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Okolo 6. týdne nitroděložniho vývoje se epitelova ztlustěni přeměňuji v srpkovitý pruh epitelu, laminu dentalis, ktera se vzapěti postupně rozděluje do řady deseti samostatných ostrůvků, zubnich pupenů, zakladů budoucich docasných zubů. Docasný chrup se prořezava od 6. do 30. měsice života a erupci doprovazi prudký narůst diferencovane enzymaticke aktivity.

The aim of this study was the analysis of WNT10A variants in seven families of probands with various forms of tooth agenesis and self-reported family history of cancer.We enrolled 60 young subjects (aged 13 to 17) from the Czech Republic with various forms of tooth agenesis. Dental phenotypes were assessed using Planmeca ProMax 3D (Planmeca Oy, Finland) with Planmeca Romexis software (version 2.9.2) together with oral examinations. After screening PAX9, MSX1, EDA, EDAR, AXIN2 and WNT10A genes on the Illumina MiSeq platform (Illumina, USA), we further analyzed the evolutionarily highly conserved WNT10A gene by capillary sequencing in the seven families.All the detected variants were heterozygous or compound heterozygous with various levels of phenotypic expression. The most severe phenotype (oligodontia) was found in a proband who was compound heterozygous for the previously identified WNT10A variant p.Phe228Ile and a newly discovered c.748G A variant (p.Gly250Arg) of WNT10A. The newly identified variant causes substitution of hydrophobic glycine for hydrophilic arginine.We suggest that the amino acid changes in otherwise highly conserved sequences significantly affect the dental phenotype. No relationship between the presence of WNT10A variants and a risk of cancer has been found.Screening of PAX9, MSX1, EDA, EDAR, AXIN2 and WNT10A genes in hope to elucidate the pattern of inheritance in families.

Tooth agenesis is one of the most common craniofacial disorders in humans. More than 350 genes have been associated with teeth development. In this study, we enrolled 60 child patients (age 13 to 17) with various types of tooth agenesis. Whole gene sequences of PAX9, MSX1, AXIN2, EDA, EDAR and WNT10a genes were sequenced by next generation sequencing on the Illumina MiSeq platform. We found previously undescribed heterozygous nonsense mutation g.8177G>T (c.610G>T) in MSX1 gene in one child. Mutation was verified by Sanger sequencing. Sequencing analysis was performed in other family members of the affected child. All family members carrying g.8177G>T mutation suffered from oligodontia (missing more than 6 teeth excluding third molars). Mutation g.8177G>T leads to a stop codon (p.E204X) and premature termination of Msx1 protein translation. Based on previous in vitro experiments on mutation disrupting function of Msx1 homeodomain, we assume that the heterozygous g.8177G>T nonsense mutation affects the amount and function of Msx1 protein and leads to tooth agenesis.

Tooth agenesis is one of the most common developmental anomalies in humans. To date, many mutations involving paired box 9 (PAX9), msh homeobox 1 (MSX1), and axin 2 (AXIN2) genes have been identified. The aim of the present study was to perform screening for mutations and/or polymorphisms using the capillary sequencing method in the critical regions of PAX9 and MSX1 genes in a group of 270 individuals with tooth agenesis and in 30 healthy subjects of Czech origin. This screening revealed a previously unknown heterozygous g.9527G>T mutation in the PAX9 gene in monozygotic twins with oligodontia and three additional affected family members. The same variant was not found in healthy relatives. This mutation is located in intron 2, in the region recognized as the splice site between exon 2 and intron 2. We hypothesize that the error in pre-mRNA splicing may lead to lower expression of PAX9 protein and could have contributed to the development of tooth agenesis in the affected subjects.

Tooth development (odontogenesis) is a very complicated and complex process involving interplay between oral epithelium and adjacent ectomesenchyme. These reciprocal interactions are mediated by more than 300 signaling molecules (e.g. PAX9, AXIN2 and MSX1). Mutations in the genes for these two regulation proteins were associated with isolated (non-syndromic) form of hypodontia and for this reason; we decided to study gene mutations for MSX1 gene. Upon receipt of approval from the Ethical Commission of University Hospital Ostrava (No. 638/2011), DNA was isolated from patients using Ultra Clean Blood Spin DNA Isolation Kit (Mo-Bio). Blood samples or buccal swabs from 200 Czech patients with various types of tooth agenesis were used for this purpose. PCR products for exons of MSX1 gene were prepared using Kapa 2G Robust Hot Start polymerase (KapaBio Systems) and sequenced using ABI 3130 Prism (Life Technologies) genetic analyzer. Finally, sequences were compared to standard sequences of the genes and mutations were described according to their possible effect to the protein structure. We identified novel heterozygous MSX1 gene polymorphism that may cause amino acid substitutions Ala40Gly. This mutation was found both in the group of patients suffering from hypodontia and in the control group. Furthermore, intron and exon mutations without influence on amino acid change have been identified. These changes in nucleotide sequence have no direct effect on protein structure; however they can affect stability and regulation of gene expression. Therefore, detailed analysis of the results and the future research has to be focused on familiar studies and relationship between mutations in MSX1 gene and tooth agenesis (hypodontia, oligodontia) as well as on analysis of other genes coding proteins participating in odontogenesis, such as PAX9 or AXIN2. The project was supported by the IGA MZ CR, project no. NT 114206 6/2010.