*Contributed equally
Mutations in the SCN5A gene has been recognized as resulting in a series of life-threatening arrhythmias. However, it also causes idiopathic ventricular fibrillation (IVF) with J wave in inferior leads and prolonged S-wave upstroke in precordial leads, which has not been previously reported. The present study aimed to study the mechanisms of a patient with IVF manifested with J wave in inferior leads and prolonged S-wave upstroke in precordial leads. The electrocardiograms (ECG) of the proband were recorded and genetic testing was conducted. Patch-clamp and immunocytochemical studies were performed in heterologously transfected 293 cells. The VF attacks was documented in a 55-year-old male proband with syncope episodes. 12-lead ECG shown the transient J wave in the inferior leads and prolonged S-wave upstroke in precordial V1-V3 leads in the same timeframe. Genetic analysis revealed a novel 1 base deletion (G) at position 839 in exon 2 in SCN5A gene (C280S*fs61), which causes a severe truncation of the sodium channel. The functional study revealed that in 293 cells transfected with mutant channel, no sodium current could be recorded even though the immunocytochemical experiment confirmed the truncated sodium channel existed in cytosol. The kinetics of the wild-type (WT) channel were not altered when co-transfected with C280S*fs61 mutant which suggested a haploinsufficiency effect of sodium channel in the cells. The present study identified a novel C280Sfs*61 mutation that caused the ‘loss of function’ of the sodium channel by haploinsufficiency mechanism. The reduced sodium channel function in the heart may cause conduction delay that may underlie the manifestation of J wave and prolonged S-wave upstroke associated with IVF.
Idiopathic ventricular fibrillation occurs in patients with structural normal heart and causes unexpected cardiac death (
Although there are clinical studies that reveal the relationship between ER and higher risk of VF (
The present study presented the electrophysiological characteristics of an novel frame-shift mutation of C280Sfs*61 in the SCN5A gene in a male proband with IVF and transient appearance of J wave in the inferior leads and prolonged S-wave upstroke in precordial leads.
The present study was approved by the Ethics Committee of Fuwai Hospital (approval no. 080133) and all experiments conformed to the principles outlined in the Declaration of Helsinki. Blood samples were obtained after the patient volunteered to participate in this study and provided written informed consent for publication.
The genomic DNA was isolated from leukocytes with TIANamp Blood DNA isolation kit (Tiangen Biotech Co., Ltd.) according to the manufacturer's instructions. The known arrhythmia syndrome suspected genes, such as KCNQ1, KCNH2, KCNE1, KCNE2, KCNE3, KCNJ8, CACNA1C, CACNB2, GPD1L, SCN5A, SCN1B and SCN3B, underwent comprehensive open-reading frame/splice site mutational screening using denaturing high-performance liquid chromatography (DHPLC) and verified by direct DNA sequencing as previously described (
The full-length wild-type (WT) human SCN5A cDNA (GenBank ID: NM198056) was subcloned into pcDNA3.1 vector for mammalian expression in the Pathophysiology Laboratory of Fuwai Hospital (Invitrogen; Thermo Fisher Scientific, Inc.). The mutation (C280Sfs*61) was constructed using a QuikChange site-directed mutagenesis kit (Stratagene; Agilent Sumitomo Dainippon Pharma Co., Ltd.) on the WT-SCN5A background, and verified by direct sequencing. 293 cells were transfected with 0.6 µg cDNA of WT or mutant channels using the Effectene method (Qiagen GmbH) (
Sodium current was measured using whole-cell patch clamp techniques with Axonpatch 700B amplifiers (Molecular Devices, LLC.) at a room temperature of 22-24˚C (
The immunocytochemical experiments were performed in 293 cells transfected with WT or mutant SCN5A plasmid as previously described. Briefly, cells were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Nonspecific binding was blocked with 5% bovine serum albumin (BSA; Beyotime Institute of Biotechnology) in PBS. Then cells were incubated with anti-Nav1.5 N-terminal monoclonal antibody (1:50; Abcam) and anti-Nav1.5 C-terminal polyclonal antibody (1:200; Alomone Labs) overnight at 4˚C (
INa data were analyzed using Clampfit 10.0 (Molecular Devices, LLC.) and non-linear curve fitting was performed with OriginPro 8.5 software (OriginLab Corporation). Data were presented as the means ± standard error of the mean (SEM). Student's t-test analysis was used for the comparison of two means. All statistical analyses were performed with SPSS, version 17.0. (SPSS Inc.). P<0.05 was considered to indicate a statistically significant difference.
The proband, a 55-year-old otherwise healthy man suffered from agonal respiration during sleep. After being woken by his family, his respiration became smooth. However, the next day he suddenly lost consciousness when talking with his colleagues at work and was admitted to the local emergency room in Weixian People's Hospital. There was no prior syncope episode and no family history of sudden cardiac death (SCD). In the hospital, six episodes of spontaneous VF (
During the hospitalization, the repeated baseline ECG with sinus rhythm exhibited wide and notched P wave (138 msec) and prolongation of QRS wave (140 msec) without signs of bundle branch block. There were also no signs of QT prolongation (QTc interval, 389 msec). The transient J waves, however, were recorded in the inferior leads (II, III and aVF), and prolonged S-wave upstroke appeared in the precordial leads of V1-V3 in the same time-frame (
A novel heterozygous 1 base (G) deletion at position 839 (c.839delG) locating in exon2 of SCN5A gene was revealed in the proband (
The transfected 293 cells transiently expressing the Nav1.5-WT or Nav1.5-C280Sfs*61 were voltage clamped after 24-48 h incubation. Given the nature and severity of the truncation mutation, the C280Sfs*61 mutant produced no detectable sodium current as expected, which conformed that the truncated protein was nonfunctional.
To mimic the heterozygous state of the proband, the INa was also recorded in cells co-transfected with NaV1.5-WT and NaV1.5-C280Sfs*61 at a 1:1 ratio. Comparing with WT channels, the peak current density of heterozygous state showed a significant reduction by ~55%, but the kinetics of steady-state activation and inactivation were not altered (
To investigate the cellular localization of the WT and mutant channels, immunocytochemical experiments were performed. 293 cells transfected with the WT or C280Sfs*61 channels were double-stained by anti-Nav1.5 N-terminal and anti-Nav1.5 C-terminal antibodies. As expected, cells expressing the mutant channels showed no fluorescence staining identified using the anti-C-terminal antibody, indicating that the truncated peptide of mutant channel was located in the plasma and not functioning (
In the present study, a novel SCN5A frame-shift mutation of C280Sfs*61 was identified in a patient suffered from spontaneous VF episodes. The patch clamp studies revealed that C280S*fs61 mutant channel failed to produce any sodium current. The ECGs of the proband revealed the distinguishing electrocardiographic anomalies of transient J wave in inferior leads along with prolonged S-wave upstroke in precordial leads. In the absence of type I Brugada ECG and other well-defined electrophysiological disorders, this patient was diagnosed as ERS according to the expert consensus statement (
SCN5A encodes the α-subunit of cardiac sodium channel, which drives the impulse conduction in the heart. ‘Loss-of-function’ in SCN5A is associated with a wide range of inherited arrhythmia syndromes, such as Brugada syndrome, progressive cardiac conduction disease and sick sinus syndrome (
As a risk factor of SCD, J wave or early repolarization is attracting more attention. The association between J-waves and idiopathic VF has been described in case-control studies (
Since Miyazaki
The prolongation of S-wave upstroke in leads V1-V3 in our patient, which could be a minor diagnostic criteria of arrthymogenic right ventricular cardiomyopathy (ARVC) (
In the present study, the C280Sfs*61 mutation causing haploinsufficiency of sodium channel has been identified. In a study of animal model of SCN5A+/- haploinsufficiency mice, the increased fibrosis was more severe in the right ventricle, which may cause conduction delay and prolonged S-wave in right precordal leads (
Moreover, Papadotos
The present study identified a novel frame-shift mutation of C280Sfs*61 in SCN5A gene in a patient manifested transient J wave in the inferior leads and prolonged S-wave upstroke in precordial leads follow by ventricular fibrillation. Functional study revealed that the mutation caused haploinsufficiency effect of the sodium channel. The results suggested that the depolarization delay may underly the mechanism of the J wave in the inferior leads and prolonged S-wave upstroke in the precordial leads.
The authors also warmly thank technicians Mr Jian Huang and Ms Yinhui Zhang, both technicians at Fuwai Hospital, Chinese Academy of Medical Science & Peking Union Medical College (Beijing, China), for carefully preparing each experiment and helping to collect data.
All data generated and/or analyzed during the present study are included in this published article.
JP conceived and designed present study. YC was responsible for administrative and financial support. LR and JP were responsible for the provision of study materials and the patient. LR, JH and YZ were responsible for collection and assembly of data. LR, XZ, YC and JP were responsible for data analysis and/or interpretation. XZ was responsible for writing the manuscript. LR and XZ confirm the authenticity of all the raw data. All authors read and approved the final manuscript. The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.
This study was approved by the Ethics Committee of Fuwai Hospital (approval no. 080133) and all experiments conformed to the principles outlined in the Declaration of Helsinki. The patient volunteered to participate in this study and provided written informed consent for publication
The patient volunteered to participate in this study and provided written informed consent for publication.
The authors declare that they have no competing interests.
12-Lead ECG recordings of the proband. (A) The baseline ECG of the proband (10 mm/mV, 25 mm/sec, 62 beats/min) showing a borderline QRS duration of 120 msec, the P-R interval was 184 msec and the QT/QTC was 384/389 msec. (B) ECG monitor strip of a spontaneous onset of ventricular fibrillation recorded in Lead II in a Weixian People's Hospital (Xingtai, China) (10 mm/mV, 25 mm/sec). ECG, electrocardiogram.
Dynamic changes of the J wave. (A) At baseline routine ECG during the hospitalization (10 mm/mV, 25 mm/sec, 70 beats/min) exhibited a relatively wide P wave (124 msec) and s wave in the inferior leads. (B) In another ECG (10 mm/mV, 25 mm/sec, 74 beats/min), the J wave appeared in leads II, III and AVF (asterisks) accompanied by newly appearing prolonged S-wave upstroke (arrows). ECG, electrocardiogram.
DNA sequence analysis of the proband and schematic representation of the α subunit of the sodium channel. (A) DNA sequence analysis of the proband showing a G base deletion at position 839 of the gene SCN5A, which resulted in the replacement of amino acid cysteine at position 280 by serine followed by a 61 frame-shift amino acids before a premature stop codon. (B) Location of C280Sfs*61 in the predictive topologic structure of the SCN5A channel. Grey circles and zigzag line denote the locations of the mutations.
Electrophysiological characterization of Nav1.5 channels. (A) Representative sodium current (INa) recordings of WT, C280Sfs*61 and WT + C280Sfs*61. (B) Current-voltage relationships for WT, C280Sfs*61 and WT + C280Sfs*61 channels. (C) Voltage-dependence of activation for WT (n=8) and WT + C280Sfs*61 (n=10). The line represents a fit to the Boltzmann function GNa=[1+exp(V1/2-V)/κ]-1, where V1/2 and κ are the midpoint and the slope factor, respectively, and GNa=INa(norm)/(V-Vrev), where Vrev is the reversal potential and V is the membrane potential. The results showed the mutant channel had no effect on the steady state activation. (D) Steady-stage inactivation relationships for WT (n=10) and WT + C280Sfs*61 (n=10) fitted with Boltzmann function: Ina=INa-max[1+exp(Vc-V1/2)/κ]-1, where the V1/2 and κ are the midpoint and the slope factor, respectively, and Vc is the membrane potential. and there was no significant difference between WT and WT + C280Sfs*61 channels. All data points are shown as the mean value, and the bars represent the standard error of the mean. WT, wild-type.
Representative confocal imaging of WT and mutant channels. Each cell was double-stained by anti-Nav1.5 N-terminal antibody (green) and anti-Nav1.5 C-terminal antibody (red). Cells expressing WT channel showed both of the fluorescence, whereas cells transfected with C280Sfs*61 only exhibited the green fluorescence. Scale bar, 10 µm. WT, wild-type.