MSX1 and PAX9 Polymorphisms and Their Association With Lateral Incisor Morphology in Patients With Maxillary Canine Impaction

Introduction

Maxillary canine impaction (MCI) is relatively common dental anomaly with significantly increasing incidence in recent years [1], varying from 0.97% to 6%, with a slightly higher rate of 6.30% observed in orthodontic patients [2]. This increase is probably related to improvement of socio-economic level of the population and rapid development of radiological methods, which enables more frequent dental examinations and acquisition of radiograms in a simple and cheap way [3]. Research indicates that MCI occurs twice as often in females as in males [3–7]. Epidemiologically, maxillary canines are the second most frequently impacted teeth after third molars, and impactions occur nearly 10 times more often in the maxilla than in the mandible [4]. Several classification of impacted maxillary canines have been proposed based on many parameters, including their buccal or palatal position, vertical, horizontal, or oblique angulation, and their spatial relationship to adjacent teeth [5–7]. Furthermore, one-third of impacted maxillary canines are located buccally, while approximately two-thirds are located palatally [8,9]. Buccally impacted canines often exhibit a more favorable vertical inclination, whereas palatally impacted canines are often positioned horizontally. As a result, buccal impactions have a higher chance of spontaneous eruption, while palatal impaction often require a multidisciplinary orthodontic–surgical approach for successful management due to the greater thickness of the palatal cortical bone [4]. The diagnosis of this anomaly is based on oral inspection and palpation, two-dimensional radiographic methods, and three-dimensional cone-beam computed tomography (CBCT) methods [10]. Panoramic radiography is most commonly used radiography for the detection and diagnosis of impacted teeth because it provides a convenient overview with a low radiation dose and lower cost compared to three-dimensional methods [11]. However, CBCT enables three-dimensional visualization of the affected area with high precision, allowing clinicians to evaluate tooth position, inclination, and its relationship with adjacent anatomical structures, thereby providing critical information for diagnosis and treatment planning [12]. Impacted canines can be associated with various pathological changes, most commonly follicular cysts and, in rare cases, ameloblastomas [13]. If left untreated, follicular cysts can lead to root resorption of adjacent teeth and displacement of neighboring structures [14,15]. Treatment options depend on clinical findings, the position of the impacted canine, and patient preference, and include observation, extraction, surgical exposure followed by orthodontic alignment, or surgical repositioning of the impacted tooth [16,17]. The etiology of canine impaction is multifactorial and can be linked to position of the canine. Buccal impaction of the canines is often related to inadequate arch space and crowding [18–20]. On the contrary, palatally impacted canines often occur in maxillary arches with sufficient space and good transversal dimensions [20–22]. Two theories have been proposed to explain palatal impaction of canine: the guidance theory [23, 24] and the genetic theory [25]. The guidance theory states that the distal surface of the lateral incisor root serve as a “guide” for the eruption and redirection of the maxillary canine tooth downward [23]; consequently, any anomalies in root size and shape of lateral maxillary incisor can lead to disturbances in eruption and impaction of canines [24,26]. According to the genetic theory, impaction of the canine in the palatal direction is due to genetics, as it is often accompanied by dental abnormalities in tooth shape, size, number, and structure, and it tends to run in families [25,27–29]. Also, tooth agenesis of lateral maxillary incisors is strongly related to palatally impacted canines [30]. Genes like MSX1 (muscle segment homeobox 1) and PAX9 (paired box gene 9) are known to play significant roles in dental development and patterning [31]. Thus, mutations or variations in these genes can disrupt normal tooth development, which can lead to dental anomalies, such as impaction or displacement of teeth [32]. Specific single-nucleotide polymorphisms (SNPs) in MSX1 and PAX9 can increase the risk of impacted teeth [31,33,34], lateral incisor morphology, and agenesis [35–38].

Therefore, this study aimed to compare gene polymorphisms of PAX9 (rs2073247) and MSX1 (rs12532) in buccal swabs in 130 adults with (n=65) and without (n=65) maxillary canine impaction using real-time polymerase chain reaction (RT-PCR).

Material and Methods

ETHICS COMMITTEE APPROVAL AND ETHICS STATEMENT:

This study was conducted in accordance with the principles of the Declaration of Helsinki and was approved by and the Ethics Committee of the Clinic of Dental Medicine, Niš, Serbia (decision no. 14\1-2023-4 EO, January 19, 2024), and by the Ethics Committee of the Faculty of Medicine, University of Niš (decision no. 12-1760-1/2-3, February 20, 2024).

This ethics approvals explicitly cover research involving minors and adolescents, including buccal swab for DNA genotyping analysis, cone-beam computed tomography (CBCT) imaging, and the collection, storage, and analysis of genetic and clinical data.

All details regarding study methodology (buccal swab sampling for genotyping analysis, CBCT imaging, data handling, and patient age) were thoroughly described in the application form submitted as part of the required documentation for the Ethics Committee.

All participants, or their parents/legal guardians in case of minors and participants under 18 years of age, were first fully informed about the study procedures, potential risk, and data handling. They were then provided with a patient information sheet containing essential details about the study. In addition, separate written consent was obtained for patients or their parents/legal guardians. Only patients who signed the written consent form were included in the study. Participants or their parents/legal guardians were explicitly informed about all study procedures, including buccal swab sampling for genetic analysis, CBCT imaging, and the measures implemented to ensure confidentiality and protection of participants’ personal and genetic data. Participants and their parents or legal guardians were given the opportunity to ask any questions regarding the study and were informed of their right to refuse participation or to withdraw from the study at any time without any consequences.

STUDY DESIGN AND PARTICIPANTS:

This prospective, 1-year observational study was conducted at the Department of Orthodontics and the Department of Oral Surgery, Clinic of Dental Medicine, Niš, Serbia. All phases of data collection were supervised by the senior investigator and study coordinator (G.F.), who ensured strict adherence to the approved study protocol. CBCT measurements were performed by a single calibrated examiner (A.T.) under this supervision.

A total of 130 participants (50 males and 80 females; mean age 17.3 years) were included in the study. The experimental group consisted of 65 patients with unilateral maxillary canine impaction, while the control group included 65 individuals without maxillary canine impaction, matched for age and sex. All participants underwent standardized clinical examination and medical history assessment. For minors, medical history was obtained from parents or legal guardians.

Maxillary canine impaction was diagnosed based on clinical criteria (absence of eruption at the expected time or persistence of a deciduous canine surrounded by fully erupted permanent teeth) and confirmed by panoramic radiography. Only patients with completed root development, indicated by a closed apex and continuous lamina dura, were referred for further procedures, including CBCT imaging and genotyping.

Inclusion criteria were: (1) presence of 1 impacted maxillary canine, (2) high-quality CBCT images of the maxilla, (3) absence of other impacted maxillary teeth except canines and third molars, (4) absence of mechanical obstacles (eg, supernumerary teeth or odontomas), (5) absence of systemic diseases, (6) absence of restorations on maxillary lateral incisors, (7) absence of congenital dentofacial anomalies, (8) completed root development of maxillary incisors and canines, and (9) healthy oral mucosa without soft-tissue pathology.

Each participant was assigned a numerical code, and participant identity was known only to the first author (A.T.). CBCT imaging was performed prior to buccal swab sampling. Genotyping and CBCT analyses were conducted using anonymized codes, and laboratory personnel were blinded to clinical group allocation.

CBCT IMAGING AND MEASUREMENT PROCEDURE:

During the study period, approximately 400 patients were clinically examined, of whom around 150 were referred for CBCT imaging. From this initial pool, 130 CBCT scans met the inclusion criteria and were included in the final analysis. Scans were excluded due to insufficient image quality, incomplete visualization of the lateral incisor root, evidence of root resorption, or incomplete canine root development not detected on initial panoramic radiographs.

All CBCT scans were obtained using the Sirona Axeos CBCT Ceph system (Sirona Dental Systems GmbH, Bensheim, Germany) with standardized parameters (85 kVp, 10 mA, 11 s exposure time, voxel size 0.2 mm, field of view 11×10 cm, SD mode). Patients were positioned with the Frankfurt plane parallel to the ground. Image analysis was performed using Sidexis 4 software (Galileos Viewer; Dentsply Sirona, USA).

Morphological parameters of maxillary lateral incisors were assessed in axial, panoramic, and sagittal planes. Measurements included total tooth length (TL), crown length (CL), root length (RL), and mesio-distal crown width (MDW). CL was measured from the cemento-enamel junction (CEJ) to the incisal edge, RL from the CEJ to the root apex, and TL from the root apex to the incisal edge. MDW was measured in the axial plane at the widest mesio-distal dimension of the crown.

The buccopalatal position of impacted maxillary canines was classified according to the Ericson and Kurol criteria [39], adapted for CBCT evaluation. Multiplanar reconstructions were used to assess the spatial relationship between the canine crown and adjacent anatomical structures.

All measurements were performed twice by the same examiner (A.T.), an experienced orthodontist, with a 2-week interval between measurements. Prior to study initiation, the examiner underwent a calibration and training procedure consisting of repeated measurements on a pilot set of CBCT scans using predefined anatomical landmarks and standardized measurement protocols to ensure consistency and accuracy. Intra-observer reliability was assessed using the intra-class correlation coefficient (ICC), which demonstrated very high reliability for all measured parameters.

BUCCAL SWAB SAMPLING AND DNA EXTRACTION:

Buccal swab samples were collected during clinical examination using sterile cotton swabs (Sam-plast d.o.o., Loznica, Serbia). Participants were instructed to rinse their mouths with water prior to sampling. Each swab was rubbed and rotated against the inner cheek for 20–30 s, and the procedure was repeated twice per participant to ensure adequate DNA yield.

Swab tips were placed into Eppendorf tubes containing 300 μL of sterile phosphate-buffered saline (PBS) and stored at −20°C until processing. Genomic DNA was extracted using the PureLink Genomic DNA Mini Kit (Invitrogen, Thermo Fisher Scientific, USA) according to the manufacturer’s protocol. DNA concentration and purity were assessed using a NanoDrop spectrophotometer. Samples were stored at −20°C for short-term use and −80°C for long-term storage, with aliquoting to minimize freeze–thaw cycles. DNA extraction and PCR preparation were performed in separate laboratory areas to prevent contamination.

GENOTYPING OF PAX9 RS2073247 AND MSX1 RS12532:

Genotyping of PAX9 rs2073247 (C>T; assay ID: C__2178751_30) and MSX1 rs12532 (A>G; assay ID: C__26933394_10) was performed using TaqMan^® SNP Genotyping Assays (Applied Biosystems, Thermo Fisher Scientific, USA) on the 7500 Fast Real-Time PCR System (Applied Biosystems), following the manufacturer’s instructions.

Each reaction (10 μL) contained 5 μL TaqMan Universal Master Mix, 0.5 μL of the commercial assay, 2.5 μL nuclease-free water, and 2 μL genomic DNA. The PCR program consisted of a pre-read at 60°C for 1 min, initial denaturation at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min, and a post-read at 60°C for 1 min.

Allelic discrimination was performed automatically by the system software and confirmed by visual inspection of allelic discrimination plots. Homozygous genotypes clustered along a single fluorescence axis, while heterozygous samples were located between VIC and FAM clusters. Samples with ambiguous signals were reanalyzed. Each sample was run in duplicate, and no-template controls were included in all runs. The system was calibrated for dye-specific fluorescence prior to genotyping. Primer and probe sequences were proprietary and supplied within the commercial assays by the manufacturer. Laboratory personnel performing DNA extraction and genotyping were blinded to the clinical grouping of the participants.

The primer and probe sequences were not disclosed because they are proprietary components of the commercially available TaqMan^® SNP Genotyping Assays. According to the manufacturer’s documentation, assay design, optimization, and validation are performed by Applied Biosystems to ensure high specificity and reproducibility; therefore, individual primer sequences are not publicly available.

STATISTICAL ANALYSIS:

The data are presented as arithmetic means and standard deviations, as well as in the form of absolute and relative frequencies. Data distribution was estimated using the Shapiro-Wilk test. Based on these results, numerical variables between the 2 groups were compared using the t test, while categorical variables were compared using the chi-square test. The intra-class correlation coefficient (ICC) was used to measure agreement between measurements of the dependent variables (TL, CL, RL, MDW) taken within 2 weeks. Reliability of measurements, which was assessed by a two-way mixed model, was very high (TL: 0.984 (95% CI 0.954–0.995), CL: 0.902 (95% CI 0.734–0.966), RL: 0.938 (95% CI 0.827–0.979), MDW: 0.971 (95% CI 0.917–0.990). The null hypothesis was tested at a significance level of P<0.05. Statistical analysis was performed using R software, version 4.3.0 [40].

Results

DEMOGRAPHIC AND CLINICAL CHARACTERISTICS:

A total of 130 participants (50 males, 80 females) were included in the study: 65 patients with a unilaterally impacted maxillary canine and 65 control participants. The mean age of the study population was 17.32±4.63 years (range: 12–39 years). The groups were matched in terms of age (P=0.667) and sex (P=0.207) (Table 1).

With respect to the location of the impacted canine tooth, palatal impaction was observed in 45 patients (69.2%), whereas 18 patients (27.7%) had buccal impaction. In only 2 cases (3.1%), the impacted tooth was located centrally within the alveolar ridge. Additionally, the impacted maxillary canine was observed more often on the right side (69.2%) than on the left (30.8%).

MORPHOLOGICAL ANALYSIS OF THE LATERAL INCISOR USING CBCT:

All measured morphological parameters of the lateral incisors – total length (TL), crown length (CL), root length (RL), and mesio-distal width (MDW) – were significantly reduced in the experimental group compared with the control group (P<0.001) (Table 2; Figures 1, 2).

GENOTYPE DISTRIBUTION OF PAX9 RS 2073247:

The PAX9 rs2073247 polymorphism exhibited 3 genotypes (CC, CT, and TT), whereas CC is considered a wild-type or reference genotype. In the experimental group, out of a total of 65 subjects, 19 had the wild-type CC homozygous genotype (29.2%), 30 had the heterozygous CT genotype (46.2%), and 26 had the mutated TT genotype (16%). In the control group, out of a total of 65 subjects, 29 were wild-type homozygotes CC (44.6%), 20 were heterozygotes CT (30.8%), and 16 were mutated TT homozygotes (24.6%) (Table 3). Chi-square test analysis revealed no statistically significant differences in genotype distribution between the groups (P=0.1298)

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The MSX1 rs12532 polymorphism exhibited 3 genotypes (AA, AG, and GG), with AA as wild-type or “normal” genotype served as referenced in this research. In the experimental group, out of a total of 65 subjects, 28 had the wild-type AA homozygous genotype (43.1%), 37 had the heterozygous AG genotype (47.7%), and 6 had the mutated GG genotype (9.2%). In the control group, out of a total of 65 subjects, 33 were wild-type homozygotes AA (50.8%), 29 were heterozygotes AG (44.6%), and 3 were mutated GG homozygotes (4.6%) (Table 3). As for PAX9, chi-square test analysis revealed no statistically significant differences in genotype distribution between the groups (P=0.4975).

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Analysis was also conducted to assess a patient’s susceptibility to maxillary canine impaction when SNPs of both genes were present. Upon evaluating the combined association of both the genes with MCI, no statistical significance was observed with CC/CT+TT, AA/AG+GG genotype combinations. However, a comparison between individuals with the combined wild-type genotypes (CC/AA) and those carrying at least 1 mutant allele in both genes (CT+TT/AG+GG) revealed a statistically significant association (P=0.01893) (Table 4, Figure 3).

LATERAL INCISORS MORPHOLOGY AND POLYMORPHIC ALLELES OF PAX9 RS2073247 AND MSX1 RS12532 CORRELATION:

A comparative analysis of lateral incisor dimensions was conducted based on combined genotypes (CC/AA vs CT+TT/AG+GG) using the independent t test. No statistically significant differences were observed between the groups for TL, crown length CL, or root length RL, with P values of 0.1412, 0.0703, and 0.3774, respectively (Table 5). However, a statistically significant difference was found in mesio-distal width (MDW), with individuals carrying at least 1 mutant allele (CT+TT/AG+GG) exhibiting a significantly reduced MDW compared to those with wild-type genotypes (5.58±0.76 mm vs 6.18±0.51 mm; P=0.0375). These results suggest that the presence of mutant alleles, previously linked with increased risk of canine impaction, could be associated with a shorter mesio-distal dimension of the lateral incisor.

Discussion

STUDY LIMITATIONS:

The limitations of this study include the relatively small sample size (130 participants) and the lack of longitudinal follow-up, both of which may have affected the results regarding the correlation between PAX9 and MSX1 polymorphism and maxillary canine impaction. Also, we only analyzed the PAX9 rs 2073247 and MSX1 rs 12532 polymorphisms, although these genes contain other known rs variations. Furthermore, this study was performed in the Serbian population; therefore, future research including other nationalities is needed. In addition, this research analyzed patients with both buccal and palatal impactions; future research should include larger samples with separate groups.

Conclusions

Taken together, these results reinforce the hypothesis that canine impaction occurs due to a combination of genetic predisposition and local morphological factors. The presence of at least 1 mutant allele in PAX9 and MSX1 genes and altered lateral incisor morphology may inrease the risk of maxillary canine impaction. This could be crucial for the early diagnosis of this common anomaly and the timely implementation of preventive and interceptive measures.