The Pilot Study Of Susceptibility Genes Of Temporomandibular Joint Osteoarthritis

Osteoarthritis (OA) is one serious subtype of temporomandibular joint disorder (TMD). Its etiology remains unclear. It has self-limitation and susceptibility. Genetic factor, especially single nucleotide polymorphism (SNP) as a marker of susceptibility has been studied widely in large synovial joints. However, this kind of studies in TMD, especially in OA, is in the initial stage. There are few studies have been published about TMJ OA. Only one report is about the relation of MMP-1 rs 1799750 polymorphism and the susceptibility to TMJ OA in Brazil people. Therefore, collagen type one, the most common component of fibrocartilage and subchondral bone of TMJ draws our attention. The SNP of collagen type one relevant genes, the polymorphism of MMP-1 promoter (-1607 1G/2G), and the G/T polymorphism within the COL1A1 gene’s regulatory region, were assumed to be associated with TMJ OA. Firstly, in order to find the most suitable genomic DNA extraction method, three classical methods were compared. Secondly, used high resolution melting method to genotype these two SNPs, and compared their alleles and genotpyes’ frequencies with multivariation logical regression model to study the association of these SNPs with cartilage and subchondral bone degeneration. These studies contained four parts:the comparison of three kinds of genomic DNA extraction methods (research 1); the relation of MMP-1 rs 1799750 polymorphism with TMJ OA (research 2); the relation of COL1A1 rs 1107946 polymorphism with TMJ OA (research 3) and the relation of rs 1799750 and rs 1107946 with osteophyte of TMJ OA and clinical observation (research 4). The summaries of above researches are as follow:Research 1Background and objective:Genetic factors play important role in the susceptibility of TMD. Extracting qualified genomic DNA is the basis of genetic research. So it is necessary to choose a scientific and effective extraction method.Method:Venous blood was collected from inpatients. And genomic DNA was extracted with phenol-chloroform (method 1) or magnetic beads isolation technique (method 2). Buccal mucosal cells were collected with sterile swabs from outpatients. And genomic DNA was extracted with forensic DNA extraction kit (method 3). These DNA samples were test about purity, concentration, integrity, and their following genetic experiment effect.Result:There were 337 blood samples were extracted with method 1 (38 DNA samples were unqualified, accounted for 11.3%); 273 blood samples were extracted with method 2 (22 DNA samples were unqualified, accounted for 8.1%); and 397 buccal mucosal cell swab samples were extracted with method 3 (21 DNA samples were unqualified, accounted for 5.3%). According to the t-test, there was no difference in the purity of DNA samples between three methods (P>0.05). There was difference in concentration of DNA samples between method 1 and 2 (P=0.0002), no difference between method 1 and 3 (P>0.05), as well as between method 2 and 3 (P>0.05). The qualified in purity and concentration DNA samples were chosen 20 cases in each method randomly. They were tested using agarose gel electrophoresis and were observed to have no degradation, and have similar molecular weight. Then they were genotyped with high resolution melting method, and were found three genotypes of MMP-1 rs 1799750 successfully.Conclusion:These three methods are scientific. Buccal sterile swabs method is more effective.Research 2Background and objective:Temporomandibular joint (TMJ) disorders are multifactory complex diseases, which are affected by multy genetic variations. So it is necessary to investigate the relationship of the polymorphism of MMP-1 promoter (-1607 1G/2G) with the susceptibility to anterior disc displacement (ADD) and osteoarthritis (OA).Methods:A total of 185 healthy individuals (group A),141 unilateral ADDWR patients (group B), and 321 unilateral ADDWOR patients (group C).115 without TMJ OA, named group C-1; 206 with TMJ OA, named group C-2) were recruited. The genomic DNA was extracted by using high resolution melting assay. After adjustment of potential variables, these groups were pairwise compared by using multivariable logical regression assay model.Results:There were significant differences in frequency of 2G2G genotype between three pairwise comparisons (PC-A<0.0005; PC1-B= 0.049; PC2-B=0.018).2G2G genotype carriers’ susceptibility to ADDWOR with or without TMJ OA is different times higher than that of other genotypes carriers, according to our three comparisons(ORO-A= 2.455; ORC1-B=1.849; ORC2-B= 1.912). There were significant differences in frequency of 1G2G genotype between two pairwise comparisons (PC-A <0.0005; PC2-B=0.041).1G2G genotype carriers’ susceptibility to ADDWOR with or without TMJ OA also is different times higher than that of other genotypes, according to these two comparisons (ORC-A= 2.641; ORC2-B=1.896).Conclusion:This study suggested that the -16071G/2G polymorphism of MMP-1 promoter might relate with the susceptibility to ADDWOR with or without TMJ OA.Research 3Background and objective:The G/T polymorphism within the COL1A1 gene’s regulatory region exhibits a clinically significant influence on bone remodeling, leading to predisposition of degenerative diseases. Enhancement of bone turnover and further loss of bone mass, featured by low-density lesion, are thought to be the primary pathological changes in early degenerative course of temporomandibular joint (TMJ). Thus, we hypothesized that this polymorphism may also affect this kind of bone lesion in TMJ osteoarthritis (OA).Method:A total of 130 TMJ OA patients with low-density lesion (cortical bone erosion, condylar head resorption, cyst-like lesion) and 186 healthy individuals were recruited. DNA samples were extracted from buccal mucosa swabs. This polymorphism was genotyped by high resolution melting assay. The distributions of genotypes in these groups were compared using a multivariate logistic regression model.Results:No significant differences in the distributions of TT and TG genotypes were observed between healthy and TMJ OA groups (P> 0.05). Borderline significance was detected in GG homozygous carriers (P= .043); this genotype might be a risk factor of this low-density lesion (OR=1.643,95% confidence interval= 1.016-2.658).Conclusion:This study indicated that the GG genotype might be a risk factor of low-density lesion of TMJ. Therefore, phenotypic subtypes of OA should be accurately identified to individualize and optimize treatment.Research 4Background and objective:The mechanism of TMJ osteophyte is not clear until now. To assist evaluate the mechanism of osteophyte from the point of genetics, the differences betwenn osteophyte cases and non-ossteophyte patients in frequencies of carring the MMP-1 rs 1799750 and COL1A1 rs 1107946 were conducted. There are reports suggest osteophyte is associated with clinical symptoms (pain and dysfunction) in other large synovial joint, so it is reasonable to assume osteophyte of TMJ may also leads to these symptoms.Method:632 patients’data about osteophyte length (more than 2 mm), and the position of osteophyte (functional surface or non-functional surface), pain status (visual analogue scale value, VAS> 2.57±1.04), limitation of mouth opening (< 3.5 cm) were recorded.310 TMJ OA patients’ genomic DNA samples were collected randomly. And used high-resolution metling method to genotype their rs1799750 and rs 1107946. Then compared their differences in the distribution of genotpyes and alleles, and evaluated the relation between osteophyte and pain and joint dysfunction with X2-test.Results:No significant difference was found in the distribution of genotypes and alleles of osteophyte and nonosteophyte patiients in these two polymorphisms (P> 0.05). There was significant association between obvious osteophyte and pain (P= 0.002), and it can more possible increase the chance of pain (OR=1.673,95%CI= 1.213-2.309). This effect depends on the position of osteophyte (P=0.007), the osteophyte on functional surface may more possible increase the chance of pain than non-functional surface osteophyte (OR=2.046,95%CI=1.230-3.402). There was significant association between obvious osteophyte and joint dysfunction (P= 0.00001),and it can more possible increase the chance of joint dysfunction (OR= 2.044,95%CI=1.480-2.821).Conclusion:Osteophyte and non-osteophyte TMJ OA patients have the same polymorphism carring situation in rs 1799750 and rs 1107946. Obvious osteophyte may lead to pain and TMJ dysfunction.