Identification of lines with CHD

Mice from three distinct ENU mutagenesis screen cohorts were evaluated for structural heart defects: (i) Brigham and Women's Hospital (BWH); (ii) The Jackson Laboratory; and (iii) Baylor College of Medicine. From each cohort, animals that died in the perinatal period (defined as the first 5 days of life) were evaluated by cardiac pathology to ascertain CHD. From the BWH cohort, serial cardiac histology from 68 deceased animals revealed 12 with structural heart defects, or 18% of the total number that died perinatally (Table 1). Structural abnormalities observed included septal defects [atrial septal defect (ASD) (3), ventricular septal defects (VSD) (2), AVSDs (5) and routing abnormalities (double-outlet right ventricle: 1)], all analogues of human congenital heart defects (Table 1). Four of the 32 lines had multiple affected progeny. One such line demonstrated inherited AVSDs. A second line demonstrated multiple affected pups with atrial septal defects, ventricular septal defects, or both. Additional affected animals from the other two lines with affected mice were sought from four sequential litters each; however, none was identified. These results demonstrated that perinatal lethality was an effective pre-screen for ENU-induced CHD.

Table 1.

ENU mutagenesis increased the rate of perinatal lethality and generated 12 lines of heritable cardiovascular defects

Analysis of CHD by screena	Control screen	Mutagenized screen
Total lethals evaluated for CHD	4	441
Total evaluated for CHD	246	441
Number with CHD	0	79
CHD rate per lethal	0%	17.9%
CHD rate per liveborn	0%	1.3%
Total lines evaluated		321
Lines with CHD		47
CHD rate per line		14.6%
Analysis of CHD by classb		Per line
Total affected		47
Septation defects		41
Atrial septal defect, secundum		17
Ventricular septal defects		22
Membranous		7
Muscular		9
Conus		2
AVSDs		6
Atrial septal defect, primum		2
Complete common AV canal		4
Unilateral blocks		4
Pulmonary stenosis		2
Tricuspid stenosis		2
Routing abnormalities		6
Interrupted aortic arch		3
Truncus ateriosus		2
Double-outlet right ventricle		1

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^aA control screen was performed simultaneously to determine the baseline rates of perinatal lethality and CHD (left column). No defects were observed in the control screen. Simultaneously, a mutagenesis screen was performed to ascertain the rates of induction of perinatal lethality and CHD caused by ENU treatment (right column). CHD was observed in animals demonstrating perinatal lethality in 47 of 321 mutagenized lines (14.6%).

^bDistribution of CHD by class from mutagenized lines. Three classes of congenital heart defects (septation defects, unilateral blocks and routing abnormalities) were observed, corresponding to the types of defects found in human CHD. From 47 affected lines, 41 demonstrated defects in septation, 4 demonstrated unilateral blocks (stenotic lesions) and 6 demonstrated routing abnormalities of the great arteries.

Our screening for CHD was expanded via participation in two ongoing large-scale ENU screens, The Jackson Laboratory National Heart Lung and Blood Institute ENU Mutagenesis Program (23) and the Justice Laboratory at Baylor College of Medicine (24,25). Each utilized a three-generation backcross strategy to identify recessive alleles. From the Jackson Laboratory–National Heart Lung and Blood Institute ENU Mutagenesis Program, we studied 366 perinatal lethals from 282 independent lines. Based upon serial section histology, 66 had CHD structural heart abnormalities (18%). The Justice Laboratory screen utilized a chromosome 11 balancer, a strategy that permitted mutation mapping concomitant with phenotypic analysis (24,25). We evaluated the cardiac anatomy of mutant mice with perinatal lethality from seven independent lines. A heart defect was identified in mutant animals from one line, L11Jus16. The hearts of mutant mice from this line demonstrated abnormal (L) looping and AVSDs.

Each line that generated perinatal lethal mice with CHD (n = 47) was tested for heritability. G2 female parents of affected pups were mated to G1 males, and 50 or more liveborn G3 pups were screened. Ten lines with greater than one mouse affected with CHD were identified. We suggest that these 10 lines carry CHD mutations.

Taken together, examination of three independent cohorts of ENU-mutagenized mice yielded comparable rates of perinatal lethality (~18%) and produced 79 CHD lesions (Table 1). Affected mice were ascertained in 47 of 321 (14.6%) mutagenized lines (CHD lines). Septal defects (n = 67) were the most common CHD class ascertained, accounting for 85% of malformations and observed in 41 of 47 CHD lines (87%) (Table 1). Ventricular septal defects (n = 22), atrial septal defects (secundum type, n = 17) and AVSDs (n = 6) accounted for these prevalent CHD. Routing abnormalities, including interrupted aortic arch type B, truncus ateriosus and double-outlet right ventricle (n = 8), occurred in six lines (13%). Unilateral blocks including pulmonary stenosis and tricuspid stenosis occurred in four lines (9%). We estimate an overall CHD rate of 1.3% of liveborn mice from the mutagenized cohorts (7.5% perinatal lethality rate × 17.9% of lethals affected with CHD).

Epidemiology of ENU-induced mouse CHD

Notably, most defects ascertained in our screens comprised septal defects. This finding is consistent with recent human prevalence data showing that the overwhelming majority of CHD in children and adults are septal defects (28). Although many human septal defects do not threaten survival, our selection of perinatal mortality may have defined mutations that alter a distinct subset of genes in cardiac septation. Alternatively, mouse cardiopulmonary physiology may be less tolerant of cyanotic challenges than human physiology. Regardless, the prevalence of different types of septal defects closely corresponded to human prevalence data. As observed in human CHD cohorts, in the mouse screens, VSDs were ascertained more commonly than ASDs, and both were more common than AVSDs. Furthermore, it has been noted that human AVSDs are more likely to be associated with a syndrome than other forms of CHD (reviewed in 29). Consistent with this observation in humans, three of six heritable mouse lines with AVSD defects (avc1, avc2, avc5) had malformations of other organ systems.

The AVSDs identified in these mouse screens modeled patterns of AVSD presentation in humans. AVSDs are among the most common of the severe CHD lesions in children and adults (28). Patients with AVSDs have an increased recurrence rate of CHD among their offspring compared with patients with other types of CHD (6). Furthermore, AVSD is the most reproducible CHD phenotype in familial non-syndromic CHD; phenotypic concurrence between affected family members is much higher for AVSD than for all other types of CHD (7,30). We observed similar phenotypic expressivity patterns among mutant mouse lines. In avc1-6, AVSDs were uniformly expressed, facilitating the mapping of the disease-causing alleles. In contrast, heritable CHD observed in other lines consistently included more than one defect, suggesting that the mutations causing defects other than AVSDs were more variably expressed. Although AVSDs were present in only 13% of affected lines, AVSDs represented the dominant phenotype in 50% of lines with heritable CHD. We believe that the mapped mutations identified in our screen to date, avc1-6, serve as appropriate mouse models of human AVSD and provide important resources for our detailed investigation of potentially novel genes in atrioventricular septation.

None of the heritable mutations we have discovered has observable effects as heterozygotes. This is not surprising; many developmental loci that are disease causing in humans when haploinsufficient do not have comparable deleterious consequences when heterozygous in mouse models. For example, mutations in GATA4 result in dominant disease in humans (10) but are recessive in mice (31).

Genetic background

In each cohort, male C57BL/6J mice (G0) were mutagenized by three weekly doses of 90 or 100 mg ENU/kg body weight. In the BWH/HMS cohort, the G0 male was outbred to two wild-type FVB/N females, chosen for their fecundity, large litter size and high degree of polymorphism with C57BL/6J. The first-generation (G1) males were also crossed with wild-type FVB/N females, and their second-generation (G2) daughters were backcrossed to G1 males, to enrich for recessive mutations in the third-generation (G3) offspring, which were screened at birth for perinatal lethality and CHD. This breeding strategy was designed to allow expedient recovery and genetic mapping of recessive mutations (22,35). To control for the mixed genetic background, a control screen was performed in non-mutagenized mice using the same breeding strategy. Both control and mutagenized lines were housed, maintained and bred in the facility, using identical protocols approved by the University of Chicago or BWH/Harvard Medical School. The rate of perinatal lethal line recovery was calculated from those lines that showed death of mice homozygous for the mutagen-treated chromosome from birth to postnatal day 5. The Jackson Laboratory–National Heart Lung and Blood Institute ENU Mutagenesis Program screen was performed in an inbred C57BL/6J background. The Justice Laboratory cohort utilized a balancer chromosome region on mouse chromosome 11, allowing phenotype analysis concomitant with initial mapping (24,36,37).

Screening for congenital heart defects

Cages for the control and mutagen-treated screens were checked morning and night for deceased neonatal mice. All mice that died during the first 5 days of life were examined for structural heart defects. By gross cardiac examination, direct inspection of the exterior of the heart was used to reveal outflow tract or situs abnormalities. Specifically, the great vessels were inspected for routing abnormalities including transposition or malposition of the great arteries, double-outlet right or left ventricle or interrupted or right aortic arch (38). They were also evaluated for septation abnormalities such as AP window or truncus arteriosus, which can often be identified by external examination (Van Praagh, 1975). Gross evaluation of the cardiac chambers was used to suggest the presence of an underlying structural abnormality within the heart (39), although all hearts subsequently underwent serial histological analysis.

Definitive anatomic evaluation of the mutant heart in our screen was by serial section histology. After gross examination of the hearts from mice that died perinatally, the hearts were then embedded in paraffin, cut in 5 μm sections and stained with hematoxylin and eosin. The evaluation of heart-looping abnormalities during serial section histology was aided by marking the left thorax with black ink prior to embedding. When multiple animals from an individual line were found to have cardiac structural abnormalities, the DNA from each affected pup was submitted for linkage analysis.