Genetics & Rare Diseases

Harnessing progenitor cells in neonatal white matter repair

The sirtuin protein Sirt1 plays a crucial role in the proliferation and regeneration of glial cells from an existing pool of progenitor cells — a process that rebuilds vital white matter following neonatal hypoxic brain injury. Although scientists do not fully understand Sirt1’s role in controlling cellular proliferation, this pre-clinical model of neonatal brain injury outlines for the first time how Sirt1 contributes to development of additional progenitor cells and maturation of fully functional oligodendrocytes.

The findings, published December 19 in Nature Communications, suggest that modulation of this protein could enhance progenitor cell regeneration, spurring additional white matter growth and repair following neonatal brain injury.

“It is not a cure. But, in order to regenerate the white matter that is lost or damaged, the first steps are to identify endogenous cells capable of regenerating lost cells and then to expand their pool. The glial progenitor cells represent 4 to 5 percent of total brain cells,” says Vittorio Gallo, Ph.D., Director of the Center for Neuroscience Research at Children’s National, and senior author of the study. “It’s a sizable pool, considering that the brain is made up of billions of cells. The advantage is that these progenitor cells are already there, with no requirement to slip them through the blood-brain barrier. Eventually they will differentiate into oligodendrocyte cells in white matter, mature glia, and that’s exactly what we want them to do.”

The study team identified Sirt1 as a novel, major regulator of basal oligodendrocyte progenitor cell (OPC) proliferation and regeneration in response to hypoxia in neonatal white matter, Gallo and co-authors write. “We demonstrate that Sirt1 deacetylates and activates Cdk2, a kinase which controls OPC expansion. We also elucidate the mechanism by which Sirt1 targets other individual members of the Cdk2 signaling pathway, by regulating their deacetylation, complex formation and E2F1 release, molecular events which drive Cdk2-mediated OPC proliferation,” says Li-Jin Chew, Ph.D., research associate professor at Children’s Center for Neuroscience Research and a study co-author.

Hypoxia-induced brain injury in neonates initiates spontaneous amplification of progenitor cells but also causes a deficiency of mature oligodendrocytes. Inhibiting Sirt1 expression in vitro and in vivo showed that loss of its deacetylase activity prevents OPC proliferation in hypoxia while promoting oligodendrocyte maturation – which underscores the importance of Sirt1 activity in maintaining the delicate balance between these two processes.

The tantalizing findings – the result of four years of research work in mouse models of neonatal hypoxia – hint at the prospect of lessening the severity of developmental delays experienced by the majority of preemies, Gallo adds. About 1 in 10 infants born in the United States are delivered preterm, prior to the 37th gestational week of pregnancy, according to the Centers for Disease Control and Prevention.  Brain injury associated with preterm birth – including white matter injury – can have long-term cognitive and behavioral consequences, with more than 50 percent of infants who survive prematurity needing special education, behavioral intervention and pharmacological treatment, Gallo says.

Time is of the essence, since Sirt1 plays a beneficial role at a certain place (white matter) and at a specific time (while the immature brain continues to develop). “We see maximal Sirt1 expression and activity within the first week after neonatal brain injury. There is a very narrow window in which to harness the stimulus that amplifies the progenitor cell population and target this particular molecule for repair,” he says.

Sirt1, a nicotinamide adenine dinucleotide-dependent class III histone deacetylase, is known to be involved in normal cell development, aging, inflammatory responses, energy metabolism and calorie restriction, the study team reports. Its activity can be modulated by sirtinol, an off-the-shelf drug that inhibits sirtuin proteins. The finding points to the potential for therapeutic interventions for diffuse white matter injury in neonates.

Next, the research team aims to study these processes in a large animal model whose brains are structurally, anatomically and metabolically similar to the human brain.

“Ideally, we want to be able to promote the timely regeneration of cells that are lost by designing strategies for interventions that synchronize these cellular events to a common and successful end,” Gallo says.

Cleft lip and palate: caught in the web of genetic interactions

Children’s National research scientists are working to unravel the complicated web of genetic interactions that lead children to develop cleft lip and palate.

On June 26, 2000, scientists around the world hailed the first draft of the human genetic code as a scientific milestone that eventually would revolutionize the practice of medicine. By knowing the approximately 20,000 protein-coding genes for humans, many speculated that researchers and doctors eventually might elucidate the unique factors that influence thousands of diseases—and, someday, make it easier to find custom ways to treat these conditions.

“It is humbling for me and awe inspiring to realize that we have caught the first glimpse of our own instruction book, previously known only to God,” said Francis S. Collins, M.D., Ph.D., who directed the international effort to sequence the human genome and who now directs the National Institutes of Health.

Nearly two decades later, actually using this wealth of information has proven exceedingly more complicated than many envisioned. While some genetic diseases like Huntington’s disease or sickle cell anemia follow a simple pattern in which variations in a single gene lead to deleterious effects, the vast majority of other genetic health problems result from the interaction of multiple genes, from a handful to hundreds.

One condition that has proven especially tricky to understand on the genetic level is cleft lip and palate (CLP), says Youssef A. Kousa, D.O., Ph.D., a pediatric neurology resident at Children’s National Health System who has made human development a central focus of his research program. CLP, which affects about 1 in 1,000 babies born worldwide, can be devastating, Kousa explains. At some point between 6 and 12 weeks gestation, the palate and lip fail to close in some fetuses. Those children are born with a fissure that can significantly impair eating and speaking and that can complicate social interactions.

While researchers have linked some genes to CLP, Kousa says it has become increasingly clear that these genes do not exert their influence in isolation. In a review paper published recently in Developmental Dynamics, he and Brian C. Schutte, Ph.D., of Michigan State University, detail the story of three of those genes. The trio plays a role in CLP but also is implicated in another devastating congenital problem, neural tube defects.

One of these genes is IRF6, which scientists tagged as the gene responsible for inherited forms of CLP called van der Woude syndrome and popliteal pterygium syndrome more than a decade ago. In the interim, research has shown IRF6 also appears to be important in orofacial clefting that occurs independently of these syndromes. Estimates suggest that mutations in IRF6 increase the risk for CLP by 12 percent to 18 percent.

That means at least 80 percent of the risk for clefting is caused by different genes. Kousa and Schutte write that one of these is GRHL3, which also can cause van der Woude syndrome if it is mutated. GRHL3 is regulated by IRF6, Kousa explains. So, if IRF6 does not work properly, neither does GRHL3.

But what regulates IRF6? Upstream of this important gene is another called TFAP2A. The healthy operation of TFAP2A is key for IRF6 and orofacial clefting. Complicating the scenario further, several studies also have shown that TFAP2A is essential for normal development of the palate and neural tube, the embryo’s precursor to the central nervous system that eventually develops into the brain and spinal cord.

To shed light on the interplay of the full array of genes involved CLP, Kousa and colleagues recently published a paper in Birth Defects Research in which they use computer programs to analyze datasets on all genes identified thus far that are involved in orofacial and neural tube development and which molecules these genes produce and target. Their analyses showed that many of these genes are linked in associated pathways that influence vast realms of development, risk of cancer and folate metabolism. (Women who take folic acid supplements before getting pregnant and during pregnancy can reduce birth defect risks.)

By better understanding how these genes are connected into networks, Kousa says, researchers may be able to reduce the risk of both CLP and neural tube defects with a single intervention. However, like the study of genetic diseases itself, finding the right intervention might not be so simple. A drug or supplement that can alleviate one condition might exacerbate others, based on the complicated web of genetic interactions, he says.

“That’s why work from our lab and others is so important,” Kousa says. “It adds layers and layers of knowledge that, eventually, we’ll be able to put together to help prevent these devastating problems.”

Zhe Han, PhD

Key to genetic influence of APOL1 on chronic kidney disease

Zhe Han

Drosophila melanogaster nephrocytes share structural and functional similarities with human renal cells, making the fruit fly an ideal pre-clinical model for studying how the APOL1 gene contributes to renal disease in humans.

Using the Drosophila melanogaster pre-clinical model, a Children’s National Health System research team identified a key mechanism by which the APOL1 gene contributes to chronic kidney disease in people of African descent. The model exploits the structural and functional similarities between the fruit fly’s nephrocytes and renal cells in humans to give scientists an unprecedented ability to study gene-to-cell interactions, identify other proteins that interact with APOL1 in renal disease, and target novel therapies, according to a paper published November 18 in the Journal of the American Society of Nephrology.

“This is one of the hottest research topics in the kidney field. We are the first group to generate this result in fruit flies,” says Zhe Han, Ph.D., a senior Drosophila specialist and associate professor in the Center for Cancer & Immunology Research at Children’s National. Han, senior author of the paper, presented the study results this month during Kidney Week 2016, the American Society of Nephrology’s annual gathering in Chicago that was expected to draw more than 13,000 kidney professionals from around the world.

The advantages of Drosophila for biomedical research include its rapid generation time and an unparalleled wealth of sophisticated genetic tools to probe deeply into fundamental biological processes underlying human diseases. People of African descent frequently inherit a mutant version of the APOL1 gene that affords protection from African sleeping sickness, but is associated with a 17- to 30-fold greater chance of developing certain types of kidney disease. That risk is even higher for individuals infected with the human immunodeficiency virus (HIV). Drosophila renal cells, called nephrocytes, accurately mimic pathological features of human kidney cells during APOL1-associated renal disease.

“Nephrocytes share striking structural and functional similarities with mammalian podocytes and renal proximal tubule cells, and therefore provide us a simple model system for kidney diseases,” says Han, who has studied the fruit fly for 20 years and established the fly nephrocyte as a glomerular kidney disease model in 2013 with two research papers in the Journal of the American Society of Nephrology.

In this most recent study, Han’s team cloned a mutated APOL1 gene from podocyte cells cultured from a patient with HIV-associated nephropathy. They created transgenic flies making human APOL1 in nephrocytes and observed that initially the transgene caused increased cellular functional activity. As flies aged, however, APOL1 led to reduced cellular function, increased cell size, abnormal vesicle acidification, and accelerated cell death.

“The main functions of nephrocytes are to filter proteins and remove toxins from the fly’s blood, to reabsorb protein components, and to sequester harmful toxins. It was surprising to see that these cells first became more active and temporarily functioned at higher levels,” says Han. “The cells got bigger and stronger but, ultimately, could not sustain that enhancement. After swelling to almost twice their normal size, the cells died. Hypertrophy is the way that the human heart responds to stress overload. We think kidney cells may use the same coping mechanism.”

The Children’s research team is a multidisciplinary group with members from the Center for Cancer & Immunology Research, the Center for Genetic Medicine Research, and the Division of Nephrology. The team also characterized fly phenotypes associated with APOL1 expression that will facilitate the design and execution of powerful Drosophila genetic screening approaches to identify proteins that interact with APOL1 and contribute to disease mechanisms. Such proteins represent potential therapeutic targets. Currently, transplantation is the only option for patients with kidney disease linked to APOL1.

“This is only the beginning,” Han says. “Now, we have an ideal pre-clinical model. We plan to start testing off-the-shelf therapeutic compounds, for example different kinase inhibitors, to determine whether they block any of the steps leading to renal cell disease.”

‘Trojan horse’ macrophage TNF-alpha opens door for HIV-1 to enter kidney epithelial cells, causing nephropathy

macrophage

Like a Trojan horse, the macrophage sits atop the epithelial cell with HIV hidden inside, opening a doorway into the kidney cell for high levels of HIV-1 to enter.

When nephrologist Patricio Ray, M.D., began investigating human immunodeficiency virus (HIV) as a renal fellow, children infected with the virus had a life expectancy of no more than seven years, and kids of African descent curiously were developing a type of HIV-related kidney disease.

HIV-associated nephropathy (HIVAN) is a progressive kidney disease seen in people who are both HIV-positive and of African ancestry. Kids who carry a modified protein that protects them against sleeping sickness are 80 times more likely to develop this type of kidney disease. Due to the kidney damage, they can have abnormal amounts of protein in their urine, focal segmental glomerulosclerosis, and microcystic tubular dilation, which can lead to enlarged kidneys and chronic kidney failure.

“No one understood how HIV could affect kidney cells that lack the receptors expressed in T cells and white cells,” recalls Dr. Ray, Robert Parrott Professor of Pediatrics at Children’s National Health System. Virologists said kidney epithelial cells that lacked CD4, a major receptor where HIV attaches, could not be infected with the virus. Nephrologists, meanwhile, were seeing that HIV infection was damaging these cells.

It’s taken two decades to unravel the medical mystery, aided by urine samples he coaxed kids to donate by offering them the latest music from New Kids on the Block in exchange for each urine bottle. Many of the kids died years ago, but their immortalized cells were essential in determining, through a process of elimination, which renal cell types were capable of being infected by HIV-1.

The paper represents the capstone of Dr. Ray’s career.

“This is how difficult it is to get an important contribution in science,” he says. “It’s 20 years of work involving the excellent contributions of many people, but that’s why research is called research. In the end, it’s all a learning process. But, it’s amazing how the puzzle pieces begin to fit. When the puzzle fits, it’s good.”

Dr. Ray, in collaboration with lead author Jinliang Li, Ph.D., and four additional Children’s National co-authors, published a paper November 3 in the Journal of the American Society of Nephrology that establishes a new role for transmembrane TNF-alpha, that of a facilitator that makes it easier for the HIV virus to enter certain cell types and replicate there.  Like a Trojan horse, the macrophage sits atop the epithelial cell with HIV hidden inside, opening a doorway into the kidney cell for high levels of HIV-1 to enter.

As a starting point, the research team cultured podocytes from the urine of kids with HIVAN. Through a number of steps, they isolated the unique contributions of the HIV envelope, heparan sulfate proteoglycans as attachment receptors – the glue that binds HIV to podocytes – and the essential role played by TNF-a, a 212-amino acid long type 2 transmembrane protein, in regulating at least two processes, including viral entry and fusion. They used a fluorescent marker to tag HIV-1 viruses, so it lit up bright green. Thus primed with transmembrane TNF-a, the podocytes were susceptible to HIV-1 infection when exposed to high viral loads.

Additional research is needed, such as in vitro work to help understand how HIV traffics within the cell, Dr. Ray says. Those insights could winnow the list of existing therapies that could block key steps, such as attachment to the viral envelope, which could help all people of African descent carrying the genetic mutation, including underserved kids in sub-Saharan Africa.

Another open research question is that certain cells located in the placenta and cervix express TNF-a, and may be more likely to be infected by HIV. Blocking that process could help prevent pregnant HIV-positive mothers from transmitting illness to their offspring.

Minimally invasive surgery brings lasting relief to pediatric achalasia patients

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Achalasia affects only a small number of people around the world, estimated at 1.6 per 100,000, and children make up fewer than 5 percent of that total. In most cases, the causes are unknown, but it is attributed to a combination of heredity and autoimmune or nerve cell disorders. For adults, treatment might include oral medication to prevent narrowing, balloon dilation, or botulinum toxin injections to relax the muscle at the end of the esophagus. For a growing child, who faces not just months but a lifetime of injections and potential repeat procedures, these methods aren’t viable. Instead, surgical correction is the standard of care. In the past 10 years, the surgical option evolved from a traditional open procedure with weeks of recovery and pain to less-invasive approaches.

“The total number of children with achalasia is small,” says Timothy D. Kane, M.D., Division Chief of General and Thoracic Surgery at Children’s National Health System. “But Children’s National treats more of these cases than most other children’s hospitals around the world, and that gives us the ability to look at a larger population and see what works.”

Dr. Kane is senior author of a study recently published in the Journal of Pediatric Surgery that analyzed the outcomes from nearly a decade’s worth of these cases to gauge the effectiveness of two different minimally invasive surgical approaches for children with achalasia.

A look at the two surgical options

The most common surgical intervention is laparoscopic Heller myotomy, performed through small incisions in the belly. Additionally, Dr. Kane and the Children’s surgical team are one of only two teams in the country who perform a different procedure called peroral endoscopic myotomy (POEM) on children. The POEM procedure is completed entirely through the mouth using an endoscope, with no additional incision needed. The procedure is commonly used for adult achalasia cases, but is not widely available for children elsewhere as it requires specialized training and practice to perform.

“Heller myotomy works very well for most kids — that’s why it’s the standard of care,” Dr. Kane says. “Our study found that patients who underwent the POEM procedure experienced the same successful outcomes as Heller patients, and we already knew from adult data that POEM patients reported less pain following surgery — a win-win for children.”

The retrospective study included all children who had undergone surgical treatment for achalasia at Children’s from 2006 to 2015. Since achalasia cases are few and far between, with most children’s hospitals seeing maybe one to five cases over 10 years, collecting reliable data on outcomes is challenging. This study provides a large enough sample to allow doctors to use the findings as a guide to find the interventions that are the best fit for each patient.

“Now we’re very comfortable presenting families with two really good options and letting them choose the one that works best for them,” he concludes.

Imagine the feeling of food stuck in your throat. For children with esophageal achalasia, that feeling is a constant truth: The muscles in the esophagus fail to function properly and the lower valve, or sphincter, of the esophagus controlling the flow of food into the stomach doesn’t relax enough to allow in food — causing a backup, heartburn, chest pain, and many other painful symptoms. For children, surgery is the best hope for permanent relief.

hands on simulation training at AAP

At AAP: hands-on simulation training with life-saving technology

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Recent medical breakthroughs have enabled very premature infants and children with rare genetic and neurological diseases to survive what had once been considered to be fatal conditions. This has resulted in a growing number of children with medically complex conditions whose very survival depends on ongoing use of technology to help their brains function, their lungs take in oxygen, and their bodies remain nourished.

“Many pediatricians care for technology-dependent children with special health needs,” says Neha Shah, M.D., M.P.H., an associate professor of pediatrics in the Division of Hospitalist Medicine at Children’s National Health System. “These kids have unique risks – some of which may be associated with that life-saving device malfunctioning.” Because there is no standard residency training for these devices, many clinicians may feel ill-equipped to address their patients’ device-related issues. To bridge that training gap, Dr. Shah and co-presenters, Priti Bhansali, M.D., M.Ed., and Anjna Melwani, M.D., will lead hands-on simulation training during the American Academy of Pediatrics 2016 National Conference.

“Inevitably, these things happen at 3 in the morning,” Dr. Shah adds. “Individual clinicians’ skill level and comfort with the devices varies. We should all have the same core competency.”

How the training works

During the simulation, the audience is given a specific case. They have eight minutes to troubleshoot and resolve the issue, using mannequins specially fitted with devices, such as trach tubes and feeding tubes, in need of urgent attention. Depending on their actions, the mannequin may decompensate with worsened breathing and racing heartbeats. The high-stakes, hands-on demo is followed by a 12-minute debrief, a safe environment to review lessons learned. Once they complete one simulation, attendees move to the next in the series of four real-life scenarios.

“We’ve done this a few times and my heart rate still goes up,” Dr. Shah admits. After giving similar training sessions at other academic meetings, participants said that having a chance to touch and feel the devices and become familiar with them in a calm environment is a benefit.

Dr. Shah came up with the concept for the hands-on training by speaking with a small group of peers, asking about how comfortable they felt managing kids with medical complex cases. The vast majority favored additional education about common devices, such as gastronomy tubes, tracheostomy tubes, and ventriculoperitoneal shunts.  In addition to the in-person training, the team has created a web-based curriculum discussing dysautonomia, spasticity, gastroesophageal reflux disease, enteric feeding tubes, venous thromboembolism, and palliative care, which they described in an article published in the Fall 2015 edition of the Journal of Continuing Education in the Health Professions.

“Most times, clinicians know what they need to do and the steps they need to follow. They just haven’t done it themselves,” Dr. Bhansali adds. “The simulation forces people to put their hands on these devices and use them.”

AAP 2016 presentations:
Saturday, October 22, 2016

  • W1059- “Emergencies in the Technology-Dependent Child: What Every Pediatrician Should Know” 8:30 a.m. to 10 a.m. (SOLD OUT)
  • W1131-  “Emergencies in the Technology-Dependent Child: What Every Pediatrician Should Know” (Encore) 2 p.m. to 3:30PM

Biomarkers sensitive to daily corticosteroid use

Using a mass spectrometer, Yetrib Hathout, Ph.D., is able to quantify 3,000 to 4,000 proteins from a tissue sample to identify proteins associated with cancer.

Using a Somascan proteomics assay – which simultaneously analyzes 1,129 proteins in a small volume of serum – a team led by Children’s National Health System researchers identified 21 biomarkers that respond to corticosteroids taken daily by children with Duchenne muscular dystrophy (DMD) and inflammatory bowel disease.

Corticosteroids are commonly prescribed to treat inflammatory conditions. High daily doses of corticosteroids are considered the standard of care for DMD, a type of muscular dystrophy characterized by worsening muscle weakness that affects 1 in 3,600 male infants. However, depending on the age of the child and drug dosage, chronic use is associated with such side effects as changes in bone remodeling that can lead to stunted growth, weight gain, facial puffiness caused by fat buildup, mood changes, sleep disturbances, and immune suppression. The research team sought to identify blood biomarkers that could be leveraged to create a fast, reliable way to gauge the safety and efficacy of corticosteroid use by children. The biomarkers also could guide development of a replacement therapy with fewer side effects.

“Ten pro-inflammatory proteins were elevated in untreated patients and suppressed by corticosteroids (MMP12, IL22RA2, CCL22, IGFBP2, FCER2, LY9, ITGa1/b1, LTa1/b2, ANGPT2 and FGG),” Yetrib Hathout, Ph.D., Proteomic Core Director at Children’s National, and colleagues write in the journal Scientific Reports. “These are candidate biomarkers for anti-inflammatory efficacy of corticosteroids.”

The blood biomarkers sensitive to corticosteroids fit into three broad groups, according to the authors. The children taking corticosteroids were matched with children of the same age who had never taken the medicine. Five biomarkers significantly increased in this corticosteroid-naïve group and decreased in kids prescribed corticosteroids. The biomarkers generally were inflammatory proteins and included chemokine, insulin-like growth factor binding protein 2, and integrin alpha-I/beta-1 complex.

The second group of biomarkers included nine proteins associated with macrophage and T-lymphocytes that were significantly reduced in concentration in kids taking corticosteroids. According to the study, this finding hints at corticosteroids blunting the ability of the immune system’s most able fighters to respond to infection.

In the third group were five proteins that were significantly increased by corticosteroid treatment in DMD and included matrix metalloproteinase 3, carnosine dipeptidase 1, angiotensinogen, growth hormone binding protein, insulin, and leptin, a hormone linked to appetite.

What researchers learned with this study will help them more accurately design the next phase of the work, Hathout says.

“We are the first team to report a number of novel discoveries, including that growth hormone binding protein (GHBP) levels increase with corticosteroid use. This represents a candidate biomarker for stunted growth. In order to use that new information effectively in drug development, the next studies must corroborate the role of serum GHBP levels as predictors of diminished stature,” he adds. “The study finding that four adrenal steroid hormones are depressed in kids taking corticosteroids raises additional questions about the broader impact of adrenal insufficiency, including its role in the delay of the onset of puberty.”

This work was supported by National Institutes of Health grants (R01AR062380, R01AR061875, P50AR060836, U54HD071601, K99HL130035, and R44NS095423) and Department of Defense CDMRP program grant W81XWH-15-1-0265. Additional support was provided by AFM-Telethon (18259) and the Muscular Dystrophy Association USA (MDA353094).

What rare diseases teach us about common ones

Think of the urea cycle as a river. A normal river flows to where it empties, similar to the process the body uses to rid itself of harmful ammonia via the urea cycle.

Think of the urea cycle as a river. A normal river flows to where it empties, similar to the process the body uses to rid itself of harmful ammonia via the urea cycle.

I recently presented at Spotlight Health 2016, the health-focused portion of the Aspen Ideas Festival, about how studying and treating rare diseases can inform innovative treatment approaches for more common medical conditions. Our Division of Genetics and Metabolism sees more than 8,000 patients a year with rare conditions, such as urea cycle disorders and Down syndrome. Through decades of analyzing these diseases and treating children who have them, we have developed therapies that apply not only for the small numbers of patients who have rare diseases but also for more common conditions caused by environmental factors leading to a similar physical response.

For instance, we’ve demonstrated that the stress of cardiopulmonary bypass during surgery to correct congenital heart disease creates conditions similar to a critical blockage in the urea cycle, specifically the biochemical creation of citrulline, a key biochemical.

When that cycle is unable to flow, or continuing the river analogy, becomes dammed up due to a genetic defect, as in urea cycle disorders, or an environmental factor, such as the extreme stress of cardiopulmonary bypass, the body is unable to make enough citrulline which is critical for maintaining normal blood pressure. We’ve shown that replacing that citrulline can correct a lot of these problems whether caused by rare genetics or the cardiac OR.

Applying rare disease treatment approaches to more common diseases is not limited to urea cycle disorders. Work by my colleague Carlos Ferreira, MD, demonstrates how a rare genetic calcifying arterial disease (generalized arterial calcification in infancy, GACI) causes the same calcium buildup and blockages as chronic kidney disease. Dr. Ferreira hypothesizes that life-saving drugs developed for use in GACI could help patients with long-term kidney disease by averting organ damage and eventual failure caused by the buildup of calcium crystals.

The more we learn about these rare diseases, the more we come to appreciate the tremendous implications our findings have for patients with the rare disorders and potentially hundreds of thousands of others.

About the Author

Marshall Summar, MD
Research interests: The interactions between common genetic variations and the environment.

How a rare disease treatment could impact millions

Post-mortem image shows significant narrowing of the artery in an infant with GACI due to buildup of calcium crystals between the vessel wall’s inner and middle layers. Inset: Normal non-calcified artery. Patients with GACI lack the protein ENPP1, which is responsible for creating pyrophosphate. Pyrophosphate plays a critical role in preventing calcium crystallization and accumulation.

Post-mortem image shows significant narrowing of the artery in an infant with GACI due to buildup of calcium crystals between the vessel wall’s inner and middle layers. Inset: Normal non-calcified artery. Patients with GACI lack the protein ENPP1, which is responsible for creating pyrophosphate. Pyrophosphate plays a critical role in preventing calcium crystallization and accumulation.

One of the first patients I ever saw with generalized arterial calcification of infancy (GACI) was actually the third child with this condition born to the same parents. GACI is a rare genetic disease, occurring in 1 of 200,000 live births. Unfortunately, as is common in GACI, two of the family’s children previously succumbed to the disorder within the first 6 weeks of life.

GACI causes calcium to build up in the arteries, causing critical blockages that reduce blood flow to organs leading to diminished function, including stroke, heart attack, and death.

Etidronate, a pyrophosphate analog developed to treat osteoporosis, has shown limited success at replacing the pyrophosphate for patients with GACI. However, more than 55 percent of children with GACI still die before their first birthday.

We need more effective solutions. Several treatment options are in development, including the administration of ENPP1 bound to an antibody, which has shown to provide a marked survival improvement in a mouse model of the disease.

These new solutions could translate to more effective treatment of GACI but also other conditions causing calcification in the arteries, particularly the calcium buildup associated with long-term kidney disease. A treatment that potentially reduces morbidity for the estimated 20 million plus Americans with chronic kidney disease would have tremendous health and economic benefits.

Developing more targeted therapies for GACI could allow this to be the outcome for many more patients, both children with GACI and potentially also patients affected by chronic kidney disease.

About the Author

Carlos Ferreira LopezCarlos Ferreira Lopez, M.D.
Geneticist Specialist

Personalized sequencing tailors genetic tests for each patient

Changes or errors in an individual’s DNA are often at the root of many disorders. Personalized Sequencing is a fast, cost-effective way to look at a region of the genome without repeat tests and blood draws.

Changes or errors in an individual’s DNA are often at the root of many disorders. Personalized Sequencing is a fast, cost-effective way to look at a region of the genome without repeat tests and blood draws.

Until recently, doctors and patients had two choices for ordering genetic sequencing panels to identify underlying causes of disease—Individual Gene Testing (single genes and gene panels) or Whole Exome Sequencing.

Individual gene testing is the standard testing modality. Physicians identify a single gene to analyze for change or mutation. If results are negative, they order another individual test, requiring a repeat visit and another blood draw. The process is repeated again and again based on likely candidate genes for a specific disease or symptom. If a physician is very lucky, it takes only a few rounds of tests to find the culprit. More likely, however, the number of individual tests grows large, taking months of patients’ time and increasing healthcare costs significantly. By contrast, Whole Exome Sequencing includes sequencing and analyses of 25,000 genes. It is more expensive when compared with individual gene testing and takes three to six months to complete. When complete, the results often can be more than the doctor and patient bargained for: Potentially revealing a genetic problem that is unrelated to the patient’s current symptoms. A 3-year-old with seizures also may come up positive for BRCA1, the breast cancer gene. Knowing that doesn’t help understand what causes the seizures or how to best treat them. In this model, you receive everything you could ever want. Because there is so much information, however, the results are difficult to interpret or to inform treatment decisions.

We’ve come up with a different way: Personalized Sequencing Panels, a precision medicine initiative at Children’s National Health System. We offer physicians a menu of genetic regions from which to choose when they order a sequencing analysis. While a medical exome is still sequenced, we only analyze a subset of genes that the physician and geneticist think are the most likely targets, which reduces the cost and time for analysis compared to Whole Exome Sequencing. Targeting regions in this approach shortens our turnaround time for results to two or three weeks. If the first identified region shows nothing, we can return to data we’ve already collected for a second look.

We’ve been using the model for 18 months and have tested more than 1,000 patients this way. Eighty percent of physicians prefer to “create their own test” using our menu of options. Rather than bringing a one-size-fits-all test to the patient, we bring the patient their very own personalized test.

About the Author

Sean Hofherr
Laboratory Medicine Specialist

Analysis of a progressive diffuse intrinsic pontine glioma: a case report

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What’s Known
Despite multiple clinical trials testing an assortment of new treatments, the survival rate for diffuse intrinsic pontine glioma (DIPG) remains abysmal, with most children succumbing to the pediatric brainstem tumor within 12 months of diagnosis. Focal radiation therapy, the primary treatment approach, has not improved overall survival. While the majority of DIPG tumors grow within the brainstem, metastases can occur elsewhere in the brain. Due to recent availability of tissue, new data are emerging about the biologic behavior of tumors, details that could be instrumental in constructing optimal treatment strategies.

What’s New
An otherwise healthy 9-year-old girl developed weakness in the left side of her face; magnetic resonance imagining revealed T2/FLAIR hyperintensity centered within and expanding the pons. Despite various treatments, her pontine lesion increased in size and new metastases were noted. The team led by Children’s National Health System researchers is the first to report comprehensive phenotypic analyses comparing multiple sites in primary and distant tumors. All tumor sites displayed positive staining for the H3K27M mutation, a mutation described in more than two-thirds of DIPGs that may portend a worse overall survival. Persistence of mutational status across multiple metastatic sites is particularly important since the effectiveness of some therapeutic approaches relies on this occurring. mRNA analyses, by contrast, identified a small number of genes in the primary tumor that differed from one metastatic tumor. This divergence implies that a single biopsy analysis for mRNA expression has the potential to be misleading.

Questions for Future Research
Q: Because a small cohort of genes in the girl’s primary tumor were different from genes in portions of the metastatic tumor, would genomic and proteomic analyses provide additional details about this genetic evolution?
Q: How do site-specific differences in mRNA expression affect decisions about which therapies to provide and in which order?

Source: “Histological and Molecular Analysis of a Progressive Diffuse Intrinsic Pontine Glioma and Synchronous Metastatic Lesions: A Case Report.” J. Nazarian, G.E. Mason, C.Y. Ho, E. Panditharatna, M. Kambhampati, L.G. Vezina, R.J. Packer, and E.I. Hwang. Published by Oncotarget on June 14, 2016.

Why subtle cellular changes can result in dramatically different genetic disorders

cellular_changes

What’s Known
One single gene, lamin A/C, is to blame for a multitude of genetic disorders, such as premature aging and problems with nerves, the heart, and muscles. Uncertainties linger in the scientific and medical community about why subtle changes of this gene cause such dramatically different disorders, such as Emery-Dreifuss muscular dystrophy, a condition that can lead to progressive muscle weakness in childhood and heart problems by adulthood.

What’s New
The nuclear envelope is where attached regions are pulled from genetic circulation never to be used again. The process of attachment signals which parts of the genome the cell no longer considers useful. Discarding superfluous DNA keeps the cell focused on what matters more: Its future role. Proper cell differentiation hinges on “the coordinated execution of three key cellular programs,” the study authors write. Pluriopotency programs, which give primitive cells the remarkable ability to generate any cell type in the body, are inactivated. Exit from the cell cycle occurs, and cells stop dividing. Myogenesis is induced, ushering in formation of muscle tissue. Mutations in lamin A/C can disrupt this careful choreography with the cumulative effect of slowing exit from cell cycle, slowing exit from pluripotency programs, and poorly coordinating induction of terminal differentiation programs.

Questions for Future Research
Q: What are the regions of human genome that become attached to the nuclear envelope during the development of tissues other than muscle (such as fat, nerve, and heart)?
Q: Can medicines that influence epigenetic pathways help to reverse the inappropriate DNA-lamin associations in Emery-Dreifuss muscular dystrophy?
Q: Can the new knowledge of DNA-lamin associations during muscle cell differentiation help to inform stem cell therapies?

Source:Laminopathies Disrupt Epigenomic Developmental Programs and Cell Fate.” J. Perovanovic, S. Dell’Orso, V.F. Gnochi, J. K. Jaiswal, V. Sartorelli, C. Vigouroux, K. Mamchaoui, V. Mouly, G. Bonne, and E. P. Hoffman. Science Translational Medicine. April 20, 2016.

researcher using ice bucket in lab

Spatial and temporal homogeneity of driver mutations in diffuse intrinsic pontine glioma

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What’s Known
Needle biopsies help to guide diagnosis and targeted therapies for diffuse intrinsic pontine gliomas (DIPGs), which make up 10 percent to 15 percent of all pediatric brain tumors but carry a median survival of 9 to 12 months. This dismal survival rate compares with a 70 percent chance of children surviving other central nervous system tumors five years post diagnosis. In DIPG, tumors appear in the pons, an area of the brain that houses cranial nerve nuclei. Surgical options are limited. Spatial and temporal tumor heterogeneity is a major obstacle to accurate diagnosis and successful targeted therapy.

What’s New
The team sought to better define DIPG heterogeneity. They analyzed 134 specimens from nine patients and found that H3K27M mutations were ubiquitous in all 41 samples with oncogenic content, and always were associated with at least one partner driver mutation: TP53, PPM1D, ACVR1 or PIK3R1. These H3K27M mutations are the initial oncogenic event in DIPG, writes the research team led by Children’s National Health System. “Driver” mutations, such as H3K27M, are essential to begin and sustain tumor formation. This main driver partnership is maintained throughout the course of the disease, in all cells across the tumor, and as tumors spread throughout the brain. Because homogeneity for main driver mutations persists for the duration of illness, efforts to cure DIPG should be directed at the oncohistone partnership, the authors write. Based on early tumor spread, efforts to cure DIPG should aim for early systemic tumor control, rather focused exclusively on the pons.

Questions for Future Research
Q: If a larger sample size were analyzed, what would it reveal about the true heterogeneity/homogeneity status of DIPGs?
Q: “Accessory” driver mutations are not absolutely essential but do help to further promote and accelerate tumor growth. What is their precise role?

Source: Spatial and Temporal Homogeneity of Driver Mutations in Diffuse Intrinsic Pontine Glioma.” H. Nikbakht, E. Panditharatna, L.G. Mikael, R. Li, T. Gayden, M. Osmond, C.Y. Ho, M. Kambhampati, E.I. Hwang, D. Faury, A. Siu, S. Papillon-Cavanagh, D. Bechet, K.L. Ligon, B. Ellezam, W.J. Ingram, C. Stinson, A.S. Moore, K.E. Warren, J. Karamchandani, R.J. Packer, N. Jabado, J. Majewski, and J. Nazarian. Published by Nature Communications on April 6, 2016.

The role of NG2 proteoglycan in glioma

A large number of staffers contribute to the Children's National team effort to unravel the mysteries of DIPG. We photograph a few essential players in Dr. Nazarian's lab.

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What’s Known
Neuron glia antigen-2 (NG2) is a protein expressed by many central nervous system cells during development and differentiation. NG2-expressing oligodendrocyte progenitor cells have been identified as the cells of origin in gliomas, tumors that arise from the brain’s gluey supportive tissue. What’s more, NG2 expression also has been associated with childhood diffuse intrinsic pontine glioma (DIPG) an aggressive tumor that accounts for 10 percent to 20 percent of pediatric central nervous system (CNS) tumors. Radiation can prolong survival by a few months, but children diagnosed with DIPG typically survive less than one year.

What’s New
Researchers are searching for appropriate targets and effective drugs that offer some chance of benefit. A team of Children’s National Health System researchers investigated whether NG2 – which plays a critical role in proliferation and development of new blood vessels and promotes tumor infiltration – could be a potential target for cancer treatment. Of the various options, antibody-mediated mechanisms of targeting NG2 are feasible, but the size of antibodies limits their ability to cross the blood-brain barrier. “Due to its role in maintaining a pluripotent pool of tumor cells, and its role in tumor migration and infiltration, NG2 provides multiple avenues for developing therapeutics,” the research team concludes. “Moreover, the large extracellular domain of NG2 provides an excellent antigen repertoire for immunotherapeutic interventions. As such, further research is warranted to define the role and expression regulation of NG2 in CNS cancers.”

Questions for Future Research

Q: Because healthy oligodendrocyte progenitor cells are important for the child’s developing brain, how could further characterization of NG2 isoforms help prevent drugs from damaging those beneficial cells?

Q: Could NG2-binding peptides cross the blood-brain barrier to deliver anti-cancer therapies precisely to tumor sites?

Source: The Role of NG2 Proteoglycan in Glioma.” S. Yadavilli, E.I. Hwang, R. J. Packer, and J. Nazarian. Published by Translational Oncology on February 2016.

Clinicopathology of diffuse intrinsic pontine glioma and its redefined genomic and epigenomic landscape

Dr. Nazarian's lab

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What’s Known
Fewer than 150 U.S. children per year are diagnosed with diffuse intrinsic pontine glioma (DIPG), one of the most lethal pediatric central nervous system cancers. Despite an increasing number of experimental therapies tested via clinical trials, more than 95 percent of these children die within two years of diagnosis. Molecular studies have yielded additional insight about DIPG, including that mutations in histone-encoding genes are associated with 70 percent of cases. Understanding mutations that drive tumors and the genomic landscape can help to guide development of targeted therapies.

What’s New: Frequently found genetic alterations prevalent in DIPGs

dipg-gene-mutations-and-biological-consequences

Source: Clinicopathology of Diffuse Intrinsic Pontine Glioma and Its Redefined Genomic and Epigenomic Landscape.” E. Panditharatna, K. Yaeger, L.B. Kilburn, R.J. Packer, and J. Nazarian. Published by Cancer Genetics on May 1, 2015.