Epigenetics is the study of changes in gene expression, or switching genes on and off, which are not the result of changes to the primary DNA sequence. It is through these changes that the environment may affect one’s genetic predisposition to disease.1,2 In this article I aim to review some of the principles behind the science and its impact on certain ocular disease processes and the potential role optometry can play in influencing patient well-being.

Brief refresher of biochemistry and genetics

First, it may be useful for a review of important terminology.3,4,5,6

  • Cells. These are fundamental working units of every human being. All the instructions required to direct their activities are contained within the chemical deoxyribonucleic acid, most commonly described as DNA.

  • DNA. This is made up of approximately three billion nucleotide bases, or letters. There are four fundamental types of bases that comprise DNA – adenine, cytosine, guanine, and thymine, commonly abbreviated as A, C, G, and T, respectively. A single DNA strand is packaged into a chromosome (Figure 1).

  • Base sequence. The sequence, or the order, of the bases is what determines our life instructions.

  • Genes. Within the three billion bases, there are over 20,000 genes. Genes are specific sequences of bases that provide instructions on how to make important proteins – complex molecules that trigger various biological actions to carry out life functions. The process of protein expression involves two key steps: Transcription (reading) of the DNA sequence of the gene to a molecule of messenger RNA (mRNA) and then translation of the mRNA into the amino acid sequence of the protein product.5 There are tens of thousands of individual proteins coded for by tens of thousands of individual genes, required for every cell of the human body. Hence, a large amount of DNA must be stored in the relatively small volume of the cell nucleus.

  • Chromatin. To accommodate the large mass of genetic material within the nucleus, DNA is packaged into a condensed structure referred to as chromatin. Chromatin is actually composed of a combination of DNA, proteins (mainly proteins known as histones) and some RNA. The (8 core) histones form disc-like structures around which portions of the DNA wrap itself to form structural units, called nucleosomes, resembling beads along a string (Figure 1). The structure (or packaging) of the nucleosomes and ability to physically reach the DNA has direct implications on gene expression (and will be discussed later).

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Inheritance

Inherited traits, such as colour blindness for example, are characteristics or diseases that are inherited from parents due to their specific coding on the DNA of either or both parents.

Inheritance patterns are of three main types.

  • Autosomal recessive inheritance. Both parents must have a copy of the ‘faulty’ gene (they are classed as ‘carriers’). If we inherit one copy of the faulty gene, the condition will not be expressed, but we become carriers of the condition. Examples include cystic fibrosis and sickle cell anaemia.

  • Autosomal dominant inheritance. Only one parent needs to carry a faulty gene for it be expressed. Examples include tuberous sclerosis and Huntingdon’s disease.

  • X-linked inheritance. Caused by a faulty gene on the X chromosome, and generally inherited in a recessive pattern. X-linked recessive conditions generally do not affect females to a significant degree as females have two X chromosomes, one of which is likely to be unaffected and will compensate for the faulty chromosome. Females who inherit the faulty chromosome will become carriers. Thus males will exhibit the condition, but will not pass the condition to their sons (as they receive the Y chromosome from him).  Daughters will become carriers of the faulty chromosome. Examples include haemophilia and congenital colour vision deficiency, familiar to most optometrists.

Epigenetics

Epigenetics is a term describing the nature of and influences upon the variation of gene expression and is not concerned with the sequence of nucleotides in the DNA of the genes but rather everything else attached to the DNA affecting its structure and function.

Epigenetics controls genes

Environmental factors can eventually cause chemical modifications around genes that can lead to the genes being switched on or off over time. The different combinations of genes that are turned on or off are what make each one of us unique. These epigenetic changes can be inherited.

Our genetic code consists of three billion letters, in the form of the nucleotide bases A,T,C and G. These bases code for over 20,000 genes within 23 chromosomes. These genes create enzymes, which regulate the biochemistry to allow the cell to function. For example, a gene may be responsible for producing an enzyme that facilitates bringing together components A and B to make product AB. So, if the code for an enzyme in your biochemistry is perfect, then the enzyme will produce product AB at the correct speed.

The Human Genome Project (HGP) sequenced the entire genetic code in 2003. Some of the data provided evidence that our genetic code is not perfect and can have misspellings (or mutations), called single nuclear polymorphisms (SNPs). If we have an SNP, the resulting enzyme works sub-optimally, either slowing down or speeding up the process of producing product AB.

If we think of these enzymes as robots on an assembly line, an error in one enzyme has the potential to affect the entire assembly line, resulting in sub-optimal biochemistry and this can cause disease.

Every cell in the body has this critical assembly line, designed to produce molecules that have the ability to impact our:

  • DNA

  • Immune system

  • Cellular maintenance

  • Energy production

  • Production of neurotransmitters.

The main mechanisms for altering gene expression are:

  • DNA methylation

  • Histone modification

  • Chromatin remodelling.

Of these three main mechanisms, we will concentrate on DNA methylation, as a significant amount of research has focused on the methylation pathway and its effects on ocular disease.8,9 The latter two mechanisms do deserve mention as they affect the structure and amount of DNA that is available to be read by the transcriptional enzymes. Mechanisms that affect nucleosome structure or that bind up DNA could affect gene expression. Genes or gene segments that are tightly bound together (or compressed) tend to inhibit transcription and are not expressed, whereas those genes or gene segments that are more open encourage transcription and more likely to be expressed (Figure 2). If a gene does not get expressed, it does not make the protein that it codes for, so it cannot perform the function that it is supposed to.6 For example, if the gene controlling melanin production was turned off due to epigenetic factors that controlled the nucleosome structure, you would have an iris that was genetically supposed to be brown but instead is blue because the melanin was never produced.

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Methylation

The methylation pathway is a vital biochemical pathway that is required for life and controls the way our DNA operates.10 It also participates in more than 40 critically important chemical reactions.

Some of the ‘key players’ in the methylation pathway include the following:11

  • Methionine: an essential amino acid ingested as a component of food protein

  • Homocysteine: an amino acid that is an intermediate of methionine metabolism. An elevation in homocysteine levels can be considered toxic and a risk factor for many health problems including cardiovascular disease (by damaging arterial endothelial cells)

  • SAMe: a compound produced in the conversion of methionine to homocysteine, acting as a methyl donor for a variety of reactions in the body

  • Glutathione: a potent antioxidant reducing the impact of oxidative stress and free radical damage at a cellular level

  • Methylfolate: a form of vitamin B9 and plays a critical role in converting homocysteine into methionine. It is obtained through diet and ‘activated’ in the liver.

Because of the work of the HGP, we can now use genetic testing to evaluate the methylation pathways by studying homocysteine and SAMe levels as well as directly measuring glutathione. As the methylation pathways are so fundamental to cellular health, they have captured the attention and interest of researchers. It has been well documented that an abnormality in the methylation pathway can lead to a great number of major eye diseases8,9 and chronic diseases including the following:12,13,14,15,16

Coronary artery disease

  • Autism

  • Alzheimer’s

  • Depression

  • Migraine and many of the

  • Autoimmune diseases including rheumatoid arthritis.

Research suggests that by balancing the methylation pathways, we now have a major new way to prevent and treat these conditions. It is possible to measure homocysteine and glutathione as well as perform genetic testing. If abnormalities are found, treatment plans that are straightforward, safe and beneficial can be developed which are based around supplementation and supported with nutrition and use of natural hormones. Of particular interest is that by influencing the methylation pathway, we not only treat the eye but also protect the rest of the body by making the patient less susceptible to other diseases.17

Overview of methylation biochemistry

Methylation is the process by which methyl (-CH3) groups are added to DNA or proteins. Methylation of DNA and histone proteins (the proteins that package DNA) affect the transcriptions of particular genes and the expression levels of proteins made from these genes. Typically the methylation pattern of particular genes or proteins is conserved as cells divide, but sometimes disturbances in the biochemical pathway leading up to methylation can result in disruption of this delicate system and changes in gene expression. Below is a schematic of the methylation pathway and some of its key components (Figure 3).

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Dietary folic acid is converted, with the help of MTHFR to 5-methyl THF, which then acts as the methyl donor in the conversion of homocysteine to methionine. Vitamin B12 is the catalyst for this reaction. Methionine is then converted to SAM, which can donate a methyl group to DNA, protein, or other methyl acceptors. The by-product of this reaction is SAH, which is converted back to homocysteine to start the cycle again.12,17,18

Homocysteine is a key to this pathway because it is necessary for the creation of the methyl donor. However, homocysteine can also undergo another reaction, called trans-sulphuration, in which it is modified to form glutathione, a powerful and important antioxidant. The balance between these two reactions has incredible biological significance, and improper homocysteine metabolism has been linked to numerous diseases. Here we will review the current research on the methylation pathway with regard to some of the most common eye diseases and suggest that this pathway is crucial to understanding these diseases.17,18,19

Another important factor that impacts cell processes and genetic expression is steroid hormone balance. Steroid hormones, including oestrogens, androgens, progesterones, glucocorticoids and mineralocorticoids, are signalling molecules that function through binding to specific hormone receptors. Steroid hormones are primarily synthesised in the gonads and adrenal glands, but processing can also happen locally on a cell and tissue level. In the synthesis of steroid hormones, cholesterol is converted to pregnenolone, the general steroid hormone precursor. Pregnenolone is then converted into didehydroepiandrosterone (DHEA), progesterone, testosterone, cortisol, oestrogens and other hormones. Steroid hormones can act systemically and also have tissue-specific effects based on the availability of their receptors. They are known to control key body processes, including inflammation, immunity, sexual characteristics, response to illness and injury, and even cancer growth. The balance of steroid hormones often changes

with age and contributes to the development of many age-related systemic and ocular diseases.17

Methylation pathway and the eye

As mentioned earlier, disturbances in the methylation pathway have been linked to eye disease. Cataract, glaucoma, macular degeneration and diabetic retinopathy will be discussed. However, methylation disturbance has been implicated in conditions such as corneal dystrophy, pterygium, ocular neoplasia, uveitis and proliferative vitreoretinopathy.8,9

Cataract

Cataract is the leading cause of blindness in the world. Of the multiple types and classifications, we will be concentrating on age-related and diabetic cataract.

Oxidative stress is considered the main contributing factor to the development and progression of cataract.20,21,22,23 Under normal conditions, a system of antioxidant enzymes and cellular processes protect the sensitive tissues from damage by reactive oxygen species generated by UV exposure, lipid peroxidation and other stressors. However, in both ageing and diabetic individuals there is a marked decrease in these protective antioxidants, such as glutathione (GSH)24 and superoxide dismutase (SOD), leaving the eye tissues more susceptible to damage by oxidation. Low levels of GSH have been associated with cataract that this molecule is used as a biomarker to measure cataract progression.25,26

Evidence suggests that elevated homocysteine levels and insufficient oestrogen are additional risk factors that likely contribute to the tissue damage from oxidative stress27 Studies have indicated that diets rich in fruit and vegetables with natural antioxidant may be beneficial in combating cataract.28,29,30,31 Supplementation with folate and B vitamins are likely to add to these benefits.

Glaucoma

Studies indicate oxidative stress and reduced levels of GSH have a role in the pathology of glaucoma.32,33 Reduced levels of the antioxidant catalase (CAT), glutathione peroxidase (GPx) and superoxide dimutase (SOD) have been isolated in the aqueous humour of glaucoma patients.34,35

This association of oxidative stress and low GSH in glaucoma suggests alterations in the methylation pathway.36 An elevation in homocysteine has been linked to cardiovascular disease and hypertension, and it is no coincidence that elevated homocysteine has been linked to glaucoma.37,38

The link between glaucoma and steroid hormones is well established, with adminstration of synthetic corticosteroids leading to glaucoma in susceptible patients.39,40 It has also been shown that POAG patients often have abnormal levels of sex hormones.41 It has been observed that women receiving some form of hormone therapy had lower IOP than women who had never received hormone therapy.42

When we consider management options in glaucoma, most traditional therapies focus on inhibiting aqueous humour production or increasing outflow. Although treatment to reduce IOP is successful in halting the progression of glaucoma in some patients, it is not universally effective. Given the growing evidence of methylation pathway aberrations and steroid hormone involvement in glaucoma, it has been suggested that exploring the effectiveness of therapies aimed at reducing homocysteine, increasing GSH and balancing steroid hormone levels may prove beneficial.43,44

Macular degeneration

While there is strong evidence to suggest that genetics is a major factor in AMD, research is pointing to the roles that epigenetics and environment play in both forms of the disease.45,46

Genetically, SNPs in a number of genes have been linked to elevated risk of AMD, some of which include control various processes in the complement system, thereby linking the immune response in the pathology of AMD.47

Although there are a number of genetic risk factors, AMD remains difficult to predict because of epigenetic and molecular factors that contribute to the disease. There is more evidence linking AMD to disruptions in the methylation pathway.48 Drusen formation has been linked to altered DNA methylation and histone modification (H3).49 As mentioned previously, histones are proteins that ‘package’ DNA and control its accessibility for transcription, thereby affecting gene expression. Other studies have shown under expression of certain enzymes that eventually reduce the overall antioxidant activity of GSH to protect cells against oxidation damage.50,51

Many studies have shown a link between wet AMD and increased levels of homocysteine.52 Of particular interest is the association between elevated homocysteine and cardiovascular disease and problems with angiogenesis, suggesting a possible role in the vascular pathology of AMD.53,54,55

Reduced levels of vitamin B12 and folate will impact the conversion of homocysteine to methionine and have been implicated with AMD.53,54 Research has shown that supplementation with folate and vitamin B12 may be beneficial.55,56 Of particular interest is that reducing homocysteine levels through folate and vitamin B12 supplementation has been shown to reduce serum VEGF levels, suggesting that homocysteine lowering therapies may reduce VEGF and potentially prevent progression to wet AMD.57

Diabetic retinopathy

There is growing evidence that links diabetic retinopathy (DR) to problems with the methylation pathway.58,59

Patients with DR have elevated homocysteine levels in their blood, vitreous and aqueous humour.59,60,61 As mentioned previously, the folate and vitamin B12 levels play a significant role in the amount of homocysteine and is strongly linked to cardiovascular disease, shedding light on the microvascular damage that occurs in DR. It is believed that, as with cardiovascular disease, treatment with folic acid and/or vitamin B12 may be effective in lowering homocysteine levels, thereby slowing the progression of DR.62

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Of specific interest is the mutation of the gene responsible for producing the MTHFR enzyme (which aids in the conversion of homocysteine to methionine), resulting in a build-up of homocysteine and is considered as a biomarker to identify individuals at higher risk of developing DR.63,63,65 This use of biomarkers is particularly exciting as elevated homocysteine and MTHFR SNPs are associated with diabetes, so their presence would identify people likely to develop the disease.66

Putting it all together

What does all of this mean to the practicing optometrist? Epigenetics is an emerging field opening new avenues into our understanding of ocular diseases related to ageing and environment. There is a significant amount of research establishing epigenetic mechanisms as influencing gene expression without any change in the DNA sequence of the cell. Epigenetics can be considered ‘holistic’, having the potential to benefit all cells in the body simultaneously. Genetic testing by means of obtaining a simple cheek swab allows us to gain a better understanding of the imbalances to the methylation pathway. Optometry can be part of a integrated health team offering therapies through supplements, nutrition and hormones targeted to individual needs.

Model answers

Which of the following is true about gene structure?

C DNA is condensed into chromatin structures

Which of the following is an autosomal recessive innherited disorder?

B Sickle cell anaemia

Which of the following best defines epigenetics?

D A knowledge of strucutures around genes that influence their expression

Which of the following is NOT thought to be influenced by the methylation pathway?

A Conjunctivitis

Which of the following is a precursor of steroid hormones?

C Pregnenolone

Which of the following is not an established contributor to cataract formation?

D Raised oestrogen levels

 Dr Rohit Narayan is an IP optometrist practising in Nuneaton, Warwickshire