Genetic Variation of SCN5A in Korean Patients with Sick Sinus Syndrome

Conditions leading to sinus node dysfunction have considerable clinical importance. Sick sinus syndrome (SSS) manifesting as sinus bradycardia, sinus arrest, and/or sinoatrial block is the most frequent worldwide clinical indication for pacemaker implantation.1) Benson et al.2) screened SCN5A as a candidate gene in ten patients from families diagnosed with congenital SSS on the basis of disorders of cardiac rhythm and conduction during the first decade of life. Moreover, several investigations have linked variations in SCN5A, the gene encoding the pore-forming α-subunit of the cardiac-type sodium ion (Na⁺) channel, Nav1.5, with familial SSS.3), 4), 5) Furthermore, the SCN5A gene is known to be associated with different forms of arrhythmias such as long QT syndrome, Brugada syndrome, progressive cardiac conduction defect, atrial fibrillation, dilated cardiomyopathy, and overlapping syndromes.6), 7)

Voltage-gated Na⁺ channels are transmembrane proteins that produce the fast inward Na⁺ current responsible for the depolarization phase of the cardiac action potential. Inherited variations in SCN5A result in a spectrum of disease entities termed as Na⁺ channelopathies.

To date, although many variations of the SCN5A gene have been documented in various cardiac diseases, SCN5A variations in Korean SSS patients have not yet been studied in detail. Therefore, we carried out complete sequencing of the coding regions of the SCN5A gene, excluding untranslated regions, in Korean SSS patients in order to identify any potential variations associated with SSS.

In the present study, the genetic nucleotide sequence of SCN5A was determined in Korean SSS patients and compared with that of the controls. We found two novel genetic variations (A3075T-E1025D and T4847A-F1616Y). In addition, the normalized I_Na in mutant F1616Y-expressing HEK cells showed a leftward-shift in inactivation and activation, and the peak I_Na was increased.

The SCN5A gene consists of 28 exons and encodes a protein of 2016 amino acids, with a molecular mass of 227 kDa.13) In our patients with SSS, there were 9 sites of nucleotide change from exon 2 to exon 28 of the SCN5A gene. Among these, 2 sites (G87A-A29A and IVS9-3C>A) have been reported as genetic variations in a Western study,14) and T5457C-D1819D has already been reported as a variation or polymorphism without functional effects to the channel in Asian studies.15), 16), 17) Recently, Gui et al.18) reported that A1673G-H558R has variation-specific effects on SCN5A-related SSS. In addition, G3823A-D1275N, which is reported as a heterozygous loss-of-function variation, is related to reduced whole-cell currents due to impaired cell surface localization.19)

Since a synonymous nucleotide change in the SCN5A gene is unlikely to induce a functional change in the Na⁺ channel, a non-synonymous substitution was considered as a candidate for the variation associated with this disorder. The two new variations found in this study are non-synonymous and heterozygous changes. The variations may not affect the pathogenesis of SSS due to the opposite normal allele. Therefore, it is reasonable to suggest that the functional consequences of these changes should be assessed by measuring their effect on Na⁺ channel activity, allowing assessment of a direct relationship between the variations and disease.

We can deduce the functional effect of the variations by structural simulations, which provide direction for subsequent studies. In the present study, we obtained some information from a structural model of SCN5A on the functional effect of the variations S1710L and F1616Y. In the model, S1710L perturbs the Nav1.5 molecular structure from the 6^th alpha helix - 6t^th loop structural changes. It is also likely that the physiological consequences of this model would be misfolding of the Nav1.5 protein.20) However, F1616Y was located in the center of the second loop, which is well exposed. There is no significant steric hindrance or helix-packing problem with a change from phenylalanine to tyrosine at the 1616 site. Although there would be no significant structural changes with the F1616Y variation, one cannot exclude the possibility that it may be involved in protein-protein interactions, resulting in pathological consequences.

We used whole-cell configuration of the voltage-clamp technique to determine the effect of these genetic mutations. The mutation F1616Y led to a significant leftward-shift in the voltage-dependency of the Na⁺ channel. Karin et al.21) showed that some voltage-sensor Na⁺ channel mutations can lead to a 10 mV leftward shift of the steady-state fast inactivation curve, which enhances inactivation. In their study, the heterologous expression of the two Na⁺ channel mutations was shown to be important in pathogenesis by reducing the number of excitable channels. Similar to Lehmann-Horn's study,21) the mutation F1616Y makes a leftward-shift in the Na⁺ channel voltage-dependency, enhancing inactivation of the channel, and may be a cause of the pathogenesis of SSS, even in the heterologous mutation.

Most SCN5A mutations associated with sinus node dysfunction have shown a reduction in function or loss of function,4) but rare SCN5A mutations such as L212P have been reported to have gain of function.22) Mutations in SCN5A have been linked to LTQ3 due to an increase in persistent late I_Na (gain of function). However, the action potential in the sinoatrial node is largely calcium channel dependent.23) One possible explanation for how gain of function mutations in SCN5A could lead to sinoatrial node dysfunction is due to failure of the impulses to propagate into the adjacent atrial myocardium. The large hyperpolarizing shift in activation of the F1616Y channel reduces the threshold potential for activation, leading to increased I_Na. The hyperpolarizing shift of steady-state inactivation and delayed recovery from inactivation reduce Na⁺ channel availability and slow cardiac impulse conduction. Such opposing gating properties have been shown in the SCN5A mutation, G514C, responsible for isolated cardiac conduction disease.24) The G514C channel exhibits a depolarizing shift in both activation and inactivation. It is assumed that the balance of opposing gating abnormalities as demonstrated in G514C, under the influence of additional factors involved in impulse propagation, may elicit exit block in the sinoatrial node. In LQT3 families with 1795insD mutations, a persistent inward current or a negative shift in inactivation has been shown to impair the coupling of electrical events between the pacemaker cells and those surrounding them within the sinoatrial node. This led to impaired propagation of electrical activity from the sinoatrial node to atrial tissue.25)

In our study, the patients with novel genetic variations (A3075T-E1025D and T4847A-F1616Y) are all female and were diagnosed at a relative young age (<60 years), meaning that the genetic variation may be related to a clinical event. In particular, the patient with a genetic variation at T4847-F1616Y had a family history of SSS. Her mother had complained of SSS-related symptoms such as dizziness and palpitations in her forties. She was diagnosed by an electrophysiological study in her sixties. However, we could not confirm the presence of the genetic variation because she was already deceased. Therefore, we believe that this study on the functional consequences of the genetic variation is important.

A limitation of this study is the relatively small number of patients. In addition, we are not able to explain the exact relationship between the gain-of-function of F1616Y and SSS. However, Makita et al.22) reported gain of function of SCN5A in one family with congenital atrial standstill similar to our study. Finally, SCN5A may play a role in the SA node,4) therefore, we are planning to generate animal models for future research.

To our knowledge, this is the first study to determine a link between genetic variations in SCN5A and Korean patients with SSS. In this study, we demonstrated that there are 2 novel genetic variations (E1025D and F1616Y) in SCN5A in Korean patients with SSS.