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(Circulation Research. 2000;87:888.)
© 2000 American Heart Association, Inc.

Molecular Medicine

Cardiac Septal and Valvular Dysmorphogenesis in Mice Heterozygous for Mutations in the Homeobox Gene Nkx2-5

Christine Biben, Roberta Weber, Scott Kesteven, Edouard Stanley, Lachlan McDonald, David A. Elliott, Louise Barnett, Frank Köentgen, Lorraine Robb, Michael Feneley, Richard P. Harvey

From the Victor Chang Cardiac Research Institute (C.B., S.K., L.M., D.A.E., M.F., R.P.H.), Darlinghurst, Australia; Walter and Eliza Hall Institute of Medical Research (R.W., E.S., L.B., F.K., L.R.), Royal Melbourne Hospital, Parkville, Australia; Cardiology Department (S.K., M.F.), St Vincent’s Hospital, St Darlinghurst, Australia; and Faculties of Medicine and Life Sciences (R.P.H.), University of New South Wales, Kensington, Australia.

Correspondence to Richard P. Harvey, Victor Chang Cardiac Research Institute, 384 Victoria St, Darlinghurst 2010, Australia. E-mail r.harvey{at}victorchang.unsw.edu.au

	Abstract

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Abstract—Heterozygous mutations in the cardiac homeobox gene, NKX2-5, underlie familial cases of atrial septal defect (ASD) with severe atrioventricular conduction block. In this study, mice heterozygous for Nkx2-5–null alleles were assessed for analogous defects. Although ASD occurred only rarely, atrial septal dysmorphogenesis was evident as increased frequencies of patent foramen ovale and septal aneurysm, and decreased length of the septum primum flap valve. These parameters were compounded by genetic background effects, and in the 129/Sv strain, septal dysmorphogenesis bordered on ASD in 17% of Nkx2-5 heterozygotes. In a proportion of neonatal heterozygotes, as well as in adults with ASD, we found that the size of the foramen ovale was significantly enlarged and altered in shape, potentially exposing the normally thin septum primum to excessive hemodynamic forces. Therefore, defective morphogenesis of the septum secundum may be one contributing factor in the generation of patent foramen ovale, septal aneurysm, and certain ASDs. Mild prolongation of P-R interval in females and an increased frequency of stenotic bicuspid aortic valves were also features of the Nkx2-5 heterozygous phenotype. Our data demonstrate that the complex effects of Nkx2-5 haploinsufficiency in mice are weaker but convergent with those in humans. As in the mouse, the phenotype of human NKX2-5 mutations may be modulated by interacting alleles.

Key Words: atrial septal defect • bicuspid aortic valve • atrioventricular conduction block • patent foramen ovale

	Introduction

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The presence of a hole in the cardiac atrial septum, referred to as atrial septal defect (ASD), is the third most common congenital heart anomaly in humans, occurring in 5 to 7 of 10 000 live births.^{1 2} Atrial septation is a complex morphogenetic process involving several tissue components³ (see Figure 4A). Initially, a septum primum forms along the roof of the common atrium in the developing heart and grows toward the atrioventricular (AV) cushions, incorporating extracardiac mesenchyme. Before the septum primum fuses with the AV cushions, regional apoptosis creates a hole, the ostium secundum, which maintains the right-to-left atrial blood flow necessary during fetal development. A second septal wall (the septum secundum) then forms to the right of the septum primum, composed in part from an infolding of the atrial wall.³ This septum also maintains an open canal, the foramen ovale. Together, the foramen ovale and the offset ostium secundum form a one-way channel, with the extant septum primum acting as a flap valve (see Figure 4A). At birth, a higher pressure develops in the left compared with the right atrium, forcing the flap valve against the septum secundum, thus eliminating the interatrial shunt and redirecting right atrial flow into the pulmonary circulation. The flap valve fuses permanently with the rim of the foramen ovale in most individuals, but in 15% to 30% of individuals it remains open, a generally benign condition called patent foramen ovale (PFO).^{4 5}

View larger version (62K): [in this window] [in a new window]   Figure 4. Figure 4. Septal dysmorphogenesis in Nkx2-5 heterozygous and wild-type mice. A, Schematized view of the interatrial septum in a developing mammalian heart, displaying the relative arrangement of the septum primum (orange) and septum secundum (purple). Red arrow indicates blood flow. B, Normal septum in a C57Bl/6 mouse, as seen from the left atrium. Arrows highlight the flap valve edge. Dashed circle (B and C) indicates the foramen ovale within the septum secundum. C, Atrial septum with a clearly patent foramen ovale. This septum also has a flap valve aneurysm. D, Oblique view of the PFO corridor in a borderline ASD found in an Nkx2-5GFP heterozygote on a 129/Sv background. Patency is highlighted using an eyebrow hair. E, Slightly oblique view of a frank ASD in a mouse heterozygous for the Nkx2-5GFP allele. F and G, Wild-type and Nkx2-5 heterozygous neonate hearts, respectively, with the primum septum dissected away to reveal the shape and size of the foramen ovale. Fo indicates foramen ovale; fv, flap valve; LA, left atrium; lsvc, left superior vena cava; lv, left ventricle; mv, mitral valve; and RA, right atrium.

Although ASD generally produces no symptoms in children, the resulting volume overload of the right side of the heart can eventually progress to pulmonary hypertension, atrial and right ventricular dilatation, and heart failure.^{6 7} Recently, autosomal dominant mutations in the gene encoding the cardiac homeodomain transcription factor NKX2-5 have been correlated with secundum ASD in rare families in which the defect is known to be inherited.⁸ NKX2-5 mutations seem to confer a range of other cardiac abnormalities, including severe and progressive AV conduction block at high penetrance and, sporadically, ventricular septal defect, left ventricular hypertrophy, Tetralogy of Fallot, double outlet right ventricle, subvalvular aortic stenosis, tricuspid valve abnormality, and Ebstein’s anomaly.^{8 9} Nkx2-5 genes have been cloned from mouse, chick, frog, and zebrafish models and extensively studied in the context of heart development.^{10 11} In frogs, the gene is essential for heart formation,^{12 13} whereas in mice, a targeted deletion of Nkx2-5 in homozygous form causes arrest at a rudimentary unlooped heart tube stage.¹⁴

The confounding range of cardiac abnormalities seen in NKX2-5 families underscores the complexity of congenital heart disease in mammals. Mouse models of human congenital disease and disease predisposition promise to be of significant value in understanding this complexity because of the availability of transgenic, knockout, and inbred genetic strains. We report here an analysis of Nkx2-5 heterozygous-null mice for congenital heart defects using echocardiography, electrocardiography, and anatomical dissection.

	Material and Methods

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Mouse Strains and Genotyping
Nkx2-5^HDneo and Nkx2-5^GFP mice (Mus musculus) were generated using E14 and W9.5 ES cells, respectively, both from the 129/SvJ strain. The 129/Sv mice used for breeding were from the 129T2/SvEms-+^Ter? substrain (http://www.informatics.jax.org/mgihome/nomen/strain_129.shtml). In this study, targeted neomycin-resistant ES cell clones occurred at a frequency of 1 in 30. Mice carrying the Nkx2-5^HDneo allele were originally bred onto [C57Bl/6JxC57Bl/10J]F1 mice for 13 generations before being crossed to pure C57Bl/6J for an additional 2 to 4 generations before analysis. Mice carrying the Nkx2-5^GFP allele had been bred onto C57Bl/6J for 2 to 6 generations. For experiments using the Nkx2-5^GFP line, the pgk-Neo cassette had been removed by Cre recombinase–mediated deletion, achieved by crossing transmitting chimeras with deleter transgenic mice (C57Bl/6J background), which express Cre recombinase in the germ line.¹⁵ Cre-deleted Nkx2-5^GFP mice were additionally bred onto a C57Bl/6J background to segregate the transgene. We also established an Nkx2-5^GFP line on a pure 129/Sv background, although this strain remained undeleted for pgk-Neo. Tail DNA was genotyped for Nkx2-5 alleles by polymerase chain reaction using 50 ng of specific oligonucleotide primers (Nkx2-5^GFP allele: sense: 5'-ACATGAAGCAGCACGACTTCTTCAAGTCCG-3'; antisense: 5'-TTGTGGCGGATCTTGAAGTTCACCTTGATGCC-3'; Nkx2-5^HDneo allele: sense: 5'-TGCAGAAGGCAGTGGAGCTGGACAA-AGCC-3'; antisense: 5'-GCTCCAGACTGCCTTGGGAAAA-3'), 0.5 U Taq DNA Polymerase (Boehringer), and 0.2 mmol/L dNTPs in manufacturer’s buffer for 36 cycles of 30 seconds at 94°C, 30 seconds at 56°C, and 1 minute at 72°C.

Echocardiography and Electrocardiography
Echocardiographic studies were performed with a Hewlett-Packard Sonos 5500 Ultrasonograph equipped with a 12-MHz phased-array transducer. Mice were anesthetized with 20 mg/kg xylazine and 100 mg/kg ketamine intraperitoneally. Parasternal long-axis and short-axis, apical 4-chamber, and suprasternal two-dimensional scans were recorded. M-mode recordings were made from the short-axis midventricular image. Digitized velocity-time recordings were traced electronically, and the peak velocity, mean velocity, and VTI for each valve were computed with inbuilt dedicated software. Three beats were averaged for each measurement. Digitized ECG recordings were signal-averaged over 30 beats to provide very high definition of the P wave and QRS complex. Data analyses were without previous knowledge of genotype.

Heart Dissections
PFO was assigned if there was passage of blood or Orange G dye solution from right to left across the interatrial septum after pressurisation of the intact right atrium, achieved by pulsing buffer or dye into the persistent left superior vena cava using a glass pipette. Aneurysm was assigned if the flap valve was ruffled or hyperelastic after pressurising the right atrium, as described above. Relative dimensions of septal features were measured with an eyepiece graticule without knowledge of genotype.

	Results

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Construction of Nkx2-5^GFPAlleles
Two independently targeted alleles of mouse Nkx2-5 were used in this study. The allele created by Lyons et al¹⁴ (Nkx2-5^HDneo) carries a neomycin resistance expression cassette (pgk-Neo) inserted into the third helix of the homeodomain. Because the Nkx2-5^HDneo allele may not have been fully null,¹⁰ we created additional alleles in which sequences encoding an enhanced variant of the jellyfish green fluorescent protein (GFP)¹⁶ were inserted into the Nkx2-5 gene at a position corresponding to amino acid 35 of the protein (Figure 1A). A pgk-Neo cassette flanked by LoxP sites was incorporated immediately 3' of the GFP insertion. Amino acids 141 to 184 containing the N-terminal part of the homeodomain were also deleted in the targeting vector, introducing a frameshift.

View larger version (22K): [in this window] [in a new window]   Figure 1. Figure 1. A, Wild-type and targeted Nkx2-5 alleles after deletion of the pgkNeo cassette using Cre recombinase. The protein coding exons of Nkx2-5 are shown as black boxes. GFP gene is shown in gray. Arrowheads indicate LoxP sites. Asterisk represents deletion of the homeodomain between the MluI and BglII sites. The probe used in panel B is represented by a bar. B indicates BamHI; Bg, BglII; H, HindIII; K, KpnI; M, MluI (not unique); Nc, NcoI; N, Not1; S, SacI; X, XbaI; and Xh, XhoI. B, Southern analysis of wild-type and targeted ES cell DNA after NcoI digestion. C, RT-PCR detection of the Nkx2-5GFP allele (287bp) in DNA of progeny of heterozygous crosses.

Heterozygous progeny of the 4 Nkx2-5^GFP strains generated with this vector (deleted or undeleted for homeodomain sequences and the pgk-Neo cassette [see Figure 1A and Materials and Methods]) were genotyped by Southern blotting or polymerase chain reaction (PCR) (Figures 1B and 1C). All heterozygotes were viable and fertile. Furthermore, the Nkx2-5^GFP fusion protein was active, as judged by emission of green fluorescence after irradiation of embryonic or adult hearts with 530 nm UV light (Figures 2A and 2B), and the pattern of fluorescence recapitulated the known expression pattern of the gene (not shown). For studies described in this report, data were collected from the Nkx2-5^GFP strain (Figure 1A) and compared with those from the original knockout line. Nkx2-5^GFP mice were undeleted for homeodomain sequences (Figure 1A) and either deleted (C57Bl/6 background) or undeleted (129/Sv background) for the neomycin cassette (see Materials and Methods). Homozygous Nkx2-5^GFP embryos displayed abnormal heart morphology at E8.5 and E9.5 (Figures 2C through 2F) identical to that reported for the original Nkx2-5^HDneo allele¹⁴ and for a recently published allele carrying a full deletion of coding sequences.¹⁷ In situ hybridization confirmed downregulation of the myosin light chain 2V (MLC2V), Hand1, and SM22 genes (Figures 2G and 2H and data not shown), as observed in the original knockout.^{14 18 19} A low level of MLC2V expression was still evident, as seen in the original knockout line using RT-PCR,¹⁴ and in the complete deletion using in situ hybridization.¹⁷ Because of the essentially identical morphological and molecular phenotypes seen in the various Nkx2-5 mutant strains, we now suppose that all are effectively null for Nkx2-5 function. The minor differences in the degree of cardiac looping suggested to occur in the original mutant line compared with the complete deletion¹⁷ may be attributable to subtle genetic background differences or merely reflect the lack of objective criteria to assess looping.¹¹ The suggestion that the Nkx2-5^HDneo protein produced from the original allele acts in a dominant negative fashion¹⁷ now seems unlikely to be true, given that the Nkx2-5^GFP allele produces an identical phenotype despite the fact that its encoded fusion protein includes only 35 amino acids of Nkx2-5 and lacks the nuclear localization signal.²⁰

View larger version (74K): [in this window] [in a new window]   Figure 2. Figure 2. Heart development in the Nkx2-5HDneo and Nkx2-5GFP strains. A and B, GFP fluorescence in hearts of an Nkx2-5GFP/+ E10.0 embryo and adult, respectively. C, E9.0 wild-type, Nkx2-5HDneo/HDneo and Nkx2-5GFP/GFP embryos. D through F, Comparison of E9.0 wild-type heart with those of homozygous Nkx2-5HDneo and Nkx2-5GFP embryos. G through H, Whole-mount in situ hybridization of Nkx2-5GFP/GFP and wild-type embryos with MLC2V and Hand1 probes. Avc indicates atrioventricular canal; ivs, interventricular septum; la, left atrium; lv, left ventricle; oft, outflow tract; rv, right ventricle; and sv, sinus venosus.

Functional Analysis of Nkx2-5 Heterozygous Mice
Cardiac structural and functional parameters were investigated in cohorts of mice of the Nkx2-5^HDneo and Nkx2-5^GFP lines using transthoracic echocardiography and electrocardiography (ECG). A total of 67 mice were examined, although 3 were excluded from additional comparisons after they showed echocardiographic evidence of aortic stenosis (see below). For echocardiography, we confined comparisons to quantitative velocity recordings across the pulmonary and aortic valves (Figures 3A and 3B). The blood volume passing across each valve is the product of the blood velocity-time integral (VTI) and the valvular cross-sectional area. Thus, pulmonary and aortic VTIs can be used as indices of right and left ventricular stroke volumes, respectively, assuming no gross valvular abnormalities. As in humans, an increased pulmonary:aortic VTI ratio would indicate a left-to-right shunt. However, we found no significant difference between heterozygous and wild-type mice with respect to pulmonary and aortic peak blood flow velocities, mean velocities, VTI values, or pulmonary:aortic VTI ratios (Table 1). The peak aortic and pulmonary velocities were the same as those observed in normal adult human hearts²¹ and similar to those previously published for mice.²² Thus, no indications of ASD were detected in this cohort. Furthermore, none of the mice demonstrated echocardiographic evidence of ventricular septal defect or cardiac hypertrophy (not shown), as seen occasionally in human NKX2-5 families. In agreement with the latter, all mice had normal heart weight:body weight and heart weight:tibial length ratios compared with age-, gender-, and weight-matched controls.

View larger version (58K): [in this window] [in a new window]   Figure 3. Figure 3. Cardiac function and aortic valvular morphology in Nkx2-5 heterozygous and wild-type mice. A and B, Pulse-wave Doppler recordings across pulmonary and aortic valves, respectively, of a normal mouse. C, Idealized ECG trace indicating waves (P, Q, R, S, and T), diagnostic durations (dur), and intervals (int). D, ECG recordings from wild-type (+/+) and heterozygote (+/-) mice of the Nkx2-5HDneo strains, aligned at the beginning of the QRS complex (dashed line). E, Doppler recording across a stenotic aortic valve present in a mouse from the Nkx2-5HDneo strain (scale inverted). F, Normal tricuspid (TAV) and abnormal bicuspid (BAV) aortic valves dissected from the Nkx2-5HDneo strain. Arrows indicate the points of attachment (commissures) of the valve cusps to the aortic wall.

View this table: [in this window] [in a new window]   Table 1. Functional Analysis of Wild-Type and Nkx2-5 Heterozygous Mice by Echocardiography

ECG revealed no evidence of the second- or third-degree AV conduction block seen in humans carrying NKX2-5 mutations. Nevertheless, female heterozygote mice showed a mild but significant prolongation of P-R interval: 46±4 ms versus 41±4 ms in wild-type female sibling controls (P=0.001; Student’s unpaired t test) (Figures 3C and 3D).

Patent Foramen Ovale and Atrial Septal Aneurysm in Nkx2-5 Heterozygotes
Anatomical dissection of hearts confirmed that no cases of ASD were present in the echocardiography cohort. Indeed, over the course of this study, only 5 overt cases of ASD were found in 425 Nkx2-5 heterozygous mutant hearts examined by dissection (Figure 4E). None were found in 415 wild-type controls. However, in many heterozygous mice from the echocardiography cohort, we noted that blood passed easily beneath the flap valve, indicating PFO (Figures 4A through 4C). To examine the significance of this finding, we analyzed a larger cohort by dissection only (Table 2). Overall, wild-type mice had a PFO prevalence of 18.9% (29 of 153) in the Nkx2-5^HDneo line and 26.9% (28 of 104) in the Nkx2-5^GFP line. Heterozygote mutants showed 66.1% (74 of 112) and 68.2% (43 of 63) increases of 3.5- and 2.5-fold, respectively (P<0.001).

View this table: [in this window] [in a new window]   Table 2. Frequency of Septal and Valvular Defects in Nkx2-5 Mutant Strains

Another abnormal feature of septal morphology prominent in Nkx2-5 heterozygotes was aneurysm of the septum primum, recognized as a ruffling of the flap valve over the foramen ovale (Figure 4C) and by its hyperelasticity when the right atrial chamber was pressurized. Aneurysm was found in 5.9% (9 of 153) of wild-type mice in the Nkx2-5^HDneo line and 2.9% (3 of 104) of wild-type mice in the Nkx2-5^GFP line. In Nkx2-5 heterozygotes, however, the overall frequency was 34.8% (39 of 112) and 22.2% (14 of 63), increases of 6- to 7-fold, respectively (P<0.001).

Influences of the Genetic Background on Septal Morphology
To determine whether septal morphology varied with genetic background, we scored PFO and aneurysm in cohorts of gender-matched mice on several genetic backgrounds at 6.5 weeks of age (Figure 5). We additionally quantitated septal morphology by measuring the length of the flap valve from the edge of the crescent to the distal rim of the fossa ovalis as well as the area of the foramen ovale as viewed from the right atrium. In cases of PFO, the width of the open corridor measured at the edge of the flap valve was also documented.

View larger version (44K): [in this window] [in a new window]   Figure 5. Figure 5. Quantitation of septal dysmorphogenesis in Nkx2-5GFP mice on different genetic backgrounds. +/+ and +/- denote wild-type and Nkx2-5GFP heterozygosity, respectively. Numbers of mice used in A through E were as follows: Swiss QS (+/+: 53); FVB/N (+/+: 51); C57Bl/6 (+/+: 41; +/-: 36); 129/Sv (+/+: 31; +/-: 35); Swiss QSxC57Bl/6 (+/+: 50; +/-: 36); and FVB/NxC57Bl/6 (+/+: 38; +/-: 31). Mean values are indicated on the top of each column. Standard errors to the mean are indicated as bars in A through E. A, Percentage of PFO. B, Percentage of atrial septal aneurysm. C, Mean length of the flap valve in millimeters. D, Mean width of the patent corridor behind the flap valve in cases of PFO. E, Scattergram of the area occupied by the foramen ovale (mm2) in individual adult and neonatal mice. F, Plot of mean flap valve length against frequency of PFO found in the different genetic backgrounds. 129 indicates 129/Sv; B6, C57Bl/6; FVB, FVB/N; SW, Swiss QS; and SW (FVB)xB6: F1 crosses between Swiss QS (or FVB/N) and C57Bl/6 mice.

These studies revealed that the prevalence of PFO in wild-type mice varied dramatically on different genetic backgrounds, ranging from 0% (0 of 51) in the FVB/N strain to 74% (23 of 31) in the 129/Sv strain (Figure 5A). Mean flap valve length varied up to 2.5-fold in different strains (Figure 5C). Interestingly, there was an excellent inverse correlation between the mean flap valve length in a particular strain and the frequency of PFO (Figure 5E). The area of the foramen ovale and mean width of the patent corridor in hearts with PFO also varied between strains (Figures 5D and 5E). In all genetic backgrounds studied, heterozygosity for Nkx2-5^GFP conferred more severe septal dysmorphogenesis, as assessed by the above parameters (Figures 5A through 5E). Nkx2-5 mutation also induced septal aneurysms, which were not evident in wild-type mice of this age (Figure 5B). Overall, septal dysmorphogenesis in both wild-type and Nkx2-5 heterozygous mice was most severe in the 129/Sv strain. In fact, on this background, 17% (6 of 35) of Nkx2-5 mutant hearts showed particularly severe septal dysmorphogenesis, which bordered on ASD, a property that was not observed in hearts from other strains or strain combinations. In these severe cases, PFO was usually accompanied by aneurysm (5 of 6 cases), the flap valve barely covered the foramen ovale, and, in some cases, the flap valve was thickened and possibly fibrotic, causing it to stand rigidly away from the septum secundum (Figure 4D). A frank but oblique communication across the septum was evident in these hearts. Although we cannot formally classify these cases as ASD, it is possible that an interatrial blood shunt occurs in vivo.

Atrial Septal Dysmorphogenesis in Neonates
To assess whether spontaneous closure masked a higher frequency of ASD in Nkx2-5 heterozygotes, we examined septal morphogenesis in neonates of the Nkx2-5^GFP line on a C57Bl/6 background (Figure 5F). As expected, all neonates had PFO, but we found only one additional ASD among the 42 heterozygous neonates examined. Nevertheless, we observed that in 25% (11 of 42) of Nkx2-5 heterozygous neonates, the foramen ovale was up to 2-fold larger than its maximum size in wild-type littermates (n=23) (Figure 5E) and had a characteristically altered shape (Figures 4F and 4G), effects that were obscured with age (Figure 5E). A significant increase in the size of the foramen ovale was also seen in heterozygous adults of the 129/Sv strain (Figure 5E) and in heterozygotes that showed ASD (Figure 4E).

Increased Frequency of Stenotic Bicuspid Aortic Valves in Nkx2-5 Heterozygous Mice
Three of 35 heterozygous Nkx2-5 mutant mice from the echocardiography cohort displayed a 3-fold increase in blood flow velocity across the aortic valve (Figure 3E and Table 1), suggesting aortic stenosis. Velocities across pulmonary valves were normal. Postmortem examination revealed that all 3 mice had a bicuspid aortic valve with mild leaflet thickening and, in some cases, commissural fibrosis (Figure 3F). We scored valve morphology in an additional 520 mice on the C57Bl/6 background (Table 2) and found bicuspid aortic valves for the Nkx2-5^HDneo strain in 1.4% (2 of 143) of wild-type mice and 11% (11 of 100) of heterozygote mice, a difference of 7.8-fold (P<0.001). For the Nkx2-5^GFP line, there was a less striking difference: 0.5% (1 of 179) in wild-type mice and 2% (2 of 98) in heterozygote mice. The different frequencies presumably reflect subtle genetic background effects: specifically, the different ratios of C57Bl/6 and 129/Sv alleles (see Materials and Methods). Because no bicuspid aortic valves were detected in pure 129/Sv mice nor in the Swiss or FVB/N strains, we can infer the presence of at least one strong modifier of bicuspid aortic valve frequency in the C57Bl/6 genotype. Bicuspid pulmonary valves were also present in about 3% of wild-type mice, but this frequency was unaltered by Nkx2-5 mutation, supporting findings in hamsters that bicuspid aortic and pulmonary valves have distinct pathogeneses.²³

	Discussion

Top Abstract Introduction Material and Methods Results Discussion References

 
Heterozygosity for NKX2-5 mutations in human families has recently been correlated with the presence of secundum ASD at a penetrance of 77%. AV conduction abnormalities were also present at 95% penetrance. A variety of other cardiac abnormalities occurred at lower frequency.^{8 9} These data suggest that NKX2-5 mutations underlie a variety of congenital cardiac abnormalities in humans. Indeed, mutations were detected in 1 of 10 cases of idiopathic AV block unassociated with other cardiac defects and in 1 of 20 cases of Tetralogy of Fallot that were not deleted at 22q11.⁹

In contrast to humans, mice heterozygous for Nkx2-5 mutations were found to have ASD only rarely (5 of 425 cases). A mild prolongation of P-R interval in female heterozygous mice was also a feature of the adult mutant phenotype, although no cases of second- or third-degree AV conduction block were detected. The present study clearly demonstrates that the impact of Nkx2-5 mutation alone on septal development and the conduction system in mice is less severe than reported for human families. However, the observations of ASD, borderline ASD, and other septal abnormalities and a mild conduction defect suggest convergence of the mouse and human phenotypes. It is possible that there are differences between humans and mouse with respect to the impact of Nkx2-5 mutation on atrial development, which could be genetic, epigenetic, or hemodynamic in nature. It is noteworthy in this respect that atrial development in Nkx2-5 homozygous mutant mice is significantly less affected than ventricular development (C.B. and R.P.H., unpublished data, January 2000), perhaps because of expression of Nkx2-6, a close relative of Nkx2-5, in the atrial region during early stages of cardiac looping.²⁴ No human homologue of Nkx2-6 has been described thus far. Alternatively, a selection bias may have been a factor in the studies on human NKX2-5.^{2 8 9} Because both phenotypic outcome (expressivity) and penetrance of a mutant phenotype can be profoundly influenced by genetic background, it cannot be assumed that NKX2-5 mutations are the only genetic influence on the generation of ASD in the families studied. Indeed, the existence of abnormalities that are apparently attributable to NKX2-5 mutations but are not accompanied by ASD or AV block could suggest genetic background influences. A third possibility would be that the NKX2-5 mutant proteins act in a dominant-negative fashion, partially inhibiting the activity of protein produced by the normal allele.²⁰ However, it is unlikely that all NKX2-5 mutations found are dominant-negative, and no major differences in expressivity or penetrance of the phenotype have been detected between families with different mutations.² Furthermore, dominant-negative effects, as demonstrated for some alleles in vitro, are relatively mild and not significantly enhanced by an increased mutant:wild-type protein ratio.²⁰ Thus, this issue needs additional clarification.

Our own study in mice has highlighted the existence of strain-dependent genetic factors for congenital heart disease. Furthermore, in every strain and strain combination analyzed, these factors were capable of interacting with Nkx2-5 mutations to create a more severe atrial septal phenotype. The presence of these factors is best illustrated by contrasting the FVB/N strain, which shows no PFO and has robust atrial septal morphogenesis by the criteria tested, with the 129/Sv strain, in which PFO was present in 74% of individuals and septal morphogenesis was poor. In 129/Sv mice carrying an Nkx2-5 mutation, septal dysmorphogenesis was even more extreme, with 94% PFO and 17% borderline ASD. It is likely that the phenotype of some or all of the human NKX2-5 mutations is similarly modified (enhanced or suppressed) by interacting alleles.

Our findings provide the first clear evidence that the frequency of PFO in a population is a function of genetic factors. They also demonstrate that PFO, at least in mice, is an index of septal dysmorphogenesis and, potentially, part of a gradation of abnormalities that includes ASD. As far as we are aware, a link between PFO and ASD in humans has never been proposed. Although PFO is common (15% to 30% of cases)^{4 5} and generally benign, a higher prevalence is found in patients younger than 60 years of age with unexplained strokes (paradoxical embolisms),²⁵ apparently because of systemic venous thrombi entering the arterial flow using a PFO as an access port. Because of the advent of percutaneous closure procedures, PFO in young stroke victims is routinely closed in some clinical centers.^{26 27}

The precise pathogenesis of atrial septal defect and PFO is unknown. In examining neonatal mice, we found that 25% of Nkx2-5 heterozygotes on a predominantly C57Bl/6 background had a foramen ovale that was significantly enlarged and altered in shape, in some cases sufficient to void the greater area of the septal wall (Figure 4G). Although this effect was largely obscured by age in this strain, presumably because of the ongoing growth of the septum, the very same phenomenon was observed in adult heterozygotes of the 129/Sv strain, which shows the most severe septal abnormalities among the strains studied, and in adults with ASD. The septum primum was intact in all but one of the affected neonates. These findings suggest that a principal defect in Nkx2-5 adult heterozygotes is delay or maldevelopment of the septum secundum. The septum primum abnormalities observed in adults, principally PFO and aneurysm, may therefore be secondary: a large foramen ovale may expose the normally thin septum primum to excessive mechanical and hemodynamic stresses. In extreme cases (rare in mice), the septum primum could rupture, causing ASD, whereas if it remains intact, PFO or aneurysm may result. However, more direct effects of Nkx2-5 mutation on the septum primum are also possible, and, indeed, Nkx2-5 seems to be upregulated on the leading edge of the developing septum primum in the chick.²⁸ Whether septum primum abnormalities seen in Nkx2-5 mutants are the result of secondary mechanical and hemodynamic effects, local genetic or epigenetic deficiencies, or some other mechanism, an appealing, although still speculative, proposition is that PFO and septal aneurysm sit within a gradation of septal abnormalities that includes ASD.

Nkx2-5 heterozygous mice also displayed an 8-fold increase in the prevalence of stenotic bicuspid aortic valves, the most frequent congenital cardiac abnormality in humans. This defect was not reported in human families with NKX2-5.^{2 8 9} The presence of bicuspid aortic valves in mice was strictly genetic background–dependent, being found only in the C57Bl/6 strain. The existence of familial cases of bicuspid aortic valves and their occurrence at low penetrance in a variety of syndromes in humans, including Turner syndrome, hints at a multiplicity of genetic causes, which could feasibly include NKX2-5 mutations.

Given the complex genetic basis of ASD and congenital heart disease in general, identification of genetic modifiers affecting atrial septal morphogenesis in the mouse may be of significant value in understanding human congenital cardiac disease. Connecting genotype with phenotype is particularly important in the case of NKX2-5 families, because they are at high risk of sudden death attributable to conduction abnormalities. It is noteworthy that the frequency of congenital heart abnormalities, including ventricular septal defect and ASD, is increased by common drugs, such as alcohol, if taken in excess during pregnancy.²⁹ Because Nkx2-5 mutant mice may be predisposed to ASD, they could be an invaluable strain for examining genotype and environment interactions in congenital heart disease.

	Acknowledgments

 

This work was funded by the National Heart Foundation of Australia, National Health and Medical Research Council of Australia, and United Way Sydney. We thank Thomas Yeoh, Andrew Owens, Edwin Kirk, and Diane Fatkin for discussions.

Received August 23, 2000; revision received September 19, 2000; accepted September 19, 2000.

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