Extraction and Analysis of Genomic Deoxyribonucleic Acid (gDNA)

Intraoral epithelial cells were collected with the help of DNA collection swabs. Then the DNA isolation process was completed using PureLink DNA isolation kit (Invitrogen, Van Allen Way Carlsbad, Calif). The DNA samples were kept at −20°C until the relevant gene area analysis was finished.

Taqman SNP genotyping kits (cat. no. 4371355, Thermo Fisher Scientific Inc., Waltham, MA, USA) and Real-Time PCR on StepOnePlus (Thermo Fisher Scientific Inc.) were used for genotyping assays. Genotyping was performed according to the manufacturer’s protocols.

Statistical Analysis

IBM SPSS Statistics version 22 (IBM Corp., Armonk, NY, USA) was used for statistical analysis. Genotype and allele frequency distributions between Class II hyperdivergent patients with TMD and control groups were analyzed by χ² test.

Conformity of the parameters to a normal distribution was evaluated by Shapiro-Wilk tests. One-way analysis of variance was used to compare parameters between treatment groups and between gene alleles, and Tukey HSD was used to determine the group causing the difference. Significance was evaluated at P < .05.

MMP1 Polymorphism

A statistically significant difference was found among the groups for distribution of MMP1 alleles (P = .017). The rate of the 2G allele in Group 2 (72.7%) was significantly higher than in Group 1 (47.7%) and Group 3 (25.5%; P < .05; Table 2).

Statistically significant differences were found between MMP1 subgroups (1G/1G, 1G/2G, and 2G/2G) for mean ANB angle and Wits value (P = .036 and .010, respectively). The means of the ANB angle and Wits value for the 1G/1G genotype were significantly lower than for 1G/2G and 2G/2G (P < .05; Table 3).

Table 3.Evaluation of MMP1 Polymorphisms Among Subgroups

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A statistically significant difference between MMP1 subgroups was also found for mean Go-Gn (P = .030). The mean Go-Gn of 1G/1G was significantly greater than for 1G/2G (P < .05; Table 3).

TGF-β1 Polymorphism

Statistically significant differences were found between TGF-β1 subgroups (TT, TC, and CC) in SNB and GoMe-SN angles (P = .030 and .041, respectively). While mean SNB of the TT genotype was higher than TC, the mean GoMe-SN of TT was lower than in the TC genotype (P < .05; Table 4).

Table 4.Evaluation of TGF-β1 Polymorphisms Among Subgroups

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A statistically significant difference between the TGF-β1 subgroups was also found for mean facial axis angle (P = .027). The mean facial axis angle of the TC genotype was significantly lower than for CC (P < .05; Table 4).

Also, a statistically significant difference was found between TGF-β1 subgroups in mean Co-Gn distance (P = .030), which showed significantly higher values in the TT genotype than in CC (P < .05; Table 4).

MMP1 Gene Polymorphism

In the present study, a statistically significant difference was found among the three groups in terms of allelic frequency of MMP1 rs1799750 polymorphism. The rate of the 2G allele was higher in Group 2 than the other groups. Other researchers found a higher rate of the 2G allele in individuals with TMD in their studies.^14,24,25 In the current study, the heterozygous genotype 1G/2G was more common in Group 1, which agreed with Rosales et al.¹⁴ and Luo et al.²⁵ Differences among the studies may have been due to inadequacies in TMD diagnosis. In the current study, patients were diagnosed based on clinical examination, panoramic radiography, and RDC/TMD due to ethical reasons. In some previous studies, advanced imaging techniques such as MRI and computed tomography images were used.

Many hypotheses have been formed regarding the causes of TMD. However, the multifactorial etiologic approach proposed by De Boever²⁶ remains valid today due to the lack of scientific resources to support alternate hypotheses, which may have created differences between the study groups in the present study.

In the current study, although it was not statistically significant, 27.3% of Group 1 had the 1G/1G genotype, 9.1% of Group 2, and 40.9% of Group 3. Since Group 3 had Class I individuals with normal vertical dimensions, the homozygous 1G/1G genotype may be more common in individuals with normal skeletal development and a healthy TMJ.

A significant difference was found between MMP1 genotype subgroups for ANB angle and Wits value averages in the present study. The mean ANB angle and Wits value of 1G/1G were significantly lower than for 1G/2G and 2G/2G. These values provide information about the direction of skeletal development in individuals, and high values indicate the presence of a Class II skeletal relationship. The fact that these values were low in individuals with 1G/1G homozygous genotype suggests that MMP1 may provide information about skeletal development. The MMP1 1G/1G homozygous genotype increases the development of a skeletal Class I relationship.

Additionally, the Go-Gn distance of 1G/1G was significantly greater than for 1G/2G. Looking at the Go-Gn linear value, which provides information about mandibular corpus length, it could be speculated that the mandibular corpus length is longer in individuals with a homozygous 1G/1G genotype than in individuals with a heterozygous 1G/2G genotype.

This present study is the first one to examine MMP1 polymorphism and TMD in a small sample of the Turkish population. Additionally, we were the first to classify patients according to cephalometric values and to examine MMP1 polymorphism. In the future, further studies on this subject with larger sample sizes and using more accurate diagnostic tools or TMD will contribute more to the finalization of the results and the elucidation of this topic in the literature.

TGF-β1 Polymorphism

In the present study, heterozygous TC and homozygous CC individuals were more common in Group 1 than in the other groups, although the difference was not statistically significant. In the literature, Jiao et al.²⁷ suggested that TGF-β1 may be an important marker for the development of TMJ osteoarthritis. No authors in the literature have investigated the relationship between TGF-β1 rs1800470 polymorphism and joint disorders. However, the presence of the C allele has been reported to cause diseases such as osteoarthritis, hip dysplasia, and rheumatoid arthritis.^28,29 Therefore, the greater percentage of the C allele in Group 1, which had TMD patients, may support these findings.

In the TGF-β1 genotype subgroups, some of the skeletal values showed significant differences. While the SNB angle in the TT subgroup was greater than in TC, the Co-Gn distance was greater in the TT subgroup than in CC. Since these values are both related to mandibular growth, it may be concluded that the presence of the C allele negatively affects mandibular growth.

Additionally, while GoMe-SN in the TC subgroup was significantly higher than TT, the facial axis angle was significantly lower in TC. Although the other differences were not statistically significant, it can be speculated that higher ratios of vertical height were seen in the heterozygous TC subgroup.

In the literature, this article is the first one relating TGF-β1 rs1800470 polymorphism and craniofacial development. Although the results were not statistically significant, it might be suggested that the presence of the C allele negatively affects mandibular growth. To reach statistically significant results, increasing the sample size and looking at these linear and angular values may contribute to the literature on mandibular development. In the future, having more information about genetic skeletal deformities in individuals may provide further information to guide growth during treatment.