Ala) has been isolated from the healthy population. Thus, precise analyses of the RUNX2 (+3Ala) variant were needed to clarify whether the tripeptide expanded RUNX2 is a second disease-causing mutant with alanine tract expansion. We therefore investigated the biochemical properties of the mutant RUNX2 (+3Ala), which contains 20 alanine residues in the polyalanine tract. When transfected in COS7 cells, RUNX2 (+3Ala) formed intracellular ubiquitinated aggregates after 24h, and exerted a dominant negative effect in vitro. At 24h after gene transfection, whereas slight reduction was observed in RUNX2 (+10Ala), all of these mutants significantly activated osteoblast-specific element-2, a cis-acting sequence in the promoter of the RUNX2 target gene osteocalcin. The aggregation growth of RUNX2 (+3Ala) was clearly lower and slower than that of RUNX2 (+10Ala). Furthermore, we investigated several other RUNX2 variants with various alanine tract lengths, and found that the threshold for aggregation may be RUNX2 (+3Ala). We conclude that RUNX2 (+3Ala) is the cause of CCD in our current case, and that the accumulation of intracellular aggregates in vitro is related to the length of the alanine tract.

Genetic epidemiology of tooth agenesis in Japan: a population- and family-based study.

Tooth agenesis is one of the most common congenital anomalies in humans. However, the etiology of tooth agenesis remains largely unclear, as well as evidence base useful for genetic counseling. Therefore, we estimated the prevalence and sibling recurrence risk, and investigated agenetic patterns systematically. Tooth agenesis was classified into two subtypes: hypodontia (one to five missing teeth) and oligodontia (six or more missing teeth). The prevalence of these two subtypes were 6.8% [95% confidence interval (CI): 6.1–7.7%] and 0.1% (95% CI: 0.04–0.3%), respectively, and sibling recurrence risk of these were 24.5% (95% CI: 13.8–38.3%) and 43.8% (95% CI: 26.4–62.3%), respectively. This result suggests that the severe phenotype, oligodontia, might be mostly transmitted in a dominant fashion. Using a simple statistical modeling approach, our data were found to be consistent with a bilateral symmetry model, meaning that there was equal probability of missing teeth from the right and left sides.

A novel PITX2 mutation causing iris hypoplasia

Iris hypoplasia (IH) is rare autosomal dominant disorder characterized by a poorly developed iris stroma and malformations of the eyes and umbilicus. This disorder is caused by mutation of the paired-like homeodomain 2 (PITX2) gene. Here, we describe a novel PITX2 mutation (c.205C>T) in an IH family presenting with very mild eye features but with tooth agenesis as the most obvious clinical feature.

WNT10A variants isolated from Japanese patients with congenital tooth agenesis

It has been reported that dozens of WNT10A variants are associated with human isolated tooth agenesis, however, little is known about the precise phenotypes. In 50 Japanese patients with severe congenital tooth agenesis, we identified 11 patients with WNT10A variants. Comparing phenotypes between the tooth agenesis patients carrying the wild-type and variants of WNT10A, we revealed that the development of lateral incisors is relatively susceptive to insufficiency of WNT/β-catenin signaling.