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Curation, HealthCare System in the US, and Calcium Signaling Effects on Cardiac Contraction, Heart Failure, and Atrial Fibrillation, and the Relationship of Calcium Release at the Myoneural Junction to Beta Adrenergic Release
Curator and e-book Contributor: Larry H. Bernstein, MD, FCAP
Curator and BioMedicine e-Series Editor-in-Chief: Aviva Lev Ari, PhD, RN
This portion summarises what we have covered and is now familiar to the reader. There are three related topics, and an extension of this embraces other volumes and chapters before and after this reading. This approach to the document has advantages over the multiple authored textbooks that are and have been pervasive as a result of the traditional publication technology. It has been stated by the founder of ScoopIt, that amount of time involved is considerably less than required for the original publications used, but the organization and construction is a separate creative process. In these curations we amassed on average five articles in one curation, to which, two or three curators contributed their views. There were surprises, and there were unfulfilled answers along the way. The greatest problem that is being envisioned is the building a vision that bridges and unmasks the hidden “dark matter” between the now declared “OMICS”, to get a more real perspective on what is conjecture and what is actionable. This is in some respects unavoidable because the genome is an alphabet that is matched to the mino acid sequences of proteins, which themselves are three dimensional drivers of sequences of metabolic reactions that can be altered by the accumulation of substrates in critical placements, and in addition, the proteome has functional proteins whose activity is a regulatory function and not easily identified. In the end, we have to have a practical conception, recognizing the breadth of evolutionary change, and make sense of what we have, while searching for more.
We introduced the content as follows:
1. We introduce the concept of curation in the digital context, and it’s application to medicine and related scientific discovery.
Topics were chosen were used to illustrate this process in the form of a pattern, which is mostly curation, but is significantly creative, as it emerges in the context of this e-book.
Alternative solutions in Treatment of Heart Failure (HF), medical devices, biomarkers and agent efficacy is handled all in one chapter.
PCI for valves vs Open heart Valve replacement
PDA and Complications of Surgery — only curation could create the picture of this unique combination of debate, as exemplified of Endarterectomy (CEA) vs Stenting the Carotid Artery (CAS), ischemic leg, renal artery stenosis.
2. The etiology, or causes, of cardiovascular diseases consist of mechanistic explanations for dysfunction relating to the heart or vascular system. Every one of a long list of abnormalities has a path that explains the deviation from normal. With the completion of the analysis of the human genome, in principle all of the genetic basis for function and dysfunction are delineated. While all genes are identified, and the genes code for all the gene products that constitute body functions, there remains more unknown than known.
3. Human genome, and in combination with improved imaging methods, genomics offers great promise in changing the course of disease and aging.
4. If we tie together Part 1 and Part 2, there is ample room for considering clinical outcomes based on individual and organizational factors for best performance. This can really only be realized with considerable improvement in information infrastructure, which has miles to go.
Curation
Curation is an active filtering of the web’s and peer reviewed literature found by such means – immense amount of relevant and irrelevant content. As a result content may be disruptive. However, in doing good curation, one does more than simply assign value by presentation of creative work in any category. Great curators comment and share experience across content, authors and themes.
Great curators may see patterns others don’t, or may challenge or debate complex and apparently conflicting points of view. Answers to specifically focused questions comes from the hard work of many in laboratory settings creatively establishing answers to definitive questions, each a part of the larger knowledge-base of reference. There are those rare “Einstein’s” who imagine a whole universe, unlike the three blindmen of the Sufi tale. One held the tail, the other the trunk, the other the ear, and they all said this is an elephant!
In my reading, I learn that the optimal ratio of curation to creation may be as high as 90% curation to 10% creation. Creating content is expensive. Curation, by comparison, is much less expensive. The same source says “Scoop.it is my content marketing testing “sandbox”. In sharing, he says that comments provide the framework for what and how content is shared.
Healthcare and Affordable Care Act
We enter year 2014 with the Affordable Care Act off to a slow start because of the implementation of the internet signup requiring a major repair, which is, unfortunately, as expected for such as complex job across the US, and with many states unwilling to participate. But several states – California, Connecticut, and Kentucky – had very effective state designed signups, separate from the federal system. There has been a very large rush and an extension to sign up. There are many features that we can take note of:
1. The healthcare system needed changes because we have the most costly system, are endowed with advanced technology, and we have inexcusable outcomes in several domains of care, including, infant mortality, and prenatal care – but not in cardiology.
2. These changes that are notable are:
The disparities in outcome are magnified by a large disparity in highest to lowest income bracket.
This is also reflected in educational status, and which plays out in childhood school lunches, and is also affected by larger class size and cutbacks in school programs.
This is not helped by a large paralysis in the two party political system and the three legs of government unable to deal with work and distraction.
Unemployment is high, and the banking and home construction, home buying, and rental are in realignment, but interest rates are problematic.
3. The medical care system is affected by the issues above, but the complexity is not to be discounted.
The medical schools are unable at this time to provide the influx of new physicians needed, so we depend on a major influx of physicians from other countries
The technology for laboratories, proteomic and genomic as well as applied medical research is rejuvenating the practice in cardiology more rapidly than any other field.
In fields that are imaging related the life cycle of instruments is shorter than the actual lifetime use of the instruments, which introduces a shortening of ROI.
Hospitals are consolidating into large consortia in order to maintain a more viable system for referral of specialty cases, and also is centralizing all terms of business related to billing.
There is reduction in independent physician practices that are being incorporated into the hospital enterprise with Part B billing under the Physician Organization – as in Partners in Greater Boston, with the exception of “concierge” medical practices.
There is consolidation of specialty laboratory services within state, with only the most specialized testing going out of state (Quest, LabCorp, etc.)
Medicaid is expanded substantially under the new ACA.
The federal government as provider of services is reducing the number of contractors for – medical devices, diabetes self-testing, etc.
The current rearrangements seeks to provide a balance between capital expenses and fixed labor costs that it can control, reduce variable costs (reagents, pharmaceutical), and to take in more patients with less delay and better performance – defined by outside agencies.
Cardiology, Genomics, and calcium ion signaling and ion-channels in cardiomyocyte function in health and disease – including heart failure, rhythm abnormalities, and the myoneural release of neurotransmitter at the vesicle junction.
This portion is outlined as follows:
2.1 Human Genome: Congenital Etiological Sources of Cardiovascular Disease
2.2 The Role of Calcium in Health and Disease
2.3 Vasculature and Myocardium: Diagnosing the Conditions of Disease
Genomics & Genetics of Cardiovascular Disease Diagnoses
disruption of Ca2+ homeostasis cardiac & vascular smooth muscle
synaptotagmin as Ca2+ sensor & vesicles
atherosclerosis & ion channels
It is increasingly clear that there are mutations that underlie many human diseases, and this is true of the cardiovascular system. The mutations are mistakes in the insertion of a purine nucleotide, which may or may not have any consequence. This is why the associations that are being discovered in research require careful validation, and even require demonstration in “models” before pursuing the design of pharmacological “target therapy”. The genomics in cardiovascular disease involves very serious congenital disorders that are asserted early in life, but the effects of and development of atherosclerosis involving large and medium size arteries has a slow progression and is not dominated by genomic expression. This is characterized by loss of arterial elasticity. In addition there is the development of heart failure, which involves the cardiomyocyte specifically. The emergence of regenerative medical interventions, based on pleuripotent inducible stem cell therapy is developing rapidly as an intervention in this sector.
Finally, it is incumbent on me to call attention to the huge contribution that research on calcium (Ca2+) signaling has made toward the understanding of cardiac contraction and to the maintenance of the heart rhythm. The heart is a syncytium, different than skeletal and smooth muscle, and the innervation is by the vagus nerve, which has terminal endings at vesicles which discharge at the myocyte junction. The heart specifically has calmodulin kinase CaMK II, and it has been established that calmodulin is involved in the calcium spark that triggers contraction. That is only part of the story. Ion transport occurs into or out of the cell, the latter termed exostosis. Exostosis involves CaMK II and pyruvate kinase (PKC), and they have independent roles. This also involves K+-Na+-ATPase. The cytoskeleton is also discussed, but the role of aquaporin in water transport appears elsewhere, as the transport of water between cells. When we consider the Gibbs-Donnan equilibrium, which precedes the current work by a century, we recall that there is an essential balance between extracellular Na+ + Ca2+ and the intracellular K+ + Mg2+, and this has been superceded by an incompletely defined relationship between ions that are cytoplasmic and those that are mitochondrial. The glass is half full!
What is the Future for Genomics in Clinical Medicine?
Author and Curator: Larry H Bernstein, MD, FCAP
Introduction
This is the last in a series of articles looking at the past and future of the genome revolution. It is a revolution indeed that has had a beginning with the first phase discovery leading to the Watson-Crick model, the second phase leading to the completion of the Human Genome Project, a third phase in elaboration of ENCODE. But we are entering a fourth phase, not so designated, except that it leads to designing a path to the patient clinical experience.
What is most remarkable on this journey, which has little to show in treatment results at this time, is that the boundary between metabolism and genomics is breaking down. The reality is that we are a magnificent “magical” experience in evolutionary time, functioning in a bioenvironment, put rogether like a truly complex machine, and with interacting parts. What are those parts – organelles, a genetic message that may be constrained and it may be modified based on chemical structure, feedback, crosstalk, and signaling pathways. This brings in diet as a source of essential nutrients, exercise as a method for delay of structural loss (not in excess), stress oxidation, repair mechanisms, and an entirely unexpected impact of this knowledge on pharmacotherapy. I illustrate this with some very new observations.
Gutenberg Redone
The first is a recent talk on how genomic medicine has constructed a novel version of the “printing press”, that led us out of the dark ages.
In our series The Creative Destruction of Medicine, I’m trying to get into critical aspects of how we can Schumpeter or reboot the future of healthcare by leveraging the big innovations that are occurring in the digital world, including digital medicine.
We have this big thing about evidence-based medicine and, of course, the sanctimonious randomized, placebo-controlled clinical trial. Well, that’s great if one can do that, but often we’re talking about needing thousands, if not tens of thousands, of patients for these types of clinical trials. And things are changing so fast with respect to medicine and, for example, genomically guided interventions that it’s going to become increasingly difficult to justify these very large clinical trials.
For example, there was a drug trial for melanoma and the mutation of BRAF, which is the gene that is found in about 60% of people with malignant melanoma. When that trial was done, there was a placebo control, and there was a big ethical charge asking whether it is justifiable to have a body count. This was a matched drug for the biology underpinning metastatic melanoma, which is essentially a fatal condition within 1 year, and researchers were giving some individuals a placebo.
The next observation is a progression of what he have already learned. The genome has a role is cellular regulation that we could not have dreamed of 25 years ago, or less. The role is far more than just the translation of a message from DNA to RNA to construction of proteins, lipoproteins, cellular and organelle structures, and more than a regulation of glycosidic and glycolytic pathways, and under the influence of endocrine and apocrine interactions. Despite what we have learned, the strength of inter-molecular interactions, strong and weak chemical bonds, essential for 3-D folding, we know little about the importance of trace metals that have key roles in catalysis and because of their orbital structures, are essential for organic-inorganic interplay. This will not be coming soon because we know almost nothing about the intracellular, interstitial, and intrvesicular distributions and how they affect the metabolic – truly metabolic events.
I shall however, use some new information that gives real cause for joy.
Reprogramming Alters Cells’ Fate
Kathy Liszewski
Gordon Conference Report: June 21, 2012;32(11)
New and emerging strategies were showcased at Gordon Conference’s recent “Reprogramming Cell Fate” meeting. For example, cutting-edge studies described how only a handful of key transcription factors were needed to entirely reprogram cells.
M. Azim Surani, Ph.D., Marshall-Walton professor at the Gurdon Institute, University of Cambridge, U.K., is examining cellular reprogramming in a mouse model. Epiblast stem cells are derived from the early-stage embryonic stage after implantation of blastocysts, about six days into development, and retain the potential to undergo reversion to embryonic stem cells (ESCs) or to PGCs.” They report two critical steps both of which are needed for exploring epigenetic reprogramming. “Although there are two X chromosomes in females, the inactivation of one is necessary for cell differentiation. Only after epigenetic reprogramming of the X chromosome can pluripotency be acquired. Pluripotent stem cells can generate any fetal or adult cell type but are not capable of developing into a complete organism.”
The second read-out is the activation of Oct4, a key transcription factor involved in ESC development. The expression of Oct4 in epiSCs requires its proximal enhancer. Dr. Surani said that their cell-based system demonstrates how a systematic analysis can be performed to analyze how other key genes contribute to the many-faceted events involved in reprogramming the germline.
Reprogramming Expressway
A number of other recent studies have shown the importance of Oct4 for self-renewal of undifferentiated ESCs. It is sufficient to induce pluripotency in neural tissues and somatic cells, among others. The expression of Oct4 must be tightly regulated to control cellular differentiation. But, Oct4 is much more than a simple regulator of pluripotency, according to Hans R. Schöler, Ph.D., professor in the department of cell and developmental biology at the Max Planck Institute for Molecular Biomedicine.
Oct4 has a critical role in committing pluripotent cells into the somatic cellular pathway. When embryonic stem cells overexpress Oct4, they undergo rapid differentiation and then lose their ability for pluripotency. Other studies have shown that Oct4 expression in somatic cells reprograms them for transformation into a particular germ cell layer and also gives rise to induced pluripotent stem cells (iPSCs) under specific culture conditions.
Oct4 is the gatekeeper into and out of the reprogramming expressway. By modifying experimental conditions, Oct4 plus additional factors can induce formation of iPSCs, epiblast stem cells, neural cells, or cardiac cells. Dr. Schöler suggests that Oct4 a potentially key factor not only for inducing iPSCs but also for transdifferention. “Therapeutic applications might eventually focus less on pluripotency and more on multipotency, especially if one can dedifferentiate cells within the same lineage. Although fibroblasts are from a different germ layer, we recently showed that adding a cocktail of transcription factors induces mouse fibroblasts to directly acquire a neural stem cell identity.
Stem cell diagram illustrates a human fetus stem cell and possible uses on the circulatory, nervous, and immune systems. (Photo credit: Wikipedia)
Transforming growth factor beta (TGF-β) is a secreted protein that controls proliferation, cellular differentiation, and other functions in most cells. http://en.wikipedia.org/wiki/TGFbeta (Photo credit: Wikipedia)
Pioneer Transcription Factors
Pioneer transcription factors take the lead in facilitating cellular reprogramming and responses to environmental cues. Multicellular organisms consist of functionally distinct cellular types produced by differential activation of gene expression. They seek out and bind specific regulatory sequences in DNA. Even though DNA is coated with and condensed into a thick fiber of chromatin. The pioneer factor, discovered by Prof. KS Zaret at UPenn SOM in 1996, he says, endows the competence for gene activity, being among the first transcription factors to engage and pry open the target sites in chromatin.
FoxA factors, expressed in the foregut endoderm of the mouse,are necessary for induction of the liver program. They found that nearly one-third of the DNA sites bound by FoxA in the adult liver occur near silent genes
A Nontranscriptional Role for HIF-1α as a Direct Inhibitor of DNA Replication
Many of the cellular responses to reduced O2 availability are mediated through the transcriptional activity of hypoxia-inducible factor 1 (HIF-1). We report a role for the isolated HIF-1α subunit as an inhibitor of DNA replication, and this role was independent of HIF-1β and transcriptional regulation. In response to hypoxia, HIF-1α bound to Cdc6, a protein that is essential for loading of the mini-chromosome maintenance (MCM) complex (which has DNA helicase activity) onto DNA, and promoted the interaction between Cdc6 and the MCM complex. The binding of HIF-1α to the complex decreased phosphorylation and activation of the MCM complex by the kinase Cdc7. As a result, HIF-1α inhibited firing of replication origins, decreased DNA replication, and induced cell cycle arrest in various cell types. To whom correspondence should be addressed. E-mail: gsemenza@jhmi.edu
Citation: M. E. Hubbi, Kshitiz, D. M. Gilkes, S. Rey, C. C. Wong, W. Luo, D.-H. Kim, C. V. Dang, A. Levchenko, G. L. Semenza, A Nontranscriptional Role for HIF-1α as a Direct Inhibitor of DNA Replication. Sci. Signal. 6, ra10 (2013).
Identification of a Candidate Therapeutic Autophagy-inducing Peptide
Beth Levine and colleagues have constructed a cell-permeable peptide derived from part of an autophagy protein called beclin 1. This peptide is a potent inducer of autophagy in mammalian cells and in vivo in mice and was effective in the clearance of several viruses including chikungunya virus, West Nile virus and HIV-1.
Could this small autophagy-inducing peptide may be effective in the prevention and treatment of human diseases?
PR-Set7 Is a Nucleosome-Specific Methyltransferase that Modifies Lysine 20 of
Histone H4 and Is Associated with Silent Chromatin
We have purified a human histone H4 lysine 20methyl-transferase and cloned the encoding gene, PR/SET07. A mutation in Drosophila pr-set7 is lethal: second in-star larval death coincides with the loss of H4 lysine 20 methylation, indicating a fundamental role for PR-Set7 in development. Transcriptionally competent regions lack H4 lysine 20 methylation, but the modification coincided with condensed chromosomal regions polytene chromosomes, including chromocenter euchromatic arms. The Drosophila male X chromosome, which is hyperacetylated at H4 lysine 16, has significantly decreased levels of lysine 20 methylation compared to that of females. In vitro, methylation of lysine 20 and acetylation of lysine 16 on the H4 tail are competitive. Taken together, these results support the hypothesis that methylation of H4 lysine 20 maintains silent chromatin, in part, by precluding neighboring acetylation on the H4 tail.
Next-Generation Sequencing vs. Microarrays
Shawn C. Baker, Ph.D., CSO of BlueSEQ
GEN Feb 2013
With recent advancements and a radical decline in sequencing costs, the popularity of next generation sequencing (NGS) has skyrocketed. As costs become less prohibitive and methods become simpler and more widespread, researchers are choosing NGS over microarrays for more of their genomic applications. The immense number of journal articles citing NGS technologies it looks like NGS is no longer just for the early adopters. Once thought of as cost prohibitive and technically out of reach, NGS has become a mainstream option for many laboratories, allowing researchers to generate more complete and scientifically accurate data than previously possible with microarrays.
Gene Expression
Researchers have been eager to use NGS for gene expression experiments for a detailed look at the transcriptome. Arrays suffer from fundamental ‘design bias’ —they only return results from those regions for which probes have been designed. The various RNA-Seq methods cover all aspects of the transcriptome without any a priori knowledge of it, allowing for the analysis of such things as novel transcripts, splice junctions and noncoding RNAs. Despite NGS advancements, expression arrays are still cheaper and easier when processing large numbers of samples (e.g., hundreds to thousands).
Methylation
While NGS unquestionably provides a more complete picture of the methylome, whole genome methods are still quite expensive. To reduce costs and increase throughput, some researchers are using targeted methods, which only look at a portion of the methylome. Because details of exactly how methylation impacts the genome and transcriptome are still being investigated, many researchers find a combination of NGS for discovery and microarrays for rapid profiling.
Diagnostics
They are interested in ease of use, consistent results, and regulatory approval, which microarrays offer. With NGS, there’s always the possibility of revealing something new and unexpected. Clinicians aren’t prepared for the extra information NGS offers. But the power and potential cost savings of NGS-based diagnostics is alluring, leading to their cautious adoption for certain tests such as non-invasive prenatal testing. Cytogenetics
Perhaps the application that has made the least progress in transitioning to NGS is cytogenetics. Researchers and clinicians, who are used to using older technologies such as karyotyping, are just now starting to embrace microarrays. NGS has the potential to offer even higher resolution and a more comprehensive view of the genome, but it currently comes at a substantially higher price due to the greater sequencing depth. While dropping prices and maturing technology are causing NGS to make headway in becoming the technology of choice for a wide range of applications, the transition away from microarrays is a long and varied one. Different applications have different requirements, so researchers need to carefully weigh their options when making the choice to switch to a new technology or platform. Regardless of which technology they choose, genomic researchers have never had more options.
Sequencing Hones In on Targets
Greg Crowther, Ph.D.
GEN Feb 2013
Cliff Han, PhD, team leader at the Joint Genome Institute in the Los Alamo National Lab, was one of a number of scientists who made presentations regarding target enrichment at the “Sequencing, Finishing, and Analysis in the Future” (SFAF) conference in Santa Fe, which was co-sponsored by the Los Alamos National Laboratory and DOE Joint Genome Institute. One of the main challenges is that of target enrichment: the selective sequencing of genomic or transcriptomic regions. The polymerase chain reaction (PCR) can be considered the original target-enrichment technique and continues to be useful in contexts such as genome finishing. “One target set is the unique gaps—the gaps in the unique sequence regions. Another is to enrich the repetitive sequences…ribosomal RNA regions, which together are about 5 kb or 6 kb.” The unique-sequence gaps targeted for PCR with 40-nucleotide primers complementary to sequences adjacent to the gaps, did not yield the several-hundred-fold enrichment expected based on previously published work. “We got a maximum of 70-fold enrichment and generally in the dozens of fold of enrichment,” noted Dr. Han.
“We enrich the genome, put the enriched fragments onto the Pacific Biosciences sequencer, and sequence the repeats,” continued Dr. Han. “In many parts of the sequence there will be a unique sequence anchored at one or both ends of it, and that will help us to link these scaffolds together.” This work, while promising, will remain unpublished for now, as the Joint Genome Institute has shifted its resources to other projects.
At the SFAF conference Dr. Jones focused on going beyond basic target enrichment and described new tools for more efficient NGS research. “Hybridization methods are flexible and have multiple stop-start sites, and you can capture very large sizes, but they require library prep,” said Jennifer Carter Jones, Ph.D., a genomics field applications scientist at Agilent. “With PCR-based methods, you have to design PCR primers and you’re doing multiplexed PCR, so it’s limited in the size that you can target. But the workflow is quick because there’s no library preparation; you’re just doing PCR.” She discussed Agilent’s recently acquired HaloPlex technology, a hybrid system that includes both a hybridization step and a PCR step. Because no library preparation is required, sequencing results can be obtained in about six hours, making it suitable for clinical uses. However, the hybridization step allows capture of targets of up to 5 megabases—longer than purely PCR-based methods can deliver. The Agilent talk also provided details on the applications of SureSelect, the company’s hybridization technology, to Methyl-Seq and RNA-Seq research. With this technology, 120-mer baits hybridize to targets, then are pulled down with streptavidin-coated magnetic beads.
These are selections from the SFAF conference, which is expected to be a boost to work on the microbiome, and lead to infectious disease therapeutic approaches.
Summary
We have finished a breathtaking ride through the genomic universe in several sessions. This has been a thorough review of genomic structure and function in cellular regulation. The items that have been discussed and can be studied in detail include:
the classical model of the DNA structure
the role of ubiquitinylation in managing cellular function and in autophagy, mitophagy, macrophagy, and protein degradation
the nature of the tight folding of the chromatin in the nucleus
intramolecular bonds and short distance hydrophobic and hydrophilic interactions
trace metals in molecular structure
nuclear to membrane interactions
the importance of the Human Genome Project followed by Encode
the Fractal nature of chromosome structure
the oligomeric formation of short sequences and single nucletide polymorphisms (SNPs)and the potential to identify drug targets
Enzymatic components of gene regulation (ligase, kinases, phosphatases)
Methods of computational analysis in genomics
Methods of sequencing that have become more accurate and are dropping in cost
Chromatin remodeling
Triplex and quadruplex models not possible to construct at the time of Watson-Crick
sequencing errors
propagation of errors
oxidative stress and its expected and unintended effects
In a three part series: Part IIA. CRACKING THE CODE OF HUMAN LIFE: Milestones along the Way Part IIB. CRACKING THE CODE OF HUMAN LIFE: The Birth of BioInformatics & Computational Genomics Part IIC. CRACKING THE CODE OF HUMAN LIFE: Recent Advances in Genomic Analysis and Disease
Part III will conclude with Ubiquitin, it’s Role in Signaling and Regulatory Control. Part I reviewed the huge expansion of the biological research enterprise after the Second World War. It concentrated on the
discovery of cellular structures,
metabolic function, and
creation of a new science of Molecular Biology.
Part II follows the race to delineation of the Human Genome, discovery methods and fundamental genomic patterns that are ancient in both animal and plant speciation. But it explores both the complexity and the systems view of the architecture that underlies and understanding of the genome.
These articles review a web-like connectivity between inter-connected scientific discoveries, as significant findings have led to novel hypotheses and many expectations over the last 75 years. This largely post WWII revolution has driven our understanding of biological and medical processes at an exponential pace owing to successive discoveries of
chemical structure,
the basic building blocks of DNA and proteins,
nucleotide and protein-protein interactions,
protein folding, allostericity,
genomic structure,
DNA replication,
nuclear polyribosome interaction, and
metabolic control.
In addition, the emergence of methods for
copying,
removal,
insertion,
improvements in structural analysis
developments in applied mathematics that have transformed the research framework.
Part IIA:
CRACKING THE CODE OF HUMAN LIFE:
Milestones along the Way
A NOVA interview with Francis Collins (NHGRI) (FC), J. Craig Venter (CELERA)(JCV), and Eric Lander (EL). RK: For the past ten years, scientists all over the world have been painstakingly trying to read the tiny instructions buried inside our DNA. And now, finally, the “Human Genome” has been decoded. EL: The genome is a storybook that’s been edited for a couple billion years. The following will address the odd similarity of genes between man and yeast
EL: In the nucleus of your cell the DNA molecule resides that is about 10 angstroms wide curled up, but the amount of curling is limited by the negative charges that repel one another, but there are folds upon folds. If the DNA is stretched the length of the DNA would be thousands of feet. EL: We have known for 2000 years that your kids look a lot like you. Well it’s because you must pass them instructions that give them the eyes, the hair color, and the nose shape they have. RK: Cracking the code of those minuscule differences in DNA that influence health and illness is what the Human Genome Project is all about. Since 1990, scientists all over the world have been involved in the effort to read all three billion As, Ts, Gs, and Cs of human DNA. It took 10 years to find the one genetic mistake that causes cystic fibrosis. Another 10 years to find the gene for Huntington’s disease. Fifteen years to find one of the genes that increase the risk for breast cancer. One letter at a time, painfully slowly… And then came the revolution. In the last ten years the entire process has been computerized. The computations can do a thousand every second and that has made all the difference. EL: This is basically a parts list with a lot of parts. If you take an airplane, a Boeing 777, I think it has like 100,000 parts. If I gave you a parts list for the Boeing 777 in one sense you’d know 100,000 components, screws and wires and rudders and things like that. But you wouldn’t know how to put it together, or why it flies. We now have a parts list, and that’s not enough to understand why it flies.
The Human Genome (Photo credit: dullhunk)
A Quest For Clarity
Tracy Vence is a senior editor of Genome Technology Tracy Vence @GenomeTechMag Projects supported by the US National Institutes of Health will have produced 68,000 total human genomes — around 18,000 of those whole human genomes — through the end of this year, National Human Genome Research Institute estimates indicate. And in his book, The Creative Destruction of Medicine, the Scripps Research Institute’s Eric Topol projects that 1 million human genomes will have been sequenced by 2013 and 5 million by 2014. Daniel MacArthur, a group leader in Massachusetts General Hospital’s Analytic and Translational Genetics Unit estimates that “From a capacity perspective … millions of genomes are not that far off. If you look at the rate that we’re scaling, we can certainly achieve that.” The prospect of so many genomes has brought clinical interpretation into focus. But there is an important distinction to be made between the interpretation of an apparently healthy person’s genome and that of an individual who is already affected by a disease. In an April Science Translational Medicine paper, Johns Hopkins University School of Medicine‘s Nicholas Roberts and his colleagues reported that personal genome sequences for healthy monozygotic twin pairs are not predictive of significant risk for 24 different diseases in those individuals. The researchers concluded that whole-genome sequencing was not likely to be clinically useful. Ambiguities have clouded even the most targeted interpretation efforts.
Technological challenges,
meager sample sizes,
a need for increased,
fail-safe automation and most important
a lack of community-wide standards for the task.
have hampered researchers’ attempts to reliably interpret the clinical significance of genomic variation.
How signals from the cell surface affect transcription of genes in the nucleus.
James Darnell, Jr., MD, Astor Professor, Rockefeller After graduation from Washington University School of Medicine he worked with Francois Jacob at the Pasteur Institute in Paris and served as Vice President for Academic Affairs at Rockefeller in 1990-91. He is the coauthor with S.E. Luria of General Virology and the founding author with Harvey Lodish and David Baltimore of Molecular Cell Biology, now in its sixth edition. His book RNA, Life’s Indispensable Molecule was published in July 2011 by Cold Spring Harbor Laboratory Press. A member of the National Academy of Sciences since 1973, recipient of numerous awards, including the 2003 National Medal of Science, the 2002 Albert Lasker Award. Using interferon as a model cytokine, the Darnell group discovered that cell transcription was quickly changed by binding of cytokines to the cell surface. The bound interferon led to the tyrosine phosphorylation of latent cytoplasmic proteins now called STATs (signal transducers and activators of transcription) that dimerize by
reciprocal phosphotyrosine-SH2 interchange.
accumulate in the nucleus,
bind DNA and drive transcription.
This pathway has proved to be of wide importance with seven STATs now known in mammals that take part in a wide variety of developmental and homeostatic events in all multicellular animals. Crystallographic analysis defined functional domains in the STATs, and current attention is focused on two areas:
how the STATs complete their cycle of activation and inactivation, which requires regulated tyrosine dephosphorylation; and how
persistent activation of STAT3 that occurs in a high proportion of many human cancers contributes to blocking apoptosis in cancer cells.
Current efforts are devoted to inhibiting STAT3 with modified peptides that can enter cells.
Cell cycle regulation and the cellular response to genotoxic stress
Stephen J Elledge, PhD, Gregor Mendel Professor of Genetics and Medicine, Investigator, Howard Hughes Medical Institute, Harvard Medical School As a postdoctoral fellow at Stanford working on eukaryotic homologous recombination, he serendipitously found a family of genes known as ribonucleotide reductases. He subsequently showed that
these genes are activated by DNA damage and
could serve as tools to help scientists dissect the signaling pathways
through which cells sense and respond to DNA damage and replication stress.
At Baylor College of Medicine he made a second major breakthrough with the discovery of the cyclin-dependent kinase 2 gene (Cdk2), which
controls the G1-to-S cell cycle transition,
an entry checkpoint for the cell proliferation cycle and
a critical regulatory step in tumorigenesis.
From there, using a novel “two-hybrid” cloning method he developed, Elledge and Wade Harper, PhD, proceeded to
isolate several members of the Cdk2-inhibitory family.
Their discoveries included the p21 and p57 genes, mutations in the latter (responsible for Beckwith-Wiedemann syndrome), characterized by somatic overgrowth and increased cancer risk. Elledge is also recognized for his work in understanding
proteome remodeling through ubiquitin-mediated proteolysis.
they identified F-box proteins that regulate protein degradation in the cell by
binding to specific target protein sequences and then
marking them with ubiquitin for destruction by the cell’s proteasome machinery.
This breakthrough resulted in
the elucidation of the cullin ubiquitin ligase family,
which controls regulated protein stability in eukaryotes.
Elledge’s recent research has focused on the cellular mechanisms underlying DNA damage detection and cancer using genetic technologies. In collaboration with Cold Spring Harbor Laboratory researcher Gregory Hannon, PhD, Elledge has generated complete human and mouse short hairpin RNA (shRNA) libraries for genome-wide loss-of-function studies. Their efforts have led to
the identification of a number of tumor suppressor proteins
genes upon which cancer cells uniquely depend for survival.
This work led to the development of the “non-oncogene addiction” concept. This is noted as follows:
proteome remodeling through ubiquitin-mediated proteolysis
F-box proteins regulate protein degradation in the cell by binding to specific target protein sequences
and then marking them with ubiquitin for destruction by the cell’s proteasome machinery
elucidation of the cullin ubiquitin ligase family, which controls regulated protein stability in eukaryotes
Playing the dual roles of inventor and investigator, Elledge developed original techniques to define
what drives the cell cycle and
how cells respond to DNA damage.
By using these tools, he and his colleagues have identified multiple genes involved in cell-cycle regulation.
Elledge’s work has earned him many awards, including a 2001 Paul Marks Prize for Cancer Research and a 2003 election to the National Academy of Sciences. In his Inaugural Article (1), published in this issue of PNAS, Elledge and his colleagues describe the function of Fbw7, a protein involved in controlling cell proliferation (see below). Elledge studied the error-prone DNA repair mechanism in E-Coli (Escherichia coli) called SOSmutagenesis for his PhD thesis at MIT. His work identified and described
the regulation of a group of enzymes now known as error-prone polymerases,
the first members of which were the umuCD genes in E. coli.
It was then that he developed a new cloning tool. Elledge invented a technique that allowed him to approach future cloning problems of this type with great rapidity. With the new technique, “you could make large libraries in lambda that behave like plasmids. We called them `phasmid’ vectors, like plasmid and phage together”. The phasmid cloning method was an early cornerstone for molecular biology research.
Elledge began working on homologous recombination in postdoctoral fellowship at Stanford University, an important niche in the field of eukaryotic genetics. Working with the yeast genome, Elledge searched for rec A, a gene that allows DNA to recombine homologously. Although he never located rec A, he discovered a family of genes known as ribonucleotide reductases (RNRs), which are involved in DNA production. Rec A and RNRs share the same last 4 amino acids, which caused an antibody crossreaction in one of Elledge’s experiments. Initially disappointed with the false positives in his hunt for rec A, Elledge was later delighted with his luck. He found that
RNRs are turned on by DNA damage, and
these genes are regulated by the cell cycle.
Prior to leaving Stanford, Elledge attended a talk at the University of California, San Francisco, by Paul Nurse, a leader in cell-cycle research who would later win the 2001 Nobel Prize in medicine. Nurse described his success in isolating the homolog of a key human cell-cycle kinase gene, Cdc2, by using a mutant strain of yeast (8). Although Nurse’s methods were primitive, Elledge was struck by the message he carried: that
cell-cycle regulation was functionally conserved, and
many human genes could be isolated by looking for complimentary genes in yeast.
Elledge then took advantage of his past successes in building phasmid vectors to build a versatile human cDNA library that could be expressed in yeast. After setting up a laboratory at Baylor, he introduced this library into yeast, screening for complimentary cell-cycle genes. He quickly identified the same Cdc2 gene isolated by Nurse. However, Elledge also discovered a related gene known as Cdk2. Elledge subsequently found that
Cdk2 controlled the G1 to S cell-cycle transition, a step that often goes awry in cancer. These results were published in the EMBO Journal in 1991.
He then continued to use
RNRs to perform genetic screens to
identify genes involved in sensing and responding to DNA damage.
He subsequently worked out the
signal transduction pathways in both yeast and humans that recognize damaged DNA and replication problems.
These “checkpoint” pathways are central to the
prevention of genomic instability and a key to understanding tumorigenesis.
This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected on April 29, 2003.
Defective cardiovascular development and elevated cyclin E and Notch proteins in mice lacking the Fbw7 F-box protein.
The mammalian F-box protein Fbw7 and its Caenorhabditis elegans counterpart Sel-10 have been implicated in
the ubiquitin-mediated turnover of cyclin E
as well as the Notch Lin-12 family of transcriptional activators. Both unregulated
Notch and cyclin E
promote tumorigenesis, and
inactivate mutations in human
Fbw7 studies suggest that it may be a tumor suppressor. To generate an in vivo system to assess the consequences of such unregulated signaling, we generated mice deficient for Fbw7. Fbw7-null mice die around 10.5 days post coitus because of a combination of deficiencies in hematopoietic and vascular development and heart chamber mutations. The absence of Fbw7 results in elevated levels of cyclin E, concurrent with inappropriate DNA replication in placental giant trophoblast cells. Moreover, the levels of both Notch 1 and Notch 4 intracellular domains were elevated, leading to stimulation of downstream transcriptional pathways involving Hes1, Herp1, and Herp2. These data suggest essential functions for Fbw7 in controlling cyclin E and Notch signaling pathways in the mouse.
Science as an Adventure
Ubiquitins
Prof. Avram Hershko – Science as an Adventure Prof. Avram Hershko shared the 2004 Nobel Prize in Chemistry with Aaron Ciechanover and Irwin Rose for “for the discovery of ubiquitin-mediated protein degradation.”
Nipam Patel is a professor in the Departments of Molecular and Cell Biology and Integrative Biology at UC Berkeley and runs a research laboratory that studies the role, during embryonic development, of homeotic genes (the genetic switches described in this feature). “Ghost in Your Genes” focuses on epigenetic “switches” that turn genes “on” or “off.” But not all switches are epigenetic; some are genetic. That is, other genes within the chromosome turn genes on or off. In an animal’s embryonic stage, these gene switches play a predominant role in laying out the animal’s basic body plan and perform other early functions;
the epigenome begins to take over during the later stages of embryogenesis.
Beginning as a fertilized single egg that egg becomes many different kinds of cells. Altogether, multicellular organisms like humans have thousands of differentiated cells. Each is optimized for use in the brain, the liver, the skin, and so on. Remarkably, the DNA inside all these cells is exactly the same. What makes the cells differ from one another is that different genes in that DNA are either turned on or off in each type of cell.
Take a typical cell, such as a red blood cell. Each gene within that cell has a coding region that encodes the information used to make a particular protein. (Hemoglobin shuttles oxygen to the tissues and carbon dioxide back out to the lungs—or gills, if you’re a fish.) But another region of the gene, called “regulatory DNA,” determines whether and when the gene will be expressed, or turned on, in a particular kind of cell. This precise transcribing of genes is handled by proteins known as transcription factors, which bind to the regulatory DNA, thereby generating instructions for the coding region.
One important class of transcription factors is encoded by the so called homeotic, or Hox, genes. Found in all animals, Hox genes act to “regionalize” the body along the embryo’s anterior-to-posterior (head-to-tail) axis. In a fruit fly, for example, Hox genes lay out the various main body segments—the head, thorax, and abdomen. Amazingly, all animals, from fruit flies to mice to people, rely on the same basic Hox-gene complex. Using different-colored antibody stains, we can see exactly where and to what degree Hox genes are expressed. Each Hox gene is expressed in a specific region along the anterior-to-posterior axis of the embryo.
A fly’s body has three main divisions: head, thorax, and abdomen. We’ll focus on the thorax, which itself has three main segments. In a normal adult fly, the second thoracic segment features a pair of wings, while the third thoracic segment has a pair of small, balloon-shaped structures called halteres. A modified second wing, the haltere serves as a flight stabilizer. In order for the pair of wings and the pair of halteres (as well as all other parts of the fly) to develop properly, the fly’s suite of
Hox genes must be expressed in a precise way and at precise times.
During development, the fly’s two wings grow from a structure in the larva known as the wing imaginal disk. (An imago is an insect in its final, adult state.) The haltere grows from the larval haltere imaginal disk. Remember the Ubx Hox gene? Using staining again, we can detect the gene product of Ubx. This reveals that
the Ubx gene is naturally “off” in the wing disk—
and is “on” in the haltere disk.
Now you’ll see what happens when the Ubx gene—just one of a large number of Hox genes—is turned off in the haltere disk. What if a genetic mutation caused the Ubx gene to be turned off, during the larval stage, in the third thoracic segment, the segment that normally produces the haltere? Instead of a pair of halteres, the fly has a second set of wings. With the switch of that single Hox gene, Ubx, from on to off, the third thoracic segment becomes an additional second thoracic segment and the pair of halteres became a second pair of wings. This illustrates the remarkable ability of transcription factors like Ubx to control patterning as well as cell type during development.
ENCODE
A. Data Suggests “Gene” Redefinition
As part of a huge collaborative effort called ENCODE (Encyclopedia of DNA Elements), a research team led by Cold Spring Harbor Laboratory (CSHL) Professor Thomas Gingeras, PhD, publishes a genome-wide analysis of RNA messages, called transcripts, produced within human cells. Their analysis—one component of a massive release of research results by ENCODE teams from 32 institutes in 5 countries, with 30 papers appearing in 3 different high-level scientific journals—shows that three-quarters of the genome is capable of being transcribed. This indicates that nearly all of our genome is dynamic and active. It stands in marked contrast to consensus views prior to ENCODE’s comprehensive research efforts, which suggested that
only the small protein-encoding fraction of the genome was transcribed.
The vast amount of data generated with advanced technologies by Gingeras’ group and others in the ENCODE project changes the prevailing understanding of what defines a gene. The current outstanding question concerns
the nature and range of those functions. It is thought that these
“non-coding” RNA transcripts act something like components of a giant, complex switchboard, controlling a network of many events in the cell by
regulating the processes of
replication,
transcription
and translation
– that is, the copying of DNA and the making of proteins is based on information carried by messenger RNAs. With the understanding that so much of our DNA can be transcribed into RNA comes the realization that there is much less space between what we previously thought of as genes, Gingeras points out.
The full ENCODE Consortium data sets can be freely accessed through
the ENCODE project portal as well as at the University of California at Santa Cruz genome browser,
the National Center for Biotechnology Information, and
the European Bioinformatics Institute.
Topic threads that run through several different papers can be explored via the ENCODE microsite page at http://Nature.com/encode. Date: September 5, 2012 Source: Cold Spring Harbor Laboratory
1000 Genomes Project Team Reports on Variation Patterns
(from Phase I Data) October 31, 2012 GenomeWeb
In a study appearing online today in Nature, members of the 1000 Genomes Project Consortium presented an integrated haplotype map representing the genomic variation present in more than 1,000 individuals from 14 human populations. Using data on 1,092 individuals tested by
low-coverage whole-genome sequencing,
deep exome sequencing, and/or
dense genotyping,
the team looked at the nature and extent of the rare and common variation present in the genomes of individuals within these populations. In addition to population-specific differences in common variant profiles, for example, the researchers found distinct rare variant patterns within populations from different parts of the world — information that is expected to be important in interpreting future disease studies. They also encountered a surprising number of the variants that are expected to impact gene function, such as
non-synonymous changes,
loss-of-function variants, and, in some cases,
potentially damaging mutations.
ENCODE was designed to pick up where the Human Genome Project left off. Although that massive effort revealed the blueprint of human biology, it quickly became clear that the instruction manual for reading the blueprint was sketchy at best. Researchers could identify in its 3 billion letters many of the regions that code for proteins, but they make up little more than 1% of the genome, contained in around 20,000 genes. ENCODE, which started in 2003, is a massive data-collection effort designed to catalogue the
‘functional’ DNA sequences,
learn when and in which cells they are active and
trace their effects on how the genome is
packaged,
regulated and
read.
After an initial pilot phase, ENCODE scientists started applying their methods to the entire genome in 2007. That phase came to a close with the publication of 30 papers, in Nature, Genome Research and Genome Biology. The consortium has assigned some sort of function to roughly 80% of the genome, including
more than 70,000 ‘promoter’ regions — the sites, just upstream of genes, where proteins bind to control gene expression —
and nearly 400,000 ‘enhancer’ regions that regulate expression of distant genes (see page 57)1. But the job is far from done.
proteins interact with the DNA to control gene expression.
Overall, the Encode data define regulatory switches that are scattered all over the three billion nucleotides of the genome. In fact, the data suggests,
the regions that lie between gene-coding sequences contain a wealth of previously unrecognized functional elements,Including
nonprotein-coding RNA transcribed sequences,
transcription factor binding sites,
chromatin structural elements, and
DNA methylation sites.
The combined results suggest that 95% of the genome lies within 8 kb of a DNA-protein interaction, and 99% lies within 1.7 kb of at least one of the biochemical events, the researchers say. Importantly, given the complex three-dimensional nature of DNA, it’s also apparent that
a regulatory element for one gene may be located quite some ‘linear’ distance from the gene itself.
“The information processing and the intelligence of the genome reside in the regulatory elements,” explains Jim Kent, director of the University of California, Santa Cruz Genome Browser project and head of the Encode Data Coordination Center. “With this project, we probably went from understanding less than 5% to now around 75% of them.” The ENCODE results also identified SNPs within regulatory regions that are associated with a range of diseases, providing new insights into the roles that
noncoding DNA plays in disease development.
“As much as nine out of 10 times, disease-linked genetic variants are not in protein-coding regions,” comments Mike Pazin, Encode program director at the National Human Genome Research Institute. “Far from being junk DNA, this regulatory DNA clearly makes important contributions to human disease.”
Other Related Articles on this Open Access Online Scientific Journal, include the following:
Impact of evolutionary selection on functional regions: The imprint of evolutionary selection on ENCODE regulatory elements is manifested between species and within human populations s Saha
ENCODE data reveals important information from Genome Wide Association Studies relevant to understanding complex genetic diseases
Author: Ritu Saxena, Ph.D.
Introduction
“The depth, quality, and diversity of the ENCODE data are unprecedented” is what was stated by John Stamatoyannopoulos, professor of genomic sciences at the University of Washington and one of the many principle investigators of ENCODE project. ENCODE (Encyclopedia of DNA elements), indeed, was an ambitious project launched as a pilot in 2003 and then expanded in 2007 for the whole genome analysis and identification of all the functional elements of the human genome. The findings were striking as they challenged the definition of “gene” and ‘the central dogma of genetics (Gene-mRNA-protein). Infact, the non-coding part that constitutes about 80% of the genome or the so-called “junk DNA” was found to contain elements crucial for gene regulation. The elements, in large part, include RNA transcripts that are not transcribed into proteins but might have a regulatory role. For detailed reading, refer to the findings published in the issue of Nature, The ENCODE Project Consortium Nature 489, 57–74 (2012) An integrated encyclopedia of DNA elements in the human genome
Protein-coding genes — Proteins are molecules made of amino acids linked together in a specific sequence; the amino acid sequence is encoded by the sequence of DNA subunits called nucleotides that make up genes.
Non-coding genes — Stretches of DNA that are read by the cell as if they were genes but do not encode proteins. These appear to help regulate the activity of the genome.
Chromatin structure features — Complex physical structures made from a combination of DNA and binding proteins that make up the contents of the nucleus and affects genome function.
Histone modifications — Histones are the proteins that make up the chromatin structures that help shape and control the genome. In addition, histone proteins can be physically modified by adding chemical groups, such as a methyl molecule, that further regulates genomic activity.
DNA methylation — Just like histones, methyl groups can be added to DNA itself in a process called DNA methylation. Chemically attaching methyl groups to DNA physically changes the ability of enzymes to reach the DNA and thus alters the gene expression pattern in cells. Methylation helps cells “remember what they are doing” or alter levels of gene expression, and it is a crucial part of normal development and cellular differentiation in higher organisms.
Transcription factor binding sites — Transcription factors are proteins that bind to specific DNA sequences, controlling the flow (or transcription) of genetic information from DNA to mRNA. Mapping the binding sites can help researchers understand how genomic activity is controlled.
How could ENCODE be helpful in the study of complex human diseases?
Complex diseases and Genome wide association studies (GWAS)
Coronary artery disease, type 2 diabetes and many forms of cancer are complex human diseases that have a significant genetic component. Unlike mendelian disorders that have defined loci, the genetic component of complex disorders lies in the form of genetic variations in the genome making an individual susceptible to these complex diseases.
Researchers have performed Genome-wide association studies (GWAS) of the human genome, leading to the identification of thousands of DNA variants that could be linked with complex traits and diseases. However, identifying the variants, referred to as SNPs (Single Nucleotide Polymorphisms), that actually contribute to the disease, and understanding how they exert influence on a disease has been more of a mystery.
How would ENCODE solve the puzzle?
The puzzle lies in interpreting how the SNPs found in the genome affect a person’s susceptibility to a particular trait or disease and what is the mechanism behind it. As identified in the GWAS, most variants that are associated with the phenotype of the trait or disease lie in the non-coding region of the genome. Infact, in more than 400 studies compiled in the GWAS catalog only a small minority of the trait/disease-associated SNPs occur in protein-coding regions; the large majority (89%) are in noncoding regions. These variants fall in the gene deserts that lie far from protein-coding region, similar to those where cis-regulatory modules (CRMs) are found. CRMs such as promoters and enhancers are a group of binding sites for transcription factors, and the presence of transcription factors bound to these sites is a good indicator of the potential regulatory regions.
The integrative analysis of ENCODE data has give important insights to the results of GWAS studies. Investigators have employed ENCODE data as an initial guide to discover regulatory regions in which genetic variation is affecting a complex trait. Additionally, ENCODE study when examined the SNPs from GWAS that were associated with the phenotype of the trait, found that these regions are enriched in DNase-sensitive regions i.e, lie in the function-associated DNA region of the genome as it could be bound by transcription factors affecting the regulation of gene expression. Thus, the project demonstrates that non-coding regions must be considered when interpreting GWAS results, and it provides a strong motivation for reinterpreting previous GWAS findings.
Using ENCODE Data to Interpret GWAS Results
ENCODE and predisposition to CANCER:
C-Myc, a proto-oncogene, codes for a transcripton factor, when expressed constitutively leads to uninhibited cell proliferation resulting in cancer. It has been observed that common variants within a ~1 Mb region upstream of c-Myc gene have been associated with cancers of the colon, prostate, and breast. Several SNPs have been reported in this region, that although affect the phenotype, lie in the distal cis-region of the MYC gene. Alignment of the ENCODE data in this region with the significant variants from the GWAS also reveals that key variants are found in the transcription factor occupied DNA segments mapped by this consortium. One variant rs698327, lies within a DNase hypersensitive site that is bound by several transcription factors, enhancer-associated protein p300, and contains histone modifications relative to enhancers (high H3K4me1, low H3K4me3). ENCODE data indicates that non-coding regions in the human chromosome 8q24 loci are associated with cancer and as observed in the case of c-myc gene, similar studies on cancer-related genes could help explain predisposition to cancer.
ENCODE and fetal hemoglobin expression:
Another example of the use of ENCODE data is that of gene regulation of fetal hemoglobin. Several regions were predicted via ENCODE that were involved in the regulation of fetal hemoglobin. It was found that these predicted regions are close to the SNPs in the BLC11A gene that is associated with persistent expression of fetal hemoglobin.
Future perspective
As evident from the above examples, the ENCODE data shows that genetic variants do affect regulated expression of a target gene. Recently, several research groups in the UK performed a large-scale GWAS study to determine the genetic predisposition to fracture risk. The collaborative effort, published in a recent issue of the PLoS journal, was made to identify genetic variants associated with cortical bone thickness (CBT) and bone mineral density (BMD) with data from more than 10,000 subjects. http://www.plosgenetics.org/article/info%3Adoi%2F10.1371%2Fjournal.pgen.1002745 The study generated a wealth of data including the result – identification of SNPs in the WNT16 and its adjacent gene, FAM3C were found to be relevant to CBT and BMD. ENCODE data, in this case, could be helpful in interpreting more detailed information including determining additional SNPs, the regulatory information of the genes involved and much more. Thus, it could be concluded that ENCODE data could be immensely useful in interpreting associations between disease and DNA sequences that can vary from person to person.
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