Association of Masseter Muscle PITX2, ENPP1 and ESR1 Expression, Muscle Fiber Type, Temporomandibular Joint Disorders and Subclassifications of Craniofacial Asymmetry

Abstract

Craniofacial asymmetry is a dentofacial deformity with genetic influences. The genes PITX2, ENPP1 and ESR1 have multiple genetic associations with functional properties in muscle and bone. The objectives of this study are to investigate how PITX2, ENPP1 and ESR1 gene expression associates with four subclassifications of craniofacial asymmetry, temporomandibular disorders and fiber type differences compared between right and left masseter muscles. We developed an asymmetry classification that diagnosed four types of asymmetry with distinctive growth patterns: Group 1 – menton deviation without ramal difference (“mandibular body asymmetry”); Group 2 –menton deviation with shorter ramal height on the deviated side (“typical asymmetry”); Group 3 – shorter ramal height on the opposite side of menton deviation (“atypical asymmetry”); Group 4 – menton deviation with shorter ramal height and maxillary canting on the deviated side (“C-shaped asymmetry”). Some of these patients are at high risk for TMD; therefore, temporomandibular joint functioning is assessed as a routine part of the pre-surgical evaluation. TMD was diagnosed using the Diagnostic Criteria for TMD (DC/TMD). The clinical examination includes mandibular range of motion, palpation for pain, joint noise and bruxism. In addition, the Jaw Pain and Function (JPF) questionnaire was used to assess patient reported symptoms as an indication of perceived severity before and one year after orthognathic surgery. Masseter muscle samples were collected from 174 subjects undergoing surgical treatment for correction of malocclusion. Muscle serial cross-sections were mounted for immunostaining with five antibodies specific for myosin heavy chain (MyHC) isoform. We classified masseter fibers into 4 fiber type groups: type I, type I/II hybrid, type IIA and/or IIX, neonatal and atrial. With the remaining muscle samples, total RNA was isolated and PITX2, ENPP1, and ESR1 expression was quantified using TaqMan qRT-PCR. Average relative quantity gene expression values and percent differences between left and right masseter samples were calculated. In this population, there is a high prevalence of facial asymmetry (48%). Pre-surgical mean JPF scores are significantly different between symmetric (JPF=1.97) and asymmetric (JPF=6.9; p<0.001) patients; with scores ≥ 6 diagnostic for presence of TMD. ENPP1 and ESR1 expression is differentially expressed between right and left masseter muscle in patients with asymmetry. ENPP1 is differentially expressed in asymmetry group 4 (p=0.01) and ESR1 is differentially expressed in asymmetry group 1 (p=0.048), group 2 (p=0.004) and group 4 (p=0.02). Masseter fiber type properties of type I, type I/II hybrid and type II fibers associate with facial asymmetry and specific subclassifications, suggesting functional differences between type I, type I/II and type II fibers may be important factors in the development of symmetry between facial sides. There are significant differences in the left-right percent differences of fiber area of type I fibers in asymmetry group 3 (p=0.05), type I/II hybrid fibers in group 3 (p=0.02), and type II fibers in asymmetric patients (p=0.03), asymmetry group 2 (p=0.05) and group 4 (p=0.005). Additionally, there are significant differences in the left-right percent differences of percent occupancy of type I fibers in asymmetric patients (p=0.04), asymmetry group 2 (p=0.01) and group 3 (p=0.05) and type II fibers in asymmetry group 2 (p=0.04). By comparing gene expression with masseter muscle fiber type properties, we found significant results for PITX2 and ENPP1 suggesting their roles as genetic factors influencing jaw bone length and masticatory muscle strength in malocclusion. There are significant positive correlations between left-right percent differences of PITX2 and type I fiber area (r=0.86; p=0.03), type I/II hybrid fiber area (r=0.94; p=0.006), and type I/II hybrid fiber percent occupancy (r=0.90; p=0.01). Also, there are positive correlations approaching significance between left-right percent differences of ENPP1 and type I fiber area (r=0.80; p=0.06) and type I/II hybrid fiber area (r=0.75; p=0.09). Given the high prevalence of TMD in a population of patients with facial asymmetry, we compared differences in gene expression in masseter muscle of patients with specific TMD diagnostic conditions. Average PITX2 expression is significantly increased (p=0.0375) and average ENPP1 is increased, but not significantly, in all TMD patients diagnosed by the clinician. Average ESR1 is slightly increased compared to JPF scores and may be an essential factor for patient reported TMD symptoms. With these results, PITX2, ENPP1, and ESR1 should be considered biomarkers for asymmetry and TMD; however, further studies are needed to provide a more thorough understanding of the genetic influences on the craniofacial complex.