Heterozygous mutations in NKX2.5, a homeobox transcription factor, were reported to cause secundum atrial septal defects and result in atrioventricular (AV) conduction block during postnatal life. To further characterize the role of NKX2.5 in cardiac morphogenesis, we sought additional mutations in groups of probands with cardiac anomalies and first-degree AV block, idiopathic AV block, or tetralogy of Fallot. We identified 7 novel mutations by sequence analysis of the NKX2.5-coding region in 26 individuals. Associated phenotypes included AV block, which was the primary manifestation of cardiac disease in nearly a quarter of affected individuals, as well as atrial septal defect and ventricular septal defect. Ventricular septal defect was associated with tetralogy of Fallot or double-outlet right ventricle in 3 individuals. Ebstein’s anomaly and other tricuspid valve abnormalities were also present. Mutations in human NKX2.5 cause a variety of cardiac anomalies and may account for a clinically significant portion of tetralogy of Fallot and idiopathic AV block. The coinheritance of NKX2.5 mutations with various congenital heart defects suggests that this transcription factor contributes to diverse cardiac developmental pathways.
D. Woodrow Benson, G. Michael Silberbach, Ann Kavanaugh-McHugh, Carol Cottrill, Yizhong Zhang, Steve Riggs, Octavia Smalls, Mark C. Johnson, Michael S. Watson, J.G. Seidman, Christine E. Seidman, John Plowden, John D. Kugler
Submitter: James M. Gruschus | email@example.com
Nat. Insts. of Health, NHLBI
Published February 17, 2000
Of the seven NKX2.5 mutations identified by Benson et al., two of the missense mutations are especially interesting, Asn188Lys and Tyr191Cys, both occurring in the third helix, the "recognition" helix, of the NKX2.5 homeodomain. Both of the mutated residues are fully exposed to solvent, and there is no reason to believe that either of these mutations alters the structure of the homeodomain (1). In contrast, the two other missense mutations, Thr178Met and Arg189Gly, should result in destabilization of the third helix of the homeodomain (1,2). Based on homology with the vnd/NK-2 homeodomain/DNA complex structure, residues 188 and 191 of NKX2.5 should make DNA base contacts (2). Asn188, residue 51 of the homeodomain, is invariant across all homeodomains, and its specific base contact with the second adenine of homeodomain DNA binding site sequences is observed in all known homeodomain/DNA complex structures (3). Mutation of residue 51 to other residues reduces binding by factors of 20 to 1000, and in many mutants binding is no longer detectable (4-6). Tyr191, residue 54 of the homeodomain, is conserved in the NK-2 class of homeodomains, and its contact with a cytosine gives rise to the unique specific binding sequence observed for NK-2 class homeodomains, CAAGTG (2,3). In a study of the vnd/NK-2 homeodomain where residue 54 is mutated from tyrosine to methionine, the residue found in the same position in Antennapedia, binding to the CAAGTG sequence was reduced by a factor of 10, and no alteration of the homeodomain structure was observed (7). Even more interesting, the binding of the mutated vnd/NK-2 to CAAGTG became equal to that of the Antennapedia binding site TAATGG.
The possibility that the two mutations Asn188Lys and Tyr191Cys alter the DNA binding of the otherwise structurally intact NKX2.5 protein has important implications. In the case of Drosophila one study has proved that the details of the specific binding sequence measured in vitro for a homeodomain are biologically important (8). For homeodomain binding sites in humans, however, the importance of such details has not yet been firmly established (9). Thus, the specificity altering mutation Tyr191Cys provides valuable evidence that such detailed binding specificity is important in human development. The functional importance of binding specificity would further imply that mutations in the genomic DNA binding sites of the homeodomain might also lead to phenotypic modifications similar to those observed for the homeodomain mutants, the AV blockage phenotype studied in this paper, for instance. For the vnd/NK-2 homeodomain, mutation of the adenine contacted by asparagine in position 51 of the homeodomain reduces binding by a factor of 50 to 200, and the mutation of the cytosine contacted by tyrosine in position 54 reduces binding by a factor of 4 to 25 (2). A transgenic Drosophila experiment involving a tyrosine to methionine mutation in position 54 is currently underway.
Understanding the relationships between structure, DNA binding, and the observed phenotypic alterations associated with NKX2.5 not only emphasizes the importance of the continued search for additional missense mutations in the homeodomain but also raises exciting questions concerning the role of mutations in the target DNA, in other factors that interact with the homeodomain protein, and in downstream regulatory targets.
J. M. Gruschus, S. Weiler, L.-H. Wang, M. Nirenberg, and J. A. Ferretti
Laboratories of Biophysical Chemistry and Biochemical Genetics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda MD 20892.
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