Temporomandibular disorder has been associated with psychological dysfunctions , and sexual dimorphism . Its occurrence also appears to coincide with puberty . Currently, controversy exists as to the importance of underlying genetic and environmental nutritional factors, such as vitamin intake . The folate-methionine axis takes part in both factors. In this axis, there are several polymorphic variants of the enzymes involved that together with folate dietary intake determines the metabolism of the axis. Furthermore, the transfer of methyl groups to DNA and proteins is one of the limiting factors for the correct development of the proliferating tissues. Therefore the latter is key for the continuous tissue growth and epigenetic modifications.
This work studied the implication of 27 polymorphisms located on 17 genes and their relation with TMD. It has been designed under the hypothesis of that TMD is a multifactorial syndrome related to a critical period of human life that have a genetic and epigenetic basis [12–22]. The study of genes related to folate cycle is directly involved in the two ways: the genetic, by gene polymorphisms, and epigenetic by the nutritional habits because of the folate intake. Deficit of folate intake is known as the most prevalent vitamin deficit in human. The majority of the genes studied were related to folate metabolism even though there were others related to oxidative metabolism and hormonal and neurotransmission receptors. Of the17 genes studied of the folate cycle, 3 of them (SHMT, MTHFD, MTRR) showed significant associations with TMD. Significant changes in the allele and genotype frequencies between the TMD patients and controls were found.
SHMT catalyzes a reversible step of THF to 5,10-CH2-THF, which is a key substrate to obtain 5-methyl-THF and synthesize thymidylate. On the other hand, MTHFD1 provides the 5, 10CH2-THF substrate in addition to having a central role in the cycle due to its triple functionality. The 5-methyl-THF to methionine step is catalyzed by MTR in cooperation with TCN and MTRR, which are essential for the participation of vitamin B12 as a cofactor and the reactivation of MTR, respectively.
In our case, a higher significant frequency of the allele SHMT-rs1979277G was observed in TMD patients (0.67 vs 0.53). The Allele G has been related to higher levels of folates and homocysteine , this could mean that the enzyme activity is deviated to the THF and glycine synthesis versus to the serine and 5,10CH3THF substrate of the MTHFR which provide 5'CH3THF needed to the methionine synthesis by mean of homocysteine. The increase of homocysteine, classified as an oxidant, could favour inflamatory process. However the effect of this polymorphism on enzyme activity is still controverted .
Statistical differences were also observed in allelic frequency of the C allele the SNP rs638416, in the promoter region of the SHMT gene. SHMT not only provides one-carbon units for thymidylate biosynthesis but also generates a pool of methylenetetrahydrofolate for SAM synthesis by means of serine synthesis. In addition, the SHMT gene has the capacity to sequester 5-methyl-THF and therefore inhibit the synthesis of methionine and SAM, which is the methyl donor for DNA and proteins . Therefore, greater SHMT activity would produce higher 5-methyl-THF sequestration, which would reduce SAM synthesis. Cellular deficits of SAM could induce lesser methylation on the novo synthetized DNA on inflamatory tissues, which can take to higher gene expresión levels, generating a positive feedback on inflammation and pain.
TMD patients and healthy controls had very significantly different genotypic frequencies of the SNP rs2236225 of the MTHFD gene. The triple functionality of the MTHFD enzyme and the central role it plays in the folate cycle make it one of the key points for the balance of this metabolic route . Therefore, it is not difficult to believe that any genetic alteration that affects its expression or activity would have consequences for methyl transfer and consequently on epigenesis.
The results of the present work show that the polymorphic variants SHMT-rs1979277, rs638416, and MTHFD1-rs2236225 found in the folate cycle were significantly associated with TMD. They coincided with a global decrease in the availability of methyl groups via a decrease of the substrate 5,10-CH2-THF (SHMT and MTHFD1), while the SNP MTRR-rs1801394 did not follow the same pattern. The wild allele A, predominant in TMD, which means greater efficiency in the pathway of homocysteine to methionine, has the same final effect of the variants of SHMT and MTHFD, an increase of THF levels.
TMD have been associated to oxidative stress of the temporomandibular joint [40–44]. Regarding GTSM1, we found a significant number of individuals with TMD (OD = 2.21; 95%CI 1.24, 4.35; p = 0.030) with the null variant of the gene. It has been published that oxidative stress, which leads to the production of NO and peroxynitrite, is harmful to DNA in response to the excessive mechanical overload, which in turn promotes synovial hyperplasia in the temporomandibular joint . This suggests that a reduced detoxification capacity would make us more susceptible to TMD.
Previous studies on associations of TMD with alpha ESR1 polymorphisms have shown different results. One of them find a predisposition to TMD associated to ESR1 polymorphism in women  others find a relation between pain  or craniofacial morphology  in TMD women with ESR1 polymorphism, however other recent report does not find this association. In the present study there were no statistical differences in their individualized allelic and genotypic frequencies or in the distinct haplotype combinations. However, a study on the association between these polymorphisms and the predisposition to TMD has recently been published and deepens the characterization of the diplotypes using novel mathematic algorithms, which we will take into account in future studies.
In contrast to others authors [18, 21], significant variations were not found in the frequencies of the 5-HTTLPR (serotonin-transporter-linked promoter region) polymorphism of the serotonin receptor gene SCL6A4. However, we found a significant variation in the 48-bp VNTR polymorphism of the DRD4 gene. This polymorphism has been associated with pain processes, such as fibromyalgia  and migraine .