Atrial septal defect is one of the most common forms of congenital heart malformation. We identified a new locus linked with atrial septal defect on chromosome 14q12 in a large family with dominantly inherited atrial septal defect. The underlying mutation is a missense substitution, I820N, in alpha-myosin heavy chain (MYH6), a structural protein expressed at high levels in the developing atria, which affects the binding of the heavy chain to its regulatory light chain. The cardiac transcription factor TBX5 strongly regulates expression of MYH6, but mutant forms of TBX5, which cause Holt-Oram syndrome, do not. Morpholino knock-down of expression of the chick MYH6 homolog eliminates the formation of the atrial septum without overtly affecting atrial chamber formation. These data provide evidence for a link between a transcription factor, a structural protein and congenital heart disease.

Atrial septal defect (ASD) is one of the most frequent congenital heart defects (CHDs) with a variable phenotypic effect depending on the size of the septal shunt. We identified two pedigrees comprising 20 members segregating isolated autosomal dominant secundum ASD. By genetic mapping, we identified the gene-encoding alpha-cardiac actin (ACTC1), which is essential for cardiac contraction, as the likely candidate. A mutation screen of the coding regions of ACTC1 revealed a founder mutation predicting an M123V substitution in affected individuals of both pedigrees. Functional analysis of ACTC1 with an M123V substitution shows a reduced affinity for myosin, but with retained actomyosin motor properties. We also screened 408 sporadic patients with CHDs and identified a case with ASD and a 17-bp deletion in ACTC1 predicting a non-functional protein. Morpholino (MO) knockdown of ACTC1 in chick embryos produces delayed looping and reduced atrial septa, supporting a developmental role for this protein. The combined results indicate, for the first time, that ACTC1 mutations or reduced ACTC1 levels may lead to ASD without signs of cardiomyopathy.

Holt-Oram syndrome is caused by mutations in TBX5, a member of the T-box gene family. In order to identify DNA sequences to which the TBX5 protein binds, we have performed an in vitro binding site selection assay. We have identified an 8 bp core sequence that is part of the Brachyury consensus-binding site. We show that TBX5 binds to the full palindromic Brachyury binding site and to the half-palindrome, whereas Brachyury does not bind to the TBX5 site. Amino acids 1-237 of TBX5 are required for DNA binding. Analysis of the effects of specific substitution mutations that arise in Holt-Oram patients indicates that G80R and R237Q eliminate binding to the target site. DNA database analysis reveals that target sites are present in the upstream regions of several cardiac-expressed genes including cardiac alpha actin, atrial natriuretic factor, cardiac myosin heavy chain alpha, cardiac myosin heavy chain beta, myosin light chain 1A, myosin light chain 1V and Nkx2.5. Cell transfection studies demonstrate that TBX5 activates the transcription of an atrial natriuretic factor reporter construct and this effect is significantly reduced by deletion of the TBX5 binding site.