natriuretic-peptide--c-type and Syndrome

Mutations in SALL1 lead to the dominant multiorgan congenital anomalies that define Townes-Brocks syndrome (TBS). The majority of these mutations result in premature termination codons that would be predicted to trigger nonsense-mediated decay (NMD) of mutant mRNA and cause haploinsufficiency. Our previous studies using a gene targeted mouse model (Sall1-DeltaZn) suggested that TBS phenotypes are due to expression of a truncated mutant protein, not haploinsufficiency. In this report, we strengthen this hypothesis by showing that expression of the mutant protein alone in transgenic mice is sufficient to cause limb phenotypes that are characteristic of TBS patients. We prove that the same pathogenetic mechanism elucidated in mice is occurring in humans by demonstrating that truncated SALL1 protein is expressed in cells derived from a TBS patient. TBS mutant protein is capable of dominant negative activity that results in ectopic activation of two downstream genes, Nppa and Shox2, in the developing heart and limb. We propose a model for the pathogenesis of TBS in which truncated Sall1 protein causes derepression of Sall-responsive target genes.

Topics: Abnormalities, Multiple; Animals; Atrial Natriuretic Factor; Codon, Nonsense; Heart Defects, Congenital; Humans; Limb Deformities, Congenital; Mice; Mice, Transgenic; Mutation; Natriuretic Peptide, C-Type; Phenotype; Protein Precursors; RNA Stability; Syndrome; Transcription Factors; Transcriptional Activation

The cardiac homeobox protein Nkx2-5 is essential in cardiac development, and mutations in Csx (which encodes Nkx2-5) cause various congenital heart diseases. Using the yeast two-hybrid system with Nkx2-5 as the 'bait', we isolated the T-box-containing transcription factor Tbx5; mutations in TBX5 cause heart and limb malformations in Holt-Oram syndrome (HOS). Co-transfection of Nkx2-5 and Tbx5 into COS-7 cells showed that they also associate with each other in mammalian cells. Glutathione S-transferase (GST) 'pull-down' assays indicated that the N-terminal domain and N-terminal part of the T-box of Tbx5 and the homeodomain of Nkx2-5 were necessary for their interaction. Tbx5 and Nkx2-5 directly bound to the promoter of the gene for cardiac-specific natriuretic peptide precursor type A (Nppa) in tandem, and both transcription factors showed synergistic activation. Deletion analysis showed that both the N-terminal domain and T-box of Tbx5 were important for this transactivation. A G80R mutation of Tbx5, which causes substantial cardiac defects with minor skeletal abnormalities in HOS, did not activate Nppa or show synergistic activation, whereas R237Q, which causes upper-limb malformations without cardiac abnormalities, activated the Nppa promoter to a similar extent to that of wildtype Tbx5. P19CL6 cell lines overexpressing wildtype Tbx5 started to beat earlier and expressed cardiac-specific genes more abundantly than did parental P19CL6 cells, whereas cell lines expressing the G80R mutant did not differentiate into beating cardiomyocytes. These results indicate that two different types of cardiac transcription factors synergistically induce cardiac development.

Topics: Atrial Natriuretic Factor; Binding Sites; Cell Differentiation; Cell Line; Genes, Reporter; Heart Defects, Congenital; Homeobox Protein Nkx-2.5; Homeodomain Proteins; Humans; Limb Deformities, Congenital; Mutation; Myocardial Contraction; Myocardium; Natriuretic Peptide, C-Type; Promoter Regions, Genetic; Protein Binding; Protein Precursors; Syndrome; T-Box Domain Proteins; Transcription Factors; Transcriptional Activation; Xenopus Proteins