Epigenetics and Its Clinical Applications

Abstract

This introduction to the relatively new field of epigenetics—which extends the conventional understanding of genetics—summarizes epigenetics' main terms and mechanisms of action, reviews the empirical evidence base, and discusses the clinical implications of the effects of lifestyle and environmental factors on the health of patients and their descendants.

Definition and Background

The Human Genome Project, which was completed in 2003, was undertaken to shed light on the genetic basis of health and disease by sequencing the estimated 30,000 genes in the human genome.¹ Other estimates put the number of human protein-coding genes as few as 19,000.² However, genome mapping does not explain such phenomena as monozygotic twins, who result from a fertilized egg that splits in two, therefore producing twins who have the same genetic code but who are not completely identical in terms of susceptibility to health disorders.

The term epigenetics (see Glossary of Terms) was introduced in the 1940s by British developmental geneticist and biologist Conrad H. Waddington, PhD (1905–1975 ad), to explain how different phenotypes could arise from a single genotype during an organism's development through externally induced changes in gene expression.³ The field of epigenetics (epi is derived from the Greek for on, over, or beside) was developed to help explain such discrepancies in heritability, disease-causing mechanisms beyond those explained by conventional genetics, and transgenerational inheritance effects that have been observed.⁴ Researchers in the National Institutes of Health's (NIH's) Roadmap Epigenomics Program have identified many of the chemical markers on deoxyribonucleic acid (DNA) that influence gene expression.⁵ In sponsoring the interdisciplinary field of geroscience, the NIH includes epigenetics among its critical areas of research relating to aging and chronic disease.⁶

Mechanisms of Action

Epigenetic modifications of genes occur in response to lifestyle and environmental factors, including diet, stress, exposure to pollutants, and certain drugs. Epigenetic mechanisms are especially critical during early development. For example, a pregnant woman who experiences stress produces and transmits elevated levels of cortisol to her fetus through the placenta. This transmission can result in heightened responses to stressful events in the woman's offspring via a sensitized hypothalamus–pituitary–adrenal (HPA) axis.⁷ In turn, elevated HPA responses to stress predispose the next generation to a greater incidence of heart disease, diabetes, anxiety, and other disorders. Nessa Carey, PhD, a British epigeneticist (formerly at Imperial College in London) noted: “Consequently, these initial small fluctuations in histone and DNA [epigenetic] modifications may eventually become ‘set’ and get transmitted to daughter cells, or be maintained in long-lived cells, such as neurons, that can last for decades.”³

DNA Methylation

DNA methylation, involving the methyl group comprised of 1 carbon atom bonded to 3 hydrogen atoms (CH₃) attached to DNA, is the most common and extensively studied type of epigenomic marking of genes that can either switch them on or off.⁶ The more methylated a gene is, the less active it tends to be. Methylation can produce long-term gene-regulation effects either directly or indirectly.

Histone Modifications

Histones are a class of proteins bundled within DNA that promote the effects of DNA methylation on gene expression indirectly.³ In contrast to the stable effects of DNA methylation, histone modifications have greater plasticity in determining the expression or silencing of specific genes in response to stimuli. For example, histone modifications are involved when insulin signaling affects muscle cells, when brain cells respond to addictive drugs, and when cells lining the gastrointestinal tract respond to the amount of fatty acids produced by the bacteria in the intestines.

Noncoding RNA/RNA Interference

Although ∼80% of the human genome has been transcribed (i.e., coded for functionality in at least one cell type), there are currently only 1%–2% codes for proteins.⁸ Noncoding ribonucleic acid (ncRNA) plays a central role in epigenetics (and in the evolution of species). In the 1990s, the process of ribonucleic acid (RNA) interference was discovered. This process occurs when short strands of RNA can silence a gene containing the same sequence of nucleotides (the building blocks of RNA and DNA).

Imprinting

Certain genes are switched on or off during perinatal and other critical periods of development, depending on whether these genes are inherited from the mother or father.⁶ Imprinted genes are particularly vulnerable to molecular defects, because the expression of these genes is dependent on such parent-of-origin effects. Endocrine-disrupting chemicals (see the section below entitled Studies on the Effects of Endocrine Disruptors) are a prime cause of defects in imprinting. Assisted-reproductive technology has also been associated with an increasing incidence of rare imprinting disorders.⁹

Nucleosome Remodeling

The role of nucleosome remodeling—in which enzymes known as chromatin remodelers promote or repress gene expression by determining the positioning of nucleosomes (the basic unit of chromatins) by how tightly they are packed in DNA—has recently been recognized as another epigenetic contributor to neurodevelopmental and cognitive disorders including autism spectrum disorder (ASD).¹⁰ In a study of 50 monozygotic twin pairs concordant and discordant for ASD, alterations in DNA methylation were correlated strongly with quantitative measures of ASD-associated traits.¹¹

Empirical Evidence

Epidemiologic Studies

The classic epidemiologic study of the transgenerational effects of maternal stress is the Dutch Famine Birth Cohort Study of pregnant women who survived a famine in 1944–1945 during World War II and their offspring.⁶ Studies of medical records showed that offspring exposed to a low-nutrient diet prenatally during the mother's third trimester had significantly higher rates of obesity in young adulthood despite having an initially low birth weight, compared to offspring born just before or after the famine. Females exposed during the mother's first trimester had a higher risk of breast cancer in adulthood, while those exposed during the second trimester had more lung and kidney problems later in life. At age 50 and at age 58, both men and women in the cohort showed a higher incidence of obesity, high blood pressure, coronary heart disease, and type 2 diabetes. Other follow-up studies shown that there are significant increases in risks for schizophrenia, depression, and other psychiatric disorders in people who were exposed prenatally to the famine.⁶

Researchers examining the long-term consequences of the Dutch famine have focused more recently on the underlying epigenetic mechanisms. Epigenetic differences were identified in the methylation patterns of a gene that codes for insulin-like growth factor 2 (IGF₂) between people who were exposed prenatally to the famine and those who had not been exposed to it.⁶ In an assessment of 422 individuals at ages ∼59 exposed to the famine prenatally and 463 time-matched controls without exposure to the famine, researchers identified early gestation (i.e., weeks 1–10) as the critical period for adult DNA methylation changes in whole blood linked to genes involved in growth, development, and metabolism after prenatal exposure to famine.¹²

Studies on the Heritability of Post-Traumatic Stress Disorder

Studies on victims of war, child abuse, and other forms of trauma who develop post-traumatic stress disorder (PTSD) have shown that the victims' offspring were more likely to also develop PTSD in response to stress without the direct experience of the conditions that their parents had experienced, suggesting a kind of “biologic memory.”¹³ A review of articles published from 1985 to 2011 provided evidence that vulnerability to PTSD may be transmitted across generations through maternal epigenetic programming during pregnancy.¹⁴

A study of adults, each with at least one Holocaust survivor parent (n=80), compared to matched participants without parental Holocaust exposure or parental PTSD (n=15), examined vulnerability for transgenerational transmission of PTSD.¹⁵ According to an analysis of blood samples that were examined for methylation of the glucocorticoid receptor gene, DNA methylation was significantly associated with greater suppression of the hormone cortisol, which helps the body cope with stress. In the absence of maternal PTSD, offspring with paternal PTSD showed higher glucocorticoid receptor gene methylation. Transgenerational effects were highest when both parents suffered from Holocaust-related PTSD, suggesting gender-specific differential epigenetic regulation that produced stress-hormone profiles, predisposing descendants of Holocaust survivors to anxiety disorders.

A prospective study of pregnant women (N=38) who were firsthand witnesses to the September 11, 2001, attacks on the World Trade Center in New York City found similar effects.¹⁶ Based on salivary cortisol samples obtained from the mothers and their 1-year-old babies at awakening and at bedtime, women who experienced PTSD at the time gave birth to babies who were later more susceptible to anxiety, depression, and PTSD than matched women who did not experience PTSD from witnessing the destruction. Lower cortisol levels were most evident in babies born to mothers with PTSD exposed to the event in their third trimester.

Studies on the Effects of Endocrine Disruptors

Data from animal studies and some clinical research provide evidence for the epigenetic effects of exposure to a group of steroid-effect mimicking chemicals known as endocrine disruptors. Endocrine disruptors bind to estrogen and testosterone receptors, and disrupt normal hormonal processes by mimicking these hormones. Because the same brain regions and neural substrates govern reproductive and social behaviors, such behaviors as communication, mating, and aggression can be compromised.¹⁷ Endocrine-disrupting chemicals have also been linked to inheritance of metabolic and neurologic phenotypes, and transgenerational persistence of neurobehavioral disruptions.¹⁸

Naturally occurring phytoestrogens (e.g., the isoflavones genistein and daidzein in soybeans [Glycine max]) and lipid-soluble vitamins A and D (which are actually hormones) can bind to—and activate—hormone receptors. However, the main concern is compounds, including dichloro-diphenyl-trichloroethane (DDT), a pesticide; polychlorinated biphenyls (PCBs), once used as electrical coolants and lubricants; atrazine, an herbicide; polybrominated diphenyl ethers (PBDEs) used as flame retardants, phthalates, and bisphenol A (BPA), the plasticizers often used in making water bottles; benzopyrene molecules in air polluted by auto exhaust, burning coal, and smoking; and the widely used agricultural fungicide vinclozolin.¹⁹

Prenatal exposure to even low doses of vinclozolin have been found to result in decreased sperm counts and lowered male fertility in up to 3 successive generations of mice.¹⁸ Such findings are relevant to the increasing prevalence of human reproductive disorders worldwide.²⁰ Heavy metals, such as mercury, arsenic, and lead, can also alter epigenetic regulatory mechanisms.²¹

Exposure to environmental toxins and certain drugs at a critical developmental period appears to be crucial in hormonal mis-imprinting. Medical interventions in human childbirth, such as administering exogenous oxytocin for promoting delivery, can result in epigenetic effects by resulting in an “overdose” of this naturally occurring hormone.¹⁸ Soy phytoestrogens in infant formulas can also disrupt normal imprinting.¹⁸ Early contact with endocrine disruptors has been associated with maladaptive social behaviors in children.¹⁶

Epigenetics of Selected Disorders

Cancer

Much of the focus in the field of cancer genetics has shifted from cancer-associated genes to cancer-related epigenetic activity. Demethylation, in which genes lose their normal methyl attachments, is a hallmark of cancer (e.g., in acute leukemia²² and in alcohol-associated hepatocellular carcinoma).²³ Agents that reverse methylation aberrations in oncogenes and tumor suppressor genes that promote uncontrolled cell proliferation were among the first studied epigenetic drugs.⁷

Metabolic and Cardiovascular Disorders

The Developmental Origins of Health and Disease hypothesis proposes that the global epidemic of obesity and type 2 diabetes, and related cardiovascular disease (CVD), is related to intrauterine and early postnatal exposure to poor maternal nutrition.²⁴ The Dutch Famine Cohort Study is a prime example of how in utero exposure to malnutrition can increase the risk of obesity, insulin resistance, type 2 diabetes, high cholesterol levels, hypertension, and CVD in adulthood. Poor maternal nutrition may program a compensatory response epigenetically for postnatal survival in a nutrient-poor environment that leads to obesity and related comorbidities in a nutrient-rich environment; the thrifty phenotype and thrifty epigenome hypotheses—extensions of the better-known “thrifty gene” hypothesis—have been proposed to explain this phenomenon.²⁵

A mother who develops gestational diabetes transfers higher-than-normal glucose levels to her fetus, and her female children therefore have a higher probability of developing gestational diabetes; in a vicious cycle, the next generation will also be at risk of developing metabolic disease through the maternal line.²³ Preliminary studies suggest a correlation between epigenetic mechanisms in the pathogenesis of atherosclerosis, cardiac hypertrophy, myocardial infarction, and heart failure,²⁶ and nonalcoholic fatty-liver disease, which shares features of metabolic syndrome.²⁷ Maternal obesity and/or maternal overfeeding on a high-fat diet also increase the risk of obesity and other symptoms of metabolic syndrome in offspring.²³

The phenotype of the offspring is also affected by the father's environment through the paternal germline. In an epidemiologic study of variations in the food supply in Överkalix, Sweden, in the late nineteenth and early twentieth centuries, the effects of the father's food intake was studied through 3 successive generations.²⁸ Historical records indicated that the sons' (but not the daughters') risk of mortality from CVD correlated with the father's decreased risk during times of scarcity and increased risk during times of plenty. Grandsons' excessive caloric intake prior to puberty also increased their diabetes-related mortality rate. In a longitudinal study of fathers who had ever smoked (N=5376), the younger a father was (starting from age 13) when he began smoking, the more likely it was that his son's body mass index (BMI) would increase significantly. However, there were no significant BMI associations in daughters.²⁹

Neurodegenerative and Mental Disorders

Aging, neurologic disorders, and addiction are associated with several epigenetic mechanisms that regulate genes responsible for complex memory formation and storage, and behavior.³⁰ Abnormal DNA methylation patterns have been identified in key genes in brain tissues of patients with Alzheimer's disease and other dementias.³¹ These aberrations can be used as biomarkers to diagnose neurodegenerative diseases. The currently rising incidence of autism is likely the result of epigenetic factors as well as genetic mutations, which are unlikely to have increased in recent years.³²

As previously noted, children born to mothers during periods of famine have a higher risk of developing schizophrenia and affective disorders in adulthood. Exposure to alcohol during fetal and early postnatal development can involve epigenetic modification of genes that regulate cellular functions, which can be transmitted across many generations through the male germline for central nervous system (CNS) disorders.³³ Drug addiction has been shown to be mediated in part by epigenetic mechanisms of drug-induced alterations in DNA methylation and histone acetylation.³⁴

A case-control study of U.S. military service members deployed in Iraq and Afghanistan compared methylation patterns in serum DNA of service members with postdeployment diagnoses of PTSD (n=75), to a random sample of service members who did not have the PTSD diagnosis (n=75).³⁵ The groups were comparable in predeployment measures of regions of immune-related genes IGF₂; long ncRNA transcript H19; and interleukin (IL)–8, IL-16; and IL-18. Service personnel who developed PTSD had increased levels of IL-18, compared with controls. Methylation patterns, which were found to be highly correlated with cytokine gene expression, may be biomarkers of hyperarousal of the sympathetic CNS in response to sustained stress and vulnerability to PTSD.

Because the circadian rhythm has been implicated in neuropsychiatric disorders relating to sleep, anxiety, mood, and addiction as a mediator between environmental factors and regulators of the circadian rhythm, it has been suggested that the epigenetic system may play a key role in these disorders—a role that has not been explained fully by genetics.³⁶

Autoimmune Disorders

The fact that the concordance rate in identical twins is below 50% for such autoimmune diseases as type 1 diabetes, rheumatoid arthritis, multiple sclerosis, and systemic lupus erythematosus (SLE) suggests that epigenetics plays a role in their pathogenesis and potential treatment.³⁷ For example, alteration in DNA methylation has been confirmed as an epigenetic mechanism that can cause a breakdown of immune tolerance and perpetuation of SLE.³⁸ A preclinical study found that grandmaternal as well as maternal smoking (nicotine) was associated with epigenetic changes in lung function and elevated pediatric asthma risk.³⁹

Clinical Applications

Evidence from the field of epigenetics reinforces the importance of the roles of nutrition and stress at all life stages, from prenatal through old age, and the potential for transgenerational inheritance without alterations in the genetic code. Counter to the paradigm of “genetic determinism,”⁴⁰ epigenetics provides support for the potential to improve health through diet, and prevention and management of stress and diseases, as emphasized in complementary and alternative medicine (CAM). Riya R. Kanherkar—a PhD student and former research associate at the Epigenetics Laboratory, Howard University in Washington, DC—commented: “Alternative medicine (AM) approaches [such as] Ayurveda, homeopathy, yoga, t'ai chi, reiki, acupuncture, body massages, naturopathy, and hypnotherapy offer a promising approach toward improving human health and lifestyle by mediating beneficial environment—epigenetic interactions.”⁴¹

Nutri-Epigenetics

Because epigenetic effects are inheritable and potentially reversible, and because nutrients can induce changes, directly or indirectly, in gene expression that can affect metabolism and disease susceptibility, dietary interventions are a major way in which health practitioners can have a positive impact on disease prevention and treatment for patients; this application has been called nutri-epigenetics.⁴² As part of the NU-AGE Project—a multidisciplinary consortium of European Union countries that is developing dietary guidelines for healthy aging—epigenetic studies are being conducted to assess the role of individual variability in responses to diet.⁴³ Chronic low-grade systemic inflammation may lead to inflammaging, premature physiologic aging via age-associated disease.⁴⁴

Epigenetics helps explain why nutrition in early developmental stages is critical for determining later health status and susceptibility to disease.

Epigenetics helps explain why nutrition in early developmental stages is critical for determining later health status and susceptibility to disease. The epigenetic mechanism of DNA methylation is contingent upon the intake of micronutrients including folate, vitamin B₁₂, methionine, betaine, and choline.⁴⁵ For example, folate in the form of folic acid is a supplement recommended for women in the early stages of pregnancy to prevent spina bifida, a neural-tube defect, in their babies.³

Folic acid, which appears to produce beneficial effects through epigenetic mechanisms, is required for production of S-adenosyl methionine (SAMe), a methyl group donor. When rodent pups were fed a diet low in folic acid, they developed abnormalities in imprinted regulation of regions of the genome.³ However, some evidence suggests that high (unspecified) levels of methyl intake (e.g., from routine fortification of grain products and energy drinks) can have detrimental effects such as those associated with excess vitamin supplementation or high caffeine intake.⁴⁴ Yet, maternal dietary supplementation with methyl donors during gestation and/or lactation might counter deleterious epigenetic programming and prevent an obese phenotype in offspring.⁴⁶

Evidence from cell-culture studies and animal models of cancer indicate that plant-derived phytochemicals act as epigenetic modulators that help reduce the risk or progression of cancer.⁴¹ These antioxidant compounds include polyphenols in green tea (Camellia sinensis), sulforaphane (in cruciferous vegetables such as broccoli), organosulfur compounds in garlic (Allium sativum) and other Allium vegetables), and resveratrol (e.g., in red wine). In a pilot study with 3 human subjects, a single ingestion of 68 g of broccoli sprouts inhibited the activity of histone deacetylases, a class of enzymes involved in tumor expression, in blood cells 3–6 hours after consumption.⁴⁷ Polyphenols, folic acid, and cocoa (Theobroma cacao) may promote epigenetic prevention of CVD via modulation of DNA methylation status.²⁵

Glossary of Terms

Epigenetics—Modifications in chemical attachments to a cell's deoxyribonucleic acid (DNA) that regulate the way genes are switched on or off but that do not alter the genes themselves

Epigenome—all epigenetic modifications on the DNA genome and associated histone proteins

Genotype—Sequences of an organism's genes (in contrast to phenotype)

Germline—the sex cells (eggs and sperm) that pass on genetic information

Histones—a class of proteins bundled within DNA that promote the effects of DNA methylation on gene expression indirectly

Imprinting—a phenomenon in which the expression of certain genes depends on whether they are inherited from the mother or father

Methylation—one of the chemical mechanisms by which genes are epigenetically modified

Nucleosomes—molecules with DNA wrapped around them that regulate gene expression

Phenotype—the observable characteristics or traits of an organism (contrasted with genotype)

Transgenerational inheritance—a phenomenon in which changes in phenotype in one generation are passed on to the next generation without any alternation in the genetic code

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Source: Ref. 3.

Exercise Epigenetics

Current data suggest that epigenetic modifications in adipose tissue; the brain; the blood; and skeletal and cardiac muscle are responsive to aerobic and resistance exercise. Six months of aerobic exercise has altered DNA methylation in adipose tissue and skeletal muscle; influenced lipogenesis (fat formation); and had ncRNA-related effects on maximal oxygen consumption.⁴⁸ RNA expression has also correlated with gains in lean body mass. In another study, healthy young men and women (N=23) used an exercise bicycle by biking with one leg at a moderate pace for 45 minutes, 4 times per week for 3 months; the other leg served as a control.⁴⁹ Muscle biopsies were performed before and after training. Biopsies of the trained leg showed changes in methylation patterns in >5000 muscle-cell genome sites that either enhance or suppress the expression of genes that affect energy metabolism, insulin response, and inflammation in muscles.

Other Modalities

Acupuncture is among the CAM therapies that are examining epigenetics as a way to elucidate the underlying mechanisms of acupuncture (e.g., in improving cardiac function and for treating myocardial ischemia). DNA methylation, histone modification, and other epigenetic processes are involved in myocardial ischemia and cardiac repair.⁵⁰ Behavioral therapy for treating PTSD and substance-abuse disorder can draw on an understanding of epigenetic mechanisms in regulation of memory, fear, anxiety, and drug-seeking to avert stress-related relapses.⁵¹

Personalized Medicine

Epigenetic and genetic alterations should be taken into account in diagnostic testing and treatment, particularly for patients whose conditions do not respond to standard therapies for cancer and other diseases.⁵² Clinicians should be aware that epigenetic markers have been implicated in models of resilience as well as in vulnerability to stress and its health consequences.⁵³ For example, some researchers have noted that customized “antiaging epigenetic diets” may help prevent inflammaging⁵⁴ and that, in the future, epigenomic “maps” may enable doctors to tailor patients' responses to therapies.⁵⁵

Conclusion

The field of epigenetics offers a bridge between genetic and environmental factors. Epigenetics offers a new paradigm for understanding the impact of nutrition, stress, toxic chemicals, and other lifestyle and environmental factors on health and disease throughout the lifespan and in progeny. Because epigenetic changes may be reversible, insights about epigenetic mechanisms offer potential for preventing and treating the adverse effects of such factors, despite a given patient's genetic predisposition to such disorders. Because the effects of such factors can be transgenerational, patients also need to take greater responsibility for their lifestyle habits. Additional clinical trials need to be conducted to confirm evidence from epidemiologic and experimental studies, and to elucidate heritable epigenetic mechanisms further with respect to action and optimal “doses” of nutrients, exercise, and other factors conducive to health across generations.▪

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