A novel TBX5 mutation predisposes to familial cardiac septal defects and atrial fibrillation as well as bicuspid aortic valve

Abstract

TBX5 has been linked to Holt-Oram syndrome, with congenital heart defect (CHD) and atrial fibrillation (AF) being two major cardiac phenotypes. However, the prevalence of a TBX5 variation in patients with CHD and AF remains obscure. In this research, by sequencing analysis of TBX5 in 178 index patients with both CHD and AF, a novel heterozygous variation, NM_000192.3: c.577G>T; p.(Gly193*), was identified in one index patient with CHD and AF as well as bicuspid aortic valve (BAV), with an allele frequency of approximately 0.28%. Genetic analysis of the proband's pedigree showed that the variation co-segregated with the diseases. The pathogenic variation was not detected in 292 unrelated healthy subjects. Functional analysis by using a dual-luciferase reporter assay system showed that the Gly193*-mutant TBX5 protein failed to transcriptionally activate its target genes MYH6 and NPPA. Moreover, the mutation nullified the synergistic transactivation between TBX5 and GATA4 as well as NKX2-5. Additionally, whole-exome sequencing analysis showed no other genes contributing to the diseases. This investigation firstly links a pathogenic variant in the TBX5 gene to familial CHD and AF as well as BAV, suggesting that CHD and AF as well as BAV share a common developmental basis in a subset of patients.

Figure 1 -. A new TBX5 mutation responsible for familial heart defect and atrial fibrillation. (A) Sequence chromatograms illustrating the TBX5 heterozygous mutation from the proband (mutant) and its homozygous wild-type control from a healthy individual (wild type). An arrow points to the heterozygous nucleotides of G/T or the homozygous nucleotides of G/G. (B) Schematic drawings showing the structural domains of the TBX5 proteins. NH2, amino-terminus; NLS1, nuclear location signal 1; TBX, T-box; TAD, transcriptional activation domain; NLS2, nuclear location signal 2; COOH, carboxyl-terminus. (C) Pedigree structure of the family suffering from congenital heart defect and atrial fibrillation. Family members are recognized by generations as well as numbers. Circles mean female members; squares, male family member; closed symbols, affected members; open symbols, unaffected members; the symbol with a slash, the deceased member; the arrow beside the closed square, the index patient; “+”, carriers of the TBX5 mutation; “-”, non-carriers.

Figure 2 -. Functional failure of TBX5 caused by the mutation. Activation of α-myosin heavy chain 6 promoter-driven luciferase in cultured COS-7 cells by wild-type or Gly193*-mutant TBX5, singly or together, revealed that the Gly193*-mutant TBX5 protein had no transcriptional activity. Transfection experiments for each plasmid were carried out in triplicates and the results are expressed as means with standard deviations. Here ## and # indicate p<0.01 and p<0.02, respectively, in comparison with wild-type TBX5.

Figure 3 -. Disrupted synergistic transactivation between mutant TBX5 and NKX2-5 as well as GATA4. The synergistic transactivation of the promoter of natriuretic peptide precursor A in cultured cells by TBX5 and NKX2-5 as well as GATA4 was ablated by the Gly193* mutation. Transfection experiments for each plasmid were done in triplicates, with means and standard deviations shown. Here the symbols a, b and c all indicate p<0.001, in comparison with their wild-type counterparts.