Ethics statement
The study was conducted in accordance with the “Declaration of Helsinki”. The patient signed informed consent with approval for publication.
Genetic analysis
DNA was extracted from blood using salting out procedure. Genetic screening was carried out by means of Next-generation sequencing (NGS). Library preparation was performed using Nextera Flex Technology combined with the TruSight Cardio Panel (Illumina) consisting of 174 genes with known cardiac associations (supplementary information). Subsequent sequencing was carried out on a MiniSeq System (Illumina). The resulting reads were aligned to the human reference genome GRCh37/hg19. Variant calling and further evaluation was performed using the Software GensearchNGS (Phenosystems). Variants were filtered according to a pre-established prioritization protocol as described previously [15]. Evaluation was mainly based on presumed functional impact on the protein and allele frequency. Genetic aberrations expected to disrupt protein function and with minor allele frequency below 0.2% in public available databases (The Genome Aggregation and Exome Sequencing Project databases) were further evaluated. Detected sequence variants were assessed using common databases (as e.g. public population databases, Human Gene Mutation Database) and applying in silico prediction tools (PolyPhen-2 [16], MutationTaster [17], SIFT [18], CADD [19]). The identified variant was confirmed by polymerase chain reaction (PCR) and standard direct sequencing by means of BigDye Terminator v1.1 chemistry and 3130xl Genetic Analyzer (Applied Biosystems). The resulting data were evaluated using SeqScape Software.
Mutagenesis
For site-directed mutagenesis, the pcDNA3.1-EGFP vector comprising the wild cDNA sequence of SCN5A (hH1a isoform) was used (kindly provided by N. Neyroud, PhD, Inserm-Sorbonne Université Paris, France). The required sequence variant was induced using the QuikChange II XL Site-Directed Mutagenesis Kit (Agilent Technologies). The resulting constructs were verified by direct sequencing.
Electrophysiological measurements
For the functional characterization of the variant p.S106G, the mutant as well as wild type Nav1.5 channels were expressed in human embryonic kidney (HEK) cells, cell line 293 as described previously [20]. To mimic the heterozygous state of the patient, wildtype and mutant channels were also co-transfected in a 1/1 ratio (WT/MT). HEK293 cells were cultivated at 37 °C, 5% CO2 and passaged by transferring them to 35 mm plates. Transfection was performed by means of TurboFect transfection reagent (Thermo Fisher Scientific, Waltham, USA) and 3 μg of Nav1.5 constructs in the pcDNA3.1-EGFP vector. On the next day, cells were detached using accutase (PAA, GE Health Freiburg, Germany). Four hundred microliters were transferred to a new plate comprising 2 ml medium. Electrophysiological measurements were performed on single cells approximately 36 h following transfection using conventional patch clamp technique. Current measurements were carried out at room temperature in whole-cell configuration. Measurements and data acquisition were performed with an EPC-9 Patch Clamp amplifier (HEKA, Lambrecht, Germany) and the Patch Master (HEKA) software. Data were low pass filtered at 10 kHz and sampled at 20 kHz. Currents were measured in a bath solution composed of 135 mM NaCl, 4 mM KCl, 1 mM CaCl2, 2 mM MgCl2, 20 mM Glucose and 10 mM HEPES (pH 7.4, adjusted with NaOH). The pipette solution consisted of 140 mM CsCl, 5 mM NaCl, 4 mM Mg-ATP, 2 mM MgCl2, 5 mM EGTA, 10 mM HEPES (pH 7.4, adjusted with CsOH). In order to measure the peak current amplitudes and determine current/voltage (I/V) relationships, the cells were clamped from a holding potential of − 140 mV (100 ms) to test voltages ranging from − 100 mV to + 50 mV in 5 mV steps. To analyze the steady-state inactivation, cells were clamped from a holding potential of − 100 mV (100 ms) to test voltages ranging from − 140 mV to − 20 mV by 10 mV increments (500 ms), followed by a 50 ms test pulse to + 20 mV. For data analysis, we utilized Igor Pro 6.03 software (WaveMetrics, Lake Oswego, OR).
For noise analysis, Nav1.5 WT and S106G channels were activated 200 times by a depolarizing voltage step from a holding potential of − 140 mV to − 20 mV or − 40 mV. Current traces were imported into MATLAB to calculate the mean current <I > (t), the current differences between successive traces ΔIn(t), the mean current differences <ΔI>(t), and the variance of the current differences σ2(t). <I > (t), ΔIn(t), <ΔI>(t), and σ2(t) were calculated with the following equations [21]:
$$ \left\langle \mathrm{I}\right\rangle \left(\mathrm{t}\right)=\frac{1}{\mathrm{M}}{\sum}_{\mathrm{n}=1}^{\mathrm{M}}{\mathrm{I}}_{\mathrm{n}}\left(\mathrm{t}\right) $$
(1)
$$ \Delta {I}_n(t)=\frac{I_n(t)-{I}_{n+1}(t)}{2} $$
(2)
$$ \left\langle \Delta I\right\rangle (t)=\frac{1}{\mathrm{M}-1}{\sum}_{\mathrm{n}=1}^{\mathrm{M}}\Delta {I}_n(t) $$
(3)
$$ {\sigma}^2(t)=\frac{2}{\mathrm{M}-1}{\sum}_{\mathrm{n}=1}^{\mathrm{M}}{\left(\Delta {I}_n(t)-\left\langle \Delta I\right\rangle (t)\right)}^2 $$
(4)
where n is trace number and M the total number of traces collected. σ2 values were plotted against Imean and fitted with the following parabolic equation:
$$ {\sigma}^2\left(\left\langle I\right\rangle \right)=i\bullet \left\langle I\right\rangle -\frac{{\left\langle I\right\rangle}^2}{N}+{\sigma_b}^2 $$
(5)
where i is the single channel amplitude, N the total number of channels, and σb2 the background noise. Data were fitted with the constrain that σb2 is ≥0.
Data analysis
Results are reported as mean ± standard deviation (SD) of n experiments. An unpaired student’s t-test was applied to determine the statistical significance of the results obtained.