Marching to Our Own Sequences

Study finds DNA replication timing varies among people

Steven McCarroll and Amnon Koren describe their surprising findings and how they made their discovery by tapping into an existing online database of genome sequencing data. Video: Rick Groleau and Stephanie Dutchen

Imagine being asked to copy a library of books. Doing it yourself would take forever. You’d probably call some friends and come up with a plan to divide and conquer.

That’s what a human cell does when faced with the task of replicating six billion letters of DNA each time it divides. Instead of reading each chromosome in one slow pass, DNA replication machinery dives in at many origin points. Some segments get copied earlier or later than others.

A new study from geneticists at Harvard Medical School and the Broad Institute of Harvard and MIT has found that this replication plan—including where the origin points are and in what order DNA segments get copied—varies from person to person.

The study, published online Nov. 13 in Cell, also identifies the first genetic variants that orchestrate replication timing.

“Everyone’s cells have a plan for copying the genome. The idea that we don’t all have the same plan is surprising and interesting,” said Steven McCarroll, assistant professor of genetics at HMS, director of genetics for the Stanley Center for Psychiatric Research at the Broad and senior author of the paper.

“It’s a new form of variation in people no one had expected,” said first author Amnon Koren, postdoctoral fellow at HMS and the Broad. “That’s very exciting.”

Hidden orchestrator

DNA replication is one of the most fundamental cellular processes, and any variation among people is likely to affect genetic inheritance, including individual disease risk as well as human evolution, the authors said.

It’s been known that replication timing affects mutation rates; DNA segments that are copied late or too early tend to have more errors. The new study indicates that people with different timing programs therefore have different patterns of mutation risk across their genomes.

For example, McCarroll’s team found that differences in replication timing could explain why some people are more prone than others to certain blood cancers.

Researchers had previously known that acquired mutations in the gene Janus kinase 2, or JAK2, lead to these cancers. They had also noticed that people with such JAK2 mutations tend to have a distinctive set of inherited genetic variants nearby, but they weren’t sure how the inherited variants and the new mutations were connected. McCarroll’s team found that the inherited variants are associated with an “unusually early” replication origin point and proposed that JAK2 is more likely to develop mutations in people with that very early origin point.

“Replication timing may be a way that inherited variation contributes to the risk of later mutations and diseases that we usually think of as arising by chance,” said McCarroll.

Untapped riches

McCarroll, Koren and colleagues were able to make these discoveries in large part because they invented a new way to obtain DNA replication timing data. Turned out, it was hiding in plain sight.

Until now, to study replication timing, scientists needed to painstakingly “grow cells for a couple of weeks and sort them with a special machine and do a big, complicated, expensive, time-consuming experiment”—all to obtain material from just a few people at a time, said Koren.

The team suspected there was an easier way. They turned to the 1000 Genomes Project, which maintains an online database of sequencing data collected from hundreds of people around the world.

Because much of the DNA in the 1000 Genomes Project had been extracted from actively dividing cells, the team hypothesized that information about replication timing lurked within.

They were right. They counted the number of copies of individual genes in each genome. Because early replication origins had created more segment copies at the time the sample was taken than late replication origins had, they were able to create a personalized replication timing map for each person.

“People had seen these patterns before, but just dismissed them as artifacts of sequencing technology,” said McCarroll. After conducting numerous tests to rule out that possibility, “we found that they reflect real biology.”

The researchers then compared each person’s copy number information with his or her genetic sequence data to see if they could match specific genetic variants to replication timing differences. From 161 samples, they identified 16 variants. The variants were short, and most were common.

“I think this is the first time we can pinpoint genetic influences on replication timing in any organism,” said Koren.

The variants were located near replication origin points, leading the team to wonder if they affect replication timing by altering where a person’s origin points are. They also suspect that the variants work by altering chromatin structure, exposing local sequences to replication machinery. The team intends to find out. They also want to search for additional variants that control replication timing.

“These 16 variants are almost certainly just the tip of the iceberg,” said Koren.

The door is open

As more variants come to light in future studies, researchers will be better able to manipulate replication timing in the lab and learn more about how it works and what its biological significance is.

Such studies should flourish now that the team has shown that “all you need to do to study replication timing is grow cells and sequence their DNA, which everyone is doing these days,” said Koren. The new method “is much easier, faster and cheaper, and I think it will transform the field because we can now do experiments in large scale.”

“We found that there is biological information in genome sequence data,” added McCarroll. “But this was still an accidental biological experiment. Now imagine the results when we and others actually design experiments to study this phenomenon.”

This research was funded by the National Human Genome Research Institute (R01 HG 006855), the Integra-Life Seventh Framework Programme (grant #315997), the Stanley Center for Psychiatric Research, the Howard Hughes Medical Institute and the Harvard Stem Cell Institute.


New Branch Added to European Family Tree

Genetic analysis reveals Europeans descended from at least three ancient groups

This skull of a 7,000-year-old German farmer was among the ancient human bones that revealed more about the genetic heritage of present-day Europeans. Image: Joanna Drath/University of Tübingen

The setting: Europe, about 7,500 years ago.

Agriculture was sweeping in from the Near East, bringing early farmers into contact with hunter-gatherers who had already been living in Europe for tens of thousands of years.

Genetic and archaeological research in the last 10 years has revealed that almost all present-day Europeans descend from the mixing of these two ancient populations. But it turns out that’s not the full story.

Researchers at Harvard Medical School and the University of Tübingen in Germany have now documented a genetic contribution from a third ancestor: Ancient North Eurasians. This group appears to have contributed DNA to present-day Europeans as well as to the people who travelled across the Bering Strait into the Americas more than 15,000 years ago.

“Prior to this paper, the models we had for European ancestry were two-way mixtures. We show that there are three groups,” said David Reich, professor of genetics at HMS and co-senior author of the study.

“This also explains the recently discovered genetic connection between Europeans and Native Americans,” Reich added. “The same Ancient North Eurasian group contributed to both of them.”

The research team also discovered that ancient Near Eastern farmers and their European descendants can trace much of their ancestry to a previously unknown, even older lineage called the Basal Eurasians.

The study was published online Sept. 17 in Nature.

Peering into the past

To probe the ongoing mystery of Europeans’ heritage and their relationships to the rest of the world, the international research team—including co-senior author Johannes Krause, professor of archaeo- and paleogenetics at the University of Tübingen and co-director of the new Max Planck Institute for History and the Sciences in Jena, Germany—collected and sequenced the DNA of more than 2,300 present-day people from around the world and of nine ancient humans from Sweden, Luxembourg and Germany.

The ancient bones came from eight hunter-gatherers who lived about 8,000 years ago, before the arrival of farming, and one farmer from about 7,000 years ago.

The researchers also incorporated into their study genetic sequences previously gathered from ancient humans of the same time period, including early farmers such as Ötzi “the Iceman.”

“There was a sharp genetic transition between the hunter-gatherers and the farmers, reflecting a major movement of new people into Europe from the Near East,” said Reich.

Ancient North Eurasian DNA wasn’t found in either the hunter-gatherers or the early farmers, suggesting the Ancient North Eurasians arrived in the area later, he said.

“Nearly all Europeans have ancestry from all three ancestral groups,” said Iosif Lazaridis, a research fellow in genetics in Reich’s lab and first author of the paper. “Differences between them are due to the relative proportions of ancestry. Northern Europeans have more hunter-gatherer ancestry—up to about 50 percent in Lithuanians—and Southern Europeans have more farmer ancestry.”

Lazaridis added, “The Ancient North Eurasian ancestry is proportionally the smallest component everywhere in Europe, never more than 20 percent, but we find it in nearly every European group we’ve studied and also in populations from the Caucasus and Near East. A profound transformation must have taken place in West Eurasia” after farming arrived.

When this research was conducted, Ancient North Eurasians were a “ghost population”—an ancient group known only through the traces it left in the DNA of present-day people. Then, in January, a separate group of archaeologists found the physical remains of two Ancient North Eurasians in Siberia. Now, said Reich, “We can study how they’re related to other populations.”

Room for more

The team was able to go only so far in its analysis because of the limited number of ancient DNA samples. Reich thinks there could easily be more than three ancient groups who contributed to today’s European genetic profile.

He and his colleagues found that the three-way model doesn’t tell the whole story for certain regions of Europe. Mediterranean groups such as the Maltese, as well as Ashkenazi Jews, had more Near East ancestry than anticipated, while far northeastern Europeans such as Finns and the Saami, as well as some northern Russians, had more East Asian ancestry in the mix.

The most surprising part of the project for Reich, however, was the discovery of the Basal Eurasians.

“This deep lineage of non-African ancestry branched off before all the other non-Africans branched off from one another,” he said. “Before Australian Aborigines and New Guineans and South Indians and Native Americans and other indigenous hunter-gatherers split, they split from Basal Eurasians. This reconciled some contradictory pieces of information for us.”

Revised flow chart of European ancestry incorporating the new data about Ancient North Eurasians (ANE), West European hunter-gatherers (WHG), early European farmers (EEF) and Basal Eurasians. Image courtesy David Reich

Next, the team wants to figure out when the Ancient North Eurasians arrived in Europe and to find ancient DNA from the Basal Eurasians.

“We are only starting to understand the complex genetic relationship of our ancestors,” said co-author Krause. “Only more genetic data from ancient human remains will allow us to disentangle our prehistoric past.”

“There are important open questions about how the present-day people of the world got to where they are,” said Reich, who is a Howard Hughes Medical Investigator. “The traditional way geneticists study this is by analyzing present-day people, but this is very hard because present-day people reflect many layers of mixture and migration.

“Ancient DNA sequencing is a powerful technology that allows you to go back to the places and periods where important demographic events occurred,” he said. “It’s a great new opportunity to learn about human history.”

This project was supported in part by the National Cancer Institute (HHSN26120080001E and NIH/NCI Intramural Research Program), National Institute of General Medical Sciences (GM100233 and GM40282), National Human Genome Research Institute (HG004120 and HG002385), an NIH Pioneer Award (8DP1ES022577-04), National Science Foundation (HOMINID awards BCS-1032255 and BCS-0827436 and grant OCI-1053575), Howard Hughes Medical Institute, German Research Foundation (DFG) (KR 4015/1-1), Carl-Zeiss Foundation, Baden Württemberg Foundation and the Max Planck Society.


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.