Background-Mutations of the transcription factor Nkx2-5 cause pleiotropic heart defects with incomplete penetrance.This variability suggests that additional factors can affect or prevent the mutant phenotype. We assess here the role of genetic modifiers and their interactions. Methods and Results-Heterozygous Nkx2-5 knockout mice in the inbred strain background C57Bl/6 frequently have atrial and ventricular septal defects. The incidences are substantially reduced in the Nkx2-5 ϩ/Ϫ progeny of first-generation (F1) outcrosses to the strains FVB/N or A/J. Defects recur in the second generation (F2) of the F1ϫF1 intercross or backcrosses to the parental strains. Analysis of Ͼ3000 Nkx2-5 ϩ/Ϫ hearts from 5 F2 crosses demonstrates the profound influence of genetic modifiers on disease presentation. On the basis of their incidences and coincidences, anatomically distinct malformations have shared and unique modifiers. All 3 strains carry susceptibility alleles at different loci for atrial and ventricular septal defects. Relative to the other 2 strains, A/J carries polymorphisms that confer greater susceptibility to atrial septal defect and atrioventricular septal defects and C57Bl/6 to muscular ventricular septal defects. Segregation analyses reveal that Ն2 loci influence membranous ventricular septal defect susceptibility, whereas Ն2 loci and at least 1 epistatic interaction affect muscular ventricular and atrial septal defects. Conclusions-Alleles of modifier genes can either buffer perturbations on cardiac development or direct the manifestation of a defect. In a genetically heterogeneous population, the predominant effect of modifier genes is health. (Circulation. 2010;121:1313-1321.)Key Words: genetic variation Ⅲ genetics Ⅲ heart defects, congenital S ignificant progress has been made toward defining genetic origins in congenital heart disease. 1,2 Current knowledge of the few dozen genes implicated in cardiac development, however, does not explain the basis of common epidemiological and clinical observations. The incidence of heart defects in newborns is 0.5% to 1%, making it a leading cause of death in children. Still, the vast majority are born with a normal heart. Selection against fetuses that have cardiac defects does not cause the prevalence of the norm. 3,4 Insults on the embryonic heart may be rare or weak, or mechanisms may have evolved to ensure the robustness of development. Direct evidence for either hypothesis is minimal, but the latter can better explain the breadth of observations related to incomplete penetrance and phenotypic variability. 5,6 Editorial see p 1277 Clinical Perspective on p 1321Although family studies led to the discovery of mutations of prototypical cardiac developmental genes such as TBX5, Nkx2-5, and GATA4, 7-10 most cases of congenital heart disease have no identified cause or association. Such sporadic cases may be due to an unknown teratogen, spontaneous mutation, or inconsistent expression of an inherited origin. The last possibility is supported by the increased incidence of...