Digital PCR found effective and reproducible in a research study

21 independent labs quantified a rare single nucleotide variant using digital PCR (dPCR) with high reproducibility between labs, demonstrating the method’s potential for routine clinical testing, according to a study published in Analytical Chemistry.

The research study is the first to examine the reproducibility of digital PCR between such a large number of laboratories at different geographic locations. The findings illustrate the inherent potential advantages of dPCR in measuring clinically important mutations, including greater reproducibility and less variability than real-time quantitative PCR (qPCR). In addition, the findings highlight the ability of dPCR to detect relevant rare sequence variants, according to the authors.

“Digital PCR lends itself to clinical testing where precise and reproducible results are critical not only for interlab data comparability, but also for dynamic testing in the same lab to track disease progression,” said George Karlin-Neumann, co-author and Director of Scientific Affairs at Bio-Rad’s Digital Biology Group.

The method permits precise and accurate quantification of nucleic acid concentration by minimizing the variability from common sources of error that can influence qPCR results. These sources include PCR inhibitors that can alter assay efficiency and skew standard curves. Digital PCR also provides absolute quantification of target nucleic acids without the need to establish standard curves, a major source of variability in qPCR.

The blinded study was part of the BioSITrace project, coordinated by the Laboratory of the Government Chemist (LGC), a private life sciences measurement and testing company and the UK’s designated National Measurement Laboratory for chemical and biomeasurement. Researchers in 21 laboratories across North America and Europe were asked to use dPCR to quantify copy number concentrations and fractional abundance of a KRAS mutant DNA with a single nucleotide variation (G12D) in the presence of excess wild-type DNA (down to ~0.2% minor allele frequency). The laboratories performed the experiments using Bio-Rad’s Droplet Digital™ PCR (ddPCR™) technology, on either a QX100™ or a QX200™ Droplet Digital PCR System from Bio-Rad. Droplet Digital PCR is Bio-Rad’s proprietary method for performing digital PCR in which a sample is divided into 20,000 water-oil emulsion droplets.

Cancer-derived mutations that differ only slightly from a wild-type sequence present in a sample in large excess are particularly difficult to detect and quantify using qPCR, especially when fractional abundance of the mutant is less than 5%.

The results of all 21 laboratories agreed with each other within a rigorously defined margin of error. Though three laboratories initially reported differing results, further analysis revealed methodological error attributed to misclassification of positive and negative droplets rather than measurement error. When the data were reanalyzed according to the recommended guidelines, they generated results consistent with the other 18 labs, validating the potential of dPCR for clinical testing applications.

Citation: Whale, Alexandra S., Alison S. Devonshire, George Karlin-Neumann, Jack Regan, Leanne Javier, Simon Cowen, Ana Fernandez-Gonzalez, Gerwyn M. Jones, Nicholas Redshaw, Julia Beck, Andreas W. Berger, Valérie Combaret, Nina Dahl Kjersgaard, Lisa Davis, Frederic Fina, Tim Forshew, Rikke Fredslund Andersen, Silvia Galbiati, Álvaro González Hernández, Charles A. Haynes, Filip Janku, Roger Lacave, Justin Lee, Vilas Mistry, Alexandra Pender, Anne Pradines, Charlotte Proudhon, Lao H. Saal, Elliot Stieglitz, Bryan Ulrich, Carole A. Foy, Helen Parkes, Svilen Tzonev, and Jim F. Huggett. “International Interlaboratory Digital PCR Study Demonstrating High Reproducibility for the Measurement of a Rare Sequence Variant.” Analytical Chemistry 89, no. 3 (2017): 1724-733.
Adapted from press release by Bio-Rad.

Novel and unique DNA vaccine to fight effectively against cancer antigen

Scientists at The Wistar Institute and Inovio pharmaceuticals, Inc. have devised a unique deoxyribonucleic acid (DNA) vaccine approach through molecular design to boost the immune responses induced against one of the most important cancer antigen targets. Research results were published in the journal Molecular Therapy.

Cancer immunotherapy approaches, designed to harness the body’s natural immune defenses to focus on and kill cancer cells, are showing great promise for cancer treatment and prevention. DNA vaccines can induce immunity through the delivery by an intramuscular injection of a sequence of synthetically designed DNA that contains the instructions for the immune cells in the body to become activated and target a particular antigen against which an immunologic response is wanted.

Despite being specific for cancer cells, cancer tumor-associated antigens generally trigger weak immune responses as a result of they’re recognized as self-antigens and also the body has in place natural mechanisms of immune acceptance, or “Tolerance”, that forestall autoimmunity and also additionally limit the efficacy of cancer vaccines. This is often the case of Wilm’s tumor gene 1 (WT1), a cancer tumor antigen that’s overexpressed in many varieties of cancer and possibly plays a key role in driving tumor development. vaccine approaches against WT1 thus far haven’t appeared promising because of immune tolerance leading to poor immune responses against cancers expressing WT1.

Wistar scientists have developed a unique WT1 deoxyribonucleic acid (DNA) vaccine employing a strategically changed DNA sequence that tags the WT1 as foreign to the host immune system breaking tolerance in animal models.

“This is an important time in the development of anti-DNA cancer immune therapy approaches. This team has developed an approach that may play an important role in generating improved immunity to WT1 expressing cancers,”said David B. Weiner, Ph.D., Executive Vice President and Director of the Vaccine Center at The Wistar Institute and the W.W. Smith Charitable Trust Professor in Cancer Research, and senior author of the study.”These immune responses represent a unique tool for potentially treating patients with multiple forms of cancer. Our vaccine also provides an opportunity to combine this approach with another immune therapy approach, checkpoint inhibitors, to maximize possible immune therapy impact on specific cancers.”

The team lead by Weiner has optimized the dna vaccine employing a artificial dna sequence for WT1 that, while maintaining a very high similarity with the native sequence, contains new modified sequences that differ from native WT1 in an attempt to render it more recognizable by the host immune system. The novel WT1 vaccine was superior to a more traditional native WT1 vaccine because it was able to break immune tolerance and induce long-term immune memory. Significantly, the vaccine also stimulated a therapeutic anti-tumor response against leukemia in mice.

Citation: Walters, Jewell N., Bernadette Ferraro, Elizabeth K. Duperret, Kimberly A. Kraynyak, Jaemi Chu, Ashley Saint-Fleur, Jian Yan, Hy Levitsky, Amir S. Khan, Niranjan Y. Sardesai, and David B. Weiner. “A Novel DNA Vaccine Platform Enhances Neo-antigen-like T Cell Responses against WT1 to Break Tolerance and Induce Anti-tumor Immunity.” Molecular Therapy, 2017. doi:10.1016/j.ymthe.2017.01.022.
Research funding: Inovio Pharmaceuticals, Inc. Basser Center for BRCA/Abramson Cancer CenterWeiner, W.W. Smith Charitable Trust Professorship for Cancer Research.
Adapted from press release by The Wistar Institute.

Obesity genes: role of foraging gene in fruit fly

A University of Toronto study on fruit flies has uncovered a gene that could play a key role in obesity in humans. The paper published online this month in Genetics examines a “foraging gene” humans share in common with the flies, which plays multiple roles and is found in similar places, such as the nervous system, in the muscle and in fat.

“What our study does is nails the gene for being very important for the traits of moving, feeding and fat storage,” says University Professor Marla Sokolowski of the Department of Ecology & Evolutionary Biology in U of T’s Faculty of Arts & Science.

In nature, fruit flies called “Rovers” with high amounts of the gene tend to move a lot, eat very little and stay lean, while flies with low amounts of for called “Sitters” are the opposite. The same could apply to obesity in humans.

“So you could imagine if you are a fly, preferences for sugar, the tendency to store a lot of fat and the tendency to move less could all be contributing to the likelihood of being more obese if you have low levels of this gene, or to be leaner if you have higher levels.”

Such similarities between species are known as orthologs, meaning they are genes that evolved from a common ancestor eons ago.

The study involved a technique called recombineering to manipulate DNA at the molecular level, so as to remove and then reinsert the gene in various doses to see the effects on behavior and metabolism.

“To be able to take a gene of this large size and complexity and put it back in the fly so that it works is almost at the edge of what is possible,” Sokolowski says it’s particularly interesting that one gene should have multiple roles in feeding and obesity in the body, a characteristic known as pleiotropy.

The next question would be how exactly it plays multiple roles. “Lots of genes have multiple roles, but the idea here is that this gene may be involved in the coordination of roles in traits important for feeding and obesity.”

 “We don’t know much about pleiotropy, or how it happens, or how it’s regulated at the level of the molecules. So this sets the groundwork to be able to look at that in detail.”

Citation: Allen, Aaron M., Ina Anreiter, Megan C. Neville, and Marla B. Sokolowski. “Feeding-Related Traits Are Affected by Dosage of the foraging Gene in Drosophila melanogaster.” Genetics 205, no. 2 (2016): 761-73.
Research funding: Natural Sciences and Engineering Council of Canada, Canadian Institutes for Health Research.
Adapted from press release by the University of Toronto.

New approach to treating dementia using antisense oligonucleotides

In a study of mice and monkeys, NIH funded researchers showed that they could prevent and reverse some of the brain injury caused by the toxic form of a protein called tau.

Scientists used a designer compound to prevent and reverse brain damage caused by tau in mice.
Credit: Miller lab, Washington University, St. Louis, MO

The results, published in Science Translational Medicine, suggest that the study of compounds, called tau antisense oligonucleotides, that are genetically engineered to block a cell’s assembly line production of tau, might be pursued as an effective treatment for a variety of disorders. Antisense oligonucleotides are short sequences of DNA or RNA that are programmed to turn genes on or off.

In several disorders, toxic forms of tau clump together inside dying brain cells and form neurofibrillary tangles, including Alzheimer’s disease, tau-associated frontotemporal dementia, chronic traumatic encephalopathy and progressive supranuclear palsy.

Researchers tested sequences designed to turn tau genes off in mice that are genetically engineered to produce abnormally high levels of a mutant form of the human protein. Tau clusters begin to appear in the brains of 6-month-old mice and accumulate with age. The mice develop neurologic problems and die earlier than control mice.

Injections of the tau antisense oligonucleotides into the fluid-filled spaces of the mice brains prevented tau clustering in 6-9-month-old mice and appeared to reverse clustering in older mice. The compound prevented the older mice from losing their ability to build nests. Further experiments on non-human primates suggested that the antisense oligonucleotides tested in mice could reach important areas of larger brains and turn off tau. In comparison with placebo, two spinal tap injections of the compound appeared to reduce tau protein levels in the brains and spinal cords of Cynomologus monkeys. As the researchers saw with the mice, injections of the compound caused almost no side effects.

Currently, researchers are conducting early phase clinical trials on the safety and effectiveness of antisense oligonucleotides designed to treat several neurological disorders, including Huntington’s disease and amyotrophic lateral sclerosis. The U.S. Food and Drug Administration recently approved the use of an antisense oligonucleotide for the treatment of spinal muscular atrophy, a hereditary disorder that weakens the muscles of infants and children.

Citation: DeVos, Sarah L., Rebecca L. Miller, Kathleen M. Schoch, Brandon B. Holmes, Carey S. Kebodeaux, Amy J. Wegener, Guo Chen et al. “Tau reduction prevents neuronal loss and reverses pathological tau deposition and seeding in mice with tauopathy.” Science Translational Medicine 9, no. 374 (2017): eaag0481.
DOI: 10.1126/scitranslmed.aag0481
Research funding: National Institutes of Health, Tau Consortium, Cure PSP.
Adapted from press release by National Institute of Neurological Disorders and Stroke.

Obesity related Epigenetic changes in DNA

Obesity has been linked to “letter” changes at many different sites in the genome, yet these differences do not fully explain the variation in people’s body mass index (BMI) or why some overweight people develop health complications while others don’t. A large study from Boston Children’s Hospital, the University of Edinburgh, the Harvard School of Public Health, the Framingham Heart Study and the National Heart, Lung, and Blood Institute (NHLBI) provides more insight, linking obesity with epigenetic modifications to DNA that in turn are tied to an increased risk of weight-related health problems such as coronary artery disease.

The study is one of the largest to date to examine the link between BMI, obesity-related disease and DNA methylation  a type of epigenetic modification that influences whether genes are turned on or off. Findings were published in PLoS Medicine.

“Even though we’ve genetically sequenced more and more people at greater and greater breadth and depth, we haven’t completely explained who develops obesity and why,” says Michael Mendelson, MD, ScM, a pediatric cardiologist with the Preventive Cardiology Program at Boston Children’s Hospital, who shared first authorship on the paper with Riccardo Marioni of the University of Edinburgh. “We found that obesity is related to widespread changes in DNA methylation. Unlike your DNA sequence, these regulatory modifications change over time and can influence your risk of disease in later life.”

The researchers studied blood samples from 7,800 adults from the Framingham Heart Study, the Lothian Birth Cohort and three other population studies. They systematically looked for markers of DNA methylation at more than 400,000 sites in the genome. They then looked to see if these markers differed according to BMI in a predictable pattern.

Their analysis identified strong associations between BMI and DNA methylation at 83 locations in 62 different genes. Methylation at these sites was, in turn, associated with differences in the expression of genes involved in energy balance and lipid metabolism.

When Mendelson and colleagues scored people in the study for how many methylation changes they had, they found that the more changes, the greater their BMI. The methylation score captured 18 percent of the variation in BMI when tested in a separate population. For each standard deviation increase in the score, the odds ratio for obesity was 2.8 times higher.

The researchers then applied a statistical technique called Mendelian randomization, which provides supportive evidence that a detected association is causal. They concluded that 16 of the 83 identified sites in the genome were differently methylated as a result of obesity, a finding that held true across people of different ethnicities.

Difference in methylation at one gene, SREBF1, appeared to be causative of obesity and was clearly linked with unhealthy blood lipid profiles, glycemic traits (a risk factor for diabetes) and coronary artery disease. It encodes a known regulator of lipid metabolism and could be a target for a drug treatment, the researchers say.

“Taken together, these results suggest that epigenetic modifications may help identify therapeutic targets to prevent or treat obesity-related disease in the population,” says Mendelson, who is also a research fellow in the Population Sciences Branch of the NHLBI. “The next step is to understand how we can modify epigenetic modifications to prevent the development of cardiometabolic disease.”
Since the study was done in blood cells, it also suggests that with further study, methylation markers could be easily accessible biomarkers to guide therapy bringing a “precision medicine” approach to preventive cardiology, says Mendelson.

“We’ve known for a long time that people who are overweight or obese are more likely to develop metabolic risk factors like diabetes, lipid abnormalities and hypertension,” adds study coauthor Daniel Levy, MD. He is director of the Framingham Heart Study, which is supported by the NHLBI. “This study may help us understand the molecular mechanism linking obesity to metabolic risk, and that knowledge may pave the way for new approaches to prevent even more dire complications such as cardiovascular disease.”

Citation: Michael M. Mendelson, Riccardo E. Marioni, Roby Joehanes, Chunyu Liu, Åsa K. Hedman, Stella Aslibekyan, Ellen W. Demerath, Weihua Guan, Degui Zhi, Chen Yao, Tianxiao Huan, Christine Willinger, Brian Chen, Paul Courchesne, Michael Multhaup, Marguerite R. Irvin, Ariella Cohain, Eric E. Schadt, Megan L. Grove, Jan Bressler, Kari North, Johan Sundström, Stefan Gustafsson, Sonia Shah, Allan F. McRae, Sarah E. Harris, Jude Gibson, Paul Redmond, Janie Corley, Lee Murphy, John M. Starr, Erica Kleinbrink, Leonard Lipovich, Peter M. Visscher, Naomi R. Wray, Ronald M. Krauss, Daniele Fallin, Andrew Feinberg, Devin M. Absher, Myriam Fornage, James S. Pankow, Lars Lind, Caroline Fox, Erik Ingelsson, Donna K. Arnett, Eric Boerwinkle, Liming Liang, Daniel Levy and Ian J. Deary. “Association of Body Mass Index with DNA Methylation and Gene Expression in Blood Cells and Relations to Cardio metabolic Disease: A Mendelian Randomization Approach.”
DOI: 10.1371/journal.pmed.1002215

Research funding: National Heart, Lung, and Blood Institute of the NIH, Tommy Kaplan Fund (Department of Cardiology, Boston Children’s Hospital), UK Biotechnology and Biological Sciences Research Council, UK Royal Society, Chief Scientist Office of the Scottish Government, Age UK, Wellcome Trust Institutional Strategic Support Fund, UK Economic and Social Research Council, UK Medical Research Council, Australian National Health and Medical Research Council.

Adapted from press release by Boston Children’s Hospital.

Role of retroviruses in evolution of human brain

Over millions of years, retroviruses have been incorporated into our human DNA, where they today make up almost 10 percent of the total genome. A research group at Lund University in Sweden has now discovered a mechanism through which these retroviruses may have an impact on gene expression. This means that they may have played a significant role in the development of the human brain as well as in various neurological diseases.

Retroviruses are a special group of viruses including some which are dangerous, such as HIV, while others are believed to be harmless. The viruses studied by Johan Jakobsson and his colleagues in Lund are called endogenous retroviruses (ERV) as they have existed in the human genome for millions of years. They can be found in a part of DNA that was previously considered unimportant, so-called junk-DNA a notion that researchers have now started to reconsider.

The genes that control the production of various proteins in the body represent a smaller proportion of our DNA than endogenous retroviruses. They account for approximately 2 per cent, while retroviruses account for 8-10 per cent of the total genome. If it turns out that they are able to influence the production of proteins, this will provide us with a huge new source of information about the human brain”, says Johan Jakobsson.

And this is precisely what the researchers discovered. They have determined that several thousands of the retroviruses that have established themselves in our genome may serve as “docking platforms” for a protein called TRIM28. This protein has the ability to “switch off” not only viruses but also the standard genes adjacent to them in the DNA helix, allowing the presence of ERV to affect gene expression.

This switching-off mechanism may behave differently in different people since retroviruses are a type of genetic material that may end up in different places in the genome. This makes it a possible tool for evolution, and even a possible underlying cause of neurological diseases. In fact, there are studies that indicate a deviating regulation of ERV in several neurological diseases such as ALS, schizophrenia, and bipolar disorder.

Two years ago, Johan Jakobsson’s team showed that ERV had a regulatory role in neurons specifically. However, this study was conducted on mice, whereas the new study published in the journal Cell Reports was made using human cells.

The differences between mice and humans are particularly important in this context. Many of the retroviruses that have been built into the human DNA do not exist in species other than humans and our closest relatives gorillas and chimpanzees. They seem to have incorporated themselves into the genome some 35-45 million years ago when the evolutionary lineage of primates was divided between the Old and New World.

“Much of what we know about the overall development of the brain comes from the fruit fly, zebrafish, and mouse. However, if endogenous retroviruses affect brain function, and we have our own set of these ERV, the mechanisms they affect may have contributed to the development of the human brain”, says Johan Jakobsson.

Citation: Brattås, Per Ludvik, Marie E. Jönsson, Liana Fasching, Jenny Nelander Wahlestedt, Mansoureh Shahsavani, Ronny Falk, Anna Falk, Patric Jern, Malin Parmar, Johan Jakobsson. “TRIM28 Controls a Gene Regulatory Network Based on Endogenous Retroviruses in Human Neural Progenitor Cells.” Cell reports Volume 18, Issue 1, p1–11.
DOI: 10.1016/j.celrep.2016.12.010
Adapted from press release by Lund University.

Effects of telomeres length on stem cells

Ever since researchers connected the shortening of telomeres the protective structures on the ends of chromosomes to aging and disease, the race has been on to understand the factors that govern telomere length. Now, Scientists at the Salk Institute have found that a balance of elongation and trimming in stem cells results in telomeres that are not too short and not too long, but just right.

Immunofluorescence analysis of pluripotent markers Nanog (red) and TRA-1-60 (green) in human induced pluripotent stem cells derived from skin fibroblasts. DNA is shown in blue. Credit: Salk Institute.

The finding, which appears in Nature Structural & Molecular Biology, deepens our understanding of stem cell biology and could help advance stem cell-based therapies, especially related to aging and regenerative medicine.

“This work shows that the optimal length for telomeres is a carefully regulated range between two extremes,” says Jan Karlseder, a professor in Salk’s Molecular and Cell Biology Laboratory and senior author of the work. “It was known that very short telomeres cause harm to a cell. But what was totally unexpected was our finding that damage also occurs when telomeres are very long.”

Karlseder, Rivera, and colleagues began by investigating telomere maintenance in laboratory-cultured lines of human embryonic stem cells (ESCs). Using molecular techniques, they varied telomerase activity. Perhaps not surprisingly, cells with too little telomerase had very short telomeres and eventually the cells died. Conversely, cells with augmented levels of telomerase had very long telomeres. But instead of these cells thriving, their telomeres developed instabilities.

The team observed that very long telomeres activated trimming mechanisms controlled by a pair of proteins called XRCC3 and Nbs1. The lab’s experiments show that reduced expression of these proteins in ESCs prevented telomere trimming, confirming that XRCC3 and Nbs1 are indeed responsible for that task.

Next, the team looked at induced pluripotent stem cells (iPSCs), which are differentiated cells (e.g., skin cells) that are reprogrammed back to a stem-cell-like state. They looked at induced pluripotent stem cells (iPSCs) because they can be genetically matched to donors and are easily obtainable–are common and crucial tools for potential stem cell therapies. The researchers discovered that induced pluripotent stem cells (iPSCs) contain markers of telomere trimming, making their presence a useful gauge of how successfully a cell has been reprogrammed.

“Stem cell reprogramming is a major scientific breakthrough, but the methods are still being perfected. Understanding how telomere length is regulated is an important step toward realizing the promise of stem cell therapies and regenerative medicine,” says Rivera.

Citation: Rivera, Teresa, Candy Haggblom, Sandro Cosconati, and Jan Karlseder. “A balance between elongation and trimming regulates telomere stability in stem cells.” Nature Structural & Molecular Biology (2016).
DOI: 10.1038/nsmb.3335
Adapted from press release by Salk Institute.

New approach to treating Obesity with fat burning molecule ABX300

A small molecule ABX300 could provide valuable help in combating the global epidemic of obesity. When it was fed to obese mice, the animals’ metabolism sped up and their excess weight was shed. It is doing so by recruiting the help of a body’s own genes in countering the effects of a high-fat diet. The research team conducting the study believes their findings may provide a new unexplored therapeutic approach to fighting excessive weight gain in cases where diets or exercise have no effect. The study was led by Julien Santo, Celia Lopez-Herrera and Cécile Apolit of a French biotechnology company, and is published in Springer Nature’s International Journal of Obesity.

A high-fat diet may contribute to obesity in some individuals. Treatment in such situations has focused on behavioral changes, which is highly challenging to achieve for the general population on a long term basis. This study introduces the concept of recruiting the help of our genes in countering the effects of a high-fat diet, instead of focusing on reducing the intake of high-fat food.
Researchers know that the structure of some genes that help to produce certain proteins can actually change when someone constantly eats too much high-fat food. In the process, the person can become overweight or obese, or develop other lifestyle-related metabolic disorders such as diabetes or heart problems. In many cases, the same gene can produce two or more alternate proteins, based on how the translation from DNA (gene) to proteins is processed. One of these genes is LMNA, which plays a role in the development of different metabolic disorders. The LMNA RNA, which is the genetic material resulting from a process called DNA transcription, is modified by three SR proteins called SRSFI, SRSF5, and SRSF6. In this process called splicing, the genetic material encoded in the RNA is basically diced or shifted around and therefore alters the resulting proteins.

The research team found that a small molecule ABX300 is able to change the way one particular SR protein, SRSFI, works in the bodies of mice that gained excessive weight after being fed a high-fat diet. SRSFI determines which of the gene products of opposing effects could be produced from a single LMNA gene. One gene product promotes fat storage, the other opposes it. This study showed that blocking SRSFI with a compound promotes gene expression of the protein that burns calories and prevents fat gain or induces fat loss when mice are on a high-fat diet. It did not have any effect on lean mice of normal weight.

According to the research team, this approach alters the animals’ metabolic rate or energy expenditure. It means that it can speed up the metabolism of obese animals and that their bodies start to function at a higher energy level shedding the excess weight. In the process, their bodies started to burn much more fat, as especially fatty acids serve as much-needed sources of energy.

“The results of this animal study show that this molecule can abrogate or do away with the effect of a high-fat diet,” says Santo.

“Dietary management and exercise are not always successful as an intervention for obesity, underscoring the need for efficient medication to treat metabolic disorders,” adds Lopez-Herrera, who believes that this treatment represents an as yet unexplored approach to treating obesity.

According to Apolit, this compound did not seem to have any adverse effects, so more research in animals and eventual research in humans is needed. If the studies are positive, this may be a new way to treat obesity.

Citation: Santo J, Lopez-Herrera C, Apolit C, Bareche Y, Lapasset L, et. al. “Pharmacological modulation of LMNA SRSF1-dependent splicing abrogates diet-induced obesity in mice”. International Journal of Obesity 2016.
DOI: 10.1038/ijo.2016.220
Adapted from press release by Springer.

Dutch universities collaborate on big data in health to understand disease process

Patients with the same illness often receive the same treatment, even if the cause of the illness is different for each person. This represents a new step towards ultimately being able to offer every patient more personalized treatment.

Six Dutch universities are combining forces to chart the different disease processes for a range of common conditions. This represents a new step towards ultimately being able to offer every patient more personalized treatment. The results of this study have been published in two articles in the authoritative scientific journal Nature Genetics.

The researchers were able to make their discoveries thanks to new techniques that make it possible to simultaneously measure the regulation and activity of all the genes of thousands of people, and to link these data to millions of genetic differences in their DNA. The combined analysis of these ‘big data’ made it possible to determine which molecular processes in the body become dysregulated for a range of disparate diseases, from prostate cancer to ulcerative bowel disease, before the individuals concerned actually become ill.

“The emergence of ‘big data’, ever faster computers and new mathematical techniques means it’s now possible to conduct extremely large-scale studies and gain an understanding of many diseases at the same time,” explains Lude Franke (UMCG), head of the research team in Groningen. The researchers show how thousands of disease-related DNA differences disrupt the internal working of a cell and how their effect can be influenced by environmental factors. And all this was possible without the need for a single lab experiment.

The success of this research is the result of the decision taken six years ago by biobanks throughout the Netherlands to share data and biomaterials within the BBMRI consortium. This decision meant it became possible to gather, store and analyze data from blood samples of a very large number of volunteers. The present study illustrates the tremendous value of large-scale collaboration in the field of medical research in the Netherlands.

Heijmans (LUMC), research leader in Leiden and initiator of the partnership: “The Netherlands is leading the field in sharing molecular data. This enables researchers to carry out the kind of large-scale studies that are needed to gain a better understanding of the causes of diseases. This result is only just the beginning: once they have undergone a screening, other researchers with a good scientific idea will be given access to this enormous bank of anonymized data. Our Dutch ‘polder mentality’ is also advancing science.”

Mapping the various molecular causes for a disease is the first step towards a form of medical treatment that better matches the disease process of individual patients. To reach that ideal, however, we still have a long way to go. The large-scale molecular data that have been collected for this research are the cornerstone of even bigger partnerships, such as the national Health-RI initiative. The third research leader, Peter-Bram ’t Hoen (LUMC), says: “Large quantities of data should eventually make it possible to give everyone personalized health advice, and to determine the best treatment for each individual patient.”

The research has been made possible thanks to the cooperation within the BBMRI biobank consortium of six long-running Dutch population studies carried out by the university medical centres in Groningen (LifeLines), Leiden (Leiden Longevity Study), Maastricht (CODAM Study), Rotterdam (Rotterdam Study), Utrecht (Netherlands Prospective ALS Study) and by the Vrije Universiteit (Netherlands Twin Register). The molecular data were generated in a standardized fashion at a central site (Human Genomics Facility HuGE-F, ErasmusMC) and subsequently securely stored and analyzed at a second central site (SURFSara). The study links in with the Personalised Medicine route of the National Research Agenda and the Health-RI and M3 proposals on the large-scale research infrastructure agenda of the Royal Netherlands Academy of Arts and Sciences (KNAW).

1. Bonder, Marc Jan, René Luijk, Daria Zhernakova, Matthijs Moed, Patrick Deelen, Martijn Vermaat, Maarten van Iterson et al. “Disease variants alter transcription factor levels and methylation of their binding sites.” bioRxiv (2015): 033084. Nature Genetics 2016.
DOI: 10.1038/ng.3721

2.  Zhernakova, Daria V,  Patrick Deelen, Martijn Vermaat, Maarten van Iterson, Michiel van Galen, Wibowo Arindrarto et al. “Identification of context-dependent expression quantitative trait loci in whole blood”. Nature Genetics 2016.
DOI: doi:10.1038/ng.3737

Adapted from press release by Leiden University.

Pandemic strain blamed for re-emergence of Syphilis

Syphilis has plagued humankind for over 500 years. After the first reported outbreaks struck Europe in 1495, the disease spread rapidly to other continents and swelled to a global pandemic. When treatment with the antibiotic penicillin became available in the mid-twentieth century, infection rates started to decrease dramatically. Strikingly, however, infection with the bacteria Treponema pallidum subsp. pallidum (TPA) has been re-emerging globally in the last few decades; more than 10 million cases are reported annually. Yet the reason for the resurgence of this sexually transmitted infection remains poorly understood.

According to the authors of the paper, little is known about the patterns of genetic diversity in current infections or the evolutionary origins of the disease. Because clinical samples from syphilis patients only contain low quantities of treponemal DNA and the pathogen is difficult to culture in the laboratory, researchers from the University of Zurich decided in 2013 to apply DNA capture and whole-genome sequencing techniques, as used by colleagues at the University of Tübingen, to ancient DNA samples. The team collected 70 clinical and laboratory samples of syphilis, yaws, and bejel infections from 13 countries spread across the globe. Like syphilis bacteria, the closely related subspecies Treponema pallidum subsp. pertenue (TPE) and Treponema pallidum subsp. endemicum (TEN), which cause yaws and bejel, are transmitted through skin contact and show similar clinical manifestations.

Immunofluorescence photomicrograph of Treponema bacteria
in human tissue. Credit: Steven J. Norris, UTHealth
McGovern Medical School, Houston/USA
By using genome-wide data, the researchers were able to reconstruct a phylogenetic tree showing a clear separation between the TPA lineage and the TPE/TEN lineage. “There have been many questions regarding the origin of syphilis since its appearance on the world stage 500 years ago. By combining an evolutionary and an epidemiological approach, we were able to decipher the genetic relation between strains infecting individuals today, and also trace the emergence of a pandemic cluster with high frequency of antibiotic resistance”, says Homayoun C. Bagheri, former professor at the UZH Institute for Evolutionary Biology and Environmental Studies.

Current syphilis infections predominantly due to resistant strains from a pandemic cluster. The genomic analyses show the emergence of a pandemic cluster named SS14-Ω, which is present in contemporary infections around the globe and distinct from the cluster comprising the well-studied Nichols reference strain. “Our findings highlight the need to study more extensively the predominant strain type in the contemporary epidemic”, states Natasha Arora, researcher at the Zurich Institute of Forensic Medicine and first author of the study published in Nature Microbiology.

An evolutionary finding of epidemiological relevance is that the SS14-Ω cluster originated from a strain ancestor in the mid-20th century – after the discovery of antibiotics. The worrying aspect of this pandemic cluster is its high resistance to azithromycin, a second-line drug that is widely used to treat sexually transmitted infections. Natasha Arora adds: “The good news is that, so far, no Treponema strains have been detected that are resistant to penicillin, the first-line antibiotic for syphilis treatment.”

Co-author Philipp Bosshard from the University Hospital Zurich is continuing to collect Swiss patient samples in order to further study the clinical aspects of the work. The researchers are convinced that this type of analysis will open new opportunities to develop a comprehensive understanding of the epidemiology of syphilis – a devastating disease that persists to this day, despite the availability of treatment.

Citation: “Origin of modern syphilis and emergence of a pandemic Treponema pallidum cluster”. 
Natasha Arora, Verena J. Schuenemann, Günter Jäger, Alexander Peltzer, Alexander Seitz, Alexander Herbig, Michal Strouhal, Linda Grillová, Leonor Sánchez-Busó, Denise Kühnert, Kirsten I. Bos, Leyla Rivero Davis, Lenka Mikalová, Sylvia Bruisten, Peter Komericki, Patrick French, Paul R. Grant, María A. Pando, Lucía Gallo Vaulet, Marcelo Rodríguez Fermepin, Antonio Martinez, Arturo Centurion Lara, Lorenzo Giacani, Steven J. Norris, David Šmajs, Philipp P. Bosshard, Fernando González-Candelas, Kay Nieselt, Johannes Krause & Homayoun C. Bagheri. Nature Microbiology 2016 vol: 2 pp: 16245
DOI: 10.1038/nmicrobiol.2016.245