Australian Dental Journal

The official journal of the Australian Dental Association

Australian Dental Journal 2014; 59:(1 Suppl): 1–11 doi: 10.1111/adj.12155

Epigenetics: a new frontier in dentistry SD Williams,* TE Hughes,* CJ Adler,† AH Brook,*‡ GC Townsend* *School of Dentistry, The University of Adelaide, South Australia, Australia. †Institute of Dental Research, Westmead Millennium Institute, Faculty of Dentistry, The University of Sydney, New South Wales, Australia. ‡Institute of Dentistry, Queen Mary University of London, United Kingdom.

ABSTRACT In 2007, only four years after the completion of the Human Genome Project, the journal Science announced that epigenetics was the ‘breakthrough of the year’. Time magazine placed it second in the top 10 discoveries of 2009. While our genetic code (i.e. our DNA) contains all of the information to produce the elements we require to function, our epigenetic code determines when and where genes in the genetic code are expressed. Without the epigenetic code, the genetic code is like an orchestra without a conductor. Although there is now a substantial amount of published research on epigenetics in medicine and biology, epigenetics in dental research is in its infancy. However, epigenetics promises to become increasingly relevant to dentistry because of the role it plays in gene expression during development and subsequently potentially influencing oral disease susceptibility. This paper provides a review of the field of epigenetics aimed specifically at oral health professionals. It defines epigenetics, addresses the underlying concepts and provides details about specific epigenetic molecular mechanisms. Further, we discuss some of the key areas where epigenetics is implicated, and review the literature on epigenetics research in dentistry, including its relevance to clinical disciplines. This review considers some implications of epigenetics for the future of dental practice, including a ‘personalized medicine’ approach to the management of common oral diseases. Keywords: Epigenetics, methylation, acetylation, oral health, dentistry. Abbreviations and acronyms: CLP = cleft lip/cleft palate; mRNA = messenger RNA; ncRNA = non-coding ribonucleic acid; ORF = open reading format; SSC = squamous cell carcinoma.

INTRODUCTION Despite the completion of the Human Genome Project in 2003, establishing causal relationships between specific genes and complex diseases has proved challenging.1 As a consequence, the focus of researchers has shifted to identifying how variation in gene expression influences the development of disease. The field of epigenetics interrogates the molecular mechanisms that link the genetic code and the environment, and the molecular mechanisms themselves are referred to as epigenetic mechanisms. Epigenetic mechanisms have now been implicated in many disease processes and in a surprising number of other areas, including newer research that suggests epigenetics could also explain intergenerational disease susceptibility not directly hardwired into our genetic material.2 Evidence is emerging that epigenetic alterations, including DNA methylation and histone modifications, are transmitted transgenerationally, providing a potential mechanism for environmental influences on parents to be passed to their children.3 A BBC science programme has summarized these exciting discoveries as follows: © 2014 Australian Dental Association

‘At the heart of this new field is a simple contentious idea – that genes have a “memory”. That the lives of your grandparents – the air they breathed, the food they ate, even the things they saw – can directly affect you, decades later, despite your never experiencing these things yourself.’4 Two specific layers of information are encoded within the human genome, conferred by discrete chemical structures. The first, and most well-known layer, contains our genetic code that is encoded within the nucleotide sequence and base pairs of a double helix of deoxy-ribonucleic acid (DNA). Our genetic code contains all of the information to produce the elements we require to function, but it does not contain the ‘programme’ that determines when and where genes are expressed; this is encoded within the second layer. The second layer of information contains an epigenetic code for development and maintenance that dictates when and where various genes are activated and deactivated during embryogenesis, growth and throughout life. It is this epigenetic code that allows our genetically-identical cells to express different patterns of genes. So, rather than having trillions of 1

SD Williams et al. cloned cells doing the same thing, we have distinct cell populations with different phenotypes and functions working together to form different tissues that, in turn, form the organs that allow us to live. Figure 1 provides a visual metaphor that likens our genetic code to an orchestra, with the sheet music being the epigenetic code, the conductor being the epigenetic machinery, the orchestra members and their instruments the genes, and the combined resulting sounds representing the phenotype. The epigenetic code works through chemical modifications of the genetic code at different ‘levels’. This includes modifications of the linear structure of DNA itself (DNA methylation), as well as modifications of structural complexes around which DNA is packaged (histone protein acetylation in nucleosomes), and also by elaboration of extra-genetic non-coding ribonucleic acids (ncRNAs). Many epigenetic modifications affect genetic expression by switching genes on or off and preventing messenger RNA (mRNA) formation, or by affecting protein structure after translation from an mRNA template. In either case, the mechanism affects protein production and so affects genetic expression. These modifications can work in isolation, but tend to work in concert, especially methylation and histoneprotein acetylation.5 Epigenetic modifications change with time and are tissue specific, unlike the genetic

Fig. 1 The Music of Life. Conceptually, our genetic code is like an orchestra with a group of musicians and their different instruments capable of producing notes and whole melodies independently. However, without sheet music and someone to interpret the music and conduct the orchestra, the musicians are more than likely to produce a cacophony rather than a symphony! In this analogy, the sheet music is the epigenetic code, the conductor is the epigenetic machinery instructing the musicians (who are equivalent to individual genes) when to play the notes and how to play them. The resultant sound is equivalent to a phenotype and, with any luck, is pleasing to the ear. 2

code which usually does not change over time and is identical in 99% of the cells in the body. Epigenetic modifications have been shown to be responsible for our developmental programme, turning genes on and off at precise moments during embryonic development. When cells are differentiating, taking on specialized roles, methylation helps switch off genes that are not needed. Furthermore, some epigenetic modifications are stable and can be inherited from cell cycle to cell cycle and from parents (possibly even grandparents) to children.6–8 For example, the experiences of a parent, even before conceiving, can markedly influence the structure and function of the nervous system in subsequent generations. Using olfactory molecular specificity in mice, Dias and Ressler9 show parental experience influencing behaviour and neural structure in subsequent generations. Epigenetic modifications of the genetic code are caused by environmental stimuli and hence are responsible for our ability to adapt to different environments. This adaptation is not limited to physical adaptations but is also relevant to emotional adaptations; this includes how we respond to stressful situations or emotional trauma. For this reason, epigenetics is now seen as the missing piece of the puzzle, linking the environment to phenotype. As such, much research is now focusing on epigenetics to try to explain differences in phenotype that cannot be explained by conventional genetics.1,10,11 A simple way to look at this is to ask why so-called (genetically) identical twins do not ever look truly identical; or to ask why a certain population is more susceptible to a certain disease.12–15 When our epigenetic code runs awry, it causes problems and these changes have been implicated in many disease states and pathologies, including cancers, inflammatory diseases, autoimmune diseases and suicide.16,17 This paper provides a review of the field of epigenetics that is aimed specifically at oral health professionals. It considers different definitions of the term ‘epigenetics’ and ‘environment’, links epigenetics and the environment, addresses the underlying concepts, provides some details about specific epigenetic molecular mechanisms, discusses some of the key areas where epigenetics is implicated, and also reviews some of the recent literature on epigenetics research in dentistry. It also considers some implications for the future of dental practice, including a ‘personalized medicine’ approach to the management of common oral diseases. DEFINING EPIGENETICS Epigenetics is not easily defined. More recently formed definitions, while accurate, do not indicate the true scope of epigenetics, whereas the original definition, which is still applicable, does not provide any molecu© 2014 Australian Dental Association

Epigenetics: a new frontier in dentistry lar detail. As such, the history of epigenetics needs to be considered when discussing any current definition. The term epigenetics was first coined by Conrad Waddington in 1942, some 11 years before the structure of DNA was described by Watson and Crick in 1953.18 Epigenetics can be split as ‘epi-’ and ‘-genetics’. The term genetics is well-understood but defining ‘epi’ is not so simple. As a prepositional prefix it means ‘on, upon, or above’ and most modern applications take this definition. In this respect, epigenetics refers to something acting upon the genome. Whilst modern technology has shown that epigenetic mechanisms do in fact occur ‘upon the genome’, and Waddington recognized that something must be acting on the genome in order to regulate it, his definition takes ‘epi’ as an apocopation of epigenesis and provides a broader insight. Epigenesis refers to ‘development’; that is, the development of a complex being from a totipotent stem cell (Fig. 2) and Waddington’s definition points to this as follows, ‘… interactions … which bring[s] the phenotype into being’. Implicit in this definition are the temporal and spatial components of epigenetics that modern research is only just describing, over half a century after it was coined by Waddington.18–21 Taking these different perspectives into account, we propose that an acceptable working definition of epigenetics could be: a group of acquired or inherited and potentially transgenerational dynamic molecular mechanisms that are affected by the environment and act directly upon the genome and genetic machinery throughout life to regulate gene expression.

THE ENVIRONMENT AND GENETIC REGULATION In line with our definition of epigenetics, we also want to clarify what is meant by ‘environmental factors’ acting upon the genome. Environmental factors have been shown to up- and down-regulate genetic expression.22 However, the definition of what constitutes an ‘environmental factor’ is changeable. As technology has improved, explanations of what is referred to as ‘environmental’ have become more specific. For instance, Darwin’s theory of evolution described the macroscopic environment in which a plant or animal exists as affecting their evolution; similarly, cigarette smoke might be seen as an environmental factor that contributes to certain disease states. We know that our cells exist within a microscopic environment and that the products of one cell can influence other cells. However, it is now appreciated that our genetic material exists within a nanoscale environment and that molecules can also interact with our DNA. Regardless of the scale, ‘the environment’ has usually been invoked as the culprit to account for phenotypic differences that cannot be attributed to differential genetic expression. Ultimately, however, any factor that confers a change in phenotype must do so by differentially affecting genetic expression at the molecular level. Socioeconomic status is linked to health disparities, including oral health. Epigenetic changes are a potential mechanism contributing to these changes. The findings from a large community based study in the USA suggest that the DNA methylation is socially patterned.23 EPIGENETIC MOLECULAR MECHANISMS

Fig. 2 Waddington’s Epigenetic Landscape. Waddington’s epigenetic landscape is a metaphor for how gene regulation modulates development. Imagine a number of marbles rolling down a hill towards a wall. The marbles will compete for the grooves on the slope, and the ridges between the grooves represent the increasing irreversibility of cell type differentiation. Each marble will come to rest at the lowest possible point, representing eventual cell fates, or tissue types. This concept has been more formalized in the context of a systems dynamics state approach to the study of cell-fate,79 which has opened the door to the key role played by stochastic fluctuation (cellular noise), as well as physical fields, in both cell differentiation and cell proliferation. Further discussion of dynamic systems theory can be found in the paper by Brook and colleagues in this special issue. © 2014 Australian Dental Association

It is currently understood that environmentally induced epigenetic regulation of gene activity occurs by one of two methods, either by affecting chromatin condensation (DNA methylation and histone protein modification), or by preventing protein production directly (non-coding RNA).16,24–27 This section will discuss the general structure of the human genome to provide a context for later sections, which discuss the specifics of the different epigenetic modifications. The 23 pairs of human chromosomes are comprised of varying continuous lengths of double-stranded linear DNA that are wrapped around structural proteins (histone proteins) and then further coiled and supercoiled. Stretched end to end, human DNA is 2 m long, but it is condensed into a nucleus that might only be 0.5 lm in diameter. Due to the condensation required for chromosomes to be packaged into a nucleus, the genes on a chromosome are normally inaccessible for transcription and ultimately protein production; local sections of the strand must be ‘unwound’ in order to 3

SD Williams et al. provide access for transcription factors and the machinery required to commence transcription. The genetic material in the nucleus is variably packaged in different densities, reflecting the level of active transcription. The less densely packed material, euchromatin, is relatively uncondensed and allows for active transcription of the coding regions in the uncondensed areas. The more densely packed material, heterochromatin, is too condensed to allow transcription activating factors to bind to promoters on the strand to start transcription. Epigenetic mechanisms influence the level of chromatin condensation, and therefore the amount of genetic transcription. Epigenetic mechanisms have been found to affect both coding and non-coding regions of the genome. The coding regions account for 2% of the length of our DNA and contain approximately 20 000 genes (in coding regions)28 that, when spliced during post-translational modification, are capable of producing approximately 200 000 proteins. It was originally thought that the remaining non-coding regions contained only space-filling junk DNA. However, research has shown that these regions are likely to be crucial for gene regulation and the structural integrity of the strand; we know they are fundamental for epigenetic modifications. Epigenetic mechanisms act not only in the coding regions of our genetic material but also in the non-coding regions, as altering the structure of the structural (non-coding) parts of the DNA affects the remainder of the DNA.29,30 DNA methylation Each DNA strand is comprised of a sequence of four nucleotides: adenine (A), thymine (T), guanine (G) and cytosine (C). Specific nucleotides on one strand are always paired with those of the opposite: A with T, and G with C. There are approximately equal numbers of each nucleotide in the whole genome. DNA methylation is a covalent modification of cytosine in the DNA. It occurs by the addition of a methyl group to a cytosine residue on the linear DNA strand; however, methylation only occurs where cytosine is adjacent to guanine. It is important to differentiate between C being adjacent to G (in the case of a CpG group/dinucleotide) on the same strand, rather than opposite G (as in the case of C-G base pairing) on the opposing strand. This distinction is significant because adjacent Cs and Gs form a palindrome once complimentary base pairing occurs. This allows methyl groups to survive DNA replication and is fundamental for methylome stability; this is important for all models of transgenerational inheritance as it allows the genetic programme to survive from one cell generation to the next.31 CpGs are under-represented in the genome: they occur throughout the genome but are less frequent 4

than chance predicts. Furthermore, CpGs tend to occur in clusters so that when they do occur, they are vastly over-represented in these areas. These clusters of CpGs are known as CpG islands and they tend to occur in the promoter regions of genes. Studies have shown that although these CpGs do not relate directly to a gene when compared with CpG islands in gene promoters, they are associated with many disease states.32,33 Hypermethylation of CpG islands results in inhibition of gene transcription in the area, and hypomethylation results in activation of these genes; variation in DNA methylation occurs at specific genes but can also show a trend across the entire genome.34 Histone modification Histone proteins form the core proteinaceous structure around which DNA is wrapped; the histone protein along with its associated part of the DNA strand form nucleosomes. The structure of the nucleosome determines how the DNA further condenses and this ultimately affects genetic expression. The most common form of histone modification is acetylation of its eight subunits, which are referred to as octamers.35 Similar to hypo- and hypermethylation, both hyperand hypoacetylation can affect chromatin condensation and allow or prevent gene transcription respectively, although the mechanisms are different. Like DNA methylation, histone acetylation is also associated with both site-specific and genome-wide chromatin structure and therefore gene transcription. Histone acetylation is also associated with DNA synthesis and damage repair. Further, like methylation, histone modifications are heritable and survive DNA replication.36,37 DNA methylation and histone acetylation interaction DNA methylation is a chemical modification of the DNA itself, whereas histone protein modifications are chemical modifications of one of the key proteins around which DNA wraps. Both exert a major influence on chromatin structure, and therefore gene expression. Despite both acting in different regions and using different enzymes, there is likely a reflexive relationship between these two systems. Indeed, recent research has demonstrated that the enzymes from the two systems may interact directly.5 The interrelationship between DNA methylation and histone protein modification is particularly important for somatic cell reprogramming and stem cell research. During development, pluripotent stem cells lose their potency and eventually become terminally differentiated. This process is tightly regulated and involves a complex interplay between DNA methylation and histone protein modification. It is important because the process can be reversed so that somatic © 2014 Australian Dental Association

Epigenetics: a new frontier in dentistry cells can be reprogrammed back to a pluripotent state.38 Figure 3 illustrates the physical and chemical relationships between DNA and the two primary epigenetic mechanisms. Non-coding RNA RNA is the coding unit from which proteins are produced. DNA is transcribed into RNA and RNA is translated into proteins. Unlike DNA, RNA is single stranded. RNA contains the same nucleotides as DNA, aside from uracil (U) that is substituted for thymine (T). The similarity between the nucleotides in RNA and DNA allows them to share a complementary ‘language’. The two complementary strands of DNA in the double-helix are separated during transcription so that one of the strands of DNA can be transcribed to form a single strand of messenger RNA (mRNA). mRNA is one of several types of eukaryotic RNA (Table 1). Broadly, RNA can be divided into coding and noncoding RNA. Coding RNAs possess an open reading

frame (ORF) and are translated to proteins, whereas non-coding RNAs (ncRNAs) do not possess an ORF and do not elaborate proteins; however, whilst ncRNAs do not elaborate an active element (protein), they are themselves active. It is now estimated that only 2–5% of RNA codes for proteins, either structural or enzymatic. The remaining 95% of RNA is non-coding. Of this 95%, most is used as the machinery for the 2–5% of RNA that does code for proteins (e.g. transport-RNA and ribosomal-RNA). The remaining ncRNAs regulate the levels of mRNA and are considered to be part of the ‘epigenetic’ soup.39 More recent analysis of the data from the human genome project is showing that although the number of protein coding genes in the human genome has remained largely unchanged, there could be around 20 000 ‘dead genes’ hidden in the genome. It is thought that these genes do not code protein, but that the RNA generated by them exerts significant effects on the expression of protein coding genes.40 RNA regulation of gene expression can occur by preventing transcription of RNA from DNA or by destroying mRNAs after they are produced. Either way, protein production and therefore gene expression are affected. EXAMPLES FROM BIOLOGY AND MEDICINE Genetic imprinting

Fig. 3 The physical and chemical relationships between DNA and the two primary epigenetic mechanisms – methylation and acetylation. The template for construction and coordination of the ~200 000 structural and functional proteins in the human body is encoded in genes, which are comprised of double-stranded DNA and arranged in specific groups on chromosomes within the nucleus of almost all cells. Template information is encapsulated in the nucleic acid code of the DNA. DNA itself is a long, linear macromolecule that is packaged with histone proteins to form nucleosomes, and then coiled to form a chromatin strand. Chromatin is further super-coiled and arranged into chromosomes to conserve nuclear space. In periods of active genetic expression in a cell, the DNA is uncoiled locally to enable access of transcriptionally important enzymes to the relevant gene(s). Epigenetic regulation of DNA transcription within a specific cell or tissue acts at two levels of DNA organization: by control of histone acetylation regulating enzymatic access, and by methylation of specific cytosine nucleic acids in the DNA regulating mRNA transcription. Further epigenetic regulation by microRNA action on messenger RNA occurs posttranscriptionally. © 2014 Australian Dental Association

Genetic imprinting is a phenomenon where either the paternally- or the maternally-inherited gene is repressed and only the other is transcribed, whereas usually either gene copy can be transcribed. This means that for some genes to be active, they must be inherited from a specific parent. Therefore, imprinted genes act very differently to non-imprinted genes. DNA methylation profiles can survive mitosis and have been implicated as the molecular mastermind behind the process in which imprinted genes are faithfully reproduced in all daughter cells. It is thought that about 100 out of our 20 000 genes are imprinted. This number is probably conservative, with more imprinted genes being discovered, and even with such a small number, their effects are profound.41–43 Silencing of genes via imprinting has been found to have significant phenotypic effects. Prader–Willi and Angelman syndromes were the first disorders discovered to be associated with imprinting. Both are associated with the loss of a specific chromosomal region on chromosome 15 from one parent and silencing of the other copy due to sex-specific imprinting. If the loss of this chromosomal region is paternally inherited, then Prader–Willi syndrome results as a consequence of the silencing of the paternally-derived SNRPN and necdin genes, along with clusters of genes 5

SD Williams et al. Table 1. Types and functions of eukaryotic RNA RNA type

Function

Coding (relative abundance 3%) Messenger RNA Transcribed from template DNA; carries encoded nucleic acid message from nucleus to extra-nuclear sites of protein manufacture (ribosomes); nucleic acid code is read (triplet codon) and translated into an amino acid sequence to produce a polypeptide chain (protein). Non-coding (relative abundance 97%) Transfer RNA Small RNA chain of about 80 nucleotides that transfers a specific amino acid to a growing polypeptide chain at the ribosomal site of protein synthesis during translation; has sites for amino acid attachment and an anticodon region for codon recognition that binds to a specific sequence on the messenger RNA chain; complementary base pairing of triplet nucleotide sequence provides specificity. Ribosomal RNA This is the catalytic component of the ribosomes. In the cytoplasm, ribosomal RNA and protein combine to form a nucleoprotein called a ribosome. The ribosome binds mRNA and carries out protein synthesis. Nearly all of the RNA found in a typical eukaryotic cell is rRNA. Regulatory MicroRNA Small chains of 17–25 nucleotides; act through RNA interference (RNAi), where an effector complex of miRNA and enzymes can cleave complementary mRNA, block the mRNA from being translated, or accelerate its degradation. Small, interfering RNA Acts through RNA interference in a fashion similar to micro RNAs; some miRNAs and siRNAs can cause genes they target to be methylated, thereby decreasing or increasing transcription of those genes. RNA processing Small nuclear RNA Involved in modifying other RNAs. Small nucleolar RNA

coding for a series of small nucleolar RNAs. Patients with Prader–Willi syndrome usually display short stature, cognitive and behavioural problems, and chronic hunger that often leads to obesity. If the loss of the chromosomal region is maternally inherited then Angelman syndrome will result as a consequence of the silencing of the maternally-derived SNRPN gene. Patients with Angelman syndrome show severe cognitive impairment, happy excitable demeanour, and profound speech impairment. Cancer Much epigenetics research focuses on cancer. In general, cancers present with genome-wide global hypomethylation and gene-specific hypermethylation. The hypermethylation usually occurs within the promoters of tumour suppressor genes and this switches them off (silences them). Histone hypo-acetylation is also implicated in the silencing of tumour suppressor genes. The absence of tumour suppressor genes allows for the uncontrolled growth of cells and hence tumourigenesis. Tumours (malignant or benign) appear in the body fairly regularly, but the body normally detects and eliminates them quickly if they progress to this stage. Silencing of the tumour suppressor genes will have drastic implications, especially when combined with overall hypomethylation which results in increased gene expression and therefore cell growth. This relationship has been demonstrated in many cancers, including oral squamous cell carcinomas (SCC).44,45 Environmental stressors Evidence is emerging that the epigenome is affected by socially patterned factors with epigenetic differ6

ences between the most and the least socially privileged.23 It is known that certain environmental stressors can induce changes in the human body. Epigenetics, particularly methylation, has been shown to provide this link between the environment and phenotype in many cases. For example, intrauterine nutrition can cause epigenetic changes via DNA methylation in the foetus. The effects of these changes can be immediately apparent, and some changes can persist and render their effects later in life. Foetal folate deficiency is one such example. If the mother does not consume enough dietary folate, there is a lack of methyl groups available for the epigenetic machinery. As a result, certain genes do not become methylated. This also results in chromosomal instability. Both of these epigenetic changes can cause birth defects, especially of the neural tube, and are associated with problems such as spina bifida.46,47 Exposure to environmental toxins in occupational chemicals, cigarette smoke, contaminated air and drinking water, as well as fossil fuel emission, may cause epigenetic changes. Diesel fumes, pesticides and arsenic produce distinct patterns that can be identified. Smoking has a measureable effect on DNA methylation and has been associated with hypermethylation of tumour suppressor genes.48–50 Research has also shown that the variability in susceptibility to environmental and dietary toxins between people may be due to differences in how different individuals metabolize and process methyl groups generally. Differences in methyl metabolism may result in susceptibility to epigenetic changes that cause health problems. The findings of Dias and Ressler9 provide a framework for addressing how environmental information may be inherited transgenerationally at behavioural, neuroanatomical and epigenetic levels. © 2014 Australian Dental Association

Epigenetics: a new frontier in dentistry Methylation and human behaviour Research is now suggesting that ‘give me the child, I will give you the man’ might have a molecular underpinning whereby ‘nurture’ adjusts DNA methylation patterns, which concomitantly fine-tune genetic expression and therefore phenotype. Experiments in rodents have shown a strong link between adversity in early life and epigenetic profile in later life. It is hard to test this in humans as the brain is not accessible for testing in live subjects. However, studies of suicide victims have shown that those who were abused in early childhood had significantly higher levels of hypermethylation of rRNA genes in neurones, and therefore produced fewer ribosomes for protein production. Importantly, these changes were specific to the hippocampus, the part of the brain associated with memory formation.51–53 EXAMPLES FROM DENTISTRY Few articles in the dental literature relate to epigenetics. However, there are some papers published about the role of epigenetics in SSCs, but as cancer was one of the original areas of epigenetic research and the treatment of SSCs is not strictly within the purview of dentists, these studies will not be discussed further here. Aside from head and neck cancer research, much epigenetic research in dentistry relates mainly to periodontology and orthodontics. However, it is interesting to note that these disciplines approach the topic in completely different ways, as will be discussed below. Periodontology Across any given population people will display varied inflammatory and immune responses to a given stimulus. Research has shown that much of the variability is due to differences in what is a highly complex polygenic immune system. However, more recent research is demonstrating that the immune system and inflammatory responses are highly dependent upon epigenetic mechanisms to function. This has implications for all inflammatory diseases, including periodontitis.54 Cytokines are some of the biomolecules constituting the inflammatory response. These substances are small proteins that act as chemical messengers and modulate the immune response. Broadly, there are pro-inflammatory cytokines and anti-inflammatory cytokines. The balance of these cytokines determines what response is taken by the immune system to particular environmental stimuli. In the case of periodontitis in a susceptible host, toxins and breakdown products from bacteria and immune cells result in a significant predisposition towards a pro-inflammatory cytokine response to these stimuli, causing the development of © 2014 Australian Dental Association

a hyperinflammatory response with concomitant periodontal breakdown. It is now evident that epigenetic changes in the genes encoding cytokines can alter their expression, leading to either pro- or anti-inflammatory responses. Studies have shown that epigenetic changes of the genes encoding pro-inflammatory cytokines are associated with periodontitis.55 Other studies have shown an association between epigenetic changes and periodontitis.56–58 Most published studies have focused on chronic periodontitis, but a link has also been established between DNA methylation of pro-inflammatory mediator genes and aggressive periodontitis.59 One of the most revealing studies has shown a link between periodontitis and HIV-1 and AIDS progression.60 Specifically, it was shown that periodontitis can reactivate HIV-1 expression through an epigenetic mediator. This study not only shows a correlation between a systemic disease and periodontitis, but also explains at least part of the molecular mechanism linking the two. The study goes further to suggest possible future treatment options that have already received FDA approval in the USA (for treatment of other conditions). The article mentions two ‘epigenetic therapies’ approved; one involves using suberoylanilide hydroxamic acid in the treatment of T-cell lymphomas, the other involves using DNA methyl transferase DNMT inhibitors for the treatment of myelodysplastic syndrome and leukaemia. Unfortunately, these treatments are associated with significant systemic effects, and no targeted epigenetic therapies have been developed to date. Nevertheless, this research suggests that targeted therapies could play a part in the management of HIV-AIDS and, possibly, periodontitis in the future. The epigenetic changes on pro-inflammatory mediators in periodontal disease have been linked to a number of environmental stimuli, including smoking and nutrition, and the oral bacteria themselves. Iacopino61 states that these changes in the host tissues can facilitate bacterial colonization, increase inflammatory damage and also provide bacteria with increased levels of carbohydrate for metabolism. He also notes that these findings have implications for the methods used to diagnose periodontal disease and to identify patients at risk. He suggests that a new approach to management of periodontal problems in the future, based on personalized medicine, is likely to consider additional factors apart from bleeding and pocket depths, including types of bacteria present in the biofilm and epigenetic changes in the periodontal tissues.61 Orthodontics The approach to epigenetics research used in the orthodontic literature is different to that in the periodontal 7

SD Williams et al. literature. As discussed earlier, the definition of epigenetics has changed over time. Although Waddington’s definition points to some molecular mechanism, earlier usage represents a broader viewpoint. This broader view usually refers to some environmental factor causing a phenotypic change but without reference to any molecular interaction. Over time, usage of the term epigenetics began to refer to intracellular chemical environments and now the intra-genomic chemical environment is considered to be the location of epigenetic activity. While current periodontal research is interrogating the action of known (molecular) epigenetic mechanisms on specific genes, the orthodontic literature concentrates on the ‘bigger picture’ of epigenetics and tends to categorize environmental factors, such as forces acting on the jaw, as inducing growth or remodelling at the condyle. This use tends to place the environmental factor outside of the genome, rather than acting upon it. Forces acting upon the jaw may induce epigenetic changes that affect gene expression, but the orthodontic literature does not currently describe which epigenetic modifications are affecting which genes. It is interesting to note that, in his later work, Melvin Moss62–65 did distinguish between epigenetic processes (e.g. mechanical loading) and the epigenetic processes that enact changes. Moss refers to a range of epigenetic processes from the macro environment (e.g. joint loading), down to and including specific mention of DNA methylation, albeit, generally.65 Proffit notes that the major differences in theories of craniofacial growth relate to the location at which the genetic control is thought to be expressed.66 For example, if bone is considered to be a primary determinant of craniofacial growth, it is implied that genetic control is expressed at the level of bone. If cartilage is considered to be the primary determinant, then genetic control is considered to lie at the level of cartilage. If the soft tissue matrix in which the skeletal elements are embedded is considered to be the primary determinant of bone, as described in Moss’s Functional Matrix Hypothesis,61–65,67 then genetic control is assumed to reside outside the skeletal system. It is this indirect genetic control that is referred to as ‘epigenetic’ in the orthodontic literature, with changes in bone or cartilage occurring in response to signals from other tissues, i.e. epigenetic controls. The concept of ‘epigenetic orthodontics’ that is often equated to ‘functional orthodontics’ has developed from this broader view of epigenetics, with spurious explanations put forward such as ‘epigenetic orthodontics uses a person’s natural genes to correct and straighten the teeth and jaws using biomimetic DNA appliances’.68 Carlson has reviewed the various theories and concepts of craniofacial growth and development and related them to the developments in the field of genetics.69 In discussing developments in the post-genomic 8

era, he notes that there is an increasing awareness of the genes and their products that regulate craniofacial development, including the notion that these genes are turned on and off at critical times. He states that ‘the issue is not the fact that intrinsic factors within the genome regulate morphogenesis, but that the complex interaction of cells and tissues with remote extrinsic factors within the body and the environment are triggers, or switches for gene expression that influences postnatal growth and responsiveness to clinical treatment’. This demonstrates a broader view of epigenetics, but does not stress that these complex interactions result in specific molecular epigenetic changes. Carlson goes on to speculate that ‘Within the next several decades, orthodontists will be using molecular kits to diagnose growth-related problems and to determine precisely each patient’s developmental status as well as the presence or absence of key polymorphisms for growth factors and signalling molecules’. Cleft lip/cleft palate For such complex and heterogenous disorders, a multifactorial model of inheritance is favoured in which genetic risk factors interact with environmental co-variates.70 In a genome-wide study when gene-environment models were applied to three common environmental exposures in pregnancy, the significant interactions found were: MLLT3 and SMC2 with alcohol consumption; TBK1 and ZNF236 with maternal smoking; and BAALC with multivitamin supplementation.71,72 Future studies will further elucidate the role of epigenetic factors in cleft lip/cleft palate and provide a basis for sound preventive advice to reduce the frequency of this distressing condition; the dental profession will have a role in providing this advice. Enamel development and defects The clinical aspects of this topic are covered in the paper by Seow elsewhere in this issue.73 Here we note the influence of DNA methylation on the enamel protein, amelogenin, produced from genes on the X and Y chromosomes. In the somatic cells of females one of the two X chromosomes is ‘inactivated’. However, while most genes on that chromosome are inactivated, approximately 15% escape to some degree and a further 10% show variable patterns of inactivation. DNA methylation is involved in this X-chromosome inactivation, genetic imprinting and tissue gene expression.74,75 Behaviour management problems It has long been established that maternal fear of dentistry is an important factor in the anxiety and prob© 2014 Australian Dental Association

Epigenetics: a new frontier in dentistry lems that some children experience in accepting dental treatment.76 The new evidence of transgenerational transmission of fear at behavioural, neuroanatomical and epigenetic levels6 suggests that, for some anxious children, their dental anxiety may have deeper roots than learned behaviour from the parent. This implies that for some children treatment will need to aim not only at addressing learned fears but also at seeking epigenetic changes. Some insights about this come from the paper by Yehuda and co-workers77 showing that psychotherapy constitutes a form of ‘environmental regulation’ that may alter the epigenetic state. Psychotherapy influenced the activity of a stress hormone, and altered the methylation of a specific region of DNA, the FKBP5 gene. Also in this study, methylation of the GR gene (NR3C1) exon F1 promoter, assessed at pre-treatment, predicted treatment outcome. These findings are encouraging for clinical dental practice in suggesting that epigenetic tests may be developed that predict which patients with major behavioural problems are likely to respond to psychotherapy and that successful outcomes of treatment might be associated with changes in epigenetic markers.77 Genetic and epigenetic testing in dentistry could become reality, but it requires investigation at the molecular level. We argue that it is, in fact, the intrinsic factors (e.g. specific epigenetic mechanisms) that are the issue because these factors are the link, or the switch, between environmental triggers and changes in phenotype. It is these mechanisms that will be the targets of molecular kits and therapeutic interventions in the future. The finding that DNA methylation is socially patterned, as are health disparities, is relevant to dentistry since levels of oral health are also linked to socioeconomic status. CONCLUSIONS Epigenetic modifications are responsible for the differential expression of our genetic material across temporal and spatial fields. This explains how all of our cells can possess exactly the same DNA but there can be large numbers of different cell types forming different tissues and performing different functions. Although the large majority of studies have focused on the effects of negative life experiences, positive events may also alter the epigenome and some changes to the epigenome may be reversible.77 Epigenetics is proving to be a valuable and insightful arm of genomics research. Whilst there are no practical applications in dentistry at present, epigenetics may have profound influences in the future and so all clinicians should be aware of its basic principles. Even though it is early days, Australian periodontal © 2014 Australian Dental Association

research is demonstrating that epigenetic therapy might one day be an effective treatment for periodontitis.78 Furthermore, in terms of dental development, it may be possible to intervene early on to prevent hypodontia and a range of dental anomalies. In the shorter time frame though, epigenetics could be used as a reliable screening tool for a range of dental anomalies, including inherited enamel defects, as well as a means of assessing an individual’s susceptibility to dental caries and periodontal disease. Exciting times lie ahead! DISCLOSURE STATEMENT The authors have no conflicts of interest to declare. REFERENCES 1. Eichler EE, Flint J, Gibson G, et al. Missing heritability and strategies for finding the underlying causes of complex disease. Nat Rev Genet 2010;11:446–450. 2. Feinberg AP. Phenotypic plasticity and the epigenetics of human disease. Nature 2007;447:433–440. 3. Handel AE, Ramagopalan SV. Is Lamarckian evolution relevant to medicine? BMC Med Genet 2010;11:73. doi:10.1186/ 1471-2350-11-73. 4. Horizon. The ghost in your genes. BBC [online]. URL: ‘http:// www.bbc.co.uk/sn/tvradio/programmes/horizon/ghostgenes.shtml’. Accessed 11 November 2013. 5. Cedar H, Yehudit B. Linking DNA methylation and histone modification: patterns and paradigms. Nat Rev Genet 2009; 10:295–304. 6. Kadauke S. Tissue specific epigenetic bookmarking to preserve transcriptional programs through mitosis. 2012. Dissertions available from ProQuest. Paper AAI3550964. URL: ‘http: repository.upenn.edu/dissertations/AAI3550964’. Accessed 11 November 2013. 7. Wilson AG. Epigenetic regulation of gene expression in the inflammatory response and relevance to common diseases. J Periodontol 2008;79:1514–1519. 8. Grossniklaus U, Kelly B, Ferguson-Smith AC, Pembrey M, Lindquist S. Transgenerational epigenetic inheritance: how important is it? Nat Review Genet 2013;14:228–235. 9. Dias BG, Ressler KG. Parental olfactory experience influences behavior and neural structure in subsequent generations. Nature Neuroscience 2013 Dec 1. doi:10.1038/nn.3594. [Epub ahead of print]. 10. Slatkin M. Epigenetic inheritance and the missing heritability problem. Genetics 2009;182:845–850. 11. Manolio TA, Collins FS, Cox NJ, et al. Finding the missing heritability of complex diseases. Nature 2009;461:747–753. 12. Kaminsky ZA, Tang T, Wang SC, et al. DNA methylation profiles in monozygotic and dizygotic twins. Nat Genet 2009;41:240–245. 13. Saffery R, Morley R, Carlin JB, et al. Cohort profile: the peri/ post-natal epigenetic twins study. Int J Epidemiol 2012;41: 55–61. 14. Fraga MF, Ballestar E, Paz MF, et al. Epigenetic differences arise during the lifetime of monozygotic twins. Proc Natl Acad Sci USA 2005;102:10604–10609. 15. Haque FN, Gottesman II, Wong AH. Not really identical: epigenetic differences in monozygotic twins and implications for twin studies in psychiatry. Am J Med Genet C Semin Med Genet 2009;151:136–141. 9

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Address for correspondence: Associate Professor Toby Hughes School of Dentistry The University of Adelaide Adelaide SA 5005 Australia Email: [email protected]

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