Korean Circ J.  2016 Jan;46(1):63-71. 10.4070/kcj.2016.46.1.63.

Genetic Variation of SCN5A in Korean Patients with Sick Sinus Syndrome

• Lee YS
• Olaopa MA
• Jung BC
• Lee SH
• Shin DG
• Park HS
• Cho Y
• Han SM
• Lee MH
• Kim YN

• KMID: 2223771
• DOI: http://doi.org/10.4070/kcj.2016.46.1.63

Abstract

BACKGROUND AND OBJECTIVES
Due to recent studies that have shown an association between the genetic variation of SCN5A and sick sinus syndrome (SSS), we sought to determine if a similar correlation existed in Korean patients with SSS.
SUBJECTS AND METHODS
We enrolled 30 patients with SSS who showed a sinus pause (longer than 3.0 s) in Holter monitoring, in addition to 80 controls. All exons including the putative splicing sites of the SCN5A gene were amplified by polymerase chain reaction and sequenced either directly or following subcloning. Wild-type and single nucleotide polymorphisms were expressed in human embryonic kidney cells, and the peak sodium current (I(Na)) was analyzed using the whole-cell patch-clamp technique.
RESULTS
A total of 9 genetic variations were identified: 7 variations (G87A-A29A, IVS9-3C>A, A1673G-H558R, G3823A-D1275N, T5457C-D1819D, T5963G-L1988R, and C5129T-S1710L) had been previously reported, and 2 variants (A3075T-E1025D and T4847A-F1616Y) were novel; the potential structural effects of F1616Y were analyzed in a three-dimensional model of the SCN5A domain. Patch-clamp studies at room temperature demonstrated that the peak I(Na) was significantly increased by 140% in HEK cells transfected with F1616Y compared with wild-type (-335.13 pA/pF+/-24.04, n=8 vs. -139.95 pA/pF+/-23.76, n=7, respectively). Furthermore, the voltage dependency of the activation and steady-state inactivation of F1616Y were leftward-shifted compared with wild-type (V(h) activation=-55.36 mv+/-0.22, n=8 vs. V(h) activation=-44.21 mV+/-0.17, n=7; respectively; V(h) inactivation=-104.47 mV+/-0.21, n=7 vs. V(h) inactivation=-84.89 mV+/-0.09, n=12, respectively).
CONCLUSION
F1616Y may be associated with SSS.

Keyword

SCN5A protein, human; Polymorphism, single nucleotide; Sick sinus syndrome

MeSH Terms

Electrocardiography, Ambulatory
Exons
Genetic Variation*
Humans
Kidney
Patch-Clamp Techniques
Polymerase Chain Reaction
Polymorphism, Single Nucleotide
Sick Sinus Syndrome*
Sodium
Sodium

Figure

• 

  Fig. 1 DNA sequencing analysis results of the newly discovered SCN5A gene variants in this study. (A) DNA sequencing results of SCN5A exon 17. A3075T is a heterozygous nucleotide change, and causes an amino acid change from glutamine to aspartate. (B) DNA sequencing results of SCN5A exon 28. T4847A is a heterozygous nucleotide change, and causes an amino acid change from phenylalanine to tyrosine. All DNA sequence electropherograms show non-synonymous nucleotide changes in the sick sinus syndrome patients.

• 

  Fig. 2 Two variation sites (F1616Y and S1710L) within the 3D model of the SCN5A domain. F1616Y is located in the center of the second loop, which is well exposed, while the S1710L variation is located immediately following the 6th alpha helix.

• 

  Fig. 3 Gating kinetics of WT and mutant E1025D sodium channels at 25℃ in HEK293 cells. (A) Voltage-dependent kinetics of the steady-state fast inactivation and peak conductance at 25℃. Steady-state inactivation was estimated by 500-ms pre-pulse protocols from a holding potential of -140 mV. Normalized peak currents were plotted against the pre-pulse membrane potentials. Conductance G (V) was calculated by the equation: G (V)=Ipeak/(Vm-Erev), where Ipeak is the peak current, Erev is the measured reversal potential, and Vm is the membrane potential. The normalized peak conductance was plotted against the membrane potentials. Inactivation data were fitted with the Boltzmann equation: y={1+exp ([Vm-Vh]/k)}-1; conductance data were fitted with the Boltzmann equation: y={1+ exp ([Vh-Vm]/k)}-1, where y represents variables; Vh, midpoint; k, slope factor; and Vm, membrane potential. Note: Vh (E1025D-delQ)=-90.29 mv±0.31 (n=12) is slightly leftward-shifted compared with Vh (WT-delQ)=-84.89 mV±0.09 (n=10), p<0.1; k (E1025D-delQ)=7.73±0.27, k (WT-delQ)=6.33±0.08. Vh activation (E1025D-delQ)=-52.14 mv±0.43 (n=6) is significantly leftward-shifted compared with Vh activation (WT-delQ)=-44.21 mV±0.17 (n=7) (p<0.05). Data are presented as the mean±SE. (B) I-V relationships at 25℃. Ipeak (E1025D-delQ)=-178.61 pA/pF±34.75 (n=6), Ipeak (WT-delQ)=-139.95 pA/pF±23.76 (n=7) (p<0.1). (C) Superimposed macroscopic sodium current traces obtained from the cells expressing WT-delQ (upper panel) and E1025D-delQ (lower panel). The currents were induced by step-pulse protocols from a holding potential of -140 mV. WT: wild type, SE: standard error.

• 

  Fig. 4 Gating kinetics of WT and mutant F1616Y sodium channels at 25℃ in HEK293 cells. (A) Voltage-dependent kinetics of the steady-state fast inactivation and peak conductance at 25℃. Steady-state inactivation was estimated by 500-ms pre-pulse protocols from a holding potential of -140 mV. Normalized peak currents were plotted against the pre-pulse membrane potentials. Conductance G (V) was calculated by the equation: G (V)=Ipeak/(Vm-Erev), where Ipeak is the peak current, Erev is the measured reversal potential, and Vm is the membrane potential. The normalized peak conductance was plotted against the membrane potentials. Inactivation data were fitted with the Boltzmann equation: y={1+exp ([Vm-Vh]/k)}-1; conductance data were fitted with the Boltzmann equation: y={1+exp ([Vh-Vm]/k)}-1, where y represents variables; Vh, midpoint; k, slope factor; and Vm, membrane potential. Note: Vh (F1616Y-delQ)=-104.47 mV±0.21 (n=8) is significantly leftward-shifted compared with Vh (WT-delQ)=-84.89 mV±0.09 (n=10), p<0.005; k (F1616Y-delQ)=6.52±0.18, k (WT-delQ)=6.33±0.08. Vh activation (F1616Y-delQ)=-55.36 mv±0.22 (n=8) is significantly leftward-shifted compared with Vh activation (WT-delQ)=-44.21 mV±0.17 (n=7), p<0.005). Data are presented as the mean±SE. (B) I-V relationships at 25℃. Ipeak (F1616Y-delQ)=-335.13 pA/pF±24.04 (n=8), Ipeak (WT-delQ)=-139.95 pA/pF±23.76 (n=7) (p<0.005). (C) Superimposed macroscopic sodium current traces obtained from the cells expressing WT-delQ (upper panel) and F1616Y-delQ (lower panel). The currents were induced by step-pulse protocols from a holding potential of -140 mV. WT: wild type, SE: standard error.

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