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. 2013 Jun 28;8(6):e67963.
doi: 10.1371/journal.pone.0067963. Print 2013.

A heterozygous deletion mutation in the cardiac sodium channel gene SCN5A with loss- and gain-of-function characteristics manifests as isolated conduction disease, without signs of Brugada or long QT syndrome

Sven Zumhagen  1 , Marieke W Veldkamp, Birgit Stallmeyer, Antonius Baartscheer, Lars Eckardt, Matthias Paul, Carol Ann Remme, Zahurul A Bhuiyan, Connie R Bezzina, Eric Schulze-Bahr
Affiliations

A heterozygous deletion mutation in the cardiac sodium channel gene SCN5A with loss- and gain-of-function characteristics manifests as isolated conduction disease, without signs of Brugada or long QT syndrome

Sven Zumhagen et al. PLoS One. .

Abstract

Background: The SCN5A gene encodes for the α-subunit of the cardiac sodium channel NaV1.5, which is responsible for the rapid upstroke of the cardiac action potential. Mutations in this gene may lead to multiple life-threatening disorders of cardiac rhythm or are linked to structural cardiac defects. Here, we characterized a large family with a mutation in SCN5A presenting with an atrioventricular conduction disease and absence of Brugada syndrome.

Method and results: In a large family with a high incidence of sudden cardiac deaths, a heterozygous SCN5A mutation (p.1493delK) with an autosomal dominant inheritance has been identified. Mutation carriers were devoid of any cardiac structural changes. Typical ECG findings were an increased P-wave duration, an AV-block I° and a prolonged QRS duration with an intraventricular conduction delay and no signs for Brugada syndrome. HEK293 cells transfected with 1493delK showed strongly (5-fold) reduced Na(+) currents with altered inactivation kinetics compared to wild-type channels. Immunocytochemical staining demonstrated strongly decreased expression of SCN5A 1493delK in the sarcolemma consistent with an intracellular trafficking defect and thereby a loss-of-function. In addition, SCN5A 1493delK channels that reached cell membrane showed gain-of-function aspects (slowing of the fast inactivation, reduction in the relative fraction of channels that fast inactivate, hastening of the recovery from inactivation).

Conclusion: In a large family, congregation of a heterozygous SCN5A gene mutation (p.1493delK) predisposes for conduction slowing without evidence for Brugada syndrome due to a predominantly trafficking defect that reduces Na(+) current and depolarization force.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Pedigree of the family 10021.
Men are denoted by squares and women by circles. Solid symbols indicate mutation carriers, symbols with an “N” means wild type and “?” possible affected, crossed symbols denotes patients, who are already dead. A “-“ at the symbols indicates that no DNA is available, the propositus is marked with an arrow.
Figure 2
Figure 2. Clinical and genetic characterization.
(A) Electropherogram of SCN5A mutation c.4477–4479delAAG and multiple sequence alignment of amino acids of human SCN5A protein regions bearing the identified in-frame deletion mutation of lysine (p.1492delK) with corresponding SCN5A amino acid sequences of different species. (B) Electrocardiogram of patient 10021_49 shows an atrioventricular block first-degree, an increased P-wave duration and an intraventricular conduction delay (P interval 145 ms, PQ interval 208 ms, QRS interval 146 ms). (C) Ajmaline challenge of patient 10021_149, overall 54 mg ajmaline (1mg/kg) was administered within 5 minutes. No Brugada type I ECG could be unmasked, but cardiac conduction delay aggravated.
Figure 3
Figure 3. 1493delK mutant and wild-type (WT) human cardiac sodium channel current expressed in HEK293 cells.
(A) Whole-cell sodium current traces in response to increasing step depolarizations in WT (left) and 1493delK (right). (B) Voltage protocols for activation and steady-state inactivation. (C) Averaged sodium current– voltage relation for WT and 1493delK sodium channels. (D) Bar histogram showing averaged WT and 1493delK sodium peak currents at −20 mV. (E) Average voltage-dependence of activation and steady-state inactivation for wild-type (WT) and 1493delK sodium channels. For the activation curve, normalized peak conductance was plotted as a function of the membrane potential. For the inactivation curve, peak sodium currents were normalized to maximum values in each cell and plotted as a function of the voltage of the conditioning step.
Figure 4
Figure 4. Inactivation kinetics of 1493delK mutant and wild-type (WT) human cardiac sodium channels.
(A) Time course of current decay. (A-i) Fast and slow time constants of current decay for WT and 1493delK sodium channels are plotted as a function of membrane potential. Asterisks indicate statistical significance (p<0.05). (A-ii) Ratio of the amplitudes of fast and slow inactivation time constants plotted as a function of voltage for WT and 1493delK sodium channels. (B) Time course of recovery from inactivation for WT and 1493delK sodium channels. Peak sodium currents elicited by P2 were normalized (P2/P1) and plotted as a function of the recovery interval. Inset: 2-pulse protocol. (C) Development of slow inactivation for WT and 1493delK sodium channels. Peak sodium currents elicited by P2 were normalized (P2/P1) and plotted as a function of the duration of the conditioning step (P1). Inset: 2-pulse protocol.
Figure 5
Figure 5. Sodium channel membrane expression in wild-type and mutant 1493delK SCN5A-transfected HEK293 cells.
Confocal immunofluorescence of the a-subunit of cardiac sodium channel (NaV1.5) and the endoplasmic reticulum transmembrane protein calnexin in HEK293 expressing WT (left) and mutant 1493delK (right) sodium channels. Top and middle panels show staining with anti-NaV1.5 (green) and anti-calnexin (red) respectively. Bottom panels show overlay of red and green channels of double staining with anti-NaV1.5 (green) and anti-calnexin (red) antibodies. Membrane labeling for NaV1.5 is observed as a clearly distinguishable green rim surrounding the intracellularly located calnexin (red) in WT SCN5A transfected HEK293 cells, whereas mutant 1493delK SCN5A transfected HEK293 cells do not show clear cell-surface labeling, but mostly cytoplasmic NaV1.5 staining. Scale bars indicate 25 µm.
Figure 6
Figure 6. Topological model of the cardiac sodium channel (NaV1.5).
Location of the mutations in the linker region between domains DIII and DIV that is responsible for the inactivation of the channel.
See this image and copyright information in PMC

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Grants and funding

This work was supported by grants of the Fondation Leducq, Paris, France, the German Research Foundation, Bonn, Germany (DFG; SFB 656-C1) and the Interdisciplinary Center for Clinical Research, Münster, Germany (IZKF; Schu01-012-11) to ES-B as well as by grants of the Innovative medicine research, Münster, Germany (IMF; ST121119) to BS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.