RAB23 mutations and insights into craniosynostosis

Growth of the skull occurs at the cranial sutures, which are seams of tissue that separate the skull bones. Early closure of the seams is termed craniosynostosis and may cause a variety of problems – for example affecting vision, breathing, intelligence and psychological development. Although we know that craniosynostosis is sometimes caused by a misprint in a genetic instruction, many of these misprints remain unknown.

Recently we identified a new gene, termed RAB23, that is altered in craniosynostosis. This was surprising because RAB23 belongs to a set of genetic instructions that were not previously thought to be involved in this condition. We hope to find out whether changes in related instructions also cause craniosynostosis or other malformations. We will use various methods in this research, including scanning genes in humans and mice for misprints, looking at how protein components stick to each other, and altering the instructions in bones isolated from mouse skulls.

By understanding more about how the skull normally develops and how craniosynostosis arises, we aim to develop new diagnostic tests, and hence provide patients and families with better information on the causes of these conditions and the chances that they could occur again in future children.

Craniosynostosis is an important human disease caused by the premature fusion of the sutures between the skull bones (birth prevalence 1 in 2,500). Severe cases are associated with multiple complications (physical, neurodevelopmental and psychological) requiring multidisciplinary management. An underlying genetic cause can currently be identified in about 25% of cases: this provides information that is fundamental for precise diagnosis, management and counselling. In the remaining 75%, the challenge is to distinguish those that also have an unidentified single gene aetiology from others with a more complex polygenic/environmental causation.

We have recently discovered the gene that is mutated in Carpenter syndrome, a rare recessive disorder comprising complex craniosynostosis, polysyndactyly, obesity, cardiac defects and learning difficulties. Unexpectedly the gene encodes RAB23, a negative regulator of signalling by hedgehog proteins, which are involved in the development of many organs but have not previously been implicated in craniosynostosis.

This project will explore the contribution of RAB23 mutations to disease, and of hedgehog signalling proteins to craniosynostosis. First, we aim to understand the mutation spectrum in RAB23 itself. To date we have analysed 11 unrelated mutation positive families but only identified 4 distinct mutations, probably owing to founder effects. We will recruit and analyse additional Carpenter syndrome patients, especially those from different ethnic backgrounds. We will test the effects of different mutations on nonsense-mediated decay in patient cells and artificial constructs, and explore genotype/phenotype correlations in RAB23 mutation by analysing patients with acrocallosal syndrome, a possibly allelic condition, and seeking additional murine Rab23 mutations in an ethylnitrosourea mutagenesis screen. This information will assist transfer of this work into the clinical molecular diagnostic laboratory, which should be achieved by the end of year 1. Next we will screen additional candidate genes in the hedgehog pathway for mutations in a large panel of DNA samples from patients with undiagnosed craniosynostosis and/or limb malformations. We will focus on the best candidates which are (1) Indian hedgehog, the ligand expressed in cranial sutures; (2) genes that, like Rab23, exhibit activation of hedgehog signalling in mouse mutants; and (3) genes encoding proteins that interact directly with Rab23, which we will identify by a combination of yeast 2-hybrid and proteomics-based screening. Finally, we will manipulate Rab23 activity in an in vitro culture of murine cranial sutures using morpholino knockdown, and monitor the effects of this on morphology and expression of genes implicated in hedgehog signalling and craniosynostosis.