Identification of nuclear localization signals in the human homeoprotein MSX1.

MSX1 is one of the homeoproteins with the homeodomain (HD) sequence, which regulates proliferation and differentiation of mesenchymal cells. In this study, we investigated the nuclear localization signal (NLS) in the MSX1 HD by deletion and amino acid substitution analyses. The web-based tool NLStradamus predicted 2 putative basic motifs in the N- and C-termini of the MSX1 HD. Green fluorescent protein (GFP) chimera studies revealed that NLS1 (161RKHKTNRKPR170) and NLS2 (216NRRAKAKR223) were independently insufficient for robust nuclear localization. However, they can work cooperatively to promote nuclear localization of MSX1, as was shown by the 2 tandem NLS motifs partially restoring functional NLS, leading to a significant nuclear accumulation of the GFP chimera. These results demonstrate a unique NLS motif in MSX1, which consists of an essential single core motif in helix-I, with weak potency, and an auxiliary subdomain in helix-III, which alone does not have nuclear localization potency. Additionally, other peptide sequences, other than predicted 2 motifs in the spacer, may be necessary for complete nuclear localization in MSX1 HD.

A single nucleotide polymorphism associated with isolated cleft lip and palate, thyroid cancer and hypothyroidism alters the activity of an oral epithelium and thyroid enhancer near FOXE1.

Three common diseases, isolated cleft lip and cleft palate (CLP), hypothyroidism and thyroid cancer all map to the FOXE1 locus, but causative variants have yet to be identified. In patients with CLP, the frequency of coding mutations in FOXE1 fails to account for the risk attributable to this locus, suggesting that the common risk alleles reside in nearby regulatory elements. Using a combination of zebrafish and mouse transgenesis, we screened 15 conserved non-coding sequences for enhancer activity, identifying three that regulate expression in a tissue specific pattern consistent with endogenous foxe1 expression. These three, located -82.4, -67.7 and +22.6 kb from the FOXE1 start codon, are all active in the oral epithelium or branchial arches. The -67.7 and +22.6 kb elements are also active in the developing heart, and the -67.7 kb element uniquely directs expression in the developing thyroid. Within the -67.7 kb element is the SNP rs7850258 that is associated with all three diseases. Quantitative reporter assays in oral epithelial and thyroid cell lines show that the rs7850258 allele (G) associated with CLP and hypothyroidism has significantly greater enhancer activity than the allele associated with thyroid cancer (A). Moreover, consistent with predicted transcription factor binding differences, the -67.7 kb element containing rs7850258 allele G is significantly more responsive to both MYC and ARNT than allele A. By demonstrating that this common non-coding variant alters FOXE1 expression, we have identified at least in part the functional basis for the genetic risk of these seemingly disparate disorders.

p53 and ki67 as biomarkers in determining response to chemoprevention for oral leukoplakia.

BACKGROUND: We performed a randomized controlled chemoprevention trial of oral leukoplakia by administrating a low dose of beta-carotene and vitamin C supplements. 17% of subjects in the experimental arm (4/23) demonstrated clinical remission (complete or partial response) at completion of the trial. The objective of this study was to determine whether baseline expression of p53 and ki67 demonstrated any differences between those responding or not responding to our intervention. A secondary objective was to elucidate any relationship between dietary factors and clinical responses. METHODS: For this biomarker study, we included all subjects in the experimental group (n = 23) who were non-smokers. Among 16 who completed the trial for 1 year of supplementation, there were four responders and 12 non-responders at 1-year follow-up. Following immuno-staining for p53 and ki67, the percentage of positive cell nuclei were analyzed as labeling index (LI). RESULTS: Expression of p53 was greater in basal layers than in para-basal layers. Mean para-basal LI of p53 was higher in non-responding (26.0) than in responding subjects (11.2) (P = 0.028). ki67 LIs were not significantly different in the two groups. CONCLUSIONS: Expression of p53 was inversely related to clinical response to the supplements. Other biomarkers that may recognize subject's responsiveness to chemoprevention require further study.

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.

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.

Novel frameshift variant of WNT10A in a Japanese patient with hypodontia.

Congenital tooth agenesis is caused by the impairment of crucial genes related to tooth development, such as Wnt signaling pathway genes. Here, we investigated the genetic causes of sporadic congenital tooth agenesis. Exome sequencing, followed by Sanger sequencing, identified a novel single-nucleotide deletion in WNT10A (NC_000002.12(NM_025216.3):c.802del), which was not found in the healthy parents of the patient. Thus, we concluded that the variant was the genetic cause of the patient's agenesis.

Novel WNT10A variant in a Japanese case of nonsyndromic oligodontia.

Congenital tooth agenesis is one of the most common anomalies in humans. Many genetic factors are involved in tooth development, including MSX1, PAX9, WNT10A, and LRP6. Thus, mutations in these genes can cause congenital tooth agenesis in humans. In this study, we identified a novel nonsense WNT10A variant, NM_025216.3(WNT10A_v001):c.1090A > T, which produces a C-terminal truncated gene product, p.(Lys364*), in a sporadic form of congenital tooth agenesis. The variant was not found in the healthy parents and thus was considered to cause congenital tooth agenesis in the case.

FOXE1 association with both isolated cleft lip with or without cleft palate, and isolated cleft palate.

Nonsyndromic orofacial clefts are a common complex birth defect caused by genetic and environmental factors and/or their interactions. A previous genome-wide linkage scan discovered a novel locus for cleft lip with or without cleft palate (CL/P) at 9q22-q33. To identify the etiologic gene, we undertook an iterative and complementary fine mapping strategy using family-based CL/P samples from Colombia, USA and the Philippines. Candidate genes within 9q22-q33 were sequenced, revealing 32 new variants. Concurrently, 397 SNPs spanning the 9q22-q33 2-LOD-unit interval were tested for association. Significant SNP and haplotype association signals (P = 1.45E - 08) narrowed the interval to a 200 kb region containing: FOXE1, C9ORF156 and HEMGN. Association results were replicated in CL/P families of European descent and when all populations were combined the two most associated SNPs, rs3758249 (P = 5.01E - 13) and rs4460498 (P = 6.51E - 12), were located inside a 70 kb high linkage disequilibrium block containing FOXE1. Association signals for Caucasians and Asians clustered 5' and 3' of FOXE1, respectively. Isolated cleft palate (CP) was also associated, indicating that FOXE1 plays a role in two phenotypes thought to be genetically distinct. Foxe1 expression was found in the epithelium undergoing fusion between the medial nasal and maxillary processes. Mutation screens of FOXE1 identified two family-specific missense mutations at highly conserved amino acids. These data indicate that FOXE1 is a major gene for CL/P and provides new insights for improved counseling and genetic interaction studies.

Novel MSX1 frameshift mutation in a Japanese family with nonsyndromic oligodontia.

Congenital tooth agenesis is a common anomaly in humans. We investigated the etiology of human tooth agenesis by exome analysis in Japanese patients, and found a previously undescribed heterozygous deletion (NM_002448.3(MSX1_v001):c.433_449del) in the first exon of the MSX1 gene. The deletion leads to a frameshift and generates a premature termination codon. The truncated form of MSX1, namely, p.(Trp145Leufs*24) lacks the homeodomain, which is crucial for transcription factor function.

A novel LRP6 variant in a Japanese family with oligodontia.

Congenital tooth agenesis is a common anomaly in human development. We performed exome sequence analysis of genomic DNA collected from Japanese patients with tooth agenesis and their relatives. We found a novel single-nucleotide insertion in the LRP6 gene, the product of which is involved in Wnt/ß-catenin signaling as a coreceptor for Wnt ligands. The single-nucleotide insertion results in a premature stop codon in the extracellular region of the encoded protein.

Prognostic factors for keratocystic odontogenic tumor (odontogenic keratocyst): analysis of clinico-pathologic and immunohistochemical findings in cysts treated by enucleation.

BACKGROUND: The purpose of this study was to determine prognostic factors for the recurrence of keratocystic odontogenic tumors (KCOTs) following simple enucleation by examining clinico-pathologic and immunohistochemical findings. METHODS: Following enucleation, the frequency of recurrence among 32 subjects diagnosed with KCOT was analyzed for tumor site, radiographic and histologic features, and immunopositivity for Ki-67 and p53. RESULTS: Keratocystic odontogenic tumors in four out of 32 subjects (12.5%) recurred during the follow-up period (median: 33 months, range: 7-114 months). Three out of four subjects (75.0%) among recurrent group showed high expression of Ki-67 (LI >10%) in basal layer and four (4/28; 14.3%) among non-recurrence group (P = 0.025). Expression of p53 among non-recurrent group was observed in 11 subjects (11/28; 39.3%), and in three subjects (3/4; 75.0%) among the recurrent group (P = 0.295). Hazard risk for the recurrence of KCOT was 4.02 (95% CI 1.42-18.14) for high Ki-67 expression in the basal layer by the Cox proportional hazard model (P = 0.009). In our study, none of the other clinico-pathologic variables were associated with the recurrence of KCOT. CONCLUSION: The results suggested that the evaluation of Ki-67 expression in KCOT at the time of pathological diagnosis might be helpful for consideration of appropriate adjunctive surgical procedures to avoid a recurrence and may serve as a prognostic marker.

Interferon regulatory factor 6 (IRF6) gene variants and the risk of isolated cleft lip or palate.

BACKGROUND: Cleft lip or palate (or the two in combination) is a common birth defect that results from a mixture of genetic and environmental factors. We searched for a specific genetic factor contributing to this complex trait by examining large numbers of affected patients and families and evaluating a specific candidate gene. METHODS: We identified the gene that encodes interferon regulatory factor 6 (IRF6) as a candidate gene on the basis of its involvement in an autosomal dominant form of cleft lip and palate, Van der Woude's syndrome. A single-nucleotide polymorphism in this gene results in either a valine or an isoleucine at amino acid position 274 (V274I). We carried out transmission-disequilibrium testing for V274I in 8003 individual subjects in 1968 families derived from 10 populations with ancestry in Asia, Europe, and South America, haplotype and linkage analyses, and case-control analyses, and determined the risk of cleft lip or palate that is associated with genetic variation in IRF6. RESULTS: Strong evidence of overtransmission of the valine (V) allele was found in the entire population data set (P<10(-9)); moreover, the results for some individual populations from South America and Asia were highly significant. Variation at IRF6 was responsible for 12 percent of the genetic contribution to cleft lip or palate and tripled the risk of recurrence in families that had already had one affected child. CONCLUSIONS: DNA-sequence variants associated with IRF6 are major contributors to cleft lip, with or without cleft palate. The contribution of variants in single genes to cleft lip or palate is an important consideration in genetic counseling.

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/ß-catenin signaling.

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.

Two missense mutations in the IRF6 gene in two Japanese families with Van der Woude syndrome.

Van der Woude syndrome (VWS) is a common autosomal dominant disorder with cleft lip and/or palate and lower lip pits. Its prevalence is estimated to be 1/33,600 in the Finnish Population, and 1/47,813 in the Japanese. We performed mutation analysis of the IRF6 gene by direct sequencing in 2 unrelated Japanese families that consist of a total of 3 affected members with cleft lip and palate associated with lower lip pits. Consequently, we found novel base substitutions, 25C>T, in IRF6-exon 3 in a boy, his mother, and his phenotypically normal maternal grandmother in one of the families. A known mutation, 250C>T, was identified in exon 4 of a girl and her unaffected father in the other family. The same mutations were never observed among 190 healthy Japanese. The results indicate incomplete penetrance and variable expressivity in the families. Because 25C>T and 250C>T predict to lead to R9W and R84C substitutions, respectively, at the most conserved DNA binding domain of IRF6, and because arginine at positions 9 and 84 is highly conserved among IRFs, the 2 mutations may lead to abolish the DNA binding activity in the developing craniofacial region. To our knowledge, this is the first report of IRF6 mutations observed in Japanese VWS patients.

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.

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.

Clinical and functional data implicate the Arg(151)Ser variant of MSX1 in familial hypodontia.

Multiple previous reports confirm that several missense alleles of MSX1 exhibit Mendelian inheritance of an oligodontia phenotype (agenesis of more than six secondary teeth besides third molars). However, the extent to which missense MSX1 alleles contribute to common, multifactorial disorders is less certain. It is still not yet clear whether multiple non-synonomous MSX1-coding variants identified among patients with oral clefting are merely neutral polymorphisms or whether any of these might represent real mutations with mild effects. The present work steps toward resolving these issues for at least one MSX1 allele: R151S, previously identified in a single Japanese proband with unilateral cleft lip and palate. Candidate gene sequencing within a patient cohort demonstrating mild tooth agenesis (loss of six or less secondary teeth besides third molars, hypodontia), secondarily identified this same MSX1 variant, functioning as a mildly deleterious, moderately penetrant allele. Four of five heterozygous R151S individuals from one Japanese family exhibited the hypodontia phenotype. The in vitro functional assays of the variant protein display partial repression activity with normal nuclear localization. These data establish that the MSX1-R151S allele is a low-frequency, mildly deleterious allele for familial hypodontia that alone is insufficient to cause oral facial clefting. Yet, as this work also establishes its hypomorphic nature, it suggests that it may in fact contribute to the likelihood of common birth disorder phenotypes, such as partial tooth agenesis and oral facial clefting. Nevertheless, the exact mechanism in which differential pleiotropy is manifested will need further and deeper clinical and functional analyses.

Searching for genes for cleft lip and/or palate based on breakpoint analysis of a balanced translocation t(9;17)(q32;q12).

OBJECTIVE: Identification of the breakpoints of disease-associated chromosome rearrangements can provide informative clues to a positional cloning approach for genes responsible for inherited diseases. Recently, we found a three-generation Japanese family segregating balanced chromosome translocation t(9;17)(q32;q12). One of the subjects had cleft lip and palate. We examined whether regions near the breakpoint could be associated with cleft lip and/or palate. METHODS: We determined the breakpoints involved in the translocation by fluorescence in situ hybridization analysis and subsequent long-range polymerase chain reaction. In order to study the role of these disrupted regions in nonsyndromic cleft lip and/or palate, we performed mutation analysis and a haplotype-based transmission disequilibrium test using tagging single-nucleotide polymorphisms in the flanking regions of the breakpoints in white and Filipino nonsyndromic cleft lip and/or palate populations. RESULTS: Sequence analysis demonstrated that two genes, SLC31A1 (solute carrier family 31 member 1) on chromosome 9 and CCL2 (chemokine ligand 2) on chromosome 17, were rearranged with the breaks occurring within their introns. It is interesting that SLC31A1 lies closed to BSPRY (B-box and SPRY domain), which is a candidate for involvement with cleft lip and/or palate. Some of the variants in BSPRY and CCL2 showed significant p values in the cleft lip and/or palate population compared with the control population. There was also statistically significant evidence of transmission distortion for haplotypes on both chromosomes 9 and 17. CONCLUSIONS: The data support previous reports that genes on chromosomal regions of 9q and 17q play an important role in facial development.

In a Vietnamese population, MSX1 variants contribute to cleft lip and palate.

PURPOSE: To identify causes of nonsyndromic cleft lip and palate in a Vietnamese population. METHODS: In this study, 175 families with at least one case of cleft lip and/or palate were studied using the candidate genes TGFA, MSX1, and TGFB3. RESULTS: Transmission distortion for alleles of MSX1 were demonstrated for the whole population and two missense mutations were identified, including one (P147Q) that is found in approximately 2% of the population. The P147Q appears to arise from a founder individual based on shared haplotypes in unrelated families. CONCLUSIONS: MSX1 contributes to nonsyndromic clefting in a Vietnamese population, and consistent with other studies, identifiable mutations in this gene cause about 2% of cases of nonsyndromic clefting.