Genes and Immunity

T lymphocyte activation integrates immunologic and genetic history

Image: National Human Genome Research Institute

Researchers from Harvard Medical School and the Broad Institute of MIT and Harvard have uncovered unexpectedly complex patterns in the T lymphocyte responses that individual people mount, reflecting environmental influences as well as a genetic component. The study lays the groundwork for further explorations into the relative contributions of genes and their environment on immunological processes, the scientists said, which could illuminate autoimmune disease and its genetic underpinnings.

The findings are reported in Science and stem from the ImmVar Project, a wide-ranging analysis of variation in gene expression in the immune system. Christophe Benoist, Morton Grove-Rasmussen Professor of Immunohematology at HMS, and Aviv Regev, a Broad Institute core member, an associate professor at MIT, and  Howard Hughes Medical Institute investigator, led the third and final phase, which focused on CD4+ T cells, immune cells that are major players in autoimmune disease.

In this study, after the scientists accounted to the best possible extent for environmental influences and immunological history, they still found that the ancestry of the donor significantly affected T cell responses. “There is a signature of variation in adaptive immune response,” Benoist said. “In general, there is stronger activation of some genes in people of African ancestry, in particular for a type of response in T helper 17 (Th17) cells that tend to protect us from microbes that enter airways or the intestinal tract.  Those responses are also highly involved in autoimmune disease.” 

"The combination of careful immunological work, high-throughout assays, and sophisticated analytics essential to dissect such a complex system could only have happened within the partnership of the ImmVar consortium, bringing together the expertise of immunologists and clinicians in the Harvard-affiliated hospitals with genomics and computational experts at the Broad and MIT," Regev said.

In autoimmune diseases such as rheumatoid arthritis, inflammatory bowel disease and multiple sclerosis, immune cells mistakenly attack the body’s own tissues as if they were invaders. In healthy people, the immune system achieves a state of tolerance, quelling defensive measures after a threat has abated.

Scientists have previously identified genes that are important in controlling the autoimmune response, but this is the first time that differences in T cell activation between population groups have been revealed.

In the current study, the scientists analyzed blood samples collected from 348 healthy volunteers representing African, Asian or European ancestry. After the researchers genotyped the samples and isolated CD4+ T cells, the T cells were activated in cell culture to model their response to antigens. A computational analysis measured which genes were turned on or off in the cells from each person.

Activation of autoimmune-associated genes can vary between individuals in a complicated interplay of genes and environment. Each person’s immunological history is written in a constellation of events, from being vaccinated against the measles in childhood to having the flu last winter. Benoist compares it to learning and personality: All the memories you accumulate make you who you are.

In one’s immunological history, “environment” also encompasses the microbial world people inhabit. The hygiene hypothesis holds that people who have encountered more challenges to their immune system—harmful microbes—are less likely to have the runaway response that is the hallmark of autoimmune disease. People who grow up exposed to fewer microbes may have difficulty stopping the immune response when it is no longer needed.

There is a strong inherited component to autoimmune disease, but changing one’s environments is also important, Benoist noted. People who relocate to a new region tend to acquire the frequency of autoimmune disease of where they are going, observational research has reported. For example, he said, there is little autoimmune disease in India, but people of Indian origin who have lived in the US, from an early age have about the same frequency of autoimmune disease as people of European origin who also live in the US.

One possibility is that at least some of this variation may reflect evolutionary adaptations to the pathogens people encountered during human migrations out of Africa 50,000 years ago. A more robust immune response would have been advantageous in sub-Saharan Africa but deleterious at higher latitudes, with fewer microbial pathogens.

“It’s a tantalizing idea, but it’s highly speculative,” Benoist said.

This work was supported by National Institute of General Medical Sciences grant RC2 GM093080, NIH F32 Fellowship (F32 AG043267), HHMI, and a Harry Weaver Neuroscience Scholar Award from the National Multiple Sclerosis Society (JF2138A1).


Sweet Feat

How hummingbirds evolved a novel mechanism of sweet taste detection

Everything about hummingbirds is rapid. An iridescent blur to the human eye, their movements can be captured with clarity only by high-speed video.

Anna’s hummingbird (Calypte anna) in the Santa  Monica Mountains, Calif. Image: Maude Baldwin

Slowed down on replay, their wings thrum like helicopter blades as they hover near food. Their hearts beat 20 times a second and their tongues dart 17 times a second to slurp from a feeding station.

It takes only three licks of their forked, tube-like tongues to reject water when they expect nectar. They pull their beaks back, shake their heads and spit out the tasteless liquid. They also are not fooled by the sugar substitute that sweetens most diet cola.

These hummingbirds look mad.

The birds’ preference for sweetness is plain, but only now can scientists explain the complex biology behind their taste for sugar. Their discovery required an international team of scientists, fieldwork in the California mountains and at Harvard University’s Concord Field Station, plus collaborations from Harvard labs on both sides of the Charles River. 

Now, in a paper published in Science, the scientists show how hummingbirds’ ability to detect sweetness evolved from an ancestral savory taste receptor that is mostly tuned to flavors in amino acids. Feasting on nectar and the occasional insect, the tiny birds expanded throughout North and South America, numbering more than 300 species over the 40 to 72 million years since they branched off from their closest relative, the swift.

“It’s a really nice example of how a species evolved at a molecular level to adopt a very complex phenotype,” said Stephen Liberles, HMS associate professor of cell biology. “A change in a single receptor can actually drive a change in behavior and, we propose, can contribute to species diversification.”

Anna's hummingbirds (Calypte anna) reject most artificial sweeteners. In this video, slowed to 10 percent of regular speed, the birds drink from sucrose-containing feeders (feeders 3 and 6) but reject aspartame (feeder 1).  Video: Maude Baldwin.

This sweet discovery all started with the chicken genome. Before scientists sequenced its genes, people assumed that chickens and all birds taste things the same way that mammals do: with sensory receptors for salty, sour, bitter, sweet and the more recently recognized umami taste, which comes from the Japanese word for savory.

The canonical view stated there was a sweet receptor present in animals, much smaller than the large families of receptors involved in smell and bitter taste perception—vital for sensing safe food or dangerous predators.

Some animals have lost certain taste abilities. The panda, for example, feeds exclusively on bamboo and lacks savory taste receptors. Carnivores, notably cats, are indifferent to sweet tastes. The gene for tasting sweetness is present in their genomes, but it’s nonfunctional. Scientists suspect that an interplay between taste receptors and diet may effectively relegate the sweet taste receptor into a pseudogene that does not get turned on and eventually disappears.

The chicken genome is another story: It has no trace of a sweet-taste receptor gene. Faced with this all-or-nothing scenario, Maude Baldwin, co-first author of the paper, had one reaction.

“The immediate question to ornithologists or to anybody who has a birdfeeder in the backyard was: What about hummingbirds?” she recalled. “If they are missing the single sweet receptor, how are they detecting sugar?”

More bird genomes were sequenced, and still no sweet receptor.

So began Baldwin’s quest to understand how hummingbirds detected sugar and became highly specialized nectar feeders. A doctoral student in organismic and evolutionary biology and the Museum of Comparative Zoology, she is a member of the lab of Scott Edwards, Professor of Organismic and Evolutionary Biology and Curator of Ornithology in the Museum of Comparative Zoology. She sought out Liberles at a meeting of the International Symposium on Smell and Taste in San Francisco. They agreed to work together on experiments that would eventually reveal how hummingbirds evolved and diversified, based on a change in their taste receptor.

After cloning the genes for taste receptors from chickens, swifts and hummingbirds—a three-year process—Baldwin needed to test what the proteins expressed by these genes were responding to. She joined forces with another scientist at another International Taste and Smell meeting. Yasuka Toda, a graduate student of the University of Tokyo and co-first author of the paper, had devised a method for testing taste receptors in cell culture.

Together they showed that in chickens and swifts the receptor responds strongly to amino acids—the umami flavors—but in hummingbirds only weakly. But the receptor in hummingbirds responds strongly to carbohydrates—the sweet flavors.

“This is the first time that this umami receptor has ever been shown to respond to carbohydrates,” Baldwin said.

Toda mixed and matched different subunits of the chicken and hummingbird taste receptors into hybrid chimeras to understand which parts of the gene were involved in this change in function. All told, she found 19 mutations, but there are likely more contributing to this sweet switch, Baldwin and Liberles suspect.

“If you look at the structure of the receptor, it involved really dramatic changes over its entire surface to accomplish this complex feat,” Liberles said. “Amino acids and sugars look very different structurally so in order to recognize them and sense them in the environment, you need a completely different lock and key. The key looks very different, so you have to change the lock almost entirely.”

Once the mutations were discovered, the next question was, do they matter? Does this different taste receptor subunit drive behavior in the hummingbirds?

Back at the feeding stations, the birds answered yes. They spat out the water, but they siphoned up both the sweet nectar and one artificial sweetener that evoked a response in the cell-culture assay, unlike aspartame and its ilk. It’s not nectar, with its nutritional value, but it’s still sweet.

“That gave us the link between the receptor and behavior,” Liberles said. “This dramatic change in the evolution of a new behavior is a really powerful example of how you can explain evolution on a molecular level.”

This work underscores how much remains to be learned about taste and our other senses, Liberles said.

“Sensory systems give us a window into the brain to define what we understand about the world around us,” he said. “The taste system is arguably a really direct line to pleasure and aversion, reward and punishment, sweet and bitter. Understanding how neural circuits can encode these differentially gives us a window into other aspects of perception.”

The work was supported by National Science Foundation grants DDIG 1110487, SICB, Sigma Xi; the Fulbright Commission and Science Foundation Ireland Research Frontiers Program EOB2673; National Institutes of Health RO1DC013289; and JSPS, LS037.


What Grandma Ate

A new study looks at how epigenetic effects are passed down 

iStock image/ParkerDeen

When a pregnant mother is undernourished, her child is at a greater than average risk of developing obesity and type 2 diabetes, in part due to so-called ‘epigenetic’ effects.

A new study led by an HMS researcher at Joslin Diabetes Center and a scientist at the University of Cambridge demonstrates that this ‘memory’ of nutrition during pregnancy can be passed through sperm of male offspring to the next generation, increasing risk of disease for grandchildren as well. In other words, to adapt an old maxim, ‘you are what your grandmother ate.’

The study also raised questions over how epigenetic effects are passed down from one generation to the next—and for how long they will continue to have an impact.

The mechanism by which we inherit characteristics from our parents is well understood: We inherit half of our genes from our mother and half from our father. However, epigenetic effects, whereby a ‘memory’ of the parent’s environment is passed down through the generations, are less well understood.

The best understood epigenetic effects are caused by a mechanism known as ‘methylation’ in which the molecule methyl attaches itself to our DNA and acts to switch genes on or off.

In the study, published in the journal Science and funded mainly by the Medical Research Council and the Wellcome Trust, the international team of researchers showed that environmentally-induced methylation changes occur only in certain regions of our genome (our entire genetic material)—but, unexpectedly, that these methylation patterns are not passed on indefinitely.

Researchers examined the impact that under-nutrition during pregnancy had on offspring in mouse models and looked for the mechanisms by which this effect was passed down through the generations. The male offspring of an undernourished mother were, as expected, smaller than average and, if fed a normal diet, went on to develop diabetes.  Strikingly, the offspring of these were also born small and developed diabetes as adults, despite their own mothers never being undernourished.

 “When food is scarce, children may be born ‘pre-programmed’ to cope with undernourishment. In the event of a sudden abundance in food, their bodies cannot cope and they can develop metabolic diseases such as diabetes. We need to understand how these adaptations between generations occur since these may help us understand the record levels of obesity and type 2 diabetes in our society today,” said Anne Ferguson-Smith, from the department of genetics at the University of Cambridge.

To see how the effect might be passed on, the researchers analyzed the sperm of offspring before the onset of diabetes to look at the methylation patterns. They found that the mouse’s DNA was less methylated in 111 regions relative to a control sperm.

These regions tended to be clustered in the non-coding regions of DNA—areas of DNA responsible for regulating the mouse’s genes. They also showed that in the grandchildren, the genes next to these methylated regions were not functioning correctly. The offspring had inherited a ‘memory’ of its grandmother’s under-nutrition.

Unexpectedly, however, when the researchers looked at the grandchild’s DNA, they found that the methylation changes had disappeared: the memory of the grandmother’s under-nutrition had been erased from the DNA, or at least, was no longer being transmitted via methylation.

“This was a big surprise: dogma suggested that these methylation patterns might persist down the generations,” added co-author and HMS assistant professor of medicine Mary-Elizabeth Patti, director of the Joslin Genomics Core and director of the Hypoglycemia and Severe Insulin Resistance Clinic at Joslin. 

“From an evolutionary point of view, however, it makes sense. Our environment changes and we can move from famine to feast, so our bodies need to be able to adapt. Epigenetic changes may in fact wear off. This could give us some optimism that any epigenetic influence on our society’s obesity and diabetes problem might also be limited and/or reversible,” Patti said. 

The researchers are now looking at whether epigenetic effects no longer have an impact on great-grandchildren and their subsequent offspring. 

Adapted from a Joslin Diabetes Center news release


Aspirin’s Two Sides

Common genetic variant influences whether aspirin prevents cardiovascular disease—or raises risk

Aspirin is the gold standard for antiplatelet therapy and a daily low-dose aspirin is widely prescribed for the prevention of cardiovascular disease.Image: Shutterstock

Now, a new study suggests that common genetic variation in the gene for catechol-O-methyltransferase (COMT) may modify the cardiovascular benefit of aspirin and, in some people, may confer slight harm. The findings, from Harvard Medical School investigators at Beth Israel Deaconess Medical Center and Brigham and Women’s Hospital, are published in the American Heart Association journal Arteriosclerosis, Thrombosis, and Vascular Biology.

“This is one of the few cases where you can identify a single genetic polymorphism which has a significant interaction with aspirin such that it affects whether or not it protects against cardiovascular disease,” said first author Kathryn Hall, an HMS research fellow and investigator in the Division of General Medicine and Primary Care at Beth Israel Deaconess.

COMT is a key enzyme in the metabolism of catecholamines, a group of hormones that include epinephrine, norepinephrine and dopamine. These hormones are implicated in a broad spectrum of disorders, including hypertension.

“We were initially interested in finding out if the COMT gene affected people’s susceptibility to cardiovascular disease, such as myocardial infarction or ischemic stroke,” Hall said.

Knowing that aspirin is commonly prescribed for the prevention of cardiovascular disease, the investigators also wanted to learn if genetic variation in COMT would influence aspirin’s potential benefit.        

To answer these questions, the researchers used data from the Women’s Genome Health Study, a cohort of more than 23,000 women who were followed for 10 years in a randomized double-blind, placebo-controlled trial of low-dose aspirin or vitamin E for the primary prevention of cardiovascular disease. Their analysis focused on val158met, a common variant in the COMT gene. Individuals who have two copies of the gene for the enzyme’s high-activity valine form, the “val/vals,” have been shown to have lower levels of catecholamines compared to individuals who have two copies of the gene for the enzyme’s low-activity methionine form, the “met/mets.” The val/met people are in between.

“When we examined women in the placebo arm of the trial, we found that the 23 percent of the women who were ‘val/vals’ were naturally protected against cardiovascular disease,” said senior author Daniel Chasman, HMS associate professor of medicine at Brigham and Women’s. He is also a genetic epidemiologist in the Division of Preventive Medicine at the hospital. “This finding, which was replicated in two other population-based studies, was in itself of significant interest.”

The investigation further revealed the surprising discovery that when the women with the val/val polymorphism were allocated to aspirin, this natural protection was eliminated.

“As we continued to look at the effects of drug allocation, we found that val/val women who were randomly assigned to aspirin had more cardiovascular events than the val/vals who were assigned to placebo,” says Chasman. Among the 28 percent of women who were met/met, the opposite was true, and these women had fewer cardiovascular events when assigned to aspirin compared to placebo. The benefit of aspirin compared to placebo allocation for met/mets amounted to reduction of one case of incident cardiovascular disease for 91 treated women over 10 years of study follow-up. By contrast, the harm of aspirin compared to placebo allocation for the val/val women was an increase of one case per 91 treated.

The researchers further found that rates of cardiovascular disease were also reduced in met/met women assigned to vitamin E compared to those assigned to placebo.

The authors stressed that the findings will require further research and replication to understand their potential for clinical impact. Nonetheless, they note that because aspirin is preventively prescribed to millions of individuals and the COMT genetic variant is extremely common, this study underscores the potential importance of individualizing therapies based on genetic profiles.

“What this study suggests is that we can be smarter about the groups of patients that would most likely benefit from aspirin,” said study coauthor Joseph Loscalzo, chairman of the Department of Medicine and physician-in-chief at Brigham and Women’s. He is also the Hersey Professor of the Theory and Practice of Physic at HMS. “Rather than give aspirin to all patients with risk factors for heart disease, we need to use modern genomics and genetics to identify those individuals for whom aspirin has the greatest benefit and the lowest risk of adverse effects.”    

One possible reason for the val/val protection could lie in COMT’s role in the breakdown of epinephrine, the “fight or flight” hormone, which is tightly linked to regulation of the cardiovascular system.

“When epinephrine levels rise in response to stress, blood pressure goes up and high blood pressure is a precursor to heart disease,” said Hall. “One possibility is that val/val individuals have less epinephrine than met/met individuals because their COMT is more efficient at breaking it down. This might help to naturally protect them against cardiovascular disease—that’s our working hypothesis. It’s harder to explain why the effect is modified by aspirin and that’s what we’re in the lab aggressively trying to figure out.”

The Women’s Genome Health Study is supported by HL043851 and HL080467 from the National Heart, Lung, and Blood Institute and CA 047988 from the National Cancer Institute. This study was also supported by NIH grants T32A5000051; R01AT004662; K24AT004095; R21AT002860; 3R01AT004662-02S1from the National Center for Complementary and Alternative Medicine.

Adapted from a Beth Israel Deaconess news release.


A Crisper Version of CRISPR

New genome-editing tool reduces risk of off-target mutations

A next-generation genome-editing system developed by Harvard Medical School investigators at Massachusetts General Hospital substantially decreases the risk of producing unwanted, off-target gene mutations. In a paper published in Nature Biotechnology, the researchers report a new CRISPR-based RNA-guided nuclease technology that uses two guide RNAs, significantly reducing the chance of cutting through DNA strands at mismatched sites.

“This system combines the ease of use of the widely adopted CRISPR/Cas system with a dimerization-dependent nuclease activity that confers higher specificity of action,” said J. Keith Joung, HMS associate professor of pathology at Mass General and senior author of the report. “Higher specificity will be essential for any future clinical use of these nucleases, and the new class of proteins we describe could provide an important option for therapeutic genome editing.”

Engineered CRISPR-Cas nucleases are genome-editing tools that combine a short RNA segment matching its DNA target with a DNA-cutting enzyme called Cas9. The tools have been the subject of much investigation since their initial development in 2012. Easier to use than the earlier zinc finger nuclease (ZFN) and transcription activator-like effector nuclease (TALEN) systems, they have successfully induced genomic changes in several animal model systems and in human cells. But in a previous Nature Biotechnology paper published in June 2013, Joung’s team reported that CRISPR-Cas nucleases could produce additional mutations in human cells, even at sites that differed from the DNA target by as much as five nucleotides (the subunits of DNA).  

To address this situation, the investigators developed a new platform in which the targeting function of Cas9 was fused to a nuclease derived from a well-characterized enzyme called Fokl, which functions only when two copies of the molecule are paired, in a relationship called dimerization. This change essentially doubled the length of DNA that must be recognized for cleavage by these new CRISPR RNA-guided Fokl nucleases, significantly increasing the precision of genome editing in human cells. Joung and his colleagues also demonstrated that these new RNA-guided Fokl nucleases are as effective at on-target modification as existing Cas9 nucleases that target a shorter DNA sequence.

“By doubling the length of the recognized DNA sequence, we have developed a new class of genome-editing tools with substantially improved fidelity compared with existing wild-type Cas9 nucleases and nickases,” the enzymes that cleave a single DNA strand, said Joung, who is associate chief for research in the Mass General Department of Pathology. 

The research team also has developed software that enables users to identify potential target sites for these new RNA-guided Fokl nucleases. The freely available software package is ZiFiT Targeter.

The study was supported by National Institutes of Health Director’s Pioneer Award DP1 GM105378; NIH grants R01 GM088040, P50 HG005550 and R01 AR063070; and the Jim and Ann Orr Massachusetts General Hospital Research Scholar Award. Joung is a co-founder of Editas Medicine Inc., which has an exclusive option to license the new CRISPR RNA-guided Fokl nuclease technology for therapeutic applications.

Adapted from a Mass General news release.


Breast Cancer Clue

Researchers identify new mechanism of cancer caused by loss of BRCA1 and BRCA2 gene function

Inherited mutations in the BRCA1 or BRCA2 tumor suppressor genes are by far the most frequent contributors to hereditary cancer risk in the human population, often causing breast or ovarian cancer in young women of child-bearing age. Attempts to test the role that the BRCA genes play in regulating a repair process associated with genome duplication have proven frustratingly difficult in living mammalian cells.

Now Harvard Medical School investigators at Beth Israel Deaconess Medical Center report a new mechanism by which BRCA gene loss may accelerate cancer-promoting chromosome rearrangements. The new findings explain how the loss of BRCA1 or BRCA2 function impairs homologous recombination, a normally accurate repair process used to fix DNA breaks, and actually stimulates faulty, error-prone homologous recombination repair.

A ribbon diagram of the BRCA1 protein, which repairs damaged DNA. Mutations in the <i>BRCA1</i> or <i>BRCA2</i> genes increase the risk for breast and ovarian cancer. Image: Wikimedia Commons

Described online in the April 28 issue of Nature, the discovery could ultimately provide clinicians with valuable new information to help them ascertain risk and guide patient treatment when faced with BRCA mutations of uncertain significance. The finding also offers a potentially valuable new tool for the development of cancer therapeutics.

 “Mutations in the BRCA genes cause breast and ovarian cancers that affect thousands of women throughout the U.S. and around the world, often striking them in the prime of life,” said senior author Ralph Scully, HMS associate professor of Medicine at Beth Israel Deaconess in the hospital’s Breast Cancer Oncology program. “For almost two decades, scientists have been striving to better understand the tumor suppressor functions of BRCA1 and BRCA2.”

Potentially harmful breaks in DNA strands commonly occur during DNA replication, a prerequisite for cell division. These breaks occur when the replication fork that duplicates the genome stalls at sites of DNA damage. If not properly repaired, the breaks can promote genomic instability, leading to cancer and other diseases.

“Some years ago, we and others suggested that BRCA1 and BRCA2 regulate homologous recombination at sites of stalled replication,” explains Scully. “We believe that this function is critical to how these genes suppress breast and ovarian cancer. Until now, we haven’t had the tools necessary to study in molecular detail the homologous recombination processes at sites of replication fork stalling in the chromosomes of a living mammalian cell.”

To solve this problem, first author Nicholas Willis, HMS research fellow in medicine in the Scully laboratory, created a new tool by harnessing a protein-DNA complex that evolved in bacteria.

“We found that the Escherichia coli Tus/Ter complex can be engineered to induce site-specific replication fork stalling and chromosomal homologous recombination in mouse cells,” explained Willis.  “In its essence, E. coli bacteria—a standard model organism in science—has evolved a very simple system to arrest replication forks in a site-specific manner.”

This system is composed of short DNA elements called Ter sites, which are 21 to 23 base pairs long and tightly bound by the protein Tus. “Tus binds these Ter elements with extremely high affinity and, upon replication fork approach, acts as a barrier to fork progression along the DNA. Tus/Ter effectively sets up a ‘roadblock’ and stalls the replication fork.”

The acid test for the new tool, said the authors, came when this same short Ter sequence was placed into a reporter, a slightly larger DNA sequence that can undergo certain rearrangements within a chromosome when triggered to do so. “When it engaged in homologous recombination, a change in the sequence caused the cells to express green fluorescent protein,” said Willis. “When the cells glowed green, we knew we had a positive event.”

The team adapted the reporter sequence to distinguish between error-free/high-fidelity homologous recombination and an error-prone/aberrant form of homologous recombination. “Remarkably, when we studied cells lacking BRCA1 or BRCA2, we found that the frequency of aberrant homologous recombination events triggered at Tus/Ter-stalled replication forks had actually increased compared to normal cells. We knew at this point that we had discovered a new and important process by which BRCA gene loss promotes cancer.”

The discovery provides a promising bridge between basic science and the clinic, said Scully. “Sometimes a genetic sequencing test reveals a mutation in BRCA1 or BRCA2 that has not been definitively associated with cancer,” he said. Often described as “variants of uncertain significance,” these mutations are not found in high enough frequency in healthy women or in women with breast or ovarian cancers to allow the specific BRCA1 or BRCA2 mutations to be reliably classified as high risk or low risk. This is an important issue because a woman with a known high-risk BRCA gene mutation may elect to undergo potentially lifesaving prophylactic mastectomy or oophorectomy, Scully said.

“There is a growing appreciation that careful measurement of the homologous recombination functions of BRCA1 and BRCA2 variants-of-uncertain-significance mutants might help to classify them into high-risk or low-risk groups,” he said. “It would be gratifying if our system could contribute new information to help ongoing efforts to classify these mutants.”

 Furthermore, understanding the mechanisms that regulate homologous recombination at stalled replication forks might hold additional promise for the development of novel cancer therapeutics. “If we could use this tool to help develop new cancer therapies, it would be a grand slam,” said Scully. “This new system might also be useful in genome editing, which is considered a groundbreaking technology used for the development of new gene therapies.”

This study was funded, in part, by National Institutes of Health grants R01CA095175, R01GM073894, R21CA144017, and R37GM26938; by National Institutes of Health /National Cancer Institute postdoctoral fellowship 5T32CA081156; and by ACS postdoctoral fellowship PF-12-248-01-DMC.

Adapted from a Beth Israel Deaconess news release.


Aspirin Tied to Lower Colon Cancer Risk for Some

Aspirin users with high levels of colonic enzyme had half the risk of developing colon cancer

Image: Chaval Brasil via Wikimedia Commons

The humble aspirin may have another beneficial effect beyond easing the pain of headache and reducing the risk of heart attack: lowering colon cancer risk among people with high levels of a specific enzyme.

The finding comes from a multi-institutional team that analyzed data and samples from two long-term studies involving nearly 128,000 participants. The researchers discovered that people who took aspirin and had high levels of a specific enzyme in their colons had half the risk of developing colorectal cancer compared with people who also took aspirin but whose colons showed low levels of the enzyme, called 15-hydroxyprostaglandin dehydrogenase, or 15-PGDH for short. About half of the population possesses high levels of 15-PGDH.

The results appear in the April 23 edition of Science Translational Medicine. While previous trials and prospective studies have shown that aspirin use reduces colorectal cancer risk, this retrospective study may explain why aspirin benefits some people, but not others.

The research team included scientists from Brigham and Women’s Hospital, Case Western Reserve School of Medicine, Dana-Farber Cancer Institute, Harvard Medical School, Massachusetts General Hospital and University Hospitals Case Medical Center.

“If you looked at the folks from the study who had high 15-PGDH levels and took aspirin, they cut their risk of colon cancer by half,” said senior author Sanford Markowitz, Ingalls Professor of Cancer Genetics at Case Western Reserve School of Medicine. “If you looked at the folks from the study who were low for 15-PGDH, they did not benefit at all from taking aspirin. These findings represent a clean Yes-No about who would benefit from aspirin.”

According to the American Cancer Society, colorectal cancer is the second leading cause of cancer-related deaths in the United States, with predictions that 137,000 Americans will develop the disease and 50,000 will die from it in 2014.

In the current study, the scientists built on earlier research indicating that regular use of non-steroidal anti-inflammatory drugs (NSAIDs), including aspirin, reduces the risk of developing colon cancer for some but not all individuals. Markowitz, also a medical oncologist at University Hospitals Case Medical Center, joined co-senior author Andrew Chan, HMS associate professor of medicine and a gastroenterologist at Mass General, to explore whether the presence of 15-PGDH led to different outcomes in developing colon cancer.

They hope to develop a test to guide physicians and patients in determining whether aspirin would help them.

The team examined tissues of 270 colon cancer patients who were among 127,865 participants followed for more than 30 years in the Harvard-based Nurses’ Health Study and Health Professionals Follow-up Study. Previous reports from the Mass General and Dana-Farber team showed that participants in these studies who regularly took aspirin had a lower risk of colorectal cancer. In earlier research, Case Western Reserve investigators and Monica Bertagnolli, HMS professor of surgery at Brigham and Women’s, had found that 15-PGDH appeared to enhance the ability of celecoxib, an anti-inflammatory medication commonly known as Celebrex, to prevent colon tumors in mice and in 16 humans tested. But when 15-PGDH was low or absent, celecoxib did not prevent colon tumors in mice or humans.

In the latest study, the investigators combined forces in a larger study to examine whether 15-PGDH levels might also be associated with the colon cancer-preventing benefits of aspirin, which lacks the adverse cardiovascular side effects of celecoxib.

The Mass General and Dana-Farber team dissected normal colon tissue from the pathology specimens of participants who developed colon cancer over the studies’ follow-up periods. The team at Case Western Reserve then analyzed these colon tissues to identify which among them had high or low levels of colon 15-PGDH. The investigators at Mass General and Dana-Farber examined how participants’ aspirin use and levels of 15-PGDH might be related to the risk of colorectal cancer.

The study’s results could lead to a test that would allow more personalized decisions about treatment to prevent colorectal cancer. People whose 15-PGDH levels indicate aspirin would have little benefit might choose to avoid the potential gastrointestinal side effects—such as stomach ulcers—that can accompany aspirin use. The researchers plan to develop a cost-effective and accessible test for measuring 15-PGDH in the colon. Chan and Markowitz believe such a test could become part of current medical practice. 

“During a colonoscopy, a gastroenterologist could easily and safely take an additional biopsy from the colon in individuals for whom preventive aspirin treatment might be appropriate,” Chan said. 

“There would be no reason why a good hospital pathology laboratory could not establish the test for 15-PGDH,” Markowitz said.

To confirm their findings, the study authors hope to launch a randomized, prospective clinical trial in which high-risk patients would be identified, treated with aspirin or a placebo, and monitored for development of colorectal tumors.

The mechanisms of action in 15-PGDH and in aspirin make them key players in colon cancer, the scientists said. Prostaglandins promote development of colon cancer. Aspirin helps prevent colon cancer development by blocking prostaglandins from being generated, while 15-PGDH helps prevent colon cancer development by catalyzing a reaction that “chews up” prostaglandins. Markowitz refers to the gene that produces 15-PGDH as the body’s genetic form of aspirin. The study suggests that both aspirin and 15-PGDH must work together to effectively prevent colon cancer, bringing the most benefit to individuals who have high levels of 15-PGDH.

“This study highlights the benefits of the relatively new practice of molecular pathological epidemiology, ” said co-senior author Shuji Ogino, HMS associate professor of pathology at Brigham and Women’s and Dana-Farber. “The molecular pathology part relates to analysis of 15-PGDH gene expression level in the normal colon to classify cancer based on molecular pathogenesis, while the epidemiology part relates to collection and analysis of aspirin use data in a population. This is an integration of these analyses."

Other researchers involved in the study include first author Stephen Fink, an instructor at Case Western Reserve; co-authors Mai Yamauchi and Reiko Nishihara, HMS research fellows at Dana-Farber; and senior author Charles S. Fuchs, HMS professor of medicine at Dana-Farber.

The study was supported by the Entertainment Industry Foundation’s National Colorectal Cancer Research Alliance; the National Cancer Institute’s GI-SPORE program (Specialized Programs of Research Excellence in Gastrointestinal Cancers) and Early Detection Research Network; National Institutes of Health grants P01 CA87969, P01 CA55075, 1UM1 CA167552, R01 CA136950, P50 CA127003, R01 CA151993, P50 CA150964, U01 CA152756, R01 CA137178 and K24 DK 098311; the Damon Runyon Cancer Research Foundation; and gifts from the Cleveland-based Marguerite Wilson Foundation, the Leonard and Joan Horvitz Foundation, the Richard Horvitz and Erica Hartman-Horvitz Foundation, and the Boston-based Bennett Family Fund for Targeted Therapies Research.

Adapted from a joint Case Western Reserve and Mass General news release.


Genes and Galaxies

Symposium taps experts to consider how genetic research can “boldly go where no one has gone before . . .” 


If you haven’t thought about reworking the human genome so people can colonize other planets, don’t worry. Plenty of people are on it.

Scientists of many stripes have been figuring out what barriers would keep us from calling distant, inhospitable galactic real estate “home” if—or when, depending on your point of view— we damage the Earth enough to face extinction. And then there’s the whole question of whether we should try to win a stay of execution for our species. After all, what makes us so special?

Those questions were just the beginning of a free-form symposium hosted March 13 by the HMS Department of Genetics on “Genetics, Biomedicine, and the Human Experience in Space,” the standing-room-only crowd in attendance fueled by pizza and unbridled curiosity.

Speakers quickly made clear why space travel and exploration over vast, uncharted distances depends on numerous, unknown factors hidden in our genes. Living with microgravity while being bombarded with cosmic rays can affect different people different ways.  Scientists want to know why—and which genes might make it better or worse.

Also, space is just cool.

The Role of Genetics

The session unleashed uninhibited discussion, with a fairly even split between prepared presentations and informed thinking-out-loud improv from the audience.

Before the event, symposium co-organizers Ting Wu and Susan Dymecki, both HMS professors of genetics, stated their belief that genetics will play a huge part in the success of humans off the planet.

“We are a medical school. Whether or not you agree with sending people into space, we are responsible for their health on and off the planet,” said Wu.

Well-known muscle and skeletal weakness and sleep disruption are not the only problems humans encounter in space.  Physical concerns ride along with behavioral and neuropsychiatric issues aboard current spacecraft, not to mention whatever vehicles might ferry people farther away.  It’s lonely out there.

Thinking about travel to Mars, one of our nearest neighbors, is daunting for robots, much less people. Just ask symposium guest Adam Steltzner, mechanical systems lead at the Jet Propulsion Lab, about the prodigious work that brought back what we know about the planet. Or Dorit Donoviel, deputy chief scientist and industry forum lead of the National Space Biomedical Research Institute, and assistant professor in the Department of Pharmacology and Center for Space Medicine at Baylor College of Medicine, who studies astronauts and the challenges they face, including problems with vision and headaches.

Focusing on Space

HMS geneticists spoke about the intersections between their scientific focus and space. Susan Dymecki said she began thinking about why cosmonauts in the former Soviet Union’s space program were forbidden from playing chess on board space flights. The answer involves aggression and impulsivity.

For HMS professor of genetics David Sinclair, this intersection involves the potential advantages of extant human variation and rallying our genetics to counter aging during long-distance travel spanning hundreds of thousands of years.

Bruce Yankner, HMS professor of genetics, talked about protecting the brain and memory in space. Wu presented her vision for using ultraconserved elements, which some consider to be among the most mysterious sequences of the human genome, to orchestrate chromosome behavior to and thus protect genomes against cosmic radiation in space.

Mary Bouxsein, a biomechanical engineer and HMS assistant professor of orthopedic surgery at Beth Israel Deaconess Medical Center, a last minute addition to the program, showed the devastating effects of space flight on bone, and how that might be prevented in space—and on Earth -- with a newly developed therapy.

Genetics professor Gary Ruvkun, whose talk was entitled “What’s true for E. coli is true for the elephant” and our speculative kin on Gliese 667 Cf, (a potential Class M planet in the Gliese 667 star system), peppered the meeting with a positive view of the extinction of the human species and then proposed that, rather than travel to another planet, we “print” ourselves there. Conversely, we could print extraterrestial life on Earth.

George Church, the Robert Winthrop Professor of Genetics, suggested using genomics to identify and engage human protective variants, speculating that, by ridding ourselves of our microbiome and taking advantage of variants that suppress pain, we might create a habitat in which surgeries can occur without anesthesia or need for sterilization.

Space is vast, cold and hard for us humans, and outside of Earth, its planets, and moons—too hot, too cold, too toxic for life that evolved here— are not much more welcoming. Should we want to go there, and decide who is best suited to do so, a great deal more work needs to be done not only in jet propulsion but in human genetics.  



Location, Location, Location

 New method IDs working copies of genes in human cells; could help diagnose sick tissues early

George Church and Evan Daugharthy explain how fluorescent in situ sequencing works and describe potential applications, such as new diagnostics that could spot the earliest signs of disease. Credit: Wyss Institute, Allen Institute for Brain Science, and Harvard Medical School.

In biology, as in real estate, location matters. Working copies of active genes -- called messenger RNAs or mRNAs -- are positioned strategically throughout  living tissues, and their location often helps regulate how cells and tissues grow and develop. But to analyze many mRNAs simultaneously, scientists have had to grind cells to a pulp, which left them no good way to pinpoint where those mRNAs sat within the cell. 

Now a team at the Wyss Institute of Biologically Inspired Engineering at Harvard University and Harvard Medical School, in collaboration with the Allen Institute for Brain Science, has developed a new method that allows scientists to pinpoint thousands of mRNAs and other types of RNAs at once in intact cells -- all while determining the sequence of letters, or bases, that identify them and reveal what they do.

The method, called fluorescent in situ RNA sequencing (FISSEQ), could lead to earlier cancer diagnosis by revealing molecular changes that drive cancer in seemingly healthy tissue. It could track cancer mutations and how they respond to modern targeted therapies, and uncover targets for safer and more effective ones.

To develop fluorescent in situ sequencing, scientists first fix in place thousands of RNAs -- including working copies of genes called messenger RNAs -- in cells, tissues, organs or embryos. Here, RNAs are labeled red in a mouse brain. Image: HMS and Wyss.

The method could also help biologists understand how tissues change subtly during embryonic development -- and even help map the maze of neurons that wire the human brain. The researchers reported the method in today’s online edition of Science.


“By looking comprehensively at gene expression within cells, we can now spot numerous important differences in complex tissues like the brain that are invisible today,” said George Church, a  professor of genetics at Harvard Medical School and a core faculty member at the Wyss Institute. “This will help us understand like never before how tissues develop and function in health and disease.”

Locking RNAs in Place

Healthy human cells typically turn on nearly half of their 20,000 genes at any given time, and they choose those genes carefully to produce the desired cellular responses. Moreover, cells can dial gene expression up or down, adjusting to produce anywhere from a few working copies of a gene to several thousand. 

But simultaneously pinpointing the cellular location of all those mRNAs is a tall order.

Church and Je Hyuk Lee, a research fellow at Harvard Medical School and the Wyss Institute, were up for the challenge. Moreover, they wanted to simultaneously determine the sequence of those RNAs, which identifies them and often reveals their function.

Lee and his colleagues first treated the tissue chemically to fix the cell’s thousands of RNAs in place. Then they used enzymes to copy those RNAs into DNA replicas, and copy those replicas many times to create a tiny ball of replica DNA fixed to the same spot.

They managed to fix and replicate thousands of the cell’s RNAs at once -- but then became a victim of their own success. The RNAs were so tightly packed inside the cell that even a tricked-out microscope and camera could not distinguish the flashing lights of one individual ball of replica DNA from those of its neighbors.

A new way to image RNA

To solve that problem, the researchers pioneered an unconventional method to visualize tiny objects inside cells. It works like an urban postal system. If a postmaster tried to identify each home in her city by color, she would quickly run out of colors as new homes were built, resulting in undelivered mail. Instead, postmasters keep track of each home by assigning it a unique address.

The researchers realized they could assign each RNA in the cell a unique address: the sequence of “letters,” or bases, in the RNA molecule itself. They figured they could read the address using methods akin to next-generation DNA sequencing, a set of high-speed genome sequencing methods Church helped develop in the early 2000s.

In next-gen sequencing, scientists grind up tissue, extract its DNA, break the DNA into pieces, then dilute those pieces enough so that each piece of DNA sticks to a separate spot on a glass slide. They use enzymes and four different fluorescent dyes -- one each for each of the four “letters,” or bases -- to make the DNA flash a sequence of colors that reveals its sequence.

By analogy, the scientists sought to fix RNA in place in the cell, make a tiny ball with many matching DNA replicas of each RNA, then adapt next-gen DNA sequencing so it worked in fixed cells. The four flashing colors would reveal the base sequence of each replica DNA, which would tell them the base sequence of the matching RNA from which it was derived. And those sequences would in theory provide an unlimited number of unique addresses – one for each of the original RNAs.

The scientists struggled at first to visualize the flashing lights of individual balls of replica DNA from a distance where the whole landscape of the tissue remained in view.

They succeeded by selectively turning on just a fraction of those flashing dots at any given time, so they could distinguish single balls of replica DNA flashing across the cellular landscape.

The strategy would only work, however, if they could actually read enough of the base sequence to provide a unique address. At first they could not determine more than six bases in the replica DNA, which did not provide enough unique addresses to identify individual genes in the human genome.  That’s when Evan Daugharthy, a graduate student at Harvard Medical School, stepped in.

FISSEQ at Work

Daugharthy first devised an algorithm to locate the sequence of the replica DNA with the known sequence of genes in the human genome. Flashing lights that did not correspond to a real gene were erased from the image.

Then Daugharthy hacked a commercial DNA sequencing kit, which enabled the team to sequence 30 bases, more than enough to provide each replica DNA with a unique address. In this way the team could create a composite image representing the sequence, and location, of RNA corresponding to every gene in the human genome.

Lee, Daugharthy and their colleagues then tested the method to detect the genes skin cells turn on as they multiply and migrate to heal a simulated wound in a petri dish. Cells growing into the wound had 12 genes that were activated much more or much less than nearby cells sitting idly on the sidelines. Similar experiments could identify new markers of diseased tissue or new targets for targeted molecular therapies.

“What George’s team has accomplished is a technological tour de force,” said Wyss Institute founding director Don Ingber.  “By spotting incredibly subtle but incredibly important changes in gene expression and precisely defining their position inside the cell, they have helped open the door to a new age of cellular diagnostics.”

The work was funded by the National Institutes of Health, the Allen Institute for Brain Science and the Wyss Institute.  


Sharper Image

Researchers capture ultrasharp images of multiple cell components at once

Ten-color super-resolution images (artificially arranged into one composite) of synthetic DNA nanostructures displaying patterns that resemble the numerals “0” through "9,” produced by Exchange-PAINT using only a single dye and a single laser source. Image: Johannes B. Woehrstein/Wyss Institute

A new microscopy method could enable scientists to generate snapshots of dozens of different biomolecules at once in a single human cell, a team from the Wyss Institute of Biologically Inspired Engineering at Harvard University reported Sunday in Nature Methods.

Such images could shed light on complex cellular pathways and potentially lead to new ways to diagnose disease, track its prognosis, or monitor the effectiveness of therapies at a cellular level.

Cells often employ dozens or even hundreds of different proteins and RNA molecules to get a complex job done. As a result, cellular job sites can resemble a busy construction site, with many different types of these tiny cellular workers coming and going. Today's methods typically only spot at most three or four types of these tiny workers simultaneously. But to truly understand complex cellular functions, it's important to be able to visualize most or all of those workers at once, said Peng Yin, assistant professor of systems biology at Harvard Medical School and a core faculty member at the Wyss Institute.

"If you can see only a few things at a time, you are missing the big picture," Yin said.

Yin's team sought a way to take aerial views of job sites that could spot up to dozens of types of biomolecules that make up large cellular work crews.


This ultrasharp Exchange-PAINT image simultaneously spots microtubules (green), mitochondria (purple), Golgi apparatus (red), and peroxisomes (yellow) from a single human cell. Image: Maier Avendano/Wyss Institute


To capture ultrasharp images of biomolecules, they had to overcome laws of physics that stymied microscopists for most of the last century. When two objects are closer than about 200 nanometers apart — about one five-hundredth the width of a human hair — they cannot be distinguished using a traditional light microscope: the viewer sees one blurry blob where in reality there are two objects.

Since the mid-1990s, scientists have developed several ways to overcome this problem using combinations of specialized optics, special fluorescent proteins or dyes that tag cellular components.

Ralf Jungmann, now a postdoctoral Fellow working with Yin at the Wyss Institute and Harvard Medical School, helped develop one of those super-resolution methods, called DNA-PAINT, as a graduate student. DNA-PAINT can create ultrasharp snapshots of up to three cellular workers at once by labeling them with different colored dyes.

To visualize cellular job sites with crews of dozens of cellular workers, Yin's team, including Jungmann, Maier Avendano, M.S., a graduate student at Harvard Medical School, and Johannes Woehrstein, a postgraduate research fellow at the Wyss Institute, modified DNA-PAINT to create a new method called Exchange-PAINT.

Exchange-PAINT relies on the fact that DNA strands with the correct sequence of letters, or nucleotides, bind specifically to partner strands with complementary sequences. The researchers label a biomolecule they want to visualize with a short DNA tag, then add to the solution a partner strand carrying a fluorescent dye that lights up only when the two strands pair up. When that partner strand binds the tagged biomolecule, it lights up, then lets go, causing the biomolecule to "blink" at a precise rate the researchers can control. The researchers use this blinking to obtain ultrasharp images.

They then repeat the process to visualize a second target, a third, and so on. Then they overlay the resulting images to create a composite image in which each biomolecule — each cellular worker — is assigned a different color. This allows them to create false-color images that simultaneously show many types of biomolecules — far more than they could simultaneously visualize by labeling them with different colored dyes. And these false-color images allow them to spot enough cellular workers at once to capture the entire scene.

To test Exchange-PAINT, the researchers created 10 unique pieces of folded DNA, or DNA origami, that resembled the numerals 0 through 9. These numerals could be resolved with less than 10 nanometers resolution, or one-twentieth of the diffraction limit.

The team was able to use Exchange-PAINT to capture clear images of the 10 different types of miniscule DNA origami structures in one image. They also used the method to capture detailed, ultrasharp images of fixed human cells, with each color tagging an important cellular component — microtubules, mitochondria, Golgi apparatus, or peroxisomes.

Yin expects the method, with further development, to be able to visualize dozens of cellular components at once.

"Peng's exciting new imaging work gives biologists an important new tool to understand how multiple cellular components work together in complex pathways," said Wyss Institute Founding Director Don Ingber. "I expect insights from those experiments to lead to new ways to diagnose and monitor disease." Ingber is also the Judah Folkman Professor of Vascular Biology at Harvard Medical School and Boston Children's Hospital and Professor of Bioengineering at Harvard School of Engineering & Applied Sciences.

In addition to Yin, Jungmann, Avendano, and Woehrstein, the team included Mingjie Dai, a graduate student in biophysics at Harvard University and William Shih, a Wyss Institute core faculty member who is also associate professor of biological chemistry and molecular pharmacology at Harvard Medical School and associate professor of cancer biology at the Dana-Farber Cancer Institute. The work was funded by the National Institutes of Health, the Office of Naval Research, the National Science Foundation, the Humboldt Foundation, and the Wyss Institute.

Adapted from a Wyss Institute news release.