Prevalence and spectrum of Nkx2.5 mutations associated with idiopathic atrial fibrillation

Abstract

Objective: The aim of this study was to evaluate the prevalence and spectrum of Nkx2.5 mutations associated with idiopathic atrial fibrillation (AF).

Methods: A cohort of 136 unrelated patients with idiopathic atrial fibrillation and 200 unrelated, ethnically matched healthy controls were enrolled. The coding exons and splice junctions of the Nkx2.5 gene were sequenced in 136 atrial fibrillation patients, and the available relatives of mutation carriers and 200 controls were subsequently genotyped for the identified mutations. The functional characteristics of the mutated Nkx2.5 gene were analyzed using a dual-luciferase reporter assay system.

Results: Two novel heterozygous Nkx2.5 mutations (p.N19D and p.F186S) were identified in 2 of the 136 unrelated atrial fibrillation cases, with a mutational prevalence of approximately 1.47%. These missense mutations co-segregated with atrial fibrillation in the families and were absent in the 400 control chromosomes. Notably, 2 mutation carriers also had congenital atrial septal defects and atrioventricular block. Multiple alignments of the Nkx2.5 protein sequences across various species revealed that the altered amino acids were completely conserved evolutionarily. Functional analysis demonstrated that the mutant Nkx2.5 proteins were associated with significantly reduced transcriptional activity compared to their wild-type counterpart.

Conclusion: These findings associate the Nkx2.5 loss-of-function mutation with atrial fibrillation and atrioventricular block and provide novel insights into the molecular mechanism involved in the pathogenesis of atrial fibrillation. These results also have potential implications for early prophylaxis and allele-specific therapy of this common arrhythmia.

Figure 1

Nkx2.5 mutations associated with idiopathic AF. A, Sequence electropherograms showing the c.55A>G mutation of Nkx2.5 in contrast to the control. The arrow indicates the heterozygous nucleotides of A/G in the index patient from family 1 (mutant) or the homozygous nucleotides of A/A in the corresponding control individual (wild-type). The rectangle designates the nucleotides comprising a codon of Nkx2.5. B, Sequence electropherograms showing the c.557T>C mutation of Nkx2.5 in contrast to the control. The arrow denotes the heterozygous nucleotides of T/C in the proband from family 2 (mutant) or the homozygous nucleotides of T/T in the corresponding control individual (wild-type). C, Schematic representation of the Nkx2.5 protein structure, with the AF-related mutations indicated. The mutations identified in patients with idiopathic AF are shown above the structural domains. NH2, amino-terminus; TN, transcriptional activation domain; HD, homeodomain; NK, NK2-specific domain; and COOH, carboxyl-terminus. D, Pedigree structures of the families with AF. The families are designated as family 1 and family 2, and the family members are identified by generations and numbers. Squares indicate male family members; circles, female members; a symbol with a slash, a deceased member; closed symbols, affected members; open symbols, unaffected members; arrows, probands; “+”, carriers of the heterozygous mutations; and “−”, non-carriers.

Figure 2

Alignment of the multiple Nkx2.5 protein sequences across species. The altered amino acids of p.N19 and p.F186 are completely conserved evolutionarily among the various species.

Figure 3

Functional defects resulting from Nkx2.5 mutations. Activation of the atrial natriuretic factor promoter-driven luciferase reporter in COS-7 cells by wild-type (WT), N19D-mutant, or F186S-mutant Nkx2.5, alone or in combination, demonstrated significantly decreased transactivational activity of the mutant proteins. The experiments were performed in triplicate, and the mean and standard deviations are shown. *represents p<0.0005 when compared to wild-type Nkx2.5.