Mutations in the WNT mediator, DVL1, cause an osteosclerotic form of Robinow Syndrome

This project aimed to clinically and biochemically characterise a novel phenotype which we have named Robinow Syndrome – Osteosclerotic type (RS-OS). Robinow Syndrome (RS) is a rare form of mesomelic dwarfism defined by a distinctive facial gestalt known as “fetal facies” – a combination of features including hypertelorism and midface hypoplasia. RS can be caused by loss-of-function mutations in genes encoding components of planar cell polarity WNT signalling. Osteosclerosis is associated with gain-of-function mutations in mediators of a different aspect of WNT signalling, the canonical WNT pathway. This thesis details three sporadic cases of RS-OS. Before this project began a combination of next generation and Sanger sequencing identified that two of these individuals had similar heterozygous de novo mutations in gene for the pan-WNT component DVL1, which has not previously been associated with RS. A third case was identified during this project and Sanger sequencing revealed another, similar, de novo mutation in DVL1. All three DVL1 mutations fall within the 14th exon and cause a -1 frameshift which predicts a DVL1 product with the wild type C-terminal sequence replaced by a novel amino acid sequence, which is shared across all three affected individuals. We hypothesised that these DVL1 mutations cause osteosclerosis through a gain-of-function leading to an increase in canonical WNT signalling. Transcript from cell lysates of cultured dermal fibroblasts taken from an affected individual were analysed by Sanger sequencing and restriction enzyme digest revealed that the mutation-bearing mRNA was endogenously expressed. Transient transfection of mouse C2C12 cells with EGFP-tagged DVL1 constructs showed similar protein levels between mutant and wild type DVL1 with fluorescent Western blotting. Taken together these experiments showed that the mutant allele is endogenously transcribed as a persistent mRNA, and that the majority of the protein product of that transcript is stable. Canonical WNT signalling was studied with a transient transfection-based TOPFlash assay. These revealed that, paradoxical to the osteosclerotic phenotype, the mutant DVL1 was significantly less active in the canonical WNT pathway than wild type DVL1. However the co-expression of the mutant DVL1 alongside the wild type DVL1 lead to a significant (~3-fold, P < 0.01) increase in canonical WNT activity over the same amount of wild type DVL1 expressed alone. This co-expression may explain the clinical osteosclerosis: the affected individuals are heterozygous for the DVL1 mutations thus are likely to co-express mutant and wild type DVL1. This work establishes that novel mutations in DVL1, a gene previously not associated with RS, causes an osteosclerotic form of RS. These mutations are likely to lead to osteosclerosis through a gain-of-function mechanism, with an increase in canonical WNT signalling. However this gain-of-function, in vitro, depends upon the presence of wild type DVL1 alongside the mutant DVL1. This wild type-dependent gain-of-function is, to our knowledge, unique amongst Mendelian disorders