An in vitro and computational validation of a novel loss-of-functional mutation in PAX9 associated with non-syndromic tooth agenesis.

Congenital tooth agenesis (CTA) is one of the most common craniofacial anomalies. Its frequency varies among different population depending upon the genetic heterogeneity. CTA could be of familial or sporadic and syndromic or non-syndromic. Five major genes are found to be associated with non-syndromic CTA, namely PAX9, MSX1, EDA1, AXIN2, and WNT10A. Very few studies have been carried out so far on CTA on this Indian population making this study unique and important. This study was initiated to identify potential pathogenic variant associated with congenital tooth agenesis in an India family with molar tooth agenesis. CTA was investigated and a novel c.336C > G variation was identified in the exon 3 of PAX9, leading to substitution of evolutionary conserved Cys with Trp at 112th amino acid position located at the functionally significant DNA-binding paired domain region. Functional analysis revealed that p.Cys112Trp mutation did not prevent the nuclear localization although mutant protein had higher cytoplasmic retention. EMSA using e5 probe revealed that mutant protein was unable to bind with the paired-domain-binding site. Subsequently, GST pull-down assay revealed lower binding activity of the mutant protein with its known interactor MSX1. These in vitro results were consistent with the computational results. The in vitro and computational observations altogether suggest that c.336C > G (p.Cys112Trp) variation leads to loss of function of PAX9 leading to CTA in this family.

Detection of novel variant and functional study in a Chinese family with nonsyndromic oligodontia.

OBJECTIVES: To investigate the pathogenic gene of a patient with nonsyndromic oligodontia, and analyze its possible pathogenic mechanism. SUBJECTS AND METHODS: The variant was detected by whole exome sequencing (WES) and Sanger sequencing in a family with oligodontia. Bioinformatic and structural analyses were used to analyze variant. Functional studies including western blotting and immunofluorescent analyses and luciferase reporter assay were conducted to explore the functional effects. RESULTS: We identified a novel frameshift variant of PAX9 (c.491-510delGCCCT-ATCACGGCGGCGGCC, p.P165Qfs*145) outside the DNA-binding domain causing an autosomal-dominant nonsyndromic oligodontia in a Chinese family. Bioinformatic and structural analyses revealed that the variant is pathogenic and conserved evolutionarily, and the changes might affect protein stability or folding. Functional studies demonstrate dramatically reduced ability in activating transcription activity of BMP4 promoter and a marked decrease in protein production, as evaluated by western blotting and immunofluorescent analyses. CONCLUSIONS: We found a novel frameshift variant of PAX9 causing nonsyndromic oligodontia in a Chinese family. Our findings indicate that frameshift variants cause loss of function of PAX9 protein during the patterning of the dentition and the subsequent tooth agenesis, providing new molecular insights into the role of frameshift variant of PAX9 and broaden the pathogenic spectrum of PAX9 variants.

Paired Box 9 (PAX9), the RNA polymerase II transcription factor, regulates human ribosome biogenesis and craniofacial development.

Dysregulation of ribosome production can lead to a number of developmental disorders called ribosomopathies. Despite the ubiquitous requirement for these cellular machines used in protein synthesis, ribosomopathies manifest in a tissue-specific manner, with many affecting the development of the face. Here we reveal yet another connection between craniofacial development and making ribosomes through the protein Paired Box 9 (PAX9). PAX9 functions as an RNA Polymerase II transcription factor to regulate the expression of proteins required for craniofacial and tooth development in humans. We now expand this function of PAX9 by demonstrating that PAX9 acts outside of the cell nucleolus to regulate the levels of proteins critical for building the small subunit of the ribosome. This function of PAX9 is conserved to the organism Xenopus tropicalis, an established model for human ribosomopathies. Depletion of pax9 leads to craniofacial defects due to abnormalities in neural crest development, a result consistent with that found for depletion of other ribosome biogenesis factors. This work highlights an unexpected layer of how the making of ribosomes is regulated in human cells and during embryonic development.

The phenotype and genotype of PAX9 mutations causing tooth agenesis.

OBJECTIVES: The purpose of this study was to identify associations between PAX9 mutations and clinical features of non-syndromic tooth agenesis patients. MATERIALS AND METHODS: Non-syndromic tooth agenesis patients were found to have mutations by whole exome sequencing (WES). Additionally, conservation analysis and three-dimensional structure prediction were also applied to identify mutated proteins. RESULTS: Eight non-syndromic tooth agenesis probands were identified with PAX9 mutations (c.C112T; C.131_134del; c.G151A; c.189delG; c.305delT; c.C365A; c.394delG; c.A679C). All of the probands were missing more than six teeth (oligodontia). The mutations (c.131_134del,p.R44fs; c.189delG,p.T63fs; c.305delT,p.I102fs and c.394delG,p.G123fs) caused premature termination of the PAX9 protein. The c.C112T(p.R38X) mutation created a truncated protein. Bioinformatic prediction demonstrated that the three missense mutations change the PAX9 structure suggesting the corresponding functional impairments. CONCLUSIONS: We reported that eight mutations of PAX9 caused non-syndromic tooth agenesis and analyzed the relationship between PAX9 mutations and non-syndromic tooth agenesis. CLINICAL RELEVANCE: Our study revealed that PAX9 mutations might be the mutations most associated with non-syndromic tooth agenesis in humans, which greatly broadened the mutation spectrum of PAX9-related non-syndromic tooth agenesis.

The role of the histone methyltransferase SET domain bifurcated 1 during palatal development.

The histone methyltransferase SET domain bifurcated 1 (SETDB1) catalyzes the trimethylation of lysine 9 of histone H3, thereby regulating gene expression. In this study, we used conditional knockout mice, where Setdb1 was deleted only in neural crest cells (Setdb1fl/fl,Wnt1-Cre + mice), to clarify the role of SETDB1 in palatal development. Setdb1fl/fl,Wnt1-Cre + mice died shortly after birth due to a cleft palate with full penetration. Reduced palatal mesenchyme proliferation was seen in Setdb1fl/fl,Wnt1-Cre + mice, which might be a possible mechanism of cleft palate development. Quantitative RT-PCR and in situ hybridization showed that expression of the Pax9, Bmp4, Bmpr1a, Wnt5a, and Fgf10 genes, known to be important for palatal development, were markedly decreased in the palatal mesenchyme of Setdb1fl/fl,Wnt1-Cre + mice. Along with these phenomena, SMAD1/5/9 phosphorylation was decreased by the loss of Setdb1. Our results demonstrated that SETDB1 is indispensable for palatal development partially through its proliferative effect. Taken together with previous reports that PAX9 regulates BMP signaling during palatal development which implies that loss of Setdb1 may be involved in the cleft palate development by decreasing SMAD-dependent BMP signaling through Pax9.

Patterns of molar agenesis associated with p.P20L and p.R77Q variants in PAX9.

Nonsyndromic tooth agenesis is associated with variants in several genes. There are numerous genotype-phenotype publications involving many patients and kindreds. Here, we identified six Thai individuals in two families with nonsyndromic tooth agenesis, performed exome sequencing, and conducted functional experiments. Family 1 had four affected members carrying the heterozygous PAX9 variant, c.59C>T (p.Pro20Leu). The p.Pro20Leu was previously reported in two families having four and three affected members. These seven cases and Proband-1 had agenesis of at least three third molars. Family 2 comprised two affected members with agenesis of all 12 molars. Both individuals were heterozygous for c.230G>A (p.Arg77Gln) in PAX9, which has not been reported previously. This variant is predicted to be damaging, evolutionarily conserved, and resides in the PAX9 linking peptide. The BMP4 RNA levels in Proband-1's leukocytes were not significantly different from those in the controls, whereas BMP4 levels observed in Proband-2 were significantly increased. Moreover, the p.Arg77Gln variant demonstrated nuclear localization similar to the wild-type but resulted in significantly impaired transactivation of BMP4, a PAX9 downstream gene. In conclusion, we demonstrate that the PAX9 p.Pro20Leu is highly associated with absent third molars, while the novel PAX9 p.Arg77Gln impairs BMP4 transactivation and is associated with total molar agenesis.

Four Novel PAX9 Variants and the PAX9-Related Non-Syndromic Tooth Agenesis Patterns.

The purpose of this research was to investigate and identify PAX9 gene variants in four Chinese families with non-syndromic tooth agenesis. We identified pathogenic gene variants by whole-exome sequencing (WES) and Sanger sequencing and then studied the effects of these variants on function by bioinformatics analysis and in vitro experiments. Four novel PAX9 heterozygous variants were identified: two missense variants (c.191G > T (p.G64V) and c.350T > G (p.V117G)) and two frameshift variants (c.352delC (p.S119Pfs*2) and c.648_649insC(p.Y217Lfs*100)). The bioinformatics analysis showed that these variants might be pathogenic. The tertiary structure analysis showed that these four variants could cause structural damage to PAX9 proteins. In vitro functional studies demonstrated that (1) the p.Y217Lfs*100 variant greatly affects mRNA stability, thereby affecting endogenous expression; (2) the p. S119Pfs* 2 variant impairs the subcellular localization of the nuclear expression of the wild-type PAX9 protein; and (3) the four variants (p.G64V, p.V117G, p.S119Pfs*2, and p.Y217Lfs*100) all significantly affect the downstream transcriptional activity of the BMP4 gene. In addition, we summarized and analyzed tooth missing positions caused by PAX9 variants and found that the maxillary second molar (84.11%) and mandibular second molar (84.11%) were the most affected tooth positions by summarizing and analyzing the PAX9-related non-syndromic tooth agenesis positions. Our results broaden the variant spectrum of the PAX9 gene related to non-syndromic tooth agenesis and provide useful information for future genetic counseling.

Six2 regulates Pax9 expression, palatogenesis and craniofacial bone formation.

In this study, we investigated the role of the transcription factor Six2 in palate development. Six2 was selected using the SysFACE tool to predict genes from the 2p21 locus, a region associated with clefting in humans by GWAS, that are likely to be involved in palatogenesis. We functionally validated the predicted role of Six2 in palatogenesis by showing that 22% of Six2 null embryos develop cleft palate. Six2 contributes to palatogenesis by promoting mesenchymal cell proliferation and regulating bone formation. The clefting phenotype in Six2-/- embryos is similar to Pax9 null embryos, so we examined the functional relationship of these two genes. Mechanistically, SIX2 binds to a PAX9 5' upstream regulatory element and activates PAX9 expression. In addition, we identified a human SIX2 coding variant (p.Gly264Glu) in a proband with cleft palate. We show this missense mutation affects the stability of the SIX2 protein and leads to decreased PAX9 expression. The low penetrance of clefting in the Six2 null mouse combined with the mutation in one patient with cleft palate underscores the potential combinatorial interactions of other genes in clefting. Our study demonstrates that Six2 interacts with the developmental gene regulatory network in the developing palate.

Pax9 is essential for granulopoiesis but dispensable for erythropoiesis in zebrafish.

Paired Box (Pax) gene family, a group of transcription regulators have been implicated in diverse physiological processes. However, their role during hematopoiesis which generate a plethora of blood cells remains largely unknown. Using a previously reported single cell transcriptomics data, we analyzed the expression of individual Pax family members in hematopoietic cells in zebrafish. We have identified that Pax9, which is an essential regulator for odontogenesis and palatogenesis, is selectively localized within a single cluster of the hematopoietic lineage. To further analyze the function of Pax9 in hematopoiesis, we generated two independent pax9 knock-out mutants using the CRISPR-Cas9 technique. We found that Pax9 appears to be an essential regulator for granulopoiesis but dispensable for erythropoiesis during development, as lack of pax9 selectively decreased the number of neutrophils with a concomitant decrease in the expression level of neutrophil markers. In addition, embryos, where pax9 was functionally disrupted by injecting morpholinos, failed to increase the number of neutrophils in response to pathogenic bacteria, suggesting that Pax9 is not only essential for developmental granulopoiesis but also emergency granulopoiesis. Due to the inability to initiate emergency granulopoiesis, innate immune responses were severely compromised in pax9 morpholino-mediated embryos, increasing their susceptibility and mortality. Taken together, our data indicate that Pax9 is essential for granulopoiesis and promotes innate immunity in zebrafish larvae.

Tooth agenesis: What do we know and is there a connection to cancer?

Like all developmental processes, odontogenesis is highly complex and dynamically regulated, with hundreds of genes co-expressed in reciprocal networks. Tooth agenesis (missing one or more/all teeth) is a common human craniofacial anomaly and may be caused by genetic variations and/or environmental factors. Variants in PAX9, MSX1, AXIN2, EDA, EDAR, and WNT10A genes are associated with tooth agenesis. Currently, variants in ATF1, DUSP10, CASC8, IRF6, KDF1, GREM2, LTBP3, and components and regulators of WNT signaling WNT10B, LRP6, DKK, and KREMEN1 are at the forefront of interest. Due to the interconnectedness of the signaling pathways of carcinogenesis and odontogenesis, tooth agenesis could be a suitable marker for early detection of cancer predisposition. Variants in genes associated with tooth agenesis could serve as prognostic or therapeutic targets in cancer. This review aims to summarize existing knowledge of development and clinical genetics of teeth. Concurrently, the review proposes possible approaches for future research in this area, with particular attention to roles in monitoring, early diagnosis and therapy of tumors associated with defective tooth development.

Functional study of novel PAX9 variants: The paired domain and non-syndromic oligodontia.

OBJECTIVES: To investigate pathogenic variants of the paired box 9 (PAX9) gene in patients with non-syndromic oligodontia, and the functional impact of these variants. SUBJECTS AND METHODS: Whole exome sequencing and Sanger sequencing were utilized to detect gene variants in a cohort of 80 patients diagnosed with non-syndromic oligodontia. Bioinformatic and conformational analyses, fluorescence microscopy and luciferase reporter assay were employed to explore the functional impact. RESULTS: We identified three novel variants in the PAX9, including two frameshift variants (c.211_212insA; p.I71Nfs*246 and c.236_237insAC; p.T80Lfs*6), and one missense variant (c.229C > G; p.R77G). Familial co-segregation verified an autosomal-dominant inheritance pattern. Conformational analyses revealed that the variants resided in the paired domain, and could cause corresponding structural impairment of the PAX9 protein. Fluorescence microscopy showed abnormal subcellular localizations of frameshift variants, and luciferase assay showed impaired downstream transactivation activities of the bone morphogenetic protein 4 (BMP4) gene in all variants. CONCLUSIONS: Our findings broaden the spectrum of PAX9 variants in patients with non-syndromic oligodontia and support that paired domain structural impairment and the dominant-negative effect are likely the underlying mechanisms of PAX9-related non-syndromic oligodontia. Our findings will facilitate genetic diagnosis and counselling, and help lay the foundation for precise oral health therapies.

Genetic variants in tooth agenesis-related genes might be also involved in tooth size variations.

OBJECTIVE: The present study aimed to evaluate if genetic variants in PAX9, MSX1, TGFα, FGF3, FGF10, FGF13, GLI2 and GLI3 are involved in TS of permanent teeth. MATERIALS AND METHODS: Pretreatment dental records from orthodontic patients were assessed prior to recruitment. Patients with tooth agenesis and congenital anomalies (including oral cleft) and/or syndromes were excluded. Dental casts were used to measure the maximum crown dimensions of all fully erupted permanent teeth except second and third molars in mesiodistal direction. Teeth with caries, occlusal wear, mesiodistal restorations, and obvious deformities were not evaluated. Genomic DNA samples were used for genotyping. The allelic discrimination of 13 genetic variants was performed. The associations between TS and genotype were analyzed by linear regression, adjusted by gender at a significance level of p ≤ 0.05. RESULTS: Genetic polymorphisms in the tooth agenesis-related genes studied here were associated with increased and decreased TS, in both maxilla and mandible (p < 0.05). CONCLUSION: This study reported associations of novel tooth agenesis-related gene variants with permanent tooth size variations. CLINICAL RELEVANCE: The presence of some genetic variants could allow the prediction of permanent tooth size.

Pax9's dual roles in modulating Wnt signaling during murine palatogenesis.

BACKGROUND: Despite the strides made in understanding the complex network of key regulatory genes and cellular processes that drive palate morphogenesis, patients suffering from these conditions face treatment options that are limited to complex surgeries and multidisciplinary care throughout life. Hence, a better understanding of how molecular interactions drive palatal growth and fusion is critical for the development of treatment and preventive strategies for cleft palates in humans. Our previous work demonstrated that Pax9-dependent Wnt signaling is critical for the growth and fusion of palatal shelves. We showed that controlled intravenous delivery of small molecule Wnt agonists specifically blocks the action of Dkks (inhibitors of Wnt signaling) and corrects secondary palatal clefts in Pax9-/- mice. While these data underscore the importance of the functional upstream relationship of Pax9 to the Wnt pathway, not much is known about how the genetic nature of Pax9's interactions in vivo and how it modulates the actions of these downstream effectors during palate formation. RESULTS: Here, we show that the genetic reduction of Dkk1 during palatogenesis corrected secondary palatal clefts in Pax9-/- mice with restoration of Wnt signaling activities. In contrast, genetically induced overexpression of Dkk1 mice phenocopied the defects in tooth and palate development visible in Pax9-/- strains. Results of ChIP-qPCR assays showed that Pax9 can bind to regions near the transcription start sites of Dkk1 and Dkk2 as well as the intergenic region of Wnt9b and Wnt3 ligands that are downregulated in Pax9-/- palates. CONCLUSIONS: Taken together, these data suggest that the molecular mechanisms underlying Pax9's role in modulating Wnt signaling activity likely involve the inhibition of Dkk expression and the control of Wnt ligands during palatogenesis.

A novel PAX9 mutation found in a Chinese patient with hypodontia via whole exome sequencing.

OBJECTIVE: To investigate a novel gene mutation in a Chinese patient with non-syndromic hypodontia. SUBJECTS AND METHODS: Mutation analysis was carried out by whole exome sequencing. Bioinformatics tools were used for the biophysical predictions of the mutative protein. Luciferase reporter assay was performed to analyse the effects of mutation on protein function. PAX9 and BMP4 gene expression from mutant cells was detected by qRT-PCR. RESULTS: A novel heterozygous mutation (c.G1057A) was detected in the patient but was not found in the controls. The novel missense mutation led to a Val111Met substitution in the paired box domain which was completely conserved evolutionarily, as analysed by dbNSFP. The mutation was predicted to be disease-causing and harmful using MutationTaster and CADD, respectively. Protean of Lasergene showed that this mutation may lead to ß-region shortening in the mutant protein compared to the wild type. Luciferase reporter assay indicated that the mutated protein reduced the transactivation activity of PAX9. This mutation led to increased levels of PAX9 transcript and reduced levels of BMP4 transcript, likely due to compensatory activation and lower transactivation activity of mutant PAX9. CONCLUSION: This novel mutation (c.G1057A) in PAX9 caused hypodontia by altering PAX9 gene function and downregulating BMP4 gene expression.

Genetic analysis: Wnt and other pathways in nonsyndromic tooth agenesis.

Tooth agenesis (TA) is one of the most common developmental anomalies that affects the number of teeth. An extensive analysis of publicly accessible databases revealed 15 causative genes responsible for nonsyndromic TA, along with their signaling pathways in Wnt/ß-catenin, TGF-ß/BMP, and Eda/Edar/NF-κB. However, genotype-phenotype correlation analysis showed that most of the causal genes are also responsible for syndromic TA or other conditions. In a total of 198 different mutations of the 15 genes responsible for nonsyndromic TA, 182 mutations (91.9%) are derived from seven genes (AXIN2, EDA, LRP6, MSX1, PAX9, WNT10A, and WNT10B) compared with the remaining 16 mutations (8.1%) identified in the remaining eight genes (BMP4, DKK1, EDAR, EDARADD, GREM2, KREMEN1, LTBP3, and SMOC2). Furthermore, specificity analysis in terms of the ratio of nonsyndromic TA mutations versus syndromic mutations in each of the aforementioned seven genes showed a 98.2% specificity rate in PAX9, 58.9% in WNT10A, 56.6% in MSX1, 41.2% in WNT10B, 31.4% in LRP6, 23.8% in AXIN2%, and 8.4% in EDA. These findings underscore an important role of the Wnt and Wnt-associated pathways in the genetic etiology of this heterozygous disease and shed new lights on the discovery of novel molecular mechanisms associated with tooth agenesis.

Novel mutations identified in patients with tooth agenesis by whole-exome sequencing.

OBJECTIVES: To identify potentially pathogenic mutations for tooth agenesis by whole-exome sequencing. SUBJECTS AND METHODS: Ten Chinese families including five families with ectodermal dysplasia (syndromic tooth agenesis) and five families with selective tooth agenesis were included. Whole-exome sequencing was performed using genomic DNA. Potentially pathogenic mutations were identified after data filtering and screening. The pathogenicity of novel variants was investigated by segregation analysis, in silico analysis, and functional studies. RESULTS: One novel mutation (c.441_442insACTCT) and three reported mutations (c.252delT, c.463C>T, and c.1013C>T) in EDA were identified in families with ectodermal dysplasia. The novel EDA mutation was co-segregated with phenotype. A functional study revealed that NF-κB activation was compromised by the identified mutations. The secretion of active EDA was also compromised detection by western blotting. Novel Wnt10A mutations (c.521T>C and c.653T>G) and EVC2 mutation (c.1472C>T) were identified in families with selective tooth agenesis. The Wnt10A c.521T>C mutation and the EVC2 c.1472C>T mutation were considered as pathogenic for affecting highly conserved amino acids, co-segregated with phenotype and predicted to be disease-causing by SIFT and PolyPhen2. Moreover, several reported mutations in PAX9, Wnt10A, and FGFR3 were also detected. CONCLUSIONS: Our study expanded our knowledge on tooth agenesis spectrum by identifying novel variants.

Familial oligodontia and regional odontodysplasia associated with a PAX9 initiation codon mutation.

OBJECTIVE: Tooth agenesis is one of the most common craniofacial developmental anomalies. In hypodontia, one to five teeth are missing, whereas oligodontia refers to the absence of at least six teeth, excluding the third molars. Mutations in several genes including MSX1, PAX9, AXIN2, and WNT10A have been shown to cause non-syndromic tooth agenesis. Regional odontodysplasia (RO), also known as "ghost teeth," is a rare developmental anomaly of tooth formation affecting both dentitions. Some possible causes of RO have been suggested, yet the etiology remains unknown. Because the phenotypes of both oligodontia and RO co-occur in one Finnish family, the aim here was to investigate the genetic etiology of the two conditions. MATERIALS AND METHODS: A mutation screening of the genes MSX1, PAX9, AXIN2, and WNT10A was performed for the family members of a RO patient and family history of oligodontia. RESULTS: An initiation codon mutation of the PAX9 gene was found in the proband and segregating with oligodontia in the family. CONCLUSIONS: The etiology of regional odontodysplasia (RO) may be genetic and the same genes can be involved both in RO and tooth agenesis. CLINICAL RELEVANCE: Our results give new insights into the etiology of regional odontodysplasia, yet further results are needed.

Genetic study of non-syndromic tooth agenesis through the screening of paired box 9, msh homeobox 1, axin 2, and Wnt family member 10A genes: a case-series.

Non-syndromic tooth agenesis (NSTA) is the most common developmental anomaly in humans. Several studies have been conducted on dental agenesis and numerous genes have been identified. However, the pathogenic mechanisms responsible for NSTA are not clearly understood. We studied a group of 28 patients with sporadic NSTA and nine patients with a family history of tooth agenesis. We focused on four genes - paired box 9 (PAX9), Wnt family member 10A (WNT10A), msh homeobox 1 (MSX1), and axin 2 (AXIN2) - using direct Sanger sequencing of the exons and intron-exon boundaries. The most prevalent variants identified in PAX9 and AXIN2 genes were analyzed using the chi-square test. The sequencing results revealed a number of variants in the AXIN2 gene, including one novel missense mutation in one patient with agenesis of a single second premolar. We also identified one variant in the AXIN2 gene as being a putative risk factor for tooth agenesis. Only one missense mutation was identified in the WNT10A gene and this mutation was found in two patients. Interestingly, WNT10A is reported as the most prevalent gene mutated in the European population with NSTA.

PAX9 gene mutations and tooth agenesis: A review.

Paired box 9 (PAX9) is one of the best-known transcription factors involved in the development of human dentition. Mutations in PAX9 gene could, therefore, seriously influence the number, position and morphology of the teeth in an affected individual. To date, over 50 mutations in the gene have been reported as associated with various types of dental agenesis (congenitally missing teeth) and other inherited dental defects or variations. The most common consequence of PAX9 gene mutation is the autosomal-dominant isolated (non-syndromic) oligodontia or hypodontia. In the present review, we are summarizing all known PAX9 mutations as well as their nature and precise loci in the DNA sequence of the gene. Where necessary, we have revised the loci of the mutations in line with the reference sequence of the PAX9 gene as it appears in the current DNA databases.

Effects of PAX9 and MSX1 gene variants to hypodontia, tooth size and the type of congenitally missing teeth.

ooth agenesis, affecting up to 20% of human population, is one of the most common congenital disorder. The most frequent form of tooth agenesis is known as hypodontia, which is characterized by the absence of one to five permanent teeth excluding third molars. It was considered that hypodontia is especially related with gene mutations which play role in tooth formation. Additionally mutations in PAX9 and/or MSX1 have been identified as the defects responsible for missing permanent molars and second premolars. In some studies it was also found that PAX9 and MSX1 gene mutations may change tooth size. Therefore  in this study all of these factors were investigated. Thirty one patients and 30 controls were enrolled to the study. Information about tooth sizes and type of congenitally missing teeth were collected. MSX1 and PAX9 gene mutations were investigated by direct sequencing. Results were evaluated statistically. As a result, 22 variations were detected in PAX9 in which 18 of them are novel. In addition, 7 variations were found in MSX1 in which 5 of them are novel and one of them lead to amino acid change. Statistically significant relations were found between detected variations and tooth sizes. Any relation between mutations and type of congenitally missing teeth were not detected. In conclusion, especially new mutations which may cause hypodontia, effect tooth size and type of congenitally missing teeth, should be investigated with other researchers for clarifying the mechanism.