Orofacial clefts: Can genetic variants reveal how they arise?

Last Updated on April 19, 2025 by Joseph Gut – thasso

April 19, 2025 – Orofacial clefts (i.e., cleft lip and cleft palate) are among the most common birth defects, occurring in about one in 1,050 births in the United States, however, they are similarly frequent all over the world with somewhat differing frequencies in given populations. These defects, which appear when the tissues that form the lip or the roof of the mouth do not join completely, are believed to be caused by a mix of genetic and environmental factors.

Researchers at the MIT have now discovered how a genetic variant often found in people with these facial malformations leads to the development of cleft lip and cleft palate. Eliezer Calo, an associate professor of biology at MIT, is the senior author of the publication, which appeared in the American Journal of Human Genetics. Their findings suggest that the variant diminishes cells’ supply of transfer RNA, a molecule that is critical for assembling proteins. When this happens, embryonic face cells are unable to fuse to form the lip and roof of the mouth.

The researchers stated that until now, no one had made the connection that they made. This particular gene was known to be part of the complex involved in the splicing of transfer RNA, but it wasn’t clear that it played such a crucial role for this process and for facial development. Without the gene of ATP-dependent RNA helicase, known as DDX1, certain transfer RNA can no longer bring amino acids to the ribosome to make new proteins. If the cells can’t process these tRNAs properly, then the ribosomes can’t make protein anymore according to Michaela Bartusel, an MIT research scientist and the lead author of the study.

Genetic variants

Cleft lip and cleft palate, also known as orofacial clefts, can be caused by genetic mutations. Thus, many genes  have been identified to contribute to the incidence of isolated cases of cleft lip/palate. This includes in particular sequence variants in the genes IRF6, PVRL1 and MSX1. However, So far, the understanding of the genetic complexities involved in the molecular processes/pathways of the morphogenesis of the orofacial clefts remains open, mostly because they are known to also be impacted by environmental factors, the researchers noted. Trying to pinpoint what might be affected has been very challenging in this context.

To discover genetic factors that influence a particular disease, scientists often perform genome-wide association studies (GWAS), which can reveal variants that are found more often in people who have a particular disease than in people who don’t. For orofacial clefts, some of the genetic variants that have regularly turned up in GWAS appeared to be in a region of DNA that doesn’t code for proteins. In this study, the MIT team set out to figure out how variants in this region might influence the development of facial malformations.

Their studies revealed that these variants are located in an enhancer region called e2p24.2. Enhancers are segments of DNA that interact with protein-coding genes, helping to activate them by binding to transcription factors that turn on gene expression. The researchers found that this region is in close proximity to three genes, suggesting that it may control the expression of those genes. One of those genes had already been ruled out as contributing to facial malformations, and another had already been shown to have a connection. In this study, the researchers focused on the third gene, which is known as DDX1. DDX1, it turned out, is necessary for splicing transfer RNA (tRNA) molecules, which play a critical role in protein synthesis. Each transfer RNA molecule transports a specific amino acid to the ribosome—a cell structure that strings amino acids together to form proteins, based on the instructions carried by messenger RNA.

While there are about 400 different tRNAs found in the human genome, only a fraction of those tRNAs require splicing, and those are the tRNAs most affected by the loss of DDX1. These tRNAs transport four different amino acids, and the researchers hypothesize that these four amino acids may be particularly abundant in proteins that embryonic cells that form the face need to develop properly.

When the ribosomes need one of those four amino acids, but none of them are available, the ribosome can stall, and the protein doesn’t get made. The researchers are now exploring which proteins might be most affected by the loss of those amino acids. They also plan to investigate what happens inside cells when the ribosomes stall, in hopes of identifying a stress signal that could potentially be blocked and help cells survive.

Malfunctioning tRNA

While this is the first study to link tRNA to craniofacial malformations, previous studies have shown that mutations that impair ribosome formation can also lead to similar defects. Studies have also shown that disruptions of tRNA synthesis—caused by mutations in the enzymes that attach amino acids to tRNA, or in proteins involved in an earlier step in tRNA splicing—can lead to neurodevelopmental disorders.

Defects in other components of the tRNA pathway have been shown to be associated with neurodevelopmental disease, the researchers note. One interesting parallel between these two is that the cells that form the face are coming from the same place as the cells that form the neurons, so it seems that these particular cells are very susceptible to tRNA defects.

The researchers now hope to explore whether environmental factors linked to orofacial birth defects also influence tRNA function. Some of their preliminary work has found that oxidative stress—a buildup of harmful free radicals—can lead to fragmentation of tRNA molecules.

Oxidative stress can occur in embryonic cells upon exposure to ethanol, as in fetal alcohol syndrome, or if the mother develops gestational diabetes.

Th researchers  think it is worth looking for mutations that might be causing this on the genetic side of things, but then also, in the future, they  would expand this into which environmental factors have the same effects on tRNA function, and then see which precautions might be able to prevent any effects on tRNAs..

See here a sequence on orofacial clefts and the life with them:

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