Key genes driving cleft lip and palate identified

A research group headed by the University of Iowa recognized three genes that, when deleted, produce cleft lip or palate, a facial abnormality that affects around one in every 1,600 babies in the United States, according to the US Centers for Disease Control and Prevention.

The genes were discovered by a high-resolution search of the genomes of over 1,000 patients with cleft lip/palate, a repository resulting from Iowa’s long engagement in investigating cleft lip or palate disorder worldwide.

Cleft lip and palate are birth abnormalities that arise when a baby’s lip or mouth does not develop normally during pregnancy. Children with a cleft lip or palate frequently struggle to feed and speak clearly, and they are prone to ear infections.

The researchers discovered copy number variations or tiny portions of the genome that were deleted or duplicated in patients with the condition. The researchers looked for genes that were uncommon even in patients with the disorder within the deleted regions.

This is significant because looking for gene losses that are unusual in persons with the disorder—and much more rare or nonexistent in everyone else—would imply that such losses should play a fundamental role in clefting rather than simply contributing to the disorder.

By lowering the function of the genes in two species, African clawed frogs and zebrafish, the researchers demonstrated the genes’ direct link with cleft lip or palate. When the function of the target genes was reduced, each species showed symptoms of clefting.

We’ve found, and validated in experiments with vertebrates, three genes that are directly associated with this disorder. It’s going to take a long time before we can do anything about it in humans, but now we’ve added several key genes driving the disorder. Eventually, if you know the genetics behind cleft lip and palate, and the step-by-step process of how you build a face, then you might figure out how to intervene to prevent the defect.”

John Manak, Study Corresponding Author and Professor, Department of Biology, University of Iowa

The reasons for orofacial clefts in most babies are unknown, however, they are assumed to be caused by genetic alterations and maybe external events.

The researchers examined DNA from cleft patients in the United States and the Philippines. The large patient group was assembled by Jeffrey Murray, Professor in the Stead Family Department of Pediatrics at Iowa, Sandra Daack-Hirsch, professor in the College of Nursing, and several others who traveled to the Philippines for years to enroll patients with clefts and their families in order to collect samples and information about the disorder as part of Operation Smile surgical missions.

The families who generously enrolled in this study were hopeful that this work would someday lead to improved prevention or treatment of cleft lip/palate, and this work is a landmark step in that direction.”

Jeffrey Murray, Professor, Stead Family Department of Pediatrics, University of Iowa

Manak utilized DNA from those patients and a technique known as comparative genomic hybridization to look for deleted portions of DNA in the patient pool with the disorder against a control group that did not have clefting. From there, he searched deleted genes that were so unusual in the clefting group that they were found in fewer than 1% of the 1,102 patients surveyed.

I wanted to identify those incredibly rare mutations that are driving this disorder, because mutations that do bad things are reduced in frequency in the population. In other words, a copy number loss where all you need is that gene deletion, and you get the disorder. That’s exciting because it defines some really key genes in the clefting pathway. Of course, we also needed to verify that our candidate genes were actually expressed in the face and made sense for taking part in craniofacial development, before we were fully confident in our results.”

John Manak, Study Corresponding Author and Professor, Department of Biology, University of Iowa

Manak is also affiliated with the Stead Family Department of Pediatrics and the Interdisciplinary Graduate Program in Genetics at Iowa.

This approach was used by the researchers to identify three genes: COBLL1, RIC1, and ARHGEF38.

When the researchers suppressed the action of these genes in African clawed frog and zebrafish embryos, both species revealed symptoms of abnormal face development. The frog trials were particularly significant since they are more closely related to humans in evolutionary terms than zebrafish, and the frog studies created facial features that mimicked human clefts.

The study’s first author, Lisa Lansdon, who received her doctorate in genetics from Iowa in 2018, claims that the research was the main focus of her thesis. She also supervised a group of undergraduates who assisted with the analysis.

Lansdon, who is currently a Clinical Assistant Professor at the University of Missouri-Kansas City School of Medicine, states, “It was very exciting to get to see the study evolve from the early design phases to ultimately discover new genes that were supported as being important for craniofacial development when we tested them in fish and frogs.”

The observations expand on an earlier study performed by Manak, which was published in 2018, in which he employed the same gene-hunting techniques in a smaller group of individuals with cleft lip or palate to identify one gene, named ISM1, that was directly associated to the disorder. In trials with clawed frogs, such as in this research, he validated the gene’s role in clefting.

Manak says, “A highlight for me is the strategy we employed in both studies, looking for gene deletions that are rare in our disease cohort that were even rarer or absent in our controls. People haven’t generally thought along those lines. It’s a lot easier to just sequence genes and then look for more traditional mutations that alter the function of a gene.”

He is particularly delighted because the genes probably play a significant role in facial development in general.

Manak concludes, “There are multiple pathways and genes and interactions between many different cell types, so we need to identify all these components in order to understand how a face gets put together.”