Unexpected role of variants in craniosynostosis: expanding the phenotype of -related disorders.

Background: Pathogenic heterozygous variants (predominantly missense) occur in branchio-otic syndrome (BOS), but an association with craniosynostosis has not been reported.

Methods: We investigated probands with craniosynostosis of unknown cause using whole exome/genome (n=628) or RNA (n=386) sequencing, and performed targeted resequencing of in 615 additional patients. Expression of SIX1 protein in embryonic cranial sutures was examined in the reporter mouse.

Results: From 1629 unrelated cases with craniosynostosis we identified seven different variants (three missense, including two de novo mutations, and four nonsense, one of which was also present in an affected twin). Compared with population data, enrichment of loss-of-function variants was highly significant (p=0.00003). All individuals with craniosynostosis had sagittal suture fusion; additionally four had bilambdoid synostosis. Associated BOS features were often attenuated; some carrier relatives appeared non-penetrant. SIX1 is expressed in a layer basal to the calvaria, likely corresponding to the dura mater, and in the mid-sagittal mesenchyme.

Conclusion: Craniosynostosis is associated with heterozygous variants, with possible enrichment of loss-of-function variants compared with classical BOS. We recommend screening of in craniosynostosis, particularly when sagittal±lambdoid synostosis and/or any BOS phenotypes are present. These findings highlight the role of in cranial suture homeostasis.