Literature review Introduction Epigenetics is a concept identified by scientist C

Literature review
Epigenetics is a concept identified by scientist C. H. Waddington around 1942 (Ungerer, Knezovich. and Ramsay, 2013). His experiment included the application of heat to Drosophila pupae which resulted in the change in their wing-vein pattern and this change in the phenotype was observed even after the removal of the heat stimulus (Tang and Ho, 2007). This led Waddington to deduce that exposure to an environmental element during a crucial phase of development presented an alteration in the phenotype which persisted throughout the organisms’ life. He first described this phenomenon as “genetic assimilation” which was later coined as epigenetics (Tang and Ho, 2007).

Prior to the 1940s, the concept of a gene as the carrier of heritable information was hypothesised without any physical identity (Crews and McLachlan, 2006). The term epigenetics was then popularised from classical embryology and dubbed ‘the multiple means that gene communication and expression give rise to the observes phenotype’. However, this definition has been revised and made more specific and its mechanisms outlined (Crews and McLachlan, 2006). Today, epigenetics describes the alteration in the way manner in which the gene is expressed that are present without alterations in the DNA code itself. Epigenetics is a major participant in developmental occurrences including genomic imprinting, the specification of different tissue for gene expression and in maintaining stem cells (Ungerer, Knezovich. and Ramsay, 2013).
Epigenetic Mechanisms
There are two major mechanisms employed by epigenetics that enable tissue-specific gene expression which are structural chromatin modifications, which includes DNA methylation and histone modification, and RNA interactions, which is the participation of non-coding RNAs (Ungerer, Knezovich. and Ramsay, 2013).
Chromatin comprises of monomers called nucleosome which are made up of DNA coiled around histone proteins. The DNA and the protein moiety of the chromatin have the ability to be modified by various mechanisms which can alter their conformation and the ease of their availability (Ungerer, Knezovich. and Ramsay, 2013). The alteration is predominantly achieved through DNA methylation. This includes the covalent incorporation of a methyl (CH3) group to the nucleotide, specifically the 5′ end of the cytosine, by the enzymes DNA methyltransferases (DNMTs) (Ungerer, Knezovich. and Ramsay, 2013). The methyl group utilised is obtained from a cascade of reactions called folate pathways. These enzymes play a vital role in the development of a foetus. According to Li, Bestor and Jaenisch (1992) the mutation in DNMTs have been to be associated with stunned foetal development and death before midgestation. Furthermore, these enzymes have a role in parental genomic reprogramming post fertilization.
Histone modification is another vital epigenetic mechanism. The histones that hold together the nucleosome in chromatin remodelling have protuberances from which modification occurs (Ungerer, Knezovich. and Ramsay, 2013). There are several mechanisms that are employed in the modification of the tails of histone proteins including acetylation, phosphorylation, ubiquitinylation and methylation, and all these processes are reversible. These give rise to loosely packed and available euchromatin and tightly packed heterochromatin and this change in conformation affects the availability of DNA and thus leading to the silencing of some genes (Ungerer, Knezovich. and Ramsay, 2013).
Genomic Imprinting
Genomic imprinting is an epigenetic phenomenon wherein genes are passed down from parent to offspring in a parent of origin manner (Ferguson-Smith, 2011). This term has also been used to describe an instance where the gene have differential expression according which parent it came from, more especially according to the sex of the parent (Moore and Haig,1991). The manner in which genomic imprinting is achieved is through the use of epigenetic mechanism of DNA methylation (Uyar and Seli, 2014). Under Mendelian genetics, it was believed that genes from both parents were equally expressed and this point of view was challenged around the 1980s through the use of uniparental disomy (UPD) studies. The results of these studies illustrated the effect of the parent is only influential in some parts of the genome and all portions. Therefore, it was concluded that both the maternal and the paternal haplo- genomes were necessary for the normal development of a foetus.
Under imprinting, genes are tagged as either of maternal or paternal origin depending on which parent from which they come (Uyar and Seli, 2014). Within an embryo, there are some genes which promote growth (paternally expressed genes) and there are those that operate antagonistically and thus restrict growth (maternally expressed genes). In mammals, offspring obtain their food directly from their mothers, therefore imprinting occurs in order to decrease the demand (Moore and Haig,1991). The dysregulation of these genes leads to the development of some conditions.
During the early stages of development, genomic imprinting is achieved by demethylation and re-methylation. Following epigenetic methylation in order to achieve cellular differentiation from totipotent stem cells, there is removal of parental patterns of methylation and new ones are established according to the parent from which they originate (Uyar and Seli, 2014).

There are several congenital disorders associated with irregularities around gene imprinting. These include Angelman syndrome, Prader-Willi syndrome (PWS) and Beckwith-Wiedemann syndrome (BWS). Angelman syndrome results from imprinting the maternally expressed UBE3A gene that is within the SNRPN collection of imprinted genes. This disorder is classed as a neurological disorder resulting in mental retardation, learning disabilities, small distortions in facial features, and an excitable and happy demeanour. Similarly, PWS also occurs as a result of an imprinting error occurring in the SNRPN imprinting cluster. It is associated with an abnormal desire for food, compulsive behaviour and obstinance. On the contrary, BWS affects the H19 imprinting cluster, specifically in the KCNQ1QT1 gene, which presents with overgrowth (Uyar and Seli, 2014).

Epigenetic Reprogramming
The term epigenetic reprogramming refers to a phenomenon that involves the deletion and re-instalment of new chromatin modifications during the developmental process of mammals (Ungerer, Knezovich. and Ramsay, 2013). During this process, the epigenetic markers from the germ cells (sperm cell and egg cell) are erased which enables the newly fertilized egg cell to have access to all genetic information and hence be able to have tissue-specific differentiation. Epigenetic modifications are incorporated such that they operate as a reversible switch that is able to monitor gene expression through either activation or repression (Ungerer, Knezovich. and Ramsay, 2013). Furthermore, these modifications allow to zygote to be able to develop the various cell lineages that will allow it to develop into an embryo.
The process of epigenetic reprogramming is found to happen in two distinct periods of inter-uterine development. The first is immediately after fertilization of the egg cell and the second occurrence of reprogramming happens during the process of gametogenesis of the foetus (Ungerer, Knezovich. and Ramsay, 2013). The first reprograming takes place in the preimplantation blastocyst. In the interior of the blastocyst, there is re-configuration the embryonic epigenetic sequences in a manner that is specific to the various cellular lineages. Mature oocytes and sperm cells are highly compacted and methylated (Reik, Dean and Walter, 2001). Therefore, during the second phase of reprogramming that occurs in the games, there is a large-scale demethylation which removes the inherited methylation sequences from the parents succeeded by the re-instalment of de novo epigenetic markers in a parent of origin pattern (Ungerer, Knezovich. and Ramsay, 2013).

The inter-uterine environment
The environment and genetic work together in the development of a foetus. According to Gluckman et al., (2008), at various stages of development the foetus becomes sensitive to stimulus from the environment which have an effect on the succeeding phases of development. These have consequences that progress and affect postnatal development. Events that lead to developmental disruption from environmental stimuli are irreversible (Gluckman, Hanson, Cooper and Thornburg, 2008).
Developmental Plasticity
According to Gluckman et al., (2008), developmental plasticity observes the manner in which an organism develops. Thus, developmental plasticity can be defined as the ease and ability of an organisms to develop in multiple different ways depending on its set environment. This requires steady regulation of gene expression with the use of various epigenetic mechanisms including DNA methylation and the modification of histone proteins. This proves that an organisms’ development and consequently its susceptibility to diseases later in life is in part dependant on the synergy of the genome and epigenome. The use of developmental plasticity as a contributing factor to diseases risk is deduced from conducting multiple studies wherein the control of food intake and endocrine or physical obstructions at various phases of foetal development had an impact of later metabolic and cardiac function in the individual (Gluckman, Hanson, Cooper and Thornburg, 2008).

The developmental-origin hypothesis
The developmental-origin hypothesis stipulates that susceptibility to development of disease is initially prompted by adaptive response that the foetus creates from signal from the mother according to her state of health and physical state (Gluckman, Hanson, Cooper and Thornburg, 2008). That is to say, the level of physical activity of the mother during the pregnancy has direct implications to the health of the offspring (Wojtyla, Kapka-Skrzypczak, Paprzycki, Skrzypczak and Bilinski, 2012). This concept was adopted approximately over 20 years when it wa observed that how a foetus adapts to the inter-uterine conditions during its developmental stages moulds the structure and functionality of its organs in the long run (Swanson, Entringer, Buss and Wadhwa, 2009).
Folate and vitamin B12 deficiency
For years, the role of folate has only been noted for its importance in preventing the development of macrocytosis during pregnancy. It is now appreciated for its versatile role in the maintenance of a healthy pregnancy which include the prevention of the development of neural tube defects (NTD) and other birth defects (Molloy, Kirke, Brody, Scott and Mills, 2008). Conversely, the role of vitamin B12 during pregnancy has not been extensively investigated as that of folate yet they have a similar metabolic pathway.

During pregnancy, there is a gradual decrease in the level of folate in the plasma (50% decrease in folate concentration) (Molloy et al., 2008). This is in part as a result of normal physiological response to pregnancy in relation to hemodilution, changes in the rate of function of the kidneys and alterations in the hormone secretions. The increase in the demand for folate by the placenta and the foetus also contribute to this decline in the level of plasma folate. The deficiency of folate during pregnancy has been related to various conditions including preeclampsia, miscarriages and spontaneous detachment of the placenta and premature delivery. The
The uterus is equipped with several barriers that protect the growing and developing foetus, the most prominent being the placenta (Waring, Harris and Mitchell, 2016). However, the placenta can be infiltrated by toxins or harmful chemicals when the mother is subjected to unsafe environments. Some of these have immediate detrimental effects on the developing foetus but some, which are sometimes referred to as developmental disrupters, may have long lasting ill effects that continues throughout the individual’s life. These substances can be categorised into metals, solvents, endocrine disruptors and an array of other industrial chemicals. These categories are according to their mechanism of operation and how they disrupt cellular function (Waring, Harris and Mitchell, 2016).

Exposure to cigarette smoke during pregnancy exposes the developing foetus to polycyclic aromatic hydrocarbons (PAH) which is suspected to be an endocrine disrupter (Waring, Harris and Mitchell, 2016). Cigarette smoke comprises of more than 4000 chemicals, the majority of which are considered to be carcinogenic. According to Waring et al., (2016), in a study conducted on mice, exposure to cigarette smoke in utero from the first day of pregnancy thill day 21 showed that there is low birth weight in the offspring with further changes in the metabolism of carbohydrates, lipids and proteins. It was also concluded that transplacental cigarette smoke conjured up the development of lung adenomas found at about 8 months postnatally. It was found that in utero exposure to polycyclic aromatic hydrocarbons was also linked to the development of obesity in 6-11 years. Furthermore, prenatal exposure to cigarette smoke is shown to increase the risk of cryptochidism in males and early development of puberty in both sexes (Waring, Harris and Mitchell, 2016).

High concentration of alcohol exposure during pregnancy also leads to developmental problems in the foetus, the most concerning being the development of the congenital disorder foetal alcohol syndrome (FAS). Offspring with the condition incur developmental delays, craniofacial dysmorphology and central nervous system disruptions (Waring, Harris and Mitchell, 2016). As previously stated, DNA methylation depends on folate pathways for the supply of methyl groups. An increased exposure of alcohol disrupts the metabolism of folate and conversely decreases its availability (Ungerer, Knezovich. and Ramsay, 2013). Folate is involved in a cascade of reaction known as the transmethylation reaction wherein it acts as a coenzyme which donates methyl groups to form S-adenosylmethionine (SAMe) which continues the circulation of the methyl group. According to Ungerer et al., (2013), a study of the effects of alcohol conducted on rats revealed there was a reduction in the enzyme methionine transferase, S-adenosylmethionine and methionine. Also, it was proved that ethanol facilitates the loss of methyl groups which subsequently disturbs transmethylation reactions. Not only does foetal alcohol exposure cause aberrant DNA methylation, it also hinders the development of a crucial part of the brain called the denate gyrus which is linked to the development of mental retardation (Ungerer, Knezovich. and Ramsay, 2013).
During the duration of gestation, it is crucial that endocrine levels be kept at homeostasis. Compounds which disrupt the normal functioning of the endocrine system are referred to as endocrine disrupters. These compounds can mimic the activity of normal hormones, much like phenol which can simulate the hormone oestrogen (Waring, Harris and Mitchell, 2016). Prenatal exposure to both naturally occurring and synthetic hormone has carcinogenic effects and also the risk of increasing sensitivity to other carcinogens. Bisphenol A (BPA) is a known endocrine disrupter with detrimental effects. It can adhere to oestrogen-responsive-elements and result in histone methylation and acetylation which in turn leads to chromatin remodelling, resulting in the activation of some genes while others are deactivated (Waring, Harris and Mitchell, 2016). This has shown to result in cervical cancer in female offspring born of exposed mothers. According to Waring et al., (2016), another endocrine disrupter is polychlorobiphenyls (PCBs) which has been related to the development of infections in infancy which reflect thyroid-mediated dysfunction and reports of behavioural problems in school-age children.
Effects of exposure to prenatal famine
It has been assumed that there is a link between development of disease, including those that progress into adulthood, and poor in utero environmental conditions during the early stages of foetal development (Heijmans, Tobi, Stein, Putter, Blauw, Susser, Slagboom and Lumey, 2008). Several epidemiological studies hypothesise that this is as a result of the poorly regulated epigenetic mechanisms. On the DNA sequence, there is epigenetic information that is passed on from parent to offspring and it is in charge of which gene is likely to be transcribed. Methylation of genes together with the modification of histone that packages DNA are the main controls of chromatin remodelling and consequently DNA accessibility (Heijmans et al., 2008).
According to Heijmans et al., (2008), animal studies illustrate that exposure to certain environmental elements, even for a short period of time, can have long term effects on the epigenetic marks and consequently the phenotype. The main focus being on embryonic development as this is the phase in life wherein most of the epigenetic marks are established. An early study on mice confirmed that indeed the amount of nutrients that a developing embryo received had a direct influence on the epigenetic marks it maintained. This hypothesis was put to the test in a study conducted on humans who were conceived during the Dutch Hunger Winter (Heijmans et al., 2008). The Dutch Hunger Winter occurred during the winter of 1944-1945 whereby the Germans banned the transport of food in the certain parts of the Netherlands nearing the end of World War II. Infants conceived during this period were prenatally exposed to famine and tracing them was unexacting since the health care sector was unaffected by the war. Furthermore, the exact periods of lack of food and the amounts that people received were well documented. These clear and unique aspects enabled for the adequate assessment of the effect of prenatal exposure to famine on epigenetic variations found in humans.
The target gene for this particular investigation was insulin-like growth factor II (IGF2) because of its role in growth and development. Insulin-like growth factor II is an epigenetically maintained as it is a paternally expressed gene (maternally imprinted gene). As a result, the hypomethylation of this gene leads to its expression on both alleles (Heijmans et al., 2008). The methylation of the IGF2 is largely affected by environmental conditions during the early stages of development and stabilises later in life. Therefore, the changes made to the expression of the gene during embryionic development can be observed years later.
In the the Dutch Hunger Winter study, an association between the exposure of a foetus to famine and the long-term alterations in the differential expression of the IGF2 was made. The main objectives of the study were to formulate the association but also to evaluate the timing as people who were subjected to the famine in the later stages of in-utero development were included (Heijmans et al., 2008). Sixty individuals who were exposed to the famine during the early stages of their foetal development were selected in the study and they were compared to their siblings but strictly those of the same sex. An additional sixty-two individuals exposed to famine later in their gestation were included in the study in order to evaluate the effect of timing. Quantitative mass spectrometry was the used to measure the five CpG dinucleotide methylations in the IGF2 differently methylated regions.
The results of the study found that in individuals who were exposed to famine in the early stages of their gestation had 5.2% less methylation than their siblings and this occurred in 72% of the study sample (Heijmans et al., 2008). In the 62 individuals who were exposed to famine late during in-utero development it was found that there were no significant differences between them and their siblings born after the famine. This proved that the timing at which a foetus is exposed to maternal hunger is important. However, the individuals exposed to famine late in their gestation incurred a lower birth weight as compared to their siblings. Individuals exposed to famine earlier in their gestation experienced a relatively normal birth weight highlighting that birth weight is not linked to the degree of methylation of the IGF2 gene (Heijmans et al., 2008).

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