Implication of SCN5A Mutations in Brugada and Long QT Syndromes: Novel Molecular Mechanisms

M. Chahine¹, P. Guicheney², S.G. Priori^3
1 Laval University, Department of Medicine & Québec Heart Institute,
Laval Hospital, Sainte-Foy, Québec, G1V 4G5 Canada
²INSERM U523, Institut de Myologie, Groupe Hospitalier Pitié-Salpêtrière, Paris, France
³ Molecular Cardiology Fondazione S. Maugeri, Pavia, Italy.

Abstract: We characterized several novel mutations on the SCN5A gene causing either Brugada syndrome (BS) or long QT syndrome (LQTS). Missens mutations (R1432G, G1740R and H681P and a double mutant R1232W/T1620M) and nonsense mutations  E473X and N1774+12X were identified in a BS patients.  Our study revealed novel mechanisms by which BS phenotype could occur.  A V1777M change was identified in a patient displaying LQTS and a 2:1 atrioventricular block. Our data provides the first evidence for a recessive variant of LQTS related to mutation on the SCN5A channel gene.

INTRODUCTION

Brugada syndrome (BS) [1] and long QT syndrome (LQTS) are two hereditary cardiac diseases that cause ventricular fibrillation and may lead to sudden death.  Clinically, LQTS is characterized by a prolonged QT interval on 12 lead electrocardiogram (ECG). However, BS is characterized by an ST segment elevation in leads V1 to V3 a right bundle branch block  and a normal QT interval.  Mutations in six genes encoding different ionic channels or their accessory subunits were identified in LQTS patients, including the SCN5A gene [2].  However, BS mutations were identified so far in only one gene, SCN5A, the cardiac sodium channel gene [3].

METHODS

SCN5A mutations were reproduced on Na_v1.5, the cardiac sodium channel in vitro by site-directed mutagenesis. Mutant channels were expressed in tsA201 human embryonic kidney cells.  The cardiac sodium channels were then characterized using the patch clamp technique and laser confocal microscopy.

RESULTS

Using patch clamp experiments, we showed that there is an abolition of functional hNa_v1.5/R1432G expression in tsA201 cells, but not in Xenopus oocytes.  In tsA201 cells a conservative positively charged mutant, R1432K, produced sodium currents with normal gating properties, whereas other mutations at this site abolished functional sodium channel expression.  Immunofluorescent staining and confocal microscopy showed that the hNa_v1.5/WT expressed in tsA201 cells was localized to the cell surface, whereas the R1432G mutant was colocalized, with calnexin, within the endoplasmic reticulum.

Macroscopic currents, similar to the wild-type, were recorded from cells expressing each of the single mutants (hNa_v1.5/R1232W and hNa_v1.5/T1620M).  However, when both mutations were present on the same channel no currents could be recorded.  Laser confocal microscopy results show that the double mutant channels are retained in the endoplasmic reticulum (ER). No current could be recorded from cells expressing the hNa_v1.5/G1740R mutant incubated at 37^oC, however at a lower incubation temperature (22^oC), macroscopic Na⁺ currents were recorded. Confocal microscopy study confirmed that at 37^oC, hNa_v1.5/G1740R mutant channels were retained in the endoplasmic reticulum.  Characterization of the hNa_v1.5/H681P mutant showed a shift of both steady-state inactivation and steady-state activation toward more hyperpolarized potentials, with a more pronounced shift of the steady-state inactivation curve. These shifts resulted in nearly 60% reduction of the window current. Finally, the E473X and N1774+12X mutants produced truncated proteins and did not express any currents.  Co-expression of each of these mutants with wild-type channels shows 50% reduction of Na⁺ currents.

On the other hand, homo and heterozygously expressed V1777M mutant channels showed persistent inward currents. However, persistent currents in homozygous systems were nearly 2-fold enhanced compared to the heterozygous system (2.9% in homozygous versus 1.7% in heterozygous system).

DISCUSSION

Our results show that the R1432G mutation and R1232W/T1620M double mutant found in a BS patient is implicated in the localization of the hNa_v1.5 cardiac sodium channel protein, revealing a novel mechanism in the genesis of Brugada syndrome [4,5].  The lack of Na⁺ current characterized the hNa_v1.5/G1740R mutation.  This was related to trafficking defect of the Na⁺ channel protein.  In fact the hNa_v1.5/G1740R mutant protein is retained with the calnexin in the ER.  Further study demonstrated that the trafficking defect could be rescued by incubating the transfected cells at a lower temperature (22^oC versus 37^oC).  This suggests that a temperature-dependent chaperon protein is implicated in the retention of hNa_v1.5/G1740R mutant protein.

We identified also two mutations (hNa_v1.5/E473X and hNa_v1.5/N1774 + 12X) on SCN5A gene in patients with RBBB and ST segment elevation in the right precordial leads.  These mutations lead to the generation of a truncated Na⁺ channel proteins.  Similar truncated proteins have been identified in BS patients.  As expected, when these two mutations were expressed in tsA201 cells, Na⁺ channel expression is absent.  When these two mutant Na⁺ channels were co-expressed with the wild-type Na⁺ channels an approximate 50% reduction of Na⁺ currents is observed.  This suggests that there is no dominant negative effect of the truncated proteins.  The normal level of expression, normal kinetics of inactivation and recovery from inactivation resembling hNa_v1.5 wild-type Na⁺ channels characterized the hNa_v1.5 /H681P mutation.  However, one striking difference is the shifts in steady-state gating properties of hNa_v1.5/H681P channel.  We recorded 17 mV shift in steady-state inactivation and 9mV shift in steady-state activation (Gv curve) toward more negative potential, without any effect on slope factors.  These shifts display a 60% reduction in the window current.  This observation revealed a novel property by which BS phenotypes could occur and highlight again the important patho-physiological role of the window current.

A novel missense mutation, V1777M, in the early C-terminal domain of SCN5A was identified. Homozygote and heterozygote expression of the mutant channels in tsA201 mammalian cells resulted in a persistent inward sodium current of 3.96±0.83% and 1.49±0.47% at -30 mV, respectively, which was dramatically reduced in the presence of tetrodotoxin. This study provides the first evidence for a homozygous missense mutation in SCN5A and suggests that LQTS with functional 2:1 AVB in young children, a severe phenotype associated with bad prognosis, may be caused by homozygous or heterozygous compound mutations not only in HERG but also in SCN5A [6].

We conclude that the haplo-insufficiency of  SCN5A is the basis of the BS clinical phenotype; and the gain of function is the basis of LQTS.

Acknowledgements

This study was supported by the Heart and Stroke Foundation of Québec (HSFQ), the Canadian Institutes of Health Research (CIHR) MT-13181 and by Fonds de la recherche en santé du Québec (FRSQ).  Dr. M. Chahine is Edwards Senior investigator (Joseph C. Edwards Foundation).

REFERENCES

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