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Review Article iMedPub Journals http://www.imedpub.com Journal of Clinical Epigenetics ISSN 2472-1158 2015 Vol. 1 No. 1:3 DOI: 10.21767/2472-1158.100003 Can Environmentally Induced Epigenetic Changes be Trans-Generationally Inherited by Offspring, Resulting in the Expression of Psychological, Behavioural, or Psychiatric Phenotypes? A Systematic Review Abstract Background: Epigenetic modifications have been found to be associated with some psychiatric conditions. Environmental stressors can cause heritable changes to the epigenome. Thus there is a possibility that parental epigenotype could contribute to the expression of a psychiatric phenotype in offspring. Here we summarise and evaluate research about trans-generational epigenetic inheritance and its association with behavioural, psychological and psychiatric phenotypes. Methods: Databases used included EMBASE <1947-Present>, Ovid MEDLINE(R) <1946 to September Week 1 2014> & PsycINFO <1806 to September Week 1 2014>. Studies were included if at least two generations were assayed on both epigenetic outcomes and behavioural/psychological/psychiatric outcomes. Given the limited available data, a meta-analysis was currently not appropriate. Findings: Results were heterogeneous and there were many different outcomes. Generally behavioural alterations co-occurred with epigenetic modifications. There was some evidence of inheritance of both these outcomes between generations. Clinically relevant changes to gene expression levels and neuroanatomy were observed in several cases. Possible modes of epigenetic transmission from the experimentally exposed group to subsequent generations were implicated in some studies. Conclusions: Despite the idea of epigenetics mediating trans-generational effects of the environment on brain and behaviour being widely touted, there is currently very little evidence. Moreover, mechanisms mediating such processes are unknown and thus whether epigenetic alterations induce behavioural change is currently still a matter of debate. Rachel A Caddick and Anthony R Isles Behavioural Genetics Group, MRC Centre for Neuropsychiatric Genetics and Genomics, Institute of Psychological Medicine and Clinical Neurosciences, School of Medicine, Cardiff University, Cardiff, CF24 4HQ, United Kingdom. Corresponding author Rachel A Caddick [email protected] Behavioural Genetics Group, MRC Centre for Neuropsychiatric Genetics and Genomics, Institute of Psychological Medicine and Clinical Neurosciences, School of Medicine, Cardiff University, Cardiff, CF24 4HQ, United Kingdom. Citation: Caddick RA, Isles AR. Can Environmentally Induced Epigenetic Changes be Trans-Generationally Inherited by Offspring, Resulting in the Expression of Psychological, Behavioural, or Psychiatric Phenotypes? A Systematic Review. J Clin Epigenet. 2016, 1:1. Received: October 15, 2015, Accepted: November 03, 2015, Published: November 12, 2015 Background Epigenetics (or more specifically epigenetic landscape) was a term coined by Conrad Waddington (1942), originally used to describe his conceptual model of how genes might interact with the surrounding environment resulting in the expression of altered phenotypes. He used this term to denote permanent changes to the expression of genes that cause cell differentiation. Epigenetics now most commonly refers to the heritable changes of gene expression and activity that do not modify gene structure [1]. Epigenetic modifications have numerous functions but are most frequently associated with expression of genes that mediate embryonic development. Within developing embryos, epigenetic modifications influence erasure and re-establishment of DNA methylation; imprinting of the genome; inactivation of the X-chromosome; pluripotent stem cell development and control of somatic cell differentiation [2]. Recently, epigenetic alterations have also been implicated in disease pathology of psychiatric, neurodevelopmental and neurodegenerative disorders. In addition to this, some epigenetic alterations have been found © Under License of Creative Commons Attribution 3.0 License | This article is available in: http://www.clinical-epigenetics.imedpub.com/ 1 Journal of Clinical Epigenetics ISSN 2472-1158 to be heritable across several generations. As the epigenome is sensitive to environmental influences, there is a possibility that environmental factors could cause heritable epigenetic modifications that result in changes in gene expression that alter behavioural, psychiatric or psychological phenotypes in offspring. Structure of the Genome DNA exists within the nucleus of animal cells as a double helix structure usually tightly wrapped around proteins called histones. The four histones around which DNA is coiled are H2A, H2B, H3 and H4. These are repeated twice [3] making an organised octamer structure with DNA, which is known as a nucleosome. Linker histones from the H1 family prevent the DNA from becoming incorrectly wrapped by sitting on the entry and exit strands of DNA. Nucleosomes make up the repeating units that form eukaryotic chromatin - a highly ordered structure that ensures DNA can be packaged into the cell nucleus. One of the functions of chromatin is to control gene expression and replication [4]. Transcription and subsequently gene expression is dependent on the structure of chromatin. RNA polymerases are responsible for initiating transcription. However, RNA polymerase does not transcribe every gene within the genome. Areas of the DNA with a more open structure are more difficult to transcribe, and are known as heterochromatin. Open areas (known as euchromatin) allow the enzymes access to the genes and therefore they can be more easily transcribed and the genes expressed as RNA. Heterochromatin is basically tightly packed DNA, usually located on the periphery of the nucleus. It generally consists of genetically inactive satellite sequences of DNA [5], an excellent example of this is the inactivated X chromosome in females. There are two forms of heterochromatin: constitutive and facultative. Constitutive heterochromatin domains are highly condensed and usually consist of repetitive lengths of DNA that are mostly transcriptionally silent in every cell of an organism. For instance large stretches of the Y-chromosome, and the centromeric- and telomeric-regions of all chromosomes. In contrast, facultative heterochromatin refers to regions of the genome that are transcriptionally silent in some cell-types, but transcriptionally active in others. Once again the example of the inactive X chromosome and the active X chromosome is a good demonstration of this difference in chromatin structure. The structural arrangement of chromatin and subsequent gene expression is subject to reversible epigenetic changes including DNA methylation, histone post-translational modifications (PTMs) and alteration by non-coding RNAs (ncRNAs). These are the epigenetic modifications of interest in this systematic review. DNA Methylation The 5’ position in the pyrimidine ring of cytosine bases in DNA can be modified with methyl groups to create 5-methylcytosine (m5C) by methyltransferases [6]. There are broadly two classes of DNA methyltransferase (DNMT) enzymes are responsible for this cytosine conversion; these are DNMTs responsible for de novo methylation and those that copy the methylation already existing onto daughter strands during cell replication [2]. Within 2 2015 2016 Vol. 1 No. 1:3 mammals DNA methylation is localised to CpG dinucleotides. These are commonly found to cluster in sequences of 200 bases made from a minimum of 50% cytosine and guanine, which are known as CpG islands. In 40-50% of genes found in humans CpG islands can be found close to or within promoter regions of genes. However, such islands are often unmethylated and are transcription factor targets. DNA methylation is generally associated with transcriptional silencing or repression and is implicated in gene regulation and development [6]. Consistent with its commonly found location on CpG islands, DNA methylation possibly blocks promoter regions and prevents the binding of activating transcription factors. Thus, DNA methylation is commonly associated with a lack of gene expression [7]. However, there is now an increasing awareness of the importance of non-CpG DNA-methylation, at so called CpH site (H = A, C, or T) and also of hydroxymethylation (5hmC), an intermediate in the recently identified active de-methylation process [8]. The existence of 5hmC in particular points towards a more dynamic role for DNA-methylation, which contrasts with prior notions of this being the most robust and stable epigenetic mechanism [9] Histone Modifications Post translational modifications of the histone proteins (PTMs) affect the 3 dimensional structure of chromatin and therefore alter gene expression [10] PTMs are an example of epigenetic modifications and are thought to affect gene expression through changing the structure of the condensed chromatin so different genes are exposed [11]. Histones can be altered with covalent modifications including acetylation, methylation, phosphorylation, sumoylation and ubiquitylation [12].Histone acetylation and methylation regulate binding of effector molecules by altering chromatin structure [13]. Lysine residues on histones are subject to acetylation which neutralises the positive charge of lysine that attracts histones to the DNA backbone. Subsequently, acetylation of histones is associated with euchromatin and increased expression [14]. Histone methylation, in contrast, is associated with both enhancement of gene expression and repression depending on location of the modifications [15]. DNA methylation is often dependent on histone methylation of a particular lysine residue in histone H3 (H3-K9 methylation), which is another mechanism in which histone methylation is involved in gene repression. DNA methylation is also thought to be involved in the deacetylation of histones, further increasing its role in transcriptional silencing. Proteins can bind to methylated DNA to form complexes that result in deacetylation. Similarly, non-methylated DNA does not attract deacetylating enzymes thus chromatin structure remains more open and available for transcription. Non-Coding RNAs Non-Coding RNAs (ncRNAs) are RNA transcripts with no obvious protein product. ncRNAs have a role in transcriptional processes like DNA methylation and histone PTMs, but do not directly affect the structure of DNA. There are several classes based on size and function of ncRNAs including microRNAs, long RNAs (lncRNAs) and This article is available in: http://www.clinical-epigenetics.imedpub.com/ Journal of Clinical Epigenetics ISSN 2472-1158 small RNAs. Small RNAs are thought to regulate gene expression as well as defensively suppress transposons. Of the numerous types of small RNAs, microRNAs (miRNAs) are thought to be most important in setting mRNA expression levels in eukaryotes through post-transcriptional modulation [16]. Generally, miRNAs are thought to bind to untranslated regions on the 3’ strand of target mRNAs, this represses the production of protein via mRNA destabilisation and silencing of translation [17]. In addition to this, miRNAs and other small RNAs target epigenetic machinery and transcription factors. Transcription factors (TF) and miRNAs can work in pairs to regulate gene expression and suppress functionally related proteins [18]. Evidence of Trans-generational Inheritance of Epigenetic Modifications As this review has already defined epigenetics as the study of heritable changes that alter gene expression but not DNA structure, it should be elucidated that this means the epigenetic features persist through both mitosis and meiosis [19]. This systematic review will only focus on the trans-generational inheritance of epigenetic changes caused by the environment. Whilst some epigenetic markers such as DNA methylation have been established as heritable throughout these processes, others such as histone modifications have been disputed but for the purposes of this review will still be considered. There are, generally speaking, two fundamental types of cells in the body. Somatic cells, which replicate by mitosis, and germ cells, which can replicate by both mitosis and meiosis. Germ cells produce gametes (sperm and ovum) that are able to fuse during fertilisation to produce offspring. Mitotic inheritance of epigenetic marks is common, for example the DNMTs responsible for the copying of methylation of cytosines from mother strands to daughter strands. The inheritance of histone modifications appears much more complex. Whilst epigenetic information provided by histone modifications should be inherited between mitotic cell generations in order to maintain cell fate [20], such modifications (like H3K27me3 and H3K9me3) aren’t precisely transmitted from the parent histones to the new histones. On both daughter strands, histones are randomly distributed in clusters [21]. Whilst mechanisms of histone transmission between mitotic states are unknown, since daughter cells inherit some histones from parental cells, it is worth considering histone modifications as mitotically heritable. Meiosis is a type of cell division that allows gametogenesis or the production of gametes. During gametogenesis the germ cells are subjected to various chromatin remodelling events and eventually their chromatin is condensed, becoming transcriptionally inert [22]. This process is mediated by histones and transition proteins. Small proteins called protamines replace histones in the late haploid stage of spermatogenesis [23]. However, recent evidence suggests that in approximately 1-10% of the genome in mammals tested, histones within nucleosomes are retained throughout spermatogenesis [24], though precise structural nature of these nucleosomes is currently unknown. After fertilisation, it is necessary that the maternal and paternal chromatin is decompressed before transcription and other © Under License of Creative Commons Attribution 3.0 License 2015 Vol. 1 No. 1:3 somatic processes can take place. The protamines of the paternal chromatin are exchanged for histones and genome wide demethylation occurs, causing further chromatin remodelling. However, the epigenetic marks associated with imprinted genes remain intact. These processes happen rapidly after fertilisation [22]. Generally most epigenetic modifications are thought to be erased through these mechanisms between generations but DNA methylation modifications are sometimes found to be heritable. Histones maintained in sperm may persist in the zygote at least to the one-cell embryo stage [25], for example, H3K27me3 retained in sperm are thought to persist causing repression in the pre-implantation embryo. Furthermore, H3K4me3 in human sperm correlates with corresponding early gene expression in the embryo [25]. Thus such persistence of epigenetic marks could have major functional consequences in developing embryos. Epigenetic Modifications Implicated in Behavioural, Psychiatric and Psychological Phenotypes Firstly, various epigenetic modifications have been found to be associated with psychiatric illness and particular behavioural traits. In the study of disease discordant monozygotic twins, there is differential methylation on specific CpG sites between twins affected with bipolar, schizophrenia and those with psychosis symptoms and the unaffected groups [26]. In addition to this, there was decreased methylation in BDNF promotor exon IV and IX in the frontal cortex of schizophrenic participants compared to controls [27]. Increasingly, evidence suggests lncRNAs are involved in disease pathology including mental disorders and autism [28]. Whilst the association of epigenetic modifications and a disease phenotype may not be causal in these cases, they at least indicate that altered epigenetic regulation may play a role in disease pathology and could be key early warning indicators of disease risk or treatment targets. The onset and development of psychiatric illness is often attributed to being induced by an interaction of environmental with an underlying genotype, as suggested by the Diathesis Stress Model [29]. Environmental and genetic factors influence the epigenome in the brain – which could potentially be the gene-environment interaction mechanism responsible for the expression of psychiatric phenotypes [30]. Various risk factors for psychiatric disease have been found to induce epigenetic changes as well as behaviours and symptoms associated with such disorders, thus implying epigenetic modifications may have a role in the onset of psychiatric conditions. Chronic social defeat stress (CSDS), a risk factor for behavioural mal-adaptations in rodent models, has been found to decrease H3K14 acetylation and histone deacetylases (HDAC2 in this case) in the nucleus accumbens, which are features also seen in depressed humans [31]. CSDS paradigms have also been found to transiently enhance then repress global H3K14 acetylation in the hippocampus, modifications which can then be reversed by chronic imipramine treatment [32], indicating histone H3 acetylation may be a target of such antidepressant medicines and possibly involved in the symptoms are depression. Furthermore, such environmentally induced modifications of phenotype and epigenotype have been found to persist over several generations. For example, poor maternal care persists 3 Journal of Clinical Epigenetics ISSN 2472-1158 through several generations with altered DNA methylation profiles in offspring [33]. A dysmasculinized phenotype was observed in two generations of male mice after chronic prenatal stress in the first generation and this correlated with altered miRNA profiles in the second generation offspring [34]. In addition to this, altered histone PTMs have been implicated in the inheritance of a cocaine-resistant phenotype [35]. A causal link between epigenetic modifications and the production of a particular disease phenotype is plausible [36]. Injected sperm RNAs of males that had been exposed to unpredictable maternal stress and separation (MSUS), into wild-type fertilised oocytes. Behaviour observed in MSUS males were then observed in the genetically unrelated MSUS-sperm injected offspring. This has led to the development of the research question of whether transgenerational epigenetic inheritance induced by environmental factors can produce behavioural, psychiatric and psychological phenotypes in unexposed offspring. Aims of the study As research on trans-generational epigenetics is expanding rapidly, the need for a systematic review of the research already conducted is necessary. Currently, many systematic reviews on epigenetics focus on a single generation or on outcomes other than behavioural, psychological or psychiatric phenotypes. However, trans-generational epigenetic inheritance potentially has an important role on the transmission of psychiatric disease and will surely be the focus of many future studies. This is why a systematic review should be conducted, to summarise current methodologies, theories and understandings – the results of such a systematic review can be utilised by future researchers to create methodologically and theoretically sound experiments. This systematic review aims to screen current literature for relevant studies that examine the role of epigenetic inheritance in the expression of behavioural, psychological or psychiatric phenotypes. This aim can be split into several components. Firstly, can epigenetic modifications induced by external stimuli be trans-generationally inherited by progeny? Secondly, if the epigenetic modification is trans-generationally inherited by the offspring, is a change in behavioural, psychological or psychiatric phenotype also present? Subsequently, this review aims to assess whether this change in phenotype can possibly be associated with the epigenetic change and through what mechanisms this is possible. Finally, the impact of the findings will be discussed in addition to a justification for further research into the field of trans-generational epigenetics. Thus, to produce a systematic review that summarises the information from these papers in a comprehensive manner, it is necessary to assess the methodological quality of the studies featured in this review. In addition to this, the conclusions of the authors featured in these papers need to be examined. This systematic review aims to assess the hypothesis that environmentally induced epigenetic modifications can be inherited by offspring and expressed as a behavioural, psychological or psychiatric phenotype. 4 2015 2016 Vol. 1 No. 1:3 Methods PICOS Structure in the Methodological Design of This Systematic Review Whilst the research question covers a fairly novel subject area for systematic reviews, the methodology will still follow a traditional structure as recommended by the Cochrane Collaboration [37]. The research question was designed and constructed through the PICOS structure [38]. Below, the components of the PICOS structure are listed and an explanation of how each aspect pertains to the design of this systematic review is given. Types of Participants There was no particular participant group defined for this systematic review as the study of trans-generational epigenetics is a relatively new field, subsequently, it was expected that very few papers would be identified and narrowing participant group criteria would produce an extremely small yield. Thus it was also assumed that most papers found would feature animal trials rather than human-patient groups. The main participant criteria of this systematic review was that papers should feature at least two generations of participants for the measures of epigenetic modifications – which is essential for the study of the transgenerational aspect of the research question. In addition to this, this review did not accept papers studying unicellular populations or plant populations. Types of Intervention Due to predicted restriction of study yield, very few intervention requirements were stipulated. Most essentially, the experimental intervention had to be implemented with the intent of producing alterations to some aspect of the epigenome in participants. In addition to this, as this paper is interested in trans-generational inheritance, the intervention should be implemented in the parental generation i.e. there should be at least one offspring group not directly exposed to the intervention (not including controls). Types of Comparison Group No particular comparison group was outlined but it was assumed that most studies would have a control group of some description (i.e. a group of participants that did not receive the intervention expected to produce an epigenetic change and behavioural change). Studies without an appropriate comparison group will not be considered for further investigation. The conditions under which the comparison group was raised/kept should be the same for at least one epigenetic assay and one behavioural assay within each paper, so meaningful associations can be made. However, this is not a requirement and such paradigms will be discussed in the results section. Types of Outcome Measures There are two main outcome types that were required for a paper to be considered, the first being a heritable epigenetic change caused by the intervention. For example, a stressor in the first generation causes an epigenomic alteration - the study must then This article is available in: http://www.clinical-epigenetics.imedpub.com/ Journal of Clinical Epigenetics ISSN 2472-1158 investigate for similar epigenetic modifications in at least one subsequent generation. Epigenetic modifications on imprinted genes are not of interest for this systematic review. The second outcome type is the measure of phenotype change – the phenotypes of interest being either behavioural, psychological or psychiatric. A neuroanatomical, cognitive or morphological change by itself is not sufficient for inclusion, it must be linked to behaviour or to a psychological/psychiatric disorder. Types of Studies Multiple types of study were considered for this review. The study of trans-generational epigenetics has only been studied relatively recently due to methodological restrictions prior to this. As a result, many of the studies of interest are expected to be more in the primary stages of research using experimental design more familiar to psychological research than epidemiological research. Experimental rather than observational studies are more likely to be featured in this systematic review; limited theoretical knowledge would probably render large scale epigenetic assays hard to administer and evaluation of results of such studies would be quite difficult to interpret due to a large number of confounders that arise in such non-controlled conditions. As most of the papers identified are likely to use experimental laboratory-research design, it is also likely that most of the studies will use one-way designs to study the direction of epigenetic and behavioural changes e.g. performance of the experimental group vs the control group on epigenetic/behavioural outcome. However, many experiments will look at other factors affecting the outcome, in addition to effect of experimental group, such as gender; these will be factorial designs. It is also probable that some paradigms will use a within-subjects design as participants will be tested on multiple occasions or on multiple assays. 2015 Vol. 1 No. 1:3 in PubMed, which established that different terminology or synonyms often produced additional hits with possible relevance for this review. These hits would otherwise not have been found. Whilst constructing the search term procedure, known relevant studies, such as [41] were used as a reference point for searching i.e. a search procedure should be able to retrieve this study if it was in the database, if it wasn’t able to then further relevant search terms were required. Each synonym was entered into each individual database (using Ovid) and then expanded and mapped to subject headings to find further useful categories, this produced 3 different search term procedures. The 3 search term procedures were then reintegrated so one unified search procedure was produced. Search terms and subject headings that were valid in one or two databases invalid in the others were converted to text terms so scope was not reduced. Search Term Procedure The following databases were used in the identification of relevant papers: A. Ovid Medline (R) Without Revisions (1996-Present) B. Embase C. Psych Info The following search terms were used in each database for the identification of papers: 1. Epigenetic/ 2. DNA acetylation.mp. 3. DNA methylation.mp. 4. DNA phosphorylation.mp. Search Strategy 5. Epigen*.mp Though the concept of epigenetics was first mentioned in the 1940s [39] studies on epigenetics have been limited by the equipment and understanding until recent years, for example, bisulfite sequencing was implemented for chemical analysis of DNA methylation in 1994 [40]. Thus, it was deemed unnecessary to carry out a hand search of data as it is unlikely that any relevant papers would not have been written in electronic form or already converted to electronic form. 6. exp epigenetics/ Electronic Searches 11.ncRNA*.mp.untranslated RNA.mp. or long untranslated RNA.mp. or non coding RNA*.mp. The outcome criteria of this systematic review are quite broad as initial PubMed searches did not yield many studies of relevance. As the field of trans-generational epigenetics has only recently expanded, not all studies have been fully indexed to the relevant area – i.e. not all papers referring to epigenetics will be retrieved if the term “epigenetic” is used when searching in a database (this was discovered during preliminary searches on PubMed). As a consequence of this, in this review, multiple synonyms have been used for each aspect of the research question. Every possible search term was also expanded upon within each individual database, so that as many papers as possible are identified. Several preliminary searches were conducted 12.Epigenetic Inheritance.mp. © Under License of Creative Commons Attribution 3.0 License 7. Histone post translational modification*.mp. or chromatin. mp. 8. Histone PTM*.mp. or histone modification.mp. 9. MicroRNA*.mp. 10.miRNA*.mp. or small interfering RNA.mp. 13.multigener*.mp. 14.multigenerational.mp. 15.Soft Inheritance.mp. 16.transgener*.mp. 17.trans-generational.mp. or Inheritance Patterns.mp. 18.ancest*.mp. 19.descend*.mp. 5 Journal of Clinical Epigenetics ISSN 2472-1158 20.Hereditary.mp. 46.43 and 44 and 45 and 46 22.inherit* .mp. Study Yield 24.anxi*.mp. or exp anxiety disorder/ 25.exp anxiety/ 26.behav*.mp. 27.exp behavior/ or Mentally Ill Offenders.mp. or behaviour. mp. or exp Social Behavior/ 28.brain.mp. or exp brain/ 29.depress*.mp. 30.exp reactive depression/ or depression.mp. or exp endogenous depression/ or exp Depression/ or exp Treatment Resistant Depression/ 31.affect*.mp. or emotion*.mp. or stress.mp. or Arousal.mp. or exp Expressed Emotion/ 32.exp fear/ or exp conditioning/ or exp phobia/ or fear.mp. 33.habit*.mp. 34.exp neuroanatomy/ or neuroan*.mp. 35.neurobe*.mp. 36.exp neurobiology/ or neurobi*.mp. or exp attention deficit disorder/ or substance related disorders.mp. 37.exp neurology/ or neurolo*.mp. or exp Epilepsy/ or exp Neurology/ or exp Conversion Disorder/ or exp Neuropsychology/ 38.neuron*.mp. or exp Neurons/ 39.mental disease.mp. or exp posttraumatic stress disorder/ or psychiat*.mp. 40.exp child psychiatry/ or exp psychiatry/ or exp forensic psychiatry/ or psychiatry.mp. or exp Child Psychiatry/ or exp Biological Psychiatry/ or exp Psychosis/ or exp Alcohol Abuse/ or exp Drinking Behavior/ or exp Mental Health/ 41.exp coping behavior/ or exp schizophrenia/ or exp cognition/ or psychol*.mp. The abstracts and titles of the studies yielded by the search procedure were used to identify relevant papers. A. Ovid Medline (R) Without Revisions (1996-Present): a. 456 papers found, 109 were identified. B. Embase: a. 541 papers found, 124 were identified. C. Psych Info a. 72 papers found, 35 were identified. At this stage review papers were included in order to ascertain whether further relevant papers could be identified from their contents that had not been found using the search procedure. Inclusion & Exclusion Criteria The 268 total identified papers were individually screened to ascertain whether they were relevant. The same inclusion and exclusion criteria were applied but at this stage review papers were also excluded. No further relevant results were yielded and the search procedure was thought to be sufficient. It should be noted that the phenotype related outcomes of interest needed for inclusion could be any paradigm relating to behaviour, psychology or psychiatry; these included tests of locomotive activity, sucrose consumption (as a measure of depression), etc. Memory assays and brain chemical assays were not included in this review. As the outcome criteria was so wide and may not have been explicitly mentioned in the abstracts, many studies were not excluded at the initial identification stage when abstracts were scanned. However, imprinting disorders were excluded. Epigenetic modifications of interest also excluded changes at imprinted genes. Papers would not be considered unless at least two generations were studied. In addition to this, at least one generation must not have been exposed to the experimental manipulation. Furthermore, the study population could not be plants or unicellular. Books or reviews were excluded if they did not contain meaningful data. The inclusion and exclusion criteria are summarised in Table 1. Results 42.psychology.mp. or exp clinical psychology/ or exp psychology/ or exp child psychology/ or exp medical psychology/ or exp Adolescent Psychology/ or exp Trauma/ or Addiction.mp. or exp Psychopathology/ or exp Eating Disorders/ or personality.mp. or exp Motivation/ or cogniti*.mp. or exp Decision Making/ or eating*.mp. Study Selection Process 43.1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 12 or 13 or 14 or 15 or 16 or 17 Of the 268 papers (162 were duplicates) identified using the search procedure outlined in Section 2.7-2.9, only 106 were retrieved. Some papers could not be found using the references provided (N=17) and some were Non-English Language papers that could not be translated (N=24). From these 100 retrieved 44.18 or 19 or 20 or 21 or 22 or 23 45.24 or 25 or 26 or 27 or 28 or 29 or 30 or 31 or 32 or 33 or 6 Vol. 1 No. 1:3 34 or 35 or 36 or 37 or 38 or 39 or 40 or 41 or 42 21.heritable.mp. or heritability.mp. 23.progeny.mp. or Offspring.mp. or Family Members.mp. or exp Family Relations/ or Grandparents.mp. or Holocaust Survivors.mp. or Family Background.mp. or Family History. mp. or exp Mothers/ or human relation.mp. or exp family/ 2015 2016 The initial search procedure yielded 1068 papers in total. In the scanning of abstracts, 109 were found to be relevant in Embase; 124 in MedLine and 35 in PsychInfo. The articles were then retrieved and read fully. This article is available in: http://www.clinical-epigenetics.imedpub.com/ Journal of Clinical Epigenetics ISSN 2472-1158 papers, six papers met all five inclusion criteria and did not meet the parameters of the exclusion criteria. Please refer to Appendix 1 for the list of excluded papers from this stage. Description of studies All of the 6 papers identified were heterogeneous laboratory conducted experiments. Within these the following behavioural paradigms were studied: Accelerating Rotarod (n=1); Auditory Fear Conditioning (n=1); Cocaine Self-Administration (n=1); Ethanol Consumption/Preference (n=1); Elevated Plus Maze (n=2); Forced Swim Test (n=2); Free Exploratory Paradigm (n=1); Light-Dark Box (n=1); Maternal Behaviour Assay (n=3); Olfactory Fear Potentiated Startle (OPS; n=1); Olfactory Sensitivity Assay (n=1); Open Field Paradigm (n=2); Sensorimotor Assay (n=1); Sucrose Consumption (n=1); Sucrose-Self Administration (n=1); Tail Suspension Test (n=1) & Two-Bottle Choice Assay (n=1). Please note that from now on as no psychiatric or psychological outcomes found, the altered phenotype will be referred to as a behavioural phenotype. In addition to this the following epigenetic features were examined: DNA methylation (n=3); Histone H3 Acetylation (n=2); miRNA/ncRNA levels (n=2). The results of the data extraction of each paper featured in this systematic review can be seen in five parts. The behavioural and epigenetic assay results included manually assessed interpretations of visual data. Such interpretations were included as many authors did not include statistical results that were non-significant, it is generally good practise to publish nonsignificant results thus as much information pertaining to the direction of non-significant results was recorded for this review. Whilst some authors such as [42] often recorded results that were approaching significance as “a trend in” other authors did not do so. As this systematic review aims to provide information for other researchers, it was decided that any available information should be extracted should other experimenters wish to use similar experimental paradigms in the future. Design of Identified Studies As predicted, the studies yielded were not epidemiological in nature. Instead experiments conducted in laboratories were found. Whilst systematic reviews traditionally examine randomised control trial (RCT) designs and other epidemiological studies, it was still decided that a systematic review of the laboratory studies was possible as they can be viewed as the nonhuman equivalent of RCT studies (Kramer, 1998). At this stage of research into the field of epigenetics, the use of laboratory experiments is more appropriate for assaying the effects of epigenetic inheritance on behavioural phenotype as extraneous variables can be controlled, measured and their effect on the results assessed. Table 4 provides a general summary of relevant methodological design features of the identified papers. Systematic Review of Participant Groups Unfortunately, total participant number (n) for all experiments cannot be reported as all data pertaining to n is not available in identified papers (see Table S1, Appendix 2). Table 5 summarises all information available about participant groups, including species and breeds, reason for selecting these breeds and species and how © Under License of Creative Commons Attribution 3.0 License 2015 Vol. 1 No. 1:3 subsequent generations were produced. Four identified papers examined mice [41-,43, 36) and two examined rats [44,9]. All four papers using mice used a C57Bl/6J strain. [42] outbred these to Strain 1295v/lmJ mice to produce hybrids. [36] also used an addition strain for IVF component of their study (where fertilised eggs of BD62F1 mice were injected with C57Bl/6J sperm RNAs). Dias and Ressler used an additional strain of mice, M71-IRES-tauLac Transgenic Mice (M71-LacZ), which were involved in a variety of behavioural and neuroanatomical experiments but not epigenetic assays. [44]) bred Sprague-Dawley male rats who were subjected to the cocaine self-administration paradigm to naïve females 3 days after the last administration of cocaine (3/18 males bred continued cocaine-self administration but offspring of these produced results no different from the other sires thus groups were combined). Finally, [9] used Long-Evans hooded rats in their experiment. Systematic Review of Experimental Manipulation Firstly, please note that experimental manipulation is equivalent to the environmental factor aspect of the research questions. Two studies used MSUS as the experimental manipulation [43] [36] Dias & Ressler used an olfactory fear conditioning paradigm; Finegersh & Homanics used vapour ethanol exposure; [44] exposed rats to cocaine-self administration & [9] used a stress paradigm during gestation. Only the F0 generation was exposed to the experimental manipulation in studies conducted by [41,[42,44] In the MSUS studies, both F0 dams and F1 litters were exposed to the experimental manipulation. Finally, [9] had several generations exposed to the experimental manipulation: for each generation the participants were split into stressed and non-stressed groups. Only [41] and [42] used at least one control group that had followed exactly the same experimental procedure as the experimental manipulation group. [44] used a similar stressful procedure in controls (intravenous injection of saline). Franklin et al (2010) and Gapp et al (2014) merely left the control groups in the home cage with 1 cage change per week (though given the nature of MSUS alternative control group procedures are difficult). Yao et al (2014) did not specify further than “not stressed”. [41] studied multiple different groups of unexposed progeny of F0conditioned sires. [42] [44] studied only one unexposed group of progeny of F0-exposed sires each. [43] [36] studied 2 unexposed group of progeny of F1-exposed males. Yao et al (2014) examined 3 unexposed progeny of F1 females who were stressed in utero. Heritable Behavioural Phenotypes Although there are limitations associated with the tests used (see Appendix 2, Table S2), all the experimental manipulations induced a behavioural phenotype in at least one gender of offspring (Table 6). Inheritance of behavioural phenotypes was observed through the maternal, and the paternal line depending on the procedure used. Inheritance of behaviour was often sex linked, though many studies only examined behavioural transmission in one gender. Heritable Epigenetic Modifications Again, although there are limitations associated with the techniques used (see Appendix 2, Tables S3) overall, most studies have demonstrated that some epigenetic modifications can be 7 2015 2016 Journal of Clinical Epigenetics ISSN 2472-1158 Vol. 1 No. 1:3 Table 1 Inclusion and Exclusion Criteria. Inclusion Criteria • Presence of trans-generational inheritance of traits i.e. across more than one generation. • Epigenetic modification studied in 2 generations. • Behavioural, psychiatric of psychological phenotype studied. • Appropriate control group for comparison of results of epigenetic assay and behavioural/psychological/ psychiatric assay. • At least one generation must not have direct or in utero exposure to experimental manipulation. Exclusion Criteria • Any of the five Inclusion Criteria were not met. • If trans-generational inheritance refers to in utero effects between mothers and offspring and no subsequent generation is studied. • If behaviours/epigenetic modifications refers to imprinting disorders. • If behavioural assay refers to memory. • If paper was from book or other medium that does not contain meaningful data. • If study population is not animals/humans e.g. unicellular organisms and plants. Table 2 Numbers of Papers Identified and Excluded by Scanning of Abstracts. Identified Inappropriate Participant Group No Epigenetic Outcome of Interest No Trans-generational Aspect No Phenotype Outcome of Interest No Epigenetic & Trans-generational No Epigenetic & Phenotype of Interest No Phenotype of Interest & Trans-generational No Epigenetic, Phenotype or Trans-generational Book/No Data Table 3 Numbers of Papers Identified and Excluded by Reading of Full Paper. Identified Inappropriate Epigenetic Measure Inappropriate Behavioural Outcome No Trans-generational Study of Epigenetic Inheritance No Trans-generational Aspect / Generation is not Removed from Experimental Exposure Review/No Data Number of Papers 6 19 8 3 2 68 transmitted to subsequent generations. However, transmission appears complex in most cases and often a direct transmission to a subsequent generation. The alteration and inheritance of miRNA/ncRNA profiles can differ in direction between generations and between neuroanatomical area (Table 7). Experimental manipulations modified DNA methylation levels at various loci in both directions (Table 8). Altered DNA methylation levels in specific areas tend to be inherited in the same direction (i.e. either increased or decreased) as they are found in the parental generation. [44] found significantly increased AcH3 associated with Bdnf promoter IV in the medial prefrontal cortex (mPFC) of F1 males. Furthermore increased AcH3 levels were found in the testes of F0 males and specifically (though global levels were not increased) at Bdnf promoters I, IV and VI in F0 sperm. This indicates a possible mode of transmission of Histone H3 acetylation (AcH3) through the paternal germline should histones persist through spermatogenesis (Table 9). 8 Embase 109 84 14 10 145 4 27 42 21 0 MedLine 124 80 13 9 210 4 36 50 15 0 PsychInfo 35 0 3 7 2 6 0 0 5 14 Discussion This systematic review summarised all data from six papers examining trans-generational inheritance of epigenetic marks and their association with behavioural outcomes (Table 10). Specifically we focused on the role of environmentally induced modifications (i.e. those changes caused by experimental manipulation) in the production of altered behavioural phenotype. Despite much discussion in the literature, original studies were limited and most studies included are in their early stages of methodological design, thus, interpretations of the results extracted are mainly associative. Furthermore, it is difficult at this point in time to extend any findings of experimental research from laboratories to humans. For example, [44] acknowledge that their findings of a cocaine resistant phenotype varies considerably from evidence of cocaine-related behaviours found in humans, where addiction is found to be heritable (Kendler et al, 2003). Currently, there are too many confounding variables in human life to be able to draw parallels from laboratory experiments. However, some promising evidence has been provided by the identified studies. Whilst the evidence is not conclusive proof of the trans-generational inheritance of environmentally induced epigenetic modifications producing altered behavioural phenotypes, it at least provides an evidence-based foundation for future studies to expand upon. Of all the papers identified, [36] provides the most convincing support for the hypothesis that environmentally induced epigenetic modifications can be passed to subsequent generations and expressed as behavioural phenotypes. Their experimental procedure provides the only direct example of an epigenetic induced change to behaviour. Firstly, the authors found an altered non-specific behavioural phenotype was present This article is available in: http://www.clinical-epigenetics.imedpub.com/ Journal of Clinical Epigenetics ISSN 2472-1158 2015 Vol. 1 No. 1:3 Table 4 Summary of Identified Papers Dias & Ressler [41] Subject Species Mice Breed(s) of Subject M71-LacZ transgenic. Species C57Bl/6J. Environmental Stressor Paradigm Used (Experimental Manipulation of Interest) Group(s) Subjected to Environmental Stressor Manipulation for Control Group Epigenetic Modification of Interest Area Epigenetic Modification(s) Assayed. Gene(s)/Promoters Epigenetic Modification(s) Studied On. Generation Epigenetic Modifications Studies In. Initial Inheritance through parental Germline Olfactory Fear Conditioning (acetophenone) F0 Males. F0 Females. Finegersh & Homanics [42] Mice C57Bl/6J (Sires) C57Bl/6J x Strain 129Sv/ImJ (Offspring) Vassoler et al [44] Rats Franklin et al [43] Gapp et al [36] Mice Mice C57Bl6/J C57Bl/6J SpragueDawley Long-Evans Hooded. MSUS Selfadministered cocaine infusion Stress by Restraint & Forced Swim. Vapour EtOH exposure. MSUS Yao et al [9] Rats F0. F1. F2. F0 Males. F0 dams & F1 litters. F1 Litters. F0 Room air exposure. Undisturbed dams and litters Left in Home Cage. Selfadministered Saline infusion Not Stressed. DNA Methylation. DNA Methylation. miRNA expression levels. Histone H3 Acetylation. miRNAs. Sperm. mPFC. VTA. Brain. Sperm. Serum. Sperm. Hippocampus. Hypothalamus. Cortex. mPFC Sperm Brain. Placenta. Uterus. Bdnf. Dlk1. 5HT1A CB1 CRFR2 MAOA MeCP2 N/A. Bdnf exon I, IV & VI. N/A. F0 F1 F2 F0 F1 F1 F2 F1 F2 RNAinj F0 F1 F0 F1 F2 Both. Paternal. Paternal. Paternal. Paternal. Maternal. Left in Home Cage or Olfactory Fear Conditioning with Propanol. DNA Methylation. Histone H3 Acetylation. Sperm. MOE. Epididymis. Olfr151 (M71). Olfr6. Generations Not Directly Exposed to Experimental Manipulation. F1. F2. F1. F2. F2. F1. F1-SN F2-SNN F2-SSN F3-SNNN F3-SSNN F3-SSSN Gender Used in Study for Epigenetic Assays Male. Both. Both. Male. Male. Female. Maternal Behaviour. Forced Swim. Cocaine Self OPS. Accelerating Odour Sensitivity Rotarod. Sucrose Administration. Elevated Plus Maze. Consumption. Maternal Assay. Elevated Plus Maze. Light-Dark Box. Behavioural Assay(s) Elevated Plus Maze. Open Field. Tail Suspension Test. Behaviour. Forced Swim. Auditory Fear Two-Bottle Choice. Free Exploratory Sucrose Self Paradigm. Administration. Conditioning Assay. Open Field. in successive generations (this includes measures of altered responses to aversive, stress inducing and anxiogenic stimuli), © Under License of Creative Commons Attribution 3.0 License Maternal PostPartum. Sensorimotor Behaviour. the second generation of which were not directly exposed to the environmental stressor (in this case MSUS). In addition to this, 9 Journal of Clinical Epigenetics ISSN 2472-1158 2015 2016 Vol. 1 No. 1:3 Table 5 Summary of Experimental Manipulation. Study Experimental Procedure Control Group Procedure Groups Exposed to Experimental Procedure Age Exposed to Experimental Procedure Unexposed Progeny of Experimental Group F1-C57-Ace(nfost) F0 C57Bl/6J Dams F1-C57-Ace(fost) F1-IVF Trained to associate acetophenone (10s) presentation with coterminating mild footLeft in home cage or conditioned with Dias & Ressler [41] shock (0.25-s 0.4mA). propanol. Average interval: 120s. Trained on 3 consecutive days: 5 odour presentation trials/day. F1-C57-Ace F0 C57Bl/6J Sires F2-C57-Ace 2 months old F1-M71-Ace F0 M71 Dams 2 months old F1-M71IVF F0 M71 Sires Finegersh & Homanics [42] ~250μl/min vapor EtOH inhalation in custom Placed in equivalent 0.5” plexiglass vapour chamber but exposed chambers (16”x16”x24”) to room air. from 8:00-16:00 5d/5wks at 78˚F. F0 C57Bl/6J sires 2 months old Dams not specified. Litters PND1-14 Franklin et al [43] MSUS 3hr/day Undisturbed (1 cage change/week). F0 dams and F1 litters. Gapp et al [36] MSUS: 3hr/day proximal separation Undisturbed (1 cage change/week). F0 C57Bl/6J dams & F1 litters Cocaine selfadministration in operant chamber through indwelling silastic catheter in right jugular vein Vassoler et al [44] (0.25mg dose over 5s, 20s intervals). Limited to 75 infusions/2hr/d for 60d. Mean infusions at start= 3-4mg/day. Mean infusions D60=7-8mg/day. Pregnant dams GD-12-18. Restraint in plexiglass 6cm inner diameter container for 20mins (prevented turning and maintains standing) in time period Yao et al [9] 8:00-9:00. Between 16:00-17:00 forced swim in tank (45x77x50cm, water 21˚C) for 5mins. 10 Yoked intravenous saline injections. F0-Sires Dams: 2-3months Litters: PND1-14 Not specified. F1-E-Sired F2-MSUS F3-MSUS F2-MSUS (C57Bl/6J) F1-RNAinj (B6ND2F1) F1-Coc-Sired F0-S F1-SS Non-Stressed, otherwise not specified. F2-SSS (and therefore their in utero pups) F2-SNN Not specified but on GD12-18 F3-SNNN F3-SSNN This article is available in: http://www.clinical-epigenetics.imedpub.com/ Journal of Clinical Epigenetics ISSN 2472-1158 2015 Vol. 1 No. 1:3 Table 6 Summary of Behavioural Results. Overall Outcome of Interest Experimental Manipulation Paradigm Used and Results Indicate Result EPM: Decreased anxiety/reduced behavioural control. Forced Swim: Increased depressive like symptoms. Behavioural Index of Maternal Care & Manifestations MSUS of F0 Dams Index of Absence of Care of Early Life and F1 Litters Stress Generations Outcome Observed In Relative to Respective Control Group Decreased Latency to Enter Open Arm F1-MSUS male# F2-MSUS male (Gapp Only) F2-MSUS females (Franklin Only) F3-MSUS females Increased Latency to Enter F3-MSUS males (Franklin Only) No Differences in Locomotive Activity F1-MSUS male# F2-MSUS male# F1-MSUS-RNAinj males (Gapp Only) Increased Time Floating F1-MSUS male# F2-MSUS female (Franklin Only) F2-MSUS male# F2-MSUS-RNAinj (Gapp Only) Decreased time floating after desipramine treatment F2-MSUS-Des vs. MSUS-Sal (Franklin Only) Increased time floating after desipramine treatment F2-MSUS-Sal vs. F2-C-Sal (Franklin Only) Decreased time actively nursing on PND1-7 F0-MSUS females Increased time spent off nest on PND1-7 F0-MSUS females Light-Dark Box: Altered response to F1-MSUS males Increased Time in Illuminated behavioural phenotype seen in two F2-MSUS males Compartment generations of males. F1-MSUS-RNAinj males Open Field: Inheritance of MSUS induced F1 male phenotype by F2 and F3 females. Test showed reduced behavioural control. (Franklin Only) Sucrose Consumption: Showed MSUS produces anhedonia in first generation only. (Franklin Only) TST (Franklin Only): Increased depressive like symptoms. © Under License of Creative Commons Attribution 3.0 License Decreased Latency to Enter Unfamiliar Arm F1-MSUS males F2-MSUS females F3-MSUS females No Difference in Distance Travelled F1-MSUS males F2-MSUS females on Trial 3 Increased Difference Travelled F2-MSUS females on Trials 1 and 2. Decreased Sucrose Consumption F1-MSUS male Decreased time immobile after acute and chronic saline F2-MSUS-Sal vs. F2-C-Sal treatment Increased time immobile after acute and chronic desipramine F2-MSUS-Des vs. F2-MSUS-Sal treatment 11 Journal of Clinical Epigenetics ISSN 2472-1158 Behavioural Manifestations Chronic Stress of Preterm Birth Inclined Plane Test: Delayed Sensorimotor Development Increased time spent in downward position (increases with every successive generation). Maternal Post-Partum Behaviour Decreased Tail Chasing and Rotational Behaviours Cocaine Self-Administration (FR1): Cocaine Resistant Phenotype Cocaine Self-Administration (PR1): Cocaine Resistant Phenotype Sucrose Self-Administration (FR1): Cocaine-resistant phenotype Cocaine-Self Administration in in cocaine self-administration paradigms not confounded by F0 males learning deficits. Sucrose Self-Administration (PR): Cocaine-resistant phenotype in cocaine self-administration paradigms not confounded by learning deficits. Maternal Behaviour Decreased Cocaine Infusions at 0.5mg 2015 2016 Vol. 1 No. 1:3 F1-SN F2-SNN F2-SSN F3-SNNN F3-SSNN F3-SSSN F1-SN F1-SS F2-SNN F2-SSN F2-SSS F1-C females F1-C males F1-C-DMSO Decreased Cocaine Infusions at 1.0mg F1-C males Reduction of breakpoint at 1.0mg/g F1-C males No Difference Between Sucrose Self-Administration F1-Coc-Sired males vs. F1-Sal-Sired males No Differences in Breakpoint of Sucrose Administration F1-Coc-Sired males vs. F1-Sal-Sired males No Differences in Licking/ Grooming F0-Dams of Cocaine Sired Offspring No Differences in Time off Nest F0-Dams of Cocaine Sired Offspring Drug Related Behaviours Accelerating Rotarod: Paternal alcohol exposure subtly increases motor enhancement after ethanol exposure. EPM: Paternal alcohol exposure increases anxiolytic and enhanced locomotor effects of alcohol. Open Field: Basal Activity not increased in mice found to have Ethanol Exposure ethanol sensitive phenotype on other assays. in F0 males. Two-Bottle Choice Assay (Ethanol): 12 Increased Time Spent on Rotarod on 5th Trial F1-E-Sired-EtOHinj Males vs. F1-E-Siredsalineinj Increased Time in Open Arm F1-E males F1-E-ETOH-inj males Increased Proportion of Open F1-E males Arm Entries F1-E-ETOH-inj males Increased Number of Total Arm Entries F1-E-ETOH-inj males No Differences in Distance Travelled F1-E-ETOH-inj males vs F1-E-Siredsalineinj Decreased Preference at 3% Concentrations Decreased Preference at 6% Concentrations F1-E males F1-E males Decreased Preference at 9% Concentrations F1-E males Decreased Consumption at 12% Concentrations F1-E males This article is available in: http://www.clinical-epigenetics.imedpub.com/ Journal of Clinical Epigenetics ISSN 2472-1158 Olfactory Sensitivity Olfactory Conditioning to Acetophenone in F0 males. 2015 Vol. 1 No. 1:3 Two-Bottle Choice Assay (Quinine and Saccharin): No Differences in Quinine or Saccharin Preference at 0.03 and 0.06mM Concentrations F1-E males vs F1-C males Auditory Fear Conditioning No Significant Difference in Acquisition, Consolidation and Extinction Retention of Aversive Memory Cue. F1-Ace-C57 vs. F1-Home-C57 EPM Equal Time in Open & Closed Arms and Equal Entries into Open Arms All Groups Increased OPS to acetophenone F1-Ace-C57 F2-Ace-C57 F1-Ace-M71 F1-Ace-C57-nfost F1-Ace-C57-fost Increased OPS to propanol F2-Prop-C57 F1-Prop-M71 OPS Olfactory Sensitivity Assay altered miRNAs levels were found in neural areas associated with stress response in two successive generations of mice. One of the altered miRNAs (miR-375) and its predicted target catenin β1 (Ctnnb) may be particularly implicated in the altered phenotype, as both are associated in the corticotropin-releasing factor signalling pathways. These pathways are involved in stress response. They also observed modified levels of both miR-375 and Ctnnb1 in the hippocampus – an area directly involved in stress response. A possible causal link between MSUS altered miRNAs and the expression of this modified phenotype can be supported by their findings that show an injection of RNAs from F0-MSUS sperm into naïve fertilised oocytes produces the same behavioural phenotype in the adult progeny as that observed in F0 males. The MSUS RNA-injected test group, also demonstrated similar metabolic phenotypes to the MSUS mice and upregulation of miR-375 in the hippocampus. It is possible that other mechanisms also mediate the transmission of this behavioural phenotype. There was no difference, compared to controls, of sncRNA levels found in F2-MSUS sperm and serum. Furthermore, normal sncRNA profiles were found in F3 progeny. As the miRNA profile did not differ from controls, the behaviour of the F3-MSUS offspring should not be altered. However, F3 progeny still exhibit the same behaviour found in the F1 and F2-MSUS generation. The authors suggest miRNAs alter other epigenetic aspects i.e. DNA methylation, and these are responsible for further transmission. Whilst posited, these alternative modes of inheritance have not been further explored. [44] provide some interesting evidence of an environmentally induced (cocaine exposure) epigenetic modification (increased © Under License of Creative Commons Attribution 3.0 License Increased Aversion Index to 0.003% and 0.006% F1-Ace-C57 acetophenone concentration. Increased Aversion Index to 0.003% and 0.006% propanol concentration. F1-Prop-C57 AcH3) that is expressed in the sperm and testes of F0 males and subsequently found in functionally relevant areas of the brain in offspring (mPFC). The AcH3 increase was found on gene promoters that correspond to the observed cocaine-resistant phenotype found both in the F0 and F1 male test subjects. Increased AcH3 was associated specifically to Bdnf promoters (I, VI and VI), which would increase BDNF expression (AcH3 is generally thought of as facilitative to transcription). Furthermore, the authors found that decreased BDNF activity at the TrkB receptor reversed the observed cocaine-resistant phenotype in cocaine-exposed offspring, implicating a causal role of BDNF. Whilst this evidence seems fairly convincing, the data still lacks a clear mechanism for which the histone modifications could be inherited as histones are mostly replaced by protoamines during spermatogenesis. Thus such results may not be as a common occurrence as it is thought. Only 1-10% of histones persist throughout spermatogenesis [24] It is unclear whether epigenetic modifications persist in both genders as only male profiles were tested – the inheritance of epigenotype and expression of phenotype may be much more complex than the data presented in this paper would first imply. However, the results are very much worth considering for further research as potentially this paradigm could easily be used whilst controlling for confounders with multifactorial analysis. Similarly, [42] also found a male-only behavioural phenotype. Ethanol exposure induced hypomethylation of Bdnf exon IXa in F0 sperm, which provides a possible mechanism of inheritance of the epigenetic modification and therefore possible transmission of phenotype in offspring. In addition to this, Bdnf exon IXa transcripts were found to be upregulated in functionally relevant 13 Journal of Clinical Epigenetics ISSN 2472-1158 2015 2016 Vol. 1 No. 1:3 Table 7 Summary of miRNA and ncRNA Results Experimental Manipulation Area assayed Generation Change Observed In miRNA miR-24-1-5p Frontal Cortices F0-MSUS male miR-138-3p miR-145-3p miR-375 miR-375-3p F1-MSUS male Hippocampus MSUS Hypothalamus Sperm miR-466c-5p miR-375-3p F2-MSUS male miR-375-5p F1-RNA-inj F1-MSUS male miR-466c-5p miR-375-5p miR-375-5p miR-200b-3p miR-375-5p F1-MSUS male miR-466c-5p miR-672-5p miR-375-3p miR-375-3p F2-MSUS male Serum miR-375-5p F1-MSUS male miR-466c-5p miR-672-5p miR-466c-5p miR-23b F2-MSUS male F1-SS miR-200c miR-96 miR-141 miR-182 Frontal Cortices miR-183 F2-SSS miR-200a Stress GD12-18 miR-200b miR-429 Placenta miR-451 miR-181a miR-181a miR-200b F2-SNN F2-SSS F1-SS miR-429 miR-200b Uterus F2-SSS miR-429 Please note that green refers to increased levels and blue refers to decreased levels of miRNAs. 14 This article is available in: http://www.clinical-epigenetics.imedpub.com/ 2015 Journal of Clinical Epigenetics ISSN 2472-1158 Vol. 1 No. 1:3 Table 8 Summary of DNA Methylation Results Experimental Manipulation Bdnf Exon IXa Direction of Change Compared to Controls EtOH Exposure Decrease CB1 MSUS Increased CRFR2 MSUS Decreased IG DMR of Dlk1 MSUS Decreased MSUS Increased MeCP2 Location of Analysis Sperm VTA Sperm Brain Sperm Brain Sperm Sperm Brain Olfr151 Conditioning to Acetophenone Decreased Sperm No Difference MOE Generation and Gender F0 male F1 male F1 female F1 male F2 female F1 male F2 male F2 female F0 male F1 males F2 males F2 females F0 male F1 male F1 male F2 male Table 9 Summary of Histone PTM Results Experimental Manipulation Direction of Changed of Histone PTM Area Assayed Gene Promoter Assayed Olfactory Fear Conditioning Decrease Epidydymis N/A mPFC Cocaine SelfAdministration Increase Sperm Testes Generation Studied In Histone H3 Acetylation F0 male Histone H3 Methylation F0 male Bdnf Promoter Histone H3 Acetylation IV Bdnf Promoters I, IV Histone H3 Acetylation & VI N/A. Histone H3 Acetylation areas of the brain (the VTA). As Bdnf is thought to be a regulator of drinking behaviour, this supports the implications that ethanol induced hypomethylation may contribute to the behavioural phenotype observed [33]. Interestingly, hypomethylation of the Bdnf IXa promoter is found in both female and male offspring of ethanol exposed mice. This result can be interpreted in two ways. Firstly, hormonal or other biological differences between males and females may interact with epigenetic modifications to produce either no change (in the case of females) or the change observed in male offspring. Secondly, the hypomethylation observed may have little or no influence on the behavioural phenotype observed. Unfortunately, the evidence linking hypomethylation to the behaviour is still only speculative and thus interpreting this complex sex-dependent transmission of behaviour is particularly difficult. [41] also begin to provide convincing evidence of a possibly causative role of environmentally induced epigenetic modifications in the expression of an altered behavioural phenotype. An odour-sensitive behavioural phenotype specific © Under License of Creative Commons Attribution 3.0 License Histone PTM Type F1 male F0 male F0 males to the F0 conditioned odour was observed in two subsequent generations, through both the maternal and paternal line and also in offspring that were cross-fostered by non-exposed mothers. These results taken together suggest biological transmission of behaviour rather than behavioural transmission. In addition to this, in these generations and an IVF-derived generation (sperm from acetophenone conditioned males used to fertilise oocyte of naïve females) neuroanatomical changes corresponded accordingly to this odour sensitive phenotype – which would be the biological basis possibly responsible for the observed behavioural phenotype. Bisulfite sequencing also indicated hypomethylation in sperm at Olfr151. As hypomethylation was located on sperm, this indicates a mechanism that changes could be inherited from F0 sires to F1 offspring and then to F2 offspring. Hypomethylation of Olfr151 theoretically corresponds to increased Olfr151 expression that was observed in the brain. However, there was no difference in methylation status of Olfr151 in the MOE of F1 and F2 animals. Like [36] [41] attribute the lack of expected epigenetic modification in the main olfactory 15 Journal of Clinical Epigenetics ISSN 2472-1158 2015 2016 Vol. 1 No. 1:3 Table 10 Summary of All Results Providing Evidence or Negating that Environmentally Induced Epigenetic Modifications can be inherited and Contribute to the Expression of a Behavioural Phenotype in Offspring Authors Experimental Manipulation Behavioural Phenotype Evidence Supporting Epigenetic Evidence Confounding/Negating Contribution to Epigenetic Contribution to Expression of Expression of Behaviour Behaviour OIfr151 (M71) responds to acetophenone. Associated Epigenetic Mechanisms No methylation of Olfr6 which is unresponsive to acetophenone. Dias & Ressler [41] F0 odour fear conditioning to acetophenone. Olfactory sensitivity to F0 conditioned odour. Decreased DNA Hypomethylation of methylation of sperm inherited from F0 to F1. Olfr151 Transmission of behavioural phenotype through the paternal line and in F2 and crossfostered generation suggest biological transmission. Bdnf regulates drinking behaviour. Decreased ethanol Decreased DNA Finegersh & preference/consumption F0 sire exposure to methylation of Homanics and increased sensitivity to ethanol. Bdnf exon IXa [42] ethanol induced anxiolysis promoter. and locomotor enhancement. Bdnf mRNA levels in VTA and on sperm increased: corresponds to hypomethylation. Mechanism for inducing methylation change unknown. Lack of hypomethylation in MOE. Lack of alterations to histone PTMs in sperm. Mechanism for inheriting epigenetic change is unknown. Females have hypomethylation but no changes to phenotype. Reduced alcohol consumption seen. Franklin et al MSUS with F0 [43] dams and F1 litters Gapp et al [36] MSUS with F0 dams and F1 litters Non-specific altered behavioural phenotype on various tests. DNA methylation Non-specific altered behavioural phenotype on various tests. sncRNAs Vassoler et al F0 sire cocaine-self Cocaine-resistant phenotype [44] administration 16 AcH3 Mechanism for inheriting epigenetic Altered methylation change is unknown. at CB1, CRFR2 and MeCP2 in F1 sperm Normal methylation profiles in sperm and female brain. of F2 males and all areas tested in F3 CRFR2 and MeCP2 in males but still expressed anxiety related F2 sperm. phenotype. Injection of MSUSNormal miRNA profiles in sperm of F2 sperm RNAs into males and all areas tested in F3 males oocytes produces but still expression of behavioural behavioural phenotype. phenotype. Increased association of AcH3 with Bdnf exon IV in both mPFC of offspring and sperm of sires. Bdnf exon I and VI also more acetylated in sperm of sires. This article is available in: http://www.clinical-epigenetics.imedpub.com/ Journal of Clinical Epigenetics ISSN 2472-1158 Decreased postpartum Successive maternal behaviours and generations increased sensorimotor exposed to Yao et al [9] deficits. Both of which were restraint and increasingly different from forced swim stress controls with each successive on GD 12-18 generation. miRNAs Altered behavioural phenotype and altered miRNA profiles. 2015 Vol. 1 No. 1:3 Confounding due to maternal behaviour and in utero effects. Only one group removed from in utero effects had miRNA profiles measured and this was only in one area. epithelia (MOE) to alternative modes of epigenetic inheritance. This was a reasonable assumption as other types of epigenetic inheritance have been found to alter olfactory receptor loci in the MOE [34]. Based on research suggesting a relationship between DNA methylation and histone modifications [6] the authors investigated histone H3 acetylation and methylation but ChIP revealed no differences on either measure in sperm chromatin – which the authors suggest could be due to incorrect immunoprecipitation. As a result, a mechanism is needed that explains how hypomethylation produces the neuroanatomical and subsequent behavioural changes before conclusions can be drawn on this data. such epigenetic contributions to behaviour are very complex and there are many variations in possible phenotype that they could potentially produce. In order to better understand the role of altered DNA methylation of these candidate genes, an assay of the brain of F1 and F2-MSUS males and F3 offspring would have been prudent to see if this could clarify the complex sexdependent results. The authors suggest alternative epigenetic modifications such as ncRNAs could be responsible but no additional assays were conducted. Currently, the most conclusive thing that can be said of such data is there is a general association between the MSUS induced behavioural phenotype and altered DNA methylation profiles. Despite a lack of hypomethylation of the Olfr151 locus in the MOE and no evidence of histone modification mediated inheritance, [41]still imply the observed neuroanatomical and behavioural changes may be associated with epigenetic mechanisms. They suggest that ncRNA inheritance may be mediating the transmission of odour-sensitivity, citing [45] who found ncRNA inheritance was observed in the Kit locus. Whilst the evidence found in their paper is quite convincing, there are several components missing in order to conclusively say that an environmentally induced epigenetic modification has produced a behavioural phenotype in offspring. Firstly, a mechanism for odorants to produce an epigenetic change is required. [41] suggest blood-borne odorants may activate receptors in sperm due to the fear conditioning protocol [46] but there is no conclusive evidence yet. The results from [9] do not contribute much to the argument. Unfortunately, it is hard to distinguish any evidence of causality as much of their data is subject to confounding. Only the F2-SNN (stress, non-stress, non-stress), F3-SNNN (stress, non-stress, non-stress, non-stress), and F3-SSNN (stress, stress, non-stress, non-stress) generations would have not been directly affected by the experimental manipulation (to avoid in utero confounding F1-SN, F1-SS, F2-SSN, F2-SSS and F3-SSSN were excluded from consideration in this systematic review). Of these experimental groups, miRNA data was only taken for F2-SNN and this was only in the placenta. Thus, it is still unclear whether miRNAs could contribute to the increasingly distinctive altered phenotype observed in successive generations of stressed mice. Certainly at least, this study contributes results that are useful for future research to build upon but the design of the study does not allow for conclusions pertaining to this systematic review’s research question to be drawn. [43] perhaps provide the most difficult results to interpret. Their behavioural assays can only be taken as suggestive of the inheritance of a “non-specific behavioural phenotype”. Not all results are provided and this makes it hard to comment on the specific inheritance of a phenotype. For example, F1 female behavioural data is missing but sometimes referred to and statistical analysis of performance on a tail suspension test (TST) by F1 males is also absent. From the data available, it appears alterations of behaviour (in all tests) are generally observed in F1 MSUS males and their F2 female but not male progeny. The F3 MSUS generation males but not females showed inheritance of a depressive-like phenotype seen in their F1-MSUS grandsires which their F2-MSUS sires do not exhibit. In contrast F3-MSUS females but not males tend to show similar performance on locomotor assays to their F1-MSUS grandsires. Similarly, epigenetic assays show various mixed results. MeCP2 hypermethylation and CRFR2 hypomethylation is seen in F1 and F2-MSUS sperm and in F2MSUS female brains. CB1 hypermethylation is observed in F1MSUS sperm and F2-MSUS female brains but not in F2-MSUS sperm. These candidate genes are quite promising examples of areas that might have a role in inducing the observed behavioural phenotype as they are commonly associated with stress response, depressive and affective disorders [47,48,49,50,51]. However, © Under License of Creative Commons Attribution 3.0 License Conclusion This study aimed to examine the role of trans-generational epigenetic inheritance in the expression of psychiatric, psychological and behavioural phenotypes in progeny. A key take home message is that currently there is a lot of hype and overlyzealous attribution of the role of epigenetics in contributing to various phenomena [52-84]. This is clearly evidenced by the fact that of the 100 retrieved papers in this systematic review, only six original articles were identified, with 68 being review articles and/or containing no original data. However, taking all the results from the identified papers together, this systematic review has found strong associations between the inheritance of environmentally induced epigenetic modifications and the presence of altered behavioural phenotypes in offspring of experimentally manipulated subjects. Many of the neuroanatomical, physiological and behavioural outcomes also provide support for an epigenetic causality or contribution. Unfortunately, confounding is a major problem in many papers and thus definitive conclusions should not be drawn. Moreover, 17 Journal of Clinical Epigenetics ISSN 2472-1158 experimental research currently lacks clear mechanisms illuminating how environmental stressors alter the epigenotype and how this altered epigenotype can be inherited. Nevertheless, the results of this systematic review begin to provide a basis on which future research can build. Acknowledgements The authors should like to thank Paul Buckland and Holly Thomas for help with analyses, and Jan Chatting for support throughout this study. 18 2015 2016 Vol. 1 No. 1:3 Funding This work was funded by the Medical Research Council (MRC) Centre for Neuropsychiatric Genetics and Genomics (G0801418) Competing and Conflicting Interests The authors declare they have no competing or conflicting interests. This article is available in: http://www.clinical-epigenetics.imedpub.com/ Journal of Clinical Epigenetics ISSN 2472-1158 References 1 Wu, C. & Morris, J.R. (2001). Genes, genetics, and epigenetics: a correspondence. Science 239: 1103-1105. 2 Phillips, T. (2008). The Role of Methylation in Gene Expression. Nature Education 1-116. 3 Luger, K Mader, A.W, Richmond, RK, Sargent, DF. & Richmond, TJ (1997) Crystal structure of nucleosome core particle 2.8 A resolution. Nature 389: 251-260. 4 Li, Q. & Zhang, Z. (2012). 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