Directly after the creation of a zygocyte, the cell begins to divide and develop

independently of its parents. Containing a genetic “blue print” and all the relative information to develop into a normal healthy human being in the refection “mom and dad” and

Once the child began to develop, it is predetermined to look a specific way, have very unique traits, hair color eye color, etc.On the bad side, be predisposed to developing a wide range of genetic diseases and disorders, also contained in the DNA code.

General ideas and radical concepts in epigenetics and DNA methylation

Research Project, K. Randazzo

Jonathan Gutierrez12/8/2009

Introduction

In order to understand the ideas behind epigentics, it is important to understand general concepts behind basic genetics, and the inherited information received from a male and female organism during reproduction. The standard explanation of genetics explains that DNA provides the instruction to make RNA, and that RNA creates proteins that control all cellular activity through protein synthesis and regulation.In the past, the general belief in biology, was that you received a very specific genetic code(DNA) from each parent, your mother passing an X-chromosome along with her genetic information. The father passing a Y-chromosome or an X-chromosome, which determines the gender of the offspring as well as passing his own unique and specific genetic traits. After sexual reproduction and conception, the newly formed organism is created through the union of two haploid cells and combination of gametes from each contributing parent. From the point of its conception, the zygote begins to grow, develops, and divides. Eventually developing into a new generation of a particular species. Possessing similar traits of its elder generation, and many generations past.

Recent advancements and research in bio-technologies, are starting to suggest that the information that you received from your parents is only half the story. The relatively new scientific field of epigentics is suggesting that we have the power to manipulate the very genetic code passed to us by our parents that, once believe, was the ultimate determining factor in our development In a sense, our inherited destiny of hereditary disease, bad teeth, being obese or having too many freckles.Many researcher and geneticists around the world have begun research studies in the realm of epigentics for a wide range of reasons. Many using monozygotic twins (identical twins) who’s DNA is genetically identical to the other, to study the changes that occur in their genetic code and gene expression as they age. Epigenetics is a relatively new field of genetics, that studies chemical alterations on chromosomes, which result in changes in gene expression by condensing the chromosome or affecting the binding of

transcription factors. Methylation of CpG islands is one such alteration. Twin studies have opened the doors to understanding how these changes occur: They’re inherited.

Epigenetics affects many areas of biology. In animals, one of the most important changes happens during embryonic development. Genes use epigenetics to guide proper development of stem cells into different cells of the body.[11]

In some cases, different DNA methylation effects from the mother and father compete to determine which parent contributes the trait. For example, when a donkey and horse mate, the resulting mule is different depending on which species was mother or father. This also explains why individuals with the same genome, such as identical twins, exhibit different characteristics, depending on whose epigenetic effects - mom's or dad's- won out in each baby[3]

If DNA really is the predetermined map of our development and eventually create an adult from an embryo from its specific “blue print” as received from each parent. Epigentics answers and creates more questions. As well as challenges the everyday view that we are just the combined image of our parents and DNA does not have the final word.

Gene expression and general epigenetic theory

Epigentics or Epigenome translates into “above the Genome” and can be best described as anything affecting or manipulating the genome and gene

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expression that is not already encode in DNA itself.

In the past gene expression patterns and translation of genetics information was thought to be contained with in the DNA, a set of instructions telling embryonic cells or stem cell to distinguish itself by developing into a hepatocyte, skin cell or one of thousands of different cells contained in the body. Since 10-20% of genes are “active” in anyone cell, This prevents genes of one cell type from being expressed in another, for example, the gene for eye color is expressed in the eye, and not in the liver or skin cells[3] but recent research is beginning

to develop a much more interesting picture

Gene expression. The process by which information from a gene is used in the synthesis of a functional gene product. These products are often proteins, but in non-protein coding genes such as rRNA genes or tRNA genes, the product is a functional RNA. The process of gene expression is used by all known life - eukaryotes (including multicellular organisms), prokaryotes (bacteria and archaea) and viruses - to generate the macromolecular machinery for life. Several steps in the gene expression process may be modulated, including the transcription, RNA splicing, translation, and post-translational modification of a protein.[2] Gene regulation gives the cell control over structure and function, and is the basis for cellular differentiation, morphogenesis and the versatility and adaptability of any organism. Gene regulation may also serve as a substrate for evolutionary change, since control of the timing, location, and amount of gene expression can have a profound effect on the functions (actions) of the gene]

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in a cell or in a multicellular organism.[2][3

Control of gene expression can be handled in different ways. Sometimes, small molecules bind to DNA, changing its ability to give instructions. These molecules originate as proteins, protein complexes or small bits of RNA. For example, in times of drought, the body produces molecules to modify DNA and turn on or off genes that help it endure difficult circumstances.[16]

Giving the organism a very specific trait, blue or brown eyes, brown hair or a muscular build.

The Epigentic phenomenon is best represented in the process of cellular differation in normal development of a human child.

Long before the child’s birth, during the first few weeks of child development. Stem cell are going through a process of differentiation being distinguished into unique cells, some will become liver cells, other cardiac cells, among countless others. Genetically, all these cells are the same. Each containing genes that the other has. Skin cells have the necessary gene and genetic information to become lung cell, or cardiac cell, or blood cell. With all the genes being genetically identical and contains all of the human genome, how does a cell know how to change into its desired cell? How does it know what to become? Since you can not tell the genetic difference from one cell to the next, yet have many highly specialized and complex cells with the body, the process in which each cell become unique and have very different gene expression, has been termed -Epigentics.

Through Epigenetic mechanisms, genes that the cell or tissue does not need are specifically turned off or “Silenced” and while gene expression that is necessary to the functionality and specialization of the cell are protected from silencing, by the

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same epigenetic mechanisms. The Entire human genome is passed on to daughter cells, along with epigenetic information that keeps the same properties of the parent cell. re: a liver cell divides into another liver cell and not a lymphatic cell. Turns out that there are two different modifications that can affect DNA. One is a Biochemical modification that attaches directly to the DNA, silencing a specific area of genetic information, The other control the physical shape of the DNA strand itself by effecting the protein in which DNA is wrapped, again, effectually expressing or silencing the specific trait or gene. In sense a second genome.... The Epigenome,

Gene Expression through methylation and histone markers in mice

DNA methyltransferase is an enzyme in cells that recognizes CpG islands, strings of cytosines and guanines, and attaches a methyl group to cytosine. This DNA methylation is absent during embryonic development due to the high level of replication and gene expression. The alteration is thought to condense the chromatin around histone, closing off access to the DNA for transcription factors as gene expression wanes and is shut off. In fact, altered methylation patterns are thought to play a role in the development of some cancers, which result from aberrant cellular programming.[22,23][1]

Methylation denotes the addition of a methyl group to a substrate or the substitution of an atom or group by a methyl group. Methylation is a form of alkylation

with specifically a methyl group, rather than a larger carbon chain, replacing a hydrogen atom. These terms are commonly used in chemistry, and biological sciences.In biological systems, methylation is catalyzed by enzymes; such methylation can be involved in modification of heavy metals, regulation of gene expression, regulation of protein function, and RNA metabolism. Methylation of heavy metals can also occur outside of biological systems. Chemical methylation of tissue samples is also one method for reducing certain histological staining artifacts.[22]

These Methyl groups are very common and can be found in foods, house hold chemicals and environmental pollutants. These methyl groups if acquired and introduced to the DNA structure can interfere and affect the way that DNA translates into RNA, and in turn proteins and cellular function. The process is referred to as DNA methylation and in simple terms, turns gene on and off. Methyl groups attach to a specific point in the DNA Strand and inhibit its ability to translate or express, in a way mask it from sharing its genetic information.[22]

“If the genome were like the hardware of a computer, the epigenome would be the software that tells the computer, how to work and when.” Randall Jirtle PhD., a professor of radiation oncology at Duke University.[24]

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Through his work in the field of epigentics and the epigenome, Randall Jirtle of Duke has been able to manipulate the epigenome in genes of the mice that cause obesity and hair color, through feeding the mice food with specific nutrients and toxins. He has been able to change accumulation of adiposetissue and hair color in mice that are genetically identical. Through controlling the variable in which the mice are introduced to, mainly diet and nutrition, he has been able to manipulate the agouti gene or agouti signaling peptide.

In mice, the agouti gene encodes a paracrine signaling molecule that causes hair follicle melanocytes to synthesize pheomelanin, a yellow pigment, instead of the black or brown pigment, eumelanin. Pleiotropic effects of constitutive expression of the mouse gene include adult-onset obesity, increased tumor susceptibility, and premature infertility. This gene is highly similar to the mouse gene that encodes a secreted protein that may (1) affect the quality of hair pigmentation, (2) act as an inverse agonist of alpha-melanocyte-stimulating hormone, (3) play a role in neuroendocrine aspects of melanocortin action, and (4) have a functional role in regulating lipid metabolism in adipocytes.[25]

By feeding a group of mice (group A) a diet high in methyl groups, he was able to silence the agouti gene, creating mice with average body weight and mass (approx 34 grams) and who had dark brown hair pigmentation. The mice were also less prone to genetic encourage disease such as cancer and diabetes. The genetic traits were passed on to further generations, even of the newer generations that were feed a normal diet. They retained the gene expression and methylation characteristics from parent mice. In contrast by feeding a group (group B) of mice a diet rich in toxins that disrupted and destroyed the methyl groups given to group (A) mice, he recorded that they were of a much lighter pigment and skin color, although of a normal and average weight in young mice, as they developed into adult-hood, almost 100%of mice suffered from adult-onset obesity. Weighing an average of 70 gram, almost twice the weight of group (A). They had much higher occurrence of

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diabetes and other diseases related to a dramatically increased BMI, Secondary conditions such as reduced cardiovascular efficiency and increase in cardiac disease. As well as arthritis, decreased life expectancy and other diseases associated with high levels of adipose tissue.

In a interview with NOVA, and PBS broadcasting company, Dr. Jirtle, stated, “this research shows that this potentially has severe repercussions on our health, and essentially, the old phrase “you are what you eat” is true, but you are also what your parents eat and your grandparents and so on ate”. He goes on to encourage, healthy life style changes for future generations, not only through learned and influenced habits in our children, but what is genetically passed on.

Phenotype expression through Histone

Modification

The second way on which Gene expression occurs is through DNA physical relationship with the histone. In biology, histones are strong alkaline proteins found in eukaryotic cell nuclei, which package and order the DNA into structural units called nucleosomes.[1] [2] They are the chief protein components of chromatin, act as spools around which DNA winds, and play a role in gene regulation. Without histones, the unwound DNA in chromosomes would be very long.

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For

example, each human cell has about 1.8 meters of DNA, but wound on the histones it has about 90 millimeters of chromatin, which, when duplicated and condensed during mitosis, result in about 120 micrometers of chromosomes.[3]Histones act as spools around which DNA winds. This enables the compaction necessary to fit the large genomes of eukaryotes inside cell nuclei: the compacted molecule is 40,000 times shorter than an unpacked molecule. Histones undergo posttranslational modifications which alter their interactin with DNA and nuclear proteins.

The H3

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and H4 histones have long tails protruding from the nucleosome which can be covalently modified at several places. Modifications of the tail include, methylation, acetylation, phosphorylation, ubiquitination, sumoylation, citrullination and ADP-ribosylation. The core of the histones H2A and H3 can also be modified. Combinations of modifications are thought to constitute a code, the so-called "histone code."[9][10] Histone modifications act in diverse biological processes such as gene regulation, DNA repair and chromosome condensation (mitosis).

Through the introduction of methyl group and other proteins, Scientists have been able to change the structure of the DNA by changing or “loosening” the histone. Differing from the Methyl groups and DNA methylation, which attach directly to the DNA strand and inhibit the DNAs’ ability to produce RNA and functional proteins? Histone formation is controlled by several proteins that when introduced to the histone, change the general structure of the histone, relaxing or Contracting it. Allowing DNA to unwind and be “loose “against the histone. When DNA is in this state, the gene are easily expressed and are turned “on” where as the lack of these proteins cause the histone to contract and tighten up, Hiding a gene from being expressed. In retrospect, Turning the gene “off” by manipulating the protein that cause this effect and change the tension and mechanical properties of the histone structure we can control gene expression. Ultimately choosing which genes we turn “on” and allow to be expressed. Genes such as obesity, longevity, or hair color. Turning gene off that cause the

over production in cholesterol and other substances which cause harm in excess or scarcity.

Epigenetic manipulation through environmental variables

A certain laboratory strain of the fruit fly Drosophila melanogaster has white eyes. If the surrounding temperature of the embryos, which are normally nurtured at 25 degrees Celsius, is briefly raised to 37 degrees Celsius, the flies later hatch with red eyes. If these flies are again crossed, the following generations are partly red-eyed – without further temperature treatment – even though only white-eyed flies are expected according to the rules of genetics.Environment affects inheritanceResearchers in a group led by Renato Paro, professor for Biosystems at the Department of Biosystems Science and Engineering (D-BSSE), crossed the flies for six generations. In this experiment, they were able to prove that the temperature treatment changes the eye color of this specific strain of fly, and that the treated individual flies pass on the change to their offspring over several generations. However, the DNA sequence for the gene responsible for eye color was proven to remain the same for white-eyed parents and red-eyed offspring.The concept of epigenetics offers an explanation for this result. Epigenetics examines the inheritance of characteristics that are not set out in the DNA sequence

This change in phenotype and gene expression is a direct result on the environmental stresses placed in the fruit flies Histone configuration, believing that

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the introduction of temperature variations inhibited the proteins involved with histone expression and tension.

Monozygotic twins and Epigenome Theories,

Since the early 1990s, the scientific literature has seen studies begin to appear regarding the disproportionate occurrence of disease in identical twins, something that was not completely explained by genetics alone and leads to questions about how identical monozygotic twins actually are.

Monozygotic twins arise with an incidence of 1 in every 250 births worldwide. For reasons yet unknown, a fertilized egg cell can clone itself and give rise to separate embryos. Each will begin and end life with the same genetic make-up, but as they grow and develop they will experience differences in their environment, some of which might alter their appearance and behavior. 

If one committed a crime and unwittingly left samples for forensic analysis, it would be impossible to determine the baddie from DNA fingerprint analysis. However, closer inspection of their molecules may reveal significant differences. Although the lads share the same genes, recent evidence suggests that some genes might be active in one twin and not the other. They might be identical genetically but not epigenetically.[12] Biochemical fine-tuning of the genome determines which genes get switched on, so twins are not necessarily destined to share the same fate.

Recent research on monozygotic twins has revealed that their DNA is marked in different ways by a tiny molecule called methyl. So it’s not really true to say that they are identical. What’s more, these differences were much more pronounced in older than younger twins.

As in the studies done at Duke University under Dr. Jirtle, and knowing that all organisms on the earth reproduce through the use of DNA. The biology of this has implications to that of human health. Through understanding of how we develop

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disease and how environmental factors affect our bodies and our development.

Since, the human race is genetically different, each having genetic information form our parents, we run into difficulties testing theories in genetics with everyone being genetically different and complex but through homozygotic twins, epigentics has had some interesting results, all of which are less then Subtle.

( Identical twins are so alike that, they have difficulty understanding mirrors and reflected images until much later in child development compared to other children)

In the case of iIdentical twins (homozygotic twins) are siblings that are also genetically identical; they are the perfect Lab “humans” in the case of genetics.

In 2005, Dr. Manel Esteller Director of the Cancer Epigenetics and Biology Program (PEBC) of the Bellvitge Institute for Biomedical Research (IDIBELL) in Barcelona and leader of the Cancer Epigenetics Group genetic study in Madrid, Spain. Began a Research study in Identical twins, ranging from age 3 to age 74. His

research was aimed to discover how similar the Homozygotic twins really were...or were not.

After Collecting tissue Samples from over 40 identical twins and Through DNA amplification and electrophoresis, they were able to compare the genes of these twins and analyze differences between a twins sibling and other identical twins.

DNA electrophoresis is an analytical technique used to separate DNAfragments by size. The DNA molecule to be analyzed is cut up into variously sized fragments by reacting it with a restriction enzyme. These fragments are set upon a viscous medium, the gel, where an electric field forces the fragments to migrate toward the positive potential, the anode, due to the net negative charge of the phosphate backbone of the DNA chain. The separation of these fragments is accomplished by exploiting the mobility with which different sized molecules are able to traverse the gel. Longer molecules migrate more slowly because they experience more drag within the gel.

Because the size of the molecule affects its mobility, smaller fragments end up nearer to the anode than longer ones in a given period. After some time, the voltage is removed and the fragmentation gradient is analyzed. The DNA fragments of different lengths are visualized using a fluorescent dye specific for DNA, such as ethidium bromide.

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The gel shows bands corresponding to different DNA molecules populations with different molecular weight. Fragment size is usually reported in "nucleotides", "base pairs" or "kb" (for thousands of base pairs) depending upon whether single- or double-stranded DNA has been separated. Fragment size determination is typically done by comparison to commercially available DNA ladders containing linear DNA fragments of known length.Gels have conventionally been run in a "slab" format such as that shown in the figure, but capillary electrophoresis has become important for applications such as high-throughput DNA sequencing.

“The Twin Studies” and genotype

similarities

Dr. Esteller’s Study suggested that as we age, our epigenome become different and change on a regular basis due to outside influences and pressures. As we age we are exposed to environmental stresses, nutrition factors, tobacco and alcohol use, even climate change and emotional stress. All of which effect our internal bodies and homeostasis in some minor or even major ways.

When DNA was analyzed through electrophoresis, and particular genes of two sets of identical twins from various ages were compared, they showed dramatic differences in gene expression similarity.Through the process of electrophereises, several genes were collected from each sibling of a monozygotic twin pair and then over-lapped with the same genes of their sibling. And then were compared to see how identical they really were.

The diagram below show several sets of genes, over-lapped and in comparison to each sibling. The yellow represents genes that are identical, resulting in similar genotypes and phenotype expression. The alternate colors represent differences in the genotype of the individual twin.

Not only does this show that identical twins are not a perfect copy of their sibling, but it also suggest that as we age and are exposed to outside influences, our epigenome changes to adapt. In looking at the two diagrams, you can see that the similarities in

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the 3 year old homozygotic twins are much more similar in gene expression and genotype than homozygotic twins that are 50 years of age. Through much greater time period and exposure to stress, injury and repair, cellular reproduction and division, variants in nutrition, exposure to pathogens, and life style differences. They have grown

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remarkably different. The gene expression has grown different and unique, resulting in difference in phenotype of the individual. This concept helps explain why one sibling of a homozygotic twin pair, would develop a disease or disorder that has strong inherited ties. Such as forms of cancer and diabetes, when the other sibling will not. Although leaving the unaffected twin at a high risk of developing the disease, the questions still remain, why would one develop and disease and the other would not.

DNA methylation and concepts in cancer

Epigenetic inactivation of genes that are crucial for the control of normal cell growth are a hallmark of cancer cells. These epigenetic mechanisms include crosstalk between DNA methylation, histone modification and other components of chromatin higher-order structure, and lead to the regulation of gene transcription. Re-expression of genes epigenetically inactivated can result in the suppression of tumour growth or sensitization to other anticancer therapies. Small molecules that reverse epigenetic inactivation are now undergoing clinical trials in cancer patients. This, together with epigenomic analysis of chromatin alterations such as DNA methylation and histone acetylation, opens up the potential both to define epigenetic patterns of gene inactivation in tumours and to use drugs that target epigenetic silencing.

[6]

Epigenomic analysis of chromatin alterations in tumours opens the potential to define mechanisms of epigenetic gene silencing, and to develop drugs that reverse

transcriptional repression of tumour suppressor genes and genes associated with resistence to anticancer therapies[6]

Alterations in DNA methylation are regarded as epigenetic and not genetic changes, although epigenetic changes affect the structure of DNA, they do not materially affect the genetic code. In recent years, numerous studies have demonstrated that a close correlation exists between methylation and transcriptional inactivation, supporting the notion that not only genetic changes, but also epigenetic changes can contribute to the carcinogenic process (Strathdee et al., 2002; Yan et al., 2001). The pattern of methylation observed in cancer generally shows a dramatic shift compared with that of normal tissue. The methylation pattern in tumors consists of a global hypomethylation, in conjunction with localized hypermethylation at CpG islands. This regional hypermethylation at CpG islands is associated with the transcriptional inactivation of cancer related genes.

Recent studies have demonstrated that hypermethylation of CpG islands may be implicated in tumorigenesis, acting as a mechanism to inactivate specific gene expression of a diverse array of genes. (Baylin et al., 2001). Genes that have been reported to be regulated by CpG hypermethylation, include tumor suppressor genes, cell cycle related genes, DNA mismatch repair genes, hormone receptors, and tissue or cell adhesion molecules. For example, tumor-specific deficiency of expression of the DNA repair genes MLH1 and MGMT[8] and the tumor suppressors,

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p16, CDKN2 and MTS1, has been directly correlated to hypermethylation.Increased CpG island methylation can result in the inactivation of these genes, resulting in increased levels of genetic damage, predisposing cells to later genetic instability which then contributes to tumor progression.[5]

Hypermethylation is now the most well characterized epigenetic change to occur in tumors, and it is found in virtually every type of human neoplasm. Promoter hypermethylation is as common as the disruption of classic tumor-suppressor genes in human cancer by mutation and possibly more so. Approximately 50% of the genes that cause familial forms of cancer when mutated in the germ line are also known to undergo methylation-associated silencing in various sporadic forms of cancer.[9]

The more recent work concentrated on isolating the difference between the gene sequence and the epigenetic pattern, which would have a wide reaching affect on inherited forms of disease. Epigenetics is a complexity not taken into

consideration until recent years, and may complicate preventative therapy, prognosis, and treatment.

(Breast cancer cells)

Conclusion and personal thoughts

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This concept was very interesting to me for many personal reasons. Being a twin, I have always been fascinated in our differences, although we are only fraternal twins, we have very little in common, from physical appearance to personal preference.

Also having a work related background in oncology and a family that have suffered loss of a sister to ovarian cancer, when she was a child, gave me a curiosity in the concept of epigentics and DNA methylation, and its role in tumor genesis

I currently work in the field of cardiovascular genetics and on a daily basis interact with people that either suffer from strong inherited disease and disorder, or are leaders in the field of hyperlipidemia

and lipid science, and there genetic influences on cardiovascular disease. I found a great deal of interest in DNA

methylation and lipid disorders, stemmed from my research on this interesting subject and plan to continue exploring the subject.

The prevalence of aberrant epigenetic inactivation of genes in tumors makes it an attractive target for novel anticancer therapies and a concept which needs more extensive research. Several small molecules are now entering early clinical trials, that give hope to the belief that we may be able to cure cancer in the future or promote longevity. The concepts behind epigentics research are new frontiers in genetics research, show us just how complex we are, from a DNA perspective, and how little we really know.

In after thought, since writing this paper, I find myself overly excited to think of all the implications and profound effects in the world today with strong evidence that shows us that our lives and the nutrition and environments that we expose ourselves to, are in a way, rewriting our DNA, and the way we pass that on to our children and grand children.

In thoughts toward the future, what if the science community was to learn how to control the phenotype expression through DNA methylation and Histone configuration with increased accuracy? Would we be able to cure inherited disease, would cancer be the next history concept similar to smallpox, only existing in a test tube? Would we be able to “ turn-on” the longevity gene and increase the life expectancy of the average human, that we would be able to shake the hand of one’s grandchildren’s’ great grandchildren.

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Do new questions in ethics arise if we gain the ability to “plan” a child through Epigenetic influences, giving our offspring a desired eye color, physical appearance or gender specific attributes that are desired by a general view from society. Would epigentics bring up the moral dilemma of “playing god” in contrast to the progression of science?

I am excited to follow the development into such a interesting field of science and hope that we can harness this for the many benefits that this may offer.

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