Genetic Factors of Teeth Impaction: Polymorphic and Haplotype Variants of PAX9, MSX1, AXIN2, and IRF6 Genes

In recent research, there has been a growing awareness of the role of genetic factors in the positioning and eruption of teeth in the maxilla and mandible. This study aimed to evaluate the potential of specific polymorphic markers of single nucleotide polymorphisms (SNPs) located within the PAX9, MSX1, AXIN2, and IRF6 genes to determine the predisposition to tooth impaction. The study participants were divided into two groups: the first group consisted of individuals with at least one impacted secondary tooth. In contrast, the second group (control group) had no impacted teeth in their jaws. To analyze the genes, real-time PCR (polymerase chain reaction) and TaqMan probes were utilized to detect the selected polymorphisms. The findings suggest that disruptions in the structure and function of the mentioned genetic factors such as polymorphic and haplotype variants of PAX9, MSX1, AXIN2, and IRF6 genes, which play a direct role in tooth and periodontal tissue development, might be significant factors in tooth impaction in individuals with genetic variations. Therefore, it is reasonable to hypothesize that tooth impaction may be influenced, at least in part, by the presence of specific genetic markers, including different allelic variants of the PAX9, AXIN2, and IRF6 genes, and especially MSX1.


Introduction
Extensive research has revealed that tooth development in humans and other mammals is influenced by both genetic and environmental factors [1][2][3].Interactions between hundreds of genes control the number, shape, and location of teeth during embryogenesis [1][2][3].The genetic basis of tooth developmental disorders, including agenesis, extra teeth, and structural anomalies, has been extensively documented [4][5][6][7].Specific genetic factors have also been associated with abnormalities in tooth location and eruption [7].Additionally, the impact of genetical, pre-, peri-, and postnatal risk factors on enamel development disorders should be considered.These risk factors include severe illness or complications during pregnancy, low birth weight or premature birth, breastfeeding issues or illness, and early childhood infections [8].
Studies in animals have shown that dental defects, including impacted teeth, are often influenced by multiple genes [9,10].Currently, an impacted tooth is defined as a fully developed tooth embedded in the jawbone or mandible, with closed and mineralized root apices.It can be categorized as complete if fully covered by bone, or partial if the crown has perforated the bone plate and remains adjacent to the soft tissues.Impacted teeth no longer exhibit growth tendencies [11,12].
Researchers are continuously delving into the intricate relationship between genetic and environmental factors in tooth development, advancing our understanding of dental defects and their underlying mechanisms.Impacted teeth are commonly found in the maxilla, specifically the canines and incisors, as well as in the mandible, including the third molars and second premolars.The prevalence of impacted teeth in the anterior region ranges from 0.9% to 4.3% of the population, with canines accounting for 70% and incisors for 11.5% of cases [13,14].The diagnosis of lower third molar impaction varies across populations and time periods.In the 1960s, the prevalence ranged from 17% to over 70% among young Europeans, while more recent studies in the early 2000s have reported varying figures [15].
The PAX9 gene, located on chromosome 14 in humans, is linked to various dental abnormalities.It consists of four exons, with the first exon acting as a start codon and exon 2 encoding a highly conserved region of the pairing domain [31].Most mutations in the PAX9 gene have been identified in exon 2, or less frequently, in exon 4.These mutations are associated with different forms of agenesis, non-syndromic hypodontia, and oligodontia [29,32,33].Specific alleles at polymorphic sites in exon 2 or 4, as well as the deletion of one copy of the PAX9 gene, can lead to selective tooth agenesis, particularly in molars.The observed phenotype is often seen in heterozygotes, indicating that haploinsufficiency of the PAX9 gene contributes to tooth agenesis [30,34].
The MSX1 gene, located on chromosome 4 in humans, consists of two exons, 2332 and 1229 bp in length, separated by a 2332 bp intron.It produces two distinct transcripts, resulting in two polypeptides of different lengths of 303 and 297 amino acids [35,36].The transcription factor encoded by the MSX1 gene regulates the expression of multiple target genes and interacts with proteins involved in odontogenesis, such as PAX9, Dlx family proteins, and Tbp [37].Genes like MSX1, which code for proteins containing a homeodomain, play a crucial role in odontogenesis, controlling various stages of tooth development and influencing the final dentition pattern [38].The AXIN2 gene is responsible for encoding a factor in the WNT signaling pathway, which regulates the stability of beta-catenin.Disruptions in this pathway can contribute to the development of certain neoplasms [39][40][41].The WNT pathway controls the levels and localization of beta-catenins.Activation of the WNT-dependent pathway stabilizes beta-catenin, allowing it to bind to transcription factors from the TCF family and regulate target gene expression.In the absence of WNT signals, beta-catenin undergoes phosphorylation and degradation through a multiprotein complex involving the APC protein, AXIN1, and AXIN2 proteins [42][43][44][45].AXIN proteins, including AXIN2, facilitate the formation of beta-catenin-degrading complexes [46].AXIN2 gene expression is induced by the WNT signaling pathway, indicating that AXIN2 acts as a negative regulator in a feedback loop [47,48].The SHH and WNT pathways, along with proteins like MSX1 and PAX9, play crucial roles in normal tooth morphogenesis and contribute to the complex regulation of tooth development [49,50].The IRF6 gene is associated with van der Woude syndrome (VWS) and Bartsocas-Papas congenital anomaly syndrome.VWS is characterized by cleft lip and palate, mucous cysts of the lower lip, and tooth agenesis [51].Certain SNP mutations in the IRF6 gene have been significantly correlated with the presence of cleft lip with or without cleft palate (CL/P) in various populations [52][53][54][55][56][57].The IRF6 gene may also contribute to tooth developmental disorders, especially when other craniofacial abnormalities are present.The development of teeth, palate, and lips is interconnected, and the severity of cleft symptoms is often associated with the number of affected teeth [58][59][60][61][62][63][64][65][66][67].Therefore, the IRF6 gene is considered important in the study of dental development disorders due to its confirmed role in the etiology of cleft lip and palate.This research's main objective was to assess the usefulness of selected polymorphic markers of single nucleotide polymorphism (SNP) characters located within the PAX9, MSX1, AXIN2, and IRF6 genes in the context of their applicability to determine the predisposition to the occurrence of the tooth impaction phenomenon.The protein products of the PAX9, MSX1, AXIN2, and IRF6 genes directly participate in the processes of tooth formation.Therefore, it has been hypothesized that the polymorphism of these genes may contribute to the phenomenon of impacted teeth in humans.

Genotypes
Alleles For genotypes and alleles for which statistical significances were demonstrated, a comparison of allele and genotype frequencies between the study and control groups is also presented in Table 5.

Discussion
When examining the phenotypic impact of mutations, certain circumstances can associate an allele reducing functional protein levels with favorable traits.For instance, the reduction in C-allele teeth at the rs4904210 locus in the PAX9 gene, primarily affecting the development of the third molars, has been observed in studies and considered not only neutral but even beneficial [68].
The shift in modern lifestyles, with a preference for highly processed and cooked foods over the raw and hard diet of our ancestors, has rendered third molars unnecessary remnants of our species' evolutionary history [69].In cases of impacted or abnormally erupted molars, their presence can pose health hazards, serving as sites for inflammation and bacterial impaction, leading to cavities in neighboring teeth and infections in the bone tissue, potentially weakening or even destroying them.Additionally, bacteria multiplying in these areas can spread systemically, attacking other organs and posing health risks [70].
In this context, the Ala240Pro mutation in the PAX9 gene is described by some authors as a beneficial change aimed at eliminating unnecessary third molars from the modern human population [68].
Functional analysis of the Arg196Pro polymorphism in the MSX1 gene, involved in hypodontia, showed that the structurally defective protein with reduced thermal stability resulted in impaired DNA binding and protein interactions.This mutation was associated with a higher frequency of missing second premolars and third molars, and in some cases, upper premolars and lower first molars [71].
In a study of the Chinese population, a new C662A mutation in the MSX1 gene's homeodomain region was identified, leading to an Ala221Glu substitution.This mutation disrupts the binding to DNA and interactions with other proteins, such as those from the DLX family, hindering the initiation of epithelial-mesenchymal signal cascades during tooth development [72].Carriers of the Ala221Glu mutation exhibited selective tooth loss, including premolars, third molars, lateral incisors, and canines [73].
Another mutation in the MSX1 gene's homeodomain region is the T671C substitution, resulting in a Leu224Pro substitution.Predictive analysis suggests that this mutation may completely abolish the activity of the MSX1 protein, leading to the congenital absence of second premolars and third molars in carriers [74].
Bearing in mind the information presented above on the influence of various mutations in the coding area of the MSX1 gene homeodomain, it is worth emphasizing that the functional analysis of the remaining five missense mutations in the same region of the MSX1 gene, correlated with hypodontia (Met61Lys, Ala194Val, Arg196Pro, Ala219Thr, and Ala221Glu), revealed none of the previously known molecular mechanisms (such as a disturbance of the interaction of MSX1 with PAX9 or other regulatory proteins).Considering the influence of various mutations in the coding region of the MSX1 gene homeodomain, it is important to note that functional analysis of five missense mutations (Met61Lys, Ala194Val, Arg196Pro, Ala219Thr, and Ala221Glu) associated with hypodontia revealed that previously known molecular mechanisms could not fully explain the significance of these amino acid substitutions in tooth agenesis [22].
Furthermore, it was demonstrated that most of these missense mutations did not impair the crucial interaction between MSX1 and PAX9, which activates the expression of the BMP4 gene, suggesting that the role of MSX1 in tooth development is not directly linked to the interaction with PAX9 [22].This implies that potential molecular targets responsible for dental abnormalities may lie in other regions of the MSX1 gene beyond the homeodomain.
Promising variants of the MSX1 gene, relevant to various dental development disorders, include polymorphic sites rs8670 and rs12532, located in the non-coding UTR region of exon 2 [75].These variants were found in the Polish population and primarily associated with non-syndromic sporadic oligodontics in humans [76].
The non-coding regions of the MSX1 gene may also contribute to the development of dental disorders, independent of the homeodomain's direct functioning.The rs8670 variant in the MSX1 gene was found to potentially correlate with non-syndromic hypodontia in the upper lateral incisors, suggesting an impact on mRNA stability, antisense RNA-mediated interactions, or translation processes [77].However, larger studies in Brazilian patients did not show significant differences in the distribution of genotypes and alleles in the rs8670 and rs12532 sites between the agenesis group and the control group [78].
In patients with severe familial hypodontia, direct sequencing of the AXIN2 gene revealed a nonsense mutation (Arg656Stop) in exon 7, which led to premature translation termination [79].This mutation may result in mRNA degradation through the nonsensemediated decay mechanism, as the mRNA transcript with a premature stop codon fulfills the conditions for degradation [80,81].The protein produced in individuals with the C1966T mutation lacks the C-terminal domain responsible for oligomerization and interaction with other proteins [42,82,83].Consequently, individuals with this mutation experience a decrease in the function of the AXIN2 protein, leading to the accumulation of beta-catenin [84].Sequence analysis of the AXIN2 gene in Polish patients with selective tooth agenesis identified several polymorphic sites, including two new variants in the intron region and one variant in exon 7 [85].Statistical analysis revealed that carriers of the c.956 + 16G and c.2062T alleles had an increased risk of selective tooth agenesis, indicating a potential association [85].The synonymous change Leu688Leu may disrupt the exonic splicing enhancer sequence, affecting the recognition of cleavage sites in splicing processes [81].The c.956 + 16A>G mutation could also impact splicing processes, creating an additional donor site within exon 2, potentially interfering with odontogenesis [85].
In studies involving Brazilian and Turkish patients with selective tooth agenesis, three intragenic polymorphic sites (rs7591, rs11867417, and rs2240308) in the AXIN2 gene were analyzed [86].The presence of the rs2240308 variant, either in isolation or as part of a haplotype, correlated with the absence of at least one incisor in the studied populations [86].Although in silico analysis did not confirm this variant as a direct etiological factor, it is suggested that it may interact with another variant to contribute to the development of tooth developmental disorders [86].
Associations between selected markers (rs7802, rs861019, and rs2235371) within the IRF6 gene and preferential agenesis of premolars were confirmed in the Brazilian population [87].The non-synonymous mutation rs2235371 (Val274Ile) was found to have an attributable fraction (AF) coefficient value of 16.4%, indicating its contribution to agenesis cases [87].Haplotypes reconstructed for the analyzed polymorphic sites in the IRF6 gene, containing the more common allele at rs2235371, were consistently found in agenesis patients, particularly in relation to premolar agenesis [87].
However, it was suggested that the rs2235371 variant may not be an etiological factor in itself, but rather interacts with other variants that directly impact the appearance of agenesis [88].To further investigate, additional markers within the IRF6 gene (rs4844880, rs2235371, rs2013162, rs861019, rs2073487, rs642961, and rs658860) were analyzed.The presence of rs658860 variants and the haplotype [A; T; A] reconstructed for sites [rs861019; rs2073487; rs642961] showed a correlation with abnormal development of at least one incisor [88].
In patients with impaired premolar development, the association was observed for the rs2013162 and rs642961 variants, as well as the haplotypes [G; T; A] in [rs861019; rs2073487; rs642961] and [T; AND; T] in [rs2073487; rs642961; rs658860] [88].The presence of the rs642961 site in all statistically significant haplotype systems suggests its potential functional role.In silico analyses indicate that the rs642961 mutation may alter the AGAAT sequence near the IRF6 gene to the AGGAT sequence, which is responsible for binding to the product of the heat shock transcription factor (HSF) gene, a transcription factor activated in response to temperature stress.Additionally, the rs861019 mutation was found to cause a change in another sequence (GTTCT → ATTCT) within the HSF binding domain [88].However, the precise mechanism by which disruption of HSF binding affects normal tooth development remains unknown.

Subjects
Participation in the research project was offered to a consecutive group of 392 patients who visited the Department of Oral Surgery.The inclusion criteria for the study group required the presence of at least one fully impacted tooth in either the maxillary or mandibular bone.An impacted tooth was defined as a tooth surrounded by bone in X-ray images but absent in the dental arch during eruption.The study group consisted of 204 individuals, including 82 males and 122 females, who had one to seven impacted teeth.Patients under the age of sixteen and those who were unable to undergo a panoramic radiograph or had multiple missing teeth that prevented a diagnosis of hypodontia were not eligible for the project.Relatives of the patients were also excluded.
The study participants were divided into two groups based on the presence or absence of a diagnosis of tooth/teeth impaction provided by a medical specialist: The study group included patients with at least one permanently impacted tooth in the maxillary or mandibular bone, as well as patients with supernumerary teeth.
The control group consisted of patients who had no impacted teeth in the maxillary bones and whose permanent teeth had properly emerged and aligned in the dental arches.
All patients were fully informed about the purpose, benefits, and research methods of the study.After providing written consent to participate, they were assigned individual identification numbers to ensure anonymity.
The study group included 204 patients (82 males and 122 females) in whom one to seven impacted teeth were identified (median 3, interquartile range 2-4).A histogram of patients in the study group with a specific number of impacted teeth is shown in Table 5.The most significant number of patients in the study group were observed to have four impacted teeth, while patients with two impacted teeth were the second most numerous subgroup.
It was assumed that the size of the Phi effect would be 0.25 with a group of 204 tested people, and the power of the statistical test for the polymorphism of the tested genes would be 0.83.However, for alleles (n = 408) the Phi effect size was assumed to be 0.16 and the power of the statistical test to be 0.83.The actual results obtained for the size of the Phi effect and the power of the statistical test are presented in Table 6.

Genotyping
Genomic DNA was extracted from venous blood using standard procedures.Genotyping was conducted with the real-time PCR method.
LightCycler ® 480 II System (Roche Diagnostic, Basel, Switzerland) was applied to perform the fluorescence resonance energy into the genotypic data.The data relating to the DRD2 gene polymorphism were obtained under the following conditions: PCR was performed with 50 ng DNA of each sample in a final volume of 20 µL containing 2 µL reaction mix, 0.5 mM of each primer, 0.2 mM of each hybridization probe, and 2 mM MgCl2 according to the manufacturer's instructions with initial denaturation (95 • C for 10 min) and then 35 cycles of denaturation (95 • C for 10 s), annealing (60 • C for 10 s), and extension (72 • C for 15 s).After amplification, a melting curve was generated by holding the reaction at 40 • C for 20 s and heating slowly to a level of 95 • C. The fluorescence signal was plotted against temperature to provide melting curves for each sample.

Conclusions
Abnormalities in the structure and function of the previously described genetic factors directly involved in the development of the tooth, including its hard tissues and periodontal tissues, may therefore have a role in the appearance of retained teeth in individuals with such alterations in genetic material.On the basis of this study, it can be assumed that the impaction of teeth will also depend, at least in part, on the presence of certain genetic markers, particularly AXIN2 rs2240308 and MSX1 rs12532.

Table 1 .
Number of impacted molars in maxilla and mandible together, and in maxilla and mandible separately in respondents with impacted teeth.

Table 2 .
Number of impacted premolars in maxilla and mandible together, and in maxilla and mandible separately in respondents with impacted teeth.

Table 3 .
Number of impacted canines in maxilla and mandible together, and in maxilla and mandible separately in respondents with impacted teeth.
p a -values calculated for allele and genotype frequency comparisons between control and study groups; pb-values calculated for genotype Hardy-Weinberg proportions in all participants (control and study groups analyzed together); pc-values calculated for genotype Hardy-Weinberg proportions in control group; pd-values calculated for genotype Hardy-Weinberg proportions in study group; HWE-Hardy-Weinberg equilibrium.

Table 6 .
Number of impacted teeth in study group patients.