February 1, 2009 (Vol. 29, No. 3)
Richard A. A. Stein M.D., Ph.D.
Emerging Approach Offers Additional Avenue to Better Understand Mechanisms of Disease
The nature-nurture relationship has attracted fascination throughout history. Roman mythology tells us that in 753 B.C. Romulus slew his twin brother Remus, in a fight over who would become king of the city known today as Rome—and this is far from being the only example of dissimilar phenotypes despite close genetic resemblance.
Modern medicine revealed that even monozygotic twins are discordant for many conditions and characteristics such as diabetes, schizophrenia, major depression, alcoholism, and body weight. In addition, the first report of monozygotic twins who developed Huntington’s disease over seven years apart, despite harboring the same number of genomic CAG repeats, was recently published.
It is increasingly evident that genetics alone cannot explain the complexity of phenotypes in the living world. Heritable phenotypic characteristics that are not caused by DNA sequence alterations represent the object of epigenetics and include potentially reversible changes such as histone modifications, DNA methylation, and imprinting. At the interface between epigenetics and genomics, a new discipline that is emerging, epigenomics, promises to profoundly change the way we envision phenomena in the biological and medical sciences.
In an elegant experiment, Randy L. Jirtle, Ph.D., professor of radiation oncology and director of the epigenetics and imprinting laboratory at Duke University, demonstrated how events from early development influence the phenotype by modifying the epigenome.
Two inbred mice, despite being genetically identical and having the same sex and age, were found to be phenotypically distinct. While the mother of one of the mice received a normal diet, the other mother’s diet was supplemented with methyl donors such as choline, betaine, folic acid and vitamin B12. Since the mice were genetically identical, phenotypic differences were the result of epigenetic, as opposed to genetic changes and the investigators demonstrated that in the brown offspring, specific DNA regions became hypermethylated.
Animal models have guided fundamental questions relevant for human biology. Particularly while exploring the epigenome, however, it is important to remember and appreciate the differences between species. As Dr. Jirtle points out, “mice are not humans; there are big differences between their epigenomes.” Genetic imprinting, in which either the maternal or the paternal copy of a specific gene is silenced, provides a relevant example.
The overlap between the repertoires of imprinted genes between mice and humans is estimated to be only about 30%. Relevant examples are provided by the HOX genes, of which 23% are predicted to be imprinted in humans but not in mice. On the other hand, the Igf2r tumor suppressor gene is imprinted in mice, whereas it is expressed from both parental copies in humans. Thus, evidence indicates that imprinting plays a disproportionately important role in disease formation that is species dependent.
Consequently, ongoing efforts in the Jirtle lab are channeled toward mapping all human-imprinted genes and their ensemble of imprint regulatory elements: the imprintome. One region of interest, on chromosome 22, contains imprinted genes that map into a genomic region associated with the maternal inheritance of schizophrenia. Interestingly, the same genomic region in mice contains no imprinted genes.
The newly identified imprinted potassium channel gene KCNK9 is frequently amplified in breast cancer; however, it is unknown whether overexpression can also occur through loss of imprinting. “I honestly do not believe we can fully understand cancer without identifying the imprinted genes in humans and determining how they are epigenetically regulated,” says Dr. Jirtle. “This is the information that is absolutely required to improve our ability to diagnose, prevent, and treat human diseases and neurological disorders.”
Although recent years revolutionized our understanding of the human genome, the mechanisms that different cell types employ under different circumstances to perform their genetic program still remains a mystery—yet these mechanisms hold the key to the essence of life.
An international collaborative effort called the Human Epigenome Project, initiated in 2000, provided the first insight into the methylation landscape of three human chromosomes: 6, 20, and 22. This initial effort relied on capillary sequencing, and once microarray-based techniques became available, profiling of the entire human genome became possible in a large number of tissues.
“Together with our collaborators, we did this in about 16 tissues, and it currently represents the largest human methylation reference dataset that is available for the human genome,” explains Stephan Beck, Ph.D., professor of medical genomics at the University College London Cancer Institute and one of the investigators in the project.
Most recently, a five-year initiative called the NIH Epigenome Roadmap, involving four U.S. genome centers, was funded and promises to map a variety of epigenetic marks in about 100 cell types, to provide a powerful reference for the scientific community. “This is, I think, the most exciting initiative that is currently going on in the world in terms of epigenomics advances,” says Dr. Beck.
Next-generation sequencing facilitated the characterization of the first mammalian methylome, and opened opportunities to screen entire methylomes in the context of specific diseases. Research in the Beck lab uses both computational and experimental approaches to understand cancer and common diseases for which genetic screens have already been performed.
Single nucleotide polymorphisms (SNPs) explain only a small proportion of the phenotypic variance and, as Dr. Beck explains, “that is a major problem to the field, because obviously there is a lot missing.” For example, SNP-based genome-wide association studies that have recently been carried out for a number of common diseases only explain up to 5% of the observed phenotype. “One possibility,” says Dr. Beck, “is that there are undiscovered genetic variants that we simply have not yet found, but the chances of finding new ones is becoming smaller and smaller over time, because each screen finds less and less; and the most likely alternative is that there are nongenetic contributions, of which epigenetic variations must be high on the list.”
Epigenetic modifications can provide an astronomic number of distinct signatures, with huge diagnostic and prognostic value, but it is essential to consider all the different sources of information. “Genetics is powerful, but the real power comes by layering the information on top of each other, using genetics and epigenetics, and including other layers of information such as proteomics and transcriptomics. Integrate all the different layers that you can measure, and then you will probably come up with the most powerful marker. The best way to understand the system is by looking at the entire system, not at the individual components,” says Dr. Beck.
Annotating Regulatory Regions
As one of the outcomes of the ENCODE (Encyclopedia Of DNA Elements) pilot project, which used a combination of experimental and computational approaches to generate functional data from a 1% region of the human genome, Paul Flicek, D.Sc., team leader in vertebrate genomics at the European Bioinformatics Institute, created the ENSEBL regulatory build, which intends to automatically annotate all functional regulatory regions within the human genome.
Since its first release in 2007, the regulatory build was updated three times, and it currently contains about 175,000 genomic regions in several cell types. The overall goal of the lab is to perform an integrated analysis of multiple different data types and, by using data from epigenetic assays and the genome sequence in combination, feed them into a computational model and ultimately obtain a region-by-region annotation of the human genome. This will facilitate a survey of the entire genome and, subsequently, allow specific questions relevant to disease state to be explored.
“It may turn out that the most effective way to answer a question about a disease is to assay the epigenetic state in certain diseases, and looking at the DNA sequence itself in other disease states,” says Dr. Flicek, and it is important to reflect on his analogy with medical tests: “for some diseases the most effective test is a blood test, for other diseases the most effective test is an X-ray. I think in the future, the most effective test for some situations will be an epigenetic test, and for other things the most effective test will be a genetic one. I think this is what the future holds.”
“Research in the field has positioned epigenomics as an essential player in understanding a disease,” says Manel Esteller, M.D., Ph.D., director of the Cancer Epigenetics and Biology Program (PEBC) in Barcelona. “Cancer has been the tip of the iceberg, but knowledge in cancer epigenetics is going to translate to other diseases. It is clear to me that there is no disease that is pure genetics, and there is no disease that is pure epigenetics. All diseases, from cancer to neurological disorders to cardiovascular conditions, are mixtures of genetics, epigenetics, and the environment.”
The Esteller lab focuses on enzymes that methylate particular genomic sequences and transcription factors binding these sequences. Insight into these aspects will ultimately allow us to comprehend the relationship between epigenetic modifications and human cancer. A smaller effort examines other human disorders with an epigenetic component such as Alzheimer’s disease and Rett syndrome.
Research in the Esteller lab revealed that O6-methylguanine-DNA methyltransferase (MGMT) promoter hypermethylation occurs early in certain malignant tumors, and identified this as a favorable independent predictor of the response to chemotherapy. These findings subsequently became the basis of an MGMT methylation assay that LabCorp recently introduced to assess responsiveness to cancer therapy.
Epigenomics-based diagnostic tools for early cancer detection represent an exciting development. Tumors shed their DNA into the blood, and epigenetic changes that occur early during tumorigenesis, sometimes even in premalignant lesions, can provide valuable biomarkers. “We think that methylation, as such, has a very good chance of revolutionizing early cancer diagnosis,” says Geert Nygaard, CEO of Epigenomics.
Previous research at Epigenomics identified Septin 9 as a single gene in which DNA methylation changes occur very early in colorectal cancer development and are present in the vast majority of tumors of all stages.
Epigenomics, in collaboration with Abbott Molecular Diagnostics, intends to launch a CE-Marked version of this blood test for colorectal cancer. “This represents a very concrete example of how epigenetics is going to change the diagnostics area,” says Achim Plum, Ph.D., svp for corporate development at Epigenomics.
It is essential to appreciate epigenetic testing for an additional benefit they provide over genetic tests. Specific mutations point toward an increased disease risk over an individual’s entire lifetime, but fail to provide additional information. “It is much more important to have a cancer detection biomarker that can actually find colorectal cancer early, rather than have markers that assess the lifetime risk of developing colorectal cancer,” says Nygaard, “That is not very actionable information.”
Medications that modify the epigenome are currently in various phases of testing. By using a technique that combines a genome-wide methylation assay, Methylated CpG island Amplification, with the Agilent promoter CpG array, Guillermo Garcia-Manero, M.D., associate professor of medicine at the MD Anderson Cancer Center and chief of the section of myelodysplastic syndromes in the department of leukemia, recently identified promoter-associated CpG islands that are hypermethylated in leukemias. This finding promises to improve our understanding of molecular pathways that are deregulated.
“We have been doing genome-wide methylation analysis, trying to identify new targets of DNA methylation. Once we identify those genes and validate them, we believe that this may give us new pathways that may be deregulated in this particular disease,” explains Dr. Garcia-Manero. Research in his lab is focusing on compounds such as vorinostat, 5-azacytidine, LB589, and MGCD0103, hypomethylating agents and histone deacetylase inhibitors that act as single or combination agents and are all in Phase I or II studies for treating leukemia and myelodysplastic syndrome.
While cancers represent the group of diseases with perhaps the strongest epigenetic component unveiled so far, epigenetic mechanisms extend to a much broader group of conditions and biological pathways. “We think that studies of epigenetic mechanisms in psychiatry are important for several reasons,” explains Eric J. Nestler, M.D., Ph.D., professor of neuroscience at Mount Sinai School of Medicine and director of the Mount Sinai Brain Institute.
“First, studying these mechanisms makes it possible, for the first time, to identify the ways in which drugs or stress regulate gene transcription in the brain in vivo. All prior research has focused on in vitro data, and studying transcriptional regulation in the brain was not possible. With the advent of chromatin biology, it is now possible to study transcriptional mechanisms in the brain, in vivo, in behaving animals. Second, we believe that changes in the epigenetic level may provide a unique mechanism by which stable and long-lasting changes can be induced in the brain.”
Research in the Nestler group focuses on epigenetic changes in two models. One of them, the social defeat model for stress, has great relevance to our understanding of molecular mechanisms involved in depression. Previous work conducted in the lab revealed that repeated aggression in mice leads to long-lasting aversion to social contacts, and gene profiling helped gain further insight into the molecular mechanisms involved. The other effort gravitates around the epigenetics of drug addiction, and revealed that both acute and repeated cocaine administration induce distinct modifications in the H3 and H4 histones associated with specific promoters in the brain.
In neuropsychiatric conditions, understanding epigenetic mechanisms raise hurdles beyond the regular challenges encountered for other medical conditions. “The challenge for psychiatry is that, in living patients, it is possible to study epigenetic changes only in peripheral tissues such as blood or fibroblasts, but we won’t be able to know what is going on epigenetically in one of our patients’ brains. And we don’t know yet the extent to which epigenetic changes in peripheral tissues will be an accurate marker of epigenetic changes in the brain,” explains Dr. Nestler.
It is widely accepted that both genetic and environmental contributions shape our development, behavior, or predisposition to diseases, but what exactly is the environment? Often, we immediately envision the chemical environment, the discrete chemical compounds that abound around and within our bodies, but we forget what could be the most important aspect. It appears that the social environment plays a crucial role, particularly early in life.
Part of the research in the laboratory led by Moshe Szyf, Ph.D., professor of pharmacology and therapeutics at McGill University School of Medicine, tries to elucidate how social environment impacts the epigenome.
“We believe that it is a kind of an adaptive process, in which the environment early in life anticipates the kind of life the person is going to live, for example whether it is going to be a stressful life or a calm life,” explains Dr. Szyf. “The mother can convey to the offspring the type of world they are going to live in; that changes DNA methylation in the brain, and now we know, also in peripheral cells.”
The Szyf lab recently demonstrated that hypermethylation of ribosomal RNA genes in the brain is intimately linked to the pathophysiology of suicide. Moreover, previous work in the lab revealed that offspring of mothers who had increased interactions with their pups exhibited epigenetic traits that persisted into adulthood.
“I think that what we are starting to see is that the social environment is much more powerful than the chemical environment. When we look at toxicology, we always consider toxicology as chemicals, but I think that social environment can be as toxic as the chemical environment, if not more so,” explains Dr. Szyf.
Understanding the importance of the social environment becomes even more important when examining animal models of disease in which many studies are conducted with animals deprived of their natural social interaction network.
Epigenomics is a testimony to the great strides witnessed in recent years, which helped bring this discipline into existence; it is has also become a harbinger of the immense unknown still waiting for answers in science. This blooming discipline provides the ideal opportunity to reflect on Lewis Thomas’ words which, although written decades ago, remain as meaningful today: “…now that we have begun exploring in earnest, doing serious science, we are getting glimpses of how huge the questions are, and how far from being answered.”