Mutated Gene Identified That Causes a Form of Cleft Lip and Palate

A genetic finding by University of Iowa investigators may help determine the causes of the most common form of cleft lip and palate. This common, or non-syndromic, form affects nearly 70 percent of all people who have a cleft lip and palate birth defect. Syndromic forms, of which there at least 350 different types, account for the other 30 percent of cleft lip and/or palate cases.

The team of researchers identified the gene whose mutated form causes Van der Woude syndrome, a dominantly inherited form of cleft lip and palate that accounts for about 2 percent of all cleft lip and palate cases. The facial abnormalities in the syndrome are very similar to the birth defects caused by the non-syndromic condition. Thus, the discovery may be applicable to this common form. The investigation was significantly aided by the analysis of DNA taken from a pair of monozygotic, or identical, twins, one of whom has Van der Woude syndrome. The findings appear Sept. 2 in the advance online edition of Nature Genetics.

"Because of the similarities between Van der Woude syndrome and the most common form of the condition, we are hopeful that finding the gene related to Van der Woude is now going to allow us to identify the causes of the common form," said Jeff Murray, M.D., UI professor of pediatrics, pediatric dentistry and biology and one of the study's authors.

Overall, one in 1,000 infants is born with some form of cleft lip or cleft palate. People with Van der Woude syndrome, in addition to having a cleft lip and/or palate, also have indentations in their lower lip, known as lip pits. Cleft lip and palate makes nursing or bottle-feeding difficult for infants. As children with the condition age, they also develop speech and dental problems and are prone to ear infections. The common form of cleft lip and palate is non-syndromic, meaning it does not cause any other facial defects except for the clefting.

Previous studies have shown that the non-syndromic form is caused by a combination of many genetic and environmental factors. This complexity makes it very difficult for researchers to discover the genetic factors in this disorder.

"One approach we are using to discover the genetic factors in non-syndromic cleft lip and palate is to identify the gene that causes a simple clefting disorder yet looks like the complex one. Van der Woude fits the bill," said Brian Schutte, Ph.D., UI assistant professor of pediatrics and lead investigator on the project.

Although the Van der Woude syndrome causes more physical problems, it is "genetically simple" compared to the common form.

"Finding the gene that causes this syndrome will help us understand the genes involved in facial development and contribute to a new diagnostic tool for determining the cause of some cases of cleft lip and palate," Schutte said.

The team will build on their genetic finding by looking more closely at the function of the affected gene and its role in Van der Woude as well as in the common form of clefting. They also are pursuing the environmental factors that may interact with the genetic causes.

"Environmental factors may be easier to manipulate or prevent rather than trying directly to do something about the gene," Murray said. "Gene therapy is an eventual possibility but it is likely going to be decades before we could deliver gene therapy to human embryos as there are many scientific and ethical issues to study first.

"Instead, we already know that changing the environment in utero -- for example, using folic acid to prevent spina bifida -- can be effective. We hope studying this gene will lead to a similar environmental finding for cleft lip and palate," he added.

The journey to the finding was complex and long. Murray, who has studied cleft lip and palate conditions for 16 years, led a team that in 1990 confirmed that the affected genetic region for Van der Woude syndrome was on the long arm of chromosome one. That study involved families worldwide.

Schutte then joined the effort and began to determine the DNA sequence of that particular chromosomal region. The DNA sequencing was performed in collaboration with the Sanger Centre in England, as part of the Human Genome Project. Analysis of the DNA sequence showed that the region of interest was 350,000 base pairs in length and contained 15 genes.

"We were very confident that one of these 15 genes was the Van der Woude gene," said Schutte, whose next task was to look for mutations among those 350,000 base pairs of DNA.

Researchers at Genetico da Universidade Estadual Paulista in Brazil provided access in 2001 to the DNA of twins, with one affected by Van der Woude syndrome. At the same time, Shinji Kondo, M.D., Ph.D., a UI postdoctoral fellow in pediatrics, joined the team and began sequencing the DNA samples from the twins to look for a mutation in the affected twin.

In a DNA comparison of any two people who are not identical, up to one of every 1,000 letters can be different. However, with almost no exceptions, the DNA of monozygotic twins will be identical, making a genetic search much easier.

"Usually when you think of identical twins, they are just that -- genetically the same in every way," Murray said. "But sometimes, although rarely, after the twins split into two embryos and start developing separately, a single mutation occurs in one that isn't found in the other."

The team theorized that such an event had occurred in the Brazilian twins and that analyzing their DNA sequences would reveal the one mutation that had caused the disease. Kondo found that difference by comparing the twins' DNA, which revealed the difference lay in just one base pair of DNA among more than 30,000 he had studied.

"The direct sequence analysis technique allowed us to find the one needle in the haystack," Schutte said. "Using data from the twins, we found the genetic problem related to Van der Woude syndrome was literally one letter of difference -- a T instead of a G in a base pair in a particular gene. That represents one base pair out of three billion in the human cell."

For confirmation, the team then looked at the same gene in the DNA samples taken from other individuals with Van der Woude syndrome and found other mutations. Work by Michael Dixon, professor of biological sciences at the University of Manchester in England, also helped confirm the discovery by showing that the gene product, a protein, is normally made at high levels to ensure proper development of the lip and palate.

Instead, the aberration causes the gene, known as interferon regulatory factor 6 (IRF6), to lose its function and not produce sufficient amounts of the protein.

"We're not sure exactly what the protein does -- whether it helps 'tell' cells to grow, or not to grow or to change into different kinds of cells, but we do know that without the full amount of the molecule, the cells don't do what they're supposed to do," Schutte said.

Murray and Schutte agreed that without the twins the team eventually would have found the difference "through brute force," but the monozygotic twins significantly boosted the investigation.

"The study involved a new way of using data from monozygotic twins," Murray said. "It turns out that identical twins who differ for a single genetic trait are quite common. So we are proposing that other people who study, say, hypertension or diabetes, might think about looking at monozygotic twins in the way we did to see if they can find genetic differences that would explain at least a genetic component of the disorder."

Murray added that scientists prefer the term "monozygotic" twins because while such twins may genetically be nearly identical, many other factors such as the environment ensure that the twins also differ in many ways.

The team also recognized how much it benefited from the dedication of families and clinicians around the world who have shared DNA samples related to clefting since 1986.

"The finding really is a nice testament to the cooperation of the families and doctors who made it possible for us to do this study," Murray said.

In addition to Murray's laboratory, the UI houses one of the foremost cleft lip and palate treatment teams, led by John Canady, M.D., UI associate professor of surgery and otolaryngology. Other UI researchers study the psychological challenges faced by individuals with cleft lip and palate conditions.

Most children in the United States with cleft lip or palate have the birth defect repaired before age one. However, cleft lip and palate treatment in other parts of the world is not routine. Thus, increasing prevention and making surgery more effective and available are important next clinical steps.

The non-syndromic form of the condition is most common in Asian populations and least common in African populations. The Van der Woude syndrome seems to occur with equal distribution worldwide.

The UI-led study was supported in part by grants from the National Institute of Dental and Craniofacial Research, an institute of the National Institutes of Health. In addition, Michael Dixon received support from Wellcome Trust, Action Research, the Biotechnology and Biological Sciences Research Council, The European Union and Fundacao Lucentis.