Observations in this study provide additional evidence for a role of NKX2.5
beyond early cardiac progenitor commitment; the coinheritance of heterozygous NKX2.5
mutations with various congenital heart defects suggests that this transcription factor contributes to diverse cardiac developmental pathways including atrial, ventricular, and conotruncal septation, AV conduction, and AV valve formation. As described previously, AV block and ASD are common in kindreds in whom congenital heart disease is due to NKX2.5
). Additionally, NKX2.5
mutation causes AV block without associated congenital heart defects, and, as demonstrated in the patients we studied, AV block was the principal clinical finding in 23% of genotype-positive individuals. Further, based on results of the present study, NKX2.5
mutation may account for a clinically significant portion of idiopathic AV block.
However, cardiovascular defects resulting from NKX2.5
mutations extend beyond AV block and ASD. For example, VSD was present in 31% of genotype-positive individuals, including conoventricular VSD associated with tetralogy of Fallot and double-outlet, right ventricle and muscular VSD that closed spontaneously in infancy (24
). Results of this study suggest that in individuals without a del22q11, NKX2.5
mutations may account for a significant portion of conotruncal defects (25
). Abnormalities of the tricuspid valve were also present (15%), and for the first time, we have identified a genetic cause of Ebstein’s anomaly. Based on the diversity of cardiac phenotypes, we hypothesize that many additional forms of congenital heart disease may result from NKX2.5
mutations. The small sample size we studied does not allow estimation of a portion of cardiac anomalies resulting from NKX2.5
; our study is further limited by concentration on mutations in coding sequences only.
The 7 NKX2.5
mutations described in this study, and the 3 reported previously, are most often located in the region encoding the homeodomain and less often in the 5′ and 3′ ends of the gene (Figure ). Of the 10 total mutations, 5 are predicted to result in truncated proteins that have the potential for dominant-negative or deleterious gain of function effects. The other 5 missense mutations are predicted to alter single, highly conserved amino acids in the homeodomain. The mechanism by which these mutations produce diverse cardiac malformations remains to be determined. Pauli et al. (26
) reported a patient with haploinsufficiency of NKX2.5
) resulting from a distal chromosome 5q deletion that manifested ASD, AV block, and ventricular noncompaction. Because of the large number of our patients who had ASD and AV block, many of the mutations we have identified may function as null alleles. However, some specific genotype/phenotype correlation can be speculated. For example, the 4 individuals with tricuspid valve abnormalities, including Ebstein’s anomaly, come from 2 families where missense mutations in adjacent codons (Asn188Lys and Arg189Gly) were identified.
Schott et al. (19
) described 2 families with an identical NKX2.5
mutation and an identical haplotype indicating common ancestry. In subjects we evaluated, each proband had a novel mutation. Out of the 7 mutations, 3 occurred in CpG islands, which have an elevated mutation rate compared with other dinucleotides (27
). The mutation in BEP II-11 is a sporadic occurrence as neither parent carries the Tyr191Cys mutation. TOF7 and CHB3 may be examples of sporadic occurrence, but we could not confirm this possibility because both parents could not be genotyped.
Previous studies have identified Nkx2.5
as an upstream regulator and/or transcriptional activator of other genes expressed during cardiac development including Hand1 (eHand
), myocyte enhancer factor-2 (MEF2) (9
), myosin light chain 2V (MLC2V) (9
), atrial natriuretic factor gene (9
), brain natriuretic peptide (BNP) (9
), α-cardiac actin gene (14
), cardiac ankyrin repeat protein gene (15
), N-myc (9
), and MSX2 (9
binding to target DNA may occur in conjunction with other factors including GATA-4
) and serum response factor (14
). The demonstration of synergistic transcriptional activation mediated by Nkx2.5
), and the elucidation of the HoxB1-Pbx1 and Ultrabithorax-Extradenticle structure of heteromeric complexes (29
), suggests ways in which mutations may alter the specificity and affinity of DNA binding, while also disrupting the tightly regulated spatial and temporal gene expression that is essential during cardiac development.
The severe phenotypes associated with heterozygous NKX2.5
mutations in humans are surprising, given the phenotypes of heterozygous mutations modeled in other organisms. For example, ablation of Nkx2.5
in mice was embryonic lethal, but heart defects were not observed in heterozygous mutant mice (8
). The differences between mice and humans may reflect a bias resulting from observing only severely affected humans. Alternatively, these differences may reflect different cardiac development in mice and humans, genetic redundancy in humans, and/or unrecognized phenotypes in heterozygous mutant mice.
Diverse cardiac malformations are recognized in other monogenic human disorders, e.g., heterozygous TBX5
mutations in Holt-Oram syndrome (31
), as well as gene-targeted mice, e.g., heterozygous mutations in retinoic X receptor–deficient mice (34
). Although variable expressivity is not a feature predicted by classic embryological models of cardiac development, heritable monogenic mutations can clearly cause pleiotropic cardiovascular defects. Based on results of the present studies, NKX2.5
appears to be a likely candidate gene for a number of forms of cardiovascular disease in the young.