Epigenetics and oral disease

7 Epigenetics and oral disease Simon A. Fox 1, Lalima Tiwari 1, Camile S. Farah 1, 2 1

2

UWA Dental School, University of Western Australia, Nedlands, WA, Australia Australian Centre for Oral Oncology Research & Education, Nedlands, WA, Australia

Abstract Recent advances in molecular cell biology have demonstrated that there are additional levels of complexity beyond changes in DNA sequence whereby epigenetic mechanisms have extensive effects on gene transcription and function through modifications of DNA, histones, and chromatin structure. These mechanisms have a critical role in cellular and organismal development and have led to a profound change in our understanding of these processes and their aberrations in disease. The epigenetic mechanisms which have been studied most intensively in medicine and oral health are DNA methylation and covalent modification of histone proteins although our understanding of their biology and regulation is incomplete. In oral health, the most research evidence has been reported for the impact of epigenetics in craniofacial development including odontogenesis, dental caries, periodontal disease, salivary gland disease, oral mucosal lesions, orofacial pain, and most extensively malignancy. In these areas, there is significant evidence showing a role for epigenetics in disease mechanisms and consequently a prospect of intervention through epigenetic-targeted therapies. Here, we discuss the evidence to date and the prospects for future research driven by advances in technology and improved biological understanding of epigenetics.

General introduction What is epigenetics? It is now well recognized that epigenetic mechanisms play a significant role in normal human development, and that epigenetic aberrations contribute to both developmental and other disease processes. While somatic cells carry the same DNA information, epigenetic mechanisms modulate the expression of this information to produce the complex patterns of gene regulation.

Translational Systems Medicine and Oral Disease. https://doi.org/10.1016/B978-0-12-813762-8.00007-4 Copyright © 2020 Elsevier Inc. All rights reserved.

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These are required not only for development but also for maintenance of the patterns which define different tissues and cell types within the body. The term “epigenetics” itself derives from the work of Conrad Waddington in 1942 who used it in the context of developmental biology to describe the interaction between genes and their products which lead to the manifestation of phenotype. Subsequently, the term has become more broadly and diversely applied in different fields leading to ambiguity in the literature. Epigenetics has been more recently specifically defined as focusing on the inheritance of gene expression patterns not based on DNA sequence.1 Deans and Maggert2 extensively discuss the definition of the term and define epigenetics as “the study of phenomena and mechanisms that cause chromosome-bound heritable changes to gene expression that are not dependant on changes to DNA sequence.” Thus, mechanisms that more broadly fall into the area of general gene regulation are not included within this definition. Although the heritable nature of epigenetic patterns is part of this definition, it is also clear that they are subject to dynamic change in response to regulation. DNA methylation is the most widely studied and best understood epigenetic mechanism in the genome. The principle mechanism is considered to be inhibition of the binding of transcription factors to regulatory regions and suppression of transcription. DNA methylation is a key mechanism regulating spatial and temporal gene expression during mammalian development.3 The basic structural unit of chromosomes is the nucleosomes which consist of genomic DNA wound around octamer structures of histone proteins. Covalent modifications to these histones by specific enzymes modify their structures leading to differences in the chromatin conformation. Numerous histone modifications have been identified including acetylation, methylation, phosphorylation, and ubiquitination.4 The function of these modifications is to influence the physical accessibility of particular genomic regions to regulatory transcriptional proteins. In addition to DNA methylation and histone modification, there is evidence that higher-order 3D chromatin structures are subject to epigenetic regulation by accessory proteins via mechanisms which are the subject of significant interest but are yet to be fully understood.5 These layers of epigenetic regulation operate in concert, and there is considerable cross talk and cooperation between them.6 The major means by which epigenetic mechanisms exert their influence is via patterns of DNA methylation, covalent modification of histone proteins, and higher-order chromosomal structures. The essential effects of these mechanisms are to influence the way that transcriptional regulatory proteins interact with the controlling regions of their target genes. A key outcome of these processes is that they enable the coordinated regulation of multiple genes. Epigenetic mechanisms tend to enable transcription of genes which are

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required by a particular tissue, cell type, or developmental stage. Conversely, they tend to disable genes which are not required. This epigenetic programming contributes to the stability of gene expression patterns within cells and the maintenance of homeostasis; however, it is not fixed and there is plentiful evidence for dynamic changes in response to environmental stimuli. Epigenetics research is providing useful insights into biological and medical phenomenon with key insights into the pathogenesis of specific diseases. In this chapter, we discuss how epigenetic mechanisms may impact on oral health and disease based on evidence from studies to date and parallels with other fields. There is evidence for a contribution by epigenetic modifications to oral health from studies of craniofacial development, abnormal tooth development, dental caries, periodontal disease, salivary gland disease, oral mucosal lesions, orofacial pain (OFP), and most extensively malignancy. Furthermore, evidence from other fields demonstrates epigenetic programs are influenced by environmental agents including the effects of environmental toxins and the intestinal microbiome on the gut,7 and this indicates the oral epigenome may similarly be modulated.

DNA methylation Methylation of DNA generally involves transfer of a methyl group to the cytosine of CpG dinucleotide sequences in genomic DNA (Fig. 7.1). The CpG dinucleotides are not evenly distributed in the genome but tend to be concentrated in regions known as CpG “islands” which are often located in the promoter regions of genes.8 In somatic cells, most promoter regions of active genes are unmethylated, whereas inactive genes are hypermethylated. There are three mechanisms by which DNA methylation can influence transcription1: changing the binding affinity of promoter regions for transcription factors2; providing a target for methylation-specific binding proteins to regulatory sequences3; and altering the structure of chromatin because methylation tends to compact genomic DNA. DNA methylation patterns are established and maintained in meiosis/mitosis by a family of specific proteins called DNA methyltransferases (DNMTs) which knockout experiments have shown to be essential for normal embryonic development in mammals.9 There are also both passive and active demethylation mechanisms in mammalian cells. Inhibition of DNMT activity can lead passively to a gradual loss of methylation during cell division. In addition, there is an active pathway for demethylation which involves a number of enzymes and is thought to play an important role in germ cell and early embryonic development.10 It is well recognized that abnormal or dysregulated methylation patterns contribute to disease development. Most intensively studied in this regard is carcinogenesis. In cancer, DNA is broadly hypomethylated contributing to chromosome instability, whereas tumor suppressor gene expression is frequently downregulated by promoter hypermethylation.

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(A)

RNA polymerase

Transcription factor RNA polymerase

Transcription factor

(B)

RNA polymerase

Transcription factor

Methyl binding protein

RNA polymerase Transcription factor

(C)

Methylation Transcription factor RNA polymerase

Figure 7.1 Mechanisms by which DNA methylation can influence gene expression. (A) Transcription factors are unable to bind to methylated target sequences and cannot initiate transcription when methylated; (B) Methylation binding proteins can bind specifically to methylated DNA and block the binding of transcription factors; (C) DNA methylation can induce chromatin remodelling and restrict the access of transcription factors to their targets.

Histone modifications It is now well understood that modification of histones inhibits the secondary and tertiary structural folding of nucleosome arrays resulting in chromatin decondensation and allowing access to transcription factors and coactivators (Fig. 7.2). Large-scale analysis of posttranslational modification of histones have demonstrated that they can dictate active and passive states of chromatin, and that there are specific modifications associated with active gene transcription.11 It has also been found that developmentally critical genes exhibit both activating and repressive histone modifications simultaneously, a feature which may function to enable rapid switching between activation and repression during differentiation.12 Although the interplay between histone modifications and other regulatory mechanisms is

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ACTIVATOR

EPIGENETIC ACTIVATION

EPIGENETIC SILENCING

- histone acetylation - histone methylation

Figure 7.2 Epigenetic mechanisms of gene activation and silencing by histone modification. Post translational modification of histone proteins by acetylation can result in relaxation of chromatin structure allowing access of transcriptional activators. Conversely, histone methylation can condense chromatin structure restricting access.

highly complex, the fundamentals of the mechanisms are relatively simple. Numerous amino acid residues of histones are subject to modifications which are performed by specific proteins such as acetylases, methyltransferases, and kinases.4 These protein modifications are recognized by other classes of proteins which initiate or stabilize the formation of regulatory complexes to influence transcription, DNA processes (repair and replication), and chromatin conformation.13 Finally, there are a group of demodifying enzymes which can remove these modifications including histone deacetylases, demethylases, and phosphatases. The most intensively studied histone modifications for their biological consequences are methylation, acetylation, and phosphorylation. These modifications are typically performed by large multisubunit complexes.14 Histone posttranslational modifications have been reported at many amino acid residues, although the most common region is the N-terminal tail. Histone methylation has diverse effects on cellular function including both transcriptional repression and activation and DNA replication and repair.15 Histone acetylation, in contrast, is generally associated with transcriptional activation because acetylation of histones relaxes DNA binding to facilitate accessibility for transcriptional complexes.4 Phosphorylation of histones has been implicated as a regulatory mechanism in a broad range of biological

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processes. In particular, it plays a key role in the initiation and regulation of DNA repair mechanisms by acting as a recruitment signal for DNA damage response proteins.16

Epigenetic cross talk There is significant cross talk between different histone modifications with one modification influencing the execution or recognition of others. This enables the cell to integrate diverse cellular signals via chromatin organization.4 Similarly, while DNA methylation and covalent histone modification are distinct mechanisms with their own effectors, they are coordinately regulated in epigenetic processes. For example, modification of histones may trigger DNA methylation and conversely DNA methylation may also serve as a signal for histone modification by binding histone deacetylases.6

Craniofacial development Introduction The structures of the human head are highly complex, and craniofacial development requires the coordinated spacial and temporal activity of elaborate signaling networks. In normal embryogenesis, the first and second branchial arches give rise to craniofacial tissues and skeletal structures including the mouth.17 The neural crest is an important population of embryonic progenitor cells which significantly contributes to cranial structures including the mouth.17 Given the complexity of craniofacial structures and developmental programs, it is not surprising that abnormalities are among the most common congenital disorders with significant impact on affected individuals. Understanding the morphogenetic processes and molecular signaling mechanisms which determine craniofacial development is the key to establishing the etiological basis of dental developmental disorders and the potential for early diagnosis and intervention. Animal models including notably frog, zebrafish, and mouse have provided essential tools to investigate the genetic and epigenetic drivers of craniofacial development and the basis of abnormal phenotypes.18 Many of the mechanisms involved in these processes are evolutionarily conserved, and this has allowed the application of these developmental models to the study of human disorders.17,18 Technical advances in imaging technologies have enabled quantitative phenotyping of craniofacial and dental development, which when combined with advances in large nucleic acid sequencing have raised the prospect of genome-based prediction of disease phenotype. The faceBase consortium (www.facebase. org) is a National Institutes of Health initiative aimed at improved understanding of craniofacial development and abnormality which integrates data from animal models and human studies.19 The results of the studies which have

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arisen from this initiative have demonstrated the complexity of genotypee phenotype relationships and suggested that predictions based on genotype alone may not be feasible.20e22 For example, even in genetically identical mouse populations, there are subtle variations in craniofacial structures that indicate the influence of other mechanisms including environment and epigenetics.23 These findings when viewed in the light of data from human twin studies24e26 and mechanistic studies of epigenetic modifications in animal developmental models27 strongly support the likely impact of epigenetic mechanisms in dental abnormalities. Historically, there has been an intensive focus on the genetic control and developmental role of transcription factors and signaling mechanisms; however, there is an increasing focus on epigenetic mechanisms particularly with regard to the identification of epigenetics dysregulation in specific developmental defects.27

Orofacial clefts Orofacial clefts are among the most common birth defects, being present at an estimated 1 in 700 births.28 Orofacial clefts are typically subcategorized on the following basis: cleft palate only, cleft lip only, and both cleft lip and palate. About 30% of orofacial clefts are associated with abnormalities in other tissues/organs and are termed syndromic. For these syndromic forms, the genetic etiology is relatively well characterized.29 In contrast, the majority of cases are nonsyndromic, not associated with other defects, and the causal etiology is much less well understood being diverse and complex with both genetic and environmental components.29 Orofacial clefts have detrimental consequences for individuals in feeding, speech, dental development, hearing, and psychosocial problems. Although surgical repair is achievable, often multiple craniofacial and dental procedures may be required. As a result, there is a significant interest to address the deficiencies in understanding the underlying disease mechanisms because this may allow early detection and potentially therapy. Twin studies and genome-wide association studies in affected families have clearly demonstrated a genetic component to nonsyndromic forms of orofacial clefts; however, inheritance is nonmendelian and many cases are apparently sporadic.29,30 Thus far, polymorphisms in multiple genes have been implicated in the etiology of nonsyndromic orofacial clefts,31 many of which have been validated as having roles in orofacial development through functional screens in animal models. Even so, studies of monozygotic and dizygotic twins have shown that factors other than conventional genetics alone are involved in determining phenotypes.29 Until now, these nongenetic factors have been variously ascribed to environmental factors such as maternal smoking and alcohol consumption, antiepileptic drugs, and retinoic acid.30 However, we know that epigenetic regulation is critical to the development of the neural crest, a key tissue in craniofacial development.27 Furthermore, it is well recognized that environmental influences can manifest themselves through

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epigenetic mechanisms.32 Maternal smoking has frequently been associated with orofacial clefts30 and is also associated with changes in the fetal epigenome,33e35 although to date potential specific mechanisms have not been explored. Similarly, the antiseizure drug valproate has been associated with craniofacial abnormalities,36 and this drug has been subsequently shown to act as a histone deacetylase inhibitor and has been repurposed as an epigenome-modifying drug in cancer therapy.37 Despite the intensity of reported research activity examining genetic factors for orofacial clefts, the literature for epigenetic mechanisms is remarkably sparse. Given that epigenetic mechanisms may well explain the impact of environmental factors on genotypeephenotype relationships, this perplexing deficiency will undoubtedly change.32 In murine models of development, there is ample evidence that epigenetic mechanisms regulate the formation of orofacial structures and perturbation of these leads to abnormality.38e40 More recently, clinical studies have begun to provide evidence regarding the role of epigenetic mechanisms with several groups reporting genome-wide methylation studies in different cohorts. Alvizi et al.41 identified differential methylation at multiple genomic sites associated with cleft lip and palate including regulatory regions of genes with an established role in orofacial development. Furthermore, methylation analysis of familial cleft lip and palate with a previously demonstrated genetic component in CDH1 gene mutation showed that CDH1 gene methylation correlated with phenotype.41 Thus, the epigenetic mechanism modulated the phenotypic expression of the mutated gene in different individuals. In a distinct methylation profiling study, the epigenetics of orofacial cleft subtypes has been explored. It was demonstrated that there were distinct methylation profiles in specific genomic regions, including genes previously associated with orofacial clefts, between the three subtypes.42 Earlier genetic studies have identified histone deacetylase 4 (HDAC4) mutation or deficiency as associated with orofacial clefts43 and subsequent mechanistic studies in animal models have shown the significant role of HDAC4 in development of the palate.44 The human craniofacial development protein 1 (CFDP1) originally identified through animal models as linked to craniofacial development has recently been shown to function as a higher-order chromatin organizer in human cells.45 The way forward in the application of functional genomics to this important area is illustrated by the recent publication of a high-resolution map of early human embryonic craniofacial development including profiling of histone modifications.46 Such mapping will be a crucial resource for ongoing research into the etiology of orofacial clefts and improvements in their clinical management through testing and intervention.

Odontogenesisddental anomalies The morphological aspects of tooth development are well understood, and investigations of the molecular and mechanistic aspects of the process have

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been greatly facilitated by the fact that it is highly evolutionarily conserved. Odontogenesis is a tightly regulated process which proceeds through temporally controlled bidirectional signaling between the dental epithelium and underlying neural crest-derived mesenchymal tissue (recently reviewed in 47). Mutual signaling between cells derived from these two cell layers results in their differentiation into the specialized structures of the teeth. The availability of well-characterized animal models and more recently in vitro models of human and animal cells has benefited advancement of this field. The progress in this area has been driven in recent years by advances in stem cell technology and the prospect of clinical application to regenerative dentistry. Genetic studies using both experimentation and large-scale gene expression profiling have identified the key signaling pathways of BMP, FGF, SHH, and WNT molecules that coordinate tooth development.48 Research using conditional knockout transgenic mouse and correlations with mutations in humans associated with tooth agenesis or hypodontia has supported this evidence.48 The significance of the Wnt signaling pathway in particular has been demonstrated by the identification of WNT10A mutations in >50% of cases of nonsyndromic hypodontia.49 The function of these genes and pathways has been studied in many laboratories including their likely regulation through epigenetic mechanisms. The findings of studies of monozygotic twins show that there are differences in dental anomalies as well as tooth size, number, and morphology are evidence for the influence of environment and epigenetics on tooth development.26,50,51 Both DNA methylation and specific histone modification have been studied for their impact on odontogenesis. In a mouse model of tooth development, molar formation was associated with increased methylation of CpG islands in the insulin like growth factor - 2 (IGF2) and delta like non-canonical notch ligand 1 (DLK1) genes52 which resulted in downregulation of expression of these genes. Similarly, Yoshioka et al.53 demonstrated both spatial and temporal changes in the distribution of methylation and hydroxymethylation of DNA in a mouse amelogenesis model. Furthermore, they found an associated temporal decline in the expression of the DNA methyltransferase DNMT1 during development, suggesting that methylation is involved in the maintenance of undifferentiated cells in the dental epithelium. Other global analyses have supported the importance of DNA methylation in this regulation. In an in vitro model of the differentiation of human dental pulp cells, pharmacological inhibition of DNMT activity resulted in decreased proliferation and increased gene expression of markers of odontogenic differentiation as well as other phenotypic changes.54 Interestingly, a recent pilot study of patients with tooth agenesis using genome-wide methylation profiling showed significant differences in the methylation of certain gene promoters compared with unaffected individual.55 This study requires further investigation in a larger cohort but suggests that DNA methylation modulates the effects of gene mutations in human tooth developmental anomalies.

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Histone modification through both methylation and acetylation has been implicated in tooth development. Recent evidence has been reported for coordinated dynamic expression of a family of histone H3K9 methyltransferases during mouse tooth development.56 Bivalent temporal changes in other H3 histone methylation states have also been described in mouse models of tooth development with changes in H3K4 and H3K27 methylation states and expression of the relevant methyltransferases and demethylases.57 Histone H3K4 trimethylation and H3K27 trimethylation have been associated with transcriptional activation and repression, respectively, at developmentally active promoters.15 Subsequently, these authors were able to show in an in vitro model of human dental mesenchymal cell odontogenic differentiation that a key driver of tooth development WNT5A gene transcription was epigenetically regulated by the balance between H3K4 and H3K27 trimethylation.58 Mutation of the BCL-6 corepressor (BCOR) gene is responsible for oculo-facio-cardio-dental syndrome, and recent studies in both animal models and human cells have shown that the most likely mechanism involved was epigenetic. BCOR was temporally expressed in a mouse model and silencing resulted in aberrant tooth development,59 and in human MSC BCOR repressed transcription through histone H3K4 and H3K36 demethylation, although it did not possess intrinsic demethylating activity.60 Subsequently, evidence for a possible mechanism has been described whereby BCOR participates in an activating complex with the histone demethylase KDM2A to remove activating methylations and suppress target gene transcription in dental stem cells.61 The histone demethylase FBXL11 has also been implicated in tooth development using human mesenchymal stem cell (MSC) models in a study which found that FBXL11 repressed odontogenic differentiation and this effect was mediated through repression of expression of the epidermal growth factor, epiregulin62. Similarly, expression of another histone demethylase KDM6B has been shown to be a critical driver of differentiation of dental MSC and contributes to the transcriptional activation of bone morphogenic protein 2 (BMP2)63 and insulin-like growth factor binding protein 5 (IGFBP5),64 both regulated during odontogenesis. The histone acetyltransferase p300 has been shown in human dental pulp cells to regulate the expression of the key regulators of stem cell differentiation NANOG and SOX2 and modulate odontogenic differentiation as indicated by the expression of key marker genes.65 A number of studies have explored the prospect of using pharmacological histone deacetylase inhibitors (HDACi) to epigenetically regulate the differentiation of cultured dental pulp stem cells with a view to their application to regenerative dentistry.66e69 HDACi essentially replicate the effects of histone acetyl transferases by increasing histone acetylation, and these studies showed that HDACi treatment could induce stem cell differentiation and stimulate mineralization.66 To date, these studies have used broad-acting HDACi; however, in the future, application of inhibitors targeted to specific HDAC will allow finer dissection of

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the role of histone acetylation in dental development and provide better targeted drugs for pharmacological intervention.70 These studies have demonstrated that odontogenic differentiation can be manipulated in vitro through epigenetic mechanisms.

Dental caries Introduction Dental caries is a dynamic disease involving a multifactorial interaction between tooth structure, microbial biofilm, sugars, salivary composition, and genetics.71 Carious lesions result when net demineralization is achieved at a certain anatomical tooth site over rapidly alternating periods of tooth demineralization and remineralization.71 The mechanisms and pathophysiology underlying the development of dental caries are now increasingly well understood as the ecological plaque hypothesis.71 A fine balance between pathogenic factors such as cariogenic dental biofilm and frequent exposure to dietary carbohydrates and protective factors such as fluoride, normal salivary function, and good oral hygiene play a crucial role in the progression to demineralization with resultant caries or promoting remineralization and arresting the process.71 Appreciation of these principles have opened new avenues for caries prevention. However, despite the huge efforts in implementing dental caries prevention programs in many populations, a large part of the world’s population still suffers from this disease, retaining its title as the most common chronic childhood disease.72,73

Epigenetic risk factors for dental caries An individual’s susceptibility to oral disease is dependent on many factors: socioeconomic, environmental, political, availability of oral health care facilities and their utilization, and an individual’s oral health knowledge, biological factors, and genes.73 The importance of inherited factors in susceptibility to dental caries has been recognized for decades. Direct evidence is present for a genetic component in the etiopathogenesis of dental caries; however, the specific causal pathways remain unknown.73 The study of epigenetics with respect to dental caries initiation or progression may be the missing link to these unanswered questions. This area of research is relatively new in the dental field and is required to completely understand DNA-based factors in the development of oral diseases. An Australian group recently outlined novel protocols for assessing maternal, environmental, and epigenetic risk factors for dental caries in children.73 An oral health subset study of the main “Environments for Healthy Livingdthe Griffith Birth Cohort Study” began in 2012 recruiting a subset of 6- to 7-yearold children and their mothers in a prospective, multiyear longitudinal study.73 The mother and child are asked not to brush their teeth 2e3 h before

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examination, not to consume sweet food or beverages, smoke, or use a mouth wash as per the oral examination criteria.73 Each child’s overall caries experience is calculated, and mother and childerelated variables such as oral health knowledge and practices, anthropometric measurements, saliva characteristics, periodontal status, demographic, and environmental data are collected using questionnaires, clinical examinations, and laboratory tests.73 At present, a pilot study with 12 motherechild pairs have been assessed (6 with high caries experience and 6 with low caries experience) and DNA extraction, purification, and sequencing protocols have been established for identifying target genes for dental caries.73 This study may pioneer research into epigenetic variation for dental caries in children and potentially provide evidence on interrelationship of epigenetic variations with other social and environmental predictors for dental caries.

Tooth structure The four types of dental tissues, enamel, dentin, cementum, and dental pulp, are formed through specialized cellular and biochemical pathways that are controlled by genes and influenced by epigenetic and environmental factors.74 Abnormalities of developmental pathways can result in reduced quantity of tissue and/or poor quality of mineralization, predisposing the individual to dental caries.74 Many genes have been identified and implicated in these developmental defects.74 If the affected genes are expressed predominantly in dental tissues, such as the amelogenin, produced by AMELX in amelogenesis imperfecta or dentin sialophosphoprotein (DSPP) gene mutation in dentinogenesis imperfecta, the teeth are the main structures affected.74 Alternatively, some genes can also be involved in the formation of other tissues, such as laminin-332 and type XVII collagen.74 Mutations in these genes can have generalized effects involving other organs in addition to dental malformations.74 Numerous types of mutations have been noted in these genes; however, which gene is expressed while the other silenced, resulting in its clinical dental malformation, remains unknown.74 Epigenetic patterns may explain part of the solution to this puzzle; however, studies in this field are in their infancy and require deeper exploration to establish a relationship between epigenetic and genetic factors with respect to these developmental defects.75

Dental pulp inflammation Dental caries is one of the most commonly described etiological factors in dental pulp inflammation (pulpitis).76 It is characterized by local accumulation of inflammatory mediators, including cytokines and chemokines, which actively participate in destructive and reparative processes in the pulp, involving multiple signaling pathways.76 Epigenetics has been shown to play a key role in pulpitis. Several cells and inflammatory mediators have been

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involved in initial pulpal responses to caries, including IFN-g.76 Inflammation in human dental pulp has been shown to be characterized by a totally methylated to partially methylated status of IFN-g.77 Hypomethylation of the promoter region on TLR2 gene has also been associated with increased proinflammatory response and has been noted in TLRs in inflamed pulp tissues, potentially influencing dental pulp repair.78 However, no significant difference of TLR2 hypomethylation in inflamed pulp compared with healthy pulp has been noted, suggesting that the unmethylated profile in TLR2 gene is a usual feature in human dental pulp.78 Furthermore, histone methylation might also be involved in dental pulp inflammation and reparative processes. Hui and colleagues demonstrated that H3K27me3 (a histone responsible for inactivating transcription) was decreased in inflamed pulp tissue and pulp cells and is thought to potentially induce the regenerating processes of dental pulp.79 Other histones such as EZH2 have also been investigated, with EZH2 noted in inhibition of human dental pulp cell osteogenic differentiation and proliferation of inflammatory response.79 Histone acetylation mechanisms have also been highlighted to play important roles in pulp inflammatory processes with histone deacetylase inhibitors (HDACi) potentially promoting reparative events in dental pulp cells by reducing proliferation and increasing mineralization, opening an avenue for applying low concentrations of HDACi in vital pulp treatment.66

Periapical lesions As dental caries progresses, bacterial penetration of the tooth structure results in pulpal inflammation/necrosis/infection and, if left untreated, results in the formation of apical periodontitis. Apical periodontitis represents a local immune response to the progression of microorganisms from an infected root canal space to the periapical area resulting in bone resorption.80 This complex cascade of events is mediated by innate and adaptive immune response mechanisms, and epigenetic mechanisms have shown a role in programming the expression and responsiveness of several inflammatory mediators.80 The forkhead box P3 (FOXP3) gene (involved in decreasing T-cell proliferation and cytokine production) has been identified to have the highest DNA methylation levels in apical periodontitis samples.80 A variation in methylation profiles of FOXP3 in active and latent periapical lesions was also observed, suggesting it to be key in determining the balance between lesion progression and/or latency status.80 Similarly, matrix metalloproteinases (MMPs) were analyzed for epigenetic modifications as they have been associated with the pathogenesis of periapical inflammatory lesions via degradation of extracellular matrix components.81 Some studies have observed unmethylated profiles of MMP9 in periapical lesions suggestive of their contribution to an individual’s predisposition to periapical disease or treatment response; however, methylation profiles of MMP2 have

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been difficult to assess.81 Unfortunately, research in this area is still relatively new, and additional functional studies are required to enhance the relevance of the epigenetic profiles in periapical conditions and aid in the potential development of effective therapies.

Periodontal disease Introduction “Periodontal diseases” encompass a wide variety of chronic inflammatory conditions of the periodontal tissues (gingiva, bone, and periodontal ligament (PDL)) resulting in attachment loss and destruction of alveolar bone.82,83 The natural progression and consequences of periodontal disease manifests in the affected dentition commonly resulting in tooth loss, but additional consequences including OFP, impaired function, tooth mobility, and esthetics can also impact an individual’s physical and psychosocial functions.83,84 In 2010, the estimated global prevalence of severe periodontitis amounted to 10.8%, affecting 743 million people worldwide, representing the sixth most prevalent condition and creating a significant public health concern.85 Clinical manifestations of periodontal diseases are heterogeneous in nature contributing to difficulty in correct diagnoses.83 Studies suggest the clinical outcome of the disease is influenced by genetics and epigenetics as many patients with the same clinical symptoms respond differently to the same therapy.86 Currently, the diagnosis of periodontitis relies on clinical inspection of the oral cavity for the disease status via probing depths, attachment levels, bleeding, plaque index, and the use of radiographs. The currently accepted classification for periodontal diseases and conditions divides these into gingival conditions and periodontal conditions, and further into plaque related and nonplaque related.87 Conditions can be localized or generalized and may be related to systemic or genetic conditions. Studying epigenetic changes and their relationship with inflammation may provide not only new diagnostic methods but could also be useful in developing personalized treatments.

The role of epigenetics in the pathogenesis of periodontal disease Periodontal diseases are currently considered to share similar etiopathogenesis in the form of bacterial interactions with host cells (via innate immune activation) initiating chronic inflammation and cytokine networks to modulate the connective tissue response and periodontal destruction.82,88e90 It is characterized by intermittent periods of activity and inactivity.90 Recent research demonstrates that the immune system and inflammatory responses are highly dependent on epigenetic mechanisms to function, providing deeper insights into the development of periodontal conditions.86 Understanding the

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epigenetic modifications on periodontal tissues from the initial periodontopathogenic infections at the biofilmegingiva interface, and related inflammation, may eventually allow for the development of a biomarker panel aiding the prevention and treatment of periodontal disease.

Bacteria The conversion of junctional epithelium to pocket epithelium is regarded as a hallmark in the development of periodontitis88 and is initiated by the presence of gram-negative bacteria accumulating in the gingival crevicular region, in particular Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia.90,91 The presence of other bacteria including Aggregatibacter actinomycetemcomitans and Fusobacterium nucleatum has also been linked to synergistically infect gingival tissues contributing to the pathogenesis of periodontitis.91 The exposure to these oral bacteria has been shown to result in epigenetic modifications in gingival epithelial cells, in turn differentially expressing downstream innate immune markers and contributing to periodontal pathogenesis.89,92 Gingival epithelial cells play a vital role in protection against bacterial infection and have been shown to widely express antimicrobial peptides such as human b-defensins (hBDs).92,93 Human beta-defensin 1 (hBD1) is distributed extensively in epithelial cells, whereas Human beta-defensin 2 (hBD-2) and Human beta-defensin 3 (hBD-3) are expressed in response to bacterial stimuli or inflammation.94 P. gingivalis and F. nucleatum have both been shown to induce hBD-2 by different epigenetic pathways in induction.92,94 F. nucleatum induces hBD-2 via both demethylation and acetylation mechanisms, whereas P. gingivalis only utilizes histone acetylation.92 Lipopolysaccharide (LPS) derived from P. gingivalis has also been observed to cause epigenetic changes via DNA hypermethylation of extracellular matrixrelated genes, inducing a significantly downregulated expression of their mRNA and has also been noted to influence interleukin-10 (IL-10) gene expression via histone modifications of IL-10 genotypes.95,96 Chemokine ligand-2 (CCL-20), another epithelial innate immune marker, demonstrated similar results when exposed to these microbes.92,93 Epigenetic mechanisms have also been considered responsible for the persistent activation of matrix metalloproteinase-2 (MMP-2) in PDL cells via upregulation of MMP-2-related genes when exposed to T. denticola, supporting the theory that epithelial innate immune responses are regulated by epigenetic modifications, but each response is specific to individual bacterial species.97 Tumor necrosis factor (TNF-a), interleukins (IL-1a, IL-6, and IL-12), and chemokines (CXCL-1 and CCL-33) are key cytokines implicated in the pathogenesis of periodontal disease and determine what response is taken by the immune system to particular environmental stimuli.86 Zhang et al. demonstrated that altering DNA methylation of THP-1 monocytes led to an increase

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in transcription of TNF-a following bacterial exposure.98 DNA methylation has also been shown to differentially affect cytokine secretion from epithelial cells in response to P. gingivalis and F. nucleatum93 suggesting this to be an important regulatory mechanism in controlling further cytokine transcriptional expression in pro- or antiinflammatory responses in periodontal disease.86,98 Furthermore, the overgrowth of periodontal pathogens drives activation of specific pathogen recognition receptors including nucleotide-binding oligomerization domain proteins (NOD1) and Toll-like receptors.89,99 Evidence suggests bacterial commensals prime NF-kB activity through histone modifications activated by NOD1 and Toll-like receptors (TLR1/2 and TLR4).99 Chronic activation of NF-kB signaling has been shown to cause osteoclast differentiation and bone resorption promoting periodontal disease progression.99 A hypermethylated TLR2 profile has also been positively correlated with increased probing depths, further supporting the possible participation of DNA methylation in the development of periodontitis.100 Recent evidence also points to another interesting aspect of bacteriainduced epigenetic alterations. Specifically, Imai et al. demonstrated that periodontitis can reactivate HIV-1 expression by periodontopathogenic bacteria through an epigenetic mediator.101 Butyric acid and TNF-a produced by P. gingivalis has been shown to promote gene expression of latent HIV-1 virus via histone acetylation of HIV-1 LTR promoter, making P. gingivalis a potential risk factor for AIDS progression due to its ability to upregulate HIV-1 replication either systemically or locally.101

Gingivitis Mechanisms or factors causing gingivitis to develop into a tissue-destructive periodontitis are not completely understood.102 It has been proposed that an individual’s genotype and its immunological defense against bacteria determine susceptibility for progression from gingivitis to chronic periodontitis.102 Studies investigating epigenetics in other inflammatory conditions such as systemic lupus erythematosus and rheumatoid arthritis (RA) indicate that chronic inflammatory processes may be a result of a decrease in histone deacetylase activity and changes in methylation, subsequently leading to an increase in gene expression of inflammatory factors.102 Zhang and colleagues consequently investigated methylation profiles of interferon-gamma (IFNgda cytokine molecule elevated in inflamed gingival tissues and responsible for periodontal disease progression) in healthy tissues, gingivitis, and chronic periodontitis.103 Surprisingly, no significant methylation difference was noted between gingivitis and healthy controls specific to IFN-g-promoter, although significantly higher IFN-g transcription levels (1.96-fold) were noted in tissues with periodontitis.103 While the findings from this study suggest that progression from gingivitis to periodontitis is unlikely related to IFN-g

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polymorphisms specifically, epigenetic influences through prolonged local environmental exposures cannot be overlooked.103 This field of research is still in its prematurity and further research is required to explore this area.

Chronic periodontitis Chronic persistence of biofilm and inflammation is the principal component in the pathogenesis of periodontitis and has been associated with epigenetic changes in local tissues at the biofilm-gingival interface.104,105 Although the presence of bacteria is essential for initiating and perpetuating periodontal disease, changes to DNA methylation patterns and cytokine gene expression as a host response contribute to the variation in the etiology and course of the disease and will be discussed in the context of epigenetic modifications below.86,106 The CpG hypermethylation of E-cadherin and prostaglandin-endoperoxide synthase 2 (PTGS-2 or COX-2 gene promoter) results in silencing of these genes in individuals with chronic periodontitis.105e107 E-cadherin is a transmembrane glycoprotein responsible for epithelial intercellular adhesion. The decreased expression of E-cadherin protein in periodontally diseased oral gingival epithelium supports its role in disease progression and periodontal destruction.107 Transcription of PTGS-2 mRNA directly results in the synthesis of inducible COX-2, a gene responsible for controlling the production of prostaglandins that promote inflammation.107 Interestingly, lower levels of prostaglandin E2 (PGE2) have been noted in deeper pockets than that seen in noninflamed individuals with shallow sites, suggesting that the chronic state of deep pockets may reflect a historical episode of disease activity with a reinstatement of a new steady-state equilibrium of COX-2 expression.105 These findings may suggest a future role for E-cadherin and PTGS-2 epigenetic changes as diagnostic markers for periodontitis; however, larger studies are required to validate these findings. Although previously investigated in gingivitis, IFN-g has been shown to enhance secretion of proinflammatory molecules such as PGE2, IL-1b, and TNF-a, playing a key role in bone loss and disintegration of soft tissue in the periodontium.103 Previous molecular studies have determined high levels of IFN-g in association with deep periodontal pockets and severe gingival bleeding.108 Epigenetic studies have identified a hypomethylated state of the IFN-g promoter region in a chronic inflammatory periodontal disease, supporting its crucial role in the progression of the disease.103 Among several kinds of cytokines involved in periodontal disease, interleukin6 (IL-6) appears increasingly in the gingival crevicular fluids and in gingival tissues of individuals with periodontitis.109 IL-6 is an inhibitor of bone formation and is thus regarded to play an important role in the development of the disease.109 Although epigenetic studies confirm the high expression of

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IL-6 in individuals with periodontitis, no association has been noted with DNA methylation status, suggesting the role of other mechanisms involved in this gene transcription regulation.109 Interleukin-8 (IL-8) is produced and secreted by a wide range of cells including epithelial cells. It can be induced by varying stimuli such as LPS, bacterial species, and early proinflammatory cytokines and has been observed to play a significant role in tissue destruction in periodontitis.110 Oliviera and colleagues noted statistically significant levels of hypomethylation of the IL-8 gene in oral epithelial cells of individuals with chronic periodontitis irrespective of their smoking status, proposing that inflammation in the oral mucosa may be responsible for the epigenetic changes of the IL-8 gene.110 Interleukin-1 (IL-1) plays a key role in the pathogenesis of periodontitis, through its involvement in the regulation of the host’s inflammatory response and bone resorption. Genes that encode for IL-1 products have received significant attention as potential genetic markers of periodontal disease progression.111 Specifically, gene polymorphisms IL-1a and IL-1b have been identified as significant contributors to chronic periodontitis in Caucasian adults, whereby individuals carrying these gene variants demonstrated an odds ratio of 1.48 and 1.54 odds, respectively, in developing chronic periodontitis.112 Surprisingly, there is limited research investigating epigenetic influences on IL-1 gene and its association with periodontal disease. However, studies investigating IL-1 and epigenetic influences with chronic inflammation have demonstrated exaggerated proinflammatory cytokine gene expression with DNA hypomethylation and histone acetylation of CBP/p300 on IL-1b promoter regions.113 It can be speculated that similar outcomes would be observed in chronic periodontitis. Given the already established significant association between IL-1 and chronic periodontitis, investigating epigenetic modifications with this gene family may provide further insight into the development of chronic periodontitis and presentation of varying clinical phenotypes.

Aggressive periodontitis Aggressive periodontitis (AgP) typically affects healthy juveniles and young adults.114,115 It is characterized by rapid attachment loss and bone destruction and has a strong familial association.114,115 The inflammatory response in this condition is complex, requiring signal-specific and gene-specific responses. One of the most important intracellular signaling pathways triggered by cytokines is the Janus-kinase-signal transducer and activator of transcription (JAK-STAT) pathway.115 This pathway is tightly regulated by a family of proteins known as the suppressors of cytokine signaling (SOCS), acting as modulators of signal transduction in health and disease.115 DNA hypomethylation of SOCS-1 and Long interspersed element-1 (LINE-1) genes allowing gene

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transcription is observed at significantly higher levels in healthy patients than individuals with AgP, suggesting that this epigenetic mechanism may participate in the complex inflammatory mechanisms that contribute to periodontitis.115 Although these observations are noted in oral epithelial cells rather than cells specifically retrieved from the gingival tissues, they can provide an overview on the epigenetic changes occurring in the oral cavity in the presence of inflammation. Further research is necessary in this area to accurately understand the epigenetic modifications in relation to AgP. A pilot genome-wide study performed by Schulz et al. aimed to detect differentially methylated genes involved in the immune response between patients with aggressive periodontitis and controls.116 A selected panel of inflammatory genes consisting of chemokines (CCL25, CXCL14, CXCL3, CXCL5, CXCL6), cytokines (IL12A, IL12B, IL17C, INHA), cytokine receptors and associated proteins (IL10RA, IL12B, IL13RA1, IL15, IL17RA, IL4R, IL6R, IL6ST), and other inflammatory genes (iron-regulated transcriptional activator (AFT2), Fas-associated protein with death domain (FADD), GATA binding protein 3 (GATA3), interleukin 13 (IL13), interleukin 7 (IL7), and tyrosine kinase 2 (TYK2)) associated with periodontal diagnosis were investigated.116 Out of this panel, cytokines CCL25 and IL17C exhibited a significant decrease in promoter methylation in gingival tissues of AgP patients.116 CCL25 and IL17C have been associated with inflammatory host reactions to bacterial infections, playing a vital role in Th17 cell-mediated immune response.116 Induction of chemokine ligand
25 (CCL25) gene expression due to P. gingivalis infection and costimulation with its LPS has also been shown.116 Further downstream stimulation of inflammatory cytokines in gingival tissues is suggestive of epigenetic modifications in CCL25 gene, being associated with gingival inflammation in individuals with aggressive periodontitis.116 Similarly, interleukin-17C (IL17C) gene upregulation has been associated with periodontitis-associated bone resorption.116 Epigenetic changes to this gene may explain part of the rapid destruction seen in individuals with aggressive periodontitis. Comprehensive large-scale studies are still required to analyze the functional importance of these preliminary findings. Less frequent than chronic forms of the disease, localized aggressive periodontitis (LAP) has been noted to exhibit an elevated immune response when compared with healthy individuals.117 The strong familial aggregation further suggests that genetics may play a role in the so-called hyper responsiveness.117 The exact cause for the exaggerated inflammatory response seen in LAP patients is unknown; however, some evidence suggests that methylation status of specific genes plays a role in this disease via TLR-mediated signaling.118 Hypomethylation of upregulating genes of TLR-mediated inflammation was noted in severe LAP patients when compared with healthy controls and subjects with moderate disease.118 Subjects with moderate levels of LAP showed the most hypermethylation of inflammatory

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genes, in some instances even higher than healthy controls.118 One may speculate that there may be some attempts at controlling inflammation by epigenetically downregulating expression of proinflammatory genes on the initial onset of disease.118 This may explain why some individuals with mild and moderate levels of disease do not progress. Interestingly, genes associated with downregulation of inflammation were also significantly hypomethylated in individuals with severe LAP, further suggesting that overexpression of downregulatory molecules are required to suppress the proinflammatory cascade.118 Furthering such findings by correlating these events at varying disease stages with actual gene expression and resulting cytokine profile will provide deeper insight into these theories.

Clinical application of epigenetics within the field of periodontics Knowledge of epigenetics contributes to a better understanding of the interactions between genes and the environment, potentially providing insight into why patients with the same clinical phenotype respond differently to similar treatments. This also provides the potential for translating findings into a personalized medicine approach as opposed to considering only bleeding on probing and pocket depths.119 The ability to correlate an epigenetic pattern or marker with a clinical phenotype of periodontitis may eventually be used as a clinical tool for diagnosis or identification of patients at risk of developing the disease.119 At present, very few studies combine epigenetic findings with clinical measurements for periodontal disease and this provides an avenue for future-focused research. Another major clinical challenge is that traditional treatments such as scaling, root planing and bone grafting do not efficiently restore periodontal tissues. Because of the reversible nature of epigenetic mechanisms, the opportunity to develop epigenetic targeted treatment models for periodontitis has now become available. Drugs that interfere with the epigenetic control of gene expression known as “epidrugs” are being developed and have shown significant promise as treatment for cancer, neurodegenerative diseases, RA, and recently also periodontitis.120,121 A novel histone deacetylase inhibitor (HDACi) has recently been shown to suppress alveolar bone loss in periodontitis.121 Using a mouse model of P. gingivalis induced periodontitis, oral administration of this broad-acting HDACi 1179.4b suppressed alveolar bone loss with an associated reduction in the number of tartrate resistant acid phosphatase positive cells in the gingiva and alveolar bone.121 Interestingly, while inhibiting bone resorption, HDACi 1179.4b did not suppress gingival inflammation.121 In the same animal model, HDAC-1 selective inhibitor MS-275 administered orally reduced levels of inflammation but had no effect on bone loss.121 Furthermore, the study of epigenetics may provide a potential tool for improving wound healing and tissue regeneration in periodontal disease.119

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The treatment of human PDL cells with HDACi trichostatin A demonstrated a decrease in HDAC3, inducing osteogenic differentiation. Increased responsiveness of gingival fibroblasts was also noted, suggesting a good cell source for periodontal tissue regeneration.122 The differentiation of human gingival fibroblasts into osteoblasts by inducing demethylation of osteogenic factors Runt related transcription factor-2 (RUNX2) and alkaline phosphatase (ALP) was further demonstrated by Cho and colleagues. Subsequent treatment with bone morphogenetic protein-2 (BMP2) resulted in fibroblast differentiation toward the osteoblast lineage and bone formation.123 Further studies are needed to determine the best combination of inhibitors to use and whether drug delivery directly to the gingiva is possible.

Salivary gland disease ¨ gren’s syndrome Sjo Sjögren’s syndrome (SS) is a systemic chronic autoimmune disease in which the principle organs affected are the exocrine glands with symptoms seen mainly in the salivary and lacrimal glands. Thus, the major oral manifestations of this disease are salivary gland hypofunction leading to xerostomia although patients also exhibit increased incidence of caries and oral candidosis.124 As with many systemic rheumatic diseases, the pathogenesis of SS is unclear and its etiology is believed to be multifactorial through interaction of genetic predisposition, epigenetic mechanisms, hormonal influences, and environmental factors. The role of epigenetic mechanisms in autoimmune disease is the subject of increasing interest and their contributions to SS have been recently reviewed demonstrating a significant role for DNA methylation.125,126 The implication of DNA hypomethylation in SS is longstanding and arose based on evidence that demethylating agents can induce SS-like symptoms in humans and animal models.125 Subsequently, studies have investigated DNA methylation in peripheral CD4þ T cells, B cells, and salivary glands using targeted candidate gene methods and more recently epigenome-wide profiling.126 A common finding across the global DNA methylation analysis of peripheral B and T cells which have been reported is the hypomethylation of interferon (IFN)-induced genes.127e129 These studies also demonstrated differential methylation more broadly across the genome compared with healthy controls which is consistent with an association with aberrant demethylation.126 In peripheral B cells in particular, promoter hypomethylation has been associated with genetic risk loci128 and differential gene expression.129 Genome-wide methylation profiling has also been performed on salivary gland tissue from SS as well as isolated salivary gland epithelial cells.129e131 The interpretation of such analysis of salivary biopsies is complicated because they are cellularly complex samples and will include lymphocytic infiltrates and other cells. With the proviso of considering this

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cellular heterogeneity, these studies found the most commonly hypomethylated genes in SS salivary glands were consistent with those reported in peripheral lymphocytes.126 To address these issues of cellular complexity, Charras et al. performed a similar global methylation analysis using isolated cultures of salivary gland epithelial cells derived from SS patients and healthy controls.131 Importantly, this study of isolated salivary gland cells showed significant overlap with the salivary biopsy data in hypomethylation of IFN-induced genes giving credibility to those results.129,130 Overall, these findings are consistent with a common feature in SS of hypomethylation of IFN-induced signaling pathways in both infiltrating T and B cells as well as salivary gland epithelial cells, although the exact methylation sites may vary between cell types.126 Two studies have investigated overall global methylation levels in salivary glands and isolated salivary gland cells.132,133 Interestingly, these studies suggest that infiltrating B cells contribute to the demethylation seen in salivary gland epithelial cells because hypomethylation was more pronounced in cells from patients with more pronounced B-lymphocytic infiltrates.132,133 Furthermore, this global demethylation was associated with downregulation of the demethylating enzyme DNMT1133 and correlated with pathogenic features.132 Although a more comprehensive inventory of epigenome variation in SS and precise delineation of their functional effects at the molecular and clinical level is required, these studies suggest a prospect in SS for therapeutic manipulation of DNA methylation. While not currently feasible, if the demethylation of IFN-responsive genes reported in a number of studies proves to be mechanistically important, then this may be a suitable target for intervention.126

Oral mucosal lesions Introduction Investigation of the genomics of oral mucosal disorders has been driven mainly because they are potential precursors to oral cancer. Understanding of the molecular basis of oral cancer has progressed greatly in recent years, advanced by developments in sequencing technology and large-scale cancer genome mapping initiatives. Oral cancer frequently manifests itself through the appearance of precancerous lesions which may progress to cancer and this has prompted significant effort with the goal of predicting progression of these lesions. Cancer is believed to arise through stepwise accumulation of genetic and epigenetic change which leads to the dysregulation of signaling pathways and networks resulting in the phenotypes associated cancer: the hallmarks of cancer.134 Although genetic studies of mutations and gene expression in cancer have led to the development of the hallmarks of cancer model, the investigation of the temporal order of these events is only in its infancy. Evidence to date is based largely on the analysis of the relative frequency of gene

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mutations in multiple tumors and from multiple sampling of single tumors. This has shown that while the temporal order of pathway dysregulation varies between different cancers, if they are considered with reference to the hallmarks of cancer among the first to appear are resistance to cell death, insensitivity to antigrowth signals, and sustained proliferation.135 Environmental factors are well established as etiological agents in the initiation of oral cancers136,137 and in particular smoking and alcohol; however, the precise molecular mechanisms by which these factors contribute to oral cancer development are not well defined. One proposal for which there is significant evidence is that the environmental factors exert their carcinogenic effects through the interface of epigenetic mechanisms.138 We know from numerous studies that epigenetic mechanisms contribute to every stage of tumor initiation and progression139,140 and that both genetic and epigenetic changes are able to induce oncogene expression or inactivate tumor suppressor genes leading to neoplasia.141 The development of understanding of the cancer epigenome has lagged behind the corresponding genomic and transcriptomic characterization; however, what is clear is that epigenetic change contributes to tumor development and aggressiveness.142 Indeed, the significance of the role of epigenetic change is illustrated by analyses of pediatric tumors where the intratumoral mutational burden is remarkably low and tumors harbor few somatic mutations indicating a prominent role for epigenetics in driving tumorigenesis.143 For example, in retinoblastoma, the genome is relatively stable apart from RB1 inactivation and there are few mutational events targeting known oncogenic drivers or suppressors.144 Indeed, epigenetic reprogramming through changes in DNA methylation, histone modification, and chromatin reorganization modulate the expression of oncogenes and are essential to tumorigenesis.144 Supportive evidence can also be found in studies of pediatric medulloblastoma which displays a relatively low mutation rate and where the identification of key genetic drivers has proven problematic.145 In contrast, genome-wide methylation analysis demonstrated specific patterns of methylation that were present at high frequency and correlated with changes in the transcriptome.145 The extent to which epigenetic mechanisms contribute to the pathology of oral mucosal disorders and their malignant progression is unknown. However, there is evidence from experimental models and human studies in other cancers that epigenetic mechanisms can have a direct causative role in carcinogenesis.139,146 A key question in the context of oral mucosal lesions is the extent to which epigenetic change induced by environmental stimuli precede or coincide with genetic change in known drivers of head and neck squamous cell carcinoma (HNSCC).

Key genetic drivers of oral carcinogenesis The molecular analysis of HNSCC has greatly advanced in recent years following the publication by TCGA of a comprehensive molecular analysis

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including the mutational landscape of head and neck cancer.147 This work and subsequent analyses have provided key insight into the disease, provided evidence for molecular subgroupings with clinical relevance, and shown that these tumors harbor frequent somatic mutations. The cellular mechanisms most influenced by these mutations were cell cycle regulation, cell growth mechanisms, survival signaling, WNT signaling, and epigenetic modifiers.147 While numerous genes were identified as mutated across the 279 tumors investigated, a number were mutated with high frequency and identified as drivers of oncogenesis in HNSCC. Tumor suppressor genes TP53 and CDKN2A were prominent among those frequently inactivated or mutated genes in oral cancer supporting earlier studies.148 Interestingly, CDKN2A has also been identified as a significant target of promoter methylation in OSCC149,150 indicating targeting of this gene by both genetic and epigenetic mechanisms. Wnt signaling is a key pathway in the determination of tissue morphogenesis and stem cell homeostasis, and in this pathway the FAT1 gene is a frequent target of somatic mutation in HNSCC.147 FAT1 is believed to function to inhibit Wnt signaling in an analogous way to its molecular homologue, E-cadherin, by binding to and sequestering the key molecule b-catenin, thereby reducing the free cytosolic pool.148 Interestingly, the E-cadherin gene is a frequent target of promoter methylation and transcriptional repression in oral cancer which may also contribute to this mechanism.151,152 Genes involved in epigenetic programming are also frequently mutated in HNSCC,148 a feature which is common to many malignancies.143 The histone methyltransferases KMT2D and NSD1 were frequently mutated147 in addition to the previously reported PRDM9 and EZH2,153 although the specific downstream effects of these mutations have yet to be explored.

Epigenetic mechanisms as transducers of environmental carcinogens Feinberg and coauthors143 recently described a framework for the classification of the principal actors in genes and molecules which effect remodeling of the epigenome during oncogenesis. This framework distinguishes between “modifiers” which effect changes in methylation or chromatin structure and the “modulators” which regulate them as well as the downstream “mediators” which transform the cellular phenotype toward a stemlike state. Both modifiers and modulators have been frequently described as mutated in cancers such as the modifying histone methyltransferases described above in HNSCC. However, these same authors have previously proposed that environmental stimuli can directly influence epigenetic modifiers promoting the appearance of altered oncogenic signaling and a phenotypically plastic cell population before the advent of genomic mutations.139 Because tobacco smoking and alcohol consumption are among the most prominent risk factors identified for oral cancer,

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epigenetic mechanisms may provide an additional means beyond mutagenesis by which these exposures can contribute to oral carcinogenesis. It is well recognized that tobacco use can lead to aberrant methylation of genes involved in neoplastic processes which have been reported in the upper aerodigestive tract and lung epithelium of smokers including genes implicated in oral carcinogenesis CDK2NA, MGMT, and DAPK.154,155 In the oral mucosa, similar aberrant methylation events have been reported in longterm smokers indicating that epigenetic mechanisms are likely to contribute to tobacco-induced carcinogenesis.156e158 For alcohol, the picture with regard to the oral mucosa is less clear, although the significance of the epigenetic effects of alcohol exposure in other organs and the developing fetus is unambiguous.159 In a mouse model of oral carcinogenesis, a combination of ethanol and carcinogen 4-Nitroquinoline 1-oxide induced global changes in histone acetylation and methylation including some which correlated with changes in expression of genes involved in alcohol metabolism and oxidative stress.160 Inflammation is another mechanism which is recognized to contribute to carcinogenic mechanisms via the epigenome in other cancers143 and may also do so in the oral mucosa. There is ample evidence that activation of signaling pathways in chronic inflammation can lead to reprogramming of the epigenome supporting a role in cancer initiation.161e163 Numerous animal models and human studies have demonstrated that diet can influence the epigenome. In particular, the effect of a methyl-deficient diet through low dietary intake of folate leads to aberrant patterns of promoter methylation which can contribute to cancer risk and that this is exacerbated by chronic alcohol consumption.164 Evidence for a direct effect in oral mucosa is limited; however, in one human study, diet was found to influence the expression of the antioxidant superoxide dismutase enzyme in the buccal mucosa via promoter methylation.165 If the oncogenic driver mutations in oral carcinogenesis induced by environmental exposure are preceded by epigenetic reprogramming events, then characterization and analysis of these early events in oral mucosa is immensely important because it presents an opportunity not only for early detection but also preventative intervention as therapeutic manipulation of epigenetic change is feasible.

Epigenetic mechanisms in oral premalignant disorders Investigation of the role of epigenetics in oral cancer is ongoing but for practical reasons many studies to date have focused on DNA methylation and tended to target a small number of specific genes. More recently, global profiling has begun to be reported; however, the overall picture remains incomplete and fragmented with particular deficiency in epigenomic

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profiling of histone modifications and chromatin states. Recent advances in technology will undoubtedly make global epigenome profiling more feasible and greatly advance this field.166 Generally, the great majority of reports to date have investigated DNA methylation in oral cancers, and oral mucosal lesions have received much less attention probably in part due to limited availability of samples. While some inferences can be made regarding likely early epigenetic change in oral premalignant conditions based on profiling data from cancer samples using the same reasoning applied to the identification of driver mutations in genomic data,147 it should be considered that epigenetic change can occur throughout tumorigenesis138 and such inferences need to be experimentally confirmed. Most DNA methylation studies of oral premalignant lesions have examined promoter methylation of known oral cancereassociated tumor suppressor genes in case-control studies of oral lesions exhibiting dysplasia.149 As reviewed by Shridhar et al.150 the most commonly reported hypermethylated promoters among cross-sectional studies (n ¼ 9) were CDK2NA (p16(INK4a)), CDK2NA (p14(ARF)), MGMT, and DAPK. The product of these genes are tumor suppressors involved in cell cycle regulation (CDK2NA), DNA repair (MGMT), and apoptotic signaling (DAPK). In a more comprehensive study, Towle et al.167 performed epigenome-wide methylation analysis of dysplastic oral lesions, adjacent normal tissue, and OSCC, identifying 605 hypermethylated and 90 hypomethylated genes between dysplastic lesions and controls.167 The results of this study were consistent with earlier studies demonstrating in dysplasia the differential methylation of genes previously associated with high frequency of methylation in OSCC including CDK2NA, MGMT, and DAPK, as well as identifying a number of candidates for further investigation.167,168 Interestingly, this study also suggests that methylation profiles appeared to cluster according to dysplastic grade and used network analysis to identify the WNT and MAPK signaling pathways as targets of aberrant methylation.167 Several studies have attempted to establish predictive methylation markers by conducting longitudinal analysis of promoter methylation in oral carcinogenesis based on retrospective analysis of mucosal lesions. Two candidate gene studies have reported differential CDK2NA promoter hypermethylation in oral dysplastic lesions which progressed to cancer in comparison with those which did not.169,170 These studies are supportive of the concept that epigenetic inactivation of CDK2NA may be an early event in oral carcinogenesis which may precede inactivating mutations. Whether such epigenetic change precedes mutational events in premalignant lesions will require parallel global analysis of both methylation profiles and somatic mutations as has been recently reported for HNSCC.171 The search for predictive methylation markers of oral mucosal transformation has been addressed more recently using DNA methylome retrospective profiling of oral premalignant lesions to investigate differential methylation between lesions which progressed to

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OSCC relative to those which had not.172 A panel of 86 genes were identified which were differentially methylated between the groups with further investigation of candidate genes suggesting that differential methylation of the promoters of the AGTR1, FOX12, PENK, and LINE1 genes may be associated with increased risk of OSCC development from preexisting lesions.172 The functional relevance of these methylation profiles and their potential for clinical risk assessment remains to be determined. In addition to reported investigations of the epigenome in oral mucosal transformation, several studies have investigated epigenetic mechanisms in the context of inflammatory signaling and therapeutic response in oral lichen planus (OLP), which is considered by many to be a potentially malignant disorder.173 In a pilot investigation of a panel of immune response genes, the STAT5A gene promoter was hypomethylated in OLP relative to normal mucosa, although whether this was reflected by changes in gene expression was not reported.174 Histone modification has been linked to therapeutic outcome in OLP where it was found that patients with high levels of histone H3K9 acetylation experienced a poor response to therapy.175 These reports suggest that epigenome reprogramming is an early event in OLP. Broadly, the focus of epigenetic investigations in oral mucosal lesions to date has been directed toward understanding carcinogenic mechanisms and developing predictive diagnostic tools for early detection of malignancy or malignant potential. However, it should not be ignored that these disorders represent pathogenic processes in their own right which may have significant impact on patients’ quality of life irrespective of their malignant potential. Any assessment of epigenetic mechanisms in oral lesions may reflect epigenetic patterns which are characteristic of pathological processes distinct from early signs of malignancy. Furthermore, if these epigenetic mechanisms contribute to pathological events in the future, they may be targeted using epigenome-directed therapies.

Orofacial pain Introduction OFP is defined as pain whose origin is below the orbitomeatal line, above the neck and anterior to the ears, including pain within the mouth.176 This region is richly innervated and has an extensive somatosensory representation in the central nervous system.177 It is also the site of some of the most common acute and chronic pain conditions (pain lasting more than 3 months duration).177,178 Pain can result from diseases of these regional structures such as dental pain, temporomandibular joint disorders (TMDs), nervous system dysfunctions (neuralgias), referred pain from distant sources, or as idiopathic pain178. Many of these disorders can be viewed as a set of transitional phenotypes that are

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temporarily influenced by environmental events such as history of injury, infections, life stressors, and a variety of chemical exposures.179 It is these environmental events or exposures that influence the expression patterns of multiple genes and the activity of a variety of signaling pathways that manifest as the signs and symptoms that define these disorders.179

Temporomandibular joint disorders TMDs encompass a variety of conditions in one or both jaw joints and their associated structures (cartilage, muscles) accompanied by pain and dysfunction.180 Mild cases generally resolve over time, whereas severe cases associated with a number of other comorbid pain conditions generally become chronic and represent the most common chronic OFP condition.180 The pathophysiology of TMD is considered multifactorial with numerous risk factors being implicated for its onset and persistence. These include joint and muscle trauma, anatomical factors, hormonal differences, bone and connective tissue disorders, sensitization of peripheral and central nervous pain processing pathways, and psychological factors.181 Genetic variants that impact pain sensitivity and psychological traits when combined with environmental factors such as physical or emotional stress may interact to produce an individual’s state that is vulnerable to TMD.181 Research in epigenetic mechanisms underlying TMD onset are currently underway but very premature as the identification of genetic variants in TMD has only been recently established in the last few years. The Orofacial Pain Prospective Risk Evaluation and Assessment Study (OPPERA) is the first, large prospective study that was designed specifically to examine and identify biopsychosocial, environmental, and genetic factors contributing to the onset and chronicity of TMD.181 One of the specific aims of this study was to determine if genetic variations in individuals’ genes associated with pain amplification and psychological profiles are associated with elevated risk of first-onset and chronic TMD.181 358 genes known to contribute to nociceptive pathways, inflammation, and affective distress were assessed in a cohort study of 2737 individuals.182 Five main gene polymorphisms were observed to show associations with predictive TMD onset.182 Voltage-gated sodium channel-type 1-alpha subunit (SCN1A) and angiotensin I-converting enzyme 2 (ACE2) were associated with nonspecific orofacial symptoms, whereas Prostaglandin-endoperoxide synthase 1 (PTGS1) and amyloid-beta precursor protein (APP) were associated with overall psychological symptoms and stress, respectively.182 Finally, multiple PDZ domain protein (MPDZ) was observed to be associated with heat pain temporal summation.182 The identification of these key gene polymorphisms in pain susceptibility may provide a foundation for epigenetic research in the field of acute and chronic TMD onset. Some preliminary studies have evaluated epigenetic mechanisms in the progression of specific TMDs. Recently, Xiao and colleagues evaluated DNA

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methylation patterns in varying phases of experimentally induced temporomandibular joint osteoarthritis (TMJOA) in rats.183 Osteoarthritis (OA) is a common form of temporomandibular disorder, resulting in joint structure remodeling, articular cartilage deterioration with associated changes in the subchondral bone and soft tissues of the temporomandibular joint. Tumor necrosis factor (TNF), A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS5), and RUNX family genes were observed to have hypomethylated DNA profiles in early-stage TMJOA.183 These genes are associated with inflammatory or immune responses and have been previously identified as the most differentially methylated genes in both knee and hip osteoarthritis.183 These genes were also observed to remain hypomethylated in late-stage TMJOA.183 Additional genes involved with angiogenesis such as vascular endothelial growth factor A (VEGFA) and connective tissue growth factor (CTGF) and genes associated with the invasion of articular cartilage and advancing endochondral ossification such as matrix extracellular phosphoglycoprotein (MEPE) and osteomodulin (OMD) were also observed to be hypomethylated in late-stage TMJOA.183 Furthermore, a new subset of genes (claudin family (CLDN) and growth arrest and DNA-damage-inducible 45 family (GADD45)) exhibited consistently hypomethylated states in DNA throughout all stages of TMJOA.183 It is speculated that these genes may be involved in the intracellular signaling that controls proliferation, hypertrophy, or disorganization of chondrocyte during TMJOA contributing to the progression of TMJOA; however, this hypothesis still requires further validation.183 Results from this novel study suggest that epigenetic events may play a key role in the progression of TMJOA. Further functional studies investigating epigenetic influences in human TMJOA are required to increase our knowledge on the progression of TMJOA and perhaps lead to novel clinical interventions on DNA methylation which may prevent progression of this disorder.

Orofacial neuropathic pain The International Association for the Study of Pain defines neuropathic pain as pain caused by a lesion or disease of the somatosensory nervous system.184 It is a clinical description (and not a diagnosis) which requires a demonstrable lesion or a disease that satisfies the established neurological diagnostic criteria.184 The orofacial region is innervated predominantly by the three branches of the trigeminal nerve: ophthalmic, maxillary, and mandibular.177 Clinical symptoms of neuropathic pain can manifest itself as paresthesia, dysesthesia, hyperalgesia, allodynia, hypoesthesia, or hypoalgesia.185 These symptoms can be associated with trigeminal neuropathy and are seen across multiple facial pain conditions such as postherpetic neuralgia, trigeminal neuralgia, and oral dysesthesia.185 Etiologies of trigeminal neuropathic pain include systemic diseases, viral infections, physical trauma, and neurotoxic agents.185 Recently, there is 191

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convincing evidence that altered gene expression within peripheral and central nervous systems is a key mechanism for neuropathic pain. Numerous studies assessing epigenetic modifications responsible for gene alterations exist for the spinal somatosensory system, however, studies pertaining to trigeminal neuropathy have only just begun.185 Brain-derived neurotrophic factor (BDNF) is one of the most characterized genes shown to be regulated by epigenetic mechanisms in the spinal dorsal horn.185,186 It is responsible in modulating GABAergic and glutamatergic neurotransmissions, contributing to hyperalgesia in neuropathic pain.185 The role of epigenetic modulations responsible for correlating BDNF and neuropathic pain remains unclear.187 A novel study by Nakae and colleagues evaluated epigenetic mechanisms of BDNF gene in the trigeminal ganglion using an infraorbital nerve injury model of orofacial neuropathic pain in rats.187 Significant methylation differences were noted in the profiles of exon III of the BDNF gene between nerve-injured and naïve rats.187 While histone demethylation of k4 and k9 were seen to be increased and k29 acetylation was decreased in injured rats compared with sham and naïve rats.187 These observations were noted to strongly correlate with lower pain thresholds of injured rats compared with sham and naïve rats 14 days after injury, suggesting that epigenetic changes may occur in the early phases of injury in the orofacial neuropathic pain model with resultant transcriptional suppression of the BDNF gene.187 Furthermore, a similar study was able to classify nerveinjured and naïve animals based on the methylation profiles of the BDNF gene in the trigeminal ganglion, concluding that classification based on the DNA methylation profiles of the BDNF gene may be a valuable diagnostic biomarker for neuropathic pain.188

Epigenetics in chronic pain Chronic pain is characterized by persistent nociceptive hypersensitivity lasting for more than 3 months and presents in patients with allodynia, where nonpainful stimuli elicit pain, and hyperalgesia, where noxious stimuli elicit an amplified response to pain at the site of injury and surrounding tissues.189 It is often a result of peripheral tissue damage and persistent inflammation resulting in central sensitization of the central nervous system.189 Chronic pain conditions appear to predominantly affect women in their child-bearing years.180 These are often overlapping conditions and are frequently associated with chronic TMDs. They can be categorized as functional somatic syndromes and include chronic headache, fibromyalgia, interstitial cystitis, irritable bowel syndrome, low back pain, chronic fatigue syndrome, and vulvodynia resulting in an overall hypersensitized state.190 Presently, the biopsychosocial model is the most analytical approach to chronic pain. Unfortunately, current pain management interventions are

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insufficient to provide adequate pain relief, consequently having a substantial negative impact on a patient’s daily activities and quality of life.189 This may be in part due to our incomplete understanding of the mechanisms involved in chronic pain. Epigenetics may play a role in furthering our understanding of the onset of chronic pain following nerve injury, with mounting evidence suggesting a link between disease expression and environment.191 The development and maintenance of chronic pain involves long-term changes in multiple areas of the central nervous system, which are characterized at the cellular and molecular levels.189 It has been shown that a patient’s gene expression profile changes rapidly, with over 1000 genes activated in the dorsal root ganglion alone, soon after nerve injury.191 Evidence also exists that this gene activation is controlled via epigenetic modifications contributing to the transition from acute to chronic pain.191 There is evidence that injury-induced changes in chromatin structure via histone modifications drive changes in gene expression and neural function, leading to symptoms such as allodynia, hyperalgesia, anxiety, and depression.189 Many animal models of chronic pain utilize rodents to investigate the molecular and cellular adaptations of inflammatory pain and nociceptive pathways. These models have been shown to result in reliable nociceptive sensitization, imitating key components of the chronic pain experience in humans.189 Utilizing this model, Zhang and colleagues identified glutamic acid decarboxylase-65 (GAD65) gene as an important target of histone modifications in the development of chronic pain.192 Decreased histone acetylation mechanisms are involved in the suppression of GAD65 gene, which have been shown to impair the inhibitory function of GABAergic synapses in central pain-modulating neurons and further the development of persistent pain sensitization.192 The group also observed that by inducing a hyperacetylated state of the histones, GAD65 activity was upregulated, resulting in increased GABA function, pharmacological activation of inhibitory GABA function, and an overall reduced pain state.192 In the case of DNA methylation, chronic inflammation has also been shown to induce hypermethylation at promoter regions of antinociceptive genes, consequently silencing these genes and hypomethylation of pronociceptive genes, increasing expression of these genes.193,194 The majority of research on chronic pain focuses on spinal cord studies, as induction occurs due to peripheral injury; however, maintenance of the condition occurs in the central nervous system. The prefrontal cortex is an area of the brain shown to be of critical importance in both the affective and sensory components of chronic pain.195 Changes in this area have been reported across many pain conditions and pain-related comorbidities such as anxiety, depression, and cognition.195 There is now evidence that decreased DNA methylation levels across thousands of genome-wide promoters in the prefrontal cortex are implicated in chronic pain resulting in dysregulation of

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dopaminergic, glutaminergic, opioid, and serotoninergic systems.195 DNA methylation alterations have also been seen with neuropathic pain in circulating T cells, in a novel study by Massarat and colleagues.195 Interestingly, the majority of differentially methylated T cells 9 months post nerve injury were also noted to affect the brain.195 Massarat and colleagues also identified 11 subsets of T-cell promoters that were sufficient to predict chronic pain in rats with 80% accuracy.195 Furthermore, DNA methylation status of two genes in particular (signal recognition particle 54 (SRP54A) and exportin-4 (XPO4) were identified to predict mechanical hypersensitivity threshold intensity to an accuracy of 99%.195 These findings suggest that changes in DNA methylation in T cells and prefrontal cortex may be an avenue of DNA methylation biomarkers of chronic pain not only in diagnosis but possibly identifying individuals with a higher susceptibility of developing chronic pain.

Role of epigenetics in management of chronic pain The goals of pain management are relief of pain and improvement in function and general health. Currently available treatment for chronic pain states provides insufficient pain relief (nonsteroidal antiinflammatory agents (NSAIDs), paracetamol, opioids, anticonvulsants, antidepressants, etc.), with up to 40% of sufferers reporting inadequate pain control.196 In particular, the prescribing of opioid analgesics for chronic pain management has increased more than fourfold in the United States since the mid-1990s.197 Although systematic reviews demonstrate an approximately 30% reduction in reported pain with the use of opioids compared with placebo, the alarming increase in misuse of opioids has now become a public health problem in countries such as United States and Australia.197 Novel epigenetic studies are demonstrating that pain patients chronically treated with opioids have higher levels of global DNA methylation at repetitive long DNA-interspersed nucleotide elements (LINE1 elements) seen distributed across the whole human genome.198 It is speculated that opioids may trigger DNA hypermethylation, in a similar way to cannabinoid receptors (which are also G protein-coupled like m-opioid receptors) increasing global methylation status in differentiating human keratinocytes.198 Interestingly, the global DNA methylation at the LINE-1 site was also correlated with increased pain, suggesting that functional hypermethylation of unspecified pain-relevant genes was triggering hyperalgesia.198 This novel finding by Doehring and colleagues is supported by the observation of pain patients on opioid treatment becoming more sensitive to certain painful stimuli.198 So far, molecular pathways point to peripheral and central sensitization as an explanation. However, results from this study provide a new insight of epigenetic mechanisms contributing to suppression of endogenous pain inhibitory pathway transcription with subsequent development of opioid tolerance. The application and availability of epigenetic-based therapies for clinical use in the management of pain is limited. The most commonly investigated

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“epidrugs” in the context of chronic pain are HDAC inhibitors. As previously described, Zhang et al. demonstrated restoration of GAD65 gene function after repeated infusion of HDAC inhibitors such as trichostatin A (TSA) or suberoylanilide hydroxamic acid (SAHA) into the nucleus raphe magnus of hyperalgesia-induced rodents.192 Utilization of demethylation agent 50 -aza20 deoxycytinide (5-aza) has also been shown effective in reducing pain behavior and peripheral sensitization.193 Administration of HDAC inhibitors in the neuropathic pain model has also produced positive outcomes in regard to pain behaviors.199 Although no studies are specific to chronic OFP, use of HDAC inhibitors valproate and TSA have demonstrated prevention of hypoesthesia in post sciatic nerve injury and may be a possible avenue for use in chronic orofacial neuropathic pain. Similarly, use of HDAC inhibitor MS-275 as an intrathecal administration prevented mechanical and thermal hyperalgesia in the same model.199 In this regard, HDAC inhibitors targeting genes involved in chronic pain may serve as a new promising class of analgesics. Unfortunately, research on the therapeutic potential of DNA methylation remains limited and is an avenue requiring further investigation. Identification of a specific epigenetic marker that is unique to the disease is vital for developing an effective therapy.199 An inviting thought is the possibility of epigenetic therapy repairing the underlying cause of chronic pain as opposed to treating the symptom alone. Presently, epigenetic therapies are nonspecific and nonselective, acting centrally and peripherally. Therapies that manipulate single epigenetic marks that target genes involved in nociception should be considered for development.

Conclusion In many areas of oral health and disease, there is evidence for involvement of aberrant epigenetic mechanisms. More broadly in medicine and biology, the understanding of these mechanisms, their regulation, and the effects of the perturbations seen in disease have arisen out of the most intensively researched areas of developmental and cancer biology. However, as the field broadens, researchers are beginning to apply this knowledge and the new methodologies to other areas such as infection and inflammation. Currently, the understanding of molecular details of epigenetic mechanisms is far from complete and this particularly applies to interactions between the layers of DNA methylation, histone modification, and chromatin structure. The pace of discovery in the field suggests that we have much more to learn. Many of the earlier studies of epigenetics have employed candidate gene methylation analysis, and while this has yielded useful insight, it is clear the more complete picture yielded by global analysis is far more valuable. Apart from the benefits of understanding disease processes and the potential identification of diagnostic biomarkers, one of the key features of epigenetic

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modifications is that they are reversible. Therefore, identifying the epigenetic modifications associated with disease represents an opportunity for therapeutic intervention. Such therapies are currently in use especially in cancer and many more are under development for application to this and other diseases.200,201 Current epigenetic therapies often display a broad spectrum of cellular effect; however, as the molecular mechanisms are more precisely defined, these therapies will become more effectively targeted with less adverse effects. These applications are already being explored in oral diseases and there is an excellent prospect that the research into epigenetics in oral disease will yield novel and effective therapies in the future.

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