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little girl with spina bifida

Oral clefts may stem from a shared genetic cause as neural tube defects

little girl with spina bifida

Research by an international team that includes Children’s National faculty, published online Jan. 25, 2019 in Human Molecular Genetics, suggests that genetic mutations that cause cleft lip and palate also may contribute to neural tube defects, such as spina bifida.

Oral clefts are some of the most common birth defects worldwide, affecting about one in every 700 births. In the U.S., more than 4,000 babies are born each year with cleft lip, with or without cleft palate.

This defect isn’t simply a cosmetic manner: Oral clefts can severely affect feeding, speech and hearing, and they cause about 3,300 deaths annually worldwide.

To better understand these conditions, researchers have isolated a number of genetic mutations that appear to play contributing roles. These include those in a gene known as Interferon Regulatory Factor 6. New research by an international team that includes Children’s National faculty, published online Jan. 25, 2019 in Human Molecular Genetics, suggests that these mutations also may contribute to neural tube defects such as spina bifida.

In the first weeks of fetal development, the neural plate curves, creating a neural tube that, once fused shut, becomes the fetal brain and fetal spinal cord. Neural tube defects, which can range from mild to severe, are characterized by incomplete development of the brain, spinal cord or meninges. These defects can potentially result in paralysis or even fetal or neonatal demise. According to the National Institutes of Health, spina bifida, which affects the spinal cord, is the most common neural tube defect in the U.S., affecting up to 2,000 infants each year.

“Despite its high frequency, spina bifida remains among the least understood structural birth defects,” says Brian C. Schutte, an associate professor of Microbiology and Molecular Genetics, Pediatrics and Human Development at Michigan State University and the study’s senior author. “There is strong evidence that genetic factors are a leading cause of such structural birth defects, but in most cases, the cause is unknown. Our team’s study is the first published research to demonstrate that DNA variants in the gene IRF6 can cause spina bifida,” Schutte says.

What’s more, the research team identified a mechanism to explain how altering IRF6 leads to neural tube defects. This mechanism links IRF6 function to two other genes – known as transcription Factor AP2A (TFAP2A) and Grainyhead Like 3 (GRHL3) – that are also known to be required for the development of the neural tube, lip and palate.

“We’re all on the hunt for the reasons when, how and why birth defects happen,” adds Youssef A. Kousa, MS, D.O., Ph.D., a clinical fellow in the Division of Child Neurology at Children’s National Health System and the study’s lead author. “Our main goal is prevention. This paper is a significant development because our team has identified a group of genes that can potentially contribute to very common types of birth defects: craniofacial as well as neural tube defects.”

The scientific odyssey is a wonderful example of serendipity. Kousa, then working in Schutte’s lab, was studying the effects of a new mutant experimental model strain on development of the palate. But one day, he walked into Schutte’s office holding a deformed preclinical embryo and said: “Brian, look at this!”

“Weird things happen in biology,” Schutte replied and counseled him to return if it happened again. Less than two weeks later, Kousa was back with several more of the deformed preclinical embryos, saying: “OK, Brian. It happened again.”

Within hours Kousa had unearthed recently published research that included an image of a similarly affected preclinical embryo. The pair then sketched out possible intersecting genetic pathways, as they brainstormed the myriad ways to end up with that specific phenotype. Initially, they tested their hypotheses in experimental models and eventually corroborated findings through human genetic studies.

The human studies could only be performed by collaborations. Schutte shared their initial observations with human genetics researchers scattered across the country. Those labs then generously agreed to test whether DNA variants in IRF6 were associated with neural tube defects in samples from patients that they had collected over decades of research.

The team found that Tfap2aIrf6 and Grhl3 are components of a gene regulatory network required for neurulation, a folding process that results in the neural tube bending and then fusing to become the basis of the embryo’s nervous system, from brain to spinal cord.

“Since this network is also required for formation of the lip, palate, limbs and epidermis, which develop at different times and places during embryogenesis, we suggest that the Tfap2aIrf6Grhl3 network is a fundamental pathway for multiple morphogenetic processes,” the researchers write.

Interferon Regulatory Factor 6 functions best when there is neither too much expression nor too little. Overexpression of Irf6 suppresses Transcription Factor Activation Protein 2A and Grainyhead Like 3, causing exencephaly, a neural tube defect characterized by the brain being located outside of the skull. Counterintuitively, experimental models that had too little Irf6 also ended up with reduced levels of Tfap2a and Grhl3 that led to a structural birth defect, but at the opposite end of the neural tube.

To test whether the experimental model findings held true in humans, they sequenced samples from people who had spina bifida and anencephaly – the rare birth defect that Kousa spotted in the experimental models – and found IRF6 function was conserved in people. Because of the genetic complexity of these birth defects, and the challenges inherent in collecting samples from cases of severe birth defects, many research teams were invited to participate in the study.

As testament to their collegiality, researchers from Stanford University, University of Texas at Austin, University of Iowa, University of Texas at Houston and Duke University agreed to share precious samples from the California Birth Defects Monitoring Program, from the Hereditary Basis of Neural Tube Defects study and from their own institutional sample collections.

“As we get better at personalized medicine, we could use this information to one day help to counsel families about their own risk and protective factors,” Kousa adds. “If we can identify the genetic pathway, we might also be able to modify it to prevent a birth defect. For example, prenatal supplementation with folic acid has led to a decrease in babies born with neural tube defects, but not all neural tube defects are sensitive to folic acid. This knowledge will help us develop individual-based interventions.”

Financial support for the research covered in this post was provided by the National Institutes of Health under grants DE13513, F31DE022696, DE025060, P01HD067244 and GM072859; startup funding from Michigan State University and the UT-Health School of Dentistry in Houston; and the Centers for Disease Control and Prevention under award number 5U01DD001033.

In addition to Kousa and Schutte, study co-authors include Huiping Zhu, Yunping Lei and Richard H. Finnell, University of Texas at Austin; Walid D. Fakhouri, University of Texas Health Science Center at Houston; Akira Kinoshita, Nagasaki University; Raeuf R. Roushangar, Nicole K. Patel, Tamer Mansour, Arianna L. Smith, and Dhruv B. Sharma, Michigan State University; A.J. Agopian and Laura E. Mitchell, University of Texas School of Public Health; Wei Yang and Gary M. Shaw, Stanford University School of Medicine; Elizabeth J. Leslie, Emory University; Xiao Li, Tamara D. Busch, Alexander G. Bassuk and Brad A. Amendt, University of Iowa; Edward B. Li and Eric C. Liao, Massachusetts General Hospital; Trevor J. Williams, University of Colorado Denver at Anschutz Medical Campus; Yang Chai, University of Southern California; and Simon Gregory and Allison Ashley-Koch, Duke University Medical Center.

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.”

Youssef A. Kousa

Generating a conditional allele of Irf6

Youssef A. Kousa

A research team that includes Youssef A. Kousa, M.S., D.O., Ph.D., has created a novel tool to delete Interferon regulatory factor 6, which regulates how epidermal cells differentiate, multiply and migrate.

A research team has created a novel tool to delete Interferon regulatory factor 6 (Irf6), which regulates how epidermal cells differentiate, multiply and migrate.

Mutations of this critical transcription factor are implicated in two orofacial clefting disorders. As with other transcription factors, the IRF6 protein binds to specific regions of DNA and plays a role in that specific gene’s activity. With van der Woude syndrome, a rare disease that occurs in 1 in 35,000 individuals, the National Institutes of Health (NIH) says mutations to the IRF6 gene inhibit production of the IRF6 protein. That protein shortfall lies at the heart of incomplete development and stalled maturation of tissues in the skull and face. For popliteal pterygium syndrome, IRF6 mutations can trigger facial abnormalities, webbed skin, and fused fingers and toes.

According to the NIH, the IRF6 protein is active in embryonic skin cells that later become tissue in the head, face and tongue. The study authors write that DNA variation in the IRF6 gene (which issues the marching orders to make the IRF6 protein) significantly heightens risk for developing non-syndromic cleft lip and palate, one of the most common congenital defects.

Studying the function of this critical gene in preclinical models has been hobbled by the fact that experimental models created without the Irf6 allele are born with severe skin, limb and craniofacial defects and die shortly after birth.

To overcome this hurdle, the research team did a bit of creative genetic shuffling to make a conditional allele of Irf6 to test in specific tissues at specific times as the experimental animals matured.

“The experimental models with the Irf6 conditional allele were viable after birth and, in fact, showed no developmental or reproductive defects when compared with their litter mates – which provides a reassurance that this specific change does not appear to affect overall normal gene function,” says Youssef A. Kousa, M.S., D.O., Ph.D., a pediatric resident in the child neurology track at Children’s National Health System and co-lead author of the technology report published online May 8, 2017 in Genesis.

To drill down into how the conditional allele affected the experimental models, the research team bred them with other animals specially designed to illuminate the function of the conditional allele. Some genotypes were lost, as was expected. Litters that were hypothesized to experience certain rates of severe limb, skin and craniofacial abnormalities did so. Immunostaining revealed IRF6 expression throughout the spinous layer and basal ­­­– or deepest ­­­– layer of the epidermis, but such expression was lacking in wildtype and knockout embryos.

In a different group of experimental models, the researchers added the deleter strain Ella-Cre. Nineteen resulting embryos were positive for the conditional allele but showed no evidence of recombination. Eight normal embryos showed incomplete recombination.  Nine embryos showed complete recombination in tail tissue. Just one embryo phenocopied the wild type embryos.

“Our research team successfully created the conditional allele for IRF6, which will open the door to future studies of gene function in neonatal experimental models,” Dr. Kousa and colleagues conclude. “Even though the allele is capable of recombination, we saw that efficacy varied and is linked to specific cell types. One possible explanation is variation in chromatin structure at the IRF6 locus.”

Future research will explore the utility of other Cre-drivers, such as Gdf9-Cre or CAG-Cre, to provide additional clarity about the functionality of the newly derived conditional alleles.

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.”