The Role of Disordered Ubiquitination in Pre- and Post-natal Growth Restriction

There are many children born each year who are small and then grow very slowly in childhood; in some this even continues when they are given treatment with growth hormone. We know how important it is for a child to grow normally so they can get the best from their lives. Often the cause for these children being born small is not known. We have been working on a condition called 3M syndrome, which is a rare cause of being born small and then not growing well after birth. Children with 3M also respond poorly when given growth hormone. We have found that the cause of 3M is a fault in a gene called Cullin 7.

Cullin 7 is found in cells in the body and helps to clear away waste proteins. However we do not know exactly how Cullin 7 affects growth. We intend to work out how Cullin 7 results in 3M children being born small and growing poorly. Knowing the mechanism behind the slow growth in 3M may also help us to find out why other children are born small and to develop new treatments to help more of these children grow better.

Low birth weight and being born small for gestational age (SGA) are associated with increased neonatal mortality, small stature in adulthood and cardiovascular mortality in later life. There are over 1300 children each year who qualify for treatment with recombinant human growth hormone (rhGH) due to being SGA with failure of catch up growth. We have focused on 3M syndrome (an autosomal recessive disorder characterised by facial dysmorphism, significant pre- and post-natal growth restriction and normal intelligence) which could be a model disorder to improve our understanding of mechanisms that control growth. Autozygosity mapping studies identified a locus on chromosome 6, and 25 distinct mutations in Cullin 7 (Cul7) in 29 families were found. Cul7 is believed to have a role as a scaffold protein in assembling an E3 ubiquitin ligase complex with Skp1, ROC1 and Fbx29 (1). 3M syndrome may be the result of failure to form this complex and the first demonstration that an abnormality in ubiquitination can cause growth failure.

We have identified that children with 3M syndrome respond very poorly to rhGH and have biochemical features of GH insensitivity.

This project aims to: 1) Describe the clinical phenotype of a cohort of children with 3M syndrome 2) Identify the prevalence of Cul7 mutations in 3M syndrome and more generally in SGA children, who show a poor response to rhGH treatment 3) Elucidate the underlying mechanism by which Cul7 mutations may impair growth and potentially indicate avenues for novel growth-promoting interventions 4) Assess the effects of knocking down Cul7 and its binding partners in a non-placental vertebrate.

Clinical, anthropometrical and biochemical data along with samples for DNA will be collected from children with 3M syndrome and SGA children who have a poor response to rhGH. Cul7 will be sequenced in all 3M patients and in our SGA children who demonstrate abnormal waveforms on heteroduplex wave analysis. To identify the mechanism of growth impairment in 3M children studies will be undertaken in normal and Cul7 mutant fibroblasts examining apoptosis and cell growth, basal gene expression, thymidine incorporation, GH receptor trafficking and GH signalling. Xenopus tropicalis will be used to study the effects of knocking down Cul7 using morpholino ologonucleotides in a non-placental vertebrate and to assess gene expression in vivo.

The opportunity exists to examine the role of ubiquitination in human growth and to identify and areas for potential therapeutic intervention for a common serious growth disorder.