Mutations in cardiac T-box factor gene TBX20 are associated with diverse cardiac pathologies, including defects of septation and valvulogenesis and cardiomyopathy

Abstract

The T-box family transcription factor gene TBX20 acts in a conserved regulatory network, guiding heart formation and patterning in diverse species. Mouse Tbx20 is expressed in cardiac progenitor cells, differentiating cardiomyocytes, and developing valvular tissue, and its deletion or RNA interference-mediated knockdown is catastrophic for heart development. TBX20 interacts physically, functionally, and genetically with other cardiac transcription factors, including NKX2-5, GATA4, and TBX5, mutations of which cause congenital heart disease (CHD). Here, we report nonsense (Q195X) and missense (I152M) germline mutations within the T-box DNA-binding domain of human TBX20 that were associated with a family history of CHD and a complex spectrum of developmental anomalies, including defects in septation, chamber growth, and valvulogenesis. Biophysical characterization of wild-type and mutant proteins indicated how the missense mutation disrupts the structure and function of the TBX20 T-box. Dilated cardiomyopathy was a feature of the TBX20 mutant phenotype in humans and mice, suggesting that mutations in developmental transcription factors can provide a sensitized template for adult-onset heart disease. Our findings are the first to link TBX20 mutations to human pathology. They provide insights into how mutation of different genes in an interactive regulatory circuit lead to diverse clinical phenotypes, with implications for diagnosis, genetic screening, and patient follow-up.

Figure 1.

Pedigrees of families with TBX20 mutations, with relevant sequence profiles of probands’ DNA and unaffected control individuals. The arrow under the sequence indicates the detected single-nucleotide change. In family 2, individual II:4, the only surviving unaffected member who was the descendent of an affected parent (and therefore a likely carrier), was not available for genotyping. For two deceased members (II:5 and II:7), there were no records available for the cause of death, although anecdotal evidence suggests a cardiac cause for both.

Figure 2.

Structural impact of TBX20 mutations illustrated with a model of the TBX20 T-box (blue ribbon) bound to DNA (gray surface) on the basis of the x-ray crystal structure of the TBX3 domain. a, Space-filling representation of affected residues showing Ile152 (yellow) located in the core of the T-box and Thr209 (green) at the DNA-interaction face. b, Side chain of Ile152, packed within the hydrophobic core. The extra length and possibility of additional rotation within the side chain of methionine may disrupt the hydrophobic packing in this region and destabilize the structure. c, Q195X, which results in truncation of the TBX20 protein within the T-box. The region of the T-box expressed in the Q195X variant is shown in blue, with the remainder of the domain shown in gray. d, Thr209, involved in a stabilizing network of H bonds with residues that direct contact DNA. The loss of these H bonds would be expected to reduce stability of the chain in this region and have a significant effect on DNA binding.

Figure 3.

Functional analysis of TBX20 mutations. a, 293T cell–transfection assay measuring activation of the Nppa promoter in the presence of Tbx20c (short isoform lacking C-terminal trans activation and trans repression domains). b, COS cell–transfection assay measuring activation of the Nppa promoter in the presence of Tbx20a (full-length isoform), Nkx2-5, and Gata4, alone or in combination. Synergistic activation is seen only in the presence of all factors. c, Ability of WT and mutant Tbx20a proteins to disturb gastrulation movements in Xenopus laevis embryos after microinjection of respective mRNAs (1 ng) into fertilized eggs. WT protein likely disturbs gastrulation by dysregulating the function of endogenous T-box proteins, including brachyury and eomesodermin, and/or Gata factors, involved in mesoderm and endoderm formation. WT, I152M, and T209I are potent inhibitors, whereas Q195X is inactive. d, Comparison of the ability of Tbx20a WT and mutant proteins to activate expression of the endogenous Actc1 gene (encoding cardiac α-actin) in frog ventral-marginal-zone (VMZ) explants removed from gastrula embryos after microinjection of the indicated mRNAs into ventral cells of four-cell–stage embryos. Amounts of injected mRNA are indicated. Control tissue was the dorsal marginal zone (DMZ) that includes cardiac tissue. The histogram indicates normalized Actc1 levels, as determined by quantitative RT-PCR, with statistical significance (P) of indicated comparisons. e, Western blot (WB) showing Tbx20a protein expressed from injected mRNAs used in panel d, relative to levels of tubulin. Tbx20a proteins are linked to a C-terminal hemogluttinin (HA) epitope tag and are detected with anti-HA antibody, except for Tbx20a-Q195X, which lacks the tag and is detected with an anti-Tbx20 antibody.

Figure 4.

The I152M mutation, which affects the structure of the TBX20 T-box and its affinity for DNA. a, Far-UV CD spectra from WT (solid line) and I152M (dotted line). The spectra are broadly similar, although alterations to the two minima indicate some change to structure. b, ¹H NMR spectra from WT and I152M T-boxes, which exhibit narrow lines and good resonance dispersion, confirming that the two domains are folded, with defined tertiary structure. The small amount of reordering within the hydrophobic core of the I152M domain is reflected in the differences in methyl resonances within the up-field region of the spectra (0.5 to −0.5 ppm). c, Thermal denaturation of Tbx20 WT and I152M T-boxes, followed as a function of secondary structure content, which demonstrates that the I152M mutation destabilizes the domain. The midpoint of thermal denaturation is reduced by ∼3°C, relative to the WT domain. d, Surface plasmon-resonance profiles of the binding of WT and I152M T-boxes to the T-half site DNA sequence, which indicate that the mutation reduces the on rate for the T site by fourfold but does not reduce the off rate.