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ECONOMICS OF ORPHAN DRUGS CROWDFUNDING FOR GENE THERAPY DIAGNOSIS THROUGH EXOME SEQUENCING
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RARE DISEASES THE REALITIES OF STUDYING UNCOMMON CONDITIONS
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VOLUME 32 NUMBER 5
ON THE COVER: THE SCIENTIST STAFF/© ISTOCK.COM/RAWPIXEL
Researchers, clinicians, and families make the most of gene sequencing data to transform the way rare and mysterious diseases are diagnosed.
While orphan drugs have become incredibly successful within the pharmaceutical industry, many academic researchers who work on rare diseases still struggle for monetary support.
Families of children with rare disorders bolster gene therapy research.
Answers in the Exome
BY AMANDA B. KEENER
Crowdfunding for a Cure
BY THE SCIENTIST STAFF
BY DIANA KWON
05 . 201 8 | T H E S C IE N T IST
The Scientist wins more kudos for editorial excellence
CREATIVE CONTENT FOR CURIOUS MINDS AMERICAN SOCIETY OF BUSINESS PUBLICATION EDITORS (AZBEE) 2017 • March 2016 issue—Print, Single Topic Coverage by a Team—National Gold and Northeast Regional Gold • Modus Operandi—Print, Regular Department— Northeast Regional Bronze • Magazine of the Year, More Than $3 Million Revenue—Honorable Mention FOLIO AWARDS 2017 • March 2017 issue—Winner B-to-B Full Issue • B-to-B Single Article, Overall— Honorable Mention
Department Contents 15
FROM THE EDITOR
Sorting the Brain’s Signals
Researching the rare disease of gun violence in America will take a concerted political effort.
A tour of computer programs that swiftly sift through terabytes of neuron activity data
BY BOB GRANT
BY ASHLEY YEAGER
Selected Images of the Day from the-scientist.com
Family constraints and communications technology are making remote research positions more popular, but there are costs, scientists say. BY ASHLEY YEAGER
Rare to the Rescue
Mitochondrial Isolation System
Rarity becomes a strength when lessons from rare-disease patients are applied to more common diseases.
BY RUTH WILLIAMS
RANJA ANDRIANTSOA; © KIMBERLY BATTISTA; © JOHN DAVIS PHOTOGRAPHY
FOUNDATIONS BY DIANA KWON
A new facet of neuromuscular coordination; geneological proteomics unearths an unexpected connection; a 3-D view of gene variation
Among the Amish, c. 1960s
46 THE LITERATURE +
AND MICHAEL YEAMAN
Rare Disease: By the Numbers
BY JIM DALEY
BY VICTORIA JACKSON
A statistical look at uncommon maladies
A transgenic approach allows researchers to collect the organelles from specific cells in nematodes with unprecedented efficiency.
Attack of the Clones; The Key to Crowdfunding; I, Robot Surgeon; The Sight of Blood
Bullets and Ballots
IN EVERY ISSUE
9 11 60 62
CONTRIBUTORS SPEAKING OF SCIENCE THE GUIDE RECRUITMENT
The March article “The Transgender Brain” incorrectly stated that Lea Davis is organizing a study to look for genetic variants linked to gender dysphoria. Davis is focused on understanding the genetic contribution to gender identity, not specifically gender dysphoria. The Scientist regrets the error.
48 PROFILE Decoding Rarities
Uta Francke has spent her career linking genes to uncommon pediatric disorders, advancing the field of molecular diagnostics along the way. BY ANNA AZVOLINSKY
SCIENTIST TO WATCH
Valerie Arboleda: Data Miner BY SHAWNA WILLIAMS
PUZZLE ON PAGE 11
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05 . 2018 | T H E S C IE N T IST
THIS MONTH AT THE-SCIENTIST.COM: VIDEO
Fighting Canavan Disease
Watch the viral video that helped the family of a child with Sanfilippo syndrome raise more than $2 million.
Meet the family seeking to fund research into a rare disease that afflicts their two boys.
Ali Guthy, the daughter of cosmetics entrepreneur Victoria Jackson, discusses NMO, the rare autoimmune disease she suffers from.
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Contributors Journalist Amanda Keener became interested in science as a 12-year-old, when her youngest brother was diagnosed with a rare genetic disease. “The internet was becoming more available at the time so I would do online searches about his disease, and became really interested in genetics,” she says. She continued to pursue biology in high school and college, and eventually entered a PhD program at the University of North Carolina, Chapel Hill. The program combined microbiology and immunology, and she began studying autoimmune diseases and the connections between autoimmunity and Staphylococcus infections. The “ups and downs” of the graduate program eventually led her to look for career options beyond academia. Without telling her graduate advisor, she started sitting in on an undergraduate journalism class. This led to writing for local newspapers and for a friend’s blog, which gave Keener the confidence she needed to start pursuing a career in science writing. With her advisor’s blessing, she landed an internship at Nature Medicine during her final summer at Chapel Hill. “That was huge because the editor there really helped me learn about the journalism side” of science writing, she says. She has been a freelance writer ever since. Her brother, she reports, is doing fine. Keener writes about whole-exome sequencing and rare disease on page 24.
VICTORIA WILLIAMS; STEVEN BUSKEN
Victoria Jackson describes herself as a “fearful warrior” who has “always been a survivor in what has come up in [her] life.” In the 1990s, Jackson began marketing a line of cosmetics via infomercials, and over 13 years the company made close to a billion dollars in sales. “That was my entrée into the beauty world, and from there, the business world.” In 2008 her whole world was rocked when her daughter Ali was diagnosed with neuromyelitis optica (NMO), a rare autoimmune disorder that affects the optic nerves and spinal cord. Jackson started the Guthy-Jackson Charitable Foundation with her husband, Bill, to fund research into prevention and treatment for the disorder. As with her business, running the foundation draws on her skills as a human being and as a survivor, says Jackson. “Everything that I did was putting one foot in front of the other,” she says. “You can pretty much do anything that you put your mind to.” She has written four books, including Saving Each Other, which she coauthored with Ali in 2012, and The Power of Rare: A Blueprint for a Medical Revolution, which she wrote with physician Michael Yeaman. Jackson and Yeaman discuss the foundation and how it has driven the evolution of NMO research and treatment on page 59. “Growing up, all I did was draw,” says Erin Lemieux. These days, she isn’t doing as much sketching and painting as she used to, but as The Scientist’s art director, Lemieux’s talent for design and eye for aesthetics shines on the pages of the magazine each month. Born and raised in Midland, Ontario, Lemieux headed south to Toronto for college, studying graphic design at OCAD University. She was interested in branding and typography, and in her first job after graduating, Lemieux tried her hand at editorial work, doing page layout as a designer at Fizzz Design Corp. In 2012, Lemieux returned to Midland as a graphic designer at LabX Media Group, which publishes The Scientist, filling in for someone on maternity leave. But she made herself indispensible and was hired permanently to the production team. Her role has changed considerably in the intervening years. Initially she focused on getting the print issue online at the-scientist.com each month, and now she plays a major role in developing infographics, doing page layout, and commissioning illustrators and photographers. In March, Lemieux was promoted to art director. “Seeing the ideas come to life from the people we work with, that’s been really cool.” 05 . 2018 | T H E S C IE N T IST
FROM THE EDITOR
Bullets and Ballots Researching the rare, but all-too-common, disease of gun violence in America will take a concerted political effort. BY BOB GRANT
10 T H E SC I EN TIST | the-scientist.com
nate fiscal reality may be emerging. In March, Congress inserted into a spending bill language essentially freeing up funding for the CDC to conduct gun violence research. President Donald Trump subsequently signed that bill and the attached report into law. I sincerely hope that with more attention and activism drawn to the senseless and random blight of gun violence in America, this rare disease can be addressed, understood, and prevented using the strength of research and scientific inquiry. As Dickey himself told The Washington Post, before dying early last year at age 77: “We need to turn this over to science and take it away from politics.” g
his issue of The Scientist is devoted to the science, funding, and drug development directed toward rare diseases. As illustrated on page 23, various countries define “rare” in this context differently: in Chile, for example, a disease is rare if it afflicts at most 50 out of 100,000 citizens, while in South Korea, no more than 5 people out of 100,000 may suffer from the malady for it to be categorized as rare. But in the U.S.—where diseases are deemed rare if they affect 64 out of 100,000 people or fewer—recent tragedies demand that we take a look at another scourge that claims lives at an alarming rate: the disease of gun violence. According to researchers at the US Centers for Disease Control and Prevention (CDC), more than 32,000 Americans die and more than 67,000 people are injured by firearms each year (Prev Med, 79:5-14, 2015). Those same researchers more recently estimated that 1,300 US children die from gunshot wounds every year, and almost 5,800 are injured by firearms. Using just a year’s worth of these statistics, almost 10 Americans out of every 100,000 suffer the ill effects of gun violence— meaning one could think of gun violence as a “rare disease” in the U.S. Gun violence also shares qualitative characteristics with other rare diseases. The majority of rare diseases result from genetic mutation, many at a single locus in the human genome. According to Global Genes, a nonprofit rare-disease patient advocacy group, 50 percent of the people affected by rare diseases are children. Much like random mutations to crucial genes, bullets strike without warning and sometimes hit the young. As with the study of rare diseases that affect Americans, gun violence research lacks adequate support from the US government. Though securing federal or private research funding to study any rare disease is challenging, as editor Diana Kwon documents on page 30, the dearth of governmental dollars devoted to gun violence is programmatic. For 22 years, the CDC has been severely restricted from conducting gun-related research by the Dickey amendment, named for former Congressman and National Rifle Association supporter Jay Dickey (R-AR), who authored the legislation in 1996. After the tragic mass shooting that took 17 lives at Marjory Douglas Stoneman High School in Florida earlier this year, the political will to reverse this unfortu-
Speaking of Science 1
Note: The answer grid will include every letter of the alphabet.
—Brenda Cooperstone, Pfizer’s chief development officer for rare disease, talking about latestage clinical trial success of tafamidis, the firm’s experimental treatment for transthyretic cardiomyopathy (April 2)
BY EMILY COX AND HENRY RATHVON
1. 8. 9. 10.
1. Event of interest to 3 Down 2 Matrix, as of solar cells 3. Eponym of a seismic scale 4. Split apart—or stick together 5. Pony with a piebald coat 6. Case for a dermatologist 7. Breed of dairy cow 12. Axilla 13. Producers of gametes 15. What an electron occupies in an atom 16. Taurine or elephantine roar 18. Poison produced biologically 20. Youngster coming through in the clutch? 21. Works as an anesthetic
Powerful extensors of the knee joint Out-of-your-head part of the ear Abalone’s shell lining Subject of study for Jean-François Champollion 11. Blooms that may be African 12. Author of Earth in the Balance (2 wds.) 14. Asa Gray’s field 17. Grazer in estuaries of Florida 19. Viscount called “the father of empiricism” 22. Sort of finger or fossil 23. #3 on the periodic table 24. Products of a supersaturated environment
We’ve been working on it for a long time so to have it read out positively, it’s exciting but also moving. It’s a really underserved patient population and a uniformly fatal disease.
We developed a way to computationally find matches between rare disease protein structures and functions and existing drug interactions that can help treat patients with some of these orphan diseases. —Louisiana State University PhD student Misagh Naderi, in a press release announcing her recently published paper on drug repurposing in npj Systems Biology and Applications (March 13)
Answer key on page 5 05 . 2018 | T H E S C IE N T IST 1 1
Caught on Camera
Selected Images of the Day from the-scientist.com
Listeria monocytogenes bacteria (red), which can cause the rare disease listeriosis, infecting cells Posted: December 5, 2017
12 T H E SC I EN TIST | the-scientist.com
This immunostain of a 16.5-day-old mouse embryo shows endothelial progenitor cells (red) proliferating into tumors between muscle fibers (green). Posted: Jan 10, 2018
1 month old mouse cortex Brd4 »
The protein Brd4 (green) is present in abnormally high amounts in the brain of a mouse model of Fragile X syndrome. Posted: October 3, 2017
The interior lining of the retina in a patient with the rare eye disease retinitis pigmentosa, in which dark deposits accumulate as the retina atrophies
O MARBURG VIRUS
A micrograph of the Marburg virus, which can cause hemorrhagic fever Posted: April 6, 2017 Listeria: M. Kortebi et al., PLOS Pathog, doi:10.1371/journal.ppat.1006734, 2017. Institut Pasteur, Paris; Fragile X: E. Korb et al., Cell, 170:1209-23, 2017; Rhabdomyosarcoma: Catherine Drummond, St. Jude Children’s Research Hospital; Marburg virus: The University of Texas Medical Branch at Galveston; Retinitis pigmentosa: © 2006 Christian Hamel/Biomed Central Ltd.
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NEWS AND ANALYSIS
Attack of the Clones
t first glance, the marbled crayfish (Procambarus virginalis), a medium-size decapod crustacean with a speckled brownish-green shell, seems rather unexceptional. But since it first appeared on the German hobby-aquarium scene around two decades ago, the creature has been making waves. The clonally reproducing, all-female animals have been spotted in numerous freshwater habitats across Europe, and, according to a recent analysis, they’re spreading swiftly through the island nation of Madagascar. While this rapid expansion has alarmed scien-
tists due to the crayfish’s ability to alter aquatic ecosystems by outcompeting native species, the organism captured the attention of some researchers for another reason: the mystery of its origin. The first scientific description of the marbled crayfish was published in the early 2000s. Shortly before that, rumors about an “enigmatic animal able to reproduce without a male” had started to circulate among the internet community of hobby aquarists, says Gerhard Scholtz, a zoologist at Humboldt University in Germany. These rumors piqued the interest of Scholtz and his colleagues, who obtained some of the mysterious creatures for analysis in the lab. As advertised, the crustaceans, when placed in isolation, reproduced without males—a phenomenon known as parthe-
WHO’S YOUR CRAWDADDY? The highly
invasive marbled crayfish, pictured here in Madagascar, might trace its origins to a German aquarium.
nogenesis—and all the offspring had female gonads. The team named the newly identified organism the marbled crayfish, or Marmorkrebs in German, due to the patterning on its shell (Nature, 421:806, 2003). Although parthenogenesis is prevalent among crustaceans, this was the first known instance of the reproductive strategy in a decapod, a subgroup that includes lobsters, crabs, and prawns. Immediately, the researchers realized the potential threat the creature could pose to aquatic life—one specimen, if introduced to a freshwater habitat, could rap05 . 2018 | T H E S C IE N T IST 1 5
idly expand and outcompete local populations of crayfish and fish. So after unveiling the findings, Scholtz says, “we alerted the public, saying that a single individual animal could colonize a lake or a river because it doesn’t have to find a partner—so this might be a problem for the European environment.” Crayfish, in general, seem to be good invaders for a few reasons, says Brian Roth, a fish ecologist at Michigan State University. For one, they have wide-ranging feeding habits, which include munching on plants, capturing insects, and scavenging for dead animals. Secondly, Roth says, researchers have recently realized that the crustaceans can adapt to colder temperatures than scientists previously realized. In the U.S., for example, the red swamp crayfish (P. clarkii), a species native to the southern states, has spread northward to the Great Lakes. But being able to reproduce without a mate allows P. virginalis to take invasion to another level. Starting around 2005, researchers began noticing these animals being sold in food markets in Madagascar—and when another team of scientists compared these creatures to other crayfish in the region, it discovered that P. virginalis had “insanely high fecundity,” says Julia Jones, a conservation science professor at Bangor University in the U.K. who participated in the study (Biological Invasion, 11:147582, 2009). “We were worried that they were going to spread very fast—and sure enough, they have.” Although scientists were tracking the crayfish’s rapid spread, where, exactly, it came from remained a puzzle. Scholtz’s 2003 study provided some hints: when they sequenced the animal’s mitochondrial genes, the researchers found that its closest relative was P. fallax, a similar-looking, sexually reproducing species from Florida. Although it’s possible that the first marbled crayfish was shipped to Germany from the U.S., to date no wild American populations have been found, and some scientists point to another theory—that P. virginalis was born in captivity. 16 T H E SC I EN TIST | the-scientist.com
This latter hypothesis stems from the marbled crayfish’s earliest known appearance, recently established by molecular biologist Frank Lyko of the German Cancer Research Center who scoured the internet to track down the first known person that possessed one of the clonally reproducing creatures (Zootaxa, 4363:544-52, 2017). As the story goes, a German biologist and hobby aquarist purchased a bag full of crustaceans labeled “Texas crayfish” from an American trader at a fair in Frankfurt in 1995. He took the creatures back home and added them to his aquarium; a year later, he had hundreds, which he distributed among other aquarists. “We can trace the animal back to 1995, back to the trade fair,” says Lyko. “[But] we don’t know what the American trader actually sold, whether it was already a marbled crayfish or the parents of the first marbled crayfish.”
He purchased a bag full of crustaceans labelled “Texas crayfish” from an American trader. A new study by Lyko and his colleagues offers a clue, and suggests that, rather than being born in a wild population in the U.S., the first marbled crayfish may have actually been conceived in this German biologist’s aquarium. After sequencing and analyzing the crustacean’s entire genome, the team discovered that the animal, which possessed a triploid genome as determined previously by Scholtz and his colleagues, also carried an AA’B genotype, meaning that of its three sets of chromosomes, two were similar and one was unique (Nat Ecol & Evol, 2:567-73, 2018). “We interpret the genome to reflect the situation that [the original] father and mother were both from the same species but distantly related populations,” Lyko says. In addition, he adds, because genetically distinct groups of the same species rarely share the same habitat—genetic
exchange through sexual reproduction would result in a relatively homogeneous population—it’s likely that such a situation would have occurred in an aquarium, rather than out in the wild. This hypothesis is “reasonable, but very speculative,” says Scholtz, who did not take part in this work. Most documented cases of triploid genomes are thought to be a consequence, rather than a cause, of parthenogenesis, he notes: because asexual reproduction can make offspring more susceptible to genetic mutations, it can be advantageous to evolve three sets of chromosomes as a “buffer against damaging changes.” Lyko and his team also compared the genomes of marbled crayfish from various regions in the world—Germany, Madagascar, and the U.S.—and found remarkably little genetic variation. “The conclusion from that was that the global population of marbled crayfish is clonal, with one origin,” Lyko says. “And now it’s spreading globally.” Indeed, when the team examined the crustaceans’ spread in Madagascar, the researchers discovered that between 2007 and 2017, the marbled crayfish’s geographic range increased 100-fold, and by the last count in March 2017, the country harbored millions of these animals. While the sale and possession of this crayfish are currently banned in Europe, such regulations do not yet exist in Madagascar. There, the extent of the spread is “absolutely shocking,” says Jones, who has collaborated with one of the current study authors, Jeanne Rasamy of the Université d’Antananarivo in Madagascar. Although unraveling the marbled crayfish’s origins may not help curb its spread, Lyko and his colleagues hope that by further analyzing this crustacean’s biology, they will be able to better understand how, exactly, the creature is able to adapt so well to different environments, despite having such low genetic diversity. “Our hypothesis is that the adaptation is facilitated by epigenetic mechanisms,” Lyko says. “[That’s] the next thing on the agenda.” —Diana Kwon
The Key to Crowdfunding? It was a parent who first approached Romina Ortiz, the COO and vice president of patient advocacy at the Rare Genomics Institute (RGI), about crowdfunding. The mother of Maya Nieder, a developmentally disabled 4-year-old, was looking for a way to raise money for her daughter’s whole-exome sequencing, which reveals the intricacies of protein-coding genes (see “Answers in the Exome” on page 24). Ortiz had cofounded the nonprofit in 2011 to connect physicians, researchers, and rare-disease patients to laboratories that could conduct diagnostic genome sequencing, and to help scientists in those labs find funding. It wasn’t easy at first. “We by no means were experts at raising funds, so we really wanted to see how else we could help our [patients’] families,” says Ortiz. In 2012, the RGI man-
aged to raise $3,550 through crowdfunding to sequence Nieder’s exome. The genetic results revealed that the child had a mutation in a single gene that researchers thought was responsible for her disor-
der. The finding was the first example of a crowdfunded gene discovery. Although the cost of DNA sequencing has plummeted in the last decade, the price of an exome analysis—still in
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18 T H E SC I EN TIST | the-scientist.com
Four of the 11 crowdfunding campaigns met their goals before the 30 days were up. the thousands of dollars—remains out of reach for many families in the Nieders’ situation. And while some insurance companies now cover whole-exome sequencing, families are often left to find alternate sources of funding themselves. It’s becoming increasingly common for patients to turn to crowdfunding, raising money by gathering dozens of small donations to reach a goal in a set time frame, typically with the help of social media (see “Crowdfunding for a Cure” on page 38). Now, more than 80 percent of the RGI’s patients who can’t obtain grant funding or insurance coverage take this approach. Three years ago, to identify consistently successful crowdfunding strategies, Ortiz and her colleagues asked rare-disease advocacy groups and genetic counselors snafamilies muHinro f hclient ctaM a era seid to offer their populations the opportunity to take part in an rev86 ocinitial sid ydo bitna cinegs11nart ylno eh experiment.yOf respondents, elbcommitted airav ydobito tnaparticipate. namuh fo yThe teritne eht sr families . study’s requirements made it difficult to amass a larger cohort, Ortiz notes: particelbairav eht tub ,ciremihc era esuoM inn ipants had to want to use crowdfunding si tluser ehT .namuh yleritne era esuom e to pay for whole-exome sequencing for a rof dezimitpo seneg ydobitna morf childethat undiagnosed, romhas a sian esu oM innairT erare hT ,sdismret tselpmis ease, and they had to have a physician’s referral for the analysis. “You can already imagine ishadlittle evathat el uopopulation y pleh nacsize ti wo na mroftalp evi smaller,” she says. researchers tisiv The ,tnem poleved dand na yrevocsid y participants agreed upon a crowdfunding goal of $5,000—the cost of wholeexome sequencing for one young patient INNAIRT and both parents—in 30 days. The results after one month were revealing. Four of the 11 crowdfunding campaigns met their goals before the 30 days were up, and Ortiz says that participants who engaged in lots of preparation before the launch date were among the most successful (Interact J Med Res, 7:e3, 2018). These participants noitisop noitisop dicA onimA tended to be more involved with the RGI eht dna the selpcampaigns, mas namuh morf devired seido prior to starting requested )3H-RDCand ( 3Rkept DC in niacontact hc yvawith eh ,ethe suoRGI, M innairT evian resources, .esuoM innairT ehT ni dna snamuh ni emas eht y she adds. “We saw how they prepared. . . . They would send emails out to their networks ycneuqerf dicA onimA
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before the launch date.” Some even organized in-person events as well, before officially launching their crowdfunding campaign. Ortiz and her colleagues collected data about the participants’ social media engagement, measured by interactions with campaign-oriented Twitter and Facebook posts. They found that messages containing specific references to individual patients garnered more clicks than those tailored to general audiences. Information collected from donors via anonymous online surveys also provided insight into the factors that influenced people’s decision to give. “A lot of the donors were more likely to donate if they saw that a campaign was close to raising the amount of funds needed,” she says, adding that engaging donors with educational videos had a significant positive impact. And although 89 percent of donors said they heard about the campaigns via Facebook, contributions were higher when the donor learned about the campaign by word of mouth, email, or a phone call. Nora Kenworthy, a sociomedical scientist at the University of Washington who was not involved in the work, tells The Scientist that there’s likely more to these results than participants’ preparedness, and points out that the RGI researchers failed to include potentially important factors in their analysis. “There’s an implicit message in this study that a lot of crowdfunding success boils down to effort and engagement,” she says. “I think that is a misrepresentation of the crowdfunding marketplace.” While effort definitely plays a role, Kenworthy notes that many other factors can influence campaign success, such as age, race, disease type, and even the attractiveness of a campaign’s photos. “I would love to see more research that looks at some of those intractable factors,” she says. There’s also more to be explored regarding the long-term effects of crowdfunding medical costs on patients and on the broader healthcare landscape, says Valorie Crooks, a health geographer at Simon Fraser University in British Columbia who was not involved in the work. “There’s not been a lot of research regarding crowdfunding,” she tells The Scientist. “Meanwhile, this has become, in many ways, a
widely popular social phenomenon.” One possible outcome is the exacerbation of existing disparities in access to healthcare, says Kenworthy. Crowdfunding has “normalized the idea that getting healthcare can be a competition . . . which really flies in the face of norms around equity and fairness and equal access to healthcare.” There are other social and economic factors at play too—factors that raise a number of challenges from both ethical and equity perspectives in crowdfunding medical costs, says Crooks. “Campaigns that tend to be successful are not always, but often, coming from people who have some degree of social capital,” she notes. “They have the social network and resources that enable them to propel the campaign forward.” Some families may be less media-savvy, or lack the equipment necessary to create a video for their campaign. “That’s not within everyone’s capacity,” Crooks says. Ortiz adds that not everyone the RGI works with wants to use crowdfunding, and even among those who do, there’s substantial variation. “All families are different,” she says. “Some are more open to talking to their network, and others are a lot more private.” Whatever one’s resources, preparing and executing a successful crowdfunding campaign can be “pretty intensive,” says Ortiz. “There are chronic, very significant medical costs and emotional tolls on the family. It is asking a lot to do a whole campaign.” In the long term, Ortiz hopes to reduce the need for crowdfunding in the first place. With this goal in mind, she has begun reaching out to companies that provide whole-exome
sequencing to negotiate reduced prices. In 2016, the RGI “created a philanthropic program” with Illumina Inc., she tells The Scientist. “We’re actually offering it for free now.” —Jim Daley
I, Robot-Surgeon Parents from all over the world bring their babies and young toddlers to Boston Children’s Hospital for an operation that surgeon Russell Jennings wishes he didn’t have to perform. These children have been born with a disconnected esophagus, the upper section of which ends in a blind pouch a few centimeters from a lower portion that protrudes from the top of the stomach. The condition, called long-gap esophageal atresia, affects around one in 2,500 newborns, and is fatal if left untreated. But the procedure that Jennings and his colleagues currently perform to fix it, called the Foker process, takes its own toll. To repair the digestive tract, Jennings makes an incision in the child’s back and stitches a few tiny sutures into the end of each of the two sections of esophagus. Then, he ties the sutures onto button-like wheels on the child’s back. The operation has to be conducted under anesthesia, and once it’s finished, depending on the particulars of the child’s condition, he or she PAGING DR. ROBOT: The esophagus-
stretching device is connected by a cable to a battery-powered, wifi-capable controller.
may be kept sedated, on a ventilator, and fed intravenously for a few weeks to ensure the sutures stay in place. Every few days, Jennings or another doctor tightens the buttons a bit more, tugging at the sutures, until both sections of the esophagus grow long enough that they can reconnect to form a continuous tube. Jennings estimates he’s performed the surgery more than 100 times. But although he says that the procedure gets better results than transplanting a section of colon to replace the missing esophagus tissue, the need for long-term sedation doesn’t sit right with him. “The use of anesthesia in babies is very controversial now,” he says. “There’s evidence that use of the sedative drugs that are either morphine-based or benzodiazepine-based, like valium, causes some brain apoptosis . . . so we’d like to avoid them.”
The team stretched the rings about 2.5 mm farther apart each day for a week. As it turned out, Jennings had a colleague hankering for just such a challenge. Harvard University robotics engineer Pierre Dupont was interested in creating an implant “to assist in performing physiological functions or to help heal the body,” Dupont explains. After learning about the esophageal atresia problem, he and his colleagues started working on an implantable device that could do the job of the surgeon and the sutures, but from inside the body—thus constraining the need for anesthesia to the duration of a single operation. Building the esophageal atresia prototype presented some unique difficulties, Dupont explains, in part because any leakage of body fluids into the robot could damage the electronics or lead to infection. “Just creating something that would reliably work inside the body for multiple weeks was perhaps the biggest challenge,” he says. The team settled on a bot with a waterproof skin made of silicone rubber and a polyester mesh, and a body of electronics with two open metal rings along its 20 T H E SC I EN TIST | the-scientist.com
side that would slowly spread apart, elongating the tissue in between the rings. The internal device connects through a cable to an external, battery-powered, wifi-capable controller on the patient’s back. Once the device was ready to test, the team implanted its esophagus elongator into five healthy pigs, where, rather than treating esophageal atresia, the robot was tasked simply with lengthening the animals’ normally developed esophagi. The researchers initially set the rings 2 cm apart. Then, using a laptop to control the devices, the team stretched the rings about 2.5 mm farther apart each day for a week. The pigs seemed untroubled by the gizmos, and continued eating normally; the portion of their esophagi between the rings grew in length by an average of 77 percent over the course of the experiment (Sci Robot, 3:eaaq0018, 2018). As the rings moved apart, the robots also measured the forces acting on them, and the researchers noticed that resistance in the tissue rose just after each stretching episode, then declined over the ensuing hours, apparently as new cells grew in the space between the two rings. And when the researchers later examined the geometry, composition, and density of nuclei of the sections of esophagus that had been stretched between the rings, they found them comparable to those of unstretched parts of the organ, indicating the addition of new cells rather than elongation of existing ones. “I think it’s a really exciting demonstration of how applying mechanical forces, in this case with an implantable robot, can lead to tissue regeneration,” says Harvard University engineer Conor Walsh, who was not involved in the study. The study’s combination of robot systems, tissue engineering, and bioengineering is “pretty cool to see from an interdisciplinary perspective,” he adds. Dongwook Kim, who was also not involved in the new study, tells The Scientist that the robots’ mechanical action is a novel and impressive achievement, and stands in contrast to other implantable robotics devised to gather data or
deliver electrical stimulation. Kim, a graduate student studying nanoelectronics at Korea Advanced Institute of Science and Technology in Daejeon, notes that one potential way to improve the device would be to make it smaller by powering it wirelessly rather than with a battery. Dupont hopes a variation on the device could be used to treat a condition similar to esophageal atresia that affects the other end of the digestive tract: short bowel syndrome, in which the gut is too short to allow patients to fully process food. He and colleagues are now testing the robots’ ability to stimulate the growth of bowel tissue in pigs. As for the fate of each device, Dupont won’t reveal details at this stage, but says the researchers want find a way to engineer “the equivalent of having the whole thing disappear on us” once its work is done, so that a second surgery wouldn’t be needed to remove it. Still, Dupont worries that overcoming this and other remaining technical challenges may be the least of the team’s problems in getting the tissue-tugging robots into patients. “I just wish there were more resources available to translate these pediatric solutions to clinical use,” he says, noting that the relatively small potential market for the device may make it difficult to raise the funds needed for commercialization. “Hopefully, we’ll get there.” —Shawna Williams
The Sight of Blood In 2015, pathologist Jonathan Lin at the University of California, San Diego, School of Medicine started to receive skin and blood samples in the mail. Lin and his colleagues had recently discovered a gene linked to a congenital disease known as achromatopsia. People with achromatopsia have damage to a part of the retina known as the fovea, and also to the photoreceptors in the retina that create color vision. Consequently, they see the world in a black-andwhite blur. “This is a rare, inherited disease,” Lin says. “There is no cure.”
SEEING CLEARLY: Researchers are piecing
MODIFIED FROM © ISTOCK.COM/SIMCH
together the genetics of achromatopsia, a rare form of colorblindness that can render the world a blur of black and white.
Earlier that year, Lin and his collaborators had linked the condition to mutations in the gene Activating transcription factor 6 (ATF6), which codes for a key protein regulator of cellular stress—specifically, a process known as the unfolded protein response—and of homeostasis in cells’ endoplasmic reticulum. A missense mutation, which results in an arginine amino acid being replaced by a cysteine in the ATF6 protein, compromises the transcription factor’s activity, the team found (Nat Genetics, 47:757– 65, 2015). Now, patients were sending in their samples to see whether Lin and his colleagues could help determine if mutations to their ATF6 genes were underlying their vision problems. “The 2015 paper was eye-opening,” says Gustavo Aguirre, a medical geneticist and ophthalmologist at the University of Pennsylvania who was not involved in the research. There had been no a priori reason to think ATF6 would have anything to do with the retina. Achromatopsia had been linked to five other genes previously, several with associations to the development of the cone photoreceptors in the retina, but never to a gene involved in protein folding. Dysfunction of ATF6, meanwhile, had been implicated in other diseases, including diabetes and stroke. Curious whether activating the ATF6 transcription factor would change the fate of cells harboring these mutations,
Lin teamed up with chemical physiologist Luke Wiseman of the Scripps Research Institute in La Jolla, California, and his colleagues to sift through millions of potential small-molecule treatments. Wiseman’s team homed in on a compound called AA 147, which activated ATF6 in various cell types, and handed it over to Lin. Lin’s team collected skin samples from three siblings in New York who had achromatopsia, and used viral vectors to reprogram their skin cells into induced pluripotent stem cells, which the researchers then treated with AA 147. “We had no idea what would happen,” Lin says. A surprise awaited: activating ATF6 in the stem cells with high concentrations of AA 147 spurred the cells’ differentiation into endothelial cells destined to become blood vessels (Sci Signal, 11: eaan5785, 2018). No one had made the connection between ATF6 and blood vessel development, Lin says, adding that activating ATF6 in young achromatopsia patients might stimulate growth of the delicate bed of capillaries that rings the fovea. Those capillaries are essential for the fovea’s development, so spurring their growth could possibly improve achromatopsia patients’ vision. What’s more, “AA 147 restores ATF6 function,” he says. “So, if we can restore ATF6 function with AA 147 in these patients with mutations, then we could help their disease.” “They made a wonderful discovery,” Fumihiko Urano, a medical geneticist at
Washington University Medical Center in St. Louis, tells The Scientist. The finding is important, he says in an email, because it reveals a link between the unfolded protein response and tissue differentiation. “ATF6 serves as a node linking protein folding to tissue differentiation, especially mesodermal tissue differentiation,” Urano adds. “This is a very nice paper,” Aguirre tells The Scientist. The research is “intriguing and solid” and in terms of experimental protocol, the team “dotted their i’s and crossed their t’s,” he says. However, he notes, it overreaches in its claim for a possible therapeutic for achromatopsia. Once the cone cells are damaged, they’re gone. It would be impossible to target them and restore their function with AA 147 activating ATF6, as Lin suggests. Aguirre explains that gene therapy would be a more focused treatment for achromatopsia, targeting, early on, just the genes in the cells that would become damaged rather activating ATF6 in all cells. AA 147, however, could have implications for treatments for diseases other than achromatopsia, Lin and colleagues note in the paper. It may treat diseases that involve insufficient blood supply to the heart and brain, such as stroke. “We know that ATF6 is an attractive target for such diseases,” Urano says. With that in mind, Wiseman is now working with his colleagues to create a more potent version of the small molecule— one that is just as effective but doesn’t have to be administered in the potentially toxic high doses that the scientists used in their in vitro study. “The hope is that compounds like AA 147 can be developed and translated into potential treatments. Of course, that’s a long way away,” Wiseman says. “And maybe it won’t be these compounds, maybe it will be a next-generation compound of 147 or something like it.” —Ashley Yeager 05 . 2018 | T H E S C IE N T IST 2 1
Mitochondrial Isolation System A transgenic approach allows researchers to collect the organelles from specific cells in nematodes with unprecedented efficiency. BY RUTH WILLIAMS
gene for hemagglutinin epitope
gene for mitochondrial membrane protein promoter
itochondria power eukaryotic cells, but they do more than produce energy. These organelles, which contain their own genomes, RNAs, and protein-synthesizing machines, also regulate other cellular processes, including programmed cell death and calcium signaling. So it’s not surprising that mutations to mitochondrial DNA (mtDNA) can cause a range of debilitating and deadly diseases. Studying these ailments is complicated, says University of Massachusetts molecular biologist Cole Haynes. “You might get a mutation in a respiratory chain gene, which should be important, but it only affects a handful of cells. And then another mutation that doesn’t look so different may cause severe neurodegeneration or muscle defects.” This unpredictability is thought to stem from the differing ratios of mutant and wildtype mitochondria that can occur in different cell types, says Steven Zuryn of the University of Queensland in Brisbane, adding that even genetically identical mitochondria can behave differently in terms of morphology and activity in different cells. Techniques for analyzing such cell-specific differences are limited, however, says Zuryn. It is possible to mash up the cells of a particular organ and isolate mitochondria by centrifugation, but organs consist of multiple cell types, which muddies results. It’s also possible to sort cells based on specific markers and then isolate mitochondria, but this is time-consuming, requires large amounts of material, and in some organisms, such as Caenorhabditis elegans, is technically challenging. To achieve more accurate, sensitive, and rapid cell-specific mitochondrial isolation, Zuryn and colleagues first created a fusion of three genes: one that codes for the mitochondrial membrane protein TOMM20, one for a fluorophore (for visualization), and one that expresses the human influenza hemagglutinin (HA) epitope. The researchers then stably integrated the fusion gene into the C. elegans genome, using tissue-specific promoters to control expression. This resulted in worms with cell-specific, HA-tagged mitochondria that could be purified from the animals by homogenizing
gene for fluorophore
SORTED: With CS-MAP, researchers genetically label mitochondria in cells
of interest, then use antibodies to isolate them for analysis.
the cells, mixing the homogenate with anti-HA antibody-coated magnetic beads, and then isolating the bead-bound mitochondria with a magnet. The technique, called cell-specific mitochondrial affinity purification (CS-MAP), allowed for the collection of mitochondria that were intact and functional—and it revealed unexpected differences in the mutation loads of different cells, such as high numbers of mtDNA mutations in germline cells, says Zuryn. All in all, says Haynes, who was not involved in the research, “if you are interested in [questions relating to] individual cell types, this is certainly the best way to go.” (Nat Cell Biol, 20:352-60, 2018) g
HOW IT WORKS
GENETIC ENGINEERING REQUIRED?
Fluorescence activated cell sorting (FACS)
Prepare suspension of cells from target tissue, label cells of interest with a fluorescent antibody, sort cells based on fluorescence, isolate mitochondria via affinity purification with a mitochondrial antibody
Approximately 4 hours
Potentially any, but technically difficult in C. elegans
A stably expressed fusion gene under the control of a cellspecific promoter results in mitochondria carrying an HA tag, allowing the organelles to be isolated via affinity purification
Approximately 1 hour
Only C. elegans so far, but should be applicable to others
22 T H E SC I EN TIST | the-scientist.com
© GEORGE RETSECK
AT A GLANCE
Rare Disease: By the Numbers
MAP: DATA FROM T. RICHTER ET AL., VALUE IN HEALTH, 18:906-14, 2015; PIE CHART: DATA FROM C.E. DESANTIS ET AL., CA CANCER J CLIN, 67:261-72, 2017.
ALTHOUGH CANCERS ARE COMMON IN THE U.S., SOME UNCOMMON SUBTYPES ARE CONSIDERED RARE DISEASES.
>25 CASES PER 100,000 PER YEAR <6 CASES PER 100,000 PER YEAR <0.5 CASES PER 100,000 PER YEAR
Funding figures from NIH. US dollars.
0.002 0.31 0.007 10.0 4.6 11.7 9.1
Maximum number of cases per 100,000 people for a disease to be categorized as "rare" in different countries:
All Rare Diseases
05.2018 | THE SCIENTIST 2 3
24 T H E SC I EN TIST | the-scientist.com
Answers in the Exome Researchers, clinicians, and families make the most of gene sequencing data to transform the way rare and mysterious diseases are diagnosed. BY AMANDA B. KEENER
CREDIT © ISTOCK.COM/XFZSY LINE
n a sunny day near Perth, Australia, two-yearold Scarlett Whitmore stares intently at her left shoulder. With absolute concentration, she raises her head to look at her physical therapist, who is holding onto Scarlett’s arm to keep her steady. “I’m so proud of you,” her mother, Kate Whitmore, cheers as she films the session with her camera phone. Looking proud herself, Scarlett rolls onto her back, stretches out her arms and legs, and smiles broadly. Her smile is infectious. Her green eyes grow wide whenever she flashes her toothy grin—the inspiration for “Scarlett’s Smile,” the name of the foundation her parents started to raise money for Scarlett’s medical expenses. Scarlett has poor hearing and vision and hasn’t learned to sit up on her own, stand, walk, or speak. And for the first year of her life, her parents had no idea why. Just after Scarlett was born, “I
remember my husband saying in the hospital, ‘She doesn’t cry,’ and I just said, ‘She’s a good baby,’” says Kate. After five months, however, Scarlett failed to meet typical milestones, such as making eye contact with her parents. And then the tests began. Full workups on her blood and spinal fluid didn’t suggest anything amiss. Neither did a test for large-scale chromosomal abnormalities. A viral screen revealed that Scarlett had been exposed to cytomegalovirus, a known cause of brain damage when contracted during development. But a blood sample from Scarlett’s newborn screens showed she was clear of the virus at birth. “So, we were back to the drawing board,” says Kate. Months passed, and more tests came back negative. The Whitmores focused on early intervention therapies for Scarlett, trying to stay positive and enjoy spending time with their daughter. But whenever Scarlett cried, Kate agonized, not knowing if 05 . 2018 | T H E S C IE N T IST 2 5
her daughter’s pain resulted from expected things, such as teething, or from her mysterious illness. “It was just eating me up not knowing what was wrong with her,” she says. While Scarlett slept, Kate researched her symptoms, which ranged from visual impairment to hypotonia (muscle weakness), trying desperately to figure out what was causing them in her child. Finally, Kate came across information about an organization in Seattle, Washington, called MyGene2 that was offering to sequence and analyze the genomes of patients with undiagnosed diseases for about $700 per sample. Kate had heard of this type of sequencing before, but it was nearly impossible for the family to access it in Australia, and Scarlett’s geneticist had recommended against it—the approach is not routine in Australian hospitals, making it more expensive and the data more difficult to interpret. Nevertheless, just after Scarlett’s first birthday, the Whitmores sent saliva samples to MyGene2, where scientists sequenced each family member’s exome—the 1.5 percent of the genome that encodes proteins. Researchers at Washington University in Seattle then compared Scarlett’s exome sequence to databases containing thousands of sequences in search of a mutation that could explain her symptoms.
It was just eating me up not knowing what was wrong with her. —Kate Whitmore In January 2017, a verdict emerged: Scarlett had a rare mutation in the gene encoding G protein subunit beta 1 (GNB1), a component of a molecular switch protein complex known to regulate some neuronal functions. The Whitmores learned that Scarlett had not inherited the mutation from them, and that the disease will most likely spare her heart and lungs, giving them huge peace of mind. “This was worth its weight in gold,” says Kate. Along with just 30 other patients with GNB1 mutations worldwide, the Whitmores enrolled in a research study describing the mutation’s effects, and a paper reporting the findings is now being prepared for publication. Just ten years ago, the Whitmores’ story would have been very different. Back then, sequencing and analyzing a single exome cost between $70,000 and $80,000 and took months to complete. These days, clinicians can easily order an exome sequence and analysis, and at a commercial cost of around $700-$5,000 the test has become widely available and is often covered by insurers. Organizations such as MyGene2 and larger, national organizations, such as the Centers for Mendelian Genomics (CMG) and the Undiagnosed Diseases Network (UDN), are using the approach to help diagnose rare diseases, and to end what clinicians call the “diagnostic odyssey” for hun26 T H E SC I EN TIST | the-scientist.com
dreds of families every year. “Exome sequencing has really been revealing,” says Robert Kliegman, a neonatologist and rare disease specialist at Children’s Hospital of Wisconsin in Milwaukee. Helpful as it’s been, however, exome sequencing only resolves 25 percent to 50 percent of undiagnosed cases. Researchers and clinicians are now exploring new tools, such as whole-genome sequencing and RNA analysis, developing better techniques to analyze sequence data, and finding ways to get patients with the same diseases connected faster. This effort is making rare disease diagnosis likely to experience another revolution in the next decade.
Exome explosion About 15 years ago, Kliegman and his colleagues started noticing a huge unmet need at Children’s Hospital of Wisconsin. Families would end up there after years of searching for a diagnosis, and there was no system in place to settle their cases. Then chair of the pediatrics department at the Medical College of Wisconsin (MCW), Children’s Hospital’s academic partner, Kliegman began bringing together specialists to discuss undiagnosed cases in detail. But the team wasn’t galvanized until it came up against the case of Nic Volker, a young boy with severe inflammatory bowel disease. By the time Volker turned four, his intestines were dotted with holes, he’d had a colostomy, and he mainly ate through a feeding tube. The hospital’s gastrointestinal specialist couldn’t make sense of the disease, leaving Volker’s doctors with no options beyond treating his symptoms. In 2009, at the request of Volker’s pediatrician, a team at MCW sequenced the boy’s exome. The $75,000 bill was covered by funds raised by Howard Jacob, the founding director of MCW’s genetics center, who hadn’t expected to implement exome sequencing there for at least another five years. Analysis of Volker’s genetic data picked up more than 16,000 gene variants, and four months of sifting through those variants revealed that a mutation in X-linked inhibitor of apoptosis protein (XIAP), a gene on the long arm of the X chromosome, was the likely culprit behind his illness.1 XIAP mutations were already associated with X-linked lymphoproliferative disease, an immunodeficiency disorder that leaves boys unable to fight off Epstein-Barr virus. Because the gene only affects immune cells, a cord blood transplant to replace Volker’s immune cell progenitors was enough to essentially cure him, says Kliegman. The case became nationally renowned as the first time DNA sequencing saved a patient’s life. In a paper describing the research, the Wisconsin team noted that a thorough study of the available medical literature turned up a list of more than 2,000 gene variants that could have been responsible for Volker’s condition on the basis of his symptoms alone, and XIAP wasn’t on it. The boy’s case “was profound for all of the people in the hospital,” says Kliegman. “That was one of those eureka moments.” The experience led to a shift in the mindset of the hospital’s board, and now genetic sequencing is a cornerstone of the center’s diagnostic approach. By 2014, the MCW’s Human and Molecular Genetics Center (now the Genomic Sciences and Precision Medicine Center) was sequencing more than 700 patients per year.
Even as that project was getting started, other research teams were already putting together studies about how the approach could help diagnose rare genetic conditions on a larger scale. In 2009 and 2010, for example, a team led by geneticists at the University of Washington in Seattle demonstrated that exome sequence analysis alone could reveal disease-causing mutations, first in a group of people with a known disease2 and then in patients with undiagnosed diseases.3 Meanwhile, Duke University geneticists Vandana Shashi and David Goldstein were working to answer the practical question of how often exome analysis could be expected to provide a diagnosis. Goldstein recalls thinking that “if it resolves even just one out of ten of these really difficult cases, that’d be a remarkable new contribution.” The team enrolled 12 undiagnosed patients— all with different symptoms—into a pilot program at Duke, and identified disease-causing gene variants in the exomes of six. This success rate, combined with the steadily dropping costs of DNA sequencing, made it clear that exome sequencing would be a costeffective way to end the frustration experienced by so many clinical geneticists and families searching for a answers, Shashi says. “It’s energized people like me.” Although for most cases, a whole-exome sequencing diagnosis doesn’t lead to a cure as it did for Nic Volker, it usually opens a treatment path, says Kliegman. For example, many diseases historically known as “seizure disorders” now have names and mutations associated with them, allowing doctors to use targeted drugs “rather than shooting an ant with an elephant gun,” he says. In one case Kliegman worked on, the diagnosis delivered by exome sequencing made his patient eligible for deep brain stimulation. “That’s rewarding,” he says. “But we don’t find that in everyone.” He notes that whatever the outcome, however, families are always glad to have some kind of answer.
Find the variant A key factor in propelling exome sequencing into clinical diagnostics is the recent expansion of genome databases. In 2009, researchers working with Nic Volker’s sequence had to ask other scientists for access to sequences to compare against his. Now, accessing thousands of samples is simple thanks to efforts such as the 1000 Genomes Project, which ran from 2008 to 2015, and the Exome Aggregation Consortium (ExAC), launched in 2014 by researchers at the Broad Institute in Boston. With 60,706 exome sequences deposited by more than 100 research projects mainly being run at the Broad, ExAC was the most comprehensive exome database at the time of its release. Its successor, the Genome Aggregation Database (gnomAD), already contains 123,136 exome sequences and 15,496 whole-genome sequences. Such databases are vital because the diagnostic power of exome sequencing depends on clinicians’ ability to sift through variants and locate the pathogenic ones. “We all have thousands of rare variants, and most of them are completely benign,” says ExAC cofounder Anne O’Donnell-Luria, a geneticist and associate director of the Broad Institute’s Center for Mendelian Genomics (CMG), one of four centers funded by the US National Human
LOOKING FOR CLUES: Sequencing
of Scarlett Whitmore’s exome identified a rare mutation that could explain her symptoms.
Genome Research Institute (NHGRI) to pinpoint causative mutations for genetic diseases. ExAC and gnomAD only contain data from individuals 18 and older unaffected by severe pediatric disease, making them particularly handy for diagnosing children, such as Scarlett Whitmore, who have very rare genetic diseases. “We know what variants occur in the human population, and we can toss all those out,” Goldstein explains. “We can really narrow in on the candidates pretty quickly and effectively now.” There are also several searchable online databases, such as the National Institutes of Health (NIH) archive ClinVar and the Wellcome Sanger Institute’s DECIPHER, which both contain variants and the phenotypes associated with them. Canada will soon have its own repository of rare disease variant data and clinical phenotypes called Genomics4RD. If a patient’s variant appears in any of these databases, a diagnosis is on its way. Otherwise, researchers have to dig further to find out if a variant of unknown significance (VUS) is pathogenic. One approach is to try to predict how a variant impacts the function of the protein coded by the gene containing it. The recently developed Model Organism Aggregated Resources for Rare Variant Exploration (MARRVEL) database integrates information from other repositories with data from animal models of specific variants.5 And MCW’s Genomic Sciences and Precision Medicine Center (GSPMC) often performs molecular dynamics simulations to visualize how variants cause proteins to move differently in three dimensions. Statistical analyses can also help determine pathogenicity. ExAC’s size has made it possible for researchers at the Broad Insti05 . 2018 | T H E S C IE N T IST 27
tute to develop constraint scores for genes by comparing how often one type of variant is expected to appear in a gene, and how often it actually shows up. For example, if the algorithm predicts that a certain gene should occur with a loss-of-function variant 20 times in a population of 60,000, but that gene never shows up with such variants, the gene is assigned a high score. The higher the score, the more likely that mutation-gene combination is to cause disease. Researchers and clinical labs use multiple different tools to get detailed information about variants of interest, and more are being developed all the time (see “The Genetic Components of Rare Diseases,” The Scientist, July 2016). “Everyone tries everything they can to solve cases,” says O’Donnell-Luria. Even then, though, an analysis may fail to return a verdict. The report may come back with no candidates, or with one or more VUS. “There’s a fraction of cases where you have to work harder to determine whether you have a diagnosis,” says Goldstein. O’Donnell-Luria says that when one of her own patients gets back a negative clinical exome sequence report, she will offer to enroll him in a study and reanalyze his data using newly developed programs. She may also encourage families to apply for free exome sequencing and analysis by the CMG. Occasionally, a case may call for whole-genome sequencing, which can reveal pathogenic mutations in noncoding regions of the genome, such as those that affect transcription. Beyond that, RNA sequencing may be performed to search for things like splice variants. MCW’s GSPMC is also optimizing methods to analyze DNA methylation patterns and may even go so far as to reproduce a VUS in a zebrafish or mouse model to determine its effect. That said, sometimes getting an answer simply requires rechecking variant databases over time. “You may find that today it’s a VUS, and tomorrow someone reports another child, and then bingo,” says Kliegman. Scarlett Whitmore’s variant, for example, was caught during a second analysis because it took time for data from a recent study by Goldstein and others that described 13 other cases to make it into the variant databases.6 The two analyses were done only a few weeks apart. What makes all the difference in solving such cases is not just the available technology, Kliegman says, but a thorough and team-based approach. For each case, his team collects all of the patient’s primary medical test results and reanalyzes every detail. “We don’t ignore anything,” he says. In 2008, the NIH initiated the Undiagnosed Diseases Program (UDP) to employ this kind of thorough workup to make diagnoses and improve research on rare diseases. In six years, the UDP received more than 10,000 inquiries.7 In 2014, the program expanded into the UDN, which includes seven academic medical centers across the U.S. that are funded to pay for patients’ travel expenses, run tests (including exome and RNA sequencing), and to gather a diverse panel of specialists to work on cases. Bret Bostwick, a clinical geneticist at Baylor College of Medicine in Houston, one of the UDN sites, says that most families accepted into the UDN’s program have seen dozens of specialists over the years, but have never before had a team work in one concerted effort on their behalf. “Families really find a niche they’ve been looking for,” he says. Of the 685 patients evaluated since 2014, 177 have received a diagnosis. 28 T H E SC I EN TIST | the-scientist.com
Matching Game The UDN’s job doesn’t end at identifying a potentially pathogenic variant. “Just having a gene discovery doesn’t help anybody,” Bostwick says. “We also need to make sure that when we discover a new gene we take the time to gather patients who have the disease and study them so that they can teach [us] about what the gene does. That in turn teaches us how to treat them.” When he and his colleagues determined that a recent patient of theirs carried a mutation in the cell cycle control gene CDK13, Bostwick searched for published case reports on the gene, and called up diagnostic laboratories to ask if they had sequenced others with the same mutation. But he made more headway by entering the patient’s information into a network called Matchmaker Exchange, which links several variant-phenotype databases and networks to help researchers and clinicians find multiple individuals with the same variant. In two months, the network connected Bostwick to eight other patients with the same CDK13 variant. “I could spend a lifetime, and I would never have found another patient by myself who has this gene change,” he says. Thanks to this accessible cohort, Bostwick and his colleagues were able to publish an updated description of the disease, which could help with future diagnoses.8 This process, however, is not always so expedient, and many patients wait years for a diagnosis because the clinical literature or variant databases haven’t yet caught up with their disease. “One of the major barriers right now to new gene discovery, and to how to use that information clinically, is data sharing,” says Michael Bamshad, a clinical geneticist at the University of Washington in Seattle who helped lead the institution’s early exome sequencing work and co-runs its CMG. Soon after the CMG launched in 2011, Bamshad says, it became clear that researchers were identifying gene variants faster than they could publish on them. “In essence, we were sitting on hundreds of discoveries, and any one of those discoveries could be useful for a family,” he says. The CMG initiated a database called GeneMatcher to link researchers and clinicians interested in the same genes, but Bamshad says this didn’t ease his frustration. “If two researchers shared data and made a match, neither were under the obligation to contact one another, much less to put together a manuscript to publicize the discovery,” he says.
Everyone tries everything they can to solve cases. —Anne O’Donnell-Luria Center for Mendelian Genomics, Broad Institute
For Bamshad and CMG colleague Jessica Chong, this frustration came to a head when they were contacted by a couple who had created a website and Facebook page to connect with families whose children had the same VUS as their son. “Gene discovery by social networking,” Bamshad called it. After connecting to another family, the couple needed help to get access to the
family’s data, and Bamshad and Chong ended up coauthoring a report on the gene with the couple. The experience made the researchers wonder if something could be done to help families share their data on their own. “That was the birth of MyGene2,” says Bamshad. MyGene2, which currently holds over 1,200 profiles, allows families to upload as much or as little information about their family member’s undiagnosed disease as they like. That may include medical records, annotated gene information from clinical exome sequencing reports, or actual exome or genome sequences. Doctors and researchers may also upload de-identified data from patients or participants in research studies. The platform automatically matches and notifies individuals who report the same variants, allowing families to utilize data that might otherwise stagnate. “This data is much more valuable if shared publicly,” says Chong. The system not only aids diagnosis, it also helps families learn about the details and prognosis of their family member’s condition. That couldn’t be truer for Kate Whitmore. Being in touch with other families whose children also have GNB1 mutations has helped the Whitmores learn how best to manage Scarlett’s disorder, what sorts of symptoms to expect, and which therapies to try. “I don’t worry so much, I don’t second-guess everything,” says Kate. These days, the Whitmores are freer to simply enjoy being with their daughter. “Scarlett’s a beautiful, happy little girl. She’s worth all the effort and then a hundred times more.” g Amanda B. Keener is a freelance science journalist living in Denver, CO.
References 1. E.A. Worthey et al., “Making a definitive diagnosis: Successful clinical application of whole exome sequencing in a child with intractable inflammatory bowel disease,” Genetics in Medicine, 13:255-62, 2011. 2. S.B. Ng et al., “Targeted capture and massively parallel sequencing of 12 human exomes,” Nature, 461:272-76, 2009. 3. S.B. Ng et al., “Exome sequencing identifies the cause of a mendelian disorder,” Nat Genetics, 42:30-35, 2010. 4. A.C. Need et al., “Clinical application of exome sequencing in undiagnosed genetic conditions,” J Medical Genetics, 49:353-61, 2012. 5. J. Wang et al., “MARRVEL: Integration of human and model organism genetic resources to facilitate functional annotation of the human genome,” Am J Hum Genet, 100:843-53, 2017. 6. S. Petrovski et al., “Germline de novo mutations in GNB1 cause severe neurodevelopmental disability, hypotonia, and seizures,” Am J Hum Genet, 98:1001-10, 2016. 7. C.J. Tifft and D.R. Adams, “The National Institutes of Health undiagnosed diseases program,” Curr Opinion Pediatrics, 26:626-33, 2014. 8. B.L. Bostwick et al., “Phenotypic and molecular characterisation of CDK13related congenital heart defects, dysmorphic facial features and intellectual developmental disorders,” Genome Medicine, 9:73, 2017. 9. Y. Cui, J. Han, “Defining rare diseases in China,” Intractable and Rare Diseases Research, 6:148-49, 2017. 10. T. Richter et al., “Rare disease terminology and definitions—A systematic global review: Report of the ISPOR Rare Disease Special Interest Group,” Value in Health, 18:906-14, 2015.
HOW RARE IS RARE ENOUGH? Although they are uncommon by definition, rare diseases affect around 350 million people worldwide in total. “The magnitude is much bigger than what is perceived,” says Duke University clinical geneticist Vandana Shashi. Nonprofit organizations such as Global Genes and the National Organization for Rare Disorders often report that there are about 7,000 known rare diseases. But the precise definition of “rare” may vary depending on who you ask. In the European Union (E.U.), a disease is “rare” if it affects fewer than 5 in 10,000 people. The World Health Organization has defined rare diseases as those affecting “less than 6.5–10 people in 10,000.” Meanwhile China’s official definition, which remains controversial, is a disease affecting one person in 500,000 or one newborn in 10,000.9 (See “Rare Disease: By the Numbers” on page 23.) One recent survey performed by researchers at the Canadian Agency for Drugs and Technologies in Health in Ontario found that, across 1,109 organizations worldwide, there were 296 different definitions for rare diseases and thresholds for orphan drugs, ranging from 5 cases to 76 cases per 100,000.10 The average definition was 40 cases per 100,000 people, close to the E.U.’s definition. These definitions matter to patients and their families waiting for drugs to be developed to treat rare diseases. In the U.S., for example, the Orphan Drug Act grants pharmaceutical companies various incentives, including tax cuts, for developing drugs meant to treat diseases that affect fewer than 200,000 Americans at any given time. The US Food and Drug Administration’s Office of Orphan Products Development funds several grants and for companies working on drugs or medical devices that can benefit patients with rare diseases. The European Medicines Agency offers similar incentives, such as reduced fees and market exclusivity for drugs developed for diseases that meet the E.U.’s definition of rare. 05 . 201 8 | T H E S C IE N T IST 2 9
Rare Funding While orphan drugs have become incredibly successful within the pharmaceutical industry, many academic researchers who work on rare diseases still struggle for monetary support.
hen Eileen Shore, a geneticist at the University of Pennsylvania, started investigating a rare bone disease in the early 1990s, it was a small group of patients that helped make her work possible. Along with Frederick Kaplan, an orthopedic surgeon and molecular geneticist at the university, Shore was focused on finding the cause of fibrodysplasia ossificans progressiva (FOP), an extremely uncommon disorder that transforms soft tissues to bone. “One person in particular, Jeannie Peeper, just decided that it was really important to have research done,” Shore says. “One person can make a pretty big impact in pushing things along.”
30 T H E SC I EN TIST | the-scientist.com
In the 1980s, Peeper, who was born with FOP, started gathering a small group of individuals with the same disorder. This was not a trivial task, given the low prevalence of FOP—it affects an estimated one in 2 million people worldwide. Still, with the help of Michael Zasloff, a geneticist at the National Institutes of Health (NIH), Peeper connected enough people to establish the International FOP Association in 1988. Together, members and their families raised money to aid research efforts into the disease. “The funding we had initially was very small,” Shore says. “But it gave us a start.” That money helped Shore and Kaplan, after more than a decade of research, to
BY DIANA KWON
CREDIT Â© ISTOCK.COM/POGONICI LINE
05 . 2018 | T H E S C IE N T IST 3 1
More than meets the eye To date, around 7,000 rare diseases have been identified. In the U.S., they are defined as conditions that affect fewer than 200,000 Americans (in the E.U., disorders occurring in fewer than one in 2,000 Europeans fall into this category). (See “Rare Disease: By the Numbers” on page 23.) In total, these conditions end up being quite common—they affect an estimated 32 T H E SC I EN TIST | the-scientist.com
ORPHANS FIND A HOME Legislation passed by the US government in 1983 provides financial incentives for pharmaceutical companies to develop drugs for rare diseases. Orphan designation for a product comes with perks such as tax credits for clinical research and eligibility for seven years of market exclusivity after the drug is approved. However, orphan designation does not guarantee marketing approval, which requires firms to establish that the treatment is safe and effective in controlled trials. The data presented here come from the FDA.
Orphan Drug Designations
Orphan Drug Approvals
2017: The FDA approves Spark Therapeutics’s Luxturna, the first gene therapy for a rare disease. The drug, which treats a rare form of blindness, is priced at $425,000 per eye.
2013: The FDA passes a second amendment, which attempts to further clarify what defines an “orphan subset” of a more common condition. For example, it requires sponsors to demonstrate why the drug can’t be used outside of the specific group of patients.
1991: Genzyme receives marketing approval for Ceredase, an enzyme replacement therapy for patients with Gaucher disease. Initially priced at $150,000 per year, the drug was one of the most expensive on the market at the time.
1991: The FDA makes the first amendment to the ODA, which clarified that a disease subset would need to be “medically plausible,” in an attempt to curb “salami slicing.” (See “Abusing Incentives,” page 33.)
2000: The E.U. passes legislation incentivizing the development of orphan products.
1983: The US Congress passes the Orphan Drug Act (ODA). 150
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pinpoint the gene underlying FOP, ACVR1.1 This discovery set the stage for tracking down a potential treatment, palovarotene, a compound that inhibits a signaling pathway involved in bone formation and has been shown to prevent abnormal growth in the soft tissues of mice.2 In 2015, Clementia, a Canadian biotech firm, raised USD $60 million from investors to develop the drug for FOP, which is currently in a Phase 3 clinical trial. Rare disease research has undergone some changes since the early years of Shore’s work. Not only have advances in sequencing made it faster and cheaper to find associations between genetic mutations and diseases, but other developments, such as the growth of gene therapies, have drawn increased commercial interest to the realm of rare, or orphan, disorders. In addition, legislation incentivizing the pharmaceutical industry to invest in therapeutics for rare diseases has been incredibly successful. “When I started working on rare disorders, the first question was, ‘Why study a rare disease that impacts so few people?’” Shore recalls. “But in recent years, the pharmaceutical industry has become very interested in studying rare diseases as a drug discovery strategy.” As a result, hundreds of new rare-disease treatments have entered the market over the past few decades, and orphan drug development has become a highly profitable industry. While this has undoubtedly helped patients, there are downsides to this trend. Some economists and scientists suggest that companies have abused the financial incentives for rare-disease drug development, and they predict a coming backlash to the hefty price tags of these medications.
The result of this sea change is that nowadays, firms with marketing authorization for orphan products are more profitable than those without. 25 million people in the U.S. and 30 million in Europe. “In your circle of friends and family, there is certainly somebody who is affected that may not have told you about it,” says Heather Etchevers, a developmental biologist who studies rare congenital malformations, such as giant congenital melanocytic nevus, a large, pigmented birthmark, at the French National Institute of Health and Medical Research (INSERM). “And not all diseases are easily visible.” Still, while rare diseases are common in the aggregate, each condition is unique. And before the mid-1980s,
when governments began passing legislation that encouraged companies to invest in these uncommon conditions, industry was reluctant to pour its money into products with such minuscule markets. “If you look at it from an economic perspective, at the time there was a clear market failure—there was no incentive for companies to develop [an orphan] drug, it just didn’t make sense,” says Dyfrig Hughes, a health economist at Bangor University in the U.K. who is involved in clinical trials for rare conditions. “That was in the era of highvolume and low-cost treatments.” In the decades since, the pharmaceutical industry has changed in numerous ways. For one, some studies suggest that the cost of drug development has increased across the board—one analysis by researchers at the Tufts Center for the Study of Drug Development estimated that the average research and development (R&D) costs per drug went up from $802 million for products approved in the 1990s to $2.6 billion for those approved between 2005 and 2013.3 It’s
ABUSING INCENTIVES While incentives provided for pharmaceutical companies by the Orphan Drug Act (ODA) have helped hundreds of treatments for rare diseases enter the market, ethicists, scientists, and many others argue that some pharmaceutical companies have exploited the law to gain profits. A key provision of the ODA is that each time a medication gets approved by the FDA to treat a rare disease, it gains an additional seven years of market exclusivity for the specified condition, giving companies the ability to charge high fees for an extended period of time. In 2015, a Kaiser Health News (KHN) investigation revealed that a number of pharmaceutical companies gamed the system to sell orphan drugs at astronomical prices by using two key strategies: repurposing commonly used drugs and getting approval to use one product for multiple orphan diseases. For example, AbbVie’s Humira, which was FDA-approved in 2003 to treat rheumatoid arthritis, a condition that affects around 1 million adults in the U.S. alone, later gained additional approvals for multiple indications with orphan designation, including juvenile rheumatoid arthritis and pediatric Crohn’s disease—giving the company market exclusivity for some of these conditions until the early 2020s. Peter Saltonstall, president of the National Organization for Rare Disorders, told KHN in 2015 that Humira is “not a true orphan drug.” In fact,
important to note, however, that the price tag of drug R&D is a contentious subject with little consensus: a subsequent examination by another group generated a much lower estimate—a median of $648 million per drug, based on medications approved between 2006 and 2015.4 In addition to rising prices, advances in science have yielded a greater understanding of the complexity of diseases—which, according to Andrew Lo, an economist at the MIT Sloan School of Management, has led to a trend in recent years of investors shying away from drug development in general, particularly in the early stages. “The irony is that as we’ve gotten smarter about the nature of these diseases, that’s actually caused the risk for investing in these therapies to increase,” Lo says. Despite higher costs and less-certain returns, investments in drug development on the rare disease side appear to be bucking the trend affecting the greater biomedical industry, Lo says. “Rare diseases have actually done well, thanks to the incentives that the Orphan Drug Act provides.”
Humira is currently one of the world’s best-selling medications: in 2017, it raked in $18 billion in sales. Another technique is to identify additional populations to gain orphan drug approvals in a practice dubbed “salami slicing,” in which a more common condition is divided into smaller, biomarker-defined categories. A 2016 study found that 13 of the 84 drugs approved with orphan designation between 2009 and 2015 were for subsets of more prevalent diseases and that some of those medications were also approved for other, related conditions (PLOS Med, 14:e1002190, 2017). For example, pharma firm Boehringer Ingelheim received FDA approval for afatinib (Gilotrif) to treat non-small cell lung cancer (NSCLC) patients with an EGFR mutation in 2013. Then, in 2016, the company received approval to use the same drug to treat NSCLC patients with squamous histology. The firm was awarded seven years of market exclusivity for both of the specified indications. “[The ODA] doesn’t discriminate between genuinely rare conditions where there’s [usually a] hereditary component, almost always in children, versus personalized approaches to cancer where clearly they still are rare but they are a different end of the spectrum,” says Dyfrig Hughes, a health economist at Bangor University in the U.K. “Arguably, the legislation wasn’t really drawn up to cater for [the latter].”
RARE DISEASES, COMMON INSIGHTS Rare-disease research—like rare diseases themselves—doesn’t occur in a biological vacuum. It’s often intertwined with investigations into fundamental cellular activities. “I think it’s now a reflex of anybody working on any rare condition to see what else might be represented by the same mechanisms but in a different part of the body, at a different time in life, or [in the context of] cancers,” says Heather Etchevers, a developmental biologist at the French National Institute of Health and Medical Research (INSERM). For instance, her work on giant congenital melanocytic nevus (CMN)—a large, pigmented birthmark—could be helpful in understanding common cancers, such as adult-onset melanoma, as well as other developmental disorders. “There are a couple of genes [known to cause CMN] whose proteins work together to tell cells when to proliferate,” she explains. “When those mutations happen early in development and in a particular set of lineages, it turns out that it can lead to a whole bunch of different disorders, including many other kinds of skin malformations, vascular malformations, and brain malformations.” She adds that the reason those genes initially appeared on scientists’ radars was because they “came up over and over again in cancers.” Such unexpected associations between rare and common diseases are uncovered “more often than [those outside the orphan disease community] realize,” says Ellen Sidransky, a physician and molecular geneticist at the National Institutes of Health (NIH). Her work revealed that mutations in the gene encoding glucocerebrosidase (GBA), which causes Gaucher disease, were also present in many individuals with Parkinson’s disease—a discovery that launched more than a decade of research into the link between the two conditions (Neuron, 93:73746, 2017). For most rare disease researchers, this is one of the key arguments justifying investments in conditions that affect far fewer people than common ailments such as heart disease, lung cancer, and obesity. There are dozens of examples of rare disease–related insights that have led to breakthroughs for more frequently occurring conditions. Perhaps the most-cited case is that of familial hypercholesterolemia, which is caused by an extremely rare mutation in the gene encoding the low-density lipoprotein (LDL) receptor that can lead to fatally high cholesterol levels. Research into this condition, conducted in the 1970s by geneticist Michael Brown and biochemist Joseph Goldstein, led to a greater understanding of LDL’s role in cholesterol synthesis (PNAS, 71:788-92, 1974). These findings helped reveal the mechanism of action of statins, the widely used cholesterol-lowering drugs that help prevent cardiovascular disease, and earned the duo the 1985 Nobel Prize in Physiology or Medicine. “That, to me, is a perfect example of how the worst aberration of a pathway has revealed how you can intervene for milder aberrations and interfere with a population risk that’s enormous,” says William Gahl, head of the Undiagnosed Diseases Program at the NIH. Statins went on to become some of the bestselling drugs of all time, still earning billions of dollars in sales every year. Biotechs such as Perlara, based in the Bay Area, are seeking to develop treatments for both rare and common conditions by mapping the genetic connections between them. To accomplish this goal, the company is creating genetically engineered animal models of ultra-rare monogenetic conditions with the aim of generating new treatments and identifying how the genetic mutations are associated with more prevalent maladies. For example, insights into NGLY1 deficiency, a rare, congenital condition Perlara researchers are investigating, may lead to better treatments for certain cancers, such as multiple myeloma (ACS Cent Sci, 3:1143-55, 2017). “Our premise is that we’re not scared of the economics of these diseases, because if you realize that these rare diseases are connected to something more common, there is not an economic problem anymore,” says Ethan Perlstein, the company’s CEO. “You just have to figure out what that connection is.”
THE LDL RECEPTOR PATHWAY IN MAMMALIAN CELLS: LDL receptors
are first synthesized in the endoplasmic reticulum 1 . After maturing in the Golgi apparatus, the receptors are transported in vesicles to the cell membrane 2. Once an LDL molecule has bound to the receptor 3 , the complex is endocytosed into the cell 4 . Then, the resulting endosome is split, and the LDL is gobbled up by a lysosome while the receptor returns back to the cell surface 5. In the lysosome, LDL is degraded into cholesterol and amino acids 6. Increased levels of cholesterol in the cell suppress the transcription of the gene encoding HMG-CoA reductase, a key enzyme required for cholesterol synthesis within the body 7 , and the production of more LDL receptors.
The most common cause of familial hypercholesterolemia is a mutation in the gene for the LDL receptor (LDLR), crippling the receptor’s function and leading to a buildup of LDL in the blood.
Statins lower cholesterol levels by inhibiting HMG-CoA reductase. 7
© CATHERINE DELPHIA
PIECING THE CHOLESTEROL PUZZLE Research into familial hypercholesterolemia (FH), a rare disease in which the body is unable to rid itself of excess low-density lipoproteins (LDLs), provided fundamental insights into cholesterol metabolism. In 1974, geneticist Michael Brown and biochemist Joseph Goldstein reported that LDLs are less likely to bind to cells from FH patients than to cells from healthy individuals. This provided the first evidence that LDL receptors existed on the cell surface and set the stage for later work by Goldstein, Brown, and their colleagues to describe the ways that cholesterol derived from LDL helps regulate the production of the fatty molecule within the cell.
With a little help from the feds The US Orphan Drug Act (ODA), enacted in 1983, was a game changer for rare diseases. Before the law passed, only 10 orphan drugs had entered the market. By the end of 2017, more than 450 products for 668 orphan indications were FDA-approved. The European Union passed a similar policy in 2000. Both pieces of legislation created incentives for pharmaceutical companies—which would normally be averse to investing in a drug that might benefit only a tiny patient population—such as market exclusivity (seven years in the U.S. and 10 years in Europe, plus extra time for pediatric indications), reduced regulatory fees, and, in the U.S., subsidies for clinical trials.
The very nature of rare diseases—many are severe and linked to a single gene—have made them attractive targets for drug development. Prior to the introduction of this legislation, “there was no motivation for industry to invest in treatments for rare conditions,” Hughes explains, whereas afterwards, firms were driven to create more products. “There’s a stark contrast.” One study reported that 41 percent of the products green-lighted by the FDA in 2014 had orphan designation—the growth in popularity, the authors noted, had also corresponded with mounting evidence that some companies were gaming the ODA for their own benefit (See “Abusing Incentives” on page 33).5 The result of this sea change, according to a 2016 analysis of 86 publicly listed pharmaceutical companies by Hughes and his colleague Jannine PolettiHughes of the University of Liverpool, is that nowadays, firms with marketing authorization for orphan products are more profitable than those without. Between 2000 and 2012, orphan drug companies had a 9.6 percent higher 36 T H E SC I EN TIST | the-scientist.com
return on investment than non-orphan drug producers.6 “I think the traditional model of a blockbuster drug, [such as] statin, where you’ve got low cost and [you’re] prescribing to hundreds of millions of people, has changed,” Hughes says. While government incentives certainly played a role, other factors, such as the characteristics of the diseases themselves, may have contributed as well, he adds, “because with rare conditions it’s arguably somewhat easier to find a pharmacological target and an effective drug to interrupt [it].”
The revival of gene therapy Although commercial incentives codified by the ODA and laws like it may be the primary drivers of orphan drugs’ success in industry, other factors have contributed as well. The very nature of rare diseases— many are severe and linked to a single gene—have made them attractive targets for drug development. An oft-cited statistic suggests that approximately 80 percent of rare diseases are monogenic (or Mendelian), which means they arise from a single mutated gene. “The rare, Mendelian genetic disorders have a very well-defined cause,” says Stylianos Antonarakis, a geneticist at the University of Geneva Medical School in Switzerland. As a result, he adds, researchers often have a more clearly defined path toward a disease-modifying treatment. For instance, phenylketonuria, which is caused by a mutation that makes the body unable to break down the amino acid phenylalanine, was once a devastating disease that could lead to issues ranging from intellectual disability to severe brain damage. Nowadays, physicians can screen for the condition at birth and prevent complications by prescribing a phenylalanine-free diet. Recent advances in gene-editing technologies and improvements in gene therapies have widened the range of possibilities for treating monogenic rare diseases. “The revival, in a way, of gene therapy because of the progress in making it safer has given us new tools that have created new avenues of investigation,” says David Adams,
a medical geneticist studying rare pigmentation disorders at the NIH. “So certainly there is new work going on based on tools like CRISPR that allow editing of genes in model organisms and that, maybe in the future, will have therapeutic benefits.” Last December, Spark Therapeutics’s voretigene neparvovec-rzyl (Luxturna), a treatment for patients with a rare form of retinal dystrophy caused by a biallelic RPE65 mutation, became the first gene therapy for a genetic disease to be approved by the FDA. Of course, gene therapies are not the only approach to treating rare diseases, and some biotech companies, such as Cydan, which helps launch startups focused on orphan-drug development, have decided to steer clear of them. “It’s too expensive and too competitive right now,” says Chris Adams, the company’s founder and CEO. “We’re focusing on small molecules, peptides, proteins, and there’s still enough opportunity there, we believe, to have impact on genetic disease.” That strategy is beginning to pay off. According to Adams, the company has successfully spun out three startups. The first of these, Vtesse, launched in January 2015 to develop a treatment for Niemann-Pick type C, a rare lysosomal storage disorder, using sugar molecules called cyclodextrins. It was acquired by Sucampo Pharmaceuticals for $200 million last April.
Clinical trial cost savings In general, regardless of whether a raredisease drugmaker is designing a gene therapy or working on another treatment approach, the severity of many rare monogenic diseases means effective drugs often yield dramatic benefits. “There’s an opportunity to generate convincing clinical safety and efficacy data with very limited patient populations,” says James Wilson, director of the Orphan Disease Center at the University of Pennsylvania. “There could be a quick path to [regulatory approval], which means the cost of development would be a fraction of what it could be for more-common diseases.” According to Wilson, a company could achieve approval for an orphan drug with as
few as 20 individuals, whereas a treatment for a common cardiovascular condition or a vaccine to prevent infection might need to be tested on thousands of people. For example, the Phase 3 trial for Luxturna included only 31 participants. And the FDA greenlighted vestronidase alfa-vjbk (MEPSEVII), an enzyme replacement therapy for another rare lysosomal storage disorder, in November 2017 after testing in just 23 patients. In addition to requiring a smaller number of participants, according to a 2012 study by Thomson Reuters and Pfizer, clinical trials for orphan drugs tended to be shorter and had a 5 percent higher probability of regulatory success than those for non-orphan products.7
Pricing backlash The question now is whether patients— or their insurers—will foot the bill for the newer rare-disease treatments. Orphan drugs are already some of the most expensive medications on the market—many costs hundreds of thousands of dollars per year—but gene therapies come with some of the heftiest price tags: Luxturna, for instance, costs approximately $450,000 per eye. “There’s a raging debate right now for how you pay for that,” says Wilson. Some scientists and economists have suggested alternatives to a one-time payment. For example, Wilson, along with Troyen Brennan, the chief medical officer at CVS Health, proposed a “pay-forperformance model,” in which payments are made on a yearly basis as long as the therapy continues to be effective.8 There’s already evidence to suggest that one-time payments may not be the best choice. Last year, alipogene tiparvovec (Glybera), a gene therapy for lipoprotein lipase deficiency, a rare metabolic disease, was withdrawn from the market due to lack of demand. The treatment, which was developed by Amsterdam-based UniQure, became the first gene therapy to enter the market in Europe when the European Commission approved it in 2012. Glybera was a one-time injection that cost approximately $1 million. Spark Therapeutics, which treated the first patient with Luxturna this March, has
been considering methods to soften the financial blow, such as providing rebates or allowing payments in installments. “There is going to be a backlash to some of the ultra-high prices for some of these rare-disease therapeutics,” says Lo, who, like Wilson, has proposed alternative payment models for treatments, including gene therapies.9 “I think we’re far away from that, because there’s still a lot more we can do, given the current pricing system—but at some point we’re going to reach a limit.”
From rags to riches Despite the upswing in rare-disease drug development, researchers studying such disorders in academia say that it’s nevertheless a challenge to get their work funded (See “The Challenges of Rare-Disease Research,” The Scientist, September 2016). “It’s definitely still a struggle for most of the 6,000 to 8,000 estimated rare diseases,” Etchevers says. And although some rare diseases are targeted by a number of drugs on the market, there are plenty of conditions for which no treatment yet exists, she adds. “There are lots of promising preclinical studies ready to be translated to the clinic, in which therapeutic targets have been identified and even validated initially, that do not find industry takers.” Accordingly, many academic researchers studying rare diseases still rely heavily on patient foundations to support their work, particularly at the early stages, when it’s more difficult to receive funding from agencies such as the NIH (See “Crowdfunding for a Cure” on page 38). “Research on rare disorders is mainly promoted by the families and by the lobbying of associations,” Antonarakis says. “Families need to continue to lobby, organize, and drive the agenda of funding agencies to the benefit of the disorder in their family.” Still, technological advances in recent decades, especially in sequencing, have
allowed academic researchers to stretch their limited dollars. “When I started out, we were dependent on more family-based studies [and] linkage analysis. I worked on trying to identify the cause of FOP for about 15 years,” Shore says. “Now, we would just take a handful of patients, do exome sequencing, and in less than a month have the answer.” (See “Answers in the Exome” on page 24.) But even for Shore, whose work on FOP has led to a promising drug currently in development, funds from patient foundations remain crucial. “Funding for basic and preclinical research from any source has become more difficult in recent years,” she says, “It’s a continual struggle and concern for most researchers.” g
References 1. E.M. Shore et al., “A recurrent mutation in the BMP type I receptor ACVR1 causes inherited and sporadic fibrodysplasia ossificans progressiva,” Nat Genet, 38:525-27, 2006. 2. K. Shimono et al., “Potent inhibition of heterotopic ossification by nuclear retinoic acid receptor-γ agonists,” Nat Med, 17:454-60, 2011. 3. J.A. DiMasi et al., “Innovation in the pharmaceutical industry: New estimates of R&D costs,” J Health Econ, 47:20-33, 2016. 4. V. Prasad, S. Mallankody, “Research and development spending to bring a single cancer drug to market and revenues after approval,” JAMA Intern Med, 177:1569-75, 2017. 5. M.G. Daniel et al., “The Orphan Drug Act: Restoring the mission to rare diseases,“ Am J Clin Oncol, 39:210-13, 2016. 6. D.A. Hughes, J. Poletti-Hughes, “Profitability and market value of orphan drug companies: A retrospective, propensity-matched casecontrol study,” PLOS ONE, 11:e0164681, 2016. 7. K.N. Meekings et al., “Orphan drug development: an economically viable strategy for biopharma R&D,” Drug Discov Today, 17:660-64, 2012. 8. T.A. Brennan, J.M. Wilson, “The special case of gene therapy pricing,” Nat Biotechnol, 32:874-76, 2014. 9. V. Montazerhodjat et al., “Buying cures versus renting health: Financing health care with consumer loans,” Sci Transl Med, 8:327ps6, 2016.
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Crowdfunding for a Cure Families of children with rare disorders bolster gene therapy research. BY THE SCIENTIST STAFF
t’s a compelling narrative: A parent learns that his or her child has a fatal disease with no cure, and, though not a scientist, embarks on a quest to find some treatment. Such stories have played out in the plotlines of films such as Lorenzo’s Oil and Extraordinary Measures, on national morning shows and local news segments, and on crowdfunding pages to drum up support for the cause. Parent-led funding campaigns to develop gene therapies for rare diseases are especially prevalent, and for good reason. Rather than finding a drug that can fill the void left by a protein lost to a single-gene disorder, gene therapy holds the promise of replacing the defective gene itself—of a cure. Just one of the thousands of singlegene disorders has an FDA-approved gene therapy, but through hard work and determination, some parents hope to change that. Some crowdfunding campaigns have been astoundingly successful: they’ve raised millions of dollars to fund basic research and, later, clinical trials that have likely saved children’s lives. Donations can, however, only carry a therapy so far before a pharmaceutical company must grab the baton—an outcome that’s not guaranteed, even when a gene therapy shows promise in early clinical trials.
And such therapies may not be able to turn back the clock on damage that’s already done, making cinematic happy endings to these stories unlikely. Still, participation in trials gives families some hope of a longer and healthier future for their children, a hope denied to the parents of kids who don’t make it in. “Gene therapy clinical trials are relatively small in terms of number of patients who can be enrolled. . . . Whenever you design stringent criteria, you know that as a physician you have to say a number of ‘no’s’ to parents who are desperately looking for treatments for their children,” says Alessandra Biffi, a gene therapy researcher at Dana-Farber Cancer Institute in Boston. “This choice, which is very rational, is also very difficult for me.” Yet for the families raising funds to combat rare diseases, any gain—whether in prolonging the life of one’s own child, sparing other families the same heartache, or some combination of the two— counts. Thanks in part to contacts made through the National Organization for Rare Disorders, The Scientist spoke with several parents whose children’s diagnoses sparked fundraising efforts to help make gene therapies a clinical reality. 05 . 2018 | T H E S C IE N T IST 3 9
Saving Eliza that “if we could deliver [the missing] gene where it’s supposed to be, we wouldn’t have to treat 100 percent of cells—we could treat some cells, [and those could] provide other cells with the secreted protein.” A handful of patient foundations supported the research for more than a decade, even after Fu moved to NCH and began working with another researcher, Douglas McCarty, to complete the preclinical work on a gene therapy for Sanfilippo types A and B.2 Although still in the preclinical stages when the O’Neills contacted Fu, it was the only research effort that looked like it would be able to treat Eliza, who has Sanfilippo type A, in time, Glenn says. But much more money was needed to start a clinical trial—which often costs in the millions of dollars to initiate. To help raise funds, the O’Neills established the Cure Sanfilippo Foundation (Cure SFF) in 2014. While they hoped their daughter would be chosen for the study, Glenn explains, their primary purpose was simply to help get the clinical trial up and running. After raising $250,000 over the first six months, the pair realized that they needed a way to raise much more money, quickly—and decided to try making a viral video. So they reached out to Karen Cheng, a videographer in California, for advice. To their surprise, she contacted a colleague, Canadian photographer Benjamin Von Wong, who travelled to the O’Neills’ home and shot a short film that went live on a GoFundMe page in April 2014. By the end of the year, the campaign had raised a whopping $2 million. In 2016, the research team at NCH received investigational new drug (IND) approvals from the FDA to start testing the treatments, which involve an intravenous injection of an adeno-associated viral
Eliza and her family in West Virginia
THE O’NEILL FAMILY
When Eliza O’Neill was 3 years old, her parents, Glenn and Cara, noted that her development began to diverge from that of her peers. Their once fast-learning, gregarious child faced difficulties in school, and her improvements in areas such as social communication and speech began to slow. It took about six months and multiple visits to the doctor for Eliza to be diagnosed with Sanfilippo syndrome, a rare lysosomal storage disease in which sugar molecules called glycosaminoglycans build up in the central nervous system, destroying cells and eventually causing severe dementia, seizures, and a loss of mobility. The disease strikes between 1 and 9 out of 1,000,000 people, and most children affected do not survive beyond their teens. The diagnosis, which Eliza’s doctors made in July 2013, was like “a lightning bolt out of the sky,” Glenn recalls. “I didn’t even know that a disease as terrible as this could even exist.” In the weeks following Eliza’s diagnosis, the O’Neills combed the scientific literature looking for a way to save their daughter. Their research led them to a potential gene therapy for Sanfilippo under investigation at Nationwide Children’s Hospital (NCH) in Columbus, Ohio. At the time, the work was still in the preclinical stage, but “the data were amazing,” says Cara, a pediatrician.1 Once she found this study, she contacted Haiyan Fu, a scientist at NCH’s Center for Gene Therapy working on the experiments, who walked her through the research. “That was the first moment that I had a real solid hope in the science,” Cara recalls. Fu’s work on Sanfilippo had begun in the late 1990s, during her postdoctoral studies at the University of North Carolina at Chapel Hill. Her initial focus was on Sanfilippo type B, and her project was supported by the Children’s Medical Research Foundation, which was established by the Wilsons, a family in Illinois whose daughter was born with the disease. Sanfilippo, which is caused by the loss of an enzyme needed to break down glycosaminoglycans, has four subtypes, A–D, each with a different enzyme deficiency and unique genetic mutations. Fu tells The Scientist that she and her colleagues thought
I didn’t even know that a disease as terrible as this could even exist. —Glenn O’Neill
BRETT FLASHNICK PHOTOGRAPHY
vector that is able to cross the blood-brain barrier, carrying the gene encoding a replacement N-sulfoglucosamine sulfohydrolase (SGSH) enzyme. This delivers the treatment throughout the body, allowing SGSH to be produced both in the central nervous system—the main target—and in other organs, such as the liver, that are also affected by the disease. Shortly after, the INDs were transferred to Texas-based Abeona Therapeutics, which sponsored two Phase 1/2 clinical trials, one for Sanfilippo A that began in 2016, and another for Sanfilippo B that started late last year. Both are being conducted by another group of investigators, led by NCH’s Kevin Flanigan. A group of 12 foundations, including Cure SFF, granted Abeona the funds to develop those therapies. Other funds raised by Cure SFF—more than $5 million in total—have financed a variety of Sanfilippo-focused investigations around the world. Nowadays, the O’Neills are not alone: more than 50 families have joined their foundation’s fundraising efforts. Their ultimate goal, Glenn says, is to have routine newborn screening and a proven treatment within the next 10 years. Because the gradual buildup of glycosaminoglycan worsens symptoms, he adds, the sooner the disease is caught and treated, the better. “I think patient foundations play a critical role as a catalyst for research for rare diseases,” Flanigan says. But he notes that helping fund a treatment does not guarantee enrollment in a clinical trial. Eliza, however, was a good candidate for one of the Abeona trials, and in 2016, at age six, she finally received the gene therapy. “She was the first child in the world treated with this—she was pretty progressed in the disease, but we were very lucky, blessed, and thrilled that she was even getting a chance,” Glenn says. According to the latest results from the Abeona-sponsored trial of the Sanfilippo A treatment, presented this February at the WORLD Symposium for Lysosomal Diseases, 10 patients have been treated, and investigators have seen a significant reduction in glycosaminoglycan buildup in cerebral spinal fluid and urine, as well as some evidence of cognitive improvement six months
MOTHERLY LOVE: Eliza and Cara O’Neill
after treatment. “We have, most importantly, no evidence of systemic toxicity with delivery,” Flanigan says. “We’re very optimistic about it.” The results look promising, but they are preliminary, says Kim Hemsley, a neuroscientist who studies Sanfilippo at the South Australian Health and Medical Research Institute but is not involved in the Abeona study. “The true test of [gene therapies] will come with time.” Hemsley, who has also received support from patient foundations for her work on Sanfilippo, says “the real unsung heroes” of our society are the families who fundraise for this work. “Not only are they having a quite extraordinary battle in their own life with a devastating disease in their family, but they still find the strength to advocate, to raise funds, to talk to clinicians and researchers around the world to try to establish research programs that have eventually led to the trials that are ongoing today,” she adds. As for Eliza, the O’Neills say that her symptoms don’t appear to have worsened since her treatment. And although she has not regained her speech, which she had completely lost, the family has found other ways to connect, such as through eye contact, music, and TV shows. “I’d say with Eliza, we’re in this uncharted territory where her future is unknown and uncertain,” Glenn says. “And that’s a good thing for Sanfilippo, because if your child is not treated, their future is certain, and it’s not good.” —Diana Kwon
Cupcakes for a Cure By the time Maria Kefalas and Patrick Carr noticed anything wrong with their youngest daughter, it was too late. Shortly after her second birthday, Calliope Joy—Cal to her parents—started to lose her balance. The family, who live in a Philadelphia suburb, visited the Children’s Hospital of Philadelphia, and in July 2012, Cal’s doctors returned a diagnosis: metachromatic leuko-
dystrophy (MLD), a rare neurodegenerative disease that affects 1 in 40,000 infants and is caused by a genetic mutation in the ARSA gene on chromosome 22. Children with MLD cannot produce arylsulfatase-A, an enzyme that breaks down sulfatides. So the compounds accumulate in the nervous system, where they attack the myelin sheaths that cover nerve 05 . 201 8 | T H E S C IE N T IST 41
PARTY TIME: The Calliope Joy
axons, leading to loss of mobility and eventual paralysis. There was no cure, the doctors explained. Cal would be unlikely to see her sixth birthday. To help assuage their grief, Cal’s parents established The Calliope Joy Foundation (TCJF) to raise money to support research into the disease. They hosted cupcake sales, and over the course of several months built up a network of families struggling with MLD or any of the more than 50 variations of leukodystrophy, all of which involve myelin damage. Then, one year after her daughter’s diagnosis, Kefalas, a sociology professor at Saint Joseph’s University in Philadelphia, came across a paper in Science written by a group of researchers at the San Raffaele Scientific Institute in Milan.3 The paper described a radical approach to treating MLD: researchers had used a self-inactivating HIV vector to insert functional ARSA sequences into patient-derived stem cells ex vivo, and then implanted the cells back into patients’ bone marrow to produce the missing enzyme. “I have to admit, it sounded like mad science,” Kefalas says. “The idea of infecting children with an inert virus seemed to me a very frightening prospect.” But the approach had a track record: San Raffaele’s Alessandra Biffi and her colleagues had shown in the early 2000s that it slowed the progression of MLD in a mouse model, and in 2010, 42 T H E SC I EN TIST | the-scientist.com
GlaxoSmithKline (GSK) announced it would get involved in the research. In the 2013 paper, the team presented data from its first human study of three presymptomatic children—each of whom had been diagnosed after an older sibling started showing symptoms of the disease. The therapy, the team reported, stopped MLD in its tracks. “It was the best outcome we could have predicted,” Biffi, now director of the Gene Therapy Program at the DanaFarber Cancer Institute in Boston, tells The Scientist. At first, the news was difficult to swallow for Cal’s family. “I remember calling my daughter’s doctor in Philadelphia, in tears of despair over the fact we’d missed our chance to help Cal,” Kefalas says. But as Cal’s doctor pointed out, the study showed that the treatment could halt MLD’s progression—it could do little to improve the symptoms of children already displaying signs of severe nerve damage. What’s more, the trial had recruited only presymptomatic patients; Cal, by the time she was diagnosed, wouldn’t have been eligible. However, Cal’s doctor also had a suggestion: that Kefalas help recruit participants for the
THE CALLIOPE JOY FOUNDATION
Foundation organizes regular “cupcake celebrations” to raise money for research on leukodystrophy. The funds have helped send young patients (four of whom are shown above) to Milan to receive an experimental stem cell treatment for one variation of the disease, metachromatic leukodystrophy (MLD).
next trial in Milan. “You’ve mistaken me for somebody brave,” Kefalas remembers replying. Yet brave is exactly what she was. In 2014, The Calliope Joy Foundation helped raise money for the family of Cecelia Price, another child diagnosed with MLD, to travel to Milan. More families followed, from the U.S., Australia, and several European countries. By the end of last year, TCJF had raised hundreds of thousands of dollars through cupcake sales and other efforts, which helped fund travel for 10 children and establish the Leukodystrophy Center of Excellence at the Children’s Hospital of Philadelphia. In 2017, the Italian team published an update on their study results: Of the first 21 presymptomatic and early onset patients treated with the therapy, 19—some of whom had been treated more than five years previously—had survived without significant disease progression.4 “Alessandra Biffi’s work is very, very promising, and very positive,” says Dolan Sondhi, a medical geneticist at Weill Cornell Medical College who is not involved in the research. She notes that wiping out existing bone marrow stem cells by chemotherapy—a pre-treatment necessary for the stem cell therapy to work—raises the risk of infection; other groups are developing therapies that avoid this step. The biotech company Shire, for example, is trialing an enzyme replacement therapy, and Sondhi is involved in developing a therapy that uses an attenuated adenovirus to deliver ARSA directly into the central nervous system— although neither is as far along as the Italian group’s approach, and none of the treatments have shown promise in reversing the damage caused in patients with later stages of the disease.
rare-disease research. Kefalas responded with an open letter that included a photo of several children who had taken part in the San Raffaele trial, “in case you have any doubts about the lives GSK’s gene therapy for MLD has changed.” GSK currently remains involved with San Raffaele’s ongoing Phase 3 trial, but is looking to “secure an appropriate partner to take over further development and commercialization of these medicines,” GSK spokesperson Mary Anne Rhyne writes in an email to The Scientist. Calliope Joy, meanwhile, is now 8 years old. Her symptoms have worsened; she can no longer walk, see, or speak. But TCJF is a constant source of motivation for her mother. “Whenever I feel sad, I look at pictures of [children treated in the trial] playing basketball,” Kefalas says. “I’m just so happy that a child gets to
TOUCHDOWN: A fundraiser for MLD research
She will have a legacy that’s so beautiful—I’ll get to see the children that her life helped save. —Maria Kefelas
THE CALLIOPE JOY FOUNDATION
The path to regulatory approval for the Milan group’s therapy remains uncertain. Last summer, GSK’s new CEO Emma Walmsley announced that the company would be moving away from
do that.” It’s an outcome that seemed impossible only a few years ago. “I’m very proud of that,” Kefalas says. “My daughter is going to die. But at least she will have a legacy that is so beautiful—I will get to be able to see children that her life helped save.” —Catherine Offord
Good news, bad news Twenty years ago, Ilyce Randell and her husband received devastating news: their son Maxie, who was a little over four months old at the time, had Canavan disease. Maxie would never walk or talk, and he likely wouldn’t live past age 10. Not much could be done to help their son, the couple was told, though a geneticist offhandedly remarked that researchers were developing a gene therapy that might lessen Maxie’s symptoms or extend his life. But
the Randells also learned that there was no funding available for a clinical trial on the gene therapy. Recently married, the couple contacted the same people they had invited to their wedding. Randell wrote a letter describing her son’s illness and included a photo of Maxie grinning. “That was my first fundraising campaign,” she says. It was also the start of Canavan Research Illinois, the Randell family’s foundation. 05 . 201 8 | T H E S C IE N T IST 4 3
THE RANDELLS: Maxie and his family at
Canavan Research Illinois’s 19th Annual Canavan Ball in October 2017
44 T H E SC I EN TIST | the-scientist.com
therapy to a Phase 3 clinical trial. The funding was there, but “there wasn’t any interest,” Leone says. The disease was too rare, with just 2,000 people affected worldwide. The response isn’t uncommon. The development of therapies for rare diseases is often funded by parents of sick children, either through individual donations or foundations like Randell’s, Leone adds: “All of this could have never happened without the support and devotion, the passion of patient advocacy groups, specifically families.”
All of this could have never happened without the support and devotion, the passion of patient advocacy groups, specifically families. —Paola Leone, Rowan University
STEPHEN NARENS, NARENS PHOTOGRAPHY
Canavan disease is caused by mutations to the ASPA gene, which encodes an enzyme, aspartoacylase, that breaks down N-acetyl-Laspartic acid. Without aspartoacylase, the acid builds up in the brain’s neurons and prevents their axons from being coated in fatty myelin sheaths. As a result, electrical signals don’t travel as efficiently from nerve cell to nerve cell. Neurons in the brain break down, leaving the organ spongy and leading to intellectual disabilities, loss of movement, abnormal muscle tone, and seizures, among other symptoms. In the first US trial of a gene therapy for Canavan, researchers tried encasing healthy copies of ASPA in liposomes and injecting them into the brain through an intraventricular catheter attached to a small, plastic, dome-shaped reservoir placed just beneath the scalp. The researchers injected the gene therapy into the reservoir, and it then diffused into the cerebrospinal fluid. In 1999, Maxie became one of 16 patients to receive the treatment. Maxie and his cohort showed some improvements in vision and movement, but the children weren’t cured.5 “We knew all along . . . that the ideal way to deliver a gene to the brain was with a viral vector,” says Paola Leone, a neuroscientist now at Rowan University who worked on the initial clinical trial at Thomas Jefferson University. As that trial proceeded, she and her collaborators began work on a second gene therapy with an adenoassociated viral (AAV) vector. Then, in 1999, a young man named Jesse Gelsinger died following an adenoviralbased gene therapy to treat a different disease. Even though Leone and colleagues were using an AAV vector, not an adenoviral vector, their research stalled along with the rest of the gene therapy field. Slowly, after working to show the safety of the AAV vectors, Leone and others pushed forward on a new gene therapy for Canavan, and started the first trial in June 2001. The treatment moved through both Phase 1 and Phase 2 clinical trials, and in 2012, the team published the first long-term study that tracked the safety of the treatment, along with some measures of efficacy. Injecting 900 million vector genomes directly into a patient’s brain appeared to decrease the levels of N-acetyl-L-aspartic acid, slow brain atrophy, and reduce the frequency of seizures, the team reported.6 Based on the results of the study, a drug company could have moved the
Randell says the reason Maxie is alive today, and gearing up for his 21st birthday, is because he received both the liposome and AAV treatments. Still, Maxie is far from cured. He is severely disabled, without the ability to move his arms and legs. He’s learned to use several different eye scan and retinalgaze computers, but his favorite mode of communication, she says, is blinking his eyes. “You’ll say, ‘Oh Maxie, I love you so much,’ and he’ll squeeze his eyes shut really tight. The longer he does it, the more meaning it has.” But there is still no FDA-approved gene therapy for Canavan disease. Randell continues to push for the development of new and improved gene therapy treatments that Maxie could try; she’s holding out hope, not just for her own son, but also for parents with children newly diagnosed with the disease. Some such parents have launched funding efforts of their own. The Landsman family, for example, launched a GoFundMe campaign to develop a treatment for Canavan after their two young sons were diagnosed with the disease; it raised $1.1 million in its first three months. Randell, the Landsmans, and others affected by Canavan could get their wish. Two groups, one that includes Leone and one led by Guangping Gao of the University of Massachusetts Medical School, are racing to get the regulatory OK for another round of gene therapy trials to treat Canavan. The treatments in development have the potential to vastly improve upon earlier versions. Leone and her collaborators are working on a viral vector targeted at the white matter of the brain, specifically the myelin-making oligodendrocytes, rather than neurons. Aspartoacylase is naturally produced in oligodendrocytes, and it’s there that the enzyme breaks down N-acetyl-L-aspartic acid, Leone explains. The vector would still be injected into the brain through holes drilled into skull, but by homing in on oligodendrocytes, the treatment could boost the myelination of neuronal axons and improve patients’ motor function and development.7 In contrast, Gao and his collaborator Dominic Gessler are developing an intravenous injection that would travel across the blood-brain-barrier. “Interestingly, if you look at the expression of ASPA, which is the defective enzyme, it’s actually expressed almost in every cell in the body,” Gessler says. “For that reason we highly advocate this intravenous injection approach, where we use a single dose to treat the entire body.” The approach has shown promising results in a mouse model of the disease.8 Crowdfunding supports both groups’ work, and the researchers involved say that their findings on Canavan disease could also be applied to more common neurodegenerative diseases, such as amyotrophic lateral sclerosis or Parkinson’s. If the vector can effectively deliver the gene to the targeted cells, it could give clues to how to deliver corrective genes in other diseases. The work could, Gessler says, “actually be beneficial for many more people than just the ones directly affected by Canavan disease.” —Ashley Yeager
BROTHERS: Maxie (left) and Alex Randell
References 1. H. Fu et al., “Correction of neurological disease of mucopolysaccharidosis IIIB in adult mice by rAAV9 trans-blood–brain barrier gene delivery,” Mol Ther, 19:1025-33, 2011. 2. F.J. Duncan et al., “Broad functional correction of molecular impairments by systemic delivery of scAAVrh74-hSGSH gene delivery in MPS IIIA mice,” Mol Ther, 23:638-47, 2015. 3. A. Biffi et al., “Lentiviral hematopoietic stem cell gene therapy benefits metachromatic leukodystrophy,” Science, 341:1233158, 2013. 4. F. Fumagalli et al., “Update on safety and efficacy of lentiviral hematopoietic stem cell gene therapy (HSC-GT) for metachromatic leukodystrophy (MLD),” Eur J Paediatr Neuro, 21:e20, 2017. 5. P. Leone et al., “Global CNS gene transfer for a childhood neurogenetic enzyme deficiency: Canavan disease,” Curr Opin Mol Ther, 1:487-92, 1999. 6. P. Leone et al., “Long-term follow-up after gene therapy for Canavan disease,” Sci Transl Med, 4:165ra163, 2012. 7. J.S. Francis et al., “N-acetylaspartate supports the energetic demands of developmental myelination via oligodendroglial aspartoacylase,” Neurobiol Dis, 96:323-34, 2016. 8. D. Gessler et al., “Redirecting N-acetylaspartate metabolism in the central nervous system normalizes myelination and rescues Canavan disease,” JCI Insight, 2:e90807, 2017.
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EDITOR’S CHOICE PAPERS
The Literature CELL & MOLECULAR BIOLOGY
Staying Strong THE PAPER
At first glance, neurons and muscle cells are the stars of gross motor function. Muscle movement results from coordination between nerve and muscle cells: when an action potential arrives at the presynaptic neuron terminal, calcium ions flow, causing proteins to fuse with the cell membrane and release some of the neuron’s contents, including acetylcholine, into the cleft between the neuron and muscle cell. Acetylcholine binds to receptors on the muscle cell, sending calcium ions into it and causing it to contract. But there’s also a third kind of cell at neuromuscular junctions, a terminal/perisynaptic Schwann cell (TPSC). These cells are known to aid in synapse formation and in the repair of injured peripheral motor axons, but their possible role in synaptic communications has been largely ignored. Problems with synaptic communication can underlie muscle fatigue, notes neuroscientist Thomas Gould of the University of Nevada, Reno, in an email to The Scientist. “Because these cells are activated by synaptic activity, we wondered what the role of this activation was.” To investigate, he and his colleagues stimulated motor neurons from neonatal mouse diaphragm tissue producing a calcium indicator, and found that TPSCs released calcium ions from the endoplasmic reticulum into the cytosol and could take in potassium ions from the synaptic cleft between neurons and muscle cells. However, TPSCs lacking the protein purinergic 2Y1 receptor (P2Y1R) didn’t release calcium or appear to take in potassium ions. 46 T H E SC I EN TIST | the-scientist.com
Motor neuron axon
MUSCLE HUSTLE: After stimulation with an action potential 1 , the synaptic terminal of a motor neuron releases acetylcholine and ATP. 2 Acetylcholine activates receptors in the muscle, which spurs voltage-gated sodium channels to open, triggering an action potential in the muscle, which contracts. At the same time, ATP or ADP stimulates P2Y1 receptors 3 , which causes calcium ions to be released from the endoplasmic reticulum of the terminal/perisynaptic Schwann cell (TPSC) 4 . In response, perisynaptic potassium ions (K+) produced by the muscle and neuronal cells move into the TPSC 5 . Regulation of perisynaptic potassium ions by TPSCs is thought to reduce the ions’ ability to inactivate voltage-gated sodium channels during repeated firing, thus reducing muscle fatigue.
Calcium release “is believed to be a very important intracellular signal initiated by neural activity,” Gould says. And the results show that TPSCs contribute to continued muscular contraction by releasing the ion and possibly taking up potassium. (Potassium ions contribute to fatigue when they accumulate in the synaptic cleft.) The results also suggest that P2Y1R or its downstream pathway help the Schwann cells to mediate muscle fatigue, he notes. “I think [the finding] reminds people that, similar to other perisynaptic glial cells like astrocytes, TPSCs may regulate extracellular ionic concentrations at sites where these concentrations matter, such as the synaptic cleft,” Gould says. He plans to examine the role in muscle fatigue of other ions, such as protons, in future studies.
Chien-Ping Ko, a neurobiologist at the University of Southern California who was not involved in the study, calls the work a “technical tour de force.” Gould’s team, he says, “does a nice job showing the connection of internal calcium ion release and potassium ion accumulation to muscle fatigue”—though it’s not yet clear whether the same mechanism applies in adult mice or in other animals. That said, Ko suggests the study points to potential drug targets for preventing muscle fatigue in diseases such as spinal muscular atrophy or hyperkalemic periodic paralysis. If therapeutics could be developed to boost internal calciumion release and potassium ion uptake in TPSCs, it might improve patients’ muscle function, Ko says. —Ashley Yeager
© KIMBERLY BATTISTA
D.J. Heredia et al., “Activity-induced Ca2+ signaling in perisynaptic Schwann cells of the early postnatal mouse is mediated by P2Y1 receptors and regulates muscle fatigue,” eLife, 7:e30839, 2018.
STRIKING A BALANCE: Errors in copper homeostasis can lead to serious problems in development.
MAPMAKING: A tool for visualizing data on metabolic pathways reveals
CELL & MOLECULAR BIOLOGY
Big Data in 3 Dimensions
S.A. Zlatic et al., “Rare disease mechanisms identified by genealogical proteomics of copper homeostasis mutant pedigrees,” Cell Systems, 6:368-80.e6, 2018.
E. Brunk et al., “Recon3D enables a three-dimensional view of gene variation in human metabolism,” Nat Biotechnol, 36:272-81, 2018.
new relationships among cancer-causing mutations.
DISPARATE DATA PARSING THE PROTEOME
Discovering the molecular mechanisms underlying disease-causing genetic mutations can be challenging. Biologists Stephanie Zlatic and Victor Faundez of Emory University took a novel approach to this problem: they compared the proteomes of patients with Menkes disease, a rare disorder, to those of healthy relatives. People with the condition have a single-gene mutation that prevents their bodies from regulating levels of copper, which is needed for normal growth and development and is integral to some metabolic pathways. The metal’s depletion results in developmental delays, intellectual disabilities, and, often, death.
© ISTOCK.COM/WARRENRANDALCARRZ; © ISTOCK.COM/SHULZ
FINDING NEW PATHWAYS
This genealogical proteomics approach allows researchers to identify disease pathways that are otherwise hard to tease apart, even if the specific gene causing the disorder is not known, says Zlatic. The research team looked for protein expression traits linked to Menkes, and found 214 proteins whose expression was altered in people with the disease. A SURPRISING FIND
Proteins that were more abundant in the proteomes of people with Menkes included some that are involved in the UCHL1/PARK5 pathway, which plays a role in ubiquitin signaling and has been linked to Parkinson’s. “We didn’t intend to find anything related to Parkinson’s—this was the surprise of the study,” says Faundez. The researchers discovered previously unreported mechanisms by which the UCHL1/PARK5 pathway is connected to defective copper homeostasis. DOUBLE IMPACT
Juan Antonio Navarro, a biochemist at Regensburg University in Germany, says the approach of using genealogical proteomics is interesting because it offers a new technique for applying insights gleaned from a rare disease to one that’s more common. “I think that this is a double impact,” he says. —Jim Daley
To make better sense of the accumulated knowledge about human metabolic pathways gathered by different research groups, researchers led by Elizabeth Brunk, a structural systems biologist at the University of California, San Diego, constructed a database that displays aggregated protein structure, pharmacogenomic associations, and phenotypic data in 3D. FILLING IN THE GAPS
Recon3D’s inclusion of protein structural information from thousands of labs in a massive searchable map is the model’s “biggest step forward from other metabolic reconstructions,” says Brunk. Often, the data may be incomplete or may contain experimental artifacts; the team filled in those gaps using homology modeling, a technique in which researchers construct a model of a protein using its amino acid sequence and hints from a structurally related protein. BIG PICTURE
The 3-D maps of protein structures gave the researchers a new perspective on cancer-causing mutations in the human proteome, revealing that many oncogenic mutations cluster near each other when proteins are folded. “We would have missed that had we not mapped them to the 3-D structure,” says Brunk. Other potential applications of the tool include determining metabolic responses to medications and studying the connections between disease, genes, and drug action. PASSING IT ON
“This is potentially very exciting, because it’s really the integration of . . . big data resources into something that is even larger than the parts,” says John Van Horn, a neuroscientist at the University of Southern California’s Institute of Neuroimaging and Informatics. By demonstrating that deleterious mutations cluster in functional hotspots, he says, Recon3D could potentially allow researchers “to identify things that are cancer-causing.” —Jim Daley 05 . 201 8 | T H E S C IE N T IST 47
Decoding Rarities Uta Francke has spent her career linking genes to uncommon pediatric disorders, advancing the field of molecular diagnostics along the way.
efore moving her lab from the University of California, San Diego (UCSD), to Yale University in 1978, Uta Francke learned how to fly. “I thought, where could you go from New Haven if you are very busy and don’t have much time? It’s hard to do with a car and even the train, so I got a license to fly a small plane and joined a flying club in New Haven,” says the professor emerita of genetics and pediatrics at Stanford University School of Medicine. In her Piper Comanche plane, Francke would zoom off to Martha’s Vineyard or Nantucket for weekend excursions or to institutions in the northeast to give invited seminars on her research. “I would agree to give talks if the institution was within flying distance. And then the researchers would take me out to dinner, and what they mostly wanted to do was talk about my flying the airplane,” she says. But Francke had much to discuss in addition to her experience as a pilot. She trained as a physician in Germany, initially driven by her interest in pediatrics. In the U.S., she entered—and helped define—the new field of medical genetics, becoming an expert in human cytogenetics and pioneering molecular diagnostics techniques.
I said from the beginning that people should have their genetic information if they want it. Her scientific accomplishments include being among the first to map specific genes to their chromosomal locations and, with that information, contribute to a detailed map of the human genome. Francke and her team also found the genes responsible for PraderWilli and Rett syndromes, laying the foundation for investigators to discover genes responsible for other rare genetic disorders. Francke’s research helped set the stage for the Human Genome Project (HGP) that began in the 1990s. What is underappreciated today, according to Francke, are the efforts by researchers like herself—who had generated detailed maps of each human chromosome—that greatly facilitated stitching together the sequences generated by the HGP. But before molecular diagnostics and her discovery of variants responsible for rare diseases, Francke was a sharp student growing up in wartime Germany with parents who initially discouraged her from following her academic talents.
FROM HER FATHER’S DEATH, AN OPPORTUNITY Francke was born in 1942 in a small town just north of Frankfurt, Germany. Her father, who had a law degree, fought for Germany in 48 T H E SC I EN TIST | the-scientist.com
World War II, and her mother was an elementary school teacher. Her parents did not think she and her sister should attend the local school that would put them on a path to a university education, as neither of her parents thought that girls needed much in the way of career options. Instead, the girls went to a local middle school that would direct them to become secretaries or technical assistants. Then, when Francke was 12, her father suffered what was assumed to be a heart attack at age 46. “It was a total shock for us. I realized that things can change suddenly, that nothing is truly stable,” says Francke. After her father’s death, Francke informed her mother that she wanted to switch schools, and her sister followed suit. From then on, Francke’s mother let her choose her own way. She chose a science track in which she was the only girl. “I didn’t feel out of place at all,” she says. After high school, Francke pursued a career in medicine. She had volunteered at a hospital, doing the unglamorous work of changing patients’ bedpans and sheets, and found the tasks gratifying. “I loved taking care of patients. I wanted to go into medicine because it was immediately useful to people,” she says. At the University of Marburg, Francke settled into a cozy student life of coursework and singing in a choir. But after two years, she realized that small-town life wasn’t preparing her well for the “real world,” so she transferred, in 1963, to the University of Munich, where she completed her clinical training. Attaining her medical degree required Francke to write a thesis, and she initially inquired about bacteriophage research at the Max Planck Institute of Biochemistry in Munich, where her first husband, whom she’d met in medical school, was working on his thesis. But a professor at the institute discouraged her from molecular genetics work, Francke recalls, likely because she was a woman. Instead, she was advised to pursue a clinically focused thesis. She followed up with patients who had been diagnosed with an appendix tumor to see whether the cancer—identified incidentally by a pathologist when they’d had their appendices removed—progressed in subsequent years. Francke and her surgeon collaborator found that the tumors were not likely to recur or metastasize.
NEW COUNTRY, NEW PROFESSION Francke completed medical school in 1967, followed by two years of postgraduate rotations in various specialties. In 1969, she applied for a pediatric fellowship at the Children’s Hospital of Los Angeles. She wanted additional pediatric training, and her husband was starting a postdoc at UCLA. The Children’s Hospital took a chance on Francke, she says, as she didn’t know much English and didn’t have a US license to practice. “I knew so little about America. I remember calling a lab and asking someone there to send me test results.
COURTESY OF STANFORD CHILDREN’S HOSPITAL
BY ANNA AZVOLINSKY
UTA FRANCKE Professor of Genetics, Emerita, Stanford University School of Medicine Professor of Pediatrics, Stanford University School of Medicine Past President, American Society of Human Genetics (1999) Past President, International Federation of Human Genetics Societies (2000-2002) March of Dimes/Colonel Harland Sanders Lifetime Achievement Award in Genetics (2001) William Allan Award, American Society of Human Genetics (2012) Association for Molecular Pathology Award for Excellence in Molecular Diagnostics (2014)
Greatest Hits • Demonstrated that quinacrine mustard staining of human and mouse metaphase chromosomes could be used to identify individual chromosomes and abnormal chromosomal translocations • Generated high-resolution banding ideograms for human and mouse chromosomes • Developed a noninvasive hair follicle genetic test to detect heterozygous carriers of variants for Lesch-Nyhan syndrome • Using positional cloning, discovered the gene underlying Wiskott-Aldrich syndrome • Created mouse models for microdeletion syndromes and identified causative genes
After saying, ‘Thank you,’ the person replied, ‘You’re welcome.’ And I thought, ‘I should write down their number, I am welcome there!’ I didn’t know about this expression and many others.” After that residency, Francke was ready for a change and applied for a new medical genetics fellowship at UCLA. In 1970, she was among the first fellows in the program, making the rounds in the hospital to identify patients with potential genetic anomalies that could be studied. Back at the lab, Francke sat at the microscope manually counting the number of chromosomes in patient samples to spot abnormal karyotypes with more or fewer than 46 chromosomes. In 1971, she found out about a new technique, developed in Sweden, in which chromosomes are stained with a fluorescent dye, quinacrine mustard, resulting in distinct chromosomal banding patterns that allowed for the identification of individual chromosomes. Francke got the method to work and started to collaborate with Muriel Nesbitt, a mouse geneticist then also at UCLA. The two published their first paper together in 1971, demonstrating the utility of the technique to identify each mouse chromosome. The same year, the pair also described a translocation involving a chromosomal fragment that moved from an autosome onto the X chromosome in mice. Francke also began staining intact human chromosomes, usually in metaphase. From the samples of 16 patients, she showed that the technique could be used to identify translocations between chromosomes, including the amount of DNA that was moved. The new approach was both quick and accurate, allowing for better characterizations of abnormal chromosomes and therefore moreaccurate diagnoses and genetic counseling for patients.
HAIR FOLLICLES TO SCREEN DISEASE CARRIERS At the end of 1971, Francke says, she had “chromosomes coming out of my ears.” She wanted to go back to pediatrics and sought out William Nyhan, a physician at UCSD who had co-discovered LeschNyhan syndrome, a recessive, X-linked disease that causes compulsive self-mutilation, cognitive deficits, involuntary muscle movement, and early death in males. The syndrome is caused by a defective gene that encodes the hypoxanthine-guanine phosphoribosyltransferase 1 (HPRT1) enzyme necessary for purine metabolism. As a postdoc in Nyhan’s lab, Francke launched into biochemical genetics to find an efficient way to identify women who were carriers of mutations in the HPRT1 gene. Because blood levels of the enzyme are normal in carriers, blood tests couldn’t identify them. Instead, doctors would take a skin biopsy and grow individual fibroblast clones. The system allowed them to measure enzyme activity, but it was a time-consuming process. 05 . 2018 | T H E S C IE N T IST 49
PROFILE Francke had read that hair follicles are essentially clonal, giving her an idea for a new carrier assay. She developed a test in which she would pluck 30 hair roots from a potential carrier, and run the roots’ dissolved proteins through a gel along with a radioactively labeled substrate, hypoxanthine, to measure activity of the HPRT1 enzyme. The test allowed for more-extensive screening of family members and provided data on inheritance and mutation rates for X-linked conditions, about which researchers knew little at the time. At UCSD, Francke also learned how to make somatic cell hybrids, combining cells from two different mammalian species, from her colleague Jerry Schneider, a pediatric rare-disease researcher. After fusing cells of an established mouse or hamster cell line to human fibroblasts, human chromosomes are randomly lost from the hybrid cells, allowing Francke to study the activity of individual chromosomes. Francke would look for abnormal human chromosomes with translocations and examine their gene expression. The analysis allowed her to map genes within a single chromosome. Francke’s expertise in making such hybrids earned her funding from the National Institutes of Health (NIH) to set up her own lab at UCSD. Using the hybridization method, Francke’s lab homed in on a region of chromosome 6, finding that it encodes the major histocompatibility complex—“a bit of a breakthrough for the immunology field,” says Francke.
IMPROVEMENTS TO IDEOGRAMS In 1978, Francke moved to Yale University, setting up her lab in the department of human genetics. She continued to map genes in both mice and humans and refined a painstaking technique that produces high-resolution chromosome diagrams. Her lab developed ideograms in which each chromosome is represented by its size, shape, and banding pattern. A dark band resulting from the method’s staining represents dense, largely nonexpressed and repetitive regions called heterochromatin, and lighter bands are enriched for euchromatin, where genes are located. Analyzing chromosomal spreads for hours a day, Francke became an expert at identifying any slight changes to banding patterns representing subtle abnormalities associated with rare genetic disorders. “When you see human chromosome banding ideograms in papers, those are often my ideograms, but no one knows that because their origin doesn’t have to be referenced,” says Francke. A decade later, in 1994, Francke developed a set of digitized ideograms. During her time at Yale, Francke worked on another X-linked rare disease, Duchenne muscular dystrophy (DMD), which affects mostly boys, causing progressive muscular degeneration and weakness. In 1983, Hans Dieter Ochs, a pediatric immunologist in Seattle, came across a particularly perplexing case of a male patient who was diagnosed with four X-linked disorders, including DMD. Ochs sought out Francke, considered to be among the best cytogeneticists, those who study chromosomes, in the U.S. at the time, for help. Francke’s lab used high-resolution chromosome banding and what were then novel complementary DNA (cDNA) probes on somatic-cell hybrids made with patients’ cells to identify the dele5 0 T H E SC I EN TIST | the-scientist.com
tion on the short arm of the X chromosome responsible for all four conditions. Harvard School of Medicine researcher Louis Kunkel had been trying to identify the gene responsible for DMD since the early 1980s, and Francke’s study provided the material that allowed Kunkel’s lab, in 1986, to clone the gene responsible for the disease.
A WINDOW INTO RARE DISEASES In 1989, Francke moved to the Stanford University School of Medicine and delved deeper into rare-disease studies. In 1992, a pediatric endocrinologist presented Francke with 20 female patients from Ecuador who had a growth hormone deficiency called Laron syndrome. It is an autosomal recessive disorder, but the girls had normal levels of human growth hormone. It turned out that the mutation was in the growth hormone receptor gene but did not change the amino acid sequence. Francke discovered that the mutation creates a new splice site that causes a deletion of part of the protein. In 1994, using positional cloning to systematically identify smaller and smaller DNA segments that likely contain the gene of interest, Jonathan Derry, a postdoc in Francke’s lab, and Ochs identified the gene responsible for Wiskott-Aldrich syndrome, an X-linked immunodeficiency. The gene, called WASP, encodes a molecule important for regulating platelet and lymphocyte functions, and was the 11th human gene to be cloned using positional cloning. In the years before and since, Francke worked on many other rare diseases, including Williams-Beuren syndrome, which, among other symptoms, manifests in a lack of stranger anxiety. Francke also studied the X-linked Rett syndrome, which affects only females. She and colleagues found that the disorder is caused by mutations within the MECP2 gene, which encodes a protein that binds methyl CpGs on DNA. The autism-like disease results, Francke and colleagues determined, when certain mutations in MECP2 affect the ability of its protein to silence certain genes in neurons.
WORKING RETIREMENT Francke collaborated with her second husband, Heinz Furthmayr, a biochemist at Stanford, to find and characterize the effects of gene mutations responsible for Marfan syndrome, a disease that weakens connective tissue. Furthmayr, whom Francke met in Colorado while skiing and married in 1986, shared her passion for flight. After 25 years of flying, the couple decided to sell their plane and committed themselves to exploring the world outside of the cockpit. Furthmayr retired in 2005 and Francke decided to close her lab in 2010. But two years later, Francke was still working part-time at Stanford and part-time at 23andMe, a direct-to-consumer genomics company. In May 2012, when Furthmayr was on trek in Nepal, a trip Francke had decided not to join, he collapsed on the trail and died of a heart attack. Francke now focuses on outpatient clinical genetics services and consulting for 23andMe. “I said from the beginning that people should have their genetic information if they want it, and I joined the company as a consultant to make sure that the information that is put out is accurate and up to date.” g
SCIENTIST TO WATCH
Valerie Arboleda: Data Miner Assistant Professor, David Geffen School of Medicine, UCLA. Age: 35 IBY SHAWNA WILLIAMS
© JOHN LINE CREDIT DAVIS PHOTOGRAPHY
alerie Arboleda traces her love of research back to time spent in a lab as an undergraduate at Columbia University. “I think I was a little bit lucky, in that early on there were a couple of fun experiments that just worked,” she says. While working on a project examining cerebral ischemia, she was able to use qPCR to confirm that microRNAs had been transfected into cultured mouse neuronal cells and successfully knocked down a targeted gene. This motivated her to further investigate the biology behind this experiment, Arboleda recalls. “I kind of got hooked after that.”
At the time, however, Arboleda wasn’t convinced a career in research was practical, and after graduation, she opted to study medicine at the University of California, Los Angeles (UCLA). But her love of the lab lingered, and between her second and third years of med school, she did a full-time research training fellowship in the lab of geneticist Eric Vilain, where she ended up staying on after her year was up, eventually earning an MD/PhD. Vilain had been instrumental in describing a rare genetic disorder called IMAGe (intrauterine growth restriction, metaphyseal dysplasia, adrenal hypoplasia congenita, and genital anomalies) syndrome in the late 1990s, when he was a resident. For more than a decade, “there had been numerous attempts by other groups [to identify the gene responsible] . . . and no one ever found it,” Vilain says. But Arboleda was able to track it down: using next-generation sequencing to compare the genomes of IMAGe patients with each other and with unaffected family members, she pinpointed variations in a specific stretch of a gene called CDKN1C.1 After earning her degrees, Arboleda started a UCLA residency in pathology that included a substantial research component. “She really was very engaged in her rotations, both intellectually and from the scientific curiosity perspective,” recalls Omai Garner, a pathologist with whom Arboleda worked regularly during her residency. Together with Garner, Arboleda spearheaded a study examining the types and quality of test results in the UCLA system that ended up in patients’ electronic medical records, drawing on statistical tools she’d used in earlier genomics work to crunch the numbers.2 “For somebody to take the initiative and to move [this kind of project] forward all the way through publication during their residency is very impressive,” Garner says.
During her transition from medical school to residency, Arboleda also worked with UCLA geneticist Stanley Nelson to investigate the KAT6A gene, which encodes a protein that acetylates the histones that package DNA. In 2015, the pair, along with more than a dozen colleagues, published the first description of KAT6A syndrome, a rare, congenital disease. The condition, which is caused by de novo mutations in KAT6A, results in a wide range of symptoms, including microcephaly, developmental delays, and cardiac defects.3 Since starting her own lab at UCLA’s School of Medicine last year, Arboleda has driven the work on KAT6A syndrome forward, using tools from computational genomics to explore the mutations’ downstream effects on chromatin folding and gene expression. Her team also continues to study the range of phenotypes associated with the mutations, and she finds it rewarding to meet families and patients affected by the condition and to give them an idea of what to expect over time. “I feel really lucky that someone pays me to do the work I do,” Arboleda says. “Every day I’m pretty excited to come to work.” g REFERENCES
1. V. Arboleda et al., “Mutations in the PCNA-binding domain of CDKN1C cause IMAGe syndrome,” Nat Genet, 44:78892, 2012. (Cited 100 times) 2. V. Arboleda and O. Garner, “Reigning in the point-of-care beast: Assessing quality of point-of-care testing within the ambulatory network of a large health system,” Am J Clin Pathol, 147:S157-58, 2017. (Cited 1 time) 3. V. Arboleda et al., “De novo nonsense mutations in KAT6A, a lysine acetyltransferase gene, cause a syndrome including microcephaly and global developmental delay,” Am J Hum Genet, 96:498-506, 2015. (Cited 28 times) 05 . 201 8 | T H E S C IE N T IST 51
Sorting the Brain’s Signals A tour of computer programs that swiftly sift through terabytes of neuron activity data BY ASHLEY YEAGER
52 T H E SC I EN TIST | the-scientist.com
tors, explains applied mathematician Rodrigo Quian Quiroga of the University of Leicester in the U.K. These include the structure of the neuron’s branched extensions, called dendrites, and the neuron’s distance to and relative positioning around the recording electrode. Clustering spikes by shape allows researchers to assign them to their respective neurons, and thereby determine neurons’ firing patterns and locations. Doing this by eye, however, is difficult and time-consuming, which it why Yger, Quiroga, and others are developing software that can automate the entire process, saving researchers weeks or even years of sifting through spike data. These computational efforts face several challenges. For one thing, explains Yger, the spike signals are noisy, making it hard to create an algorithm to characterize them. Additionally, the neurons move, or drift, relative to the electrodes during recording, which can put any
given neuron out of range of the electrode that was recording it, and in the range of a different electrode. Such shifts can change the shape of spikes, making it harder to link the electrical signals to their neurons of origin. But tools that can tease apart a multitude of such signals at once could pave the way not only for new neuroscience insights, but also for vastly improved prosthethics and neural implants. Here are several freely available software solutions for analyzing neuronal activity.
WAVES FOR SPIKES SOFTWARE: Wave_clus URL: https://github.com/csn-le/wave_clus LEAD DEVELOPER: Rodrigo Quian
Quiroga, Director of the Centre for Systems Neuroscience, University of Leicester, United Kingdom
euroscientists have been sliding tiny electrodes onto neurons to record the cells’ electrical activities for more than five decades. The delicate technique has helped researchers discover features such as grid and place cells that animals use to navigate, and so-called concept cells that humans use to recognize specific faces. In early setups, researchers used a single electrode—essentially, a pin attached to a computer chip—to record from single cells in brain tissue grown in a Petri dish or from the brains of freely moving mice or rats. But such recordings don’t capture the crosstalk between neurons that is fundamental to the complex brain processes that underlie cognitive functions, such as learning and memory. Ever more sophisticated probes being developed can simultaneously record electrical activity from hundreds or even thousands of neurons in animals as they go about their normal activities. Electrophysiology in neuroscience is “blooming,” says Pierre Yger, a computational neuroscientist at the French National Institute of Health and Medical Research in Paris. But as researchers seek to put an increasing number of electrodes into ever-smaller regions of the brain, determining which neuron is “saying what, when” becomes a challenge, he says. Large arrays of probes with multiple recording channels also produce massive amounts of data—a 30-minute recording from 4,000 channels generates half a terabyte, for example. Analyzing the resulting electrode signals is called spike sorting because it requires organizing spikes in electric potential, recorded from just outside each neuron, by shape. A neuron’s characteristic spike shape depends on several fac-
A spike-sorting algorithm must perform three basic steps: detect spikes, extract distinct features of spikes, and cluster the spikes by the identified features. In 2004, Quiroga and his colleagues first introduced Wave_clus, a spike-sorting system that relies on two components (Neural Comput 16:1661-87). The first is a mathematical tool called wavelets, which extract information from signals such as the neuronal spikes, and the second is a clustering algorithm that draws on ideas from statistical mechanics to group together spikes with similar shapes. The program first sifts through raw data and identifies major changes in the amplitude of the electrical signal; it labels those as spikes and discards the rest of the data. Then, using wavelet analysis, the spike shapes are characterized and fed into the clustering algorithm, which assigns each spike to a neuron and reveals when each neuron fired. In a final step, researchers must comb through the spike-sorting results and, by eye, search for mistakes—clusters that don’t look quite right, missing spikes, or false positives. Mistakes made by the algorithm and missed by the researcher reduce the reproducibility of results. In the 14 years since its release, Wav_ clus has undergone several updates that have improved its speed and automated some steps, reducing the time a user must spend correcting the algorithm’s mistakes. However, the software is not fully automated, and it can’t yet correct for drift. “There’s still a huge amount of room for improvement,” Quiroga says.
SORTING KILOS OF DATA SOFTWARE: KiloSort URL: https://github.com/cortex-lab/KiloSort
regions simultaneously in rodents (Nature 551:232-36, 2017). Researchers hope that the probe can reveal the networks that connect neuronal firing patterns in distant parts of the rodent brain. But its unprecedented bandwidth—previous probes could record from just dozens of neurons— complicates spike sorting by adding vast amounts of data to the analysis. To address the analytical complexity introduced by the tens of millions of spikes Neuropixels generates, Pachitariu developed and released KiloSort in 2016 (bioRxiv doi:10.1101/061481). The spike-sorting system works by stamping out a pattern, or template, for every spike based on its features, then sifting through the raw data to identify spikes that match each template and stripping those from the noise. The algorithm scours the raw data repeatedly to find spike snippets based on the templates. No data are ever discarded, as they are in other algorithms, Pachitariu says. It’s also fast. An analysis that would take two weeks to conduct with other software programs can be run in 30 minutes with KiloSort. The program runs on graphics processing units and uses the programming language MATLAB installed on the computers doing the analysis, so the spike sorting can be done on-site. On the other hand, it can’t be done in the Cloud, potentially complicating access to the software. But the biggest issue is, again, drift, Pachitariu says. “If we can fix drift, that’s when these software programs will become fully automated.”
SPYING ON SPIKES SOFTWARE: SPYKing CIRCUS
spyking-circus LEAD DEVELOPER: Pierre Yger, compu-
tational neuroscientist, French National Institute of Health and Medical Research One difficulty in designing spike-sorting algorithms that can handle massive amounts of data is having the program recognize that spikes from a single neuron will be recorded on several probes, says Yger. One neuron might register a full, well-developed spike on one probe, and its signal might also be picked up on another probe nearby. That’s especially true over time as the nerve cells drift, and probes that were recording one set of neurons are now recording another set. Another problem is analysis time. With more data, algorithms need to work more quickly to deliver results. But the time it takes for software to sort spike data cannot exponentially increase in the same way new probes exponentially increase the amount of data being collected. To address these problems, SPYKing CIRCUS, released in 2016, conducts its analysis by performing two main steps (bioRxiv, doi: 10.1101/067843). First, it develops a dictionary of spikes from a given neuron, and second, it matches the templates to spikes in the raw data. The first step reviews the pattern of activity that arises from many electrodes when a single neuron fires, and the second scans the data to find all examples of that activity. It works in a similar way to KiloSort. A user does have to review the templates in the dictionary that define each spike, ensuring that
GAINING SIGNAL: The Neuropixel probe is a next-generation electrode that can record the activity of hundreds of neurons in the brain simultaneously.
LEAD DEVELOPER: Marius Pachitariu,
Group Leader, HHMI Janelia Research Campus Last year, Pachitariu and collaborators described a new, ultrathin silicon probe called Neuropixels that records from hundreds of neurons across multiple brain 05 . 2018 | T H E S C IE N T IST 53
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MINING A DATA MOUNTAIN SOFTWARE: MountainSort URL: https://github.com/magland/ mountainlab LEAD DEVELOPER: Jeremy Magland, Senior Data Scientist, Flatiron Institute, New York City
If we can fix drift, that’s when these software programs will become fully automated. —Marius Pachitariu, HHMI Janelia Research Campus
A few years ago, when Magland and his colleagues began developing software to sort spikes, they realized that no such fully automated software existed. In designing MountainSort, the team stuck to the standard spike-sorting steps: identifying spikes, discarding excess data, sorting the remaining data into small clusters, and then evaluating whether clusters next to each other should be combined. The process is streamlined, Magland says, so users don’t have to adjust the 5 4 T H E SC I EN TIST | the-scientist.com
DECODING MESSAGES: These raw voltage data from a 16-channel probe are fed into a spikesorting algorithm to identify how neuronal interactions underly cognition in a rat.
parameters for how spikes are sorted. It also uses a generic statistical clustering method to group shapes of spikes together, keeping user intervention low. Then, the system looks at the time spikes occurred and de-mixes them if they are too close together. A neuron can fire electrical activity about once every few milliseconds, so if spikes from the same neuron appear to be closer together than that, the software might be sorting them incorrectly. The team is working on teasing apart those signals with the software. Once the sorting step is done, the program assesses the quality of its sorting. If any of the clustering has a poor quality ranking, the data for that neuron are discarded. Depending on what data a particular lab wants, researchers can set the algorithm’s evaluation criteria, Magland says (Neuron 95:1381-94, 2017). Similar to the SPYKing CIRCUS team, Magland and his collaborators are working on an update to MountainSort that can correct for drift in the data. The researchers are also building an online interface where users will be able to run several of the spike-sorting algorithms on the same datasets to see how each performs. This system will give researchers a way to validate their algorithms, compare them, even take elements from one algorithm and integrate them into another to create hybrid versions of the software. “Spike sorting is a rich and complex field,” Magland says. g
JEREMY MAGLAND, FLATIRON INSTITUTE
the shapes properly match spikes in the raw data and that there aren’t any errors. But the work is minimal compared to other programs, Yger says, because researchers don’t have to build into the software assumptions about which data to remove. The team is now comparing the software’s performance to that of other spike-sorting programs. SPYKing CIRCUS is not yet fully automated, but the team is working on an update that can handle drift.
The TelePostdoc Family constraints and communications technology are making remote research positions more popular, but there are costs, scientists say. BY ASHLEY YEAGER
© JULIA TIM/SHUTTERSTOCK.COM
n a warm September day, paleoecologist Caitlin McDonough MacKenzie and her colleagues hiked to a lake in Maine’s Acadia National Park, set up a floating research platform, and drilled deep into the sediment in the lakebed, pulling up a meter-long shaft of soil. “It took us . . . maybe three and a half days to actually get our cores out of the lake, and then we hiked everything back down,” she tells The Scientist a few months later. “Right now, that core is sitting in a cold room at the University of Maine waiting for me.” McDonough MacKenzie, who uses the pollen in these cores to reconstruct the biological responses of plants to past climate change, is a postdoc at the institution. But you might not immediately realize that from looking at her schedule. Although she typically spends weeks at a time in the Maine lab of her advisor, paleoecologist Jacquelyn Gill, in between research trips around the state, the rest of McDonough MacKenzie’s time is spent working from her hometown, Boston. After all, while she needs lab access for sample preparation and analysis, the rest of her activities—reviewing data, reading research papers, writing up notes of the results she’s gotten, and preparing for her future fieldwork—she can carry out from anywhere. This arrangement is no accident: McDonough MacKenzie officially holds a remote postdoctoral position. To ensure her “ghostdocing” gig would work, McDonough MacKenzie says, she and Gill had many detailed discussions in person and over the phone about the project before she applied for the position. “We talked about the possibility that I would not actually move to Maine. My family is in Boston, my husband works here, I have a two-year-old daughter,” she says. “I was
finishing my PhD and going on the job market, and my husband and I agreed: if I did a postdoc, we would not move, but if I got a job, we would move.” Eventually, McDonough MacKenzie was awarded a fellowship from the Society of Conservation Biology and started her remote postdoc in the fall of 2017. Her situation is hardly unique: The Scientist has found that a number of prospective postdocs and their advisors are exploring the possibility of remote work, citing the opportunities afforded in terms of work/life balance and other advantages. Although data on the number of postdocs working remotely are scarce, the phe-
Compared to traditional postdoc jobs, remote positions may offer a better work/life balance. nomenon appears to be part of a broader trend across the US workforce. According to Global Workplace Analytics, the number of Americans working at home grew by 115 percent between 2005 and 2015 among non–self-employed individuals, with 3.7 million employees now working from home at least half the time. The setup is not without challenges, from simple communication difficul05 . 2018 | T H E S C IE N T IST 5 5
ties to feelings of isolation, leaving postdocs to come up with bespoke solutions to their unusual situations. “The PI-postdoc relationship is about the same,” Brian O’Meara, an evolutionary biologist at the University of Tennessee, Knoxville, tells The Scientist by email. “But there is a cost in terms of other interactions. My postdoc in Texas is not going to bump into an ecologist in my department while getting coffee and strike up a conversation.”
Family first O’Meara’s remote postdoc is David Bapst, a paleobiologist who builds computational tools to improve the construction of phylogenies for extinct species, including dinosaurs and birds. Bapst lives in College Station, Texas, with his daughter and his wife, who has a tenure-track position at Texas A&M. Like McDonough MacKenzie, he didn’t want move away from his family for a postdoc—a situation O’Meara is sympathetic to. “A postdoctoral fellowship is of finite duration,” O’Meara says. “Not everyone can move for their career for a oneor two-year position.” When O’Meara offered Bapst the position, fully funded, Bapst says he leapt at the chance. Family considerations such as these can substantially influence a scientist’s ability to remain in academia. If a postdoc applicant has a spouse, or is caring for children or parents, it can be hard to move—an issue that tends to affect some sectors of society more than others. For example, research by Mary Ann Mason of the University of California, Berkeley, and others has shown that marriage and the birth of children are major reasons why women leave science between receiving a PhD and earning tenure—an effect that is less pronounced among male researchers. And a recent survey conducted by Nature found that many US institutions offer postdocs no parental leave at all, with parents from ethnic minorities reporting greater discouragement from taking leave than their white counterparts. Employer flexibility regarding the need for postdocs to be onsite has the potential to allow scientists from a wider 5 6 T H E SC I EN TIST | the-scientist.com
range of backgrounds to take up postdoc positions, notes Henry Sauermann, an associate professor of strategy at the European School of Management and Technology Berlin who studies the scientific workforce. “There may be a benefit of ‘flexible’ formats or arrangements in that it allows certain groups of individuals to pursue this path,” Sauermann writes in an email to The Scientist, noting the lack of data on the subject. “So, there could be a benefit in terms of increasing diversity.” O’Meara says he believes that remote postdocs may be just as beneficial for PIs as they are for early-career scientists. “The advantage [of remote postdoc positions] for me is both short-term—being able to pull from a broader pool of potential folks than just those who can move to where my lab is— and long-term: helping to reduce barriers that rob the field of important voices,” he says. Ultimately, any hiring decision should come down to choosing the right person, says Jack Williams, a paleoecologist at the University of Wisconsin–Madison. He is currently working with two remote research scientists who had been on-site in Williams’s Wisconsin lab but couldn’t continue there for family reasons. In both cases, these individuals had “a lot of deep skill sets and knowledge, so it just seemed to make the most sense that, if we could be flexible and give them a way to keep working with us, that was the best way to do it,” he says. Williams notes that for him it is not ideal to have people unable to be present regularly in the lab, “but it works, and if it lets you have someone who’s really good work with you and in the place where they need to be for their own personal life or family reasons, then it seems like a reasonable compromise.”
Expanded opportunities Sometimes, instead of restricting scientists’ contributions, remote postdoc positions can broaden their horizons. Computational microbiologist and immunologist Kevin Bonham, for example, tells The Scientist that his motivation for taking a remote position in the University of California, San Diego (UCSD),
lab of microbiologist Rachel Dutton was to continue to do bench science while pursuing teaching. In 2014, he started a curriculum fellowship at Harvard Medical School, where he designed and taught courses in immunology. He also started working with Dutton, who was then at Harvard but moved to UCSD in 2015. Bonham opted to stay with Dutton in a remote postdoc position, where he could focus on studying horizontal gene transfer in microbial communities. The distance was difficult, but the post helped Bonham realize he wanted to pursue research.
Not everyone can move for their career for a oneor two-year position. —Brian O’Meara University of Tennessee, Knoxville
By working remotely, postdocs may also end up with access to the scientific community at more than the home institution—a relative rarity for scientists working onsite. In addition to teaching at Harvard, for example, Bonham attended seminars there and at other Boston-area institutions to discuss his work with colleagues in his field—a move mirrored by several remote researchers, notes Brian McGill, an ecologist at the University of Maine. Some remote postdocs even find local labs to work with. “Every person who has worked remotely for me has been able to find a college town nearby, and find somebody who’d be happy to let them come in for free,” he says. “They’re a postdoc, so they’re already experienced.” And those networking opportunities introduce whole new groups of people to collaborate and share ideas with. Meanwhile, for some remote postdocs, the limited time spent in a supervisor’s lab brings its own advantages. McDonough MacKenzie, for example, notes that being remote makes her “super-efficient” when she’s at Gill’s lab in Maine. “Because I know I have a limited amount of time, I am way more present and productive than I probably would be looking at a two-year
postdoc versus a two-month visit,” she says. “My thinking was: I’m just here for two months—what needs to get done?”
Dealing with the downsides It’s not all positive, of course. Remote postdocs have to contend with missing out on being part of day-to-day lab life. For a start, Williams explains, you can never connect completely with your labmates, despite chatting on Skype, Slack, Zoom, and other communication platforms. “As a supervisor and mentor, there is that value of being able to go out for a cup of coffee and check in and see how things are going,” he says. “So much of communication is body language.” When someone comes into the office and has had a hard day, he can see it, he says. “Some of that stuff just doesn’t communicate well over the phone or over Skype.” This lack of personal interaction often makes the relationship between advisor and postdoc particularly important. As a postdoc, remote or not, “you’re sort of in this liminal space where you’re not really a student and not really faculty or staff, so often the university doesn’t know how to handle you—what email lists to put you on, or how to integrate you or connect you with others,” says Gill. “That can be great for sort of getting your head down and banging out a bunch of papers, but it can also be lonely, both personally, socially, and also intel-
lectually.” McDonough MacKenzie and Gill have worked out their own solutions to get around the added distance involved in remote postdoc positions; for example, they frequently text back and forth about research. It’s McDonough MacKenzie’s way of tapping her PI on the shoulder, as if she were right there in the lab, McDonough MacKenzie says. Remoteness also highlights the importance of having a support network outside the lab. At institutions where remote postdoc positions have become commonplace, researchers may find communities of people with similar situations, and feelings of isolation can be somewhat assuaged. Through her fellowship program, for example, McDonough MacKenzie notes that “you have a cohort, and even though you’re not too geographically close together, you spend three weeks of the year on these one-week professional development retreats, coming together.” The same is true of engaging in the local community, she adds. “I’ve been going to a bunch of job talks at Harvard, just because I want to see more of them. . . . Just being a part of an academic community feels really nice.” Nevertheless, there are some downsides that are difficult to get around, whatever the effort of the researchers involved. With remote positions, Sauermann notes, it’s difficult to reproduce the managerial, relationship-focused aspects of postdoc work—often seen as important
HOW TO WORK WELL FROM FAR AWAY Self-reflect first: Remote work is not for everyone, says the University of Maine’s Jacquelyn Gill. “You should really check in with yourself about whether or not you’re self-motivated, whether you have the local resources and support, whether you’ll feel isolated, or you have a strong community.” Find a local base: Brian McGill, also of the University of Maine, suggests trying to come to an arrangement with a local institution, to gain access to a university library, seminars, and other people with whom to talk about ideas face-to-face. Finding researchers with similar interests nearby can prevent a sense of isolation, he says.
preparation for a future faculty career. “Spending too much time outside the lab may mean limited opportunities to develop those skills,” he says. Bapst came up against this problem as he was applying for a second remote postdoc position. Many advisors turned him down, explaining they wanted a person on-site who could add to the culture of the lab or advise graduate students, he says. Gill hopes that such attitudes will soften. From a PI perspective, “having the right person who’s longdistance can be better for you, your lab, and your project than having a weaker fit that’s close by,” she explains. “Much as I love having a strong lab culture with people who are close and able to interact informally a lot, I’d say, at the end of the day, it’s also the projects that need to get moved forward. I would encourage people to think about it, even if they’re sort of feeling like, ‘Oh, I would never do that.’” Even so, the remote postdoc is unlikely to be a good solution for everyone. Individuals thinking of doing remote postdocs have to know themselves extremely well, and they have to be honest with themselves, Gill says. “If your mentor is super hands-off and isn’t checking in with you and you tend to need a lot of encouragement or structure and you know you don’t work well at home, then this is going to be a bad fit for you.” g
Stay connected: Reach out to your PI regularly and often, says McGill. “It’s easy to get busy and say, ‘Oh, we don’t really have anything to talk about this week.’ But then you miss the casual interactions, finding out what’s up in the person’s life, or randomly bouncing science ideas off of each other,” he says. Make time for face time: Visiting your lab every few months or so is essential, McGill says—not just for lab work, but to get to know your colleagues and to feel connected. “That’s most important at the beginning, to get the relationship established.” he says. University of Maine paleoecologist Caitlin McDonough MacKenzie agrees—as a remote postdoc, she packs her days when she’s in the lab. She’ll attend lab meetings and seminars and “dive into the lab lifestyle.”
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Rare to the Rescue Rarity becomes a strength when lessons from rare disease patients are applied to more common diseases. BY VICTORIA JACKSON AND MICHAEL YEAMAN
n 2008, a young woman named Ali Guthy experienced eye pain out of the blue. Already extraordinary in her academic, athletic, and leadership achievements, at that moment she unknowingly became an example of the power of being rare. Neuromyelitis optica (NMO) is an autoimmune disease that can cause inflammation and demyelination of the optic nerves and spinal cord. The devastating effects of NMO can include blindness, paralysis, or worse. A decade ago, fewer than 1 person out of 100,000 was thought to be afflicted with NMO worldwide. Like most autoimmune diseases, NMO predominantly afflicts females, and often strikes in the prime of life. Although NMO is rare, autoimmune diseases collectively occur with the same incidence as heart disease, and twice that of cancer. Yet, little is known about the risks or causes of autoimmune diseases. And while therapies may tame some of them, treatments can increase the risks of infection and cancer. Few, if any, cures exist. Even amid the complexities of autoimmune diseases such as NMO, exceptions prove the rule. And for Ali’s mother, cosmetics pioneer Victoria Jackson, rare in her own accomplishments, no puzzle was too hard to keep her from trying to save her 14-yearold daughter, Ali, or any patient facing the fear and uncertainty of NMO. In 2008, she launched the Guthy-Jackson Charitable Foundation, an organization that views each rare-disease patient as an essential piece in solving the cure puzzle. In just one decade, the immunology, diagnosis, and treatment of NMO has been transformed. The foundation united an International Clinical Consortium of more than 90 members representing 30 countries. This group has published dozens of state-of-the-art papers that rethink
scientific dogma and hundreds of research articles in leading journals that reveal a new understanding of NMO and related autoimmune diseases. It is now evident that autoreactive T cells drive the cascade of events leading to B cells making autoantibodies targeting the water channel protein aquaporin-4. The book The Power of Rare: A Blueprint for a Medical Revolution tells the foundation’s story and how it has positioned research on NMO as a model to solve other diseases. The foundation also built an NMO biobank called CIRCLES (Collaborative International Research in Clinical and Longitudinal Experience for Neuromyelitis Optica Studies) to harness the collective power of NMO patients and their relatives. Presently, CIRCLES has enrolled more than 1,000 patients, and the research it has enabled is opening the locks to solving NMO. With its panel of experts, the foundation also rewrote the book on NMO diagnosis. Once believed to be a rare form of multiple sclerosis (MS), it is now clear that NMO is not MS—a fact that has helped both NMO and MS patients receive more-rapid and accurate diagnoses and care. Currently, 10 out of every 100,000 people are diagnosed with NMO in some regions of the world. While there were no clinical trials focusing on NMO for more than a century after it was first identified, there are presently three Phase 3 clinical trials nearing the finish line—and more coming. The Guthy-Jackson Charitable Foundation is continues to work on new initiatives, such as identifying biomarkers that predict relapse, to prevent the life-threatening immune attacks that are hallmarks of NMO. And beyond therapies, the foundation has launched its mission for tolerization to find cures. Tolerization in auto-
Regan Arts, September 2017
immune disease aims to retrain the body’s immune cells to stop attacking it’s own tissues. In this way, rebooting the immune system to end the underlying causes of NMO may lead to a cure, sparing patients from the risks of lifelong immunosuppressive therapy. Achieving this goal will open doors to solve rare and common autoimmune diseases alike. From rheumatoid arthritis to type 1 diabetes to MS—even to stealthy cancer cells and infections that defeat immune responses and resist treatment—there has never been a more urgent need for immune health. The revolution of personalized and precision medicine is upon us—if only we take purposeful action together. We are all rare. The strength of one—raised to the benefit of all—that is The Power of Rare. g Victoria Jackson is the founder of Victoria Jackson Cosmetics and head of the GuthyJackson Charitable Foundation. Michael Yeaman is Professor of Medicine at UCLA David Geffen School of Medicine, and serves as the Chair of Advisors to the Guthy-Jackson Charitable Foundation. Read an excerpt of The Power of Rare at the-scientist.com. 05 . 2018 | T H E S C IE N T IST 59
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Cold Spring Harbor 2018 Summer & Fall Meetings
photo: course picnic at CSHL beach
Single Biomolecules August 28 - September 1
abstracts due June 8
Organizers Jennifer Lippincott-Schwarz, HHMI Janelia Campus Robert Singer, Albert Einstein College of Medicine Robert Tjian, University of California, Berkeley
Topics • Super Resolution Imaging Modalities • Live Cell Single Molecule Imaging: Biomolecular Dynamics & Forces • Tracking Single Molecules in Living Organisms • Biomolecular Self Assembly and Weak Interactions • In Vitro Single Molecule Imaging: Dynamics and Assembly • Developing Probes for Biomolecular Imaging • High Resolution Imaging of Organelle Dynamics • Biomolecular Processing and Turnover
Other Meetings this Summer & Fall The Biology of Genomes May 8 - 12 Regulatory & Non-Coding RNAs May 15 - 19 Retroviruses May 21 - 26 Brains & Behavior: Order & Disorder in the Nervous System May 30 - June 4 Glia in Health & Disease July 19 - 23 Mechanisms & Models of Cancer August 14 - 18 The CRISPR/Cas Revolution August 22 - 25 Translational Control September 4 - 8
Invited Speakers Patricia Bassereau, Institut Curie, France Anne Bertolotti, MRC LMB, UK Carlos Bustamante, University of California, Berkeley Steven Chu, Stanford University Ibrahim Cisse, Massachusetts Institute of Technology Maxime Dahan, Institut Curie, France Xavier Darzacq, University of California, Berkeley Abby Dernburg, University of California, Berkeley Johan Elf, Uppsala University, Sweden Judith Frydman, Stanford University Hernan Garcia, University of California, Berkelely Jeff Gelles, Brandeis University Thomas Gregor, Princeton University Taekjip Ha, Johns Hopkins University School of Medicine Erika Holzbaur-Howland, University of Pennsylvania Daniel Larson, NCI / NIH Wesley Legant, HHMI/Janelia Research Campus Michael Levine, Princeton University Zhe Liu, HHMI/Janelia Research Campus Markus Sauer, University of Wuerzburg, Germany Jens Schmidt, Michigan State University Laura Waller, University of California, Berkeley Bin Wu, Johns Hopkins University School of Medicine X. Sunney Xie, Harvard University Ahmet Yildiz, University of California, Berkeley Xiaowei Zhuang, HHMI/Harvard University Epigenetics & Chromatin September 11 - 15 Molecular Mechanisms of Neuronal Connectivity September 25 - 29 Mechanisms of Aging October 1 - 5 Germ Cells October 9 - 13 The Evolving Concept of Mitochondria October 18 - 21 Nutrient Signaling October 25 - 28 Transposable Elements November 1 - 4 Probabilistic Modeling in Genomics November 4 - 7 Biological Data Science November 7 - 10 Neurodegenerative Diseases: Biology & Therapeutics November 28 - December 1
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Organized in collaboration with Volkswagen Foundation The completion of the Human Genome Project in 2003 has catalyzed innovations in scientific research and health care embodied in the term “precision medicine.” Across the globe, many nations are investing in large-scale national sequencing cohort programs resulting in over one million human genomes sequenced and linked to dense phenotypic and clinical data. This Keystone Symposia conference will bring together scientists and leaders from healthcare, government and industry to: • Look at what individual countries (China, Estonia, Iceland, Israel, Thailand, UK, USA) have done
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(as of April 1, 2018): Peter Campbell Mark Caulfield Zhengming Chen J. Michael Gaziano Geoffrey S. Ginsburg Henk-Jan Guchelaar Carolina Haefliger Sue Hill* Jianjun Liu James R. Lupski Daniel MacArthur Surakameth Mahasirimongkol Teri A. Manolio Kristjan Metsalu Andres Metspalu Maja Mockenhaupt Kathryn North Martin Reese Heidi Rehm Mary V. Relling Gad Rennert Peter N. Robinson Jeong-Sun Seo Alan R. Shuldiner Kári Stefánsson Patrick Boon Ooi Tan Robyn L. Ward James S. Ware John E.L. Wong Sarah Wordsworth *Keynote speaker
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Among the Amish, c. 1960s BY DIANA KWON
6 8 T H E SC I EN TIST | the-scientist.com
SNAPSHOTS: In addition to
being a prolific scientist, Victor McKusick was an avid photographer. “He documented everything with his camera,” Francomano says. “His collection of photographs is unparalleled.” This was a bit of an issue among the Amish, she adds, because taking pictures ran counter to their community’s beliefs. However, they often agreed to have snapshots taken for medical purposes. Here McKusick photographs the hands of an Amish child with Ellis–van Creveld syndrome.
the reasons was that McKusick cared deeply for the families he studied, Francomano says. “If there were medical issues, families would come down to Hopkins and be cared for, so there was a lot of goodwill built up as a result.” The work by McKusick and his colleagues in Amish communities led to a greater understanding of the characteristics and the risk factors associated with many hereditary disorders and contributed to another of McKusick’s key accomplishments—the book Mendelian Inheritance in Man (MIM), a comprehensive catalog of human genes and hereditary disorders. Today, that collection lives on as an online database, OMIM, and contains information on more than 15,000 genes. MIM “is one of the truly great guides to our field,” says Jim Evans, a medical geneticist at the University of North Carolina. McKusick, he adds, “was a visionary—there’s no getting around that.” g
THE ALAN MASON CHESNEY MEDICAL ARCHIVES OF THE JOHNS HOPKINS MEDICAL INSTITUTIONS
n the early 1960s, Victor McKusick, an American clinician and scientist, was in the early stages of his pioneering work in the field of medical genetics. He had recently left cardiology, and, after amassing over more than a decade’s worth of data on inherited connective tissue disorders, had established Johns Hopkins University’s first medical genetics program and clinic in 1957. Around this time, a forthcoming book and an article drew McKusick’s interest to Amish communities. The book, Amish Society by sociologist John Hostetler, which McKusick reviewed for Johns Hopkins University Press before publication, made him realize that the Amish—who reside in groups founded by a small number of couples, stay isolated from the rest of society, and carefully document their genealogy—would be a perfect community in which to examine recessive phenotypes. Recessive disorders are more common in groups of genetically similar individuals because parents with shared ancestry tend to bear children with large homozygous regions in their genomes. The other publication that captured McKusick’s attention was a magazine article by a physician, David Krusen, that noted high rates of achondroplasia, a common form of dwarfism, among Pennsylvania’s Amish communities. This observation “didn’t make sense to him,” says Clair Francomano, a medical geneticist at the Greater Baltimore Medical Center who worked with McKusick for more than three decades. Achondroplasia is an autosomal dominant disorder and therefore unlikely to be more prevalent in the Amish than in the general public. His curiosity piqued, McKusick took a group of trainees and colleagues to investigate the Amish community in Lancaster County, Pennsylvania, less than two hours’ drive from Baltimore. In only a year of examining families with inherited anomalies, the team had found two different types of rare dwarfism, both recessive disorders: a newly identified form called cartilage-hair hypoplasia, which causes individuals to have fine, sparse hair, and Ellis–van Creveld syndrome, a condition also known as “six-fingered dwarfism,” which had been previously described. After making these discoveries, McKusick and his colleagues spent decades teasing apart the genetics of those and many other inherited disorders they observed among the Amish. The researchers dug through genealogical and hospital records, visited families to examine phenotypes and document symptoms, and collected samples from patients. “We spent a lot of very long days going from household to household,” recalls Francomano, who was heavily involved in these studies. Still, she says, “it was such an exciting time, and one of the most stimulating and rewarding periods of my professional career.” Despite the community’s insularity, most of the Lancaster County Amish came to welcome the researchers, and many developed close personal relationships with them over time. One of
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