A study of hundreds of new genomes from across the globe has yielded insights into modern human genetic diversity and ancient population dynamics, including compelling evidence that essentially all non-Africans today descend from a single migration out of Africa.

The multinational research effort, led by Harvard Medical School geneticists and published Sept. 21 in Nature, also suggests that no single genetic change or small group of changes is likely to explain the substantial transformations in human culture and cognition seen in the last 50,000 years.

Get more HMS news here.

The study represents the largest data set yet of high-quality genome sequences from understudied populations, adding nearly 6 million DNA base pairs to the “canonical” human genome sequence published in 2001. The data identify millions of previously unknown mutations that may help scientists develop precision-targeted diagnostics and treatments and improve health care for the world’s underserved populations.

Most genome-wide population sequencing studies to date have focused on a handful of populations of large census size. The HMS-led study, by comparison, sequenced samples from 142 smaller populations, most of which were previously unstudied.

“As humans, we are not just the people who live in industrialized countries, and we are not just the people who live in numerically large groups,” said David Reich, professor of genetics at HMS and senior author of the study. “If we want to understand who we really are, we have to realize that some of the most interesting aspects of human variation are only present in underrepresented, small populations.”

“We wanted to go out into the world and pull together as many of the ethnically, linguistically and anthropologically diverse samples as we possibly could,” said Swapan Mallick, bioinformatic systems director in the Reich lab and first author of the study.

The team’s analyses are already answering questions about various populations’ genetic origins, but, the researchers note, these insights are only a milestone on a longer journey.

“Of course, there are thousands of ethnically distinct populations in the world, and much more work needs to be done,” said Mallick.

Clarifying history

Reich, Mallick and their international team of colleagues began by selecting two genomes each from 51 populations represented in a collection called the Human Genome Diversity Project. Next, they assembled samples from members of 91 other groups, including diverse Native American, South Asian and African populations not previously included in genome-wide studies, and sent the DNA for sequencing. In all, the project analyzed the genomes of 300 people.

A key conclusion—that the vast majority of modern human ancestry in non-Africans derives from a single population that migrated out of Africa—is also supported by two other whole-genome sequencing studies appearing simultaneously in Nature. One, led by an Estonian group, focused on 379 whole genome sequences; the other, led by a Danish group, analyzed 108 Australians and New Guineans.

Together, the three studies put to rest a lingering question about whether indigenous peoples of Australia, New Guinea and the Andaman Islands descend in large part from a second group that left Africa earlier and skirted the coast of the Indian Ocean. They do not, the HMS researchers say.

“Our best estimate for the proportion of ancestry from an early-exit population is zero,” said Reich, who is also an investigator of the Howard Hughes Medical Institute and associate member of the Broad Institute. “Taken together, all three studies leave wiggle room for, at most, around two percent.”

The HMS-led study further revealed that the common ancestors of modern humans began to differentiate at least 200,000 years ago, long before the out-of-Africa dispersal occurred.

“It had been unclear whether the group that expanded out of Africa represented a large subset of the populations within Africa,” said Mallick. “This really shows that there was a lot of substructure prior to the expansion.”

The additional discovery that genetics alone can’t account for the acceleration of cultural, economic and intellectual progress in the last 50,000 years runs contrary to a popular hypothesis in the field.

“There does not seem to have been one or a few enabling mutations that suddenly appeared among our ancestors and allowed them to think in profoundly different ways,” said Reich.

Instead, the researchers say, a constellation of factors, including environment, lifestyle and possibly genetics, likely drove the great changes that occurred.

“Geneticists often search for examples where genetics is the explanation. Here, paradoxically, genetic data are showing that there will be no clear genetic answers,” Reich said.

Overcoming hurdles

Mallick and colleagues overcame significant logistical hurdles posed by sharing and processing an enormous amount of data.

Often, in studies of this size, data are collected in many laboratories that use different sequencing machines and different experimental protocols. This can create so-called batch effects that make it difficult to distinguish true differences among samples. The current study minimized batch effects by sending all of the samples to a single center to be sequenced at the same time.

The team made much of the data set publicly available in 2014; multiple research groups have already used it for their studies.

In a way, the authors say, the findings reported thus far are just the tip of the iceberg.

“It’s impossible for our group to analyze even a tiny fraction of what the data represents,” said Mallick. “Our goal is to push the data out and let people use it to consider their own questions.”

“A project of this magnitude is a combination of considerable efforts from multiple groups and creates an important resource for researchers worldwide,” Mallick added. “Such work is only possible in the environment and with the remarkable support from administration and computing groups here at HMS.”

Primary funding for the study, called the Simons Genome Diversity Project, was provided by the Simons Foundation (SFARI 280376) and the National Science Foundation (BCS-1032255).

Maintaining appropriate levels of proteins within cells largely relies on a cellular component called the proteasome, which degrades unneeded or defective proteins to recycle the components for the eventual assembly of new proteins.

Deficient proteasome function can lead to a buildup of unneeded and potentially toxic proteins, so cells usually respond to proteasome dysfunction by increasing production of its component parts.

Get more HMS news here.

Now two Harvard Medical School investigators at Massachusetts General Hospital have identified key molecules in the pathway by which cells in the C. elegans roundworm sense proteasome dysfunction. Their findings may have applications for the treatment of several human diseases.

“Proteasome inhibitors are currently being used to treat some cancers, including multiple myeloma, so future drugs targeting this pathway could enhance their activity,” said Gary Ruvkun, professor of genetics at HMS, an investigator at Mass General and corresponding author of the report in the journal eLife. 

Lead author Nicolas Lehrbach, HMS postdoctoral fellow in Ruvkun’s laboratory, added, “Boosting proteasome activity might help treat some neurodegenerative disorders–including Alzheimer’s and Parkinson’s disease, both of which are characterized by abnormal protein deposits in the brain that exceed the ability of the proteasome to respond.”

While it has been known that a transcription factor called SKN-1 was essential for the response to proteasome dysfunction in C. elegans, exactly how SKN-1 is coupled to a sensor of proteasome activity to induce expression of proteasome subunits was unknown. Lehrbach conducted a comprehensive genetic screen of C. elegans mutants that were unable to activate SKN-1 in order to identify the mutated genes.

“The genetic analysis that Nic did was extremely comprehensive, isolating across the genome of C. elegans nearly 100 new mutations that render these animals unable to sense and respond to decreased proteasome activity,” Ruvkun said. “These new mutations were mapped by determining the full genome sequence of all of the mutant strains.”

Among many mutated genes that were isolated was a protease enzyme called DDI-1, present in many different species but not previously associated with protein damage signaling. Another essential element of the pathway is a protein called PNG-1, which removes sugar modifications from other proteins.

In order for SKN-1 to be activated, DDI-1 must cleave or clip the protein, and PNG-1 must remove a sugar molecule. The mammalian versions of SKN-1 are the transcription factors Nrf1 and Nrf2, and blocking Nrf1 has been proposed as a way to improve protease inhibitor treatment of cancer.

In another paper published simultaneously in eLife, investigators from the University of Tokyo led by Shigeo Murata independently discovered that the human version of C. elegans DDI-1, called DDI-2, is needed to couple proteasome deficiency to the upregulation of proteasome genes in human cells. Taken together, these two papers show that the DDI-1/2 protease is essential to sensing proteasome stress in animals from roundworms to humans, making the enzyme an ideal target for drug development.

“We know from the successful development of protease inhibitors to treat HIV and hepatitis C infection that protease enzymes are highly amenable to the development of small-molecule inhibitors,” Ruvkun said. “In addition to potentially being powerful enhancers of the proteasome inhibitors being used to treat multiple myeloma, DDI-1/2 inhibitors may be more generally deployed against conditions in which proteasome deregulation has been implicated.”

The variety of diseases caused by aberrant protein aggregation include Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis and prion diseases, Ruvkun said.

Inactivating mutations in the human version of PNG-1, a protein called NGLY1, have recently been identified as the cause of a rare genetic disease, Ruvkun said. The new findings strongly suggest this condition could be treated by reactivation of proteasome genes.

The team’s discovery that PNG-1 acts with DDI-1 and SKN-1 in the proteasome pathway now places PNG-1/NGLY1 in a genetic pathway that is better understood. 

“We have the genetic tools now to ask in C. elegans, how can we cure NGLY1 deficiency?” said Ruvkun. “We are doing these screens right now and are anxiously awaiting what the genome sequence of the mutants will teach us.”

The research was supported by National Institutes of Health grant R01 AG016636.

Adapted from a Mass General news release.

Genetic testing has greatly improved physicians’ ability to detect potentially lethal heart anomalies among asymptomatic family members of people who suffer cardiac arrest or sudden cardiac death.

But a study from Harvard Medical School published in the Aug. 18 issue of The New England Journal of Medicine shows that over the last decade these lifesaving tools may have disproportionately misdiagnosed one cardiac condition — hypertrophic cardiomyopathy (HCM) – in black Americans.

HCM, which affects one in 500 people, is an often-asymptomatic thickening of the heart muscle that can spark fatal arrhythmias in seemingly healthy young adults.

Get More HMS News

The notion that genetic tests could misread benign genetic alterations as disease-causing mutations is not entirely new, but this study is believed to be the first one to trace the root of the problem to racially biased methodologies in early studies that defined certain common genetic variants as causes of HCM.

Indeed, the analysis reveals that in the case of HCM, the false positive diagnoses stemmed from inadequately designed clinical studies that used predominantly white populations as control groups.

White Americans harbor far fewer benign mutations on several genes implicated in HCM than black Americans. The higher rate of benign alterations in the latter group can cause test results to be misread as abnormal, the researchers say.

Using statistical simulations, the HMS team demonstrated that including even small numbers of black participants in the original studies would have improved test accuracy and, consequently, helped avert some of the false-positive diagnoses.

The findings, the researchers say, highlight the importance of interpreting genetic test results against diverse control populations to ensure that normal variations of genetic markers common in one racial or ethnic group do not get misclassified as disease-causing in another.

                                                                                                                                   
The team says their findings point to a pressing need to reevaluate decades-old genetic studies by using new racially diverse sequencing data.

“We believe that what we’re seeing in the case of hypertrophic cardiomyopathy may be the tip of the iceberg of a larger problem that transcends a single genetic disease,” said study first author Arjun Manrai, a research fellow in the Department of Biomedical Informatics at Harvard Medical School. “We hope our study motivates a systematic review of this issue across other genetic conditions.

“Ensuring that genomic medicine benefits all people and all populations equally is nothing short of a moral imperative, not only for scientists and clinicians but for political and health policy powers that be,” —Zak Kohane

Aside from the emotional toll that a genetic misdiagnosis can take on individuals and families, the researchers say their findings represent a cautionary tale with a broad relevance to geneticists, clinicians and policy-makers alike.

“Our study powerfully illustrates the importance of racial and ethnic diversity in research,” says Zak Kohane, senior investigator on the study and chair of the Department of Biomedical Informatics at Harvard Medical School. “Racial and ethnical inclusiveness improves the validity and accuracy of clinical trials and, in doing so, can better guide clinical decision-making and choice of optimal therapy. This is the essence of precision medicine.”

In the current study, the team analyzed more than 8,000 DNA samples stored in three national databases — the National Institutes of Health’s Mendelian Exome Sequencing Project, the 1000 Genomes Project and the Human Genome Diversity Project.

Five genetic variants — each of them benign — accounted for 75 percent of all genetic variation across populations. However, the team found, these five mutations occurred disproportionately in black Americans.

Between 2.9 and 27 percent of black Americans harbored one or more such variants, compared with 0.02 to 2.9 percent of white Americans.

Next, researchers examined records of more than 2,000 patients and family members tested at a leading genetic laboratory between 2004 and 2014. Seven patients received reports indicating they harbored disease-causing mutations that were subsequently reclassified as benign. Five of the seven patients were black, and two were of unspecified ancestry.

Researchers say it remains unclear how many of the seven patients had been re-contacted to communicate the change in test results.
The investigators caution that test results from a single genetic lab are not necessarily representative of the scope of the problem nationally, but say their findings likely point to a discrepancy that goes beyond a single laboratory and a single condition.

To trace the root of the misclassifications, researchers reviewed the five original studies that shaped early understanding of genetic variants and their role in the development of hypertrophic cardiomyopathy. All of them, the researchers found, analyzed small population sizes and none indicated that black people were included in the control groups.

But, the investigators add, even small studies can avert misclassification of genetic variants as long they include racially diverse populations.

Using statistical simulation, the team showed that a sample of 200 people that included 20 black participants would have only 50 percent chance of correctly ruling out a harmful mutation. The same sample would have more than an 80 percent accuracy if a third of patients were black and more than 90 percent accuracy if half of them were black.

Investigators say the newly created Exome Aggregation Consortium — a compilation of data from various large-scale sequencing projects that includes DNA from more than 60,000 individuals — is well-powered to discern between harmful and benign mutations even for relatively rare genetic variants and should help in the reanalysis of decades-old data.

The latest clinical guidelines urge physicians to interpret genetic test results by cross-referencing them against racially matched controls. However, with expanding efforts to sequence DNA from various ethnic and racial groups, researchers say more genetic variants will be reclassified in the next decade. Interpreting the meaning of test results within the context of such rapidly evolving knowledge will pose a serious challenge for clinicians.

One way to address the problem, the HMS team says, could be the use of point-of-care risk calculators to help clinicians and genetic counselors more precisely gauge the significance of a given genetic variant. Such risk calculators would use algorithms that incorporate statistical probability, race, ethnicity and family history to help sift variant noise from truly pathogenic mutations.

“Ensuring that genomic medicine benefits all people and all populations equally is nothing short of a moral imperative, not only for scientists and clinicians but for political and health policy powers that be,” Kohane said.

The work was funded by the National Human Genome Research Institute under grant 5T32HG002295-9, by the National Institute of Mental Health under grant P50MH094267 and by the National Centers for Biomedical Computing under grant 5U54-LM-008748.

Other investigators on the research included Birgit Funke, Ph.D., Heidi Rehm, Ph.D., Morten Olesen, Ph.D., Bradley Maron, M.D., Peter Szolovits, Ph.D., 
David Margulies, M.D., Joseph Loscalzo, M.D., Ph.D.


Combining two emerging, large-scale technologies for the first time—multiplexed mass spectrometry and a mouse population with a high level of natural genetic diversity—researchers at Harvard Medical School and The Jackson Laboratory can now crack an outstanding question in biology and medicine: How do genetic variants affect protein levels?

Proteins are chains of amino acids that comprise the structural and functional “parts list” of all cells and organisms. Understanding the regulation of protein expression is therefore critical to understanding normal development and disease.

“We can now uncover relationships among genes, transcripts and proteins not previously known.”—Steven Gygi 

The central dogma of molecular biology describes this transfer of genetic information from DNA to RNA to protein. The DNA sequence is first transcribed into messenger RNA, or mRNA, and then the cell’s protein-building machinery translates the mRNA sequences into the amino-acid sequence of the protein.

Get more HMS news here.

Given this direct relationship between RNA and proteins, it was widely assumed that protein expression would track closely with mRNA expression. Yet several studies comparing cellular mRNA levels and protein levels have shown a surprisingly high level of discordance between the two, suggesting that one or more mechanisms act to buffer protein levels from genetic variants that affect mRNA levels.

Previous experiments in mice and human cell lines aimed at identifying these mechanisms have been inconclusive.

To address this puzzle, Gary Churchill,  Jackson Lab professor and Karl Gunnar Johansson Chair and a pioneer in developing the Collaborative Cross and Diversity Outbred mouse populations, joined forces with Steven Gygi, HMS professor of cell biology, a leader in the rapidly advancing field of quantitative proteomics, which is the study of an organism’s entire complement of proteins.

"This makes an entirely new scale of analysis possible.”—Gary Churchill

Diversity Outbred mice, bred from eight founder strains, contain extensive genetic variation.

“Our mouse populations have more than 50 million SNPs,” or single nucleotide polymorphisms, which are variations in individual DNA building blocks, said Churchill.  “Steve can measure the levels of thousands of proteins instead of dozens. This makes an entirely new scale of analysis possible.”

Gygi and Churchill are co-senior authors of a paper in Nature in which they compared mRNA and protein levels in the livers of 192 Diversity Outbred mice.

The researchers identified 2,866 genetic markers that correlate with differences in protein levels across mice (protein quantitative trait loci, or pQTL) and observed two striking patterns. Most proteins with “local” pQTL—where the genetic variant influencing protein abundance is located close to the DNA sequence that encodes that protein—showed strong evidence of transcriptional regulation where protein levels tracked closely with mRNA levels.

In stark contrast, proteins with “distant” pQTL—where the genetic variant influencing protein abundance is located far away from the DNA sequence that encodes the protein—appeared completely uncoupled from their corresponding mRNA’s abundance. By applying a novel statistical approach, they showed that the post-transcriptional effects of many distant pQTL could be attributed to a second protein, revealing an extensive network of direct protein–protein interactions and tightly regulated cellular pathways.

The researchers confirmed their findings in Collaborative Cross mice.

“We can now uncover relationships among genes, transcripts and proteins not previously known,” Gygi said. “Our findings suggest a new predictive genomics framework, combining quantitative proteomics and transcriptomics to infer the proteome-wide effects of a specific genetic variant.”

Within this framework, Gygi said, researchers can explore and fine-tune pathways associated with the physical process, disease or characteristic of interest.

The study was supported by Harvard Medical School, The Jackson Laboratory and the National Institutes of Health (grants P50GM076468, F32HD074299, GM67945 and U41HG006673).

Adapted from a Jackson Lab news release.

Our cells contain two different genomes: one in the cell nucleus and another in the mitochondria. Each has its own distinct machinery and evolutionary origin.

Both genomes contribute proteins to build the power plants that fuel our cells. For years, researchers have wondered: Do the nucleus and mitochondria talk to each other to coordinate energy production? If so, how?

Get more HMS news here

To find out, members of the lab of Stirling Churchman, assistant professor of genetics at Harvard Medical School, are eavesdropping on the conversation.

They report in Nature on May 11 that in Saccharomyces cerevisiae, or baker’s yeast, the nucleus and mitochondria do work together. They discovered that the coordinated effort is directed by the nucleus and occurs at a later stage than anticipated, not when genes are read but instead when proteins are made.

“A lot of attention has gone to studying gene expression and translation in the cell nucleus and cytosol”—the fluid in which cell organelles float—“but we didn’t know much about them in the mitochondria,” said Mary Couvillion, a postdoctoral researcher in the Churchman lab and first author of the paper.

The team was able to discover “this really elegant synchronization,” Couvillion said, by taking RNA sequencing methods and translation protocols designed to study the nuclear genome and modifying them to study the mitochondrial genome.

The researchers are making these tools available so others can use them to further investigate questions such as how mitochondrial genes respond to shifting energy demands that occur during physical activity, eating, embryonic development and aging.

Although the findings still need to be confirmed in human cells, the results of this study could also help researchers better understand mitochondrial disorders as well as diseases and conditions that have been linked to mitochondrial dysfunction, such as cancer, neurodegeneration, obesity and aging.

From chaos to coordination

The team began by switching the type of sugar fed to the yeast cells, which prodded them to change how they produce energy. 

“The cells said, ‘We need to beef up our mitochondria,’ the same way our muscle cells do when we exercise—and we watched how they did that,” said Churchman.

In the cell nuclei, genes that encode proteins for various power plant components switched on and started churning out RNA copies. Mitochondrial genes did the same, but at a slower rate, to the researchers’ surprise.

Then the chaos began to coalesce.

As if an invisible conductor raised a baton, the nuclear RNAs exited into the cytosol.

The baton lowered. Ribosomes in the cytosol and ribosomes in the mitochondria went to work on all the RNAs associated with the first three power plant components. After a few hours, the ribosomes had constructed the protein parts for those components.

With another swoop of the conductor’s baton, both sets of ribosomes then translated the RNAs for the power plant’s fourth component.

“Looking back, it all makes sense—obviously the nuclear and mitochondrial genomes have to produce the protein parts synchronously,” said Churchman. “But it wasn’t understood at what level coordination occurs. Now we know.”

Nuclear conductor

One major question remained: Who was wielding the conductor’s baton? Were the cytosolic and mitochondrial ribosomes communicating directly, or were they responding to external signals?

The team used a drug to inhibit cytosolic protein translation. Mitochondrial translation was “dramatically affected,” said Couvillion.

But when the team inhibited mitochondrial protein translation, cytosolic translation continued on as usual.

“That tells us the nucleus is in charge,” said Churchman.

One aspect of the study that fascinates Couvillion is “how our environment, including the food we eat, affects gene expression,” she said. “Now that we’ve done these relatively quick experiments in yeast, we’re looking forward to moving into mammalian cells to find out what’s going on in our own bodies.”

This study was supported by a Damon Runyon-Dale F. Frey Award for Breakthrough Scientists, a Burroughs Wellcome Fund Career Award at the Scientific Interface, an Ellison Medical Foundation New Scholar in Aging Award, a National Institutes of Health Ruth L. Kirschstein National Research Service Award (F32), and a Boehringer Ingelheim Fonds PhD Fellowship.

The 2016 Warren Alpert Foundation Prize will be awarded to five scientists for their remarkable contributions to the understanding of the CRISPR bacterial defense system and the revolutionary discovery that it can be adapted for genome editing.

The recipients are:

• Rodolphe Barrangou, associate professor in the Department of Food, Bioprocessing and Nutrition Sciences and the Todd R. Klaenhammer Distinguished Scholar in Probiotics Research at North Carolina State University
• Philippe Horvath, senior scientist at DuPont in Dangé-Saint-Romain, France
• Jennifer Doudna, the Li Ka Shing Chancellor’s Chair in Biomedical and Health Sciences and professor of molecular and cell biology and of chemistry at the University of California, Berkeley
• Emmanuelle Charpentier, scientific member and director at the Max Planck Institute for Infection Biology in Berlin and professor at Umeå University in Sweden
• Virginijus Siksnys, professor, chief scientist and department head at the Institute of Biotechnology at Vilnius University in Lithuania

Get more HMS News here

​Barrangou and Horvath established that bacteria protect themselves from being killed by pathogens, such as viruses, using a system called CRISPR (short for clustered regularly interspaced short palindromic repeats) to cut up specific segments of the invading viruses’ DNA.

Building on their findings, Doudna, Charpentier and Siksnys realized that the system could be programmed to zero in on any desired genetic sequence in a broader array of organisms, including humans, and that this purposeful cutting could be used to alter or replace the targeted DNA at will.

Together, these discoveries, which were further refined and expanded by the prize recipients and other researchers, have generated a powerful tool for rapidly determining gene function and have democratized the ability to pursue clinical advances such as correcting genetic defects and designing better drugs by making gene editing faster, easier and cheaper than the technologies available previously.

“The game-changing insights achieved by these five scientists led to a technique that has been swiftly embraced across the globe, altering the way we study and understand eukaryotic genetics and offering enormous potential for developing new gene- and cell-based therapies, including treatment strategies for previously intractable genetic diseases,” said Jeffrey S. Flier, dean of the faculty of medicine at Harvard Medical School and chair of the Warren Alpert Foundation Prize Scientific Advisory Committee.

“Determining biochemically and molecularly how the CRISPR system operates and then demonstrating its potential as a gene editing tool is one of the most important technological advances that has been made during my career,” said Cliff Tabin, head of the Department of Genetics at Harvard Medical School and a member of the Alpert Foundation’s scientific advisory prize committee.

“There are a large number of researchers who have contributed to the development of the CRISPR system and in no small measure share credit for it, but in choosing this year’s Alpert Prize recipients, we recognized those who established the understanding of the system and its potential, which others were then able to perfect as a usable tool,” Tabin added.

The Warren Alpert Foundation Prize recognizes scientists whose research has led to the prevention, cure or treatment of human diseases or disorders and constitutes a seminal scientific finding that holds great promise for ultimately changing our understanding of, or ability to treat, disease.

The late Warren Alpert, a philanthropist dedicated to advancing biomedical research, established the prize in 1987. To date, the foundation has awarded more than $3 million to 54 individuals. Eight honorees have also received a Nobel Prize.

This year’s recipients will share an unrestricted award of $500,000 and will be honored at a symposium at Harvard Medical School on Oct. 6.

“These five scientists have made foundational contributions to a technique that has hit the field of genetics like a lightning bolt,” said Bevin Kaplan, director and vice president of the Warren Alpert Foundation and a member of the Harvard Medical School Board of Fellows. “The nature of this work is unprecedented in its far-reaching potential for future therapeutic advances.”

“We are confident that innumerable lives will be positively touched by this work in the years to come,” Kaplan added. “In this way, these exemplary men and women most definitely embody the spirit of the Warren Alpert Foundation Prize. As always, we anticipate a phenomenal symposium and award ceremony in the fall.”

In their own words

“I am absolutely delighted and honored to receive the Warren Alpert Foundation Prize. This is a prestigious award, which I am grateful for, and thrilled to share with my colleagues and collaborators, especially Philippe Horvath, with whom I have shared my entire CRISPR journey. It has been enjoyable to observe firsthand the evolution of the CRISPR field in the past decade, and seeing the field evolve from humble beginnings of CRISPR analysis in dairy cultures to driving the genome-editing craze has been fantastic. We could not have dreamed that trying to understand how bacteria resist viruses would eventually lead to the CRISPR revolution we have witnessed. I am thankful to the Warren Alpert Foundation and to the prize selection committee for their consideration, and I look forward to further serving the CRISPR community.” —Rodolphe Barrangou

“Our seminal work, published in Science in 2007, had a significant impact on the scientific community. It really set the stage, opened new research avenues and inspired numerous scientists to investigate further in the CRISPR field. Being awarded with the Warren Alpert Foundation Prize is an immense honor and a distinct privilege that I wish to extend to all my collaborators and colleagues at DuPont.” —Philippe Horvath

“​As an alumna of the HMS graduate program, I am particularly honored to receive this award in recognition of research conducted with my collaborator Emmanuelle Charpentier and our outstanding postdoctoral associates and students. We hope that future students are inspired by the value of curiosity-driven research and the passion for fundamental discovery​ that our work represents.” —Jennifer Doudna

“I am honored to receive this award in recognition of our seminal work published in Nature in 2011 and in Science in 2012, including the discovery of tracrRNA and the delineation of its key role in the targeting and editing of DNA by CRISPR-Cas9. This work is a wonderful example of the importance of basic research, demonstrating its relevance for translational science and important medical applications.” —Emmanuelle Charpentier

“It is a great privilege to be among such outstanding current and former awardees of the Warren Alpert Foundation. This prize makes my work even more enjoyable and challenging. I am really glad that my research, aimed in the beginning on a very basic question of how bacteria protect themselves against phages, paved the way for the development of novel tools for genome-editing applications.” —Virginijus Siksnys

Previous Alpert Prize recipients

The Warren Alpert Foundation Prize has recognized recipients for discoveries that have impacted a wide spectrum of diseases, including asthma, breast cancer, H. pylori, hepatitis B and HIV/AIDS.

Last year’s honorees were Tu Youyou of the China Academy of Chinese Medical Sciences, who went on to receive the 2015 Nobel Prize in Physiology or Medicine, and Ruth and Victor Nussenzweig of NYU Langone Medical Center for their pioneering discoveries in chemistry and parasitology and personal commitments to translate these into effective chemotherapeutic and vaccine-based approaches to control malaria.

Previous honorees include Oleh Hornykiewicz of the Medical University of Vienna and the University of Toronto; Roger Nicoll of the University of California, San Francisco; and Solomon Snyder of the Johns Hopkins University School of Medicine in 2014 for research into neurotransmission and neurodegeneration.

David Botstein of Princeton University and Ronald Davis and David Hogness of Stanford University School of Medicine received the Alpert Prize in 2013 for contributions to the concepts and methods of creating a human genetic map.

Alain Carpentier of Hôpital Européen Georges-Pompidou in Paris and Robert Langer of MIT received the prize in 2011 for innovations in bioengineering.

Harald zur Hausen and Lutz Gissmann of the German Cancer Research Center received the prize in 2007 for their research on human papillomavirus (HPV) and cancer of the cervix. Hausen and others were later honored with the 2008 Nobel Prize in Physiology or Medicine.

The Warren Alpert Foundation

Each year the Warren Alpert Foundation receives 30 to 50 nominations for the Alpert Prize from scientific leaders worldwide. Prize recipients are selected by the foundation’s scientific advisory board, composed of distinguished biomedical scientists and chaired by the dean of Harvard Medical School.

Warren Alpert (1920-2007), a native of Chelsea, Massachusetts, established the Warren Alpert Foundation Prize in 1987 after reading about the development of a vaccine for hepatitis B. Alpert decided on the spot that he would like to reward such breakthroughs, so he picked up the phone and told the vaccine’s creator, Kenneth Murray of the University of Edinburgh, that he had won a prize. Alpert then set about creating the foundation.

To award subsequent prizes, Alpert asked Daniel Tosteson (1925-2009), then dean of Harvard Medical School, to convene a panel of experts to identify scientists from around the world whose research has had a direct impact on the treatment of disease.

The Warren Alpert Foundation does not solicit funds. It is a private philanthropic organization funded solely by the Warren Alpert Estate.

A new version of the CRISPR-Cas9 nuclease appears to robustly abolish the unwanted, off-target DNA breaks that are a significant current limitation of the gene-editing technology. 

Harvard Medical School researchers at Massachusetts General Hospital describe in Nature how engineering the Cas9 enzyme to reduce nonspecific interactions with the target DNA may greatly expand applications of the CRISPR-Cas9 technology.

Get more HMS news here

“Our creation of a Cas9 variant that brings off-target effects to levels where we can no longer detect them, even with the most sensitive methods, provides a substantial advance for therapeutic applications in which you want to accurately hit your target without causing damage anywhere else in the genome,” said J. Keith Joung, HMS professor of pathology at Mass General and senior author of the Nature paper. 

“We envision that our high-fidelity variant will supplant the use of standard Cas9 for many research and therapeutic applications.”— J. Keith Joung

“Its impact will also be incredibly important for research applications because off-target effects can potentially confound the results of any experiment,” Joung added. “As a result, we envision that our high-fidelity variant will supplant the use of standard Cas9 for many research and therapeutic applications.”

The limitation

Used to create targeted DNA breaks at which genetic changes can be introduced, CRISPR-Cas9 nucleases combine a bacterial DNA-cutting enzyme called Cas9 with a short guide RNA sequence that can bind to the target DNA sequence. While easier to use than previous gene-editing tools, CRISPR-Cas9 nucleases have a well-characterized and significant limitation. 

As described in 2013 studies led by Joung and others, CRISPR-Cas9 nucleases can induce off-target DNA breaks at sites that resemble the on-target sequence. Subsequent investigations by Joung’s team and others have reduced but never completely and consistently eliminated these off-target effects.

Joung and his colleagues hypothesized that reducing interactions between Cas9 and the target DNA might more completely eliminate off-target effects while still retaining the desired on-target interaction. The team focused on the fact that certain portions of the Cas9 enzyme itself can interact with the backbone of the target DNA molecule. 

Pursuing an observation originally made by co-lead author Vikram Pattanayak, HMS clinical fellow in pathology at Mass General, the team altered four of these Cas9-mediated contacts by replacing the long amino acid side chains that bind to the DNA backbone with shorter ones unable to make those connections. 

“Our previous work suggested that Cas9 might bind to its intended target DNA site with more energy than it needs, enabling unwanted cleavage of imperfectly matched off-target sites,” said Pattanayak. “We reasoned that, by making substitutions at these four positions, we could remove some of that energy to eliminate off-target effects while still retaining full on-target activities.”

Testing the fix

Co-lead author Benjamin Kleinstiver, HMS research fellow in pathology at Mass General, and Michelle Prew, a research technician in Joung’s lab, then tested all 15 possible variants in which any combination of one, two, three or four of those amino acid side chains were altered. They found that one three-substitution and one four-substitution variant appeared to show the greatest promise in discriminating against mismatched target sites while retaining full on-target activities in human cells. 

The researchers then more fully characterized the four-substitution variant, which they called SpCas9-HF1; Sp for Streptococcus pyogenes bacteria, which is the source of this widely used Cas9, and HF for high fidelity. They found that this variant induced on-target effects comparable to those observed with the original unaltered SpCas9 when used with more than 85 percent of 37 different guide RNAs they tested.

Using GUIDE-Seq, a highly sensitive system Joung’s lab developed in 2014 to detect off-target CRISPR-Cas9 effects across the genome, the team found that, while nucleases combining unaltered SpCas9 with seven different guide RNAs induced as many as 25 off-target mutations, use of SpCas9-HF1 produced no detectable off-target effects with six of those guide RNAs and only one off-target site with the seventh. These results were further confirmed using targeted deep-sequencing experiments.

Joung’s team also found that SpCas9-HF1 could reduce off-target effects when targeting atypical DNA sites characterized by repeat sequences of one or two nucleotides—sites that are typically subject to many off-target mutations. They developed additional derivatives of SpCas9-HF1—called HF2, HF3 and HF4—which could eliminate the few residual off-target effects that persisted with the HF1 variant and a small number of guide RNAs.

Engineering new variants

“If SpCas9-HF1 using a certain guide RNA still produces a handful of off-target effects that are particularly difficult to eliminate, it may be possible to engineer new variants that get rid of even those effects,” said Joung. 

The researchers also showed that SpCas9-HF1, like its naturally occurring counterpart, could be combined with other useful alterations that extend its utility. Previous work from the Joung lab published last summer in Nature showed that introducing a series of amino acid substitutions could expand the targeting range of unaltered SpCas9.

In the current study, the authors show that introducing these same alterations into SpCas9-HF1 also extended the targeting range of the high-fidelity variant.

“These results show that these variants should be broadly useful to anyone currently using CRISPR-Cas9 technology,” said Kleinstiver. “They can easily be used in place of wild-type SpCas9 and provide a highly effective method for reducing off-target mutations to undetectable levels.”

Support for the study includes National Institutes of Health (NIH) Director’s Pioneer Award DP1 GM105378, NIH grants R01 GM107427 and R01 GM088040, the Jim and Ann Orr MGH Research Scholar Award, and the Natural Sciences and Engineering Research Council of Canada.

Adapted from a Mass General news release.

Children with significant congenital heart disease have a far better chance of surviving today than in decades past, thanks to major advances in surgery. But some infants who recover from repairs to their hearts later show the effects of delays in brain development, including impairments to cognitive, language and social functioning. Such impairments can affect how well these children do in school and in the workplace; they can even diminish their overall quality of life.

Get more Harvard Medicine news here

Epidemiological studies have given numbers to what doctors and families have long observed: The risk of neurodevelopmental delays is tenfold higher for children with moderate to severe congenital heart disease than for other children.

But why?

Over the years, those who study these phenomena have considered several possible reasons. Do the rigors of open-heart surgery so soon after birth play a role? Could heart defects limit nutrients and oxygen needed by the fetus? Or could spontaneous genetic mutations cause congenital problems that affect both the heart and the brain of a child?

Now, the “why” may have been answered by the efforts of the Pediatric Cardiovascular Genetics Consortium, led by a team of Harvard Medical School scientists. In a recent issue of Science the consortium reported exome sequence analyses of more than 1,200 children and their parents and showed that children with both congenital heart disease and neurodevelopmental delays share certain genetic mutations that thwart the normal development of both the heart and the brain.

“We’re homing in on a set of genes that have multiple different roles on multiple different tissues during development: heart tissue, brain tissue, other developing organs, limb tissue.” —Jason Homsy

Using a mathematical model created by co-authors Kaitlin Samocha and Mark Daly of the Analytical and Translational Genetics Unit at Massachusetts General Hospital, the team analyzed mutations in the protein-coding portion of the genomes of children with congenital heart disease that were not present in their parents’ genomes. They found that these children have more of these de novo mutations in genes that are highly expressed in the developing heart, compared to a control cohort of children without congenital heart disease. 

The de novo mutations were also found to be more frequent in children with congenital heart disease plus another birth defect, either neurodevelopmental delay or more-subtle abnormalities of finger or ear shape. These findings bolster the case for shared genetic causes of the cardiac and extra-cardiac abnormalities rather than surgeries or environmental factors.

“We’re homing in on a set of genes that have multiple different roles in multiple different tissues during development: heart tissue, brain tissue, other developing organs, limb tissue,” said Jason Homsy, an HMS LaDue Fellow who trained at Mass General and co-lead author of the Science paper. “Our study shows a common genetic link for the development of these diseases.”

Potential for early testing

According to Homsy and co-senior author Christine Seidman, the HMS Thomas W. Smith Professor of Genetics and Medicine at Brigham and Women’s Hospital and a Howard Hughes Medical Institute investigator, these findings could lead to early testing that would help identify newborns with congenital heart disease who are at high risk of neurodevelopmental difficulties.

“We can pretty clearly tell the parents of children with congenital heart disease what’s going to happen after the heart surgery, but there’s always a big question: Will my kid learn well in school?” Seidman said. “If we could identify children at high risk for neurodevelopmental delays, they could receive increased surveillance and earlier interventions than occur now.”  

The mutations primarily affected genes involved in three areas: morphogenesis, chromatin modification and transcriptional regulation. If any one of these processes is perturbed even slightly at a critical time in development, the heart is malformed; sometimes another developmental defect occurs, such as a missed connection in the brain.

'Master regulators'

“These genes are not just involved in shaping the heart,” Seidman said. “They are master regulators of organ development.”

One of the mutated genes is RBFOX2, which encodes a molecule that regulates RNA splicing. Although RBFOX2 has not been previously implicated in congenital heart disease, de novo mutations were identified in multiple affected children.

“There are still many unanswered questions, including why the same mutation can cause very different clinical manifestations,” Seidman said. Perhaps additional genetic variants in the multiple layers of transcriptional regulation allow compensation for some mutations but worsen the consequences of others. For now, Seidman said, knowing that a genetic mutation is present is different from knowing the outcome.

“It’s a long, long, long way down the road,” Seidman said, “but we’d like to believe that if you knew the steps by which these mutations perturbed the regulation of gene expression, there might even be ways to actually treat it.”

This work was supported by grants from the National Heart, Lung, and Blood Institute and the National Human Genome Research Institute of the National Institutes of Health, Howard Hughes Medical Institute, Simons Foundation for Autism Research, John S. LaDue Fellowship at HMS, Medical Scientist Training Program and National Research Science Award, Academy of Medical Sciences, British Heart Foundation, Wellcome Trust, Arthritis Research UK and the NIHR Cardiovascular Biomedical Research Unit at Royal Brompton and Harefield NHS Foundation Trust and Imperial College London, Leducq Foundation, Heart and Stroke Foundation of Ontario, Ted Rogers Centre for Heart Research, Kostin Family Innovation Fund, Aaron Stern Professorship at the University of Michigan, and Braylon’s Gift of Hope Fund.

WASHINGTON, D.C.—Genetics is not your destiny, George Church told an overflow audience gathered in a Congressional briefing room. This was one of many head-turning statements he and two other speakers made about advances in the rapidly progressing field of genetic technologies.

“You can change it,” Church said about the genome. “Certainly you need to know it first.”

Get more Harvard Medicine news here

In a lightning-quick 10 minutes, Church, the Robert Winthrop Professor of Genetics at Harvard Medical School, explained how gene editing can potentially eliminate malaria and allow transplants of pig organs into humans, among other innovations.

Change was the theme of a briefing designed to shed light on the frontiers of genetic technologies. Church was joined by Diana Bianchi, the Natalie V. Zucker Professor at Tufts University School of Medicine, and Jennifer Doudna, professor of molecular and cell biology and of chemistry at the University of California, Berkeley.

“This is a technology that seemed at first like science fiction: a technology for rewriting DNA itself. We can sequence DNA, we can find mutations. What if we had a way to fix those mutations? That technology is not science fiction; it is a reality.”— Jennifer Doudna, University of California, Berkeley

The Nov. 17 briefing was presented by the Personal Genetics Education Project (pgEd), a multifaceted effort based in the Department of Genetics at HMS. The mission of pgED is to educate individuals about advances in the field through school programs, libraries, religious institutions and other public forums, and to accelerate public awareness of genetics issues by advising the entertainment industry. It also seeks to engage lawmakers—the “eyes and ears of the nation”—in discussions.

pgEd takes no position on policy, preferring to educate from a neutral position so that its audience can make better-informed decisions.

Each speaker was passionate about the need to inform and educate the public about genetics and the power of new technologies to affect health and disease.

Bianchi is a leading medical geneticist in the burgeoning field of prenatal diagnosis. Doudna and her colleague Emmanuelle Charpentier, now at the Max Planck Institute for Infection Biology, are credited with translating an ancient mechanism in bacterial immunity into a powerful tool for editing genomes. Church optimized the gene-editing technology, now known by the acronym CRISPR, by developing what are known as gene drives, that is, molecular tools that can “drive” a DNA sequence to be inherited by all of an organism’s offspring. 

Doudna described the technology she and others have advanced as a genome-engineering revolution.

“This is a technology that seemed at first like science fiction; a technology for rewriting DNA itself,” she said. “We can sequence DNA, we can find mutations. What if we had a way to fix those mutations? That technology is not science fiction; it is a reality.”

The possible therapeutic applications are promising, she said, but delivery into cells and tissues remains a challenge.

Doudna also pointed out the fundamental difference between gene editing that affects only one person versus gene editing in the germ line that is passed on to future generations.

“When do we apply that? And for what kind of afflictions?” she asked. “I’m very interested myself in trying to stimulate public conversation over the implications of this technology.”

Bianchi, who both sees patients and conducts research in medical genetics, is already using genomic sequencing in her practice. Non-invasive prenatal testing takes advantage of the fact that small amounts of DNA fragments from the placenta circulate within the blood of pregnant women. Cell-free fetal DNA can be analyzed to screen for extra or missing copies of chromosomes that might indicate Down syndrome or other inherited syndromes.

Since 2011, the DNA screening tests have been done on 2 million women. The DNA tests have both a high sensitivity and specificity and most important, Bianchi said, a higher positive predictive value than the current noninvasive screening tests that measure blood proteins and fetal ultrasound markers.

“Genomic testing has really changed prenatal medicine,” Bianchi said. “We think the future is linked to treatment.”

Bianchi’s lab is testing in mice whether administering therapy during pregnancy improves fetal brain development. Treatment in pregnant women could start as early as 12 weeks following a confirmed diagnosis of fetal Down syndrome and continue until delivery at 40 weeks.

If such a strategy works, it would give pregnant women three options to consider: continuing the pregnancy aware of a high risk for a genetic abnormality, terminating the pregnancy or treating the condition before birth.

Church explained how gene drives could make mosquitoes immune to malaria and therefore unable to transmit the disease to humans. 

“That would have a huge impact. Like smallpox, once it’s gone, it’s gone,” he said.

Gene editing could also eliminate the viral components of the pig genome, thereby making organ transplants into humans more feasible. A recent paper showed that 62 genes could be altered at once, demonstrating the power of the technology.

While such projects are tantalizing, concern is also growing among scientists, bioethicists, policymakers and others over how gene-editing technologies, such as CRISPR, will be used, the speakers said, underscoring the importance of public discussion.  

The briefing on Capitol Hill was sponsored by U.S. Rep. Louise Slaughter, D-N.Y., and Sen. Elizabeth Warren, D-Mass. More than 160 people, half of them Congressional staffers, heard where the science of genetic technologies is today, where it may be going and what concerns exist over its benefits and risks.

“It’s all about scalability,” said Ting Wu, HMS professor of genetics and pgEd’s director and co-founder. She hoped to send people out of the room astounded by the science and eager to spark discussion among policymakers, thus amplifying pgEd’s reach.

After the briefing, the speakers and the pgEd team met with the White House Office of Science and Technology, where they expressed hope that all segments of society could become part of the discussion.

“We think it is critical to engage across all communities, particularly the disenfranchised,” Marnie Gelbart, pgEd director of programs, said later. “This piece is as important as the technologies themselves, for safely and fairly integrating the technologies into society.”

The briefing was the fourth presented by pgEd. The first briefing highlighted the science of genomics, personalized medicine and genetic engineering, as well as ways to reach out to the public. The second focused on two topics: the role of genetics research in the unfolding Ebola outbreak in West Africa and the issues addressed by the Genetic Information Nondiscrimination Act, also known as GINA. The third centered on law enforcement and highlighted research on how microbial genomics is being used to improve health and increase public safety. For the fourth, pgEd wanted to bring attention to the excitement at the forefront of genetics and the questions being raised about how we as a society use these new technologies.

pgEd is supported by the HMS Department of Genetics and private funding from Sigma-Aldrich, Autodesk, Genentech, IDT (targeted specifically for GETed conferences and Map-Ed) and an anonymous donor.

It used to be enough to call a serotonergic neuron a serotonergic neuron.

These brain cells make the neurotransmitter serotonin, which helps regulate mood, appetite, breathing rate, body temperature and more.

Recently, however, scientists have begun to learn that these neurons differ from one another—and that the differences likely matter in dysfunction and disease.

Get more Harvard Medicine News here

Last year, a team led by Harvard Medical School genetics professor Susan Dymecki defined a subgroup of serotonergic neurons in mice by showing that those cells specifically, among all serotonergic neurons, were responsible for increasing the breathing rate when too much carbon dioxide builds up in the body.

Now, Dymecki and colleagues have taken a first stab at systematically characterizing serotonergic neurons at the molecular level and defining a full set of subtypes, again in mice.

The researchers report in Neuron that serotonergic neurons come in at least six major molecular subtypes defined by distinct expression patterns of hundreds of genes. In many cases, the subtypes modulate different behaviors in the body.

By conducting a cross-disciplinary series of experiments, the researchers found that the subtypes also vary in their developmental lineage, anatomical distribution, combinations of receptors on the cell surface and electrical firing properties.

“This work reveals how diverse serotonin neurons are at the molecular level, which may help to explain how, collectively, they are able to perform so many distinct functions,” said Benjamin Okaty, a postdoctoral researcher in the Dymecki lab and co-first author of the paper.

“To have the list of molecular players that make each of these subtypes different from one another gives us an important handle on learning more about what that cell type does and how we can manipulate only that subtype,” said Dymecki. “It holds enormous therapeutic potential.”

“This is an ancient neurotransmitter system that’s implicated in many different diseases, and it’s starting to be cracked open,” said Morgan Freret, a graduate student in the Dymecki lab and co-first author of the paper. “We can now ask questions in a more systematic way about which serotonergic cells and molecules are important in, for example, pain, sleep apnea or anxiety.”

Crucially, the team also showed that a serotonergic neuron’s gene expression and function depend not only on its location in the adult brain stem, but also on its cellular ancestor in the developing brain.

“Earlier work had shown that you could explore the relationship between a mature neuronal system and the different developmental lineages that gave rise to it, but we had no idea whether it was meaningful,” said Dymecki. “We show that the molecular phenotypes of these neurons track quite tightly to their developmental origin, with anatomy making some interesting contributions as well.”

While the work was done in mice, Dymecki is optimistic that it will be replicated in humans because the serotonergic neuronal system is in a highly conserved region of the brain, meaning it tends to remain consistent across vertebrate species.

Because of this, researchers can look for the same molecular signatures in human tissue and begin to tease apart whether particular subtypes of serotonergic neurons are involved in conditions such as sudden infant death syndrome (SIDS) or autism.

Such research could ultimately reveal previously unknown contributions of the serotonergic neuronal system to disease, inform the development of biomarkers or lead to more targeted therapies.

The team’s findings could also inform stem cell research. “Which subtype of serotonergic neuron are we getting when we use current stem cell protocols?” asked Dymecki. “Can we drive the development of different subtypes? Can we watch how gene expression patterns change over time during development for each subtype?”

Finally, the study provides an example of a highly integrative approach to understanding brain function at multiple scales, “linking genes and gene networks to the properties of single neurons and populations of neuron subtypes, all the way up to the level of animal behaviors,” said Okaty. “I think it’s a useful template going forward. Imagine what we’d learn by applying this approach to all the neurotransmitter systems in the brain.”

This research was supported by funding from the National Institutes of Health (R01 DA034022, P01 HD036379, T32 GM007753, R21 MH083613, R21 DA023643), the American SIDS Institute, a Harvard Stem Cell Institute seed grant, a NARSAD Distinguished Investigator Grant from the Brain and Behavior Foundation, and Harvard’s Blavatnik Biomedical Accelerator, which provides resources to develop early-stage biomedical technologies toward clinical applications. Harvard’s Office of Technology Development has filed a patent application on the technology.