Phenotypic variability in Hey2 ?/? mice and absence of HEY2 mutations in patients with congenital heart defects or Alagille syndrome

Abstract

The genetic alterations leading to congenital heart defects (CHD) are still poorly understood. We and others have recently shown that in mice loss of Hey2 results in a high incidence of fatal ventricular and atrial septal defects, combined with tricuspid stenosis or atresia in some cases. The phenotype has been postulated to resemble human tetralogy of Fallot. Our analysis of CD1 outbred mice suggests that phenotypic consequences of Hey2 loss can be quite variable and dependent on modifier genes as we detected only isolated VSDs with lower prevalence and a significantly reduced mortality rate in this strain. Since Hey2 is one of the few Notch target genes, it is also conceivable that HEY2 mutations may account for cases of Alagille syndrome (AGS: variable combinations of heart, skeleton, eye, and facial malformations and cholestasis), in which the typical mutations of the Notch ligand JAG1 cannot be found. To clarify the role of HEY2 in human CHD and AGS, we screened by direct sequencing 23 children with CHD and 38 patients diagnosed with AGS, which lack mutations in the JAG1 gene. We found two types of silent changes in the coding region: a CTT-->CTG transition in exon 3 and a CTG-->CTC polymorphism in exon 5. Furthermore, a heterozygous SNP in the splice donor site of exon 4 was detected that is unlikely to disrupt splicing. Although the high incidence and variability of human congenital heart defects implies a multifactorial genetic basis, our results suggest that mutation of HEY2 is not a major contributing factor.

HEY2 mutations in malformed hearts

“…Indeed, only 2/52 patients had HEY2 sequence alterations. Likewise, after analysis of blood samples, two further studies did not find any HEY2 mutations in patients affected by CHD (Fischer et al, 2004, Sarkozy et al, 2005.…”

Hey Basic Helix-Loop-Helix Transcription Factors Are Repressors of GATA4 and GATA6 and Restrict Expression of the GATA Target Gene ANF in Fetal Hearts

“…HEK293, HEK293T, Cos7, and HeLa cells were cultured in Dulbecco's modified Eagle's medium (Gibco) with 10% fetal bovine serum. ES cells were maintained in ES medium containing Dulbecco's modified Eagle's medium with 10% ES cell qualified fetal bovine serum, 10 3 U/ml leukemia inhibitory factor, 50 mM ␤-mercaptoethanol, and minimal essential medium nonessential amino acids (Gibco) on mitomycininactivated fibroblasts. Before embryoid body formation ES cells were adapted to gelatin-coated culture dishes for three passages.…”

“…The hearts of newborn Hey2 knockout mice develop massive hypertrophy shortly after birth, and electron microscopy revealed structural defects in the myocardium of these mutants (14). In addition, there is a high incidence of ventricular septal defects, often accompanied by tricuspid atresia or dysplastic atrioventricular valves, atrial septal defects, and even pulmonary stenosis, depending on the genetic background (10).…”

Multiple gene variations contributed to congenital heart disease via GATA family transcriptional regulation

“…Notch signaling could also regulate the GATA-dependent cardiac gene expression [25]. Mutations in elements along this pathway result in cardiac malformation [26]. JAG1 is expressed in the mesocardium, pulmonary artery and aorta during embryogenesis [27].…”