SCN5A rare variants in familial dilated cardiomyopathy decrease peak sodium current depending on the common polymorphism H558R and common splice variant Q1077del

Abstract

Obtaining functional data with newly identified rare variants increases certainty that the variant identified is relevant for dilated cardiomyopathy (DCM) causation. Two novel SCN5A rare variants, R222Q and I1835T, segregated with DCM in two families with affected individuals homozygous or heterozygous for the common SCN5A polymorphism H558R. cDNAs with each rare variant were constructed in the common Q1077del or Q1077 splice variant backgrounds with and without the H558R polymorphism and expressed in HEK293 cells. Sodium current (I(Na) ) was studied for each using whole-cell voltage clamp. In the Q1077del background I(Na) densities of R222Q and I1835T were not different from wild type, but the combined variants of R222Q/H558R, I1835T/H558R caused approximately 35% and approximately 30% reduction, respectively, and each showed slower recovery from inactivation. In the Q1077del background R222Q and R222Q/H558R also exhibited a significant negative shift in both activation and inactivation while I1835T/H558R showed a significant negative shift in inactivation that tended to decrease window current. In contrast, expression in the Q1077 background showed no changes in peak I(Na) densities, decay, or recovery from inactivation for R222Q/H558R and I1835T/H558R. We conclude that the biophysical findings, dependent upon common SCN5A variants, provide further evidence that these novel SCN5A rare variants are relevant for DCM.

Figure 1

Pedigrees of SCN5A related cardiomyopathy. Pedigrees have been labeled by letter (A) and (B), which correspond to their respective mutation as shown in Table 1 . Squares represent males, circles females. An arrowhead denotes the proband. A diagonal line marks deceased individuals. Solid symbols indicate idiopathic dilated cardiomyopathy (IDC) with or without heart failure; shaded symbols represent any cardiovascular abnormality. Open symbols represent unaffected individuals. For IDC cases, current age or age at death and age at IDC diagnosis or cardiovascular abnormality finding (in parenthesis) is presented. The presence or absence of the pedigree’s SCN5A mutation is indicated by a + or – symbol, respectively, next to the identified variant (R222Q or I1835T). Obligate carriers are noted in parenthesis, (+). Genotype for the H558R polymorphism is denoted H558R+ (present) or H558R‐ (absent) or H558R* (homozygous).

Figure 2

Biophysical properties of R222Q‐SCN5A associated mutations in the Q1077del background. Standard patch clamp protocols are depicted in the inset of each figure. (A) Peak current‐voltage relationships. (B) Voltage dependence of activation for WT and variants. (C) Steady‐state inactivation for WT and variants. (D) The peak current activation data are replotted as a conductance (G) curve with steady‐state inactivation relationships to show the overlap of these relationships (window current). (E) Time course of recovery from inactivation was elicited using the protocol described in the inset. R222Q/H558R exhibited slower recovery from inactivation than WT (time is on a log scale).

Figure 3

Biophysical properties of I1835T‐SCN5A associated mutations in the Q1077del background. (A) Peak current‐voltage relationships. (B) Voltage dependence of activation for WT and variants. (C) Steady‐state inactivation for WT and variants. (D) The peak current activation data are replotted as a conductance (G) curve with steady‐state inactivation relationships to show the overlap of these relationships (window current). (E) I1835T/H558R exhibited slower recovery from inactivation than WT (time is on a log scale). (F) Decay of macroscopic current. Compared with WT, I1835T/H558R showed significantly larger fast component (t_f) values across a wide range of test potentials from −20 mV to 10 mV. *p < 0.05 versus WT.