Abstract
BackgroundAutosomal dominant Holt-Oram syndrome (HOS) is caused by mutations in the TBX5 gene and is characterized by congenital heart and preaxial radial ray upper limb defects. Most of the TBX5 mutations found in patients with HOS cause premature truncation of the primary TBX5 transcript. TBX5 missense mutations alter the three-dimensional structure of the protein and result in failed nuclear localization or reduced binding to target DNA. In this study we present our functional analyses of the novel and unusual c.1333delC mutation found in a patient with classical HOS.MethodsThe functional impact of this novel mutation was assessed by investigating the intracellular localization of the resulting TBX5 protein and its ability to activate the expression of its downstream target ANF.ResultsThe deletion of the cytosine is the first TBX5 frameshift mutation predicted to result in an elongated TBX5 protein with 74 miscoding amino acids and 62 supernumerary C-terminal amino acids. The c.1333delC mutation affects neither the nuclear localization, nor its colocalization with SALL4, but severely affects the activation of the ANF promoter.ConclusionThe mutation c.1333delC does not locate within functional domains, but impairs the activation of the downstream target. This suggests that misfolding of the protein prevents its biological function.
Highlights
Autosomal dominant Holt-Oram syndrome (HOS) is caused by mutations in the TBX5 gene and is characterized by congenital heart and preaxial radial ray upper limb defects
C.1333delC does not influence intracellular localization The entire coding sequence of human wild type and mutated TBX5 was cloned into the green fluorescent protein (GFP) expression plasmid pEGFP-N3, whereas the coding sequence of SALL4 was inserted into the FLAG expression plasmid pFLAGN3, resulting in TBX5wt-GFP, TBX5mut-GFP and SALL4FLAG fusion proteins
TBX5 was shown to display a nuclear localization during embryogenesis, thereby having two distinct nuclear localization signals working cooperatively [17,19]
Summary
Autosomal dominant Holt-Oram syndrome (HOS) is caused by mutations in the TBX5 gene and is characterized by congenital heart and preaxial radial ray upper limb defects. Most of the TBX5 mutations found in patients with HOS cause premature truncation of the primary TBX5 transcript. TBX5 missense mutations alter the three-dimensional structure of the protein and result in failed nuclear localization or reduced binding to target DNA. Holt-Oram syndrome (HOS) is an autosomal-dominantly inherited disorder caused by mutations in the gene coding for the T-box transcription factor TBX5, which is located on chromosome 12q24 [1,2]. The detection rate of mutations in the TBX5 gene in patients with familial or sporadic HOS is 74% following application of stringent clinical criteria [9]
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