IDENTIFICATION OF POTENTIAL GENETIC MARKERS OF FACIAL ASYMMETRY AND TMD IN ORTHOGNATHIC SURGERY PATIENTS

Abstract

Temporomandibular disorders (TMD) are comorbid conditions. Most are related to anxiety-induced muscular pain, but some are associated with facial asymmetry resulting from condylar resorption (CR) or condylar hyperplasia (CH). The etiology of the most common forms of CH and CR are still unknown. CR can be caused by rheumatoid arthritis (RA) or more commonly osteoarthritis (OA) of the TMJ, and inflammatory mediators have been previously implicated. Previous studies have identified pain/inflammatory genes related to chronic TMD while others have demonstrated potential genetic markers for RA. Similarly, genome-wide association (GWA) studies have identified genes associated with height, some of which may participate in craniofacial growth, CH, and the development of asymmetry. Masseter muscle is frequently involved in TMD of muscular origin, and left/right fiber-type differences have been previously found in subjects with facial asymmetry. A human transcriptome microarray was used to evaluate whether genes involved with height, pain, or inflammation were differentially expressed in masseter muscle from facially asymmetric patients with and without TMD. This study evaluated orthognathic surgery patients with varying skeletal malocclusions, including subjects with and without facial asymmetry and TMD (n= 93). Masseter muscle samples were collected from ten orthognathic surgery patients treated to correct skeletal malocclusions. Two of whom were classified with facial asymmetry with or without TMD, with one of the two showing positive evidence of CR. Samples were disrupted in QIAzol Lysis Reagent, RNA was isolated using a Qiagen miRNeasy Mini Kit according to the manufacturer's instructions, and quality of the total RNA was tested by Agilent Bioanalyzer and Nanodrop spectrophotometry. Samples were used for quantitative Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) and protocols for microarray analysis were conducted as described in the Ambion WT Expression Manual and the Affymetrix GeneChip Expression Analysis Technical Manual. Principal Components Analysis (PCA) was completed to detect fold-changes for each transcript to determine differences in global gene expression between the two asymmetric and eight remaining subjects. To find differentially expressed transcripts step-up t-tests were performed to correct for false discovery rate (FDR) comparing the two asymmetric samples to the eight symmetric samples. Differences were considered significant if step-up p-values were ±2 between groups. This study evaluated 847 height-related genes and 551 genes associated in pain/inflammatory processes. Genes of interest were determined a priori from GWA studies and the Algynomics Pain Research Panel v.2.0 partially derived from the Orofacial Pain: Prospective Evaluation and Risk Assessment (OPPERA) study. Two hundred and eight transcripts of 847 height associated genes and 132 of 551 pain/inflammatory genes were significant for expression (P±2.0 fold differences in facial asymmetry and/or TMD specimens. Among genes specifically reported to be associated with pain/inflammation, NPY5R (+2.11 fold), GABRA6 (+2.14 fold), CACNA2D1 (-12.51 fold) and EREG (+2.12 fold) showed significantly different (P<0.001) expression levels in the two asymmetric versus the remaining eight symmetric patients. CACNA2D1 expression was significantly increased in symmetric male subjects versus symmetric females (P < 0.05) as well as in asymmetric females versus asymmetric males (P < 0.05). CACNA2D1 expression was also significantly increased in symmetric male subjects versus symmetric females (P <0.05) and was differentially expressed at lower levels, however not significantly, in asymmetric males (p = 0.51). Based on the results collected, the following conclusions were drawn. These methods provide a novel approach to study TMD and/or facial asymmetry in human subjects. To our knowledge, this is the first study to demonstrate that significant expression variation in human height genes may contribute to facial asymmetry with or without TMD, possibly through decreased expression of CACNA2D1. These data suggest TMD patients with facial asymmetry associated with condylar resorption may show significant differential expression of certain inflammatory marker genes such as EREG and CACNA2D1. These data support that gender may play a key role in the development of TMD, possibly through increased CACNA2D1 expression providing protective effects in TMD-free males but deleterious effect in females with TMD. These results support previous findings of pain/inflammatory genes associated with TMD derived from muscular pain. Further studies are needed to understand the genetic contributions to TMD, which may play an important role in future clinical intervention.