Functional characterization of SCN10A variants in several cases of sudden unexplained death

Abstract

Background: Multiple genome-wide association studies (GWAS) and targeted gene sequencing have identified common variants in SCN10A in cases of PR and QRS duration abnormalities, atrial fibrillation and Brugada syndrome. The New York City Office of Chief Medical Examiner has now also identified five SCN10A variants of uncertain significance in six separate cases within a cohort of 330 sudden unexplained death events. The gene product of SCN10A is the Nav1.8 sodium channel. The purpose of this study was to characterize effects of these variants on Nav1.8 channel function to provide better information for the reclassification of these variants.

Methods and results: Patch clamp studies were performed to assess effects of the variants on whole-cell Nav1.8 currents. We also performed RNA-seq analysis and immunofluorescence confocal microcopy to determine Nav1.8 expression in heart. We show that four of the five rare 'variants of unknown significance' (L388M, L867F, P1102S and V1518I) are associated with altered functional phenotypes. The R756W variant behaved similar to wild-type under our experimental conditions. We failed to detect Nav1.8 protein expression in immunofluorescence microscopy in rat heart. Furthermore, RNA-seq analysis failed to detect full-length SCN10A mRNA transcripts in human ventricle or mouse specialized cardiac conduction system, suggesting that the effect of Nav1.8 on cardiac function is likely to be extra-cardiac in origin.

Conclusions: We have demonstrated that four of five SCN10A variants of uncertain significance, identified in unexplained death, have deleterious effects on channel function. These data extend the genetic testing of SUD cases, but significantly more clinical evidence is needed to satisfy the criteria needed to associate these variants with the onset of SUD.

Keywords: Channelopathies; Na(+) channels; SCN10A; Sudden unexplained death.

Figure 1:

Schematic topological representation of the human sodium channel α-subunit, Nav1.8. Several of the mutations fall within the transmembrane α-helical domains (L388M, R756W, L867F, V1518I) while the P1102S mutation is in the DII-DIII intracellular linker region. All the mutations occurred at evolutionarily conserved sites.

Figure 2:

Representative Nav1.8 current traces of WT channel expressed in HEK293 in the absence (A) or presence (B) of Navβ3 subunit, using the voltage protocol as shown inset in (C). The Nav1.8 current densities are plotted as a function of the test voltage (C). Data points represent mean ± SEM, with n = 7 in absence and n = 7 in the presence of Navβ3. *P < 0.05 versus wild-type (Student’s t-test).

Figure 3:

Effects of SCNA10 variants on the rate of inactivation of Nav1.8 currents (A) Representative Nav1.8 current traces of WT and P1102S channels co-expressed Navβ3 subunit in HEK293 cells. The recordings were made at 0 mV. (B) The inactivating portions of currents at −20 to +20 mV were subjected to curve fitting to a single exponential function and time constants are plotted as a function of the voltage. *P < 0.05 versus wild-type (1W-ANOVA, followed by Dunnet’s t-test).

Figure 4:

Effects of SCNA10 variants on Nav1.8 current density and total protein expressing. (A) Representative Nav1.8 current traces of WT, L388M and R756W channel co-expressed Navβ3 subunit in HEK293 cells. (B) Representative immunoblot of total proteins from HEK293 cells transfected with WT or mutant Nav1.8. Input lysates were probed with a pan-Nav antibody. GAPDH antibody was used as a protein loading control. (C) The peak currents at 0mV was normalized to the cell surface size by dividing by the cell capacitance and plotted. Compared to WT, the L388M mutant current density was significantly reduced, −11.08 ± 3.66 pA/pF; n = 7 vs −2.43 ± 1.74 pA/pF; n=7. The R756W (−18.94 ± 5.35 pA/pF; n = 5), L867F (−14.55 ± 1.62 pA/pF; n = 5), P1102S (−19.03 ± 4.36 pA/pF; n=5) and V1518I (−12.12 ± 3.28 pA/pF; n=5) mutants all showed non-significant increased peak currents at 0 mV when compared to WT. *P < 0.05 versus wild-type (1W-ANOVA, followed by Dunnet’s t-test).

Figure 5:

(A) Steady-state activation curves for WT and mutant Nav1.8 channels. The steady-state activation curves were derived from IV curves by dividing the peak currents by the driving force (V_m-V_Na, with V_Na is the calculated equilibrium potential for Na⁺ ions, which is +60mV under our experimental conditions). (B) Half-maximal voltage of steady state activation was significantly decreased in R756W (−8.37 ± 1.96 mV; n = 5) and V1518I (−2.24 ± 0.62 pA/pF; n = 5) mutants when compared to WT (−23.90 ± 3.16 mV; n = 7). *P < 0.05 versus wild-type (Student’s t-test).

Figure 6:

(A) Representative Nav1.8 current traces of WT or L388M mutant channel with or without 24 h treatment with MG132. (B) The Nav1.8 current densities measured at voltages between −80mV to +80mV, normalized by the cell capacitance and plotted as a function of test voltage. Data points represent mean ± standard error of the mean. (C) Representative Nav1.8 immunoblot of WT and L388M mutant channel after 24 h treatment with 10UM MG132 or DMSO.

Figure 7:

Expression of Nav1.8 in rat ventricular tissue. Immunofluorescence images of adult rat DRG (top) and left ventricle (bottom) were taken using a confocal microscope. Immunostaining was performed using mouse monoclonal anti-Nav1.8 antibody followed by AlexaFlour 594 conjugated secondary antibody. Rabbit polyclonal anti-Neurofilament heavy polypeptide (NF) and anti-Nav1.5 were used as positive controls for DRG neurons and left ventricular myocytes respectively. In the negative controls for DRG and left ventricle normal donkey serum was used in place of the primary antibody. Nuclei are stained with DAPI (blue). Images were taken at 20X magnification

Figure 8:

RNA-seq analysis of VGSC expression in human heart. We analyzed available RNA-seq datasets obtained adult human ventricle (NCBI GEO accession: GSE71613). Shown are SCN5A (chr3:38,587,553–38,693,164) and SCN10A (chr3:38,736,837–38,837,501) mapped to the human hg19 reference build. The respective dark blue lines at the bottom of each panel represent the gene, with exons being thicker in width. Note that these two genes are anti-parallel, with exons 28 and 27 to the left respectively for SCN5A and SCN10A. The colored tracks depict Sashimi plots (reads within and spanning the exons) for the four ‘control’ human heart samples (SRR2138381, SRR2138382, SRR2138383 and SRR2138384). The figure was produced using Integrative Genomics Viewer v2.3.68.