| Background: Atrial fibrillation is the most common form of cardiac arrhythmia in clinical practice and a major contributor to cardiovascular morbidity and mortality. Some researches have demonstrated that genetic basis exists in a part of patients with atrial fibrillation. Recently, studies revealed that mutations in the gene coding for hyperpolarization-activated cyclic nucleotide-gated potassium channel type 4(HCN4) were involved in the pathogenesis of sinus node dysfunction, and a part of mutation carriers were accompanied with atrial fibrillation. These findings indicate that genetic variation in HCN4 may be responsible for atrial fibrillation.Objective: The aim of this study was to identify a novel HCN4 mutation associated with atrial fibrillation, and further to reveal the cellular electrophysiological mechanism by which the mutation contributes to atrial fibrillation.Methods: A cohort of 120 unrelated index patients with idiopathic atrial fibrillation and 200 ethnically matched healthy individuals used as the controls were enlisted. Clinical data and blood samples from the study participants were collected. Genomic DNA from all participants was extracted from blood leucocytes using genomic DNA purification Kit. The entire coding region and flanking splice junction sites of the HCN4 gene was amplified by polymerase chain reaction(PCR). The amplified products were purified and then PCR-sequenced. By comparison of the acquired HCN4 sequences with those released from Gen Bank, a HCN4 sequence variation may be identified. In order to identify a novel HCN4 mutation responsible for idiopathic atrial fibrillation, the frequency distributions of HCN4 variation in patients with idiopathic atrial fibrillation and healthy control individuals were analyzed by sequencing, and alignment of multiple HCN4 proteins across various species was performed for the evolutionary stability. Once a HCN4 mutation was identified, we would promptly clone the wild-type HCN4 gene by using standard molecular cloningtechnique. The mutant HCN4 was produced by site-directed mutagenesis. The recombinant eukaryotic expression plasmids for both wild-type HCN4 and mutant HCN4 were constructed. The liposome Lipofectamine was used to transfect the plasmids to COS-7 cells. Approximately 24~36 hours following transfection, the COS-7 cells transfected with above-mentioned DNAs were collected. Electrophysiological recordings by patch clamp were carried out to analyze the effect of a HCN4 mutation on the electrophysiological characteristics of the hyperpolarization-activated cyclic nucleotide-gated potassium channel, so as to unveil the cellular electrophysiological mechanism by which the mutation predisposes to atrial fibrillation.Results: A novel heterozygous missense mutation in the HCN4 gene was identified in a patient with paroxysmal lone atrial fibrillation and bradycardia. The mutation resulted in a substitution of glutamic acid for aspartic acid at amino acid position 600 of the HCN4 protein(p.D600E). No variant in HCN4 was found in 200 healthy control subjects, and alignment of multiple HCN4 protein sequences among various species showed that the aspartic acid at amino acid 600 of HCN4 was highly conserved evolutionarily. Additionally, the identified HCN4 mutation was predicted to be pathogenic by softwares. Studies by patch clamp showed that this mutation led to impaired hyperpolarization-activated cyclic nucleotide-gated channel function, by accelerating deactivation of the If channel and decreasing the magnitude of the If channel availability. These effects reduce the automaticity of sinus node and thus explain bradycardia and the occurrence of lone atrial fibrillation in the mutation carrier.Conclusions: A novel heterozygous missense mutations(p.D600E) in the HCN4 gene is identified in an idiopathic atrial fibrillation patient. The p.D600 E mutation exerts an effect of accelerating deactivation of the If channel, which may contribute to bradycardia and atrial fibrillation. |
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