When I wrote my first blog entries on epigenomics and epigenetics eleven months ago(ref)(ref) , it was clear that these were active areas of extremely interesting academic research. However, my impression was that it would be years before the knowledge being accumulated could be put to practical work in medicine. I was wrong. Not only has the depth and amount of research in these areas increased significantly but epigenetics has entered the commercial medical marketplace in the forms of new therapeutic drugs and diagnostic tools. This blog entry provides an update.
An introduction to epigenetics and epigenomics and their relevance to aging is in the Feb 2009 blog entry Epigenetics, Epigenomics and Aging. And, of course, the 13th theory of aging in my treatise is “Epigenetics is the study of changes in phenotype and gene expression arising from mechanisms other than changes in a gene’s DNA sequence.– Over 25 years ago, Surani et al. showed that certain regions of a cell’s genome carry markers over and above the actual gene sequence. This imprint conveys information on differential gene expression, and therefore, shapes the fate of the cell. Epigenetic information is passed from one cell to another, but the epigenetic code can change through life by interacting with environmental factors. Moreover, unlike gene-sequence mutations, epigenetic changes may be reversible(ref).”
Since cells of all types in an individual have the same DNA, epigenetic information is what gives a cell memory as to the type of cell it is, and many events in the lifetime of a cell are also encoded epigenetically. And some epigenetic information can be inherited. The main mechanisms of encoding epigenetic information are DNA methylation and chromatin modifications, such as histone acetylation. See the blog posts DNA methylation, personalized medicine and longevity and Histone acetylase and deacetylase inhibitors.
“DNA methylation involves the addition of a methyl group to the 5 position of cytosine (one of the four bases of DNA), which occurs in the context of CpG (cytosine followed by guanine) dinucleotides. This modification can be inherited through cell division. DNA methylation is typically removed during zygote formation and reestablished through successive cell divisions during development. DNA methylation is a crucial part of normal organismal development and cellular differentiation in higher organisms. DNA methylation stably alters the gene expression pattern in cells such that cells can “remember where they have been”; in other words, cells programmed to be pancreatic islets during embryonic development remain pancreatic islets throughout the life of the organism without continuing signals telling them that they need to remain islets. In addition, DNA methylation suppresses the expression of viral genes and other deleterious elements which have been incorporated into the genome of the host over time. DNA methylation also forms the basis of chromatin structure, which enables cells to form the myriad characteristics necessary for multicellular life from a single immutable sequence of DNA. DNA methylation also plays a crucial role in the development of nearly all types of cancer(ref).”
Of particular interest from the viewpoint of DNA methylation in mammals are the so-called CpG islands, gene promoter sites that are not methylated when a gene is being expressed. “CpG islands typically occur at or near the transcription start site of genes, particularly housekeeping genes, in vertebrates. — “Unlike CpG sites in the coding region of a gene, in most instances, the CpG sites in the CpG islands of promoters are unmethylated if genes are expressed(ref).”
Testing for DNA methylation
A standard approach has been developed for checking out the DNA methylation patterns of humans known as bisulfite sequencing. “Bisulfite sequencing is the use of bisulfite treatment of DNA to determine its pattern of methylation. — Treatment of DNA with bisulfite converts cytosine residues to uracil, but leaves 5-methylcytosine residues unaffected. Thus, bisulfite treatment introduces specific changes in the DNA sequence that depend on the methylation status of individual cytosine residues, yielding single- nucleotide resolution information about the methylation status of a segment of DNA.” Once a DNA sample has been pre-treated with bisulfite, it can be sequenced just like any other DNA. Software comparison(ref) of a sequenced output against that for a normal human genome will reveal the GpC promoter sites that are methylated. Thus, orders-of-magnitude increases in power of gene sequencing such as discussed in the blog post My personal longevity – the race between death-stalker and life-prolonger equally benefit testing for DNA methylation.
Some sequencing platforms have been used extensively for bisulfite sequencing for methylation patterns, for example the Illumina GoldenGate bead array platform. “Through an adaptation of the GoldenGate genotyping assay implemented on a BeadArray platform, the methylation state of 1536 specific CpG sites in 371 genes (one to nine CpG sites per gene) was measured in a single reaction by multiplexed genotyping of 200 ng of bisulfite-treated genomic DNA. The assay was used to obtain a quantitative measure of the methylation level at each CpG site. After validating the assay in cell lines and normal tissues, we analyzed a panel of lung cancer biopsy samples (N = 22) and identified a panel of methylation markers that distinguished lung adenocarcinomas from normal lung tissues with high specificity. These markers were validated in a second sample set (N = 24). These results demonstrate the effectiveness of the method for reliably profiling many CpG sites in parallel for the discovery of informative methylation markers. The technology should prove useful for DNA methylation analyses in large populations, with potential application to the classification and diagnosis of a broad range of cancers and other diseases(ref).”
The June 2009 report CpG Methylation Analysis—Current Status of Clinical Assays and Potential Applications in Molecular Diagnostics provides a snapshot of the technology at that time, a picture that is rapidly changing with the arrival of new ever-more powerful sequencing platforms.
Several methylation studies have been concerned with basic science. An example is described in a report dated February 3, 2010 International Team Maps Methylation Changes During Cellular Differentiation. “Using bisulfite sequencing with the Illumina Genome Analyzer, researchers from Singapore and the US mapped and compared DNA methylation patterns in human cells during three progressive stages of differentiation: embryonic stem cells, skin-like cells derived from embryonic stem cells, and primary neonatal skin cells. In the process, the team identified shared and cell type-specific methylation patterns, providing insights into how gene regulation shifts during development. — With these comprehensive DNA methylome maps, scientists now have a blueprint of key epigenetic signatures associated with differentiation,” co-corresponding author Chia-Lin Wei, a researcher affiliated with the Genome Institute of Singapore and the National University of Singapore, said in a statement.”
DNA methylation and diseases
“DNA methylation is widespread and plays a critical role in the regulation of gene expression in development, differentiation, and diseases such as multiple sclerosis, diabetes, schizophrenia, aging, and cancers (Li et al. 1993; Laird and Jaenisch 1996; Egger et al. 2004). Methylation in particular gene regions, for example in promoters, can inhibit gene expression (Jones and Laird 1999; Baylin and Herman 2000; Jones and Baylin 2002). Recent work has shown that the gene silencing effect of methylated regions is accomplished through the interaction of methylcytosine binding proteins with other structural components of chromatin (Razin 1998), which, in turn, makes the DNA inaccessible to transcription factors through histone deacetylation and chromatin structure changes (Bestor 1998). Hypermethylation of CpG islands located in the promoter regions of tumor suppressor genes is now firmly established as the most frequent mechanism for gene inactivation in cancers (Esteller 2002; Herman and Baylin 2003 Changes in DNA methylation are recognized as one of the most common forms of molecular alteration in human neoplasia (Baylin and Herman 2000; Balmain et al. 2003; Feinberg and Tycko 2004))(ref).”
Many studies have been performed using high-throughput bisulfate sequencing to identify methylation patterns of disease conditions, so many that I can cite only a few examples here.
A December 22 2009 report Twin Epigenetics Study IDs Methylation Differences in Lupus. “– Epigenetic changes, specifically differences in DNA methylation, may contribute to environmental factors involved in systemic lupus erythematosus risk, according to an online study in Genome Research today. — A Spanish, German, and American research team used bead arrays and targeted bisulfite sequencing to compare DNA methylation patterns in the genomes of more than a dozen sets of identical twins who were discordant for SLE or two other autoimmune diseases. While they did not detect methylation differences for two of the conditions, the team did detect intriguing epigenetic changes when one twin had SLE and the other did not. — “Our study suggests that the effect of the environment or differences in lifestyle may leave a molecular mark in key genes for immune function that contributes to the differential onset of the disease in twins,” Esteban Ballestar, a researcher at the Bellvitge Biomedical Research Institute in Barcelona, said in a statement.”
The September 2009 study Epigenetic silencing of death receptor 4 mediates tumor necrosis factor-related apoptosis-inducing ligand resistance in gliomas had the purpose “To identify and characterize epigenetically regulated genes able to predict sensitivity or resistance to currently tested chemotherapeutic agents in glioma therapy.” The conclusion is “DR4 promoter methylation is frequent in human astrocytic gliomas, and epigenetic silencing of DR4 mediates resistance to TRAIL/DR4-based glioma therapies.”
Other recent research reports on the epigenetics of disease processes include The ADAMTS12 metalloprotease gene is epigenetically silenced in tumor cells and transcriptionally activated in the stroma during progression of colon cancer, New insights into the biology and origin of mature aggressive B-cell lymphomas by combined epigenomic, genomic, and transcriptional profiling, Epigenetic profiling reveals etiologically distinct patterns of DNA methylation in head and neck squamous cell carcinoma, Aberrant DNA methylation is a dominant mechanism in MDS progression to AML, Large-Scale Profiling of Archival Lymph Nodes Reveals Pervasive Remodeling of the Follicular Lymphoma Methylome, Allele-specific gene expression patterns in primary leukemic cells reveal regulation of gene expression by CpG site methylation, and Epigenetic Profiles Distinguish Pleural Mesothelioma from Normal Pleura and Predict Lung Asbestos Burden and Clinical Outcome. And this is just a starter list.
A Feb 1 2010 report in Gen Epigenetics Offers Strategies for New Drugs states “According to a recent report from Business Insights, “Innovations in Epigenetics: Advances in Technologies, Diagnostics & Therapeutics,” epigenetic medicine is already here. — The company puts the epigentic market at over $560 million, derived from the sale of three anticancer products (Dacogen™ from Eisai, Vidaza® from Celgene, and Zolinza® from Merck), which target two epigenetic pathways—DNA methyltransferase (DNMT) and histone deacteylase (HDAC). — Approximately 30 epigenetic drugs are under development by more than a dozen biotechnology companies, according to the Business Insights study. These drugs focus mainly on the treatment of cancer, and neurodegenerative and infectious.” The $560 million market mentioned above is only for drug sales. It does not include epigenetics R&D expenditures and the market for epigenetic disease tests. Whatever the market size is now, I estimate it will double in size every 12 to 18 months being driven by the factors that drive Giuliano’s Law until it gets up into the hundreds of billions of dollars in less than 15years. Epigenetics and epigenomics are destined to be very big biz.
Epigenetic diagnostic tools
Finally, as DNA methylation patterns are discovered and confirmed for more and more cancers and other diseases, and as the costs of bisulfite epigenomic sequencing continue to drop, it is likely that we will see many methylation diagnostic and predictive tests being integrated into mainline medical practice. There is evidence, for example, that cancers develop in stages and that methylation tests can provide predictors of premalignant conditions long before the cancers themselves emerge. “One of the advantages of using epigenomic biomarkers is that, in most cases, DNA methylation changes precede clinical symptoms. “If there is a small abnormality that is not yet an invasive cancer, but a precancerous lesion, or a small tumor, many genes will have abnormal methylation, and that is probably true in many tissues,” says Dr. Baylin(ref) .
A number of small biotech companies are moving into this diagnostic area. As reported in Gen the first of this month “Epigenomics specializes in DNA-methylation technologies and biomarkers and is developing a number of cancer diagnostics based on differences in DNA methylation between healthy and diseased tissue. Constellation Pharmaceuticals is focused on developing therapeutics based on epigenetics. It is currently establishing a preclinical pipeline and developing a technology platform for histone modification. Initial applications will be in oncology. Finally, Epizyme is looking at histone methyltransferases and is developing a pipeline of inhibitors for cancer.”
Like the computer industry was in 1959, the genetic, genomic, epigenetic and epigenomic areas are multifaceted and messy. They involve far-out ideas, scientific research, hype, university gurus, technology industry developments, entrepreneurial ventures, commercial activities in big companies and sizeable investments. Some approaches and companies will succeed, others will fail. Some will succeed for a while and then fail. That is what progress looks like. Epigenetics, genomics and sequencing are built on the back of the computer and communications revolution. Without extremely powerful and cheap computers, genetic or epigenetic sequencing would be impossible. This time around, one net result will be that people will live longer.