Pawlik, Barbara (2010) Molecular mechanisms of congenital limb malformations. PhD thesis, Universität zu Köln.
Congenital limb malformations occur in 1 in 500 to 1 in 1000 human live births and are diverse in their epidemiology, aetiology and anatomy. The molecular analysis of disturbed gene function in inherited limb malformations provides essential information for the understanding of physiological and pathophysiological limb development in humans as well as in other vertebrates. The following Ph.D thesis focussed on the identification and molecular characterisation of disease causing genes and their pathophysiological mechanism for selected human limb defects such as Cenani-Lenz syndrome (CLS), Werner mesomelic syndrome (WMS), Bardet-Biedl syndrome (BBS), Split hand/ foot malformation (SHFM) and Temtamy preaxial brachydactyly syndrome (TPBS). In this context, we were able to identify novel limb specific genes and causative mutations in different components of evolutionary highly conserved pathways and, furthermore, to elucidate their role in physiological as well as in pathophysiological limb development. In detail, we found (i) alterations in the low-density-lipoprotein-related protein 4 (LRP4), an antagonistic receptor of Wnt signalling, causing the rare autosomal recessive CLS, (ii) specific mutations in the cis-acting limb-specific enhancer of the sonic hedgehog (SHH) gene being causative for WMS, and (iii) mutations in CHSY1 to be responsible for TPBS. Furthermore, we could show that mutations in the ciliary protein BBS12 can cause a very mild BBS phenotype. Moreover, we used in vitro studies to obtain insights into the molecular pathogenesis of these limb malformations. We studied the effect of five LRP4 mutants on the transduction and activation of canonical Wnt signalling by using a Dual-Luciferase Reporter Assay and showed that co-expression of each of the five missense mutations with LRP6 and WNT1 abolish the known antagonistic effect of LRP4 on LRP6-mediated activation of Wnt/ß-catenin signalling and thus conclude that homozygous LRP4 mutations in CLS cause a loss of protein function. Additionally, we functionally characterized the first autosomal recessive p.R332W mutation in the WNT10b gene causing SHFM6 and rise evidence that p.R332W causes loss of function of Lrp6-mediated Wnt signalling. In this regard we examined the role of the SHFM3 candidate gene Fgf8 in altering Wnt signalling and demonstrated that Fgf8 is a novel putative Wnt signalling antagonist which functions by direct interaction with Wnt10b. Hence, we present the first direct cross-talk between Fgf and Wnt signalling pathways and, therefore, physically link two important signalling pathways involved in limb initiation and outgrowth.
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