This study focused on a small group of candidate genes from a signaling pathway important in cardiac development. We identified an association between ERBB4, a gene encoding a receptor tyrosine kinase of the EGFR family, and LVOT defects. The association with LVOT defects is noted not only for the whole group of defects, but also individually for AVS, COA, and HLHS.
The EGFR signaling pathway is important in cardiac development. ERBB4
(Gassmann et al., 1995
) and NRG1
(Meyer and Birchmeier, 1995
) mouse knockouts demonstrate decreased ventricular trabeculation, whereas ERBB3
knockout animals have valve abnormalities (Erickson et al., 1997
is a single pass transmembrane receptor tyrosine kinase (Fuller et al., 2008
). It homodimerizes or heterodimerizes with other ERB receptors, and is activated by binding with a number of ligands, including Neuregulin1. The gene is located on chromosome 2, covering 1.16 Mb, and contains 28 exons coding for a 1308 amino acid protein. At least four splice variants are known (Junttila et al., 2003
). Isoforms JM-a and JM-b are formed by the excision of exon 15 or exon 16, respectively. The CYT-1 isoform contains exon 26, whereas in CYT-2, this exon is spliced out. The presence of JM-a allows cleavage of the receptor and release of an intracellular domain, with subsequent degradation of the receptor. CYT-1 contains a PI3K docking site. Recently, a set of three SNPs around exon 3 were found to be associated with the amount of CYT-1 expression in the brain of patients with schizophrenia (Law et al., 2007
The precise LVOT defect susceptibility variant in ERBB4
is not yet known. No coding sequence changes were identified. The associated haplotype lies within intron 3 of the gene, thus a mutation in this area would not have been identified by our sequencing strategy. Investigation of altered splice variant ratios in lymphoblastoid cell lines from individuals with LVOT defects did not reveal any obvious changes. However, ERBB4
is not well expressed in this cell type (Sei et al., 2007
) and may not be indicative of gene expression patterns in the developing heart. Bioinformatics analysis of haplotype block 5 suggests this region may be important based on areas of conservation and presence of enhancer marks contained within the block region.
Recent work from our laboratories, along with the current report, suggests a possible endothelial/endocardial development defect in the pathogenesis of LVOT defects. We have found NOTCH1
mutations among sporadic cases of AVS, COA, and HLHS, which seem to confer an increased susceptibility to these defects via altered signaling through the NOTCH pathway (McBride et al., 2008
is required for both endocardial (Timmerman et al., 2004
) and myocardial differentiation (Grego–Bessa et al., 2007
). Conditional knockouts of NOTCH1
in the mouse cause abnormal endothelial to mesenchymal transformation in the developing heart and alter NRG1
expression in the developing ventricular trabeculae (Grego–Bessa et al., 2007
), highlighting the interaction between the NOTCH and EGFR pathways in heart development.
The involvement of this pathway may explain other phenomena observed in LVOT malformations. NRG1
is becoming more appreciated in cardiovascular physiology through its role in cardiac sympathetic response (Pentassuglia and Sawyer, 2009
). This may contribute to postrepair hypertension in individuals with COA (O’Sullivan et al., 2002
seem to be cardioprotective, and dysregulation causes heart failure, particularly in the setting of drug-induced cardiomyopathy such as with herceptin for breast cancer therapy (Perez, 2008
). Defects in this signaling cascade may thus contribute to poor response due to ventricular failure seen in many individuals between stages 1 and 2 for HLHS palliation (Simsic et al., 2005
A possible weakness of our study is inherent to all candidate gene studies: success depends on selection of the correct genes. Although the candidates represented are known to be involved in cardiac development, it is estimated that there may be at least 500 genes critical for normal cardiac development. Our study was also small, with a higher risk of false-negative results.
In summary, we have identified an association between SNPs in the gene ERBB4 and LVOT defects, collectively and by individual anatomic defect. ERBB4 joins NOTCH1 as a susceptibility gene for LVOT defects. Replication of these results in other cohorts of subjects will be required.