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Cholera's Evolution

Cholera’s Evolution

Study identifies genomic regions associated with cholera susceptibility in Bangladesh

By SUE McGREEVEY
Tue, 2013-07-16

People in the Ganges River Delta of Bangladesh have been affected by cholera for centuries. Image: NASA World Wind screenshot via Wikimedia Commons

An international research team has used a novel approach to identify genetic factors that appear to influence susceptibility to cholera, a disease that affects from 3 to 5 million people each year and causes more than 100,000 deaths. The findings indicate the importance of pathways involved in regulating water loss in intestinal cells and highlight the innate immune system’s key role in the body’s response to the bacteria that causes cholera.

The work involved investigators from Harvard Medical School and Massachusetts General Hospital; the Broad Institute; and the International Center for Diarrhoeal Disease Research, Bangladesh.

“We sought to understand cholera by studying the genetics of a population that has been affected by the disease for centuries: people in the Ganges River Delta of Bangladesh,” said Regina LaRocque, an assistant professor of medicine at Mass General and a co-senior author of the report published in Science Translational Medicine. “Our findings are just a first step, but they demonstrate how combining ancient history with the current impact of an infectious disease can be a powerful way of identifying human genes that are important to disease outcome.”

People contract cholera by consuming water or food contaminated with the bacteria Vibrio cholerae, which releases a toxic protein upon reaching the small intestine. This toxin binds to the intestinal surface, causing severe diarrhea and sometimes death from dehydration. Cholera or cholera-like illnesses have been reported in the Ganges Delta for centuries, and most recent global outbreaks of the disease originated in that region.

A potential fingerprint of cholera’s genetic impact could be the relative rarity in the region of people with blood type O, which confers an increased risk of severe cholera symptoms. The persistence of cholera in the Ganges Delta would be expected to exert an evolutionary force on the population, since individuals with gene variants that reduce their susceptibility to the disease would be more likely to survive and pass those variants along to their children.

To search for genomic regions that affect cholera susceptibility, the team employed a new two-step approach. The first step, developed by the Broad team, used a method called Composite of Multiple Signals (CMS) to scan the genomes of 126 individuals from the Ganges Delta for patterns that signal a long-term increase in the prevalence of particular DNA segments, indicating the effects of natural selection. That scan identified 305 regions under selective pressure, many of which are involved in two important biologic functions. One is regulation of the passage of water through intestinal cells via structures called potassium channels and the other is a signaling pathway involved in both the innate immune system and the maintenance of the intestinal lining.

The second step directly tested the potential impact of these selected regions on cholera susceptibility by comparing the genomes of 105 cholera patients from the region with the genomes of 167 individuals who did not contract the disease, despite being exposed to it in their homes. That comparison found that the genomic region most strongly associated with cholera susceptibility in this population was one that the CMS scan indicated was under strong selection pressure. Genes in this region relate to an innate immune signaling pathway. LaRocque’s team had previously shown this pathway to be activated by exposure to cholera toxin, and the current study identified the potential involvement of several additional genes in that pathway

“Understanding the basic biology of a disease is fundamental to making clinically relevant advances in treatment,” said LaRocque. “Our laboratory is now working on further studies of the innate immune response to cholera, and we believe this work will be highly relevant to developing improved vaccines.”

Support for the study includes National Institutes of Health grants TW007409, AI058935, AI079198 and NIH Innovator Award DP2-OD006514-01; grants from the Howard Hughes Medical Institute, the American Cancer Society and the Packard Foundation; and an MGH Claflin Distinguished Scholar Award.

Adapted from a Mass General news release.

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After Tamoxifen

After Tamoxifen

Biomarker predicts likelihood of ER-positive breast cancer recurrence after treatment

By SUE McGREEVEY
Tue, 2013-07-16

Breast cancer cell. Image: National Cancer Institute

A biomarker reflecting expression levels of two genes in tumor tissue may be able to predict which women treated for estrogen-receptor-positive (ER positive) breast cancer should receive a second estrogen-blocking medication after completing tamoxifen treatment. In their report published in the Journal of the National Cancer Institute, HMS researchers at the Massachusetts General Hospital Cancer Center describe finding that the HOXB13/IL17BR ratio can indicate which women are at risk for cancer recurrence after tamoxifen and which are most likely to benefit from continuing treatment with the aromatase inhibitor letrozole.

“Most patients with early-stage, estrogen-receptor-positive breast cancer remain cancer-free after five years of tamoxifen treatment, but they remain at risk of recurrence for 15 years or longer after their initial treatment,” said Dennis Sgroi, HMS professor of pathology at Mass General and lead author of the report. “Our biomarker identifies the subgroup of patients who continue to be at risk of recurrence after tamoxifen treatment and who will benefit from extended therapy with letrozole, which should allow many women to avoid unnecessary extended treatment.”

Previous research by Sgroi’s team, in collaboration with investigators from bioTheranostics Inc., discovered that the ratio between levels of expression of two genes—HOXB13 and IL17BR—in tumor tissue predicted the risk of recurrence of ER-positive, lymph-node-negative breast cancer, whether or not the patient was treated with tamoxifen. The current study of patients from MA.17, the highly successful clinical trial of letrozole, was designed to go further. It would evaluate the usefulness of the HOXB13/IL17BR ratio for predicting which tamoxifen-treated patients remained at risk of recurrence, but it also sought to identify who could benefit from continued treatment with letrozole.

To answer those questions, the investigators analyzed primary tumor samples and patient data from the placebo-controlled MA.17 trial, which confirmed the ability of extended letrozole therapy to improve survival after the completion of tamoxifen treatment. Tissue samples were available from 83 patients whose tumors recurred during the study period (31 who had received letrozole and 52 in the placebo group) and 166 patients with no recurrence (91 who had received letrozole and 75 who got the placebo).

Analysis of the tumor samples revealed that a high HOXB13/IL17BR ratio—meaning the expression level of HOXB13 is greater than that of IL17BR—predicts an increased risk for tumor recurrence after tamoxifen therapy, but that elevated risk drops significantly if a patient receives letrozole.

“This discovery means that about 60 percent of women with the most common kind of breast cancer can be spared unnecessary treatment with the concomitant side effects and costs,” said Paul Goss, HMS professor of medicine, director of the Breast Cancer Research Program at the MGH Cancer Center and a co-author of the report. “But more important, the 40 percent of patients who are at risk of recurrence can now be identified as needing continued therapy with letrozole, and many will be spared death from breast cancer.” 

Goss and Sgroi noted that their findings need to be validated by additional studies before they can be put into clinical practice.

The study was supported by National Institute of Health grant R01-CA112021, Department of Defense Breast Cancer Research Program grant W81XWH-04-1-0606, and grants from the Avon Foundation, the Breast Cancer Foundation, the NCI SPORE in breast cancer at MGH, and Novartis.

Adapted from a Mass General news release.

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Off-Target Gene Editing

Off-Target Gene Editing

Unwanted mutations indicate need to improve precision of CRISPR-Cas RNA-guided nucleases

By SUE McGREEVEY
Tue, 2013-06-25

Image: iStock

In the past year a group of synthetic proteins called CRISPR-Cas RNA-guided nucleases (RGNs) have generated great excitement in the scientific community as gene-editing tools. Exploiting a method that some bacteria use to combat viruses and other pathogens, CRISPR-Cas RGNs can cut through DNA strands at specific sites, allowing new genetic material to be inserted. 

Now a team of HMS researchers at Massachusetts General Hospital has found a significant limitation to the method’s use: CRISPR-Cas RGNs produce unwanted DNA mutations at sites other than the desired target.

“We found that expression of CRISPR-Cas RGNs in human cells can have off-target effects that, surprisingly, can occur at sites with significant sequence differences from the targeted DNA site,” said J. Keith Joung, HMS associate professor of pathology at Mass General and associate chief of pathology, research in the Mass General Department of Pathology. He is co-senior author of the report published online in Nature Biotechnology. “RGNs continue to have tremendous advantages over other genome-editing technologies, but these findings have now focused our work on improving their precision.”

Consisting of a DNA-cutting enzyme called Cas9, coupled with a short, 20-nucleotide segment of RNA that matches the target DNA segment, CRISPR-Cas RGNs mimic the primitive immune systems of certain bacteria. When these microbes are infected by viruses or other organisms, they copy a segment of the invader’s genetic code and incorporate it into their DNA, passing it on to future bacterial generations. If the same pathogen is encountered in the future, the bacterial enzyme Cas9, guided by an RNA sequence that matches the copied DNA segment, inactivates the pathogen by cutting its DNA at the target site. 

About a year ago, scientists reported the first use of programmed CRISPR-Cas RGNs to target and cut specific DNA sites. Since then several research teams, including Joung’s, have successfully used CRISPR-Cas RGNs to make genomic changes in fruit flies, zebrafish, mice and in human cells—including induced pluripotent stem cells that have many of the characteristics of embryonic stem cells. The technology’s reliance on such a short RNA segment makes CRISPR-Cas RGNs much easier to use than other gene-editing tools called zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). RGNs can also be programmed to introduce several genetic changes at the same time.

The possibility that CRISPR-Cas RGNs might cause additional, unwanted genetic changes has been largely unexplored, so Joung’s team set out to investigate the occurrence of “off-target” mutations in human cells expressing CRISPR-Cas RGNs. Since the interaction between the guiding RNA segment and the target DNA relies on only 20 nucleotides, they hypothesized that the RNA might also recognize DNA segments that differed from the target by a few nucleotides. 

Although previous studies had found that a single-nucleotide mismatch could prevent the action of some CRISPR-Cas RGNs, the MGH team’s experiments in human cell lines found multiple instances in which mismatches of as many as five nucleotides did not prevent cleavage of an off-target DNA segment. They also found that the rates of mutation at off-target sites could be as high as, or even higher than, those at the targeted site, something that has not been observed with off-target mutations associated with ZFNs or TALENs.

“Specificity is important both for research and especially for gene therapy,” George Church, the Robert Winthrop Professor of Genetics at HMS, said about Joung’s report. “This is the first paper to seriously address this topic. The next big question is how to reduce the off-target ratio to on-target.”

In January, Church reported in Science on research using the genome-editing tool. While he was not involved in the current study reported in Nature Biotechnology, Church and Joung are collaborators. Together with George Daley, HMS professor of biological chemistry and molecular pharmacology, and Kun Zhang, associate professor of bioengineering at the University of California at San Diego, they are co-principal investigators of a National Human Genome Research Institute Center for Excellence in Genomic Science.

Joung said RGNs remain valuable.

“Our results don’t mean that RGNs cannot be important research tools, but they do mean that researchers need to account for these potentially confounding effects in their experiments.  They also suggest that the existing RGN platform may not be ready for therapeutic applications,” said Joung. “We are now working on ways to reduce these off-target effects, along with methods to identify all potential off-target sites of any given RGN in human cells so that we can assess whether any second-generation RGN platforms that are developed will be actually more precise on a genome-wide scale. I am optimistic that we can further engineer this system to achieve greater specificity so that it might be used for therapy of human diseases.”

Support for the study includes National Institutes of Health (NIH) Director’s Pioneer Award DP1 GM105378; NIH grants R01 GM088040 and P50 HG005550, DARPA grant W911NF-11-2-0056, and the Jim and Ann Orr MGH Research Scholar Award.

Adapted from a Mass General news release.

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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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