Clefting is a malformation in humans in which a gap can form during development in the palate, lip, or both. This gap can lead to several problems, including difficulty feeding, problems in overall speech function, ear infections, and social impacts. It could also require fairly extensive surgeries.

The birth defect affects about one in every 1,600 babies born in the U.S., according to Centers for Disease Control and Prevention data.

This research took place over a 10-year time span, beginning around 2010. The study was published on Jan. 5 in the American Journal of Human Genetics.

By searching through the genomes of more than 1,000 patients with cleft lip/palate, researchers found small sections in the genome that were deleted or duplicated, known as copy number variants.

The copy number variants also occur in the genomes of healthy individuals, which meant the researchers needed to come up with a strategy to identify those that might be involved in clefting.

“I thought what might be a first step in identifying genes driving the disorder would be first to only focus on deletions in the genome, rather than both deletions and duplications,” John Manak, UI biology professor and the study’s corresponding author, said.

The researchers looked for genes that were removed by the deletions at a rare frequency for people with the disorder. For people without the disorder, which was the control group, researchers looked for the deletions at an even rarer frequency.

“I thought this would be a really cool strategy because genetic variants that do bad things, including deletions of genes, tend to be quite rare in a population,” Manak said. “So, we were hoping that this strategy would select for deletions that really drove the disorder, deletions that were really critically involved in causing the cleft lip and/or palate.”

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The study’s first author, Lisa Lansdon, earned her doctorate in genetics from the UI in 2018 and is a clinical assistant professor at the University of Missouri-Kansas City School of Medicine. She performed most of the human data analysis, and collaborators then used African clawed frogs and zebrafish to test the genes by disrupting their function in the two species.

“By using fish and frogs, our group was able to show that we could successfully use our data to find plausible new genes that, when deleted, could cause a cleft,” she said. “That was the really exciting part of our study, identifying these new candidate genes.”

The fish and frog testing models ultimately contributed greatly to the results, Lansdon said.

“They’re really useful tools for modeling these deletions,” Lansdon said. “They have pretty rapid development from fish eggs or frog embryos to when you can actually detect craniofacial changes.”

Another reason these animals were chosen for the study is that the developmental pathways in cleft lip and palate and face formation are consistent across humans to fish and frogs.

“This means that if we disrupt a gene and it leads to a cleft-like face in a fish or a frog, then it is possibly applicable to humans as well,” Lansdon said.

Moving forward, Lansdon believes the next steps in this research would be further investigating these candidate genes.

“Clearly, when disrupted, [the genes] cause craniofacial abnormalities,” she said. “Now the next question is, well, why? What pathways are being disrupted, and at what developmental time points?”

Because they only specifically highlighted three candidate genes, the researchers also want to continue to look into the long list of other genes identified in their study that was deleted at a higher frequency.

“All of those genes could be further investigated in animal models to see if there’s additional support for their involvement in clefting,” Lansdon said.

Even though there is a lot of work to do for researchers, Manak emphasized the importance of continuing to identify as many genes involved in craniofacial development as possible.

“Craniofacial defects are among the most common birth defects on planet Earth,” he said. “Making a face is an incredibly complex process that involves a lot of signaling pathways, and we need to understand how these pathways are integrated before we stand a chance at preventing these kinds of defects.”