Genotyping analysis of the Pax9 Gene in patients with maxillary canine impaction

Background: Paired-box gene 9 ( PAX9) mutation is potentially associated with impaction in some patient populations. Here, we analyzed the relationship between PAX 9 polymorphism and the occurrence of maxillary canine impaction. Methods: Patients with and without maxillary canine impaction were selected based on specific inclusion criteria, and samples of genomic DNA were obtained from a buccal mucosa swab. DNA was amplified by polymerase chain reaction and sequenced for further bioinformatics analysis to identify single nucleotide polymorphism (SNP) genotypes. Genotype and allele counting was performed in both case and control groups prior to conducting statistical analysis. Results: Four SNPs were identified in patients with maxillary canine impaction, with relative confidence determined based on chromatogram-peak assessment. All SNPs were located in exon 3 of PAX 9 and in the region sequenced by the primer pair −197Fex3 and +28Rex3. Three of the SNPs (rs375436662, rs12881240, and rs4904210) were reported previously and are annotated in NCBI (dbSNP version 150), whereas another SNP mapped to chromosome 14 has not been reported. Patients with a CC genotype at SNP 3 [odds ratio (OR): 2.61 vs. TT; 1.28 vs. CT] and a CC genotype at SNP 4 [OR: 0.71 vs. GG; 0.79 vs. CG] were more likely to have maxillary canine impaction. Conclusions: These results demonstrated that the presence of SNPs 3 and 4 is associated with increased likelihood of suffering from maxillary canine impaction.

Maxillary canine teeth are the second most common targets of impaction after the third molars 1 . An impacted maxillary canine occurs in 1% to 3% of the general population and is twice as common in females as males 2,3 . It is commonly presented in clinics by patients often arriving with an aesthetic-related complaint.
Maxillary canine impaction in the palatal position is possibly caused by genetic factors and often accompanied by dental abnormalities in tooth shape, size, number, and structure. Abnormalities, such as agenesis, oligodontia, and peg-shaped teeth, have a genetic link to the presence of impacted teeth and generally manifest in developmental disorders during growth 4-8 . There is a relationship between malposition of certain teeth, such as palatal canines, and teeth agenesis. Similar to dental agenesis, canine-tooth-position anomalies affect several family members and are considered to be under strong genetic control 9-15 . Paired-box gene 9 (PAX9) is most commonly involved in affecting the odontogenesis process and thought to determine the localization of tooth seeds 16 . In this study, we identified an association between PAX9 genotype and the occurrence of maxillary canine impaction.

Patients
Patients were recruited from the Dental Hospital Faculty of Dentistry, Universitas Indonesia, and three different junior high schools in South Jakarta, and the study was conducted through clinical observations from May 2018 to August 2018. Those meeting the inclusion criteria (male or female, 10-25-years old, no systemic disease, and no hereditary disease) were either diagnosed with maxillary canine impaction (group I) or diagnosed as without (group II; control). Diagnosis was based on clinical examinations and radiographic interpretations performed by radiologists and orthodontists who were experts in their fields. Comprehensive clinical data were obtained for 132 patients (see Underlying data 17 ). All participants gave their written informed consent to participate in this study, which was approved by the Ethics Committee of the Faculty of Dentistry, Universitas Indonesia (No. 07107105/Ethical Approval/ FKGUI/2018).
Polymerase chain reaction (PCR) amplification Genomic DNA was collected from the buccal mucosa via swabbing and extracted using the Gene Jet whole blood genomic DNA purification kit (Cat. No. K0781; Thermo-Biogen, Karlsruhe, Germany). The area of the buccal mucosa/cheek to be treated was dried with a cotton roll to prevent salivary contamination. Samples from the buccal mucosa were obtained using a cytobrush (#C0104; Medscan; Cooper Surgical, Trumbull, CT, USA) on the bilateral buccal mucosa, with each side swabbed 10 to 15 times. The cytobrush swab was inserted into a screw-capped Eppendorf tube (Cat. No. SPL-60015; Extragene, Taichung City, Taiwan) containing 200 µL of 1% phosphate-buffered saline. Eppendorf tubes were labeled and stored in a freezer at −4°C. DNA concentration was measured using a Qubit Fluorometer 3 (#Q33216; Invitrogen, Carlsbad, CA, USA) at standard fluorescence wavelengths (excitation/emission: ~480/530 nm) with Qubits assay reagent (MP423-#Q32851; Qubit dsDNA HS assay kit; Invitrogen).

Bioinformatics analysis
To detect sites containing potential single nucleotide polymorphisms (SNPs), sequencing data were converted to FASTQ format, and chromatogram-peak of each patient DNA sequence was performed as can be seen in the representative data from one sample ( Figure 1). Heterozygous SNPs were determined by visually identifying sites containing two overlapping peaks in both the forward and reverse sequences.
For each SNP identified, genotypes across all samples were obtained, annotated, and cross-checked against the NCBI SNP reference database (dbSNP v.150). Both genotype and allele counting were performed before conducting statistical analysis for both the case and control groups.

Statistical analysis
Statistical analysis of the data was performed using SPSS (v.20.0; SPSS, IBM Corp., Armonk, NY, USA). Analysis was initiated with a quality check of the variables. Analysis of genotype data was conducted using a chi-squared test to compare SNP frequency between patients with maxillary canine impaction and controls. A P < 0.05 was considered statistically significant.

SNP identification
Four SNPs were identified, with all of these located in exon 3 of PAX9 sequenced using primer pair 3 (−197Fex3 and +28Rex3). Sequencing data for this region were available for 121 of 132 samples, and no SNPs were identified in regions sequenced using the other primer pairs.  and are annotated in NCBI dbSNP (v.150; rs375436662, rs12881240, and rs4904210). The fourth SNP in exon 3 maps to chromosome 14, position 36,666,530, and has not been previously reported. Table 3 lists the SNPs identified in this study along with their annotation/reference identification and the location of the nucleotide substitutions. Nucleotide changes included 640A>G in SNP 1, 700C>T in SNP 2, 717C>T and 718G>C in SNP 3.

Genotype calling
Genotype assessment was performed for all of the identified SNPs, as well as controls, focusing on counts of homozygous (wild-type) and heterozygous and homozygous (mutant) alleles (Table 4-Table 7).
For SNPs 3 and 4, all genotype and allele variations were observed in both the case and control samples, whereas for SNPs 1 and 2, the homozygous and heterozygous mutants were present in only one of the cases (Table 6 and Table 7).

Statistical analysis
We then analyzed associations between SNPs 3 and 4, which were present in both case and control samples, and maxillary canine impaction. We verified Hardy-Weinberg equilibrium (HWE) for both SNPs in case and control samples (

Genotype efficacy for clinical diagnosis
The use of patient genotype information for clinical assessment represents a possible diagnostic strategy for predicting disease likelihood. Patient characteristics and genotype information were available from 121 of 132 samples, with Table 9 summarizing the clinical information associated with both the case and the control groups.
Stratification of cases with SNP 3 (rs12881240) showed that 21 of 53 males and 24 of 68 females in the case group harbored the CT genotype, whereas only four males and two females harbored the TT genotype, with the difference between males and females in this group not statistically significant (P = 0.3996       Although the frequency of genotype variation was higher in females than in males, the difference was not statistically significant (P = 0.7725). Consequently, these findings suggested that gender was not a major influence on the occurrence of maxillary canine impaction.

Discussion
The etiology of canine impaction might be multifactorial and involve external factors, such as environmental input (e.g., trauma), local factors (e.g., lack of space, prolonged retention of primary teeth, trauma to permanent tooth seeds, rotation of permanent seed teeth, and the presence of pathological lesions, such as dentigerous cysts or odontoma), genetic factors, and systemic disease 18-24 . Dentistry is increasingly making use of genetic information, which plays an important role in addressing clinical problems, especially with regard to dental abnormalities or anomalies, including impaction of the maxillary canines. Previous studies show that tooth development is regulated by >200 genes 23 . PAX9 encodes a transcription factor and is among the most frequently identified genes affecting the odontogenic process and involved in the occurrence of dental anomalies, such as agenesis teeth, congenital missing teeth, and variabilities in tooth size and position. The identification of genetic risk factors associated with canine impaction has recently become the subject of intensive research.
In this study, DNA sequencing of 121 of 132 patient and control samples identified four SNPs located in a similar region of PAX9 exon 3 (Table 2). SNPs play a role in determining disease characteristics, including etiology and the incidence and risk of disease development. Subsequent analysis revealed that all of the identified SNPs would result in missense mutations (Table 3). These findings suggest that SNPs might be efficacious for determining dental anomalies, specifically the impaction of maxillary canines.
Although we found no statistically significant association between PAX9 genotype and maxillary canine impaction (Table 8), there were variations between patients with and without this condition according to the presence of SNP 3 and SNP 4, which carried a greater risk for maxillary canine impaction. A previous study reported that the genes involved in the impaction or displacement of canines into the palate are also responsible for controlling the growth and eruption of teeth 25 , and Klein et al. 26 showed that dental anomalies (size, shape, and position of teeth and agenesis of teeth or supernumerary teeth) were determined by a set of genes involved in tooth development. The results of the present study suggested a potential role for PAX9 in tooth growth and development.
In summary, our findings showed no statistically significant association between SNP genotype and gender and demonstrated that although the frequency of impaction-related genotype variation in women was higher than that in men, the differences were not statistically significant. These results suggested that gender-associated variations in genetic profile do not contribute to the incidence of maxillary canine impaction.

Consent
Written informed consent for publication of patient details was obtained.

Data availability
Underlying data ABI files and chromatograms that support the findings of this study are available on reasonable request from the corresponding author [author initials] and by submitting the applicable request form ('Form Padia' to request access for genetic resources (available as part of the OSF deposit)). The data are not publicly available due to them containing information that could compromise research participant privacy.

Grant information
The author(s) declared that no grants were involved in supporting this work. 1.

4.
I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. This study has reported new information that is very important to explain the effect of genes mutation in canine impaction. This research is very interesting. The researcher analysed the relationship between mutations and Pax9 gene polymorphisms with cases of canine tooth impaction. This research has been carried out using gene sequencing. The results showed an association between Pax9 gene mutations (SNPs 3 and 4) with the appearance of maxillary canine teeth impaction. The abstract contains concise and clear information about the background, objectives, methods, results and conclusions, so that it is quite easy for the readers. The following are inputs to improve the quality of this manuscript: In the abstract, it is written in the background section: "Paired-box gene 9 (PAX9) mutation is often associated with impaction in some patient populations." This indicates that previous researchers may have conducted similar studies in different populations. It is advisable to write about this in the introduction, to strengthen the reasons for choosing the pax9 gene and also in the discussion, to analyse possible similarities or differences in pax9 polymorphism in different study populations. The results showed that gender has no effect on the incidence of canine impaction, gender-associated genetic variation has no contribution. It would be better if the authors explain the possible causes, or show references from previous research reports that are relevant to the phenomenon. Does this occur only in canine impaction or is it possible for other tooth impaction cases? Please add to the discussion section, factors that are likely to cause the emergence of Pax9 gene polymorphism. Material and methods: the researcher conducted a sequencing analysis, please explain whether the method used is next generation sequencing.

If applicable, is the statistical analysis and its interpretation appropriate? Yes
Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results?

Are the conclusions drawn adequately supported by the results? Yes
No competing interests were disclosed.

Competing Interests:
Reviewer Expertise: Cell Biology I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
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