TBX5 mutations contribute to early-onset atrial fibrillation in Chinese and Caucasians

Abstract

Aims: Atrial fibrillation (AF) is a common arrhythmia with an important heritable aspect. The genetic factors underlying AF have not been fully elucidated.

Methods and results: We screened six candidate genes (CAV1, KCNJ2, KCNQ1, NKX2.5, PITX2, and TBX5) for novel mutations in 139 patients of Chinese descent with early-onset AF and 576 controls. Four missense TBX5 mutations, p.R355C, p.Q376R, p.A428S, and p.S372L, were identified in evolutionarily conserved regions. We did not find any mutations in CAV1, KCNJ2, KCNQ1, NKX2.5, and PITX2. These mutations increased the expression of atrial natriuretic peptide (ANP) and connexin-40 (CX40) in the primarily cultured rat atrial myocytes but did not alter the expression of cardiac structural genes, atrial myosin heavy chain-α (MHC-α) and myosin light chain-2α (MLC-2α). Overexpression of p.R355C developed an atrial arrhythmia suggestive of paroxysmal AF in the zebrafish model. To replicate our findings, we screened TBX5 in 527 early-onset AF cases from the Massachusetts General Hospital AF study. A novel TBX5 deletion (ΔAsp118, p.D118del) was identified, while no TBX5 mutations were identified in 1176 control subjects.

Conclusion: Our results provide both genetic and functional evidence to support the contribution of TBX5 gene in the pathogenesis of AF. The potential mechanism of arrhythmia may be due in part to the disturbed expression of ANP and CX40.

Keywords: ANP; Atrial fibrillation; Connexin; Mutation; TBX5.

Figure 1

(A–E) The ECG episodes of atrial fibrillation, trace of chromatogram, and genomic localization of Tbx5 mutation obtained from each patient. (A–D), the DNA sequence from wild-type flanking point mutation is shown above the chromatogram, and the mutant allele is indicated on the top of DNA sequence. In (E), both wild-type (down) and mutant (up) sequences are presented to show the deletion. Nine exons are shown as box where T-box and transactivation functional domains are filled with blue and green colour, respectively. A conservative analysis across eight species is shown in (F).

Figure 2

Rat atrial cardiac myocytes are infected by adenoviral vector that carries wild-type (grey) or mutant (black) Tbx5 gene for 24 and 48 h, respectively. The open bars are presented as cells infected by GFP-adenoviral vector (n = 6 in each group). The expression of Tbx5, ANP, connexin 40 (Cx40), MHC-α, and MLC-2α is evaluated by real-time PCR with 18S as loading controls shown in (A–E). The protein levels of ANP and Cx40 were determined by western blot analysis in the cells infected by wild-type and mutant Tbx5, respectively, glyceraldehyde-3-phosphate dehydrogenase was used as loading control (GAPDH) (F).

Figure 3

Gross morphological observations of hearts from zebrafish with overexpression of wild-type (A, n = 38) and mutant (B, n = 27) Tbx5, respectively. (C) ECG of zebrafish with overexpression of wild-type Tbx5; the waves, P, QRS, and T are indicated in (D). (E and F) Two episodes of AF in zebrafish with overexpression of mutant Tbx5, respectively. R, R wave.