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Why ‘Good Hair’ Matters

Why ‘Good Hair’ Matters

The first animal model of recent human evolution reveals that a mutation for thick hair does much more

By R. ALAN LEO
Thu, 2013-02-14

The first animal model of recent human evolution reveals that a single mutation produced several traits common in East Asian peoples, from thicker hair to denser sweat glands, an international team of researchers reports.

The team, led by researchers from Harvard Medical School, Harvard University, the Broad Institute of MIT and Harvard, Massachusetts General Hospital, Fudan University and University College London, also modeled the spread of the gene mutation across Asia and North America, concluding that it most likely arose about 30,000 years ago in what is today central China. The findings are reported in the cover story of the Feb. 14 issue of Cell.

“This interdisciplinary approach yields unique insight into the generation of adaptive variation among modern humans,” said Pardis Sabeti, associate professor in the Center for Systems Biology and Department of Organismic and Evolutionary Biology at Harvard University, and one of the paper’s senior authors. Sabeti is also a senior associate member at the Broad Institute.

“This paper tells a story about human evolution in three parts,” said Cliff Tabin, head of the HMS Department of Genetics and co-senior author. “The mouse model links multiple traits to a single mutation, the related association study finds these traits in humans, and computer models tell us where and when the mutation likely arose and spread.”

Previous research in Sabeti’s lab had identified the mutation as a strong candidate for positive selection. That is, evidence within the genetic code suggested the mutant gene conferred an evolutionary advantage, though what advantage was unclear.

The ectodysplasin receptor EDAR is a protein linked to hair and skin glands. A single genetic mutation yields a subtle change with a host of effects. Sabeti Lab imageLike humans, mice engineered with the variant EDAR gene grew thicker hairs. Photo by Yana Kamberov/Tabin LabThe mice with the variant gene also had denser eccrine, or sweat, glands. That led the team to search for—and find—the same trait in humans. Photo by Yana Kamberov/Tabin Lab The mutation was found in a gene for ectodysplasin receptor, or EDAR, part of a signaling pathway known to play a key role in the development of hair, sweat glands and other skin features. While human populations in Africa and Europe had one, ancestral, version of the gene, most East Asians had a derived variant, EDARV370A, which studies had linked to thicker scalp hair and an altered tooth shape in humans.

The ectodysplasin pathway is highly conserved across vertebrates — the same genes do the same thing in humans and mice and zebrafish. For that reason, and because its effects on skin, hair and scales can be observed directly, it is widely studied.

This evolutionary conservation led Yana Kamberov, one of two first authors on the paper, to reason that EDARV370A would exert similar biological effects in an animal model as in humans. The HMS research fellow in genetics developed a mouse model with the exact mutation of EDARV370A — a difference of one DNA letter from the original, or wild-type, population. That mouse manifested thicker hair, more densely branched mammary glands and an increased number of eccrine, or sweat, glands.

“This not only directly pointed us to the subset of organs and tissues that were sensitive to the mutation, but also gave us the key biological evidence that EDARV370A could have been acted on by natural selection,” Kamberov said.

The findings prompted the team to look for similar traits in human populations. When co-first author Sijia Wang and the team including collaborators at Fudan examined the fingertips of Chinese volunteers at colleges and farming villages, they found that the sweat glands of Han Chinese, who carry the derived variant of the gene, were packed about 15 percent more densely than those of a control population with the ancestral variant.

At the same time, Wang and the team including collaborators at University College London were working to zero in on when and where the mutation arose. Computer models suggested that the derived variant of the gene emerged in central China between 13,175 and 39,575 years ago, with a median estimate of 30,925 years. Researchers concluded the derived variant is at least 15,000 years old, predating the migration from Asia by Native Americans, who also carry the mutation.

That time span suggests that different traits could have been under selection at different times. The mutation’s many effects, known as pleiotropy, only complicate the question. If changes to the sweat glands conferred an advantage in new climates — one of the theories the researchers plan to explore further — changes to hair and to mammary glands could have conferred other advantages at other times.

Not all of these advantages need be direct effects on fitness. “When Pardis started this work, I would not have predicted that a gene that makes good hair would top a list of mutations that confer evolutionary advantage among humans,” said Bruce Morgan, HMS associate professor of dermatology at Massachusetts General Hospital and co-senior author on the paper. “However, in this case ‘good hair’ may have a biological meaning because it is genetically linked to a physiologically adaptive trait like increased sweating capacity. A cultural preference for a physically obvious trait like hair type could have arisen because individuals with it were more successful, and this would help increase selection on the new variant.”

“That (pleiotropy) makes it harder for us to make a guess,” Wang said. “If there were only one associated trait, we could say with confidence that’s where the selective advantage comes from. But with many traits, we don’t know which is the target of selection, and which are just hitchhiking.” Wang intends to focus on that question in his new role, as a Max Planck independent research group leader in dermatogenomics at Chinese Academy of Sciences – Max Planck Partner Institute for Computational Biology in Shanghai.

By leveraging the power of diverse fields, the team is piecing together the foundation for understanding how selected mutations like EDARV370A have impacted human diversity. But, they say, this is only the beginning.

“These findings point to what mutations, when, where and how,” said Daniel Lieberman, a professor of human evolutionary biology at Harvard University and a co-senior author on the study. “We still want to know why.”

From left: Senior authors Cliff Tabin, Dan Lieberman, Bruce Morgan and Pardis Sabeti talk with first authors Sijia Wang and Yana Kamberov. Sabeti Lab photo.

The research was funded by the Harvard University Science and Engineering Committee Seed Fund for Interdisciplinary Science; NIH grants R37 HD032443 and R37 054364; NIH Innovator Award 1DP2OD006514-01; NIAMS BIRT Award AR055256-04S1; the Packard Foundation; the American School of Prehistoric Research; NSFC 30890034; MOST 2011BAI09B00; MOH 201002007; AXA Research Fund; and a LeCHE Marie Curie FP7framework.

 

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Of Men, Not Mice

Of Men, Not Mice

Mouse models fail to reproduce inflammatory genomic response to serious injuries

By SUE McGREEVEY
Mon, 2013-02-11

Hand with third-degree burn. Photo by Plougmann/iStock

Existing mouse models do not appear to accurately reproduce the human genomic response to serious traumatic injury, including major burns, according to an article appearing in PNAS Early Edition

The report from a national consortium investigating the role of inflammation in the body’s response to injury finds little correlation between the human response to burns, trauma or a bacterial toxin and that of currently used mouse models for those conditions. The authors noted that their results cannot be applied to the use of mouse models for other research purposes.

“Our findings question the validity of using mouse models to mimic inflammatory conditions in humans,” said Shaw Warren, HMS associate professor of pediatrics at Massachusetts General Hospital and a co-lead author of the report.

 “An additional finding is that the whole-genome responses to these conditions in humans correlated well with each other, suggesting that treatments developed for an inflammatory disease from one cause might also work for inflammatory diseases with different causes,” Warren said.

The study is part of the Inflammation and the Host Response to Injury consortium, established in 2001 to investigate how the human body responds to injury, with particular attention to factors that set off excessive, uncontrolled inflammation.

Based at Mass General, the program includes investigators from 20 academic research centers around the country and is led by Ronald G. Tompkins, HMS Sumner M. Redstone Professor of Surgery and director of the Sumner Redstone Burn Center at Mass General. He is a co-corresponding author of the current report.

In 2011, the group reported finding that serious injuries set off a “genomic storm” in the human body, altering around 80 percent of normal gene expression patterns. The current study drew on information from that study and others conducted by the consortium to compare the human genomic response to inflammatory disease with that of mouse models. 

The investigators from Mass General, the Stanford University Genome Technology Center and several other research centers combined data from four of their studies of genomic responses to systemic inflammation: two in burn or trauma patients and volunteers treated with a bacterial toxin that produces brief flu-like symptoms and two studies of the responses in mouse models of the three conditions. 

While the responses among human patients were very similar, showing changes in the expression of more than 5,500 genes, there was very little correlation with the expression patterns of corresponding genes in the mouse models.  Not only was the human genomic response to inflammatory injury much greater—affecting the expression of more than three times as many genes as in the models—but it also lasted longer, up to six months in humans compared with a few days at most in mice.

To confirm their findings, the investigators analyzed data from an additional 20 studies of acute inflammatory disease—10 in humans and 10 in mice—and found a similar lack of correlation between the response of human patients and the mouse models. In all the human studies, the genomic responses were very similar, despite differences in patient age, gender, type and severity of injury or illness, treatment and outcomes.

“Mice have been used in biomedical research for well over 50 years, in part because of the cost, size, convenience, ease of genetic manipulation and social acceptability. But it is often forgotten that mice appear to be much more resistant to inflammation and infection than humans,” said Warren. 

“By studying and understanding the mechanisms by which mice differ from humans, we may be able to develop treatments that help make humans more resistant to damaging inflammation. We also hope that our article will start a broader discussion among scientists, research organizations, journals and granting and regulatory agencies as to the value of mouse models in different specific circumstances,” Warren said.

The study was supported by Defense Advanced Research Projects Agency grant W9111NF-10-1-0271; National Institute on Disability and Rehabilitation Research grant H133A070026; National Institute of General Medical Sciences grants 5P50GM060338, 5R01GM056687, R24GM102656 and 5U54GM062119; and National Human Genome Research Institute grant P01HG000205.

Sue McGreevey is manager for science & research communications at Mass General Public Affairs.

Adapted from a Mass General news release.

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Complex Mysteries

Complex Mysteries

New tools and collaborative methods shed light on autism

By JAKE MILLER
Thu, 2013-01-10

It is difficult to overstate the complexity of autism.

Two children born with identical genetic mutations can have profoundly different outcomes—one with typical neural and social development, the other unable to communicate with the world around him.

One in 88 children in the U.S. has autism, according to the CDC’s Autism and Developmental Disabilities Monitoring (ADDM) Network. Hundreds of different genetic abnormalities may contribute to—or inhibit—autism. Many of the known anomalies cluster around genes that control the development of neurons and synapses, but many others are in regions of the genome that are still poorly understood.

Layered on top of the bewildering genetics, scientists are only beginning to understand how environmental factors may affect this condition. Autism, which is characterized by impaired social communication, fixated interests and repetitive behaviors, may occur alone or as part of other developmental syndromes, such as Fragile X, Rett’s, and tuberous sclerosis, conditions that also include intellectual delays, epilepsy and a number of gastrointestinal disorders.

Unraveling all of this is like trying to solve a riddle encrypted in a cipher printed on a jigsaw puzzle—with extra pieces mixed in that don’t quite match.

iStockPhoto

This complexity presents fascinating challenges for researchers, but it also makes diagnosing and treating children with autism vexingly difficult. Even with the latest genetic testing, pediatricians still can’t predict whether an at-risk child will develop autism, or how severe symptoms will be. And while some genetic analogs for autism have been reversed in animal models, there is currently no treatment for any of the core symptoms of autism for people with the disorder.  

Despite its daunting complexity, however, new technologies and techniques and an emphasis on collaborative research are helping scientists unravel the fundamental biological basis for autism, and providing suggestions for new treatments. 

A hub for autism research

In 2006, realizing that the challenges of autism’s complexity were too great to solve piecemeal, a group of researchers and patient advocates in Boston banded together to form the Autism Consortium. The Consortium, which brings together researchers from Harvard, Boston University, MIT, Tufts, UMASS Medical School and affiliated hospitals and research institutes, affords a platform for collaboration, enabling diverse groups to share samples of patient and animal data. The organization hosts an annual meeting that highlights biomedical and clinical advances.

“The history of science isn’t just a history of great ideas, it’s a history of new tools,” said Steve Hyman, HMS professor of neurobiology and Harvard professor of stem cell and regenerative biology, at a Consortium meeting at HMS last fall.

“Galileo’s novel use of the telescope allowed him to revolutionize our understanding of the workings of the solar system,” said Hyman. “Galileo was a really smart guy, but without the tool, no new conception of the universe, no excommunication—none of that stuff.”

Those new tools—including genomic resources, engineering tools, cellular models, and organizational models and partnerships to facilitate data sharing and collaboration—are opening windows for understanding the biological bases for autism.

One challenge to understanding the biology of autism is purely physiological: the disorder is hidden in the folds of the brain. Researchers can’t biopsy living brain tissue, and the image resolution of many imaging techniques is severely limited. Because autism is a disease of neural systems and circuits and a disorder of human social behavior, studying nerve cells in the lab and animal models provides limited insight.

Nicholas Lange, HMS associate professor of psychiatry at McLean Hospital and Harvard School of Public Health associate professor of biostatistics, has worked on brain imaging techniques that are beginning to shed some light on the physiology of the brains of people with autism. This is another area with a great deal of unmapped territory to explore. In a recent essay in Nature, Lange emphasized that we still need to better understand the great breadth and depth of healthy typical and atypical neurobiology and the diversity of the types of brain features in autism, and that without that foundational biology all attempts at diagnosing autism by brain imaging are futile.

“Brain scans, neuronal subtyping and genetics are the signposts showing us where and how to look, see, ponder, hypothesize and test,” Lange said. “We start where we are and delve more deeply into the biological etiology and treatment of autism.”

Some members of the Consortium are planning to grow neurons from stem cells as a way of examining how neurons from patients with different genomic profiles develop and operate.

Others are developing new techniques to look at how the neurons perform at the synaptic level, using high-resolution, high-speed imaging to visualize a single pulse of neurotransmitters in order to see what happens each time a neuron fires.

“You need that excruciating, exquisite level of detail so you can watch signals in real time,” said Mriganka Sur, director of the Simons Center for the Social Brain at MIT. Researchers will also need to scale these tiny neuronal interactions to see how they interrelate in complex neural networks, he added.

“One of the greatest challenges is determining how these genes map onto the development of the social brain,” Sur said.

Synaptic tuning

One reason autism is so complex is that the process of brain development itself is incredibly dynamic, according to Christopher Walsh, chief of the Division of Genetics at Boston Children’s Hospital and HMS Bullard Professor of Pediatrics and Neurology.

Walsh began his career as a developmental neurobiologist and focused on genetics as a method of getting to the root of neural development. In 2008, working with samples of consanguineous Middle Eastern families to find recessive genes related to developmental disorders, Walsh identified a handful of new genetic components of autism, including both protein encoding genes and the codons surrounding genes that act as on-off switches for genes.

Michael Greenberg, chair of the neurobiology department at HMS and Nathan Marsh Pusey Professor of Neurobiology, had previously identified those genes as key players in experience-dependent learning.

“When neurons become active, they turn on a lot of genes,” Walsh said. When a person is learning a new behavior, repeated experience activates genes that, over time, strengthen some connections in the brain and weaken others. This building and pruning of neural pathways shapes the networks that record and enable learned behaviors.

This kind of learning is essential to developing the complex social behaviors impaired in autism, and it turns out that several of the genomic anomalies associated with autism are related to regulators that signal these synaptic shaping and pruning genes when to turn on and off.

If the genes involved in making proteins to build those connections don’t turn on at the right time, or if they shut down too soon, they might not build an important connection; if they stay on too long, connections can be overly strengthened.

“You could think of it as synaptic tuning,” Walsh said. “When a synapse fires, it doesn’t just fire once, it fires repeatedly at a certain frequency. “Hearing” the right tune is what stimulates the next synapse,” he said. “In autism, for certain kinds of complex tasks, like social communication, you might say that you’ve lost that tuning mechanism.”

Walsh said a variety of drugs under development to treat synaptic deficiencies in far-flung disorders from Fragile X to Alzheimer’s might prove effective in treating autism.

Gene hunting

A handful of genetic anomalies related to autism have been identified, but some researchers believe that more than 100 genes may contribute to autism, and that those genes are just the beginning of a series of complex biochemical pathways, many of which remain uncharted territory.

Researchers in the Greenberg lab are studying the genetic and molecular machinery that builds brains; that is, the proteins that coax embryonic stem cells into becoming neurons as well as the switches that help promote and prune synapses. When this developmental process goes awry, conditions such as autism can result. For example, Greenberg discovered how the loss of a key enzyme related to the molding of synapses could explain the devastating developmental deficits that occur in Angelman syndrome, which causes a constellation of developmental problems in children, including mental disability and, in some cases, autism. 

There are more than 300 genes that trigger these synaptic changes. Many of the genes that Greenberg’s molecular and cellular studies have identified as playing important roles in the modification of synaptic connections have been identified as sites for genetic anomalies that may contribute to autism. Researchers probing these links include Walsh and Mark Daly, HMS associate professor of medicine at Massachusetts General Hospital’s Center for Human Genetic Research.

 “One piece of the puzzle isn’t enough to open up the biology and crack something as complex as autism,” said Daly, who is also director of computational biology for the Medical and Population Genetics Program of the Broad Institute of Harvard and MIT.

Working with the samples available through the Autism Consortium, Daly’s lab has now sequenced the genomes of hundreds of families, identifying several novel genetic contributions to autism.

 “By this time next year, we will have several thousand families sequenced. We’ll have a much bigger set of those puzzle pieces,” Daly said.

Recent technical advances have made gene sequencing much more accessible. A decade ago it took 10 years and cost $1 billion to sequence a human genome. Now it can be done in one week or less for a few thousand dollars. Still, the work of understanding sequenced genomes remains challenging, Daly said.

Differences in the genomes of any two healthy individuals are vast. When researchers sort through all that unique data, in billions of combinations of bases in the genetic code, identifying the differences that are characteristic of autism is no simple task.

“It’s a huge computational challenge,” Daly said. “We can’t yet answer basic questions for families about the potential severity of their children’s illnesses, let alone offer any treatment.”

Finding—and learning from—biomarkers

One way to solve the autism puzzle is to identify the genes that are implicated in the disorder and then study the pathways and processes that those genes regulate.

Another way is to identify measurable biological features characteristic of autism, and to compare those with the same features in the biology of typical development. If genetics are the pieces of the jigsaw puzzle, biomarkers are part of the picture on the box that shows you what the pieces will look like when they’re put together.

HMS researchers at Boston Children’s Hospital, for example, have developed a blood test for autism spectrum disorders that outperforms existing genetic tests. It also presents evidence that abnormal immunologic activity affecting brain development may help explain some of autism’s origins.


The same is true for many other projects that autism researchers are undertaking to define how the disorder manifests itself physiologically; for example, measuring electrical activity in the brain, monitoring stress levels and predicting the onset of seizures by measuring skin conductivity.

The research not only leads to more objective diagnostic tools, it can also provide crucial insight into the basic biology of the brain, which remains one of the great black boxes of biomedical science. 

As complex as the challenges are, researchers say that great progress is being made in sorting out the roles of genes and gene regulators, of toxins and the immune system, of synaptic pruning and stress. Researchers are also building a basic understanding of the function of the social brain from the molecular to the macro level, and beginning to see how the pieces fit together.

 “All of these things are in sight now,” said Gerald Fischbach, director of the Simons Foundation Autism Research Initiative. “We have the tools to do this.”

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A Sharper Tool for Genome Engineering

A Sharper Tool for Genome Engineering

New method for editing genomes may be faster, safer and more efficient than recent breakthroughs

By ELIZABETH COONEY
Fri, 2013-01-11

Green fluorescent cells show a genetic sequence that was repaired using a new method for genome engineering. Image courtesy of the Church Lab.

A new method for more easily and precisely engineering the human genome may help scientists pin subtle changes in DNA to disease, moving the needle from correlation to causality and potentially improving gene therapy techniques.

Two groups recently reported in Science their success using a tool borrowed from a bacterial immune system called Cas, short for CRISPR-associated systems, which in turn stands for Clustered Regularly Interspaced Short Palindromic Repeats. In bacteria the Cas9 enzyme system uses short stretches of RNA to target and then cut invading viral DNA. Scientists have customized this system to work in human cells, creating an RNA-guided editing tool that allows them to integrate DNA changes into the genomes of living cells, a process called genome engineering.

One of the teams, led by George Church, HMS Robert Winthrop Professor of Genetics, also tested the method in induced pluripotent stem cells (iPSCs), an important milestone on the path to human genome engineering. These cells, taken from a child or adult, have been modified to mimic embryonic cells, which means they can develop into any adult cell type. In experiments, iPSCs offer greater clarity than traditional cell lines as researchers explore gene function in different cell types.

The other team, led by Feng Zhang of the Broad Institute of Harvard and MIT, independently showed the effectiveness of Cas9.

While the Cas9 method is still a long way from the clinic, Church is hopeful.

“We need a lot more experience and optimization, but it looks very promising,” he said. “This is much easier than any previous human genome engineering method, and it is relevant to testing the flow of ideas from GWAS [genome-wide association studies] and the Personal Genome Project.”

The Cas9 approach, first developed by Harvard University graduate Jennifer Doudna of the University of California, Berkeley, could end up supplanting a technique that Science just last month named one of the top 10 scientific breakthroughs of 2012. For this technique, a class of proteins called TALENs (Transcription Activator-Like Effector Nucleases) zero in on a particular region of the genome, where they can precisely cut DNA and insert or delete a gene. TALENs followed a method from the 1990s that used enzymes called zinc finger nucleases to target specific parts of genomes.

TALENs were easier to design, but, like zinc fingers, they require about 2,000 bases of messenger RNA to encode an enzyme that would cleave DNA at a specific site in a genome.  Cas9 needs 1/100 as much RNA; as few as 20 variable bases, embedded in short constant guide RNAs, are sufficient for precise targeting, the scientists showed in human cell lines.

According to Church, also testing Cas9 in iPSCs is particularly noteworthy.

“We need to have human stem cells in culture so we can manipulate the genome and see how the cells differentiate, or fit into tissues,” he said. “To change from correlation and speculation to causality, you introduce single changes, one by one, into a test genome and ask which of those or how many of those do you need in order to see the trait. That turns correlation into causation and moves it closer to the gold standard.”

The more compact Cas9 system opens the door to engineering multiple changes in different genes and then testing them simultaneously to see what role they play in complex diseases. Large genome-sequencing studies can find a variety of genes active in people with a particular disease, but it takes the kind of multiplex testing Cas9 allows to establish which ones actually matter, Church said.

A decade ago early attempts at gene therapy failed because delivery of a new, corrected gene could inadvertently promote cancer or provoke a harmful immune system response. Newer methods, in addition to having more precise delivery, are also designed to have lower toxicity and generate only a tolerable immune response in the case of a rare off-target event.  These newer approaches also make it possible to target an old gene with new information, either knocking it out or changing it precisely.

Cas9 has the potential to accelerate these improvements in gene therapy, but much more testing is necessary to see if it will continue to display the greater efficiency and very low toxicity shown in these early experiments. “But it looks like a very promising starting point,” Church said.

This work was supported by NIH grant P50 HG005550. Church and the Science paper’s first author, research fellow Prashant Mali, have applied for a patent based on the findings of this study. 

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Breakthrough Prize for Ruvkun

Breakthrough Prize for Ruvkun

Award honors advances in gene regulation

Mon, 2014-11-17

Gary Ruvkun, Harvard Medical School professor of genetics at Massachusetts General Hospital, was named one of six winners of the 2015 Breakthrough Prizes in Life Sciences. Each winner, along with winners in Physics and Mathematics, will receive a $3 million prize.

Ruvkun and Victor Ambros, University of Massachusetts Medical School, were each recognized for the discovery of a new world of genetic regulation by microRNAs, a class of tiny RNA molecules that inhibit translation or destabilize complementary mRNA targets. The other winners in the life sciences include C. David Allis, Alim Louis Benabid, Jennifer A. Doudna and Emmanuelle Charpentier.

The Breakthrough Prizes aim to celebrate scientists and generate excitement about the pursuit of science as a career. Breakthrough Prizes are funded by a grant from  Sergey Brin and Anne Wojcicki's  foundation, The Brin Wojcicki Foundation; a grant from Mark Zuckerberg’s fund at the Silicon Valley Community Foundation; a grant from Jack Ma Foundation; and a grant from Milner Foundation.

The awards were presented at an exclusive Gala co-hosted by founders Sergey Brin and Anne Wojcicki, Jack Ma and Cathy Zhang, Yuri and Julia Milner, Mark Zuckerberg and Priscilla Chan and Vanity Fair editor Graydon Carter at NASA's Ames Research Center. Seth MacFarlane hosted The 2nd Annual Breakthrough Prize Ceremony which also featured Kate Beckinsale, Benedict Cumberbatch, Cameron Diaz, Jon Hamm and Eddie Redmayne as presenters.

 “This year’s life sciences laureates have made some spectacular discoveries, from a new kind of gene to a Parkinson’s treatment that has improved the lives of many,” Wojcicki said. “It’s energizing to be in the company of such brilliant and fertile minds.”

"The world faces many fundamental challenges today, and there are many amazing scientists, researchers and engineers helping us solve them,” Zuckerberg said. “This year's Breakthrough Prize winners have made discoveries that will help cure disease and move the world forward. They deserve to be recognized as heroes."

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Cancer Spread's Family Tree

Cancer Spread’s ‘Family Tree’

Rapid test reveals relationships between primary tumors and metastatic cells

By SUE McGREEVEY
Wed, 2014-04-23

Image: iStock

The process of metastasis—a tumor’s ability to spread to other parts of the body—is still poorly understood. It has not been easy to determine whether metastasis began early or late in the development of the primary tumor, or whether individual metastatic sites were seeded directly from the original tumor or from an intermediate site. Now a research team has developed a simple test that can reveal the evolutionary relationships among various tumor sites within a patient, information that may someday help with treatment planning.

“If we could build a ‘family tree’ of all cancer nodules in a patient, we could determine how different tumors are related to each other and reconstruct how the cancer evolved,” said Kamila Naxerova, Harvard Medical School research fellow in radiation oncology in the Steele Laboratory for Tumor Biology at Massachusetts General Hospital. She is corresponding author of the report, published in PNAS Early Edition. “Usually that would require extensive genetic analysis with complex sequencing methods, but our methodology achieves that goal quickly and with minimal experimental effort.”

Cancer researchers are just beginning to investigate the extent and significance of genetic differences among tumor cells, either among cells within a discrete tumor or between a primary tumor and metastases in other parts of the body. The authors note that there are two different models of metastasis. In one model, an advanced primary tumor disseminates metastatic cells late in its development, which would predict little genetic difference between primary and metastatic cells. In the other model, metastasis occurs early in tumor development, which would predict significant genetic differences in metastatic cells that have evolved separately from those in the primary tumor. Some studies have suggested that the two models apply to different types of cancer, but patient data so far has been limited.

Answering important clinical questions, such as whether genetic diversity is a risk factor for aggressive tumor development and how it relates to treatment resistance, requires analyzing samples from many patients with different types of cancer. Sequencing the whole genome or just the protein-coding portion of the genome (the exome) requires specialized equipment and advanced data analysis, making the process relatively expensive.

The approach developed by the Mass General team focuses on small areas of the human genome called polyguanine (poly-G) repeats that are particularly susceptible to mutation, with genetic “mistakes” occurring frequently during cell division. While these mutations do not directly relate to the development or progression of a tumor, they can reveal its lineage—how individual tumor cells are related to each other.

Poly-G repeat analysis was initially developed to study lineage relationships between single cells in mice, but in the current paper, the authors adapted it to study human cancer for the first time. Analyzing the poly-G profiles of primary and metastatic colon cancer samples from 22 patients revealed that the way the primary and metastatic tumors related to each other was different for each patient.

In some individuals there were significant genetic differences between tumor sites, suggesting early metastatic spread; in others, there was little difference between a primary tumor and its metastases. The investigators also identified instances in which the genetic profiles of metastases were similar to those of only some cells in the primary tumor, suggesting that those cells were the source of the metastases, and other cases in which the genetic profiles of metastases from the same primary tumor differed depending on their location.

“We found that there are several paths that can lead to metastatic disease,” said Naxerova. “We are now applying this methodology to address specific clinically relevant questions about the biology of metastasis in larger numbers of patients. The method is fast and inexpensive and should be applicable to other types of tumors than colon cancer.”

Co-author Elena Brachtel, HMS assistant professor of pathology at Mass General, noted that archival tissues from the files of the department were used for this study. “After diagnostic studies on tissue removed during a patient’s operation are completed, the formalin-fixed paraffin tissue blocks are stored for several years. Increasingly, new molecular tests can be performed on tissue that was removed from a patient several years earlier, at a time when these tests were not yet available.”

Rakesh K. Jain, the A. Werk Cook Professor of Radiation Oncology (Tumor Biology) at HMS and Mass General, director of the Steele Lab and senior author of the paper, added, “The assay has many potential clinical applications. For example, it could be used to reliably and quickly distinguish a metastasis from a second, independent tumor. Or it could identify the primary tumor in situations where multiple lesions are present and it is ambiguous which one is responsible for seeding metastases.”

The work was supported by Department of Defense grants W81XWH-10-1-0016 and W81XWH-11-1-0146.

Adapted from a Mass General news release.

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Connecting Crohn's and Gut Microbes

Connecting Crohn’s and Gut Microbes

Microbiome analysis reveals increase in pathogenic bacteria, drop in beneficial microbes in newly diagnosed patients

By SUE McGREEVEY
Wed, 2014-03-12

Image: iStock

A multi-institutional study led by Harvard Medical School investigators at Massachusetts General Hospital and scientists from the Broad Institute has identified how the intestinal microbial population of patients newly diagnosed with Crohn’s disease differs from that of individuals free of inflammatory bowel disease. In their paper in the March 12 issue of Cell Host and Microbe, the researchers report that Crohn’s patients showed increased levels of harmful bacteria and reduced levels of the beneficial bacteria usually found in a healthy gastrointestinal tract. 

Several studies have suggested that the excessive immune response that characterizes Crohn’s may be associated with an imbalance in the normal microbial population, but the exact relationship has not been clear. The current study analyzed data from the RISK Stratification Study, which was designed to investigate microbial, genetic and other factors in a group of children newly diagnosed with Crohn’s disease or other inflammatory bowel diseases. At 28 participating centers in the U.S. and Canada, samples of intestinal tissues were taken from 447 participants with a clear diagnosis of Crohn’s and from 221 control participants with noninflammatory gastrointestinal conditions. The researchers also analyzed samples from an additional group of about 800 participants in previous studies, for a total of more than 1,500 individuals.

Advanced sequencing of the microbiome—the genome of the entire microbial population—in tissue samples taken from sites at the beginning and the end of the large intestine revealed a significant decrease in diversity in the microbial population of the Crohn’s patients, who had yet to receive any treatment for their disease. The samples revealed an abnormal increase in the proportion of inflammatory organisms in Crohn’s patients and a drop in noninflammatory and beneficial species, compared with the control participants. The imbalance was even greater in patients whose symptoms were more severe and in those who had markers of inflammatory activity in tissue samples.

“These results identifying the association of specific bacterial groups with Crohn’s disease provide opportunities to mine the Crohn’s disease-associated microbiome to develop diagnostics and therapeutic leads,” said senior author Ramnik Xavier, the HMS Kurt J. Isselbacher Professor of Medicine in the Field of Gastroenterology at Mass General.

Other key findings of the study include:

  • identifying the niches of specific microbial strains in Crohn’s disease and their effects on other microbial community members
  • finding that rectal biopsies can indicate the presence of disease early in the course of Crohn’s, regardless of which intestinal segments are affected
  • showing that fecal samples collected at the onset of disease do not reflect changes in the bacterial communities of the intestinal lining

Antibiotics are often prescribed for symptoms suggestive of Crohn’s before a diagnosis is made. In participants who happened to be taking antibiotics at the time samples were taken, the microbial imbalance was even more pronounced, suggesting that the antibiotic use could exacerbate symptoms rather than relieve them, the authors note. Next steps will be to uncover the function of these microbes and their products and to learn how the microbiome and microbial products interact with the patient’s immune system, with the possibility that these interactions could represent the molecular basis for the disease.

“Identifying which microbial products are key to disease onset and to inflammation resolution in inflammatory bowel disease and establishing which can be effectively targeted are our best hope to uncover the first microbiome-based therapies in inflammatory bowel disease,” said Xavier, who is chief of the Mass General Gastrointestinal Unit and director of the Mass General Center for the Study of Inflammatory Bowel Disease.

Dirk Gevers of the Broad Institute is the lead author of the report and Curtis Huttenhower of the Broad and the Harvard School of Public Health is also a co-author.

The study was supported by the Crohn’s and Colitis Foundation of America, the Helmsley Charitable Trust, by Army Research Organization grant W911NF-11-1-0473, the MGH Center for the Study of Inflammatory Bowel Disease and by National Institutes of Health grants U54DE023798, R01HG005969 and R01DK092405.

Adapted from a Mass General news release.

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Testing the Whole Haystack

Testing the Whole Haystack

Genetic analysis reveals architecture of obsessive-compulsive disorder and Tourette syndrome

 

By SUE McGREEVEY
Thu, 2013-10-24

Image: iStock

An international research consortium led by investigators at Massachusetts General Hospital and the University of Chicago has provided the first direct confirmation that both obsessive-compulsive disorder (OCD) and Tourette syndrome are highly heritable. Their work, which appears in the October issue of PLOS Genetics, also reveals major differences in the genetic makeup of the two conditions that sometimes occur together.  

“Both Tourette syndrome and OCD appear to have a genetic architecture of many different genes—perhaps hundreds in each person—acting in concert to cause disease,” said Jeremiah Scharf, HMS assistant professor of neurology at Mass General and senior corresponding author of the report. “By directly comparing and contrasting both disorders, we found that OCD heritability appears to be concentrated in particular chromosomes, particularly chromosome 15, while Tourette syndrome heritability is spread across many different chromosomes.”

An anxiety disorder characterized by obsessions and compulsions that disrupt the lives of patients, OCD is the fourth most common psychiatric illness in the United States. Tourette syndrome is a chronic condition known for causing motor and vocal tics. Usually beginning in childhood, Tourette syndrome is often accompanied by OCD or attention-deficit hyperactivity disorder. Because OCD and Tourette syndrome often develop in close relatives of affected individuals, they have been considered heritable, but identifying specific genes that confer risk has been challenging. 

Scharf and several co-authors of the current study published two papers last year in the journal Molecular Psychiatry describing genome-wide association studies (GWAS) of thousands of people affected by OCD or Tourette syndrome. While those studies identified several gene variants that appeared to increase the risk of each individual condition, none of the associations was strong enough to meet the strict standards of genome-wide significance. 

The GWAS approach is designed to identify relatively common gene variants. Because OCD and Tourette syndrome might be influenced by a number of rare variants, the research team adopted a different method. Called genome-wide complex trait analysis (GCTA), the approach allows simultaneous comparison of genetic variation across the entire genome, rather than the GWAS method of testing sites on the genome one at a time. GCTA also estimates the proportion of disease heritability caused by rare and common variants.

“Trying to find a single causative gene for diseases with a complex genetic background is like looking for the proverbial needle in a haystack,” said Lea Davis, a research assistant professor of genetic medicine at the University of Chicago and co-corresponding author of the PLOS Genetics paper. “With this approach, we aren’t looking for individual genes. By examining the properties of all genes that could contribute to Tourette syndrome or OCD at once, we’re actually testing the whole haystack and asking where we’re more likely to find the needles.”

Using GCTA, the researchers analyzed the same genetic datasets screened in the Molecular Psychiatry studies: almost 1,500 people with OCD compared with more than 5,500 controls, and nearly 1,500 people with Tourette syndrome compared with more than 5,200 controls. To minimize variations that might result from slight differences in experimental techniques at more than one location, all genotyping was done by collaborators at the Broad Institute of Harvard and MIT, who generated the data at the same time using the same equipment. Davis analyzed the resulting data on a chromosome-by-chromosome basis, charting the frequency of the identified variants and the function of variants associated with each condition.

The researchers concluded that the degree of heritability for both disorders captured by GWAS variants is actually quite close to what previously was predicted based on studies of families affected by the disorders. 

“This is a crucial point for genetic researchers, as there has been a lot of controversy in human genetics about what is called ‘missing heritability,’” Scharf explained. “For many diseases, definitive genome-wide significant variants account for only a minute fraction of overall heritability, raising questions about the validity of the approach. Our findings demonstrate that the vast majority of genetic susceptibility to Tourette syndrome and OCD can be discovered using GWAS methods. In fact, the degree of heritability captured by GWAS variants is higher for Tourette syndrome and OCD than for any other complex trait studied to date.”

Nancy Cox, a professor of medicine and human genetics at the University of Chicago and co-senior author of the PLOS Genetics report, added, “Despite confirming there is shared genetic liability between these two disorders, we also show there are notable differences in the types of genetic variants that contribute to risk. Tourette syndrome appears to derive about 20 percent of genetic susceptibility from rare variants, while OCD appears to derive all of its susceptibility from variants that are quite common, which is something that has not been seen before.”

About half the risk for both disorders appears to come from variants already known to influence the expression of genes in the brain. Further investigation of those findings could identify the affected genes and show how changes in their expression contribute to the development of Tourette syndrome and OCD. Additional studies in even larger patient populations, some of which are in the planning stages, could reveal the biologic pathways disrupted in the disorders, potentially leading to new therapeutic approaches. 

The study reflects a collaboration between two consortia representing 43 institutions across 12 countries: the Tourette Syndrome Association International Consortium for Genomics and the International OCD Foundation Genetics Collaborative. Scharf is co-chair of the Tourette Syndrome Association International Consortium for Genomics steering committee and a member of the International OCD Foundation Genetics Collaborative steering committee. 

Support for the study includes National Institutes of Health grants U01 NS40024, R01 MH101820 and K23 MH085057; and grants from the Tourette Syndrome Association and the David Judah Fund.

Adapted from a Mass General news release.

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Tracking Brain Tumors

Tracking Brain Tumors

Noninvasive molecular biology tool detected a common tumor-associated genetic mutation in cerebrospinal fluid

By SUE McGREEVEY
Wed, 2013-07-24

Researchers have tracked brain tumors by searching for evidence of mutations in cerebrospinal fluid. Image: iStock

Borrowing a tool from molecular biology, HMS researchers at Massachusetts General Hospital have detected a tumor-associated genetic mutation in the cerebrospinal fluid (CSF) of a small number of patients with brain tumors.  In a paper published in the open-access journal Molecular Therapy – Nucleic Acids, the investigators described using digital versions of the gene-amplification technology polymerase chain reaction (PCR) to analyze bits of RNA carried in membrane-covered sacs. They found a common tumor-associated mutation in a gene called IDH1, a biomarker whose presence could potentially influence patient care.

“Reliable detection of tumor-associated mutations in cerebrospinal fluid with digital PCR would provide a biomarker for monitoring and tracking tumors without invasive neurosurgery,” said Xandra Breakefield, HMS professor of neurology at Mass General and corresponding author of the paper. “Knowing the IDH1 mutation status of these tumors could help guide treatment decisions, since a number of companies are developing drugs that specifically target that mutant enzyme.”

Both normal and tumor cells regularly release membrane-covered sacs called extracellular vesicles. Found in blood, CSF and other body fluids, they contain segments of RNA, DNA or proteins. A 2008 study from the Mass General team identified a relatively large tumor-associated mutation in extracellular vesicles from the blood of brain tumor patients, but most current diagnostic technologies that analyze CSF do not capture molecular or genetic information from central nervous system tumors.  

In addition, “Tumor-specific extracellular vesicles make up only a small percentage of the total number of extracellular vesicles found in either blood or cerebrospinal fluid, so finding rare, single-nucleotide mutations in a sample of blood or CSF is very challenging,” explained Leonora Balaj, an HMS research fellow in neurology and co-lead author of the paper. “These digital PCR techniques allow the amplification of such hard-to-find molecules, dramatically improving the ability to identify tumor-specific changes without the need for biopsy.”

The current study used two forms of digital PCR—BEAMing and Droplet Digital PCR—to analyze extracellular vesicles in the blood and in the CSF of brain tumor patients and healthy controls. The scientists were searching for the presence of a single-nucleotide IDH1 mutation known to be associated with several types of cancer. Both forms of PCR detected the presence and abundance of mutant IDH1 in the CSF of 5 of the 8 patients known to have IDH1-mutant tumors. 

Two of the three mutation-positive tumors that had false negative results were low grade and the third tumor was quite small, suggesting a need for future studies of more samples to determine how the grade and size of the tumors affect the ability to detect mutations. The failure to detect tumor-associated mutations in blood samples with this technology may indicate that CSF is a better source for extracellular vesicles from brain tumors. 

The ability to noninvasively determine the genetic makeup of brain tumors could have a significant impact on patient care. “The current approach for patients who may have a brain tumor is first to have a brain scan and then a biopsy to determine whether a growth is malignant,” said Fred Hochberg, HMS associate professor of neurology and a study co-author. “Patients may have a second operation to remove the tumor prior to beginning radiation therapy and chemotherapy, but none of these treatments are targeted to the specific molecular nature of the tumor.”

Having this kind of molecular diagnostic assay—whether in spinal fluid or blood—would allow clinicians to immediately initiate treatment that is personalized for a particular patient without the need for surgical biopsy, Hochberg said. 

“For some patients, the treatment could shrink a tumor before surgical removal. For others, it may control tumor growth to the point that surgery is not necessary, which in addition to keeping patients from undergoing an unnecessary procedure, could save costs,” he said. “We still have a long way to go to improve survival of these malignancies, so every improvement we can make is valuable.”

Mass General has applied for a patent on the use of BEAMing PCR to analyze RNA from extracellular vesicles. Support for the study includes National Institutes of Health grants CA069246, CA141226, CA156009 and CA141150 and grants from the Brain Tumor Funders’ Collaborative  and the American Brain Tumor Association.

Adapted from a Mass General news release.

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Gene variant linked to weight-loss surgery success

Gene variant linked to weight-loss surgery success

Genetic factors appear to determine a patient’s response to gastric bypass

By SUE McGREEVEY
Thu, 2013-05-02

Image: CDC/Debora Cartagena

Massachusetts General Hospital researchers have identified a gene variant that helps predict how much weight an individual will lose after gastric bypass surgery, a finding with the potential both to guide treatment planning and to facilitate the development of new therapeutic approaches to treating obesity and related conditions like diabetes. The report, published online in The American Journal of Human Genetics, is the first to identify genetic predictors of weight loss after bariatric surgery.

“We know now that bypass surgery works not by physically restricting food intake but primarily through physiological effects—altering the regulation of appetite to decrease hunger and enhance satiety and increasing daily energy expenditure,” said Lee Kaplan, HMS associate professor of medicine at Mass General and director of the hospital’s Obesity, Metabolism and Nutrition Institute. He is a senior author of the report. “Genetic factors appear to determine a patient’s response to gastric bypass, and the identification of markers that predict postoperative weight loss could provide important insight into those physiological mechanisms.”

The research team conducted genome-wide association studies of more than 1,000 patients who had bypass surgery at Mass General from 2000 to 2011, analyzing almost 2 million gene sites for associations between specific variants and the percentage of weight lost after surgery. One specific variant at a site on chromosome 15 was most closely associated with weight loss. Individuals with two copies of the beneficial version of the gene lost an average of almost 40 percent of their presurgical weight, while those with only one copy lost around 33 percent. The single individual in the study group who had no copies of the beneficial variant lost less than 30 percent of presurgical weight.

Expression of one of the genes closest to the site of this variant was also able to predict the percentage of weight lost. In addition, experiments in a mouse model of gastric bypass indicated that expression of the corresponding version of that human gene, as well as another gene adjacent to the variant site, was altered by bypass surgery. Additional gene variants not as strongly associated with the response to bypass surgery are candidates for further study in larger groups of patients.

Two predictive models developed by Kaplan and his team have had promising initial results. One of these combines the chromosome 15 genetic variant with clinical factors such as age, gender, the presence of diabetes and exercise behaviors to predict surgical outcomes; the other includes 12 additional gene variants the investigators are studying to determine their usefulness in treatment planning. 

Notably, none of the predictive gene sites identified in this study is involved in pathways previously known to influence the development of obesity, suggesting that different genes contribute to the benefits of bypass. Development of drugs that target the activity of those genes might produce some of the same benefits without the need for surgery, Kaplan said.

“The fact that genetics appears to play such an important role in how well bypass surgery works in an individual patient gives us even more evidence that obesity results from dysfunction of the biological mechanisms that regulate fat mass and body weight and not solely from aberrant behavior or limited willpower,” he adds. “Identifying the involved genes opens up the potential for new classes of antiobesity therapies that mimic or exploit the molecular mechanisms so effectively used by gastric bypass.”  

The study was supported by National Institutes of Health grants DK093257, DK088661 and DK090956, along with grants from Merck Research Laboratories and Ethicon Endo-Surgery.

Adapted from a Mass General news release.

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