Discovery of TBX20 as a Novel Gene Underlying Atrial Fibrillation

Figures

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  Figure 1. Haplotype analysis of microsatellite markers at chromosome 7p14.2-p14.3 in Family 1 suffering from atrial fibrillation. A vertical bar represents the chromosomal segment delimited via genotypic analysis using polymorphic genetic markers. The chosen eight markers covering the linked locus on chromosome 7p14.2-p14.3 are given to the left of the pedigree, and the family members’ genotypes (symbolized with Arabic numbers) for these microsatellite markers are exhibited next to the chromosomal bars. A blackened vertical bar denotes a haplotype segregating with atrial fibrillation, in contrast to an open one indicating a normal haplotype.

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  Figure 2. A typical electrocardiograph from the index patient (III-1) of Family 1. The representative electrocardiograph illustrates atrial fibrillation.

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  Figure 3. Pedigree of Family 2 suffering from atrial fibrillation. A “⁺” symbol denotes a heterozygote for the identified TBX20 mutation of c.862G>C (p.D288H), in contrast to a “−” symbol denoting a homozygote for wild-type TBX20.

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  Figure 4. A typical electrocardiogram from the proband (II-3) of Family 2. The representative electrocardiogram documents atrial fibrillation.

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  Figure 5. Sequence chromatograms illustrating the TBX20 c.695A>G mutation in a heterozygous status. An arrow directs to G/A from the proband of Family 1 (mutant) or A/A from a healthy individual of Family 1 (wild type).

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  Figure 6. Sequence electropherograms illustrating the TBX20 c.862G>C mutation in a heterozygous status. An arrow directs to C/G from the proband of Family 2 (mutant) or G/G from an unaffected person (wild type).

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  Figure 7. Decreased transactivation effect of TBX20 on KCNH2 by the mutations. (A) In cultured HeLa cells expressing various recombinant plasmids, including different doses (from 25 ng to 400 ng) of wild-type TBX20-pcDNA3.1 (WT), reporter analysis unveiled that TBX20 transcriptionally activated KCNH2 in a dose-dependent mode from 25 ng to 100 ng. (B) In cultivated HeLa cells expressing various recombinant plasmids, including WT, His232Arg-mutant TBX20 (H232R), and Asp288His-mutant TBX20 (D288H), dual-luciferase reporter assay unveiled that both H232R and D288H, singly or together with WT, had significantly decreased transactivation of KCNH2. For each expression vector used, reporter assay experiments were repeated three times in triplicate. Here * indicates p < 0.05; a, b, c, and d indicate p = 0.0005, 0.0010, 0.0003, and 0.0007, respectively, when compared with WT (100 ng).

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  Figure 8. Reduced ability of the mutant TBX20 proteins to bind to the KCNH2 promoter. Wild-type TBX20 (WT) normally bound the KCNH2 promoter, while the ability of His232Arg-mutant TBX20 (H232R) or Asp288His-mutant TBX20 (D288H) to bind the KCNH2 promoter was significantly diminished.

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  Figure 9. Normal intracellular distribution of the TBX20 mutants. Subcellular distribution of wild-type TBX20 (WT), His232Arg-mutant TBX20 (H232R), and Asp288His-mutant TBX20 (D288H) was observed in cultured HeLa cells by immunofluorescence. The HeLa cells expressing WT, H232R, or D288H were stained by utilizing anti-FLAG (red) and DAPI (blue). Magnified cellular images with FLAG and DAPI stained and merged cellular images were shown. Cellular immunofluorescent images demonstrated that WT, H232R, or D288H was distributed predominantly in the cellular nuclei. Bar = 50 μm.