The Genetics of Skeletogenesis
2012
Holman, Sarah Katherine Fay
WTX is a member of a protein family bearing no homology to proteins with known functional domains with in silico analysis, but is of substantial interest because germline and somatic mutations lead to the developmental disease osteopathia striata congenita with cranial sclerosis and the childhood renal malignancy, Wilms tumour. The full-length spliceform, WTXS1 contains binding domains for ß-catenin, axin, axin2, WT1, APC, axin, p53, KEAP1, ß-TrCP2 and a domain that binds PIP2 that localises the protein to the plasma membrane. The alternatively spliced transcript, WTXS2, excludes residues that are represented in WTXS1 that mediate the binding of PIP2. These binding domains for multiple proteins facilitate the role of WTX in many cellular functions including the canonical WNT signalling pathway, relocalisation of the subcellular distribution of APC, cell cycle regulation and transcriptional regulation. The aim of this study was to characterise the functions and mechanistic properties of WTX that relate to the generation of OSCS using a combination of genotype-phenotype analysis, investigation of the phenotype in wtx morphant zebrafish and mRNA rescue experiments utilising mutated WTX alleles.
A comprehensive genotype-phenotype analysis in a cohort of individuals with OSCS resulted in the characterisation of two subsets of the male phenotype, a severe disorder with multiple structural and patterning malformations resulting from mutations in WTX located 5’ to the first APC binding domain, and a milder manifestation that has additional neuromuscular anomalies and results from mutations in WTX located more 3’. The identification of three affected individuals with nephroblastomatosis, a precursor toward Wilms tumour, indicates that mutations in WTX may predispose toward formation of precursor lesions however additional mutations are probably required for progression to tumourigenesis.
Utilisation of the zebrafish as a model organism for the function of wtx during development resulted in the definition of a novel role for wtx in the specification of neural crest cells. Knockdown of Wtx using morpholino oligonucleotide technology resulted in reduced specification of and a decrease in number of neural crest cells entering the branchial arches resulting in malformed craniofacial cartilage, loss of the enteric nervous system and pigmentation abnormalities. The wtx morphant phenotype was then used as readout for phenotypic rescue following injection of mRNA encoding modified WTX alleles. From these experiments it was concluded that WTXS2 is not necessary for correct embryogenesis and alleles that result in a severe OSCS phenotype in males are non-functional. Site-directed mutagenesis of residues involved in localisation of WTXS1 to the plasma membrane results in a non-functional allele that is unable to rescue the wtx morphant phenotype, suggesting this ability is essential for its function. In aggregate these studies have facilitated the definition of the critical domains required for correct function of WTX during development.
In summary this thesis defines osteopathia striata congenita with cranial sclerosis as a neurocristopathy through identification for a role of wtx in neural crest development. It presents the first integrated view on the function of wtx during the development of a whole organism and has contributed information on the human phenotype to aid in the counselling of affected families.