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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
CRACKING THE CODE OF HUMAN LIFE: Recent Advances in Genomic Analysis and Disease – Part IIC
Author: Larry H. Bernstein, MD, FCAP, Triplex Medical Science
Article 1.4 CRACKING THE CODE OF HUMAN LIFE: Recent Advances in Genomics Analysis and Disease – Part IIC
Part I: The Initiation and Growth of Molecular Biology and Genomics – Part I From Molecular Biology to Translational Medicine: How Far Have We Come, and Where Does It Lead Us?
Part IIB. “CRACKING THE CODE OF HUMAN LIFE: The Birth of BioInformatics & Computational Genomics” lays the manifold multivariate systems analytical tools that has moved the science forward to a groung that ensures clinical application.
Part IIC. “CRACKING THE CODE OF HUMAN LIFE: Recent Advances in Genomic Analysis and Disease “ will extend the discussion to advances in the management of patients as well as providing a roadmap for pharmaceutical drug targeting.
This final paper of Part II concludes a thorough review of the scientific events leading to the discovery of the human genome, the purification and identification of the components of the chromosome and the DNA structure and role in regulation of embryogenesis, and potential targets for cancer.
The first two articles, Part IIA, Part IIB, go into some depth to elucidate the problems and breakthoughs encountered in the Human Genome Project, and the construction of a 3-D model necessary to explain interactions at a distance.
Part IIC, the final article, is entirely concerned with clinical application of this treasure trove of knowledge to resolving diseases of epigenetic nature in the young and the old, chronic inflammatory diseases, autoimmune diseases, infectious disease, gastrointestinal disorders, neurological and neurodegenerative diseases, and cancer.
Recently, large studies have identified some of the genetic basis for important common diseases such as heart disease and diabetes, but most of the genetic contribution to them remains undiscovered. Now researchers at the University of Massachusetts Amherst led by biostatistician Andrea Foulkes have applied sophisticated statistical tools to existing large databases to reveal substantial new information about genes that cause such conditions as high cholesterol linked to heart disease.
Foulkes says, “This new approach to data analysis provides opportunities for developing new treatments.” It also advances approaches
to identifying people at greatest risk for heart disease. Another important point is that our method is straightforward to use with freely
available computer software and can be applied broadly to advance genetic knowledge of many diseases.
The new analytical approach she developed with cardiologist Dr. Muredach Reilly at the University of Pennsylvania and others is called “Mixed modeling of Meta-Analysis P-values” or MixMAP. Because it makes use of existing public databases, the powerful new method
represents a low-cost tool for investigators.
MixMAP draws on a principled statistical modeling framework and the vast array of summary data now available from genetic association
studies to formally test at a new, locus-level, association.
While that traditional statistical method looks for one unusual “needle in a haystack” as a possible disease signal, Foulkes and colleagues’
new method uses knowledge of DNA regions in the genome that are likely to
contain several genetic signals for disease variation clumped together in one region.
Thus, it is able to detect groups of unusual variants rather than just single SNPs, offering a way to “call out” gene
regions that have a consistent signal above normal variation.
The LPA gene codes for apolipoprotein(a), which, when linked with low-density lipoprotein particles, forms lipoprotein(a) [Lp(a)] —
a well-studied molecule associated with coronary artery disease (CAD). The Lp(a) molecule has both atherogenic and thrombogenic effects in vitro , but the extent to which these translate to differences in how atherothrombotic disease presents is unknown.
LPA contains many single-nucleotide polymorphisms, and 2 have been identified by previous groups as being strongly associated with
levels of Lp(a) and, as a consequence, strongly associated with CAD.
However, because atherosclerosis is thought to be a systemic disease, it is unclear to what extent Lp(a) leads to atherosclerosis in other arterial beds (eg, carotid, abdominal aorta, and lower extremity),
as well as to other thrombotic disorders (eg, ischemic/cardioembolic stroke and venous thromboembolism).
Such distinctions are important, because therapies that might lower Lp(a) could potentially reduce forms of atherosclerosis beyond the coronary tree.
To answer this question, Helgadottir and colleagues compiled clinical and genetic data on the LPA gene from thousands of previous
participants in genetic research studies from across the world. They did not have access to Lp(a) levels, but by knowing the genotypes for
2 LPA variants, they inferred the levels of Lp(a) on the basis of prior associations between these variants and Lp(a) levels. [1]
Their studies included not only individuals of white European descent but also a significant proportion of black persons, in order to
widen the generalizability of their results.
Their main findings are that LPA variants (and, by proxy, Lp(a) levels) are associated with
CAD,
peripheral arterial disease,
abdominal aortic aneurysm,
number of CAD vessels,
age at onset of CAD diagnosis, and
large-artery atherosclerosis-type stroke.
They did not find an association with
cardioembolic or small-vessel disease-type stroke;
intracranial aneurysm;
venous thrombosis;
carotid intima thickness; or,
in a small subset of individuals, myocardial infarction.
English: Structure of the LPA protein. Based on PyMOL rendering of PDB 1i71. (Photo credit: Wikipedia)
Micrograph of an artery that supplies the heart with significant atherosclerosis and marked luminal narrowing. Tissue has been stained using Masson’s trichrome. (Photo credit: Wikipedia)
Scientists at the Gladstone Institutes have revealed the precise order and timing of hundreds of genetic “switches” required to construct a fully
functional heart from embryonic heart cells — providing new clues into the genetic basis for some forms of congenital heart disease.
In a study being published online today in the journal Cell, researchers in the laboratory of Gladstone Senior Investigator Benoit Bruneau, PhD,
employed stem cell technology, next-generation DNA sequencing and computing tools to piece together the instruction manual, or “genomic
blueprint” for how a heart becomes a heart. These findings offer renewed hope for combating life-threatening heart defects such as arrhythmias (irregular heart beat) and ventricular septal defects (“holes in the heart”).
They approach heart formation with a wide-angle lens by
looking at the entirety of the genetic material that gives heart cells their unique identity.
The news comes at a time of emerging importance for the biological process called “epigenetics,” in which a non-genetic factor impacts a cell’s genetic
makeup early during development — but sometimes with longer-term consequences. All of the cells in an organism contain the same DNA, but the
epigenetic instructions encoded in specific DNA sequences give the cell its identity. Epigenetics is of particular interest in heart formation, as the
incorrect on-and-off switching of genes during fetal development can lead to congenital heart disease — some forms of which may not be apparent until adulthood.
the scientists took embryonic stem cells from mice and reprogrammed them into beating heart cells by mimicking embryonic development in a petri dish. Next, they extracted the DNA from developing and mature heart cells, using an advanced gene-sequencing technique called ChIP-seq that lets scientists “see” the epigenetic signatures written in the DNA.
Map of Heart Disease Death Rates in US White Males from 2000-2004 (Photo credit: Wikipedia)
Estimated propability of death or non-fatal myocardial-infarction over one year corresponding ti selectet values of the individual scores. Ordinate: individual score, abscissa: Propability of death or non-fatal myocardial infarction in 1 year (in %) (Photo credit: Wikipedia)
simply finding these signatures was only half the battle — we next had to decipher which aspects of heart formation they encoded
To do that, we harnessed the computing power of the Gladstone Bioinformatics Core. This allowed us to take the mountains of data collected from
gene sequencing and organize it into a readable, meaningful blueprint for how a heart becomes a heart.”
For each of the above datasets, an upstream analysis from the identified transcription factors correctly identified the stimulus. IPA’s tools were very
easy to use and the
analysis time for the above experiments was less than one minute.
The performance, speed, and ease of use can only be characterized as very good, perhaps leading to breakthroughs when extended and used creatively. Ingenuity’s new transcription factor analysis tool in IPA, coupled with Ingenuity’s established upstream grow tools, should be strongly considered for every lab analyzing differential expression data.
NF-E2-related factor 2 (Nrf2) is an important transcription factor that
activates the expression of cellular detoxifying enzymes.
Nrf2 expression is largely regulated through the association of Nrf2 with Kelch-like ECH-associated protein 1 (Keap1), which
results in cytoplasmic Nrf2 degradation.
Conversely, little is known concerning the regulation of Keap1 expression. Until now, a regulatory role for microRNAs (miRs) in controlling Keap1 gene expression had not been characterized. By using miR array-
based screening, we observed miR-200a silencing in breast cancer cells and
demonstrated that upon re-expression, miR-200a
targets the Keap1 3′-untranslated region (3′-UTR), leading to Keap1 mRNA degradation. Loss of this regulatory mechanism may
contribute to the dysregulation of Nrf2 activity in breast cancer. Previously, we have identified epigenetic repression of miR-200a
in breast cancer cells. Here, we find that treatment with epigenetic therapy, the histone deacetylase inhibitor suberoylanilide hydroxamic acid, restored miR-200a expression and reduced Keap1 levels. This reduction in Keap1 levels corresponded with
Nrf2 nuclear translocation
and activation of Nrf2-dependent NAD(P)H-quinone oxidoreductase 1 (NQO1) gene transcription.
Moreover, we found that Nrf2 activation inhibited the anchorage-independent growth of breast cancer cells. Finally, our in vitro observations were confirmed in a model of carcinogen-induced mammary hyperplasia in vivo. In conclusion, our study demonstrates
that miR-200a regulates the Keap1/Nrf2 pathway in mammary epithelium, and we find that epigenetic therapy can restore miR-200a
regulation of Keap1 expression,
reactivating the Nrf2-dependent antioxidant pathway in breast cancer.
Nuclear factor-like 2 (erythroid-derived 2, also known as NFE2L2 or Nrf2, is a transcription factor that in humans is encoded by the NFE2L2 gene.[1]) NFE2L2 induces the expression of various genes including those that encode for several antioxidant enzymes, and it may play a physiological role in the regulation of oxidative stress. Investigational drugs that target NFE2L2 are of interest as potential therapeutic interventions for
oxidative-stress related pathologies.
4. Highly active zinc finger nucleases by extended modular assembly
Zinc finger nucleases (ZFNs) are important tools for genome engineering. Despite intense interest by many academic groups,
the lack of robust non-commercial methods has hindered their widespread use. The modular assembly (MA) of ZFNs from
publicly-available one-finger archives provides a rapid method to create proteins that can recognize a very broad spectrum of DNA sequences.
However, three- and four-finger arrays often fail to produce active nucleases. Efforts to improve the specificity of the one-finger archives have not increased the success rate above 25%, suggesting that the MA method might
be inherently inefficient due to its insensitivity to context-dependent effects.
Here we present the first systematic study on the effect of array length on ZFN activity. ZFNs composed of six-finger MA arrays produced mutations at 15 of 21 (71%) targeted
loci in human and mouse cells. A novel Drop-Out Linker scheme was used to rapidly assess three- to six-finger combinations,
demonstrating that shorter arrays could improve activity in some cases. Analysis of 268 array variants revealed that half of
MA ZFNs of any array composition that exceed an ab initio
B-score cut-off of 15 were active.
MA ZFNs are able to target more DNA sequences with higher success rates than other methods.
These insightful reviews are based on the strategic data and insights from Thomson Reuters Cortellis™ for Competitive Intelligence. (A Review of April-June 2012).
The majority of diseases are complex and multi-factorial, involving multiple genes interacting with environmental factors. At the genetic level,
information from genome-wide association studies that elucidate common patterns of genetic variation across various human populations,
in addition to profiling, technologies can be utilized in discovery research to provide snapshots of genes and expression profiles that are controlled
by the same regulatory mechanism and are altered between healthy and diseased states.
The characterization of genes that are abnormally expressed in disease tissues could further be employed as
diagnostic markers,
prognostic indicators of efficacy and/or toxicity, or as
targets for therapeutic intervention.
As the defining catalyst that exponentially paved the way for personalized medicine, information from the published genome sequence revealed that much of the genetic variations in humans are concentrated in about 0.1 percent of the over 3 billion base pairs in the haploid DNA. Most of these variations involve substitution of a single nucleotide for another at a given location in the genetic sequence, known as single nucleotide polymorphism (SNP).
Combinations of linked SNPs aggregate together to form haplotypes and
together these serve as markers for locating genetic variations in DNA sequences.
SNPs located within the protein-coding region of a gene or within the control regions of DNA that regulate a gene’s activity could
have a substantial effect on the encoded protein and thus influence phenotypic outcomes.
Analyzing SNPs between patient population cohorts could highlight specific genotypic variations which can be correlated with specific phenotypic variations in disease predisposition and drug responses.
Prior to the genomic revolution, many of the established therapies were directed against less than 500 drug targets, with many of the top selling drugs acting on well defined protein pathways. However, the sequencing of the human genome has massively expanded the pool of molecular targets that could be exploited in unmet medical needs and currently, of the approximately 22,300 protein-coding genes in the human code, it has been estimated that up to 3000 are druggable. Furthermore, genomic technologies such as
high-throughput sequencing
and transcription profiling,
can be used to identify and validate biologically relevant target molecules, or can be applied to cell-based and mice disease models or directly to in vivo human tissues,
helping to correlate gene targets with phenotypic traits of complex diseases.
This is particularly important, as
insufficient validation of target gene/proteins in complex diseases may be a contributing factor in the decline in R&D productivity.
Personalized medicine no doubt is already having a tremendous impact on drug development pipelines. According to a study conducted by the Tufts Center for the Study of Drug Development, more than 90 percent of biopharmaceutical companies now utilize at least some
genomics-derived targets in their drug discovery programs.
However, pipeline analysis from Cortellis for Competitive Intelligence suggests that there is still a scientific gap that has resulted in difficulty optimizing these novel genomic targets into the clinical R&D portfolios of major pharmaceutical companies, particularly outside the oncology field. Selected examples of personalized medicine product candidates in clinical development include (see TABLE 4).
Mutations in Melanomaare in regions that control genes, not in the genes themselves. The mutations are exactly the type caused by exposure to ultraviolet light. The findings are reported in two papers in http://Science.com/ScienceExpress/
The findings do not suggest new treatments, but they help explain how melanomas – and possibly – other cancers – develop and what drives their growth. This is a modification found in the “dark matter”, according to Dr. Levi A. Garraway, the 99 percent of DNA in a region that regulates genes. A small control region was mutated in 7 out of 10 of the tumors, commonly of one or two tiny changes. A German Team led by Rajiv Kumar (Heidelberg) and Dirk Schadendorf (Essen) looked at a family whose members tended to get melanomas. Their findings indicate that those inherited with the mutations might be born with cells that have taken the first step toward cancer.
The mutations spur cells to make telomerase, that keeps the cells immortal by preventing them from losing the ends of their chromosome, the telomere. Abundant telomerase occurs in 90 percent of cancers, according to Immaculata De Vivo at Harvard Medical School.
The importance of the findings is that the mechanism of telomerase involvement in cancer is now within view. But it is not clear how to block the telomerase production in cancer cells.
A slight mutation in the matched nucleotides can lead to chromosomal aberrations and unintentional genetic rearrangement. (Photo credit: Wikipedia)
Comment
This discussion addresses the issues raised about the direction to follow in personalized medicine. Despite the amount of work necessary to bring the clarity that is sought after, the experiments and experimental design is most essential.
The arrest of ciliogenesis in ovarian cancer cell lines compared to wild type (WT) ovarian epithelial cells, and
The link to suppressing ciliogenesis by AURA protein and CHFR at the base of the cilium, which disappears at mitosis or with proliferation.
There is no accumulation by upregulation of PDGF under starvation by the cancer cells compared to the effect in WT OSE.
Here we have a systematic combination of signaling events tied to changes in putative biomarkers that occur synchronously in Ov cancer cell lines.
These changes are identified with changes in
proliferation,
loss of ciliary structure, and
proliferation.
In this described scenario,
WT OSE cells would be arrested, and
it appears that they would take the path to apoptosis (under starvation).
Even without more information, this cluster is what one wants to have in a “syndromic classification”. The information used to form the classification entails the identification of strong‘signaling-related’ biomarkers. The Gli2 peptide has to be part of this.
In principle, a syndromic classification would be ideally expected to have no less than 64 classes. If the classification is “weak”, then the class frequencies would be close to what one would expect in the WT OSE. In this case, in reality,
several combinatorial classes would have low frequency, and
others would be quite high.
This obeys the classification rules established by feature identification, and the information gain described by Solomon Kullback and extended by Akaike.
Does this have to be the case for all different cancer types? I don’t think so. The cells are different in ontogenesis. In this case, even the WT OSE have mesenchymal features and so, are not fully directed to epithelial expression. This happens to be the case in actual anatomic expression of the ovary. On the other hand, one would expect shared features of the
ovary,
testes,
thyroid,
adrenals, and
pituitary.
There is biochemical expression in terms of their synthetic function – TPN organs. I would have to put the liver into that broad class. Other organs – skeletal muscle & heart – transform substrate into energy or work. (Where you might also put intestinal smooth muscle).
They have to have different biomarker expressions, even though they much less often don’t form neoplasms. (Bone is not just a bioenergetic force. It is maintained by muscle action. It forms sarcomas. But there has to be a balance between bone removal by osteoclasts and refill by osteoblasts.)
Viewpoint: What we have learned
The Watson-Crick model proposed in 1953 is limited for explaining fully genome effects
The Pauling triplex model may have been prescient because of a more full anticipation of molecular bonding variants
A more adequate triple-helix model has been proposed and is consistent with a compact genome in the nucleus
The structure of the genome is not as we assumed – based on the application of Fractal Geometry. Current body of evidence is building that can reveal a more complete view of genome function.
transcription
cell regulation
mutations
Summary
I have just completed a most comprehensive review of the Human Genome Project. There are key research collaborations, problems in deciphering the underlying structure of the genome, and there are also both obstacles and insights to elucidating the complexity of the final model.
This is because of frequent observations of molecular problems in folding and other interactions between nucleotides that challenge the sufficiency of the original DNA model proposed by Watson and Crick. This has come about because of breakthrough innovation in technology and in computational methods.
Radoslav Bozov •
Molecular biology and growth was primarily initiated on biochemical structural paradigms aiming to define functional spatial dynamics of molecules via assignation of various types of bondings – covalent and non-covalent – hydrogen, ionic , dipole-dipole, hydrophobic interactions.
Lab techniques based on z/m paradigm allowed separation, isolation and identification of bio substances with a general marker identity finding correlation between physiological/cellular states.
The development of electronic/x-ray technologies allowed zooming in nano space without capturing time.
NMR technology identified the existence of space topology of initial and final atomic states giving a highly limited light on time – energy axis of atomic interactions.
Sequence technology and genomic perturbations shed light on uncertainty of genomic dynamics and regulators of functional ever expanding networks.
Transition state theory coupled to structural complexity identification and enzymatic mechanisms ran up parallel to work on various phenomena of strings of nucleotides (oligomers and polymers) – illusion/observation of constructing models on the dynamics of protein-dna-rna interference.
The physical energetic constrains of biochemistry were inapplicable in open biological systems. Biologists have accepted observation as a sole driver towards re-evaluating models.
The separation of matter and time constrains emerged as deviation of energy and space constrains transforming into the full acceptance of code theory of life. One simple thing was left unnoticed over time –
the amount of information of quantum matter within a single codon is larger than that of a single amino acid. This violated all physical laws/principles known to work with a limited degree of certainty.
The limited amount of information analyzed by conventional sequence identity led to the notion of applicability of statistical measures of and PCR technology. Mutations were identified over larger scale of data.
Quantum chemistry itself is being limited due discrete space/energy constrains, thus it transformed into concepts/principles in biology that possess highly limited physical values whatsoever.
The central dogma is partially broken as a result of
regulatory constrains
epigenetic phenomena and
iRNA.
Large scale code computational data run into uncertainty of the processes of evolution and its consequence of signaling transformation. All drugs were ‘lucky based’ applicability and/or discovery with largely unpredictable side effect over time.
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