Identification of a novel variant of the ciliopathic gene FUZZY associated with craniosynostosis

William B Barrell,Marieke Van Dooren,Elena Torban,Hadeel Adel Al-Lami,Peter J Van Der Spek,Irene M J Mathijssen,Sigrid M A Swagemakers,Karen J Liu,Jacqueline A C Goos

doi:10.1038/s41431-021-00988-6

Abstract

Craniosynostosis is a birth defect occurring in approximately one in 2000 live births, where premature fusion of the cranial bones inhibits growth of the skull during critical periods of brain development. The resulting changes in skull shape can lead to compression of the brain, causing severe complications. While we have some understanding of the molecular pathology of craniosynostosis, a large proportion of cases are of unknown genetic aetiology. Based on studies in mouse, we previously proposed that the ciliopathy gene Fuz should be considered a candidate craniosynostosis gene. Here, we report a novel variant of FUZ (c.851 G > C, p.(Arg284Pro)) found in monozygotic twins presenting with craniosynostosis. To investigate whether Fuz has a direct role in regulating osteogenic fate and mineralisation, we cultured primary osteoblasts and mouse embryonic fibroblasts (MEFs) from Fuz mutant mice. Loss of Fuz resulted in increased osteoblastic mineralisation. This suggests that FUZ protein normally acts as a negative regulator of osteogenesis. We then used Fuz mutant MEFs, which lose functional primary cilia, to test whether the FUZ p.(Arg284Pro) variant could restore FUZ function during ciliogenesis. We found that expression of the FUZ p.(Arg284Pro) variant was sufficient to partially restore cilia numbers, but did not mediate a comparable response to Hedgehog pathway activation. Together, this suggests the osteogenic effects of FUZ p.(Arg284Pro) do not depend upon initiation of ciliogenesis.

Highlights

Craniosynostosis is the premature fusion of one or more sutures of the skull vault and has an incidence of 1:2000 live births [1,2,3]
Using an Osx-1::GFP-cre reporter line to lineage label the osteoblast precursors, we showed that the frontal bone mesenchyme was expanded at the expense of the parietal bone [30], which could manifest as craniosynostosis, or an absence of the coronal suture in late gestation animals
Novel missense mutation in FUZ identified in twins presenting with craniosynostosis We performed whole genome sequencing and identified a novel homozygous variant in FUZ

Summary

INTRODUCTION

Craniosynostosis is the premature fusion of one or more sutures of the skull vault and has an incidence of (approximately) 1:2000 live births [1,2,3]. Recent work has shown that Fuz depletion leads to a gradual loss of the cilium, due to a failure of the retrograde intraflagellar transport machinery [19] This explains why Fuz loss-of-function mouse mutants do not demonstrate early embryonic lethality seen in core ciliogenesis or IFT pathway genes. Using an Osx-1::GFP-cre reporter line to lineage label the osteoblast precursors, we showed that the frontal bone mesenchyme was expanded at the expense of the parietal bone [30], which could manifest as craniosynostosis, or an absence of the coronal suture in late gestation animals These studies identified a clear role for FUZ in early establishment of cell fate and cranial bone tissue boundaries. At birth 2125 grams, skull circumference 31 cm, gestation 36 weeks and 4 days embryonic fibroblasts, suggesting that this variant may reveal cilia dependent and independent functions of FUZ

RESULTS

DISCUSSION

MATERIALS AND METHODS

ETHICAL APPROVAL