The research, led by Alexandros Rammos and Evie Stergiakouli at the University of Bristol together with colleagues at Cardiff University and the University of Exeter, drew on two of the United Kingdom’s most valuable population resources. The team analysed genetic and phenotypic data from 2,313 children with cleft lip and/or palate recruited through the Cleft Collective, the world’s largest cleft-specific cohort study, and compared them with 7,913 unaffected children from the Millennium Cohort Study, a nationally representative birth cohort. This scale matters, because questions about subtle genetic correlations demand thousands of participants before any signal can be distinguished from statistical noise.

The investigators deployed a battery of complementary genomic techniques, each probing a different aspect of the relationship between cleft biology and neurodevelopment. First, they used linkage disequilibrium score regression, a method that estimates the overall genetic correlation between two traits by examining whether the same stretches of the genome show association signals in both. If cleft liability and psychiatric conditions shared a common genetic architecture, this technique would detect it across hundreds of thousands of genetic variants simultaneously. Instead, the analysis revealed little evidence of genetic correlations between cleft lip and/or palate and any of the eight traits examined, which spanned cognitive, neurodevelopmental, and psychiatric domains.

Second, the team constructed polygenic risk scores for each child. These scores summarise the cumulative effect of thousands of common variants, each contributing a tiny amount, into a single number that estimates an individual’s inherited liability to a given trait. The researchers calculated scores for ADHD, autism, depression, anxiety, schizophrenia, bipolar disorder, educational attainment, and intelligence, and then asked two separate questions. Did children with clefts carry higher polygenic risk than controls? And within the cleft population, did polygenic scores predict actual developmental and behavioural outcomes measured through validated instruments such as the Strengths and Difficulties Questionnaire, the Ages and Stages Questionnaire, and the Moods and Feelings Questionnaire?

The results were revealing precisely because they diverged. Children with cleft lip and/or palate did not show elevated polygenic risk scores for any of the psychiatric or neurodevelopmental traits compared with population controls, nor lower scores for educational attainment or intelligence. Yet within the cleft group itself, the polygenic scores behaved exactly as they do in the general population: children with higher inherited liability to ADHD, for example, showed more ADHD-type behaviours, and children with higher educational attainment scores performed better developmentally. In other words, the genetic factors that shape neurodevelopment in children with clefts appear to be the same ones that shape it in everyone else, operating independently of cleft status rather than through a shared cleft-neurodevelopment pathway.

Third, the researchers turned to Mendelian randomization, a technique that exploits the random allocation of genetic variants at conception to test whether an exposure plausibly causes an outcome. Because genetic variants are inherited independently of the environmental and social confounders that plague observational research, they can serve as natural proxies. The team used genetic variants associated with cleft liability to ask whether genetic predisposition to clefts causally influences neurodevelopmental outcomes. The analysis provided no robust evidence that it does, further undermining the hypothesis that the cleft itself, through shared genetic roots, drives the increased rates of neurodevelopmental difficulty.

The study also examined the relationship between common and rare genetic variation, an area where prior findings had been much more positive. Previous work has established that rare neurodevelopmental copy number variants, large deletions or duplications of DNA segments that remove or multiply dozens of genes at once, are enriched among children with clefts and are associated with poorer developmental outcomes. If common and rare variants were acting through convergent biological pathways, one might expect children carrying these rare variants to also carry a heavier burden of common risk alleles. The team found no such difference: neurodevelopmental copy number variant carriers did not differ from non-carriers in their polygenic burden, suggesting the two classes of variation contribute independently.

The implications for clinical practice are significant. Because polygenic risk scores predict behavioural and developmental outcomes within the cleft population just as they do in the general population, these scores may retain clinical relevance for children with clefts, potentially helping to identify those who would benefit from early developmental monitoring and support. At the same time, the findings make clear that screening for clefts alone does not identify a population with elevated common variant liability to psychiatric illness. Clinicians and families should instead understand the increased neurodevelopmental risk in this group as arising from other sources, which may include the rare variants already identified, environmental exposures, perinatal complications, the social and surgical burdens associated with cleft care, or factors yet to be discovered.

The negative result also carries a broader lesson for psychiatric genetics. Comorbidities between apparently distinct conditions are often assumed to reflect shared genetic architecture, and in many cases that assumption holds. But this study demonstrates that a well-documented clinical association, between a major congenital malformation and neurodevelopmental difficulty, can exist without any detectable common genetic overlap. The mechanisms linking clefts to neurodevelopmental outcomes may be heterogeneous, involving rare mutations in some children, environmental and medical factors in others, and possibly the cumulative experience of living with a visible craniofacial difference and repeated surgical interventions. Disentangling these threads will require longitudinal data of the kind the Cleft Collective was designed to provide.

The research was funded by the Medical Research Council, with additional support from The Scar Free Foundation, The Underwood Trust, and the Vocational Training Charitable Trust, and it relied on the participation of thousands of families and the United Kingdom’s NHS cleft teams. By rigorously ruling out one of the most plausible genetic explanations for neurodevelopmental comorbidity in cleft lip and/or palate, the study sharpens the field’s focus on the mechanisms that remain. For the roughly one in seven hundred children born with an orofacial cleft, the message is cautiously reassuring: their inherited common genetic risk for psychiatric and cognitive difficulties looks much like anyone else’s, and the developmental challenges some of them face are not written into the same variants that shaped their cleft. Finding what does explain that risk is now the field’s central task.

Subject of Research: Common and rare genetic variant contributions to neurodevelopmental risk in children with orofacial clefts

Article Title: Common variant contributions to neurodevelopmental risk in orofacial clefts

Article References: Common variant contributions to neurodevelopmental risk in orofacial clefts. (n.d.). https://doi.org/10.1186/s12916-026-05171-6

Image Credits: AI Generated

DOI: 10.1186/s12916-026-05171-6

Keywords: orofacial clefts, cleft lip and palate, neurodevelopmental disorders, polygenic risk scores, Mendelian randomization, copy number variants, genetic correlation, ADHD, autism spectrum disorder, Cleft Collective, psychiatric genetics, BMC Medicine

The research team, led by Matthias Weider and Lina Gölz of University Hospital Erlangen together with colleagues in Bonn, began by making IRF6 visible in developing mouse embryos. Using transgenic animals in which all neural crest-derived tissue glows yellow thanks to a Wnt1::Cre-driven fluorescent reporter, the scientists stained embryonic sections at days 9.5, 10.5 and 11.5 of development. They found IRF6 protein in neural crest-derived cells of the pharyngeal arches, the embryonic structures that give rise to the jaw, palate and other facial components. Intriguingly, the fraction of neural crest cells carrying high levels of IRF6 shrank as development progressed: by embryonic day 11.5, only about 2.6 percent of these cells retained signal intensity comparable to that of epithelial cells, hinting at a narrow, precisely timed window of activity.

To confirm that this was not a quirk of the mouse or of the staining method, the team mined single-cell RNA sequencing data from embryonic craniofacial tissue of both mouse and human origin. The computational analysis revealed a subset of mesenchymal cells expressing IRF6 in both species. Crucially, many of these IRF6-positive cells also expressed SOX9, with 56 percent of IRF6-positive mesenchymal cells co-expressing the gene, and an even larger share, 86.8 percent, co-expressing TFAP2A, the transcription factor already known to regulate IRF6 in epithelium. The overlap placed IRF6 squarely inside the regulatory world of cranial neural crest cells, a transient, stem cell-like population whose migration, proliferation and differentiation must be choreographed with extraordinary precision for the face to form correctly.

SOX9 was a compelling suspect for several reasons. It is a bona fide cranial neural crest transcription factor, essential for assigning cells to the cartilage lineage, and it is the major gene mutated in Pierre Robin sequence, a craniofacial condition characterized by an underdeveloped lower jaw, a tongue that falls back into the throat, and frequently a cleft palate. Moreover, a single nucleotide variant called rs76145088, which genetic studies had previously linked to non-syndromic orofacial clefting, sits within a DNA sequence in the IRF6 enhancer MCS-9.7 that resembles a SOX9 binding site. That enhancer, located thousands of base pairs upstream of IRF6, was already famous: a different variant within it, rs642961, disrupts binding of TFAP2A and increases cleft risk, helping to establish MCS-9.7 as a hotspot of cleft-associated regulatory variation.

To test whether SOX9 really drives the enhancer, the researchers cloned MCS-9.7 into a luciferase reporter plasmid, a standard tool that glows when the enhancer is active. In neural crest-derived Neuro-2a cells, they first reproduced the known activation by TFAP2A, validating the system. They then added SOX9 and found that it, too, switched the enhancer on. The striking result concerned the rs76145088 variant: the ancestral allele, which is the risk allele for orofacial clefting, supported robust SOX9-dependent activation, while the derived allele, whose guanine base breaks the SOX9 consensus sequence, drove drastically weaker activation. Dose experiments sharpened the point, as 250 nanograms of SOX9 expression plasmid sufficed to activate the ancestral version of the enhancer, whereas three times that amount was needed for the derived version.

The team then moved from artificial reporters to endogenous biology. In O9-1 cells, a stable cell line derived from mouse cranial neural crest, they silenced Sox9 with short hairpin RNAs and watched the activity of the ancestral enhancer allele drop, confirming that normal cellular levels of Sox9 sustain MCS-9.7 activity. Electrophoretic mobility shift assays showed that purified SOX9 protein binds directly to the DNA sequence spanning rs76145088, and that it binds the ancestral allele more strongly than the derived one, exactly as the reporter experiments predicted. Chromatin immunoprecipitation in O9-1 cells, pulling down DNA bound by Sox9 inside living cells, enriched the region corresponding to MCS-9.7 relative to a negative control, albeit modestly, consistent with an enhancer that is active in only a small subset of cells.

The decisive experiment came next. Using CRISPR/Cas9 gene editing, the researchers knocked out Sox9 in O9-1 cells, generating two independent clones in which the protein was completely absent, one through a frameshift mutation and the other through a dramatic rearrangement including an inversion around the start codon. Quantitative PCR revealed that Irf6 transcript levels were virtually eliminated in both knockout clones. Other known upstream regulators of Irf6, including Tfap2a, Notch1, Foxe1, Trp63, the Pbx genes and several Wnt genes, showed only mild and inconsistent changes, arguing that SOX9 acts directly on the IRF6 enhancer rather than through intermediate regulators. Notably, the well-established IRF6 target gene Grhl3 was not expressed in these neural crest cells, suggesting that IRF6 may control a different set of downstream genes in cranial neural crest cells than it does in epithelium.

Perhaps the most thought-provoking twist is the direction of the risk effect. Usually, loss-of-function mutations in IRF6 cause clefting by reducing gene activity in epithelium. Here, the ancestral, risk-associated allele of rs76145088 is the one that binds SOX9 more strongly and drives higher enhancer activity. The authors therefore propose a gain-of-function mechanism: prolonged or excessive IRF6 expression in craniofacial mesenchyme could contribute to clefting, in the same way that enhanced binding of the transcription factor ETS1 to an SHH enhancer causes polydactyly. Because rs76145088 is common across human populations while clefts remain relatively rare, the finding fits the multifactorial threshold model of non-syndromic clefting, in which risk accumulates from many variants and environmental influences before a developmental threshold is crossed. The authors also note that the derived allele may be protective rather than the ancestral allele harmful, and that transcription factors like IRF6 must be kept within tight dosage windows, since both too little and too much activity can derail embryonic development.

The study does not close the case. Only a small fraction of cranial neural crest cells express IRF6, the enhancer carries both active and repressive histone marks suggestive of a poised or heterogeneous state, and previous mouse experiments deleting Irf6 in neural crest lineages produced bone mineralization defects but no palatal clefts. Still, by connecting a classic epithelial cleft gene to SOX9, the gene behind Pierre Robin sequence, the work draws two major cleft-associated pathways into a single regulatory axis and reveals that the developing face depends on IRF6 operating in more cellular neighborhoods than anyone had appreciated. Dysregulation of the SOX9-IRF6 axis in cranial neural crest cells, the authors conclude, may be a genuine contributor to the origins of orofacial clefting, and the heterogeneous mesenchymal cells that carry this signal are now an obvious target for the next generation of studies.

Subject of Research: Transcriptional regulation of the orofacial cleft risk gene IRF6 by SOX9 in cranial neural crest cells

Article Title: The orofacial cleft risk gene IRF6 is a target gene of SOX9 in cranial neural crest cells

Article References: Weider, M., Wagner, M. C., Schmid, T., Gehlen-Breitbach, S., Rodrian, G., Peschel, N., Schneider, H., Ludwig, K. U., Wegner, M., & Gölz, L. (2026). The orofacial cleft risk gene IRF6 is a target gene of SOX9 in cranial neural crest cells. Journal of Molecular Medicine, 104(1), Article 113. https://doi.org/10.1007/s00109-026-02720-7

Image Credits: AI Generated

DOI: 10.1007/s00109-026-02720-7

Keywords: orofacial cleft, IRF6, SOX9, cranial neural crest cells, cleft lip and palate, Pierre Robin sequence, MCS-9.7 enhancer, TFAP2A, CRISPR/Cas9, gene regulation, craniofacial development, single-cell RNA sequencing