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New MATH for Cancer

New MATH for Cancer

In head and neck tumors, genetic diversity predicts outcomes

By SUE McGREEVEY
Mon, 2013-05-20

Oral cancer squamous cell carcinoma, one of several types of head and neck cancer. Image: Wikimedia Commons

A new method of measuring the variety of genetic mutations found in cells within a tumor appears to predict treatment outcomes of patients with the most common type of head and neck cancer. In the May 20 issue of the journal Cancer, HMS investigators at Massachusetts General Hospital and Massachusetts Eye and Ear described how their way of measuring tumor heterogeneity was a better predictor of survival than are most traditional risk factors in a small group of patients with squamous cell carcinoma of the head and neck. 

“Our findings will eventually allow better matching of treatments to individual patients, based on this characteristic of their tumors,” said Edmund Mroz, HMS associate professor of physiology in the Department of Otology and Laryngology at Mass General’s Center for Cancer Research and lead author of the Cancer paper. “This method of measuring heterogeneity can be applied to most types of cancer, so our work should help researchers determine whether a similar relationship between heterogeneity and outcome occurs in other tumors.”

For decades investigators have hypothesized that tumors with a high degree of genetic heterogeneity—the result of different subgroups of cells undergoing different mutations at different DNA sites—would be more difficult to treat because particular subgroups might be more likely to survive a particular drug or radiation or to have spread before diagnosis.  While recent studies have identified specific genes and proteins that can confer treatment resistance in tumors, there previously has been no convenient way to measure tumor heterogeneity.

Working in the laboratory of James Rocco, who is the HMS Daniel Miller Associate Professor of Otology and Laryngology at Mass Eye and Ear, Mroz and his colleagues developed their new method by analyzing advanced gene-sequencing data to produce a value reflecting the genetic diversity within a tumor—not only the number of genetic mutations but how broadly particular mutations are shared within different subgroups of tumor cells. They first described this measure, called mutant-allele tumor heterogeneity (MATH), in the March 2013 issue of Oral Oncology.  But that paper was able to show only that patients with known factors predicting poor outcomes—including specific mutations in the TP53 gene or a lack of infection with the human papillomavirus (HPV)—were likely to have higher MATH values. 

In the current study, the investigators used MATH to analyze genetic data from the tumors of 74 patients with squamous cell head and neck carcinoma for whom they had complete treatment and outcome information. Not only did they find that higher MATH values were strongly associated with shorter overall survival—with each unit of increase reflecting a 5 percent increase in the risk of death—but that relationship was also seen within groups of patients already at risk for poor outcomes. For example, among patients with HPV-negative tumors, those with higher MATH values were less likely to survive than those with lower MATH values. Overall, MATH values were more strongly related to outcomes than were most previously identified risk factors. MATH values also improved outcome predictions based on all other risk factors the researchers examined.

The impact of MATH values on outcome appeared strongest among patients treated with chemotherapy, which may reflect a greater likelihood that highly heterogeneous tumors contain treatment-resistant cells, Mroz said. He also noted that what reduces the chance of survival appears to be the subgroups of cells with different mutations within a tumor, not the process of mutation itself. 

“If all the tumor cells have gone through the same series of mutations, a single treatment might still be able to kill all of them,” he said. “But if there are subgroups with different sets of mutations, one subgroup might be resistant to one type of treatment, while another subgroup might resist a different therapy.”

In addition to combining MATH values with clinical characteristics to better predict a patient’s chance of successful treatment, MATH could someday help determine treatment choice—directing the use of more-aggressive therapies against tumors with higher values, while allowing patients with lower values to receive less-intense standard treatment, Mroz noted. While MATH will probably be just as useful at predicting outcomes for other solid tumors, that remains to be shown in future studies, the investigators said. 

“Our results have important implications for the future of oncology care,” said Rocco, who is director of the Mass Eye and Ear/Mass General Head and Neck Molecular Oncology Research Laboratory and senior author of the Cancer paper. “MATH offers a simple, quantitative way to test hypotheses about intratumor genetic heterogeneity, including the likelihood that targeted therapy will succeed. The results also raise important questions about how genetic heterogeneity develops within a tumor and whether heterogeneity can be exploited therapeutically.”

The study was supported by National Institute of Dental and Craniofacial Research grants R01DE022087 and RC2DE020958, National Cancer Institute grant R21CA119591, Cancer Prevention Research Institute of Texas grant RP100233, and the Bacardi MEEI Biobank Fund.  Mass General has filed a patent application for the MATH measure.

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Mind-body Genomics

Mind-body Genomics

Relaxation response immediately alters gene expression tied to inflammation, metabolism and insulin

By SUE McGREEVEY
Wed, 2013-05-01

Image: National Center for Complementary and Alternative Medicine

A new study from investigators at the Benson-Henry Institute for Mind/Body Medicine at Massachusetts General Hospital and Beth Israel Deaconess Medical Center finds that eliciting the relaxation response—a physiologic state of deep rest induced by practices such as meditation, yoga, deep breathing and prayer—produces immediate changes in the expression of genes involved in immune function, energy metabolism and insulin secretion.

“Many studies have shown that mind/body interventions like the relaxation response can reduce stress and enhance wellness in healthy individuals and counteract the adverse clinical effects of stress in conditions like hypertension, anxiety, diabetes and aging,” said Herbert Benson, HMS professor of medicine at Mass General and co-senior author of the report.

Benson is director emeritus of the Benson-Henry Institute.

“Now for the first time we’ve identified the key physiological hubs through which these benefits might be induced,” he said.

Published in the open-access journal PLOS ONE, the study combined advanced expression profiling and systems biology analysis to both identify genes affected by relaxation response practice and to determine the potential biological relevance of those changes.

“Some of the biological pathways we identify as being regulated by relaxation response practice are already known to play specific roles in stress, inflammation and human disease. For others, the connections are still speculative, but this study is generating new hypotheses for further investigation," said Towia Libermann, HMS associate professor of medicine at Beth Israel Deaconess and co-senior author of the study.

Benson first described the relaxation response—the physiologic opposite of the fight-or-flight response—almost 40 years ago, and his team has pioneered the application of mind/body techniques to a wide range of health problems. Studies in many peer-reviewed journals have documented how the relaxation response both alleviates symptoms of anxiety and many other disorders and also affects factors such as heart rate, blood pressure, oxygen consumption and brain activity. 

In 2008, Benson and Libermann led a study finding that long-term practice of the relaxation response changed the expression of genes involved with the body’s response to stress. The current study examined changes produced during a single session of relaxation response practice, as well as those taking place over longer periods of time.

The study enrolled a group of 26 healthy adults with no experience in relaxation response practice, who then completed an 8-week relaxation-response training course.

Before they started their training, they went through what was essentially a control group session: Blood samples were taken before and immediately after the participants listened to a 20-minute health education CD and again 15 minutes later. After completing the training course, a similar set of blood tests was taken before and after participants listened to a 20-minute CD used to elicit the relaxation response as part of daily practice. 

The sets of blood tests taken before the training program were designated “novice,” and those taken after training completion were called “short-term practitioners.” For further comparison, a similar set of blood samples was taken from a group of 25 individuals with 4 to 25 years’ experience regularly eliciting the relaxation response through many different techniques before and after they listened to the same relaxation response CD.

Blood samples from all participants were analyzed to determine the expression of more than 22,000 genes at the different time points.

The results revealed significant changes in the expression of several important groups of genes between the novice samples and those from both the short- and long-term sets. Even more pronounced changes were shown in the long-term practitioners. 

A systems biology analysis of known interactions among the proteins produced by the affected genes revealed that pathways involved with energy metabolism, particularly the function of mitochondria, were upregulated during the relaxation response. Pathways controlled by activation of a protein called NF-κB—known to have a prominent role in inflammation, stress, trauma and cancer—were suppressed after relaxation response elicitation. The expression of genes involved in insulin pathways was also significantly altered.

“The combination of genomics and systems biology in this study provided great insight into the key molecules and physiological gene interaction networks that might be involved in relaying beneficial effects of relaxation response in healthy subjects,” said Manoj Bhasin, HMS assistant professor of medicine, co-lead author of the study, and co-director of the Beth Israel Deaconess Genomics, Proteomics, Bioinformatics and Systems Biology Center.

Bhasin noted that these insights should provide a framework for determining, on a genomic basis, whether the relaxation response will help alleviate symptoms of diseases triggered by stress. The work could also lead to developing biomarkers that may suggest how individual patients will respond to interventions.

Benson stressed that the long-term practitioners in this study elicited the relaxation response through many different techniques—various forms of meditation, yoga or prayer—but those differences were not reflected in the gene expression patterns.

“People have been engaging in these practices for thousands of years, and our finding of this unity of function on a basic-science, genomic level gives greater credibility to what some have called ‘new age medicine,’ ” he said.

“While this and our previous studies focused on healthy participants, we currently are studying how the genomic changes induced by mind/body interventions affect pathways involved in hypertension, inflammatory bowel disease and irritable bowel syndrome. We have also started a study—a collaborative undertaking between Dana-Farber Cancer Institute, Mass General and Beth Israel Deaconess—in patients with precursor forms of multiple myeloma, a condition known to involve activation of NF-κB pathways," said Libermann, who is the director of the Beth Israel Deaconess Medical Center Genomics, Proteomics, Bioinformatics and Systems Biology Center.

The study was supported by Centers for Disease Control and Prevention grants H75 CCH123424 and R01 DP000339, by National Center for Complementary and Alternative Medicine grant R01 AT006464-01, and by National Center for Research Resources grant M01 RR01032.

Adapted from a joint Mass General and Beth Israel Deaconess news release.

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Learning from Lassa Virus

Learning from Lassa Virus

Researchers discover gene mutations that can result in a congenital disorder

Thu, 2013-03-21

An MRI of a child with Walker-Warburg syndrome shows abnormal brain features. Image courtesy of Hans van Bokhoven of Radboud University Medical Center. Researchers have known that two seemingly distant human maladies—a devastating set of hereditary disorders called Walker-Warburg syndrome and infection with the virus that causes hemorrhagic Lassa fever—both involve a cellular protein involving sugar.

Now an international team has discovered new genetic mutations that cause the severe brain, muscle and eye defects found in children with Walker-Warburg syndrome but also make cells insensitive to the Lassa virus.

The scientists found all known gene defects and identified new mutations in genes critical for coupling sugar groups to a particular protein receptor for Lassa virus, called glycosylated alpha-dystroglycan.

The team, which includes Sean Whelan, HMS professor of microbiology and immunobiology; Hans van Bokhoven of Radboud University Medical Center; and Thijn Brummelkamp of the Netherlands Cancer Institute, published their findings this week in Science.

Defects in how sugar modifies the dystroglycan complex causes Walker-Warburg syndrome, in which affected children usually die at an early age. The Lassa virus is one of many pathogens that hijacks this protein complex in order to enter cells.

Studying Lassa leads to syndrome clues

Viruses need to get inside host cells to amplify and cause disease. The Lassa virus gains entry by binding to dystroglycan, a protein complex on the outside of the cell. This protein complex is heavily decorated with sugars that anchor the cell to its surroundings.

To generate a detailed map of how Lassa virus enters human cells, as well as to identify genes required for successful infection, the scientists infected a unique human cell line in a lab dish. Because these genes are present only in a single copy, they can be readily inactivated. Until recently this genetic trick was possible only in model organisms, such as yeast and fruit flies.

Using the genetic approach of inactivating host gene function by inserting new genetic material, a technique called insertional mutagenesis, the team found human cells that were resistant to Lassa virus infection.

The scientists were able to do the Lassa work safely by replacing the surface protein on another non-lethal virus with the surface protein of Lassa and then testing the ability of that virus to infect and kill the haploid cell-line in which genes had been inactivated by insertional mutagenesis. Only those cells that lacked the components essential for Lassa virus entry survived the infection, whereas others died.

Walker-Warburg mutations identified

The identity of the genes associated with resistance was revealed by deep genetic sequencing of the surviving cells. Among them were genes that encode the known Lassa virus receptor alpha-dystroglycan as well as machinery required to add sugars to this receptor. Mutations in several of these genes are associated with Walker-Warburg syndrome, but not all of the genes had previously been linked with the syndrome.

Based on these findings, researchers scanned the genome of Walker-Warburg patients for defects in these newly discovered genes. Strikingly, several families affected by hereditary Walker-Warburg syndrome of previously unknown cause carried mutations in these genes.

Lassa virus is endemic in regions of Africa and causes thousands of deaths every year. The researchers said it will be interesting to see if, in communities exposed to Lassa virus, the human genome shows traces of the ongoing struggle in the sections encoding the identified host factors that are involved in building the sugar trees on the dystroglycan protein.

Adapted from a Netherlands Cancer Institute news release.

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Mitochondrial Quality Control

Mitochondrial Quality Control

Researchers define how a gene mutated in Parkinson’s disease may normally function to ensure neuronal health

By ELIZABETH COONEY
Thu, 2013-03-21

The VDAC3 mitochondrial pore is ubiquitylated on 3 lysine residues, shown in red, on the cytoplasmic surface on mitocohndria. The membrane protein makes a channel through which molecules can be transported. Ubiquitylation could potentially affect this trafficking. Image courtesy of the Harper Lab.

Cell biologists studying Parkinson’s disease are training their sights on mitochondria, the energy source of the cell, whose activity in neurons appears to go awry in this devastating neurodegenerative illness. A neuron needs its mitochondria to be healthy and mobile, particularly during their continual cycles of fission and fusion in which damaged bits are removed and healthy mitochondria are renewed.

A particular gene that is mutated in early-onset Parkinson's—a subset of the disease that can strike people in their 30s—has opened a window into a mechanism called mitophagy, an important component in this form of cellular housekeeping. When mitochondria are damaged, they must first be identified and then cleared away so they don’t fuse with and poison “good” mitochondria. In mitophagy, an unwanted mitochondrion is engulfed and degraded.

Fundamental biochemical pathway

The gene PARKIN and its regulatory companion PINK1 work together in this process, one that involves multiple proteins on the mitochondrial outer membrane that may ultimately serve as potential targets for treatments in Parkinson’s. PARKIN was discovered a dozen years ago, but only within the last couple of years have scientists pinpointed its role in mitochondrial quality control.

Wade Harper, the Bert and Natalie Vallee Professor of Molecular Pathology, together with his collaborator Steven Gygi, HMS professor of cell biology, led an HMS Department of Cell Biology team that has identified a fundamental biochemical pathway in which PARKIN resculpts the mitochondrial proteome—the full complement of mitochondrial proteins produced—to promote mitophagy. Harper and his colleagues published their results in Nature this week and share a web portal with a wealth of information about the proteins in this pathway.

The pathway that eradicates damaged mitochondria begins with a process called ubiquitylation. Ubiquitin is a small protein that modifies other proteins in many aspects of biology, including the response to damage that ultimately ends in ridding the cell of such waste. Understanding the sites of modification in target proteins is a key step in elucidating the role of ubiquitin in specific pathways, but this has been a technically challenging area of research. The Gygi and Harper labs recently developed a mass spectrometry-based proteomic methodology to identify ubiquitylation sites in target proteins, potentially on a global scale, and in the current study extended this to the PARKIN system.

Resculpting the mitochondrial proteome

“Identifying actual sites of ubiquitylation provides us not only a visual way to look at the structure and mechanisms of how the PARKIN system works, but it also gives you the potential in the future to make reagents that will let you look in tissue of the brain, for example, and determine whether the pathway is on or off,” said Harper.

In defining the near complete repertoire of PARKIN substrates—which they call the PARKIN-dependent ubiquitylome—the researchers reveal how the structure and function of the mitochondrial proteome is resculpted by PARKIN. Indeed, their work identified hundreds of ubiquitylation sites on dozens of proteins.

While this contribution does not have immediate clinical implications, it does serve as a key steppingstone toward a greater understanding of Parkinson’s and perhaps other neurodegenerative diseases involving mitochondrial quality control. This challenging work is necessary before contemplating ways to somehow overcome defects in genes such as PARKIN.

“We want to be able to use the data to understand in more detail how PARKIN is regulated and how it is activated and how it is capable of ubiquitylating a dizzying array of proteins on the mitochondrial surface,” Harper said. “In addition, we also want to try to test the hypothesis that ubiquitylation of specific mitochondrial proteins is critical for mitophagy.”

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Clues to Fetal Alcohol Risk

Clues to Fetal Alcohol Risk

Molecular switch promises new targets for diagnosis and therapy

By R. ALAN LEO
Wed, 2013-02-20

Fetal alcohol syndrome is the leading preventable cause of developmental disorders in developed countries. And fetal alcohol spectrum disorder (FASD), a range of alcohol-related birth defects that includes fetal alcohol syndrome, is thought to affect as many as 1 in 100 children born in the United States.

Any amount of alcohol consumed by the mother during pregnancy poses a risk of FASD, a condition that can include the distinct pattern of facial features and growth retardation associated with fetal alcohol syndrome as well as intellectual disabilities, speech and language delays, and poor social skills. But drinking can have radically different outcomes for different women and their babies. While twin studies have suggested a genetic component to susceptibility to FASD, researchers have had little success identifying who is at greatest risk or what genes are at play.

Research from Harvard Medical School and Veterans Affairs Boston Healthcare System sheds new light on this question, identifying for the first time a signaling pathway that might determine genetic susceptibility for the development of FASD. The study was published online Feb. 19 in the journal Proceedings of the National Academy of Sciences.

“Our work points to candidate genes for FASD susceptibility and identifies a path for the rational development of drugs that prevent ethanol neurotoxicity,” said Michael Charness, chief of staff at VA Boston Healthcare System and HMS professor of neurology. “And importantly, identifying those mothers whose fetuses are most at risk could help providers better target intensive efforts at reducing drinking during pregnancy.”

The discovery also solves a riddle that had intrigued Charness and other researchers for nearly two decades. In 1996, Charness and colleagues discovered that alcohol disrupted the work of a human protein critical to fetal neural development—a major clue to the biological processes of FASD. The protein, L1, projects through the surface of a cell to help it adhere to its neighbors. When Charness and his team introduced the protein to a culture of mouse fibroblasts cells, L1 increased cell adhesion. Tellingly, the effect was erased in the presence of ethanol (beverage alcohol).

A model of the L1 adhesion protein shows the binding pocket for alcohol molecules (top center). Charness Lab image

Charness and his team went on to develop multiple cell lines from that first culture, and that’s where they encountered the riddle: In some of those lines, alcohol disrupted L1’s adhesive effect, while in others it did not.

“How could it be possible that a cell that expresses L1 is completely sensitive to alcohol, and others that express it are completely insensitive?” asked Charness, who is also faculty associate dean for veterans hospital programs at HMS and assistant dean at Boston University School of Medicine.

Clearly, something else was affecting the protein’s sensitivity to alcohol — but what? Studies of twins provided one clue: Identical twins are more likely than fraternal twins to have the same diagnosis, positive or negative, for FASD. “That concordance suggests that there are modifying genes, susceptibility genes, that predispose to this condition,” Charness said.

In the current study, Charness’ team and collaborators at the University of North Carolina School of Medicine in Chapel Hill conducted cell culture experiments to identify specific molecular events that contribute to the alcohol sensitivity of L1 adhesion molecules. They focused on what was happening to the L1 molecule inside a cell that could affect an event outside the cell such as disruption by alcohol.

“We found that phosphorylation events that begin inside the cell can render the external portion of the L1 adhesion molecule more vulnerable to inhibition by alcohol,” said Xiaowei Dou, HMS instructor in neurology in the Charness Lab and first author on the new study. “Phosphorylation was controlled by the enzyme ERK2, and occurred at a specific location on the internal portion of the L1molecule.”

Phosphorylation plays a significant role in a wide range of cellular processes. By adding a phosphate group to a protein or other molecule, phosphorylation turns many protein enzymes on and off, and thereby alters their function and activity.

The researchers also found that variations in ERK2 activity correlated with differences in L1 sensitivity to alcohol that they observed across cell lines and among different strains of mice.  “Dou showed that he could take these cells that had been insensitive to alcohol for 13-14 years, and make them sensitive by ramping up the activity of this kinase” Charness said.

These variations suggest that genes for ERK2 and the signaling molecules that regulate ERK2 activity might influence genetic susceptibility to FASD.  Moreover, their identification of a specific locus that regulates the alcohol sensitivity of L1 might facilitate the rational design of drugs that block alcohol neurotoxicity.

“The only thing this modification blocked was alcohol’s ability to inhibit L1,” Charness said. “If you’re looking for a drug, ideally you’re looking for it to block the effects of the toxin without interfering with the target molecule of the toxin.”

The findings will also help guide an international consortium in its search for genes linked to families with fetal alcohol spectrum disorders.

“Prenatal alcohol exposure is the leading preventable cause of birth defects and developmental disorders in the United States,” said Kenneth Warren, acting director of the National Institute on Alcohol Abuse and Alcoholism (NIAAA),which supported the study.  “These new findings are yet another important contribution from researchers who have been at the forefront of scientific discovery in FASD.”

This work was supported by the NIAAA Grant R01-AA12974; NIAAA Grant U24-AA014811, as a component of the Collaborative Initiative on Fetal Alcohol Spectrum Disorders (CIFASD); the Medical Research Service, Department of Veterans Affairs; NIAAA Grant AA017124 (CIFASD); NIAAA Grant AA011605; and NIAAA Grant K99/R00-AA018697.

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Why ‘Good Hair’ Matters

Why ‘Good Hair’ Matters

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

 

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Of Men, Not Mice

Of Men, Not Mice

Mouse models fail to reproduce inflammatory genomic response to serious injuries

By SUE McGREEVEY
Mon, 2013-02-11

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Adapted from a Mass General news release.

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Complex Mysteries

Complex Mysteries

New tools and collaborative methods shed light on autism

By JAKE MILLER
Thu, 2013-01-10

It is difficult to overstate the complexity of autism.

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

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

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

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

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

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

A hub for autism research

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

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

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

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

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

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

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

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

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

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

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

Synaptic tuning

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

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

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

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

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

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

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

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

Gene hunting

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

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

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

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

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

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

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

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

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

Finding—and learning from—biomarkers

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

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

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


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

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

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

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

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A Sharper Tool for Genome Engineering

A Sharper Tool for Genome Engineering

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

By ELIZABETH COONEY
Fri, 2013-01-11

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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Cancer Spread's Family Tree

Cancer Spread’s ‘Family Tree’

Rapid test reveals relationships between primary tumors and metastatic cells

By SUE McGREEVEY
Wed, 2014-04-23

Image: iStock

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

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

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

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

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

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

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

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

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

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

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

Adapted from a Mass General news release.

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