Wednesday, April 6, 2016

One of a kind. What do you do if your child has a condition that is new to science?

Matt Might and Cristina Casanova met in the spring of 2002, as twenty-year-old undergraduates at the Georgia Institute of Technology. Cristina was an industrial-design major with an interest in philosophy; Matt was a shy computer geek obsessed with “Star Trek.” At first, Cristina took no notice of him, but the two soon became friends, and that fall they began dating. Within a year, they were married.

The couple had their first child, a son, on December 9, 2007, not long after Matt completed his Ph.D. in computer science and Cristina earned her M.B.A. They named him Bertrand, in honor of the British philosopher and mathematician Bertrand Russell. After a few blissful weeks, the new parents began to worry. Matt and Cristina described Bertrand to friends as being “jiggly”; his body appeared always to be in motion, as if he were lying on a bed of Jell-O. He also seemed to be in near-constant distress, and Matt’s efforts to comfort him “just enraged him,” Matt says. “I felt like a failure as a father.” When the Mights raised their concerns with Bertrand’s doctor, they were assured that his development was within normal variations. Not until Bertrand’s six-month checkup did his pediatrician agree that there was cause for concern...

In April of 2009, the Mights flew to Duke University, in Durham, North Carolina, to meet with a range of specialists, including a geneticist named Vandana Shashi, whose clinical practice focusses on children with birth defects, intellectual disabilities, and developmental delays. After five days of tests and consultations, the Duke team told the Mights that there was widespread damage to Bertrand’s nervous system and that some of his odd behavior—wringing his hands, grinding his teeth, staring into space—was likely due to the fact that his brain appeared to be suffering from spikes of seizure-like activity.

When Bertrand was a newborn, Matt joked to friends that he would be so relaxed as a parent that he wouldn’t care which technical field his son chose to pursue for his Ph.D. In May of 2009, the Mights closed Bertrand’s college savings accounts so that they could use the money for medical care. That fall, Bertrand was rushed to the emergency room after suffering a series of life-threatening seizures. When the technicians tried to start an I.V., they found Bertrand’s veins so scarred from months of blood draws that they were unable to insert a needle. Later that evening, when Cristina was alone with Matt, she broke down in tears. “What have we done to our child?” she said. “How many things can we put him through?” As one obscure genetic condition after another was ruled out, the Mights began to wonder whether they would ever learn the cause of their son’s agony. What if Bertrand was suffering from a disorder that was not just extremely rare but entirely unknown to science?... 

In early 2010, the couple had decided to try to have a second child. This was a gamble: if Bertrand’s condition was indeed new to science, there was a chance that it was caused by a spontaneous, or de novo, mutation in the egg or sperm cell, and was not in Matt’s or Cristina’s DNA. On the other hand, if the condition had a genetic history, the Mights could pass it on to other children. That summer, Cristina learned that she was pregnant, and on April 14, 2011, she gave birth to a girl, Victoria. Within minutes of the delivery, Matt and Cristina knew that their daughter was healthy; she moved with a fluidity that Bertrand never had. When I arrived at the Mights’ house, Victoria was bouncing around and grabbing at her mother’s sleeve. “Victoria, you need to wait for Mommy to say hello,” Cristina said. To me, she added, “I had no idea how easy we had it with Bertrand.”...

At first, they said, he seemed to represent a challenging problem for each new specialist to solve. But, as one conjecture after another was proved wrong, the specialists lost interest; many then insisted that the cause of Bertrand’s illness lay in someone else’s area of expertise. “There was a lot of finger-pointing,” Cristina said. “It was really frustrating for us—our child hot-potatoed back and forth, nothing getting done, nothing being found out, nobody even telling us what the next step should be.”

Then, in the summer of 2010, Vandana Shashi, the Duke geneticist, contacted the Mights about a new research project that was exploring whether genetic sequencing could be used to diagnose unknown conditions... 

Then there was Bertrand. The Duke team thought it was likely that mutations on one of his candidate genes, known as NGLY1, were responsible for his problems. Normally, NGLY1 produces an enzyme that plays a crucial role in recycling cellular waste, by removing sugar molecules from damaged proteins, effectively decommissioning them. Diseases that affect the way proteins and sugar molecules interact, known as congenital disorders of glycosylation, or CDGs, are extremely rare—there are fewer than five hundred cases in the United States. Since the NGLY1 gene operates in cells throughout the body, its malfunction could conceivably cause problems in a wide range of biological systems… 

That November, Goldstein shipped Freeze a supply of Bertrand’s cells. Freeze was unable to find evidence of a functioning NGLY1 gene. He soon reported back: Goldstein’s hypothesis—that Bertrand suffered from a new glycosylation disorder caused by NGLY1 mutations—was almost certainly correct.

On May 3, 2012, nearly two years after the sequencing study began, the Mights met with the Duke team in an examination room of a children’s hospital in Durham. Shashi explained that Bertrand’s condition was probably not caused by a de-novo mutation, as the Mights had thought; rather, Matt and Cristina each had a different NGLY1 mutation, and Bertrand had inherited both. Matt and Cristina had only to look at their daughter playing on the floor to realize how lucky they’d been: Victoria had had a twenty-five-per-cent chance of being born with the same disorder as Bertrand. (Later testing showed that she had not inherited either parent’s NGLY1 mutation.)

Goldstein, who was meeting the Mights for the first time, spoke next. He explained that, until other patients with the same condition were found, there was a chance, however remote, that Bertrand’s disorder was caused by something else. Moreover, without additional cases, there was virtually no possibility of getting a pharmaceutical company to investigate the disorder, no chance of drug trials, no way even to persuade the F.D.A. to allow Bertrand to try off-label drugs that might be beneficial. The Duke researchers estimated that there might be between ten and fifty other patients in the country with Bertrand’s condition, which would make it one of the rarest diseases in the world. “That’s basically what they left us with—‘You need more patients,’ ” Matt told me. “And I said, ‘All right, we’ll get more.’ ”...

But a number of factors prevent sequencing from reaching its full diagnostic potential. As a matter of protocol, researchers typically avoid sharing test results with subjects until the research is published; the Mights didn’t learn that NGLY1 was the likely cause of Bertrand’s condition until months after the Duke team reached that conclusion. 

“If you want to be charitable, you can say there’s just a lack of awareness” about what kind of sharing is permissible, Kohane said. “If you want to be uncharitable, you can say that researchers use that concern about privacy as a shield by which they can actually hide their more selfish motivations.”...

The Mights couldn’t wait for the culture of scientific research to change: they had been told that Bertrand could have as little as a few months left to live. The same day that they learned about NGLY1, they began plotting ways to find more patients on their own. Several years earlier, Matt had written a blog post, called “The Illustrated Guide to a Ph.D.,” that became a worldwide phenomenon; it was eventually translated into dozens of languages, including Serbian, Urdu, and Vietnamese. The popularity of the post, combined with Matt’s rising profile among computer programmers, meant that almost anything he put online was quickly re-posted to Hacker News, the main social news site for computer scientists and entrepreneurs. He decided to use his online presence to create what he referred to as a “Google dragnet” for new patients.

For the next three weeks, Matt worked on an essay that described Bertrand’s medical history in clinical detail. Matt called the result, which was more than five thousand words long, “Hunting Down My Son’s Killer,” and on May 29, 2012, he posted it to his personal Web site. It began: “I found my son’s killer. It took three years. But we did it. I should clarify one point: my son is very much alive. Yet, my wife Cristina and I have been found responsible for his death.”

Half an hour after Matt hit “publish,” Twitter began to light up. By the end of the day, “Hunting Down My Son’s Killer” was the top story on Reddit. The next morning, an editor from Gizmodo, a tech blog owned by Gawker Media, asked Matt for permission to republish the essay. In less than twenty-four hours, the post had gone viral. The more it was shared and linked to, the higher it rose in search engines’ rankings, and the easier it would be for parents of other children to find.

Eight days later, the co-founder of a commercial genetic-testing company in San Francisco e-mailed the piece to a friend, Matt Wilsey. The Wilseys are one of the most prominent families in San Francisco, famous both for their philanthropic generosity and for the complicated marital life of Alfred Wilsey, Matt’s grandfather, who died in 2002. Matt Wilsey, who is thirty-six, graduated from Stanford in 2000. After working on George W. Bush’s election campaign and spending five months as an aide in the Pentagon, he returned to Northern California to work as a tech entrepreneur. In the fall of 2007, he married a former classmate at Stanford. Two years later, Matt and Kristen Wilsey had their first child, a girl they named Grace.

Last fall, I met Matt Wilsey at the annual conference of the Society of Glycobiology, in St. Petersburg, Florida. He has a wide smile and black hair that is flecked with gray. Over lunch at an outdoor café, he told me that Grace’s problems began before she was born: she was delivered by emergency Cesarean section after her heartbeat dipped dangerously low. Almost immediately after Grace’s birth, he and Kristen began to worry. “She just seemed out of it,” Matt said. Within days, Grace was admitted to the neonatal I.C.U. Her doctors collected a number of samples, including cerebrospinal fluid from a lumbar puncture. Three weeks later, when she was discharged from intensive care, the Wilseys still did not know what was causing their daughter’s problems.

During the next two years, Matt Wilsey used his networking skills to set up meetings with specialists at institutions around the country, including Baylor College of Medicine, in Houston; the Broad Institute of M.I.T. and Harvard; Johns Hopkins; Columbia; and the University of California, San Francisco. “We’d talk to one great doctor and say, ‘Who’s the best liver person in the country?’ ” he told me. “And then that would lead us to one person and then that person would lead us to two more. That’s just kind of how we did it.”

When the Wilseys first read Matt Might’s blog post, it didn’t occur to them that Grace and Bertrand might be suffering from the same disease. “Their phenotypes were too different,” Matt Wilsey said. Grace could crawl and pull herself up to a standing position, while, at age four, Bertrand wasn’t even able to roll over. She also had a vocabulary of more than two dozen words, and was able to follow one-step directions, while Bertrand could only make indistinguishable grunts. The most striking difference, Kristen said, had to do with Bertrand’s seizures. “At the time, we didn’t think Grace was having seizures,” she said. “And so we thought, Oh, no, no—she’s completely different from Bertrand. So we just ruled it out.” (Later testing showed some abnormal activity in Grace’s brain.)

By the spring of 2012, Grace’s genome had already been sequenced twice: once at Baylor and once at Stanford. As it happened, Stanford geneticists had identified NGLY1 as a candidate gene, but they set it aside because Enns believed that Grace was suffering from an unidentified mitochondrial disorder. By the time Grace turned three, that October, the Wilseys had consulted more than a hundred researchers around the world, yet they were still without a diagnosis. Around this time, Kristen said, “I told Matt, ‘I don’t want to do this anymore. I’m just exhausted.’ ”

Matt asked Kristen if they could make one final trip to Baylor, and in February of 2013 the Wilseys took Grace back to Houston. They were introduced there to a young geneticist named Matthew Bainbridge. When he looked through Grace’s genome, he ignored mitochondrial genes entirely—“I figured Stanford had that covered,” he told me—and soon narrowed his search to three genes: one known to cause intellectual disability, one associated with a movement disorder, and NGLY1. “NGLY1 stuck out, because I’d never seen it before,” Bainbridge said. When he searched a Baylor database of more than seven thousand people, he found that a handful of them had a single NGLY1 mutation, but none had two.

Bainbridge next looked online for information about the gene. He quickly found “Hunting Down My Son’s Killer.” After reading about one of Bertrand’s more unusual symptoms, Bainbridge e-mailed the Wilseys a question: Did Grace produce tears? Kristen replied almost immediately: Grace could produce tears but not very often. Then, four and a half hours later, Kristen wrote back, “After thinking about it this afternoon, it is actually very rare that Grace will make a tear. I have only seen it a handful of times in her three years.” As soon as Bainbridge read that, he told me, he thought, “Oh, we fucking got it.”
Inspired by a conference I am attending.


  1. Need AC, Shashi V, Hitomi Y, Schoch K, Shianna KV, McDonald MT, Meisler MH, Goldstein DB. Clinical application of exome sequencing in undiagnosed genetic conditions. J Med Genet. 2012 Jun;49(6):353-61.

    There is considerable interest in the use of next-generation sequencing to help diagnose unidentified genetic conditions, but it is difficult to predict the success rate in a clinical setting that includes patients with a broad range of phenotypic presentations.
    The authors present a pilot programme of whole-exome sequencing on 12 patients with unexplained and apparent genetic conditions, along with their unaffected parents. Unlike many previous studies, the authors did not seek patients with similar phenotypes, but rather enrolled any undiagnosed proband with an apparent genetic condition when predetermined criteria were met.
    This undertaking resulted in a likely genetic diagnosis in 6 of the 12 probands, including the identification of apparently causal mutations in four genes known to cause Mendelian disease (TCF4, EFTUD2, SCN2A and SMAD4) and one gene related to known Mendelian disease genes (NGLY1). Of particular interest is that at the time of this study, EFTUD2 was not yet known as a Mendelian disease gene but was nominated as a likely cause based on the observation of de novo mutations in two unrelated probands. In a seventh case with multiple disparate clinical features, the authors were able to identify homozygous mutations in EFEMP1 as a likely cause for macular degeneration (though likely not for other features).
    This study provides evidence that next-generation sequencing can have high success rates in a clinical setting, but also highlights key challenges. It further suggests that the presentation of known Mendelian conditions may be considerably broader than currently recognised.

  2. Enns GM, Shashi V, Bainbridge M, Gambello MJ, Zahir FR, Bast T, Crimian R,
    Schoch K, Platt J, Cox R, Bernstein JA, Scavina M, Walter RS, Bibb A, Jones M,
    Hegde M, Graham BH, Need AC, Oviedo A, Schaaf CP, Boyle S, Butte AJ, Chen R, Chen
    R, Clark MJ, Haraksingh R; FORGE Canada Consortium, Cowan TM, He P, Langlois S, Zoghbi HY, Snyder M, Gibbs RA, Freeze HH, Goldstein DB. Mutations in NGLY1 cause an inherited disorder of the endoplasmic reticulum-associated degradation pathway. Genet Med. 2014 Oct;16(10):751-8.

    Erratum in
    Genet Med. 2014 Jul;16(7):568. Chen, Rui [added].
    The endoplasmic reticulum-associated degradation pathway is responsible for the translocation of misfolded proteins across the endoplasmic reticulum membrane into the cytosol for subsequent degradation by the proteasome. To define the phenotype associated with a novel inherited disorder of cytosolic endoplasmic reticulum-associated degradation pathway dysfunction, we studied a series of eight patients with deficiency of N-glycanase 1.
    Whole-genome, whole-exome, or standard Sanger sequencing techniques were employed. Retrospective chart reviews were performed in order to obtain clinical data.
    All patients had global developmental delay, a movement disorder, and hypotonia. Other common findings included hypolacrima or alacrima (7/8), elevated liver transaminases (6/7), microcephaly (6/8), diminished reflexes (6/8), hepatocyte cytoplasmic storage material or vacuolization (5/6), and seizures (4/8). The nonsense mutation c.1201A>T (p.R401X) was the most common deleterious allele.
    NGLY1 deficiency is a novel autosomal recessive disorder of the endoplasmic reticulum-associated degradation pathway associated with neurological dysfunction, abnormal tear production, and liver disease. The majority of patients detected to date carry a specific nonsense mutation that appears to be associated with severe disease. The phenotypic spectrum is likely to enlarge as cases with a broader range of mutations are detected.

  3. Mutations in NGLY1 gene linked with new genetic disorder: parents' reports of children's symptoms help facilitate the discovery. Am J Med Genet A. 2014 Jul;164A(7):viii-ix.

  4. Hunting down my son's killer

  5. Nearly four years ago, Hugh Rienhoff watched as his baby girl was pulled from a small incision in his wife's belly. It was their third child — the two boys had also been delivered by caesarean — and Rienhoff was there for all three births. But this child seemed different. He remembers her looking a little dark and sort of floppy, possibly attributable to the stress of delivery. Then he caught a glimpse of her feet, which were just a little longer than normal. For an instant, his training as a clinical geneticist kicked in. Could she have Marfan's syndrome?...

    "I didn't really think about anything from that point on medically, at least for that day," says Rienhoff. "I did all the usual things you do when you have a baby, which is cry and call my family." When the paediatrician handed his new daughter to Rienhoff, she offered some technical terms — nevus flammeus for a port-wine-stain birthmark down the middle of her face and arthrogryposis for the reluctance of her tiny fingers to extend all the way. Rienhoff had to write them down to remember them.

    Although in the weeks and months after his daughter's birth the port-wine stain receded, it soon became clear to Rienhoff that she wasn't developing normally. Her fingers and toes wouldn't uncurl. More worryingly, in spite of ample feeding, the girl just wasn't gaining weight...

    But for Rienhoff that wouldn't do. Although he had largely left his practice, he had trained as a physician under Victor McKusick, the father of clinical genetics. Rienhoff knew genes, and he wanted to know his daughter's. For almost four years he has been trying to understand what makes her different at a molecular level, hoping that such knowledge could inform her care and treatment. He's quizzed experts, gone to meetings, and even set up gene-amplification equipment at home so that he can test his hypotheses with sequence data. He has also begun sharing the information he's found, telling his story on the Internet in the hope of helping others and of learning more. He may even have found a treatment that improves his daughter's condition...(continued)

  6. (continued)Rienhoff's need for clarity was not purely intellectual. About five months after their daughter's birth, Rienhoff and Hane became very concerned about her failure to thrive. Although growing taller, she wasn't putting on weight. "She was just melting away," says Rienhoff. The gastrointestinal specialists they went to see advised them to stuff her with calories, but it didn't do any good. The doctors drew up a list of things that might be causing her problems — disorders of the metabolism, of the way nutrients were absorbed from gut and stomach, of the way that mitochondria in her cells produced energy. One possibility that arose was an unusual form of cystic fibrosis, but her symptoms looked quite unlike this condition.

    Rienhoff thought that a mitochondrial disorder was a particularly plausible cause. The typical symptom is muscle weakness, which his daughter clearly had, but making a precise diagnosis is very tricky. Rienhoff dove into the literature and talked with the experts, quickly finding himself in what he calls a very messy field. "That really ate up a lot of time — eight or nine months," says Rienhoff.

    As Rienhoff studied the murky world of the mitochondriacs, his daughter had her first birthday and took her first steps. She was developing — and, as a result, so was what could be said about her condition. When she stood up from a squatting position, she needed to brace her hands on her thighs. This behaviour, known as Gowers' sign, is common in children with muscle-wasting diseases such as Duchenne's muscular dystrophy. Sometimes, says Rienhoff, in a hard-to-determine diagnosis, you try to find a guiding principle. The inability to form muscle mass and tone, he says, "became the North Star of the case"...

    But when Valle was looking at Rienhoff's daughter with a couple of colleagues, something clicked. Her widely spaced eyes and marfanoid features, which are admittedly common in genetic disorders, looked strikingly similar to a syndrome that had just been defined. They asked the girl to open her mouth wide, and when they looked down her throat, they thought they'd cracked the case...

    Loeys and Dietz found that some people who seemed to have Marfan's harboured mutations not in the gene for fibrillin-1, but in the genes for two TGF-β receptors. Exactly how the mutations, which seem to disable the TGF-β receptors, have an activating effect on the pathway is an ongoing puzzle. But the result is a syndrome that, because it disrupts the same bodily system, is quite similar to that caused by the fibrillin-1 defects in Marfan's.

    In addition to the molecular details, Loeys and Dietz had found three obvious bodily symptoms for their syndrome: the widely spaced eyes; a cleft in the palate and/or the uvula (the soft tissue that hangs down at the back of the throat); and severe structural defects in the arteries...

    Strikingly — although it had never been noticed before, despite a great deal of medical and parental inspection — Rienhoff's daughter had a forked uvula...(continued)

  7. (continued)On the plane trip back Rienhoff read the Dietz and Loeys paper, which showed detailed pictures of the patients and their devastating aortic defects. His own heart sank...

    The sequence data on the TGF-β receptors arrived a few weeks later; they showed none of the mutations Loeys and Dietz had identified for the syndrome...

    Rienhoff threw himself into the literature on TGF-β activation, once again guided by his North Star, his daughter's inability to build muscle...

    Myostatin works through three activin receptors: ACVR1B, ACVR2A and ACVR2B. These look similar in sequence to the TGF-β receptors mutated in Loeys–Dietz. Rienhoff thought that a mutation in one of these specific receptors might explain why his daughter's skeletal muscle was so dramatically affected while her blood vessels were not...

    So he bought a used PCR machine, a microcentrifuge, some small-volume pipettes and a brand new gel box. All told, the equipment cost him about $2,000. With these simple tools and some sequence-specific DNA primers of his own design, he could pick the relevant genes out of his daughter's genome and amplify them enough for sequencing. Freezing the samples and packing the tiny tubes on ice, Rienhoff sent them off for sequencing at about $3.50 a pop. He prepared upwards of 200. If he was right, the data he got back would show a mutation in one of the genes for the activin receptors analogous to the mutations seen in Loeys–Dietz...

    When he got the sequences, Rienhoff compared them to the human reference sequences in GenBank. In the gene encoding the ACVR1B receptor he found a variant. But it was a long way upstream of where he would have expected it to be, far from the active domain where many of the Loeys–Dietz mutations are found on the TGF-β receptor genes...

    An obvious way to clear the mutation of any blame is for Rienhoff to sequence the copies of the gene in both his genome and his wife's. If one of them has the mutation too it is probably irrelevant — a harmless change, not one that explains the syndrome, because if it did the parentwith the faulty copy would share the symptoms. Rienhoff says that he plans to sequence his and Hane's genes when he gets the time.

    In May, based on the hypothesis that errantly activated signals might account for her inability to build muscles, Rienhoff, Hane and their daughter's cardiologist decided to put her on losartan, a drug for treating high blood pressure. Recent evidence suggests that it reduces the activity of secondary messengers triggered by TGF-β receptors4, and that marfanoid mice are helped by the drug...

    First, if his daughter does have some aberrant form of Loeys–Dietz or Marfan's, the drug could forestall the vascular disease associated with the condition. Although he may eventually sequence her fibrillin and other genes for these disorders, the most definitive answers will come from regular cardiograms. The side effects of losartan are minor and reversible, he says, but vascular disease isn't. Second, her muscles might get a little better...

    Meanwhile he devours any literature on TGF-β signalling he can find. He has begun looking for scientists with whom he might collaborate on related projects, such as finding other patients with similar symptoms who might have mutations in the genes he's been looking at, or creating knock-out mice. He's waiting for more people to start using his website.
    Inspired by a conference I am attending.

  8. Corrected link for the above comment:

  9. Rienhoff recognizes that he has benefited from his training and connections. But he told me part of his mission is to empower others. "I think probably the most important thing that people could take away from this is that the process is not mysterious," he says.

    His enthusiasm is not universal. In the course of my reporting, Rienhoff gave his daughter's doctors permission to speak to me, and not all of them agreed that he was doing the right thing. Dietz says he worries that Rienhoff's example may lead some parents down the wrong path, searching for answers in the genes and diverting resources from the important goal of making sure their children are receiving proper care.

    Rienhoff has heard these criticisms, and understands the discomfort. "There is a certain sense that all of this will unravel, meaning all of this will become driven by the people," he says. In deference to Dietz he has removed from his website a folder called 'How to sequence DNA' that he had never filled. "The purpose of the website is not about teaching people how to sequence DNA, at least not now," he says. But he still believes that patients and patient advocates can usher in what he calls a golden age in genetic research. It won't be for everyone. Rienhoff's search has been slow and methodical, and as yet inconclusive. Still, it has been fulfilling. "I'm really being given an opportunity, if you will, with this site and at this time in the history of genetics."

  10. Hugh Rienhoff says that his nine-year-old daughter, Bea, is “a fire cracker”, “a tomboy” and “a very sassy, impudent girl”. But in a forthcoming research paper, he uses rather different terms, describing her hypertelorism (wide spacing between the eyes) and bifid uvula (a cleft in the tissue that hangs from the back of the palate). Both are probably features of a genetic syndrome that Rienhoff has obsessed over since soon after Bea’s birth in 2003. Unable to put on much muscle mass, Bea wears braces on her skinny legs to steady her on her curled feet. She is otherwise healthy, but Rienhoff has long worried that his daughter’s condition might come with serious heart problems.

    Rienhoff, a biotech entrepreneur in San Carlos, California, who had trained as a clinical geneticist in the 1980s, went from doctor to doctor looking for a diagnosis. He bought lab equipment so that he could study his daughter’s DNA himself — and in the process, he became a symbol for the do-it-yourself biology movement, and a trailblazer in using DNA technologies to diagnose a rare disease (see Nature 449, 773–776; 2007).

    “Talk about personal genomics,” says Gary Schroth, a research and development director at the genome-sequencing company Illumina in San Diego, California, who has helped Rienhoff in his search for clues. “It doesn’t get any more personal than trying to figure out what’s wrong with your own kid.”

    Now nearly a decade into his quest, Rienhoff has arrived at an answer. Through the partial-genome sequencing of his entire family, he and a group of collaborators have found a mutation in the gene that encodes transforming growth factor-β3 (TGF-β3). Genes in the TGF-β pathway control embryogenesis, cell differentiation and cell death, and mutations in several related genes have been associated with Marfan syndrome and Loeys–Dietz syndrome, both of which have symptomatic overlap with Bea’s condition. The mutation, which has not been connected to any disease before, seems to be responsible for Bea’s clinical features, according to a paper to be published in the American Journal of Medical Genetics.

    In 2008, Jay Flatley, chief executive of Illumina, offered Rienhoff the chance to sequence Bea’s transcriptome — all of the RNA expressed by a sample of her cells — along with those of her parents and her two brothers. After drilling into the data, Rienhoff and his collaborators found that Bea had inherited from each parent a defective-looking copy of CPNE1, a poorly studied gene that seems to encode a membrane protein. It looked like the answer.

    But questions remained. The gene did not have obvious connections to Bea’s features, and publicly available genome data suggests that the CPNE1 mutations are present in about 1 in 1,000 people — an indication that there should be many more people like Bea.

    Unsatisfied, Rienhoff went back to Illumina in 2009 to ask for more help. He proposed exome sequencing, which captures the whole protein-encoding portion of the genome, and is in some ways more comprehensive than transcriptome sequencing. At the time, Illumina was developing its exome-sequencing technology, and the company again took on the Rienhoff family as a test group.

    The analysis pulled up a mutation in one copy of the gene that encodes TGF-β3 — just in Bea. In cell culture and experiments in frog eggs, the faulty gene seems to produce a non-functional protein that reduces TGF-β signalling. This mechanism would differ from what many suspect is going on in Marfan and Loeys–Dietz syndromes, in which mutations paradoxically amp up TGF-β signalling. A collaborator of Rienhoff is now engineering a mouse that shares Bea’s gene variant, which could help to clarify whether the mutation revs up signalling or suppresses it.

  11. Rienhoff HY Jr, Yeo CY, Morissette R, Khrebtukova I, Melnick J, Luo S, Leng N, Kim YJ, Schroth G, Westwick J, Vogel H, McDonnell N, Hall JG, Whitman M. A mutation in TGFB3 associated with a syndrome of low muscle mass, growth retardation, distal arthrogryposis and clinical features overlapping with Marfan and Loeys-Dietz syndrome. Am J Med Genet A. 2013 Aug;161A(8):2040-6.

    The transforming growth factor β (TGF-β) family of growth factors are key regulators of mammalian development and their dysregulation is implicated in human disease, notably, heritable vasculopathies including Marfan (MFS, OMIM #154700) and Loeys-Dietz syndromes (LDS, OMIM #609192). We described a syndrome presenting at birth with distal arthrogryposis, hypotonia, bifid uvula, a failure of normal post-natal muscle development but no evidence of vascular disease; some of these features overlap with MFS and LDS. A de novo mutation in TGFB3 was identified by exome sequencing. Several lines of evidence indicate the mutation is hypomorphic suggesting that decreased TGF-β signaling from a loss of TGFB3 activity is likely responsible for the clinical phenotype. This is the first example of a mutation in the coding portion of TGFB3 implicated in a clinical syndrome suggesting TGFB3 is essential for both human palatogenesis and normal muscle growth.