Unexpected role ofSIX1variants in craniosynostosis: expanding the phenotype ofSIX1-related disorders

Abstract

BackgroundPathogenic heterozygous SIX1 variants (predominantly missense) occur in branchio-otic syndrome (BOS), but an association with craniosynostosis has not been reported.MethodsWe investigated probands with craniosynostosis of unknown cause using whole exome/genome (n=628) or RNA (n=386) sequencing, and performed targeted resequencing of SIX1 in 615 additional patients. Expression of SIX1 protein in embryonic cranial sutures was examined in the Six1nLacZ/+ reporter mouse.ResultsFrom 1629 unrelated cases with craniosynostosis we identified seven different SIX1 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 SIX1 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.ConclusionCraniosynostosis is associated with heterozygous SIX1 variants, with possible enrichment of loss-of-function variants compared with classical BOS. We recommend screening of SIX1 in craniosynostosis, particularly when sagittal±lambdoid synostosis and/or any BOS phenotypes are present. These findings highlight the role of SIX1 in cranial suture homeostasis.

Generation of a new six1-null line in Xenopus tropicalis for study of development and congenital disease

“…Mutations in SIX1 occur in the N-terminal protein-protein interaction domain (SD) or in the DNA-binding homeodomain (HD). A recent study also found Six1 mutations in patients with craniosynostosis and hearing defects (Calpena et al, 2021). Because Six1 has important roles in both normal and abnormal development of many tissues, we used Crispr/Cas 9 technology to create a null line in Xenopus tropicalis, an animal model highly suited for studying the cell and molecular regulation of gene function (Tandon et al 2017, Kakebeen and Wills 2019, Bhattacharya et al 2015.…”

“…Since mutations in human SIX1 underlie some cases of BOR and craniosynostosis (Smith, 2018;Calpena et al, 2021), this line also will be very useful for elucidating the role of this gene in these craniofacial syndromes.…”

Generation of a new six1‐null line in Xenopus tropicalis for study of development and congenital disease

“…This line of X. tropicalis will be a useful resource for the community seeking to elucidate the role of Six1 in the development of several tissues, including olfactory sensory epithelium, inner ear, cranial sensory ganglia, kidney, BAs, and somitic and hypaxial muscle. Since mutations in human SIX1 underlie some cases of BOR and craniosynostosis (Calpena et al, 2021;Smith, 2018), this line also will be very useful for elucidating the role of this gene in these craniofacial syndromes.…”

“…the N-terminal protein-protein interaction domain (SD) or in the DNA-binding homeodomain (HD). A recent study also found Six1 mutations in patients with craniosynostosis and hearing defects (Calpena et al, 2021). Because Six1 has important roles in both normal and abnormal development of many tissues, we used Crispr/Cas9 technology to create a null line in Xenopus tropicalis, an animal model highly suited for studying the cell and molecular regulation of gene function (Bhattacharya, Marfo, Li, Lane, & Khokha, 2015;Kakebeen & Wills, 2019;Tandon, Conlon, Furlow, & Horb, 2017).…”

Manzamine-A Alters In Vitro Calvarial Osteoblast Function

“…Strengthening this argument are data from the murine knockout model that present with multiple anomalies including those related to the musculoskeletal system [ 61 , 66 ]. SIX1 has also been shown to segregate in birth defects related to bone development leading to excess bone formation [ 86 ]. Importantly, if manzamine-A use is suspected of affecting bone health, additional patient populations may be implicated including those susceptible to bone loss diseases.…”