other families and individuals,90,91 but overall they are rare in patients with congenitally malformed hearts Mouse models previously would not have predicted that heterozygous mutations in GATA4 could cause congenital cardiac disease, as mice heterozygous for Gata4 null alleles were normal Recent results have shown that, when bred in a particular strain, loss of one copy of Gata4 does indeed result in significant congenital cardiac disease, which resembles to a certain degree the malformations seen in humans with mutations of GATA4.91 Investigation of human cardiomyocytes derived from induced pluripotent stem cells from patients with GATA4 mutations and septal defects has begun to reveal the mechanisms by which mutations in this gene cause congenital heart disease.92 A combination of transcriptional and epigenetic studies suggests that GATA4 and TBX5 cooperate to activate and repress thousands of genes in the developing heart Specifically related to congenital heart defect (CHD), they activate genes that allow cardiomyocytes to respond to the morphogen, sonic hedgehog, which is secreted by the neighboring pulmonary endoderm during development, and properly develop tissue required for septation Inability to respond to this critical signal may underlie the septal defects observed in patients with GATA4 and possibly TBX5 mutations FIG 4.3 GATA4 mutations cause septal defects Family A is a fivegeneration kindred with several family members afflicted with congenital malformations of the heart, as listed in the table to the right of the family tree Family B has inherited defects across four generations; defects are listed in the table to the right of the family tree In both families, affected members with a + sign carry a GATA4 mutation An echocardiogram of one patient shows atrial and ventricular septal defects AR, Aortic regurgitation; ASD, atrial septal defect; CHD, congenital heart defect; LA, left atrium; LV, left ventricle; MR, mitral regurgitation; PDA, patent arterial duct; PS, pulmonary stenosis; RA, right atrium; RV, right ventricle; VSD, ventricular septal defect (Modified from Garg V, Kathiriya IS, Barnes R, et al GATA4 mutations cause human congenital heart defects and reveal an interaction with TBX5 Nature 2003;424:443–447.) Mutations of NOTCH1 Cause Bicuspid Aortic Valve Malformations of the valves are due to improper valvogenesis, and can vary in severity As such, they may not be recognized until adulthood, when the valves begin to malfunction The most common cause of valvar disease is seen in patients with aortic valves having two instead of three leaflets, the so-called bicuspid aortic valve Found with a prevalence of 1% to 2% in the population, it is the most common congenital cardiac anomaly, affecting more patients than all other defects combined The bifoliate valve can cause disease in childhood if the valvar abnormality is severe, and can be part of the hypoplastic left heart syndrome Indeed, one-sixth of first-degree relatives with hypoplasia of the left heart have bifoliate aortic valves not necessarily producing symptoms, suggesting a common genetic etiology The bifoliate aortic valve is more frequently asymptomatic until later decades of life, when premature calcification, prolapse, or bacterial endocarditis occurs In those with calcification, the valve becomes stenotic or regurgitant, leading to dysfunction that eventually requires valvar replacement This disease is the third most common form of cardiac disease seen in adults, and over 50,000 bifoliate aortic valves are replaced annually in the United States of America alone Recently, through investigation of families with autosomal dominant disease, mutations in NOTCH1 were identified as a genetic cause of the valvar malformations and calcification.17 Linkage studies mapped the disease locus to mutations in NOTCH1 in two unrelated families with a similar valvar phenotype (Fig 4.4), specifically severe premature calcification of a bifoliate valve Interestingly, a subset of family members who harbored mutations in NOTCH1 had trifoliate aortic valves, but still developed calcification that required subsequent valvar replacement Thus NOTCH1 signaling may be required to suppress calcification of the mesenchymal cells of the valve under normal circumstances Indeed, experimental studies showed that NOTCH1 represses the osteoblast phenotype that is typical of valvar and vascular calcification Similar to the approach for GATA4 mutations, study of endothelial cells derived from human iPS cells made from patients with NOTCH1 mutations revealed new insight into the mechanism of the calcification process.93 In a dose-sensitive manner, osteogenic and inflammatory genes normally repressed by NOTCH1 were de-repressed in patient-derived cells, suggesting a constitutive process that is necessary to keep valve leaflets in the thin, membranous state rather than the thick, calcified state typical of disease As for other familial disease, a wide spectrum of lesions was observed within family members, including tetralogy of Fallot, hypoplasia of the left heart, and ventricular septal defect, likely due to genetic background features