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Objective Inherited congenital anomalies in tooth number, particularly hypodontia are relatively common. Although substantial progress has been made that permits a better understanding of the causes of tooth agenesis, overall knowledge of the phenotype:genotype correlations in this anomaly are still lacking. The aim in this study was to identify the causal gene mutation(s) in a family of two sisters with severe hypodontia (oligodontia) including 2nd premolars and 1st and 3rd molars, using whole exome sequencing (WES). Methods WES was performed using in-solution hybridization, followed by massively parallel sequencing. Results A frameshift insertion of 7 basepairs (GCAAGTT) in the homebox of MSX1 gene located in the exon 2 in heterozygous state has been identified in both sisters ( NM_002448 :exon2:c.572_573ins GCAAGTT: p.F191fs). Conclusion We conclude that this frameshift mutation in the homeodomain (which plays an essential role in DNA binding) of MSX1 gene is responsible for tooth agenesis in this family. This expands the phenotype-genotype correlation associated with MSX1 mutations. [ABSTRACT FROM AUTHOR]

Objectives In this study, the aim was to investigate a consanguineous Saudi family with non-syndromic premolars and third molars agenesis and to identify the causal mutation(s) using whole exome sequencing. Design Family phenotype and family pedigree were constructed from clinical and radiographic examinations. Whole exome sequencing was performed in two affected members of the Saudi family using the SureSelect Human all Exon 50 Mb kit (Agilent Technologies, Inc., Santa Clara, CA) and then sequenced on an Illumina HiSeq. SNP and indel calling were performed using samtools version 0.18 and were annotated using the software ANNOVAR. Results The family pedigree showed that the inheritance was autosomal dominant. Whole exome sequencing revealed that the affected members in this family were heterozygous with a novel frameshift mutation in exon 2 of the MSX1 gene, (NM_002448:c.750_751insACCGGCTGCC, p.F251PfsX92). Conclusions The novel MSX1 frameshift mutation was linked to a family with moderate to severe tooth agenesis phenotype affecting second premolars and third molars in both arches. This expands the genotype–phenotype of MSX1 associated conditions. [ABSTRACT FROM AUTHOR]

LIM homeobox transcription factor Lhx2 is known to be an important regulator of neuronal development, homeostasis of hair follicle stem cells, and self-renewal of hematopoietic stem cells; however, its function in skeletal muscle development is poorly understood. In this study, we found that overexpression of Lhx2 completely inhibits the myotube-forming capacity of C2C12 cells and primary myoblasts. The muscle dedifferentiation factors Msx1 and Msx2 were strongly induced by the Lhx2 overexpression. Short interfering RNA-mediated knockdown of Lhx2 in the developing limb buds of mouse embryos resulted in a reduction in Msx1 and Msx2 mRNA levels, suggesting that they are downstream target genes of Lhx2. We found two Lhx2 consensus-binding sites in the −2097 to −1189 genomic region of Msx1 and two additional sites in the −536 to +73 genomic region of Msx2 . These sequences were shown by luciferase reporter assay to be essential for Lhx2-mediated transcriptional activation. Moreover, electrophoretic mobility shift assays and chromatin immunoprecipitation assays showed that Lhx2 is present in chromatin DNA complexes bound to the enhancer regions of the Msx1 and Msx2 genes. These data demonstrate that Msx1 and Msx2 are direct transcriptional targets of Lhx2. In addition, overexpression of Lhx2 significantly enhanced the mRNA levels of bone morphogenetic protein 4 and transforming growth factor beta family genes. We propose that Lhx2 is involved in the early stage of skeletal muscle development by inducing multiple differentiation inhibitory factors. [ABSTRACT FROM AUTHOR]

The homeobox gene Msx1 encodes a transcription factor that is highly expressed during embryogenesis and postnatal development in bone. Mutations of the MSX1 gene in humans are associated with cleft palate and (or) tooth agenesis. A similar phenotype is observed in newborn mice invalidated for the Msx1 gene. However, little is known about Msx1 function in osteoblast differentiation and bone mineralization in vivo . In the present study, we aimed to explore the variations of individualized bone shape in a subtle way avoiding the often severe consequences associated with gene mutations. We established transgenic mice that specifically express Msx1 in mineral–matrix-secreting cells under the control of the mouse 2.3 kb collagen 1 alpha 1 (Col1α1) promoter, which enabled us to investigate Msx1 function in bone in vivo . Adult transgenic mice (Msx1-Tg) presented altered skull shape and mineralization resulting from increased Msx1 expression during bone development. Serial section analysis of the mandibles showed a high amount of bone matrix in these mice. In addition, osteoblast number, cell proliferation and apoptosis were higher in Msx1-Tg mice than in controls with regional differences that could account for alterations of bone shape. However, Von Kossa staining and μCT analysis showed that bone mineralization was lower in Msx1-Tg mice than in controls due to alteration of osteoblastic differentiation. Msx1 appears to act as a modeling factor for membranous bone; it stimulates trabecular bone metabolism but limits cortical bone growth by promoting apoptosis, and concomitantly controls the collagen-based mineralization process. [ABSTRACT FROM AUTHOR]

Abstract: Objectives: Oligodontia is defined as the congenital absence of 6 or more permanent teeth excluding the third molar. Tooth agenesis may be classified as syndromic/non-syndromic and as familial/sporadic. To date, more than 300 genes have been found to be involved in tooth development, but only a few of these genes, such as MSX1, PAX9 and AXIN2, are related to the condition of non-syndromic oligodontia. The objective of the present work was to investigate the disease-causing gene of non-syndromic oligodontia in a Han Chinese family and analyse the pathogenesis of mutations that result in oligodontia. Design: We examined all individuals of the oligodontia family by clinical and radiographic examinations. Based on the clinical manifestations, the candidate genes MSX, PAX9 and AXIN2 were selected to analyse and screen for mutations. Results: The clinical evaluation suggested that the family might show non-syndromic oligodontia. DNA sequencing of the MSX1 gene revealed two mutations in the two patients with oligodontia: a heterozygotic silent mutation, c.348C>T (P.Gly116=), in exon 1 and a homozygotic deletion of 11 nucleotides (c.469+56delins GCCGGGTGGGG) in the intron. However, the silent mutation and the deletion mutation were thought to be known polymorphisms (rs34165410 and rs34341187) by bioinformatics analysis. We did not detect any mutations in the PAX9 and AXIN2 genes of oligodontia patients. Conclusion: Our finding suggests that identified polymorphisms (c.348C>T and c.469+56delins GCCGGGTGGGG) may be responsible for the oligodontia phenotype in this Chinese family, but the association requires further study. [Copyright &y& Elsevier]

The Witkop syndrome is a rare autosomal dominant disorder characterized by the absence of several teeth and abnormalities of the nails. This is the first report of a rare genetic tooth and nail syndrome diagnosed in a 2.5-year-old boy with early exfoliation of the primary canine, absence of the primary incisors, and nail dysplasia. A homozygous mutation was identified in 3'-UTR of MSX1 gene in the proband. The parents of the patient had no dental and nail anomalies. [ABSTRACT FROM AUTHOR]

Abstract: Objectives: The goal of our research was to look into the clinical traits and genetic mutations in nonsyndromic oligodontia in a Chinese family and to gain insight into the role of mutations of PAX9, MSX1 and AXIN2 in oligodontia phenotypes. Materials and methods: 6 subjects from a family underwent complete oral examination, including panoramic radiographs. Retrospective data were reviewed and blood samples were collected. PCR primers for PAX9, MSX1, and AXIN2 were designed through the Oligo Primer Analysis Software. PCR products were purified and sequenced using the BigDye Terminator Kit and analysed by the 3730 DNA Analyzer. Results: The proband missed 4 permanent canines, 2 permanent maxillary lateral incisors, 2 permanent mandibular lateral incisors, and 2 permanent mandibular central incisors, whilst his maternal grandfather lacked only 2 permanent mandibular central incisors. Moreover, the size of some permanent teeth appeared smaller than normal values of crown width of Chinese people. Oligodontia and abnormalities of teeth were not present in other family members. Radiographic examination showed that the proband and the rest of family members retained all germs of the third molars. There was one known mutation A240P (rs4904210) of PAX9 in the coding region in the proband and the maternal family members (II-2, II-3, and II-4), which possibly contributed to structural and functional changes of proteins. No mutations were identified in MSX1 and AXIN2. Conclusions: Our findings may imply that the PAX9 A240P mutation is a risk factor for oligodontia in the Chinese population. A240P is likely to be a genetic cause of oligodontia though previous literature suggested it as a polymorphism only. [Copyright &y& Elsevier]

Msx homeogenes play an important role in the epithelial-mesenchymal interactions leading development. Msx1 is relevant for dental and craniofacial morphogenesis, as suggested by phenotypes of Msx1 mutations in human and Msx1 KO mice. Our group showed that Msx1 gene expression can be regulated by a bidirectional transcription generating long noncoding antisense (AS) RNA the expression which is linked to the Msx1 sense (S) RNA level. Thus, the aim of the present study was to analyze the synthesis of Msx1 (AS) RNA. In vivo Msx1 AS expression analysis showed that (i) the putative promoter sequence but not the transcribed sequence was important and necessary for its expression, (ii) 2 different areas of alveolar bone can be distinguished depending on Msx1 S and AS expression, and (iii) Msx1 presence was necessary for Msx1 AS RNA full expression. In silico analysis of the Msx1 AS putative promoter region showed the presence of 4 Msx response elements possibly involved in Msx1 regulation. Msx1 constitutes an example of a bidirectionally transcribed gene giving rise to an S/AS RNA pair included in the big and growing family of AS noncoding RNAs. Our results contribute to defining a link between Msx1 S and AS RNAs resulting in a fine-tuned regulatory loop of Msx1 expression. The significance of this finding is that disturbance of the balance between Msx1 S and AS RNA status may be associated with tooth agenesis and bone loss. Copyright © 2011 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

For many years the molecular mechanisms governing bone morphogenetic protein 4 (Bmp4) expression in tooth bud mesenchyme could be explained by an uncomplicated model involving the interaction of the homeobox gene Msx1 and the paired domain gene Pax9 and a limited proximal promoter segment of Bmp4. New insights have led to major revisions, but we are still far from understanding the role of Msx1 and Pax9 in the complex processes that result in the expression of Bmp4 in the mesenchymal layer of the developing tooth bud. The objective of these studies was to gain further insight into the molecular relationship between Pax9, Msx1, and Bmp4 in dental mesenchyme and explore its association with nonsyndromic tooth agenesis in humans. Copyright © 2011 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Abstract: Purpose: The aim of this study was to investigate the association of a 11 nucleotide deletion, the c.469+46_56del mutation, in the intron of the homeobox MSX1 gene and breast cancer occurrence and characteristics. Methods: The mutation was genotyped in peripheral blood lymphocytes of 200 breast cancer patients and 203 controls by single-strand conformational PCR and DNA sequencing. Results: The del/del variant of the c.469+46_56del mutation increased the risk of breast cancer occurrence (OR 2.20; 95% CI 1.41–3.44, p <0.05). We did not observe any association between genotypes of this mutation and lymph node status, Bloom–Richardson grading, estrogen and progesterone receptors and HER2 expression. Conclusions: The del/del genotype of the c.469+46_56del mutation in the MSX1 gene may be associated with the increased risk of breast cancer in Polish population and may be considered as an early marker in this disease. [ABSTRACT FROM AUTHOR]

Abstract: In an effort to understand the morphogenetic forces that shape the bones of the skull, we inactivated Msx1 and Msx2 conditionally in neural crest. We show that Wnt1-Cre inactivation of up to three Msx1/2 alleles results in a progressively larger defect in the neural crest-derived frontal bone. Unexpectedly, in embryos lacking all four Msx1/2 alleles, the large defect is filled in with mispatterned bone consisting of ectopic islands of bone between the reduced frontal bones, just anterior to the parietal bones. The bone is derived from neural crest, not mesoderm, and, from DiI cell marking experiments, originates in a normally non-osteogenic layer of cells through which the rudiment elongates apically. Associated with the heterotopic osteogenesis is an upregulation of Bmp signaling in this cell layer. Prevention of this upregulation by implantation of noggin-soaked beads in head explants also prevented heterotopic bone formation. These results suggest that Msx genes have a dual role in calvarial development: They are required for the differentiation and proliferation of osteogenic cells within rudiments, and they are also required to suppress an osteogenic program in a cell layer within which the rudiments grow. We suggest that the inactivation of this repressive activity may be one cause of Wormian bones, ectopic bones that are a feature of a variety of pathological conditions in which calvarial bone development is compromised. [Copyright &y& Elsevier]

Abstract: Developmental abnormalities of craniofacial structures and teeth often occur sporadically and the underlying genetic defects are not well understood, in part due to unknown gene–gene interactions. Pax9 and Msx1 are co-expressed during craniofacial development, and mice that are single homozygous mutant for either gene exhibit cleft palate and an early arrest of tooth formation. Whereas in vitro assays have demonstrated that protein–protein interactions between Pax9 and Msx1 can occur, it is unclear if Pax9 and Msx1 interact genetically in vivo during development. To address this question, we compounded the Pax9 and Msx1 mutations and observed that double homozygous mutants exhibit an incompletely penetrant cleft lip phenotype. Moreover, in double heterozygous mutants, the lower incisors were consistently missing and we find that transgenic BMP4 expression partly rescues this phenotype. Reduced expression of Shh and Bmp2 indicates that a smaller “incisor field” forms in Pax9 +/− ;Msx1 +/− mutants, and dental epithelial growth is substantially reduced after the bud to cap stage transition. This defect is preceded by drastically reduced mesenchymal expression of Fgf3 and Fgf10, two genes that encode known stimulators of epithelial growth during odontogenesis. Consistent with this result, cell proliferation is reduced in both the dental epithelium and mesenchyme of double heterozygous mutants. Furthermore, the developing incisors lack mesenchymal Notch1 expression at the bud stage and exhibit abnormal ameloblast differentiation on both labial and lingual surfaces. Thus, Msx1 and Pax9 interact synergistically throughout lower incisor development and affect multiple signaling pathways that influence incisor size and symmetry. The data also suggest that a combined reduction of PAX9 and MSX1 gene dosage in humans may increase the risk for orofacial clefting and oligodontia. [Copyright &y& Elsevier]

Abstract: Neuroblastoma is the most common extra-cranial solid childhood cancer; it arises from neural crest-derived cells of the sympathetic nervous system. The anomalous regulation of embryonic developmental pathways like Delta-Notch and Wnt has been implicated in aberrant cell growth and differentiation in many (childhood) tumours. We have previously found regulation of Delta-Notch pathway genes by the MSX1 neural crest development gene in a neuroblastoma cell line, and significant correlations between these genes in neuroblastic tumours. However, a clear role for the Wnt pathway in neuroblastic tumours has not yet been determined. We now analyze the complete spectrum of genes regulated by inducible expression of MSX1 in the SJNB8 neuroblastoma cell line using Affymetrix expression profiling. We show that MSX1 induces the expression of four different Wnt pathway inhibitor genes: Dickkopf 1-3 (DKK1-3) and secreted frizzled-related protein 1 (SFRP1), and provide evidence that high expression of two of these genes correlates with good prognosis. We were able to demonstrate that both the canonical Wnt3 and the alternative Wnt5A ligands are highly expressed in neuroblastic tumours and cell lines, and specifically activate the DVL3 Wnt co-receptor protein in SJNB8 neuroblastoma cells. These results suggest involvement of MSX1 in Wnt signalling and demonstrate activity of the more upstream Wnt pathway in neuroblastic cells. [Copyright &y& Elsevier]

Abstract: Craniofacial morphogenesis is a complex multi-step process that involves numerous biological processes to coordinate the growth, proliferation, migration, and subsequent differentiation of the cranial neural crest cells. Members of the Fibronectin Leucine-Rich Transmembrane (Flrt) gene family have been previously reported to be widely expressed in the developing embryo. We mapped the expression of Flrt2 and Flrt3 at critical stages of craniofacial development and found that, during early craniofacial development, Flrt2 was highly expressed initially in the cranial neural crest cells and Flrt3 in the midbrain. Later both genes were expressed in the developing pharyngeal region. Flrt2 expression predominated in the neural crest-derived mesenchyme in the medial aspect of the developing frontonasal region in close relationships with the expression of Fgfr2, Shh, and Msx1, three genes shown previously to play critical roles in craniofacial development. Flrt2 was also present in the vomero-nasal organ, mandibular primodia, and the posterior aspects of the unfused and fused secondary palatal shelves. Flrt3, however, had a more restrictive expression, being present in the mesenchyme underlying the ectoderm of the medial nasal process and in the mandibular primordium and in regions undergoing outgrowth, in a pattern that overlapped with Bmp4 expression. Both Flrt2 and Flrt3 were later found to be present at sites of epithelial–mesenchymal interactions such as the developing tooth buds, hair follicles, and eye. Together the data suggested important roles for Flrt2 and Flrt3 in mediating events such as NCC migration, chondrogenesis and epithelial–mesenchymal interactions during craniofacial development. [Copyright &y& Elsevier]

Abstract: Previous studies have shown that Msx proteins control gene transcription predominantly through repression mechanisms. However, gene expression studies using either the gain-of-function or the loss-of-function mutants revealed many gene targets whose expression require functional Msx proteins. To date, investigations into the mechanisms of Msx-dependent transactivation have been hindered by the lack of a responsive promoter. Here, we demonstrated the usefulness of the mouse Hspa1b promoter in probing Msx-dependent mechanisms of gene activation. We showed that Msx protein activates Hspa1b promoter via its C-terminal domain. The activation absolutely depends on the HSEs and physical interactions between Msx proteins and heat shock factors may play a contributing role. [Copyright &y& Elsevier]

Msx homeogenes play an important role in epithelial-mesenchymal interactions leading development. Msx1 is relevant for dental and craniofacial morphogenesis, as suggested by phenotypes of Msx1 mutations in human and Msx1 KO mice. During adulthood, Msx1 is still expressed in the skeleton where its role is largely unknown. Our group showed that the Msx1 gene is submitted to bidirectional transcription generating a long noncoding antisense (AS) RNA. During tooth development, Msx1 sense (S) and AS RNAs showed specific patterns of expression. Thus, the aim of the present study was to analyze the relation between Msx1 S and AS RNAs. In vivo mapping in adult mice showed that both Msx1 RNAs were detected in tested tissues such as bone. In vitro, Msx1 AS RNA decreased endogenous Msx1 S expression and modified Msx1 protein cell distribution. Regulations of Dlx5 and Bmp4 expression involving Msx1 S and AS RNAs showed that Msx1 AS RNA could modulate Msx1 function. The study of Msx1 S and AS RNA status is interesting in the case of tooth agenesis and bone loss to see if a disturbance of this balance could be associated with a disturbance of bone homeostasis. In that sense, our current results suggest a clear involvement of Msx1 in alveolar bone. Copyright © 2008 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Abstract: Objectives: Oligodontia is defined as the congenital absence of 6 or more permanent teeth excluding the third molar. The occurrence of non-syndromic still remains poorly understood, but in recent years some cases have been reported where mutations or polymorphisms of PAX9 and MSX1 had been associated with non-syndromic oligodontia. The objective of the present work was to study the phenotype and genotype of three generations of a Han Chinese family affected by non-syndromic autosomal-dominant oligodontia. Design: We examined all individuals of the oligodontia family by clinical and radiographic examinations. Based on clinical manifestations, candidate genes MSX1 and PAX9 were picked up to analyse and screen mutations. Results: Dental evaluation showed that the most commonly missing teeth are the mandibular second premolars, followed by the maxillary second premolars and maxillary lateral incisors, and subsequently the maxillary first premolars. The probability of missing a particular type of tooth is not always bilaterally symmetrical, and differences exist between maxilla and mandible. PCR-SSCP analysis and DNA sequencing revealed a novel missense mutation c.662C>A in a highly conserved homeobox sequence of MSX1 and a known polymorphisms c.347C>G. Conclusion: Our finding suggests the missense transversion (c.662C>A) and the polymorphisms (c.347C>G) may be responsible for oligodontia phenotype in this Chinese family. [Copyright &y& Elsevier]

Abstract: Neuroblastoma is an embryonal tumour of the peripheral sympathetic nervous system (SNS). One of the master regulator genes for peripheral SNS differentiation, the homeobox transcription factor PHOX2B, is mutated in familiar and sporadic neuroblastomas. Here we report that inducible expression of PHOX2B in the neuroblastoma cell line SJNB-8 down-regulates MSX1, a homeobox gene important for embryonic neural crest development. Inducible expression of MSX1 in SJNB-8 caused inhibition of both cell proliferation and colony formation in soft agar. Affymetrix micro-array and Northern blot analysis demonstrated that MSX1 strongly up-regulated the Delta–Notch pathway genes DLK1, NOTCH3, and HEY1. In addition, the proneural gene NEUROD1 was down-regulated. Western blot analysis showed that MSX1 induction caused cleavage of the NOTCH3 protein to its activated form, further confirming activation of the Delta–Notch pathway. These experiments describe for the first time regulation of the Delta–Notch pathway by MSX1, and connect these genes to the PHOX2B oncogene, indicative of a role in neuroblastoma biology. Affymetrix micro-array analysis of a neuroblastic tumour series consisting of neuroblastomas and the more benign ganglioneuromas showed that MSX1, NOTCH3 and HEY1 are more highly expressed in ganglioneuromas. This suggests a block in differentiation of these tumours at distinct developmental stages or lineages. [Copyright &y& Elsevier]

Abstract: The product of the Msx1 gene is a potent inhibitor of muscle differentiation. Msx1 is expressed in muscle precursor cells of the limb bud that also express Pax3. It is thought that Msx1 may facilitate distal migration by delaying myogenesis in these cells. Despite the role played by Msx1 in inhibiting muscle differentiation, nothing is known of the mechanisms that support the expression of the Msx1 gene within limb bud muscle precursor cells. In the present study we have used a combination of comparative genomics, mouse transgenic analysis, in situ hybridisation and immunohistochemistry to identify a highly conserved and tissue-specific regulatory sub-domain within the previously characterised Msx1 gene proximal enhancer element that supports the expression of the Msx1 gene in Pax3-expressing mouse limb pre-muscle masses. Furthermore, using a combination of in situ hybridisation, in vivo ChIP assay and transgenic explant culture analysis we provide evidence that Msx1 expression in limb bud muscle precursor cells is dependent on the canonical Wnt/TCF signalling pathway that is important in muscle shape formation. The results of these studies provide evidence of a mechanistic link between the Wnt/TCF and the Msx1/Pax3/MyoD pathways within limb bud muscle precursor cells. [Copyright &y& Elsevier]

Abstract: The homeobox genes Msx1 and Msx2 function as transcriptional regulators that control cellular proliferation and differentiation during embryonic development. Mutations in the Msx1 and Msx2 genes in mice disrupt tissue–tissue interactions and cause multiple craniofacial malformations. Although Msx1 and Msx2 are both expressed throughout the entire development of the frontal bone, the frontal bone defect in Msx1 or Msx2 null mutants is rather mild, suggesting the possibility of functional compensation between Msx1 and Msx2 during early frontal bone development. To investigate this hypothesis, we generated Msx1−/−;Msx2−/− mice. These double mutant embryos died at E17 to E18 with no formation of the frontal bone. There was no apparent defect in CNC migration into the presumptive frontal bone primordium, but differentiation of the frontal mesenchyme and establishment of the frontal primordium was defective, indicating that Msx1 and Msx2 genes are specifically required for osteogenesis in the cranial neural crest lineage within the frontal bone primordium. Mechanistically, our data suggest that Msx genes are critical for the expression of Runx2 in the frontonasal subpopulation of cranial neural crest cells and for differentiation of the osteogenic lineage. This early function of the Msx genes is likely independent of the Bmp signaling pathway. [Copyright &y& Elsevier]

Background. Nonsyndromic orofacial clefing (NS-OFC) is among the most common malformations in humans. It is a complex multifactorial trait with a considerable genetic component. Among numerous candidate genes, those related to syndromic OFC recently have emerged as particularly strong ones. Our study was aimed to investigate whether mutations in the MSX1 and IRF6 genes contribute to NS-OFC in the population of Lithuania. Patients, materials and methods. Patients with NS-OFCs from Lithuania were tested for nucleotide sequence changes in the MSX1 gene (80 patients) and IRF6 gene (190 patients). DNA fragments covering MSX1 gene and exonic parts of the IRF6 gene were PCR-amplified, and the direct sequencing of the PCR products was performed with a subsequent comparison of sequencing results with reference DNA sequences of the genes. Results. Eight and 22 different nucleotide sequence changes were revealed in the MSX1 and IRF6 genes, respectively. While only already known functionally neutral polymorphisms were detected in the MSX1 gene, scanning IRF6 gene resulted in nine novel nucleotide sequence variants. Of them, three were missense mutations (p.S212I, p.L295P, p.R400L), which, together with the p.A61G mutation found in the case of Van der Woude syndrome, might be related to the NS-OFC phenotype in the population of Lithuania. Conclusions. Failure to find MSX1 gene mutations potentially related to NS-OFC phenotype of the probands from Lithuania does not reject the necessity to continue investigation on a larger population sample. Our study highlights the IRF6 gene sequence variability and supports the hypothesis that variation in this gene contributes to NS-OFC phenotype, encouraging further investigations to test whether IRF6 gene mutations identified in individuals from Lithuania are rare alleles causative for NS-OFC. [ABSTRACT FROM AUTHOR]

Tooth agenesis constitutes the most common anomaly of dental development in humans. In the majority of familial cases of hypodontia alone or in association with other anomalies, the mode of inheritance is autosomal dominant. In the present study, we have identified two distantly related consanguineous Pakistani kindreds with an autosomal recessive form of oligodontia with associated dental anomalies. Locus in this case has been mapped on chromosome 4p16.1–p16.3. The maximum two-point LOD score of 2.85 ( θ=0.0) was obtained at markers D4S2925 and D4S2285. A maximum multipoint LOD score exceeding 4 was obtained at the same markers. Recombination events observed in affected individuals localized the disease locus between markers D4S412 and D4S2935, spanning a 9.24-cM region on chromosome 4p16.1–p16.3. Sequence analysis of candidate gene MSX1 revealed a novel recessive missense mutation resulting in substitution of alanine to threonine amino acid (p. A219T), located in the MSX1 homeodomain, which is important for DNA binding and protein–protein interaction. The mutation, p. A219T, is the first recessive mutation identified in MSX1. [ABSTRACT FROM AUTHOR]

We have studied the pattern of programmed cell death in the neural crest and analyzed how it is controlled by the activity of the transcription factors Slug and msx1. Our results indicate that apoptosis is more prevalent in the neural folds than in the rest of the neural ectoderm. Through gain- and loss-of-function experiments with inducible forms of both Slug and msx1 genes, we showed that Slug acts as an anti-apoptotic factor whereas msx1 promotes cell death, either in the neural folds of the whole embryos, in isolated or induced neural crest and in animal cap assays. The protective effect of expressing Slug can be reversed by expressing the apoptotic factor Bax, while the apoptosis promoted by msx1 can be abolished by expressing the Xenopus homologue of Bcl2 (XR11). Furthermore, we show that Slug and msx1 control the transcription of XR11 and several caspases required for programmed cell death. In addition, expression of Bax or Bcl2, produced similar effects on the survival of the neural crest and on the development of its derivatives to those produced by altering the activity of Slug or msx1. Finally, we show that in the neural crest, the region of the neural folds where Slug is expressed, cells undergo less apoptosis, than in the region where the msx1 gene is expressed, which correspond to cells adjacent to the neural crest. We show that the expression of Slug and msx1 controls cell death in certain areas of the neural folds, and we discuss how this equilibrium is necessary to generate sharp boundaries in the neural crest territory, and to precisely control cell number among neural crest derivatives. [Copyright &y& Elsevier]

In the developing mammalian tooth, the cranial neural crest derived dental mesenchyme consists of the dental papilla and dental follicle. The dental papilla gives rise to odontoblasts and dental pulp and the dental follicle gives rise to the periodontium, including the osteoblasts that contribute to the alveolar process. The alveolar process is a specialized intramembranous bone that forms the primary support structure for the dentition. The Msx1 gene controls many aspects of craniofacial development, as evidenced by craniofacial abnormalities seen in Msx1−/− mice, including the arrest of tooth development and the absence of the alveolar bone. Previous studies demonstrated that ectopic expression of Bmp4, a downstream target of Msx1, in the Msx1−/− dental mesenchyme rescued alveolar bone formation. Here we confirm an early requirement of BMP activity for alveolar bone formation. We show that the expression of Cbfa1 and Dlx5, two genes encode transcription factors that are critical for bone differentiation, overlaps with that of Msx1 and Bmp4 in the developing tooth and alveolar process. We have demonstrated that Dlx5 and Cbfa1 expression is down-regulated in Msx1−/− dental mesenchyme and that Msx1 and Bmp4 expression are unaltered in Cbfa1−/− mice. These data place Dlx5 and Cbfa1 downstream from the Msx1/Bmp4 in the genetic pathway that regulates tooth development. Ectopic expression of Bmp4 in Msx1 mutants restores the expression of Dlx5, but not Cbfa1, in the dental mesenchyme, and rescues the expression of both Dlx5 and Cbfa1 in the developing alveolar bone. Therefore, the early expression of Cfba1 in the dental mesenchyme appears dispensable for the development of the alveolar bone. Taken together with in vitro gene induction studies, our results demonstrate that BMP4 controls Dlx5 expression in dental mesenchyme, and functions upstream to both Dlx5 and Cbfa1 to regulate alveolar bone formation during tooth development. [Copyright &y& Elsevier]

Tooth development is regulated through a series of reciprocal interactions between the dental epithelium and mesenchyme and requires protein products of a number of genes. It has been reported that selective tooth agenesis is associated with mutations in human MSX and PAX9 genes. Mutational analysis of the two genes was performed in 25 individuals with familial or sporadic form of permanent tooth agenesis. Single-stranded conformational polymorphism analysis revealed no mutations in the entire coding sequence of the MSX1 gene. In PAX9 , a novel, heterozygous G151A transition in the sequence encoding the paired domain of the PAX9 protein was detected in a patient with agenesis of third molars, second premolars and incisors, but not in her parents, the remaining patients or 162 individuals with normal dentition. This is the first de novo mutation described in PAX9 . Our results support the view that mutations in PAX9 could constitute a causative factor of oligodontia. We hypothesize that the G151A transition in PAX9 might be responsible for the sporadic form of tooth agenesis in this patient. [ABSTRACT FROM AUTHOR]

The article discusses the study "Combined Deficiencies of Msx1 and Msx2 Cause Impaired Patterning and Survival of the Cranial Neural Crest," by Mamoru Ishii, Henry M. Sucov et al. The study focused on the function of Msx1 and Msx2, homeobox genes implicated in several disorders affecting craniofacial development in humans. The change in the expression of the hindbrain markers Krox20 and Epha4 in Msx1/2 mutants suggests that defects in neural crest populations may result from defects in rhombomere identity.