Functional analysis of novel TBX5 T-box mutations associated with Holt-Oram syndrome.

AIMS: Holt-Oram syndrome (HOS) is a heart/hand syndrome clinically characterized by upper limb and cardiac malformations. Mutations in T-box transcription factor 5 (TBX5) underlie this syndrome, the majority of which lead to premature stops. In this study, we present our functional analyses of five (novel) missense TBX5 mutations identified in HOS patients, most of whom presented with severe cardiac malformations. METHODS AND RESULTS: Functional characterization of mutant proteins shows a dramatic loss of DNA-binding capacity, as well as diminished binding to known cardiac interaction partners NKX2-5 and GATA4. The disturbance of these interactions leads to a loss of function, as measured by the reduced activation of Nppa and FGF10 in rat heart derived cells, although with variable severity. Two out of the five mutations are peculiar: one, p.H220del, is associated with additional extra-cardiac defects, perhaps by interfering with other T-box dependant pathways, and another, p.I106V, leads to limb defects only, which is supported by its normal interaction with cardiac-specific interaction partners. CONCLUSION: Overall, our data are consistent with the hypothesis that these novel missense mutations in TBX5 lead to functional haploinsufficiency and result in a reduced transcriptional activation of target genes, which is likely central to the pathogenesis of HOS.

Expression of muscle segment homeobox genes in the developing myocardium.

Msx1 and Msx2 are essential for the development of many organs. In the heart, they act redundantly in development of the cardiac cushions. Additionally, Msx2 is expressed in the developing conduction system. However, the exact expression of Msx1 has not been established. We show that Msx1 is expressed in the cardiac cushions, but not in the myocardium. In Msx2-null mice, Msx1 is not ectopically expressed in the myocardium. The absence of myocardial defects in the Msx2 knock-out can therefore not be attributed to a redundant action of Msx1 in the myocardium.

Protein interactions at the heart of cardiac chamber formation.

The vertebrate heart is a muscular pump that contracts in a rhythmic fashion to propel the blood through the body. During evolution, the morphologically complex four-chambered heart of birds and mammals has evolved from a single-layered tube with peristaltic contractility. The heart of Drosophila, referred to as the dorsal vessel, is a blind sac composed of myogenic cells that contract rhythmically. The fish heart is composed of a single atrial chamber connected to a single ventricular chamber. The evolutionary development of fast-contracting chambers allowed the heart to build up high blood pressures. In amphibians two atrial chambers exist, separated by a septum, connecting to a single ventricle. The division of a common atrium and ventricle into right and left-sided chambers represents an evolutionary milestone in the development of the four-chambered heart and is necessary for separation of oxygenated and deoxygenated blood. In amphibians and reptiles, pulmonary and systemic circulations are incompletely separated allowing adaptable blood flows to both circulations. In contrast, the hearts of birds and mammals, in which septa completely separate the pulmonary and systemic circulations, both circulations have similar flows, but blood pressures can be regulated separately. In this review we focus, in a morphologically integrated fashion, on the molecular interactions that govern the intricate cardiac design.