Eric Vilain

Exploring differences of sex development

Eric Vilain, M.D., Ph.D.

Eric Vilain, M.D., Ph.D., analyzes the genetic mechanisms of sex development to give families more answers that will help them make better treatment (or non treatment) decisions for a child diagnosed with DSD.

Eric Vilain, M.D., Ph.D., is well versed in the “world of uncertainty” that surrounds differences of sex development. Since joining Children’s National as the director of the Center for Genetic Medicine Research in 2017, he’s shared with our research and clinical faculty and staff his expertise about the ways that genetic analysis might help address some of the complex social, cultural and medical implications of these differences.

Over the summer, he gave a keynote address entitled “Disorders/Differences of Sex Development: A World of Uncertainty” during Children’s National’s Research and Education Week, an annual celebration of research, education, innovation and scholarship at Children’s National and around the world. In January 2018, he shared a more clinically oriented version of the talk at a special Children’s National Grand Rounds session.

The educational objective of these talks is to inform researchers and providers about the mechanisms of differences of sex development (DSD), which are defined as congenital conditions in which the development of chromosomal, gonadal or anatomical sex is atypical.

The primary goal, though, is to really shine light on the complexity of this hot topic, and share how powerful genetic tools can be used to provide vital, concrete information for care providers, patients and families to assist with difficult treatment (and non-treatment) decisions.

“A minority of DSD cases are able to receive a genetic diagnosis today,” he points out. “But geneticists know how important it is to come to a diagnosis and so we seek to increase the number of patients who receive a concrete genetic diagnosis. It impacts genetic counseling and reproductive options, and provides a better ability to predict long term outcomes.”

“These differences impact physiology and medicine. We want to better understand the biology of reproduction, with an emphasis on finding ways to preserve fertility at all costs, and how these variations may lead to additional complications, including cancer risk.”

At conception, he explains, both XX and XY embryos have bipotential gonads capable of differentiating into a testis or an ovary, though embryos are virtually indistinguishable from a gender perspective up until six weeks in utero.

Whether or not a bipotential gonad forms is largely left up to the genetic makeup of the individual. For example, a gene in the Y chromosome (SRY) triggers a cascade of genes that lead to testis development. If there is no Y chromosome, it triggers a series of pro-female genes that lead to ovarian development.

Dr. Vilain notes that a variation of enzymes or transcription factors can occur at any single step of sex development and alter all the subsequent steps. Depending on the genotype, an individual may experience normal gonadal development, but abnormal development of the genitalia, for example.

He also noted that these genes are critical to determining the differences between men and women in non-gonadal tissues, including differences in gene expression within the brain. One study in the lab of investigator Matt Bramble, Ph.D., investigates if gonadal hormones impact sex differences in the brain by modifying the genome.

This work is a prime example of research informing the care provided to patients and families. Dr. Vilain is also a member of the multidisciplinary clinical team of the PROUD Clinic at Children’s National, a program completely devoted to caring for patients with a wide array of genetic and endocrine issues, including urogenital disorders and variations of sex development.

Human Rhinovirus

Finding the root cause of bronchiolitis symptoms

Human Rhinovirus

A new study shows that steroids might work for rhinovirus but not for respiratory syncytial virus.

Every winter, doctors’ offices and hospital emergency rooms fill with children who have bronchiolitis, an inflammation of the small airways in the lung. It’s responsible for about 130,000 admissions each year. Sometimes these young patients have symptoms reminiscent of a bad cold with a fever, cough and runny nose. Other times, bronchiolitis causes breathing troubles so severe that these children end up in the intensive care unit.

“The reality is that we don’t have anything to treat these patients aside from supportive care, such as intravenous fluids or respiratory support,” says Robert J. Freishtat, M.D., M.P.H., chief of emergency medicine at Children’s National Health System. “That’s really unacceptable because some kids get very, very sick.”

Several years ago, Dr. Freishtat says a clinical trial tested using steroids as a potential treatment for bronchiolitis. The thinking was that these drugs might reduce the inflammation that’s a hallmark of this condition. However, he says, the results weren’t a slam-dunk for steroids: The drugs didn’t seem to improve outcomes any better than a placebo.

But the trial had a critical flaw, he explains. Rather than having one homogenous cause, bronchiolitis is an umbrella term for a set of symptoms that can be caused by a number of different viruses. The most common ones are respiratory syncytial virus (RSV) and rhinovirus, the latter itself being an assortment of more than 100 different but related viruses. By treating bronchiolitis as a single disease, Dr. Freishtat says researchers might be ignoring the subtleties of each virus that influence whether a particular medication is useful.

“By treating all bronchiolitis patients with a single agent, we could be comparing apples with oranges,” he says. “The treatment may be completely different depending on the underlying cause.”

To test this idea, Dr. Freishtat and colleagues examined nasal secretions from 32 infants who had been hospitalized with bronchiolitis from 2011 to 2014 at 17 medical centers across the country that participate in a consortium called the 35th Multicenter Airway Research Collaboration. In half of these patients, lab tests confirmed that their bronchiolitis was caused by RSV; in the other half, the cause was rhinovirus.

From these nasal secretions, the researchers extracted nucleic acids called microRNAs. These molecules regulate the effects of different genes through a variety of different mechanisms, usually resulting in the effects of target genes being silenced. A single microRNA typically targets multiple genes by affecting messenger RNA, a molecule that’s key for producing proteins.

Comparing results between patients with RSV or rhinovirus, the researchers found 386 microRNAs that differed in concentration. Using bioinformatic software, they traced these microRNAs to thousands of messenger RNAs, looking for any interesting clues to important mechanisms of illness that might vary between the two viruses.

Their findings eventually turned up important differences between the two viruses in the NF-kB (nuclear factor kappa-light-chain-enhancer of activated B cells) pathway, a protein cascade that’s intimately involved in the inflammatory response and is a target for many types of steroids. Rhinovirus appears to upregulate the expression of many members of this protein family, driving cells to make more of them, and downregulate inhibitors of this cascade. On the other hand, RSV didn’t seem to have much of an effect on this critical pathway.

To see if these effects translated into cells making more inflammatory molecules in this pathway, the researchers searched for various members of this protein cascade in the nasal secretions. They found an increase in two, known as RelA and NFkB2.

Based on these findings, published online Jan. 17, 2018, in Pediatric Research, steroids might work for rhinovirus but not for RSV, notes Dr. Freishtat the study’s senior author.

“We’re pretty close to saying that you’d need to conduct a clinical trial with respect to the virus, rather than the symptoms, to measure any effect from a given drug,” he says.

Future clinical trials might test the arsenal of currently available medicines to see if any has an effect on bronchiolitis caused by either of these two viruses. Further research into the mechanisms of each type of illness also might turn up new targets that researchers could develop new medicines to hit.

“Instead of determining the disease based on symptoms,” he says, “we can eventually treat the root cause.”

Study co-authors include Kohei Hasegawa, study lead author, and Carlos A. Camargo Jr., Massachusetts General Hospital; Marcos Pérez-Losada, The George Washington University School of Medicine and Health Sciences; Claire E. Hoptay, Samuel Epstein and Stephen J. Teach, M.D., M.P.H., Children’s National; Jonathan M. Mansbach, Boston Children’s Hospital; and Pedro A. Piedra, Baylor College of Medicine.

Ashley Hill and Joyce Turner

New clues to detect rare pediatric cancers

Ashley Hill and Joyce Turner

Using germline and tumor testing and centralized pathology review, a research team that included D. Ashley Hill, M.D, and Joyce Turner found that Sertoli-Leydig cell tumor and gynandroblastoma are nearly always DICER1-related tumors.

Children’s National Health System researchers played a key role in a new study exploring the clinical and genetic qualities of a group of rare, potentially deadly cancers that affect infants, children and adolescents. The research team’s findings suggest that genetic testing for people at risk may aid in earlier, more accurate diagnoses of these cancers, leading to early-stage treatment that could greatly improve survival.

Ovarian sex cord-stromal tumors (OSCST) include juvenile granulosa cell tumors (JGCT), Sertoli-Leydig cell tumor (SLCT) and gynandroblastoma (GAB). Mutations in the DICER1 gene often have been noted in children with these cancers, as well as in those with a particularly lethal pediatric lung cancer called pleuropulmonary blastoma (PPB). All of these cancers are highly curable if caught early but, at later stages, can be aggressive and often fatal.

Using germline and tumor testing and centralized pathology review, the research team found that SLCT and GAB are nearly always DICER1-related tumors. There also may be a much stronger association between SLCT and DICER1 than was previously appreciated. The new findings have implications for earlier detection and diagnoses of these cancers, as well as for screening other family members. The study was published in the December 2017 edition of Gynecologic Oncology.

“These types of tumors are diverse, relatively rare and understudied,” says D. Ashley Hill, M.D., the study’s senior author and a professor in the Division of Pathology and Laboratory Medicine at Children’s National. “Sertoli-Leydig cell tumor, for instance, is a unique genetic and pathologic entity and this rare cancer of the ovaries can be hard to detect. Using the testing process from this study, we now may be able to classify these tumors more accurately.”

The study authors assessed the first 107 individuals enrolled in the International Ovarian and Testicular Stromal Tumor Registry. They obtained medical and family history, and they conducted central pathology review plus DICER1 gene sequencing on blood and tumor tissue. Thirty-six of 37 patients with SLCTs and all four patients with GABs they tested showed DICER1 mutations, and half of those with SLCT had germline or mosaic mutations. The team noted that individuals with predisposing DICER1 mutations had significantly better overall and recurrence-free survival.

Based on their findings, the study authors recommend:

  • Careful and ideally centralized pathologic review for all individuals with OSCST tumors
  • DICER1 testing for all those with SLCT and GAB and
  • Consideration of DICER1 testing for patients with other OSCSTs.

“Genetic testing may be useful for screening and diagnosing entire families if one family member tests positive for a DICER1 mutation, especially to determine if they are at risk for PPB. When we know who is at risk, we can protect all children in a family,” Dr. Hill says. “Ultimately we may be able to cure this deadly lung cancer, PPB, by identifying and performing computed tomography scans on people who are at risk, so we can catch these cancers early.”

Dr. Hill thinks future research may study children whose cancer was not detected early or has become resistant to chemotherapy. They also may explore ways to restore normal controls in cancer cells, so they follow normal paths of development, for the purpose of developing targeted treatments with fewer side effects than current therapies.

In addition to Dr. Hill, other Children’s National study co-authors include Amanda Field, M.P.H., Department of Pathology; Weiying Yu, Ph.D., Department of Pathology; and Joyce Turner, director of the Cancer Genetic Counseling Program in Children’s Rare Disease Institute.

Other members of the study team are experts from the International Ovarian and Testicular Stromal Tumor Registry, Children’s Minnesota, Washington University Medical Center, Carolinas Health Care System, University of Texas MD Anderson Cancer Center, Harvard Medical School, University of Colorado School of Medicine, Clinic of Pediatrics (Dortmund, Germany), National Cancer Institute and Dana-Farber Cancer Institute.

Research reported in this story was supported by the National Institutes of Health under award number NCI R01CA143167, The Parson’s Foundation, St. Baldrick’s Foundation, Pine Tree Apple Tennis Classic Foundation, Hyundai Hope on Wheels, the Randy Shaver Cancer Research and Community Fund, the German Childhood Cancer Foundation and the Intramural Research Program of the Divisions of Cancer Epidemiology and Genetics, National Cancer Institute.

Carlos Ferreira Lopez

Researchers discover new gene variant for inherited amino acid-elevating disease

Carlos Ferreira Lopez

What’s known

Hypermethioninemia is a rare condition that causes elevated levels of methionine, an essential amino acid in humans. This condition stems from genetic variations inherited from one or both parents. Some forms of hypermethioninemia are recessive, meaning that two copies of defective genes are necessary to cause this disease. Other forms are dominant, meaning that only one copy can cause hypermethioninemia. Recessive forms of the disease tend to have more serious consequences, causing elevated methionine levels throughout life and leading to changes in the brain’s white matter visible on magnetic resonance imaging that can cause neurological problems. The dominant forms are generally thought to be largely benign and require minimal follow-up.

What’s new

A research team led by Carlos R. Ferreira, M.D., a medical geneticist at Children’s National Health System, discovered a new gene variant that had not been associated with hypermethioinemia previously when an infant who had tested positive for elevated methionine on newborn blood-spot screening came in for a follow-up evaluation. While the majority of dominant hypermethioninemia are caused by a genetic mutation known as MAT1A p.Arg264His, the child didn’t have this or any of the common recessive hypermethioninemia mutations. Genetic testing showed that she carried a different mutation to the MAT1A gene known as p.Ala259Val, of which she carried only a single copy. The child fit the typical profile of having the dominant form of the disease, with methionine levels gradually declining over time. Testing of her mother showed that she carried the same gene variant, with few consequences other than a hepatitis-like illness as a child. Because liver disease can accompany dominant hypermethioninemia, the infant’s doctors will continue periodic follow-up to ensure she remains healthy.

Questions for future research

Q: Besides the potential for harmful liver effects, does dominant hypermethioninemia have other negative consequences?

Q: How common is this gene variant, and are certain people at more risk for carrying it?

Source: Confirmation that MAT1A p.Ala259Val mutation causes autosomal dominant hypermethioninemia. Muriello, M.J., S. Viall, T. Bottiglieri, K. Cusmano-Ozog and C. R. Ferreira. Published by Molecular Genetics and Metabolism Reports December 2017.

Debra Regier

U.S. leads the pack in medical genetics and genomic medicine

Debra Regier

Debra S. Regier, M.D., Ph.D., a pediatric geneticist who is the director of education in the Rare Disease Institute at Children’s National Health System.

It long has been recognized that traits can be passed down from parents to offspring in humans, just as occurs with other species. But medical genetics – the scientific field that covers the diagnoses and management of heritable diseases – didn’t get its start until recently. Only in the past century or so have researchers devoted significant resources to better understanding the patterns of inheritance or syndromes that have a genetic cause.

Although this research has taken place around the world, the United States is well established as a leader in this field, say authors of an article published in the July 2017 issue of Molecular Genetics & Genomic Medicine.

This article covers the history of the field, demographics of genetic conditions, legislation that relates to genetic disease and its burdens and highlights a long list of American researchers who have genetic diseases named after them. The list, comprising 86 scientists in a diverse array of fields including pediatrics, pathology, dermatology and oncology, is a testament to the devotion of these researchers to understanding a specific condition or, sometimes, group of related conditions.

Their dedication, often spanning the entirety of their career, contributed to the wealth of knowledge now available that’s improved the outcomes of many individuals with these diseases, says article co-author Debra S. Regier, M.D., Ph.D., a pediatric geneticist who is the director of education in the Rare Disease Institute at Children’s National Health System.

“Because these researchers spent their lives characterizing these disorders,” Dr. Regier says, “we can use that information when we find a child who fits the scheme of a particular disorder to tell families what they can expect – and in many instances – explain how best to treat them.”

Beyond tracking heritable disease traits through families, modern genomics also has led to the ability to recognize specific genes that cause various disorders, speeding the process of diagnosis and intervention.

“There are about 7,000 rare diseases, and sometimes it’s hard to know where to start with patients because it’s unclear which one they have,” Dr. Regier says. “By doing genetic testing, we can give families information, offer a prognosis and start treatments that have helped children who came before them with the same genetic mutation.”

Dr. Regier speculates that U.S. leadership in this field is largely due to the presence of large academic centers that are devoted to the study of genetic disorders, like Children’s National. Such centers give researchers dedicated time and space to better understand genetic diseases, both on a basic and an applied level. Despite the country’s stature as a frontrunner in this research arena, the United States has a relatively small medical genetics community, which researchers can use to their advantage.

“If I find a child with a rare genetic disorder, I can call up the world expert on this condition to share and receive information,” Dr. Regier adds. “That’s relatively rare in science, but it happens all the time in our field because we’re so small.”

Although the United States has contributed to many medical genetics and genomic medicine advances that have helped patients worldwide, the history of the field in this country wasn’t always laudable, Dr. Regier says. The article also addresses the eugenics movement during the early 20th century. For example, in 1907, Indiana became the first state to enact involuntary sterilization legislation, an effort to remove “flawed” individuals from the gene pool that was followed by similar laws in several other states. In 1924, Virginia enacted a law that allowed eugenic sterilization of people with intellectual disabilities that was upheld by the U.S. Supreme Court in 1927.

After atrocities committed by the Nazis during World War II, when the repercussions of these policies became more clear, these laws were gradually abolished.

More recent legislation, the article’s authors write, aims to protect individuals from discrimination for genetic disorders. Thus far, 35 states have laws on the books protecting against employment discrimination, and 48 states passed legislation against health insurance discrimination based on genetic information. Twenty-four states endorsed statutes that limit the use of genetic information for other types of insurance, including life, long-term care and disability.

The article is the first of a two-part series and was followed Nov. 26, 2017 by a second article addressing the current status of prenatal testing, reproductive options and reproductive law in the United States, as well as newborn screening, genetic services, rare disease registries, and education and training in genetics.

“We can take pride in our progress, while still acknowledging that we have a long way to go in this field,” Dr. Regier says.

boy sitting in wheelchair

Long-term glucocorticoids help patients with DMD

boy sitting in wheelchair

Glucocorticoids, a class of steroid hormone medications, have definite long-term benefits for patients with Duchenne muscular dystrophy, including extending muscle strength and function over years and decreasing the risk of death.

There is currently no cure for the devastating, progressive neuromuscular disease known as Duchenne muscular dystrophy (DMD). But clinics that treat patients with this disease have long relied on a class of steroid hormone medications, known as glucocorticoids, to ease its symptoms. Over weeks and months, these drugs help preserve muscle strength and function. Though these short-term benefits have been clear, some physicians have balked at using these medications over the long term – their benefits over years was unknown, making their potential side effects not worth the risk.

Now, a study published online Nov. 22, 2017 in The Lancet suggests that these medicines have definite long-term benefits, including extending muscle strength and function over years and even decreasing the risk of death. These findings support what has become the standard prescribing practice at many clinics and could help sway parents who are on the fence about their children receiving these therapies.

DMD is characterized by loss of muscle function and progressive muscle weakness that begins in the lower limbs and typically affects males due to the location of its causative genetic mutation. Patients with this devastating neuromuscular disease often receive glucocorticoids at some point as the disease progresses. Studies since the late 1980s have confirmed short-term benefits of treating with these drugs, including delaying the loss of muscle strength and function.

However, no prospective study had followed long-term glucocorticoid use in these patients, explains Heather Gordish-Dressman, Ph.D., a statistician at the Center for Genetic Medicine Research at Children’s National Health System and study senior author. The lack of long-term data led some physicians to delay treatment with these drugs since their use can lead to significant side effects, including weight gain, delayed growth and immunosuppression.

“Everyone had the idea that long-term use could be beneficial, but nobody had really rigorously tested that,” Gordish-Dressman says.

Craig McDonald, M.D., a University of California, Davis, professor and lead author of the study adds: “This long-term, follow-up study provides the most definitive evidence that the benefits of glucocorticoid steroid therapy in DMD extend over the entire lifespan. Most importantly, patients with Duchenne using glucocorticoids experienced an overall reduction in risk of death by more than 50 percent.”

To determine whether the short-term benefits of these drugs extend in the long term, Gordish-Dressman and researchers scattered across the country tapped data from the Cooperative International Neuromuscular Research Group’s Duchenne Natural History Study, the largest study to follow patients with DMD over time. They gathered data for 440 males with DMD aged 2 to 8 years old. About 22 percent had never taken glucocorticoids or had taken these medications for less than one year. The remainder had taken them for at least one year or longer.

By analyzing data for up to 10 years for these patients, the long-term benefits became clear, Gordish-Dressman adds. Glucocorticoid treatment for patients who received it for more than one year delayed loss of mobility milestones that affected the lower limbs by 2.1 to 4.4 years, such as going from supine to standing, climbing four stairs, and walking or running 10 meters, compared with boys who received the medications for less than one year. Long-term glucocorticoid therapy also delayed the loss of mobility milestones in upper limbs, such as hand function, performing a full overhead reach and raising the hands to the mouth.

Long-term use of these drugs also was associated with a decreased risk of death over the length of the study. Furthermore, deflazacort – a glucocorticoid recently approved by the Food and Drug Administration specifically for DMD – delayed loss of the ability to move from supine position to standing, walking and hand-to-mouth function significantly better than prednisone, the most popular glucocorticoid prescribed for DMD in the United States.

Gordish-Dressman says that glucocorticoids are currently a standard part of care for most patients with DMD, with some clinics prescribing these medications as soon as patients are diagnosed. However, because long-term data supporting their use was lacking, some physicians hesitate to prescribe glucocorticoids until the disease had progressed, when patients already had lost significant function.

Future studies will examine which medicines in this class of drugs and which regimens might offer the most benefits as well as how benefits differ with longer-term medication use.

Research reported in this news release was supported by the U.S. Department of Education/NIDRR, H133B031118 and H133B090001; the U.S. Department of Defense, W81XWH-12-1-0417; National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health under award number R01AR061875; and Parent Project Muscular Dystrophy.

Javad Nazarian

Liquid biopsy spots aggressive brainstem cancer earlier

Javad Nazarian

A Children’s National research team led by Javad Nazarian, Ph.D., M.S.C., tested whether circulating tumor DNA in patients’ blood and cerebrospinal fluid would provide an earlier warning that pediatric brainstem tumors were growing.

A highly aggressive pediatric brain cancer can be spotted earlier and reliably by the genetic fragments it leaves in biofluids, according to a study presented by Children’s National Health System researchers at the Society for Neuro-Oncology (SNO) 2017 Annual Meeting. The findings may open the door to non-surgical biopsies and a new way to tell if these tumors are responding to treatment.

Children diagnosed with diffuse midline histone 3 K27M mutant (H3K27M) glioma face a poor prognosis with a median survival time of only nine months after the pediatric brainstem cancer is diagnosed. Right now, clinicians rely on magnetic resonance imaging (MRI) to gauge how tumors are growing, but MRI can miss very small changes in tumor size. The Children’s research team led by Javad Nazarian, Ph.D., M.S.C., scientific director of Children’s Brain Tumor Institute, tested whether circulating tumor DNA in patients’ blood and cerebrospinal fluid would provide an earlier warning that tumors were growing. Just as a detective looks for fingerprints left at a scene, the new genetic analysis technique can detect telltale signs that tumors leave behind in body fluids.

“We continue to push the envelope to find ways to provide hope for children and families who right now face a very dismal future. By identifying these tumors when they are small and, potentially more responsive to treatment, our ultimate aim is to help children live longer,” says Eshini Panditharatna, B.A., study lead author. “In addition, we are hopeful that the comprehensive panel of tests we are constructing could identify which treatments are most effective in shrinking these deadly tumors.”

The researchers collected biofluid samples from 22 patients with diffuse intrinsic pontine glioma (DIPG) who were enrolled in a Phase I, Pacific Pediatric Neuro-Oncology Consortium clinical trial. Upfront and longitudinal plasma samples were collected with each MRI at various stages of disease progression. The team developed a liquid biopsy assay using a sensitive digital droplet polymerase chain reaction system that precisely counts individual DNA molecules.

“We detected H3K27M, a major driver mutation in DIPG, in about 80 percent of cerebrospinal fluid and plasma samples,” Panditharatna says. “Similar to adults with central nervous system (CNS) cancers, cerebrospinal fluid of children diagnosed with CNS cancers has high concentrations of circulating tumor DNA. However, after the children underwent radiotherapy, there was a dramatic decrease in circulating tumor DNA for 12 of the 15 patients (80 percent) whose temporal plasma was analyzed.”

Nazarian, the study senior author adds: “Biofluids, like plasma and cerebrospinal fluid, are suitable media to detect and measure concentrations of circulating tumor DNA for this type of pediatric glioma. Liquid biopsy has the potential to complement tissue biopsies and MRI evaluation to provide earlier clues to how tumors are responding to treatment or recurring.”

Support for this liquid biopsy study was provided by the V Foundation, Goldwin Foundation, Pediatric Brain Tumor Foundation, Smashing Walnuts Foundation, the Zickler Family Foundation, the Piedmont Community Foundation, the Musella Foundation, the Mathew Larson Foundation and Brain Tumor Foundation for Children.


Improving treatment success for Duchenne muscular dystrophy


Macrophages, white blood cells involved in inflammation, readily take up a new medicine for Duchenne muscular dystrophy and promote its sustained delivery to regenerating muscle fibers long after the drug has disappeared from circulation.

Chronic inflammation plays a crucial role in the sustained delivery of a new type of muscular dystrophy drug, according to an experimental model study led by Children’s National Health System.

The study, published online Oct. 16, 2017 in Nature Communications, details the cellular mechanisms of morpholino antisense drug delivery to muscles. Macrophages, white blood cells involved in inflammation, readily take up a new medicine for Duchenne muscular dystrophy (DMD) and promote its sustained delivery to regenerating muscle fibers long after the drug has disappeared from circulation.

Until recently, the only approved medicines for DMD targeted its symptoms, rather than the root genetic cause. However, in 2016 the Food and Drug Administration approved the first exon-skipping medicine to restore dystrophin protein expression in muscle: Eteplirsen, an antisense phosphorodiamidate morpholino oligomer (PMO). The drug had shown promise in preclinical studies but had variable and sporadic results in clinical trials.

The Children’s National study adds to the understanding of how this type of medicine targets muscle tissue and suggests a path to improve treatments for DMD, which is the most common and severe form of muscular dystrophy and currently has no cure, explains study co-leader James S. Novak, Ph.D., a principal investigator in Children’s Center for Genetic Medicine Research.

Because the medication vanishes from the blood circulation within hours after administration, Children’s research efforts have focused on the mechanism of delivery to muscle and on ways to increase its cellular uptake – and, by extension, its effectiveness. However, researchers understand little about how this medication actually gets delivered to muscle fibers or how the disease pathology impacts this process, knowledge that could offer new ways of boosting both its delivery and effectiveness, says Terence Partridge, Ph.D., study co-leader and principal investigator in Children’s Center for Genetic Medicine Research.

To investigate this question, Novak, Partridge and colleagues used an experimental model of DMD that carries a version of the faulty DMD gene that, like its human counterparts, destroys dystrophin expression. To track the route of the PMO into muscle fibers, they labeled it with a fluorescent tag. The medicine traveled to the muscle but only localized to patches of regenerating muscle where it accumulated within the infiltrating macrophages, immune cells involved in the inflammatory response that accompanies this process. While PMO is rapidly cleared from the blood, the medication remained in these immune cells for up to one week and later entered muscle stem cells, allowing direct transport into regenerating muscle fibers. By co-administering the PMO with a traceable DNA nucleotide analog, the research team was able to define the stage during the regeneration process that promotes heightened uptake by muscle stem cells and efficient dystrophin expression in muscle fibers.

“These macrophages appear to extend the period of availability of this medication to the satellite cells and muscle fibers at these sites,” Partridge explains. “Since the macrophages are acting as long-term storage reservoirs for prolonged delivery to muscle fibers, they could possibly represent new therapeutic targets for improving the uptake and delivery of this medicine to muscle.”

Future research for this group will focus on testing whether macrophages might be used as efficient delivery vectors to transport eteplirsen to the muscle, which would avert the rapid clearance currently associated with intravenous delivery.

“Understanding exactly how different classes of exon-skipping drugs are delivered to muscle could open entirely new possibilities for improving future therapeutics and enhancing the clinical benefit for patients,” Novak adds.

What Children’s has learned about congenital Zika infection

Roberta DeBiasi

Roberta DeBiasi, M.D., M.S., outlined lessons learned during a pediatric virology workshop at IDWeek2017, one of three such Zika presentations led by Children’s National research-clinicians during this year’s meeting of pediatric infectious disease specialists.

The Congenital Zika Virus Program at Children’s National Health System provides a range of advanced testing and services for exposed and infected fetuses and newborns. Data that the program has gathered in evaluating and managing Zika-affected pregnancies and births may offer instructive insights to other centers developing similar programs.

The program evaluated 36 pregnant women and their fetuses from January 2016 through May 2017. Another 14 women and their infants were referred to the Zika program for postnatal consultations during that time.

“As the days grow shorter and temperatures drop, we continue to receive referrals to our Zika program, and this is a testament to the critical need it fulfills in the greater metropolitan D.C. region,” says Roberta L. DeBiasi, M.D., M.S., chief of the Division of Pediatric Infectious Diseases and co-leader of the program. “Our multidisciplinary team now has consulted on 90 dyads (mothers and their Zika-affected fetuses/infants). The lessons we learned about when and how these women were infected and how their offspring were affected by Zika may be instructive to institutions considering launching their own programs.”

Dr. DeBiasi outlined lessons learned during a pediatric virology workshop at IDWeek2017, one of three such Zika presentations led by Children’s National research-clinicians during this year’s meeting of pediatric infectious disease specialists.

“The Zika virus continues to circulate in dozens of nations, from Angola to the U.S. Virgin Islands. Clinicians considering a strategic approach to managing pregnancies complicated by Zika may consider enlisting an array of specialists to attend to infants’ complex care needs, including experts in fetal imaging, pediatric infectious disease, physical therapists, audiologists, ophthalmologists and radiologists skilled at reading serial magnetic resonance images as well as ultrasounds,” Dr. DeBiasi says. “At Children’s we have a devoted Zika hotline to triage patient and family concerns. We provide detailed instructions for referring institutions explaining protocols before and after childbirth, and we provide continuing education for health care professionals.”

Of the 36 pregnant women possibly exposed to Zika during pregnancy seen in the program’s first year, 32 lived in the United States and traveled to countries where Zika virus was circulating. Two women had partners who traveled to Zika hot zones. And two moved to the Washington region from places where Zika is endemic. Including the postnatal cases, 89 percent of patients had been bitten by Zika-tainted mosquitoes, while 48 percent of women could have been exposed to Zika via sex with an infected partner.

Twenty percent of the women were exposed before conception; 46 percent were exposed to Zika in the first trimester of pregnancy; 26 percent were exposed in the second trimester; and 8 percent were exposed in the final trimester. In only six of 50 cases (12 percent) did the Zika-infected individual experience symptoms.

Zika infection can be confirmed by detecting viral fragments but only if the test occurs shortly after infection. Twenty-four of the 50 women (nearly 50 percent) arrived for a Zika consultation outside that 12-week testing window. Eleven women (22 percent) had confirmed Zika infection and another 28 percent tested positive for the broader family of flavivirus infections that includes Zika. Another detection method picks up antibodies that the body produces to neutralize Zika virus. For seven women (14 percent), Zika infection was ruled out by either testing method.

“Tragically, four fetuses had severe Zika-related birth defects,” Dr. DeBiasi says. “Due to the gravity of those abnormalities, two pregnancies were not carried to term. The third pregnancy was carried to term, but the infant died immediately after birth. The fourth pregnancy was carried to term, but that infant survived less than one year.”

Jyoti Jaiswal and Adam Horn

Antioxidants could thwart muscle repair

Science Signaling cover image 05Sept17

Science Signaling features a Research Article that describes the pathway by which mitochondria transduce the increase in cytosolic Ca2+ caused by plasma membrane injury into a ROS-dependent repair response. The image shows ROS production and actin polymerization as detected by fluorescent reporters near a plasma membrane injury site in a skeletal myofiber in an intact bicep of an experimental model. Credit: Adam Horn and Jyoti Jaiswal, M.S.C., Ph.D. Children’s National Health System and The George Washington University School of Medicine and Health Sciences

Reactive oxygen species (ROS) are a biological double-edged sword. These atoms, molecules or molecular fragments containing oxygen that is poised for chemical reactions, are a key part of the immune response, used by immune cells to kill potentially dangerous invaders such as bacteria. However, too much ROS – which also are produced as a normal part of cellular metabolism – can cause extreme damage to normal, healthy cells.

Because oxidative damage has been linked with cancer, many people make a concerted effort to consume antioxidants in food and as concentrated supplements. These compounds can neutralize ROS, stemming cellular damage. Taking antioxidants also has been thought to stem the muscle soreness from exercise since ROS are produced in excess during hard physical activity.

However, a new study led by researchers from Children’s National Health System finds that taking antioxidants could thwart the processes that repair muscle fibers. According to the study published Sept. 5, 2017 in Science Signaling and featured on the journal’s cover, oxidative species are crucial signals that start the process of repairing muscle fibers.

Cellular powerhouses known as mitochondria help injured muscle cells (myofibers) repair by soaking up calcium that enters from the site of injury and using it to trigger increased production of reactive oxygen species. Loading up mitochondria with excess antioxidants inhibits this signaling process, blocking muscle repair, exacerbating myofiber damage and diminishing muscle strength.

“Our results suggest a physiological role for mitochondria in plasma membrane repair in injured muscle cells, a role that highlights a beneficial effect of reactive oxygen species,” says Jyoti K. Jaiswal, M.S.C., Ph.D., principal investigator in the Center for Genetic Medicine Research at Children’s National Health System, associate professor of genomics and precision medicine at The George Washington University School of Medicine and Health Sciences and senior study author. “Our work highlights the need to take a nuanced view of the role of reactive oxygen species, as they are necessary when they are present at the right place and right time. Indiscriminate use of antioxidants actually could harm an adult with healthy muscles as well as a child with diseased muscle.”

Antioxidants are widely used by Baby Boomers with muscles that ache from a grueling workout or newborns diagnosed with muscular dystrophy. Jaiswal and Children’s National colleagues understand that their results buck conventional wisdom that antioxidants generally benefit muscle recovery.

“It is still a common belief within the fitness community that taking antioxidant supplements after a workout will help your muscles recover better. That’s what people think; that’s what I thought,” says Adam Horn, lead study author, a graduate student at The George Washington University who works with Jaiswal at Children’s National. “What we’ve done is figure out that mitochondria need to produce a very specific oxidative signal in response to muscle damage in order to help injured muscles repair.”Jyoti Jaiswal and Adam Horn

The oxidative signals produced by mitochondria are delicately balanced by the antioxidant defenses in healthy cells. This balance can be disrupted in diseases such as Duchenne muscular dystrophy, which is caused by the lack of a muscle-specific protein, dystrophin. Lack of dystrophin makes the muscle cell plasma membrane more vulnerable to injury. In an experimental model of Duchenne muscular dystrophy, the muscles at birth are seemingly normal but, within weeks, show obvious muscle damage and progressive weakness.

“What changes? One of the things that changes in the third and fourth week of life of this experimental model is mitochondrial functionality,” Jaiswal adds. “They end up with many dysfunctional mitochondria, which compromise repair of injured myofibers. This permits chronic and excessive oxidation of the myofibers and disruption of the proper oxidant-antioxidant balance.”

In this case, a dose of antioxidants may restore that proper balance and help to reverse muscle damage and progressive weakness.

As a next step, the research team is examining oxidation in healthy and diseased muscle to understand how the oxidant-antioxidant balance is disrupted and how it could be restored efficiently by using existing supplements. In one such study funded by the National Institutes of Health, the team is looking at the potential benefit of vitamin E supplements for patients with muscular dystrophy.

“Antioxidant supplements are made from extracts of bark, sap, chocolate and other compounds so they’re all different,” Jaiswal says. “Knowing which ones can restore balance under a specific circumstance has the potential to help the body maintain proper cellular signaling ability, which will keep muscles healthy and working properly.”

The response of actin protein following injury to a pair of muscle fibers in an intact biceps muscle.

Javad Nazarian

Advancing pediatric cancer research by easing access to data

Javad Nazarian

“This is a tremendous opportunity for children and families whose lives have been forever altered by pediatric cancers,” says Javad Nazarian, Ph.D., M.S.C., principal investigator in the Center for Genetic Medicine Research and scientific director of the Brain Tumor Institute at Children’s National.

Speeding research into pediatric cancers and other diseases relies not only on collecting good data, but making them accessible to research teams around the world to analyze and build on. Both efforts take time, hard work and a significant amount of financial resources – the latter which can often be difficult to attain.

In a move that could considerably advance the field of pediatric cancer, the National Institutes of Health (NIH), a body that funds biomedical research in the United States, recently awarded a public-private research collective that includes Children’s National Health System up to $14.8 million to launch a data resource center for cancer researchers around the world in order to accelerate the discovery of novel treatments for childhood tumors. Contingent on available funds, five years of funding will be provided by the NIH Common Fund Gabriella Miller Kids First Pediatric Research Program, named after Gabriella Miller, a 10-year-old child treated at Children’s National.

As principal investigators, researchers at Children’s Hospital of Philadelphia will lead the joint effort to build out the “Kids First” Data Resource Center. Children’s National in Washington, D.C., will spearhead specific projects, including the Open DIPG project, and as project ambassador will cultivate additional partnerships with public and private foundations and related research consortia to expand a growing trove of data about pediatric cancers and birth defects.

“This is a tremendous opportunity for children and families whose lives have been forever altered by pediatric cancers,” says Javad Nazarian, Ph.D., M.S.C., principal investigator in the Center for Genetic Medicine Research and scientific director of the Brain Tumor Institute at Children’s National. “From just a dozen samples seven years ago, Children’s National has amassed one of the nation’s largest tumor biorepositories funded, in large part, by small foundations. Meanwhile, research teams have been sequencing data from samples here and around the world. With this infusion of federal funding, we are poised to turn these data into insights and to translate those research findings into effective treatments.”

Today’s NIH grant builds on previous funding that Congress provided to the NIH Common Fund to underwrite research into structural birth defects and pediatric cancers. In the first phase, so-called X01 grantees—including Eric Vilain, M.D., Ph.D., newly named director of the Center for Genetic Medicine Research at Children’s National—received funding to sequence genetic data from thousands of patients and families affected by childhood cancer and structural birth defects.

This new phase of funding is aimed at opening access to those genetic sequences to a broader group of investigators around the globe by making hard-to-access data easily available on the cloud. The first project funded will be Open DIPG, run by Nazarian, a single disease prototype demonstrating how the new data resource center would work for multiple ailments.

DIPG stands for diffuse intrinsic pontine glioma, aggressive pediatric brain tumors that defy treatment and are almost always fatal. Just as crowd sourcing can unleash the collective brainpower of a large group to untangle a problem swiftly, open data sharing could accomplish the same for childhood cancers, including DIPG. In addition to teasing out molecular alterations responsible for making such cancers particularly lethal, pooling data that now sits in silos could help to identify beneficial mutations that allow some children to survive months or years longer than others.

“It’s a question of numbers,” Dr. Vilain says. “The bottom line is that making sense of the genomic information is significantly increased by working through large consortia because they provide access to many more patients with the disease. What is complicated about genetics is we all have genetic variations. The challenge we face is teasing apart regular genetic variations from those genetic variations that actually cause childhood cancers, including DIPG.”

Nazarian predicts some of the early steps for the research consortium will be deciding nuts-and-bolts questions faced by such a start-up venture, such as the best methods to provide data access, corralling the resources needed to store massive amounts of data, and providing data access and cross correlation.

“One of the major challenges that the data resource center will face is to rapidly establish physical data storage space to store all of the data,” Nazarian says. “We’re talking about several petabytes—1,000 terabytes— of data. The second challenge to address will be data dissemination and, specifically, correlation of data across platforms representing different molecular profiles (genome versus proteome, for example). This is just the beginning, and it is fantastic to see a combination of public and private resources in answering these challenges.”


Mitochondria key for repairing cell damage in DMD


A research team led by Jyoti K. Jaiswal, M.S.C., Ph.D., found that dysfunctional mitochondria prevent repair of muscle cells in Duchenne muscular dystrophy.

What’s known

Duchenne muscular dystrophy (DMD), one of the most severe forms of muscular dystrophy, is caused by a defect in the dystrophin gene. The protein that this gene encodes is responsible for anchoring muscle cells’ inner frameworks, or cytoskeletons, to proteins and other molecules outside these cells, the extracellular matrix. Without functional dystrophin protein, the cell membranes of muscle cells become damaged, and the cells eventually die. This cell death leads to the progressive muscle loss that characterizes this disease. Why these cells are unable to repair this progressive damage has been unknown.

What’s new

A research team led by Jyoti K. Jaiswal, M.S.C., Ph.D., a principal investigator in the Center for Genetic Medicine Research at Children’s National Health System, investigated this question in two experimental models of DMD that carry different mutations of the dystrophin gene. The researchers monitored the effects of the lack of functional dystrophin protein in these preclinical models on the level and function of muscle cell. They found that mitochondria – organelles that act as powerhouses to supply the chemical energy to drive cellular activities – are among the first to be affected. They found that the decline in mitochondrial level and activity over time in these experimental models preceded the onset of symptoms. The research team also looked at the ability of the experimental models’ muscle cells to repair damage. As the muscle cell mitochondria lost function, the cells’ ability to repair damage also declined. Efforts to increase mitochondrial activity after these organelles became dysfunctional did not improve muscle repair. This suggests that poor muscle repair may not be caused by a deficit in energy production by mitochondria.

Questions for future research

Q: Does similar mitochondrial dysfunction occur in human patients with DMD?
Q: How can the mitochondrial dysfunction be prevented?
Q: Is there a way to reverse mitochondrial dysfunction to better preserve the ability of muscle cells to repair from DMD-related damage?

Source: “Mitochondria mediate cell membrane repair and contribute to Duchenne muscular dystrophy.” Vila, M.C., S. Rayavarapu, M.W. Hogarth, J.H. Van der Meulen, A. Horn, A. Defour, S. Takeda, K.J. Brown, Y. Hathout, K. Nagaraju and J.K. Jaiswal. Published by Cell Death and Differentiation February 2017.

Zhe Han, PhD

Lab led by Zhe Han, Ph.D., receives $1.75 million from NIH

Zhe Han, PhD

A new four-year NIH grant will enable Zhe Han, Ph.D., to carry out the latest stage in the detective work to determine how histone-modifying genes regulate heart development and the molecular mechanisms of congenital heart disease caused by these genetic mutations.

The National Institutes of Health (NIH) has awarded $1.75 million to a research lab led by Zhe Han, Ph.D., principal investigator and associate professor in the Center for Genetic Medicine Research, in order to build models of congenital heart disease (CHD) that are tailored to the unique genetic sequences of individual patients.

Han was the first researcher to create a Drosophila melanogaster model to efficiently study genes involved in CHD, the No.1 birth defect experienced by newborns, based on sequencing data from patients with the heart condition. While surgery can fix more than 90 percent of such heart defects, an ongoing challenge is how to contend with the remaining cases since mutations of a vast array of genes could trigger any individual CHD case.

In a landmark paper published in 2013 in the journal Nature, five different institutions sequenced the genomes of more than 300 patients with CHD and their families, identifying 200 mutated genes of interest.

“Even though mutations of these genes were identified from patients with CHD, these genes cannot be called ‘CHD genes’ since we had no in vivo evidence to demonstrate these genes are involved in heart development,” Han says. “A key question to be answered: How do we efficiently test a large number of candidate disease genes in an experimental model system?”

In early 2017, Han published a paper in Elife providing the answer to that lingering question. By silencing genes in a fly model of human CHD, the research team confirmed which genes play important roles in development. The largest group of genes that were validated in Han’s study were histone-modifying genes. (DNA winds around the histone protein, like thread wrapped around a spool, to become packed into a higher-level structure.)

The new four-year NIH grant will enable Han to carry out the next stage of the detective work to determine precisely how histone-modifying genes regulate heart development. In order to do so, his group will silence the function of histone-modifying genes one by one, to study their function in the fly heart development and to identify the key histone-modifying genes for heart development. And because patients with CHD can have more than one mutated gene, he will silence multiple genes simultaneously to determine how those genes work in partnership to cause heart development to go awry.

By the end of the four-year research project, Han hopes to be able to identify all of the histone-modified genes that play pivotal roles in development of the heart in order to use those genes to tailor make personalized fly models corresponding to individual patient’s genetic makeup.

Parents with mutations linked to CHD are likely to pass heart disease risk to the next generation. One day, those parents could have an opportunity to sequence their genes to learn the degree of CHD risk their offspring face.

“Funding this type of basic research enables us to understand which genes are important for heart development and how. With this knowledge, in the near future we could predict the chances of a baby being born with CHD, and cure it by using gene-editing approaches to prevent passing disease to the next generation,” Han says.

Baby with Cleft Palate

Understanding genetic synergy in cleft palate

Baby with Cleft Palate

Like mechanics fixing a faulty engine, Youssef A. Kousa, M.S., D.O., Ph.D., says researchers will not be able to remedy problems related to IRF6, a gene implicated in cleft palate, until they better understand how the gene works.

Like all of the individual elements of fetal development, palate growth is a marvel of nature. In part of this process, ledges of tissue on the sides of the face grow downwards on each side of the tongue, then upward, fusing at the midline at the top of the mouth. The vast majority of the time, this process goes correctly. However, some part of it goes awry for the 2,650 babies born in the United States each year with cleft palates and the thousands more born worldwide with the defect.

For nearly two decades, researchers have known that a gene known as IRF6 is involved in palate formation. Studies have shown that this gene contributes about 12 percent to 18 percent of the risk of cleft palate, more than any other gene identified thus far. IRF6 is active in epithelial tissues – those that line cavities and surfaces throughout the body – including the periderm, a tissue that lines the mouth cavity and plays an important role during development.

According to Youssef A. Kousa, M.S., D.O., Ph.D., a child neurology fellow at Children’s National Health System, the periderm acts like a nonstick layer, preventing the tongue or other structures from adhering to the growing palate and preventing it from sealing at the midline. While researchers have long suspected that IRF6 plays a strong role in promoting this nonstick quality, exactly how it exerts its influence has not been clear.

“Gaining a better understanding of this gene might help us to eventually address deficits or perturbations in the system that creates the palate,” Dr. Kousa says. “Like a mechanic fixing a faulty engine, we will not be able to remedy problems related to this gene until we know how the gene works.”

Youssef Kousa

“Gaining a better understanding of this gene might help us to eventually address deficits or perturbations in the system that creates the palate,” Dr. Kousa says. “Like a mechanic fixing a faulty engine, we will not be able to remedy problems related to this gene until we know how the gene works.”

In a study published July 19, 2017 by the Journal of Dental Research, Dr. Kousa and colleagues seek to decipher one piece of this puzzle by investigating how this key gene might interact with others that are active during fetal development. The researchers were particularly interested in genes that work together in a cascade of activity known as the tyrosine kinase receptor signaling pathway.

Because this pathway includes a large group of genes, Dr. Kousa and colleagues reasoned that they could answer whether IRF6 interacts with this pathway by looking at whether the gene interacts with the last member of the cascade, a gene called SPRY4. To do this, the researchers worked with experimental models that had mutations in IRF6, SPRY4 or both. If these two genes interact, the scientists hypothesized, carrying mutations in both genes at the same time should result in a dramatically different outcome compared with animals that carried mutations in just one gene.

Using selective breeding techniques, the researchers created animals that had mutations in either of these genes or in both. Their results suggest that IRF6 and SPRY4 indeed do interact: Significantly more of the oral surface was adhered to the tongue during fetal development in experimental models that had mutations in both genes compared with those that had just one single gene mutated. Examining the gene activity in the periderm cells of these affected animals, the researchers found that doubly mutated experimental models also had decreased activity in a third gene known as GRHL3, which also has been linked with cleft lip and palate.

Dr. Kousa says the research team plans to continue exploring this interaction to better understand the flow of events that lead from perturbations in these genes to formation of cleft palate. Some of the questions they would like to answer include exactly which gene or genes in the tyrosine kinase receptor signaling pathway specifically interact with IRF6 – since SPRY4 represents just the end of that pathway, others genes earlier in the pathway are probably the real culprits responsible for driving problems in palate formation. They also will need to verify if these interactions take place in humans in the same way they occur in preclinical models.

Eventually, Dr. Kousa adds, the findings could aid in personalized prenatal counseling, diagnosis and screening related to cleft palate, as well as preventing this condition during pregnancy. Someday, doctors might be able to advise couples who carry mutations in these genes about whether they are more likely to have a baby with a cleft palate or determine which select group of pregnancies need closer monitoring. Additionally, because research suggests that GRHL3 might interact with nutrients, including inositol, it might be possible to prevent some cases of cleft palate by taking additional supplements during pregnancy.

“The more we know about how these genes behave,” Dr. Kousa says, “the more we can potentially avoid fetal palate development going down the wrong path.”

Mark Batshaw

Gene therapy’s slow rebirth

Mark Batshaw

A speech by outgoing American Pediatric Society President Mark L. Batshaw, M.D., explored the impact of a single clinical trial on the entire field.

Gene therapy – delivering genetic material into patients’ cells as a way to treat or cure their diseases – has immense promise to alleviate or end many lifelong and deadly conditions. This treatment has so much potential that it was a heavy focus of research and research dollars around the world in the 1980s and 1990s.

However, many of these efforts came to a screeching halt in 1999 when a teenaged patient named Jesse Gelsinger died in a gene therapy trial aimed at curing a disease called ornithine transcarbamylase deficiency, a urea cycle disorder. Gelsinger’s death triggered a number of investigations, halted gene therapy trials in the United States, and severely restricted financial support from federal, foundation and industry funders.

The tragedy also spurred Mark L. Batshaw, M.D., one of the clinical trial investigators and newly named Chief Academic Officer at Children’s National Health System, to turn in his resignation. The chief executive at the time declined to accept it, instead naming an outside panel to investigate Dr. Batshaw’s role in a study marred by conflicts of interest, delays in updating patient consent forms, lack of adherence to the study protocol and ineffective team leadership.

As Dr. Batshaw passed the gavel to the next president of the American Pediatric Society during the Pediatric Academic Societies’ annual meeting this spring, he told attendees of his Presidential Address that “not a day goes by that I don’t think of Jesse Gelsinger and his family and hope that the work our team has continued will honor him by eventually achieving success with gene therapy.” In an act of altruism, 18-year-old Jesse Gelsinger had joined the trial with the aim of helping other kids suffering from metabolic disorder.

Dr. Batshaw recognizes that his is an unusual choice, speaking about his “greatest professional failure” when predecessors have used their addresses to speak exclusively about scientific accomplishments.

“Because I was a principal, I think telling this story first of all says, hey look, this guy who is president of this organization, who has had a significant career, is willing to talk openly about a failure and how he dealt with it and how the field dealt with that failure,” Dr. Batshaw says. “Secondly, the field of gene therapy right now is starting to explode. It’s telling two different stories in an integrated way: One is of a great personal failure – and failure of an entire field. And the recovery from that, and what the future will be for a technology that holds great promise.”

More than 1,000 gene therapy trials are currently underway, 23 of them at Phase III, the pivotal stage that makes or breaks approval by the Food and Drug Administration (FDA). Dr. Batshaw estimates about a dozen of those are likely to demonstrate robust enough results to progress to a formal application for FDA approval. “After a period of virtually no growth in gene therapy trials from 2000 to 2013, there has been a marked upswing in the past two to three years,” he says.

Children’s National is a study site for one of those clinical trials, a Phase I/II adenoassociated virus (AAV)-mediated gene therapy for late-onset ornithine transcarbamylase (OTC) deficiency. Children with urea cycle disorders have enzyme deficiencies that leave them unable to adequately dispose of waste nitrogen. Often as newborns, they develop severely elevated ammonia in their brains leading to encephalopathy, an often fatal condition. The Phase I work will test escalating doses in three patients for safety. The Phase II work will explore whether the gene therapy improves outcomes like lowering ammonia levels and improving patients’ ability to convert ammonia to urea. (A precursor study in an experimental model was among the most impactful research papers published by Children’s National authors in 2016.)

“So, for both our group’s program – and viral-delivered gene therapy in general – there has been a rebirth after the disastrous outcome of the initial adenovirus trial in OTC deficiency,” Dr. Batshaw said in his prepared remarks. “This resurgence has likely been fueled by improved viral vectors, especially AAVs, and an improved economy and industry investment. The future of gene therapy is likely to be enhanced by new genetic therapy platforms including RNA interference as a means of vertically transmitted gene regulation and the CRISPR gene-editing technology. It will also be impacted by the results of the trials that will be completed in the next few years, especially those using AAV vectors in hemophilia A and B, spinal muscular atrophy and leukemia.”

Looking forward 10 years, Dr. Batshaw is hopeful that gene therapy will become part of the therapeutic tools routinely used to help patients who suffer from rare disease and cancer. Making that next leap forward will be powered by innovative research, including work by colleagues at Children’s National. Among the presenters at PAS2017, the world’s largest pediatric research meeting, were more than 100 Children’s presenters, speakers and moderators.

“It makes me very proud that there are so many clinicians who are also scientists who are not satisfied with simply doing things the way they have always been doing it but constantly questioning how can we do things better for our children?” Dr. Batshaw says. “Our whole focus at Children’s National is caring for children, and that means caring for them the very best way possible and not being satisfied with current therapy if it’s not curative.”

fruit fly

Studying fruit flies to better understand human kidneys

fruit fly

In his latest study, Zhe Han and co-authors zeroed in on Rab genes to determine their role in fruit fly renal function.

It’s a given that fruit flies and humans are different. Beyond the obvious are a litany of less-apparent distinctions. For example, fruit flies have hemolymph instead of blood. Arranged around a single cardiac chamber, compared with humans’ four-chamber hearts, are a group of cells called nephrocytes that serve the same function as human kidneys, filtering toxins and waste from hemolymph.

But despite the dissimilarities between these two organisms, fly nephrocytes and human kidney cells are similar enough to allow the fruit fly, a common lab model that shares about 60 percent of its DNA with people, to provide insights on kidney disease in people. In a new study in fruit flies led by Zhe Han, Ph.D., principal investigator and associate professor in the Center for Cancer and Immunology Research at Children’s National Health System, researchers identified several new genes thought to be critical for renal function in humans. The findings could lend insight to the inner workings of this organ down to the molecular level and eventually help further the understanding or treatment of kidney disorders.

Han explains that recent research by his group tied 80 fruit fly genes to renal function. Many of these newly identified genes were Rab GTPases, a family of genes that make proteins whose job is to move substances around in cells through membrane-enclosed pouches called vesicles. For example, Rab proteins might put some substances on the path to destruction by moving them into lysosomes, vesicles with enzymes that break down all kinds of biomolecules. Rab proteins might help other substances be reused by steering them into recycling endosomes, vesicles that shuttle biomolecules that are still useful to where they will be used next.

In their latest study, published online Feb. 8, 2017 in Cell & Tissue Research, Han and co-authors zeroed in on these Rab genes to determine their role in fruit fly renal function. The researchers accomplished this by using genetic alterations to shut down each gene selectively in fruit fly nephrocytes. They then evaluated these transgenic flies on a number of different characteristics, including ability to effectively filter proteins from the blood, whether toxins placed in their food accumulated in their nephrocytes, how they developed and how they survived.

Their findings readily identified five Rab genes that seemed more important for these functions than the others: Rabs 1, 5, 7, 11 and 35, which all have analogous genes in humans.

Peering into the nephrocytes of flies in which these three Rabs had been silenced, the researchers made critical discoveries. Turning off Rab 7 appeared to block the path toward biomolecules in the cell entering lysosomes. Rather than biomolecules being destroyed, they instead were shuttled to the recycling route. Turning off Rab 11 had the reverse effect; recycling endosomes were drastically reduced, while lysosomes dramatically increased. Turning off Rab 5 had the most striking effect: All vesicles going in or out were blocked – like a cellular traffic jam – filling the cell with biomolecules that had no place to go, Han says.

Han, who has long tracked renal-related mutations in humans, says that no patients with kidney disease have turned up so far with Rab mutations. These genes are critical for functions throughout the body, he explains, so any embryos with these mutations are unlikely to survive. However, he adds, a host of other renal-related genes work in parallel or are controlled by different Rabs. So understanding the role of Rabs in renal function provides some insight into how these genes operate as well as what might happen when the function of these genes goes awry.

Han plans to study how Rabs 5, 7 and 11 fit into networks of renal genes as well as the role of the other Rabs that could play novel roles in the nephrocyte cell trafficking.

“These findings in fly Rabs provide the framework to study the major causes of kidney disease in human patients,” he adds.

Zhe Han

Fruit flies can model human genetic kidney disease

Zhe Han

Zhe Han, Ph.D., has found that a majority of human genes known to be associated with nephrotic syndrome play conserved roles in renal function, from fruit flies to humans.

Drosophila melanogaster, the common fruit fly, has played a key role in genetic research for decades. Even though D. melanogaster and humans look vastly different, researchers estimate that about 75 percent of human disease-causing genes have a functional homolog in the fly.

A Children’s National Health System research team reported in a recent issue of Human Molecular Genetics that the majority of genes associated with nephrotic syndrome (NS) in humans also play pivotal roles in Drosophila renal function, a conservation of function across species that validates transgenic flies as ideal pre-clinical models to improve understanding of human disease.

NS is a cluster of symptoms that signal kidney damage, including excess protein in urine, low protein levels in blood, elevated cholesterol and swelling. Research teams have identified mutations in more than 40 genes that cause genetic kidney disease, but knowledge gaps remain in understanding the precise roles that specific genes play in kidney cell biology and renal disease. To address those research gaps, Zhe Han, Ph.D., a principal investigator and associate professor in the Center for Cancer & Immunology Research at Children’s National, and colleagues systematically studied NS-associated genes in the Drosophila model, including seven genes whose renal function had never been analyzed in a pre-clinical model.

“Eighty-five percent of these genes are required for nephrocyte function, suggesting that a majority of human genes known to be associated with NS play conserved roles in renal function from flies to humans,” says Han, the paper’s senior author. “To hone in on functional conservation, we focused on Cindr, the fly’s version of the human NS gene, CD2AP,” Han adds. “Silencing Cindr in nephrocytes led to dramatic impairments in nephrocyte function, shortened their life span, collapsed nephrocyte lacunar channels – the fly’s nutrient circulatory system – and effaced nephrocyte slit diaphragms, which diminished filtration function.”

And, to confirm that the phenotypes they were studying truly caused human disease, they reversed the damage by expressing a wild-type human CD2AP gene. A mutant allele derived from a patient with CD2AP-associated NS did not rescue the phenotypes.

Thus, the Drosophila nephrocyte can be used to explain the clinically relevant molecular mechanisms underlying the pathogenesis of most monogenic forms of NS, the research team concludes. “This is a landmark paper for using the fly to study genetic kidney diseases,” Han adds. “For the first time, we realized that the functions of essential kidney genes could be so similar from the flies to humans.”

A logical next step will be to generate personalized in vivo models of genetic renal diseases bearing patient-specific mutations, Han says. These in vivo models can be used for drug screens to identify treatments for kidney diseases that currently lack therapeutic options, such as most of the 40 genes studies in this paper as well as the APOL1 gene that is associated with the higher risk of kidney diseases among millions of African Americans.

Teen Girl drawing a heart on an iPad

Illuminating cardiometabolic risk in Down syndrome

Teen Girl drawing a heart on an iPad

A leading researcher at Children’s National says researchers should look closely at the increased risks of obesity and thyroid disease common in patients with Down Syndrome, and determine how these long term comorbidities relate to cardiovascular and metabolic (cardiometabolic) risk, body image, and quality of life.

Over the last several decades, physicians’ improved ability to treat the common comorbidities of Down syndrome, such as congenital heart disease, has dramatically prolonged survival. Today, more than 400,000 people across the country are living with Down syndrome, and life expectancy has increased to 60 years.

New strategies to manage care for patients with Down syndrome must include preventive, evidence-based approaches to address the unique needs of these patients, according to Sheela N. Magge, M.D., M.S.C.E., Director of Research in the Division of Endocrinology and Diabetes at Children’s. She says that these efforts should include looking more closely at the increased risks of obesity and thyroid disease common in this population, and determining how these long term comorbidities relate to cardiovascular and metabolic (cardiometabolic) risk, body image, and quality of life.

An NIH-funded study from Children’s National and the Children’s Hospital of Philadelphia (CHOP), led by Dr. Magge and her colleague from CHOP, Dr. Andrea Kelly, seeks to better understand how the body composition of patients with Down syndrome impacts their likelihood for developing diabetes and obesity-related cardiovascular risks long term.

“We know that individuals with Down syndrome are at increased risk for obesity, but what hasn’t been clear is whether or not they also have the same cardiometabolic risk associated with obesity that we know holds true for other populations,” says Dr. Magge. “In this previously under-studied population, the common assumption based on very limited studies from the 1970’s was that individuals with Down syndrome were protected from the diabetes and cardiovascular risks that can develop in other overweight people. However, more recent epidemiologic studies contradict those early findings.”

The study has enrolled 150 Down syndrome patients and almost 100 controls to date, and the team is currently beginning to analyze the data. Dr. Magge believes that the findings from this study will help to provide new, research-driven evidence to inform the long term clinical management of obesity and cardiometabolic risk in adolescents with Down syndrome.

She concludes, “The goal is for our research to provide the foundation that will advance prevention and treatment strategies for this understudied group, so that individuals with Down syndrome not only have a longer life expectancy, but also a healthier and better quality of life.”

Sarah B. Mulkey

Puzzling symptoms lead to collaboration

Sarah B. Mulkey, explaining the research

Sarah B. Mulkey, M.D., Ph.D., is lead author of a study that describes a brand-new syndrome that stems from mutations to KCNQ2, a genetic discovery that began with one patient’s unusual symptoms.

Unraveling one of the greatest mysteries of Sarah B. Mulkey’s research career started with a single child.

At the time, Mulkey, M.D., Ph.D., a fetal-neonatal neurologist in the Division of Fetal and Transitional Medicine at Children’s National Health System, was working at the University of Arkansas for Medical Sciences. Rounding one morning at the neonatal intensive care unit (NICU), she met a new patient: A newborn girl with an unusual set of symptoms. The baby was difficult to wake and rarely opened her eyes. Results from her electroencephalogram (EEG), a test of brain waves, showed a pattern typical of a severe brain disorder. She had an extreme startle response, jumping and twitching any time she was disturbed or touched, that was not related to seizures. She also had trouble breathing and required respiratory support.

Dr. Mulkey did not know what to make of her new patient: She was unlike any baby she had ever cared for before. “She didn’t fit anything I knew,” Dr. Mulkey remembers, “so I had to get to the bottom of what made this one child so different.”

Suspecting that her young patient’s symptoms stemmed from a genetic abnormality, Dr. Mulkey ran a targeted gene panel, a blood test that looks for known genetic mutations that might cause seizures or abnormal movements. The test had a hit: One of the baby’s genes, called KCNQ2, had a glitch. But the finding deepened the mystery even further. Other babies with a mutation in this specific gene have a distinctly different set of symptoms, including characteristic seizures that many patients eventually outgrow.

Dr. Mulkey knew that she needed to dig deeper, but she also knew that she could not do it alone. So, she reached out first to Boston Children’s Hospital Neurologist Philip Pearl, M.D., an expert on rare neurometabolic diseases, who in turn put her in touch with Maria Roberto Cilio, M.D., Ph.D., of the University of California, San Francisco and Edward Cooper, M.D., Ph.D., of Baylor College of Medicine. Drs. Cilio, Cooper and Pearl study KCNQ2 gene variants, which are responsible for causing seizures in newborns.

Typically, mutations in this gene cause a “loss of function,” causing the potassium channel to remain too closed to do its essential job properly. But the exact mutation that affected KCNQ2 in Dr. Mulkey’s patient was distinct from others reported in the literature. It must be doing something different, the doctors reasoned.

Indeed, a research colleague of Drs. Cooper, Cilio and Pearl in Italy — Maurizio Taglialatela, M.D., Ph.D., of the University of Naples Federico II and the University of Molise — had recently discovered in cell-based work that this particular mutation appeared to cause a “gain of function,” leaving the potassium channel in the brain too open.

Wondering whether other patients with this same type of mutation had the same unusual constellation of symptoms as hers, Dr. Mulkey and colleagues took advantage of a database that Dr. Cooper had started years earlier in which doctors who cared for patients with KCNQ2 mutations could record information about symptoms, lab tests and other clinical findings. They selected only those patients with the rare genetic mutation shared by her patient and a second rare KCNQ2 mutation also found to cause gain of function — a total of 10 patients out of the hundreds entered into the database. The researchers began contacting the doctors who had cared for these patients and, in some cases, the patients’ parents. They were scattered across the world, including Europe, Australia and the Middle East.

Dr. Mulkey and colleagues sent the doctors and families surveys, asking whether these patients had similar symptoms to her patient when they were newborns: What were their EEG results? How was their respiratory function? Did they have the same unusual startle response?

She is lead author of the study, published online Jan. 31, 2017 in Epilepsia, that revealed a brand-new syndrome that stems from specific mutations to KCNQ2. Unlike the vast majority of others with mutations in this gene, Dr. Mulkey and her international collaborators say, these gain-of-function mutations cause a distinctly different set of problems for patients.

Dr. Mulkey notes that with a growing focus on precision medicine, scientists and doctors are becoming increasingly aware that knowing about the specific mutation matters as much as identifying the defective gene. With the ability to test for more and more mutations, she says, researchers likely will discover more cases like this one: Symptoms that differ from those that usually strike when a gene is mutated because the particular mutation differs from the norm.

Such cases offer important opportunities for researchers to come together to share their collective expertise, she adds. “With such a rare diagnosis,” Dr. Mulkey says, “it’s important for physicians to reach out to others with knowledge in these areas around the world. We can learn much more collectively than by ourselves.”

Lisa M. Guay-Woodford, M.D

Lisa Guay-Woodford: minimizing kidney disease effects

Lisa M. Guay-Woodford, M.D

Lisa M. Guay-Woodford, M.D., is internationally recognized for her examination of the mechanisms that make certain inherited renal disorders particularly lethal, a research focus inspired by her patients.

The artist chose tempera paint for her oeuvre. The flower’s petals are the color of Snow White’s buddy, the Bluebird of Happiness. Each petal is accentuated in stop light red, and the blossom’s leaves stretch up toward the sun. With its bold strokes and exuberant colors, the painting exudes life itself.

It’s the first thing Lisa M. Guay-Woodford, M.D., sees when she enters her office. It’s the last thing she sees as she leaves.

Dr. Guay-Woodford, a pediatric nephrologist, is internationally recognized for her research into the mechanisms that make certain inherited renal disorders, such as autosomal recessive polycystic kidney disease (ARPKD), particularly lethal. She also studies disparate health disorders that have a common link: Disruption to the cilia, slim hair-like structures that protrude from almost every cell in the human body and that play pivotal roles in human genetic disease.

Sarah, the artist who painted the bright blue flower more than 20 years ago when she was 8, was one of Dr. Guay-Woodford’s patients. And she’s part of the reason why Dr. Guay-Woodford has spent much of her career focused on the broader domain of disorders tied to just a single defective gene, such as ARPKD.

“It dates back to when I was a house officer and took care of kids with this disorder,” Dr. Guay-Woodford says. “Maybe 30 percent die in the newborn period. Others survive, but they have a whole range of complications.”

Two of her favorite patients died from ARPKD-related reasons in the same year. One died from uncontrolled high blood pressure. The other, Sarah, died from complications from a combined kidney and liver transplant.

“The picture she drew hangs in my office,” she says. “She was a wonderful kid who was really full of life, and what she chose really mirrored who she was as a person. We put up lots of those sorts of those things in my office. It’s a daily reminder of why we do the things we do and the end goal.”

ARPKD is characterized by the growth of cysts in the liver, the kidney – which can lead to kidney failure – and complications within other structures, such as blood vessels in the heart and brain, according to the National Institutes of Health. About 1 in 20,000 live births is complicated by the genetic disorder. The age at which symptoms arise varies.

“Given the way it plays out, starting in utero, this is not a disease we are likely to cure,” she says. “But there are children who have very minimal complications. The near-term goal is to use targeted therapies to convert the children destined to have a more severe disease course to one that is less complicated so that no child suffers the full effects of the disease.”

That’s why it is essential to attain detailed knowledge about the defective gene responsible for ARPKD. To that end, Dr. Guay-Woodford participated in an international collaboration – one of three separate groups that 14 years ago identified PKHD1 as the defective gene that underlies ARPKD.

“The progress has been slow, partly because the gene and its protein products are very complex,” she says. “The good news is the gene has been identified. The daunting news is the identification did not leap us forward. It is just sort of an important step in what is going to be a fits-and-starts kind of journey.”

The field is trying to emulate the clinical successes that have occurred for patients with cystic fibrosis, which now can be treated by a drug that targets the defective gene, attacking disease at a fundamental level. Patient outcomes also have improved due to codifying care.

When she was a resident in the 1980s, children with cystic fibrosis died in their teens. “Now, they’re living well into their 40s because of careful efforts by really astute clinicians to deliver a standardized approach to care, an approach now enhanced by a terrific new drug. We measure quality care in terms of patient outcomes. That has allowed us to really understand how to effectively use antibiotics, physical therapy and how to think about nutrition – which makes a hugely important contribution that previously had been underappreciated.”

Standardizing clinical approaches dramatically improved and extended patients’ lives. “For renal cystic disease, we are beginning to do that better and better,” she adds.

There’s no targeted medicine yet for ARPKD. But thanks to an international conference that Dr. Guay-Woodford convened in Washington in 2013, such consensus expert recommendations have been published to guide diagnosis, surveillance and management of pediatric patients with ARPKD.

“There is an awful lot we can do in the way we systematically look at the clinical disease in these patients and improve our management. And, if you can overlay on top of that specific insights about why one person goes one way in disease progression versus another way, I think we can boost the baseline by developing good standards of care,” she says.

“Science does march on. There are a number of related research studies that are expanding our understanding of ARPKD. Within the next decade, we probably will be able to capitalize on not just the work in ARPKD but work in related diseases to learn the entry points for targeting therapies. That way, we can build a portfolio of markers of disease progression and test how effective these potential therapies are in slowing the course of the disease.”