Researchers have found that a defect in the processing of tRNAs may contribute to the development of cleft lip and cleft palate—two of the most common craniofacial malformations.
Among congenital craniofacial malformations, cleft lip and cleft palate are the most common. These conditions involve a separation in the upper lip beneath the nose, an opening in the roof of the mouth, or both. While a cleft lip is mainly a cosmetic concern, a cleft palate can interfere with chewing, swallowing, speaking, and hearing—largely due to impaired drainage of fluid from the middle ear. This occurs because the palate lies just below the eustachian tube, a drainage canal that connects the middle ear to the nasal passages. Both conditions are typically corrected through surgical intervention.
Although dozens of genes have been linked to these malformations as part of broader syndromes, cleft lip and palate more often occur in isolation, without accompanying health issues, and the genetic or environmental causes in such cases remain unknown. A new study published in The American Journal of Human Genetics identifies a mutation that may help explain some of these cases. The mutation indirectly disrupts the processing of transfer RNA (tRNA) in embryonic cells responsible for forming the lip and palate, potentially leading to the development of cleft lip and/or palate.
A common congenital malformation characterized by a split in the upper lip beneath the nose, an opening in the roof of the mouth, or both. Left: normal lip; center: unilateral cleft lip; right: bilateral cleft lip | Pepermpron, Shutterstock
The Genome’s Long and Mysterious Regulatory Regions
Previous studies investigating the genetic basis of cleft lip and palate have mainly identified alterations in regulatory regions of DNA. DNA contains genes—segments that encode instructions for producing approximately 20,000 proteins in humans—as well as tens of thousands of RNA molecules. Between these genes lie regulatory regions: DNA sequences that control how much RNA or protein is produced from a given gene. These regulatory regions can be longer than the genes themselves, and in most cases, their function is either completely unknown, poorly studied, or both. A research team led by Eliezer Calo at the Massachusetts Institute of Technology (MIT) discovered that a mutation in a regulatory region called e2p24.2 on chromosome 2 occurs more frequently in children born with cleft lip or palate. The team set out to understand how this mutation contributes to the development of these malformations.
When the researchers investigated this regulatory region in cranial neural crest cells (cNCCs)—the cells that give rise to the lip and palate during embryonic development—they found that it is positioned near three genes: MYCN, DDX1, and CYRIA. Since it is not possible to study the activity of these genes directly in human embryos, the researchers turned to chicken embryos and confirmed that the same regulatory region lies near the same trio of genes in chickens as well.
To investigate the function of each of these genes, the researchers used CRISPR gene-editing technology to introduce individual mutations into MYCN, DDX1, and CYRIA in chicken embryos. They found that mutating CYRIA did not result in any malformations, whereas mutations in MYCN and DDX1 led to craniofacial anomalies.
The role of the MYCN gene in facial development had been established in earlier mouse studies. However, the involvement of DDX1 in this process was previously unknown. The DDX1 gene encodes a protein of the same name, which plays a role in various RNA-related processes. To investigate its function in cranial neural crest cells (cNCCs), the researchers generated cNCCs lacking the DDX1 protein.
They found that DDX1-deficient cells stopped dividing, became immobile, and showed a 34% reduction in protein synthesis. This decline in protein production likely underlies the observed halt in cell proliferation and movement—but what causes the decrease in protein synthesis?
Cutting And Rejoining
Biochemical analysis revealed that in cranial neural crest cells (cNCCs), the DDX1 protein functions alongside several other proteins involved in processing transfer RNA (tRNA) molecules. tRNAs are essential components of translation—the cellular process that produces proteins. The researchers found that DDX1 is specifically involved in the processing of 28 out of the 428 known human tRNA genes. The RNA produced from these 28 genes contains an extra fragment that is normally cut out, allowing the remaining two remaining pieces to be joined together.
However, in the absence of DDX1, although the excess fragment is successfully removed, the two remaining parts fail to rejoin. As a result, the affected molecules cannot participate in translation. Still, whether this defect in tRNA processing is directly responsible for facial malformations, or merely correlated with them, remained an open question.
To investigate this further, the researchers turned to zebrafish. They injected a tRNA cleavage enzyme into fertilized zebrafish eggs and observed that the developing embryos exhibited abnormal head development. This finding suggests a link between defective tRNA processing and craniofacial abnormalities. The researchers also noted that beyond disrupting protein synthesis, cleaved tRNA fragments may have additional roles, including gene regulation and epigenetic inheritance, as shown in other studies.
Understanding the molecular mechanism underlying cleft lip and palate could help identify mutations in genes involved in this process, such as mutations in DDX1 or in other genes critical for tRNA processing. If such mutations are identified, they may be incorporated into genetic screening panels, whether for pre-pregnancy planning, in vitro fertilization (IVF), or prenatal diagnostics such as amniocentesis.
Currently, cleft lip can often be identified by ultrasound as early as 13 to 14 weeks of gestation, whereas cleft palate is typically not diagnosed until after birth. A genetic test indicating elevated risk for cleft palate could enhance both prenatal decision-making and early postnatal care. In the longer term, understanding the genetic and molecular basis of these malformations may pave the way to preventive interventions during early pregnancy - potentially reducing their severity or eliminating the need for surgical correction altogether.