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Cardiovascular Research 2002 55(2):279-289; doi:10.1016/S0008-6363(02)00445-5
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Copyright © 2002, European Society of Cardiology

A novel SCN5A arrhythmia mutation, M1766L, with expression defect rescued by mexiletine

Carmen R Valdiviaa,1, Michael J Ackermanb,1,*, David J Testerb, Tomoyuki Wadaa, Jorge McCormackc, Bin Yea and Jonathan C Makielskia

aDepartments of Medicine and Physiology, University of Wisconsin, Madison, WI, USA
bDepartments of Internal Medicine, Pediatrics, and Molecular Pharmacology, Long QT Syndrome Clinic, Guggenheim 501, Mayo Clinic, Rochester, MN 55905, USA
cPediatric Cardiology Associates, Tampa Bay, FL, USA

* Corresponding author. Tel.: +1-507-284-0101; fax: +1-507-284-3757 ackerman.michael{at}mayo.edu

Received 8 January 2002; accepted 8 April 2002


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: Mutations in the cardiac sodium channel gene, SCN5A, cause congenital long QT syndrome (LQT3), Brugada syndrome, idiopathic ventricular fibrillation, and conduction disease by distinct cellular and clinical electrophysiological phenotypes. Methods: Postmortem molecular analysis of SCN5A was conducted on an infant who presented shortly after birth with self-terminating torsades de pointes. The infant was treated with lidocaine, propranolol, and mexiletine and was stable for 16 months manifesting only a prolonged QT interval. The infant collapsed suddenly following presumed viral gastroenteritis, was found in 2:1 AV block, and was subsequently declared brain dead. Genomic DNA was subjected to SCN5A mutational analyses and DNA sequencing revealing a novel, spontaneous germline missense mutation, M1766L. The M1766L mutation was engineered into the hH1a clone by site-directed mutagenesis, transfected into embryonic kidney cells (HEK-293), and studied by voltage clamp. Results: The M1766L mutation caused a significant decrease in the sodium channel expression. Co-expression with β1 subunit, incubation at low temperature, and most effectively incubation with mexiletine partially ‘rescued’ the defective expression. In addition to this pronounced loss of function, M1766L also showed a 10-fold increase in the persistent late sodium current. Conclusions: These findings suggest that M1766L–SCN5A channel dysfunction may contribute to the basis of lethal arrhythmias, displays an overlapping electrophysiological phenotype, and represents the first sodium channelopathy rescued by drug.

KEYWORDS hH1a, NaV1.5 clone used in this study; INa, sodium current; M1766L, mutated hH1a channel containing a leucine at position 1766 instead of the normal methionine residue; NaV1.5, nomenclature for the channel protein product of SCN5A; SCN5A, official gene designation for the cardiac sodium channel gene residing on chromosome 3p21; WT, ‘wild-type’ Na channel

This article is referred to in the Editorial by C.R. Bezzina and H.L. Tan (pages 229–232) in this issue.


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
The SCN5A gene encodes for the {alpha} subunit of the human cardiac voltage dependent sodium channel hNaV1.5 [1]. The cardiac sodium channel initiates excitability in virtually all cardiac cells except sino-atrial and atrio-ventricular nodal cells. hNaV1.5 activates to produce a depolarizing inward sodium current (INa), and then promptly inactivates nearly completely within a few milliseconds. Mutations in SCN5A cause a particular type of the congenital long QT syndrome (LQTS) designated LQT3. LQT3 mutations generally yield a ‘gain-in-function’ phenotype by increasing the late sodium current whereby repolarization is delayed, the action potential plateau is sustained, and the QT interval is prolonged on the surface ECG (see recent reviews by Ackerman [2] and Splawski et al. [3]).

Mutations in SCN5A also cause Brugada syndrome and idiopathic ventricular fibrillation. In contrast to LQT3, these syndromes stem from ‘loss-of-function’ where INa is decreased (see review by Antzelvitch [4]). We report here a novel SCN5A missense mutation, M1766L (methionine replaced with leucine at position 1766), that occurred as a spontaneous, germline mutation in an infant presenting with torsades de pointes (TdP). Voltage clamp studies of this mutated channel studied in HEK cells show both ‘gain-of-function’ (increased late INa) and ‘loss-of-function’ (decreased peak INa density). INa density was increased in vitro by incubation with the antiarrhythmic drug mexiletine. In correlation with the clinical history, this suggests a completely novel antiarrhythmic drug mechanism, namely rescue of defective Na channel expression.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Postmortem molecular analysis of SCN5A
The Mayo Foundation Institutional Review Board approved this postmortem molecular analysis. The decedent's parents provided written informed consent for the study. This investigation conforms with the principles outlined in the Declaration of Helsinki [5]. Genomic DNA was extracted from peripheral blood lymphocytes obtained at the time of death using the Purgene DNA extraction kit from Gentra (Minneapolis, MN) [6]. Protein-encoding exons of the cardiac sodium channel gene, SCN5A, containing previously published mutations were targeted and amplified from genomic DNA by polymerase chain reaction using the full-length genomic sequence and previously published intron/exon-based primers [7].

Sequence variants were detected by denaturing high-performance liquid chromatography (DHPLC) using a Transgenomic WAVETM system (Omaha, NE), as previously described [8,9]. Sequences underlying abnormal DHPLC profiles were determined by automated dye-terminator cycle-sequencing (ABI Prism 377). Two-hundred control subjects including the 100 Caucasian human variation panel and the 100 African–American human variation panel from Coriell Cell Repositories and the National Institute of General Medical Sciences were analyzed to verify the putative SCN5A mutation (Camden, NJ). The decedent's parents were analyzed for the presence of the SCN5A mutation and paternity was confirmed using six highly polymorphic markers validated for paternity determinations [10].

A single nucleotide substitution (5298 A>C) involving codon 1766 (ATG-to-CTG) was found in Exon 28 of SCN5A. This novel missense mutation was absent in 400 normal alleles as well as in the decedent's parents indicating a spontaneous germline mutation. M1766 is located at the junction of the final transmembrane spanning segment of the fourth domain (DIVS6) and the C-terminal domain: a region that appears to be critical for drug-binding interaction [11] and one containing a published LQT3 mutation [12].

2.2 Clones, M1766L site-directed mutagenesis, and cell lines
The M1766L mutation was engineered in the hNaV1.5 {alpha}-subunit clone hH1a provided by Dr. H. Hartmann (Baylor College of Medicine, Houston, TX) by site-directed mutagenesis. To introduce the M1766L mutant, a cassette of hH1a was created from nucleotides 4224 to 6209 using the Kpn and XbaI restriction sites, and sub-cloned into pGEM7 vector. Primers were designed to introduce the M1766L mutation during site-directed mutagenesis (ExSite, Stratagene). DNA encoding either the {alpha}-subunit of the hH1a (designated wild-type, WT) or the M1766L mutant {alpha} subunit in the pcDNA 5.0 vector were transiently transfected into HEK 293.

For the experiments involving co-expression with β1 subunit, the channels were transiently transfected into HEK cells stably expressing the β1 subunit with the FLAG epitope on the extracellular domain (cell line kindly provided by Dr. Jack Kyle, University of Chicago, Chicago, IL). Approximately 5x105 cells were plated on a 60-mm-diameter plate (Falcon 3001) 24 h before transfection with 3 ml culture medium (MEM-complete) supplemented with 2 mM L-glutamine, 10% fetal bovine serum, 1 mM pyruvate solution, 0.1 mM non-essential amino acids, 10 000 U of penicillin and 10 000 µg of streptomycin. Transfection was carried out using a Superfect method (Qiagen). Plasmid DNA (5 µg) was mixed with Suprafect at a ratio of 1:5 (DNA/Suprafect) and diluted with Opti-MEM (GibcoBRL) at a final volume for transfection of 150 µl. The Suprafect/DNA mixture was incubated with the cells for 5 h in 1 ml of Opti-MEM medium. After incubation, cells with Suprafect/DNA mixture were replaced with 3 ml of MEM complete medium. To select transfected cells, GFP protein was co-transfected at a ratio of 1:10 DNA/DNA. Cells were incubated for 24 h at 37 °C after transfection before study unless otherwise indicated. The cells were treated with a trypsin–EDTA solution (0.25% trypsin, 1 mm EDTA, GibcoBRL), and then placed directly in the experimental chamber.

2.3 Functional expression and electrophysiological characterization
Macroscopic INa was recorded using the whole-cell method of the patch clamp technique. The electrophysiological recordings were carried out at room temperature. The bath (extracellular) solution contained (in mM): NaCl 140, KCl 4, CaCl2 1.8, MgCl2 0.75, Hepes 5, (pH 7.4 set with NaOH). The pipette (intracellular) solution contained: CsF 120, CsCl 20, EGTA 2, Hepes 5, (pH 7.4 set with CsOH). Electrodes were made from borosilicate glass using a puller (P-87, Sutter Instrument Co.) and heat-polished with a microforge (MF-83, Narishige, Japan). Electrode resistances ranged from 1 to 2 M{Omega}. Cells were continuously perfused with the bath solution mounted on an inverted microscope (Nikon) in a Faraday cage. Voltage-clamp protocols were generated using pClamp software (v8.3 Axon Instruments, Foster City, CA). Recorded membrane currents were digitized at 100 kHz for peak currents and 16 kHz for late current and low pass filtered at 2 kHz. An Axopatch 200 patch clamp amplifier was used with series resistant compensation. Data were acquired and analyzed using pClamp. Technical issues including a discussion of the adequacy of voltage control, endogenous currents, and the rate of negative shift in kinetic parameters for this preparation and voltage clamp method have been discussed previously [13]. Steady-state inactivation midpoints shifted with time (0.3 mV/min). Consequently, the experiments were completed within the first 15 min after patch rupture to minimize the effects of this shift on the results.

2.4 Data analysis
Passive leak subtraction was performed by measuring the passive leak for hyperpolarizing voltages between –150 and –110 mV. Measurement of late INa (INa-L) was performed as previously described [13]. We again confirmed by saxitoxin (1 µM) subtraction (n = 5) that passive leak subtraction yielded results similar to the passive leak subtraction for M1766L under our conditions. Data were fit to model equations (Boltzmann relationships) using non-linear regression with pClamp v6.4 or Origin 6.0. Goodness of fit was judged both visually and by the sum of squares errors. In the figures, mean data are shown as symbols with standard error of the mean (S.E.M.) bars. Determinations of statistical significance were performed using a Student t-test for comparisons of two means or when appropriate analysis of variance (ANOVA) for comparisons of multiple means. A P value of <0.05 was considered statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Clinical case
In 1998, JT was born full term to a 31-year-old G3P2 mother after an uncomplicated pregnancy. However, during labor, irregular fetal heart tones and bradycardia were detected resulting in an emergency Caesarean section delivery of a healthy appearing infant male weighing 3.1 kg and receiving excellent APGAR scores of 9 (out of 10) and 9 at 1 and 5 min, respectively. Shortly after birth, however, frequent premature ventricular contractions were noted on the monitor and the newborn had a >30-s, self-terminating episode of torsades de pointes (TdP) (Fig. 1A). A pediatric electrophysiologist (JM) was consulted, long QT syndrome was suspected, and the infant received 1 mg/kg lidocaine and 0.1 mg/kg propranolol intravenously. The infant's rhythm stabilized to ventricular bigeminy (Fig. 1B) and the infant was transferred to the neonatal intensive care unit. Upon arrival, another self-limited episode of TdP occurred. The infant was started on a lidocaine drip and intravenous propranolol every 6 h. Shortly thereafter, the ventricular bigeminy resolved and there were no further episodes of polymorphic VT. He remained in normal sinus rhythm with bradycardia and marked QT prolongation (QTc 480–530 ms) confirmed by serial electrocardiograms (Fig. 1C). On the second day of life, the infant had a generalized tonic–clonic seizure (normal heart rhythm) and phenobarbital was added. There was no subsequent seizure activity.


Figure 1
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  		Fig. 1 Electrocardiographic tracings from V5 (A) and V1 (B–E) recorded at standard settings of 25 mm/s and 10 mm/mV demonstrating: (A) self-terminating torsades de pointes (TdP) within first hours of life followed by normal sinus rhythm with 2:1 AV block and premature ventricular contractions. (B) Sinus rhythm with QT prolongation and ventricular bigeminy recorded 4 h after initial ECG shown in A. (C) Normal sinus rhythm with sinus bradycardia (HR 81 bpm) and marked QT prolongation (QTc=513 ms) at 5 days of age. (D) Following presumed viral gastroenteritis at 16 months of age, V1 trace showing severe QT prolongation with 2:1 AV block (arrows pointing to P waves). (E) Following administration of lidocaine, resumption of normal sinus rhythm. Arrows indicate macrovoltage T wave alternans. Tracings in D and E were recorded after extensive CPR.

 
He was discharged to home at 3 weeks of age on propranolol (5.5 mg/kg/day QID), mexiletine (6.6 mg/kg/day TID), and phenobarbital. His medications were adjusted according to weight as an out-patient and he remained event-free for 15 months. At 16 months of age, JT had a 24–48 h viral gastroenteritis with vomiting and diarrhea. He was evaluated as an out-patient by a pediatrician. Appearing well hydrated and having normal electrolytes, no further action was taken. Later that day, he was running when he tripped, struck a table, and began to cry profusely. Moments later, he collapsed and was unresponsive. CPR was initiated by his mother. He was asystolic when the paramedics arrived. After further resuscitation, sinus rhythm with 2:1 AV block was obtained (Fig. 1D). He was transported by helicopter to the pediatric intensive care unit in a comatose state. In the ICU, the 2:1 AV block did not improve with atropine. Lidocaine boluses promptly but transiently improved AV conduction. Steady state was achieved within 3 h restoring normal sinus rhythm with QT prolongation and manifest T wave alternans (Fig. 1E). Two days later, he was pronounced brain dead and life support was removed. Blood in EDTA was sent for molecular genetic testing for a cardiac channelopathy. Except for his arrhythmias, JT was normal. Both parents were asymptomatic and had normal ECGs. The family history was negative for unexplained sudden death except for a paternal great uncle who died of unknown causes 40 years ago.

3.2 Postmortem molecular analysis of SCN5A
Because of the infant's dramatic clinical response to lidocaine, we speculated that a defect in the cardiac sodium channel, SCN5A, might underlie his ventricular arrhythmia. Analysis by denaturing high-performance liquid chromatography of an amplicon from the final exon (28) of SCN5A elicited an abnormal elution profile (Fig. 2A). DNA sequencing (Fig. 2B) confirmed a novel missense mutation, M1766L. This amino acid substitution resides in a highly conserved region of SCN5A adjacent to both LQT3 and Brugada mutations and was not detected in 400 control alleles. Investigation of the decedent's parents indicates that this was a spontaneous, germline mutation (Fig. 2C). Analysis with polymorphic markers confirmed paternity (data not shown).


Figure 2
Figure 2
Figure 2
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  		Fig. 2 Molecular autopsy detects a novel, spontaneous germline mutation (M1766L) in SCN5A. (A) Analysis of the PCR product from exon 28 by DHPLC depicts an abnormal elution profile indicative of the presence of a sequence variation. (B) A portion of the DNA sequence from exon 28 encoding a portion of DIVS6 is shown for a normal control and the 16-month-old child. The arrow indicates the single nucleotide substitution (5298 A>C) found in the mutant allele in the decedent's sequence. The affected codon is enclosed by a rectangle indicating the M1766L missense mutation. (C) DHPLC elution profiles indicating that M1766L is a spontaneous, germline mutation in the infant. Automated DNA sequencing confirms its absence in both parents and analysis with polymorphic markers confirmed paternity. The decedent is indicated by the black square with a slash and his parents are indicated in the open square (father) and circle (mother).

 
3.3 M1766L–SCN5A: a ‘loss-of-function’ defect rescued by mexiletine
HEK 293 cells transiently expressing the M1766L and the WT channels were investigated for functional properties using the whole cell configuration of the patch-clamp technique. Examples of current traces for the WT (Fig. 3 left panels) and M1766L-hH1a mutant (Fig. 3 right panels) channels show that the most dramatic finding was greatly reduced INa levels for M1766L when the {alpha} subunit was expressed alone (Fig. 3B compared to Fig. 3A). Co-expression with the β1 subunit is known to increase INa density for WT [14]. β1 subunit co-expression also increased INa density for the M1766L mutant but did not restore the current fully (Fig. 3D compared to Fig. 3C).


Figure 3
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  		Fig. 3 Representative whole-cell current traces from HEK 293 cells transfected with either WT- (left panel), or M1766L- (right panel) hH1a Na channel clones under different culture conditions. The labels refer to the different culture conditions. All currents were recorded at room temperature (23 °C) and in the absence of mexiletine. In control conditions, the {alpha} subunit of WT (A) or M1766L (B) was expressed alone and incubated at 37 °C. In panels C and D, the channels were co-expressed with the β1 subunit and incubated at 37 °C. In panels E and F, the channels were co-expressed with the β1 subunit and incubated at 27 °C. In panels G and H, the {alpha} subunit was expressed alone and incubated with 500 µM mexiletine (Mex) which was washed out prior to recording the currents. The voltage clamp was a 24-ms step depolarization to different potentials in increments of 10 mV from a holding potential of –120 mV.

 
Previous studies have shown that mutations in the cardiac potassium channel encoded by HERG that decrease current density can be rescued by incubation at lower temperature and with channel blocking drugs [15]. Other reports have shown that incubation with mexiletine increases mRNA levels for the Na channel [16]. When the cells were incubated at lower temperature (27 °C), indeed M1766L current level was additionally increased for both WT (Fig. 3E compared to Fig. 3C) and for M1766L (Fig. 3F compared to Fig. 3D). Furthermore, when HEK-293 cells expressing the WT or the M1766L mutant channels were incubated for 24 h with 500 µM mexiletine at physiological temperature and without the β1 subunit, current density (measured after mexiletine was washed-out) was increased for M1766L (Fig. 3H compared to Fig. 3B).

Data for current levels found for the WT and M1766L mutant channels under different experimental conditions are summarized (Fig. 4). Mexiletine achieved the greatest rescue in current levels associated with M1766L. On average, current amplitudes for M1766L were 12 times greater after incubation with mexiletine.


Figure 4
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  		Fig. 4 Summary data for INa amplitudes with the culture conditions as described in Fig. 3. All incubations were done at 37 °C except for the third set of bars designated @27. The bars from left to right represent mean data for n = 24, 112, 10, 46, 7, 14, 6, and 8. Statistical significance in the difference between WT (clear bars) and M1766L (dark bars) was present for each condition as shown (*P<0.001). Statistical significance across conditions for WT and for M1766L were determined by ANOVA and differences from control are indicated (**P<0.001).

 
3.4 M1766L–SCN5A: a ‘gain-of-function’ defect—gating kinetics and late current
‘Steady-state’ activation and inactivation properties were also studied using standard protocols. The M1766L mutation caused a nonsignificant shift in the depolarizing direction by 7 mV for activation (Fig. 5A) and a significant depolarizing shift by 11 mV for inactivation (Fig. 5B). The greater shift of inactivation over activation would tend to increase the voltage range over which ‘window’ current could be observed, a mechanism proposed for ‘gain-of-function’ in the E1295K-LQT3 mutation [17]. These results were obtained from M1766L-hH1a {alpha} subunits expressed without the β1 subunit. The current densities for M1766L under these conditions were very low requiring selection of cells with higher current densities. Even after such selection the current density was only 20 pA/pF for M1766L compared with 205 pA/pF for WT.


Figure 5
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  		Fig. 5 Steady state of activation and inactivation for the WT and the M1766L mutant channel. The left panel represents the voltage-dependence of activation for both the WT (circles) and M1766L (squares). Currents were elicited by step depolarization for 24 ms to different potentials between –110 and +30 mV from a holding potential of –120 mV and normalized to the maximal peak INa. The mid-point of activation was obtained using a Boltzmann function where GNa=[1+exp (V1/2–V)/{kappa}]–1, where V1/2 and {kappa} are the mid-point and the slope factor, respectively, and GNa=INa(borm)/(V–Vrev) where Vrev is the reversal potential, k are the midpoint and the slope factor, respectively, and V is the membrane potential. The mid-points of activation were –47 mV for WT (n = 20) and –40 mV for M1766L (n = 24), respectively and the slopes were 4.3 and 5.3. In the right panel, steady state availability from inactivation for WT (circles) and the mutant channel M1766L (squares) were obtained in response to a test depolarization to 0 mV for 24 ms from a holding potential of –140 mV, following 1 s conditioning step to the various conditioning potentials (see protocol insert). The voltage dependent availability from inactivation relationship was determined by fitting the data to the Boltzmann function: INa=INa-max [1+exp (Vc–V1/2)/k]–1, where INa-max is the maximum peak INa, and the V1/2 and k are the mid-point and the slope factor, respectively, of the Boltzmann relationship. V1/2 for WT was –85 mV (n = 16) and for M1766L was –76 mV (n = 14).

 
Similar analyses were performed for the larger currents obtained by the rescue strategies summarized in Fig. 4. A similar significant shift in inactivation was obtained for mexiletine rescue (15 mV, from –90±5 mV in WT n = 16 to –75±2.0 in M1766L n = 10) but not for β1 rescue (2 mV from –75±3.5 mV for WT n = 8 to –73±0.7 mV for M1766L n = 9). For M1766L, about 10% of INa failed to inactivate during the 1-s conditioning depolarization (Fig. 5B). This finding for this two-pulse inactivation protocol is consistent with a non-inactivating or slowly inactivating late INa that was previously described for LQT3 mutations [18]. Increased late current for M1766L was also demonstrated for a single depolarization to –30 mV (Fig. 6A). To compare late INa from different cells, the currents were normalized to peak INa. In this example where the {alpha} subunits were expressed without the β1 subunit, the current for M1776L was much smaller, and the noisy trace for M1766L represents both random noise as well as noise from single channel activity. Summary data for late INa using this protocol showed that late INa was 12% of peak INa when the mutant M1766L-hH1a {alpha} subunit was expressed by itself (Fig. 6B). Late INa was also significantly increased for M1766L over WT with the greater current amplitudes obtained with the β1 and mexiletine rescue. The relative amount of late INa was not statistically different from M1766L-hH1a expressed alone without β1 or mexiletine. Note that unlike the {Delta}KPQ mutant [13] where late INa inactivates over time, decay of late INa in M1766L after 250 ms appears minimal (Fig. 6A). For three experiments with mexiletine rescued current, late INa at 750 ms was 98±1% of the late INa at 250 ms.


Figure 6
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  		Fig. 6 Late INa is increased relative to peak under different culture conditions. (A) Example of late currents in control culture conditions for WT and M1766L elicited by a depolarization from –140 mV to –30 mV for 750 ms. The currents were normalized to the peak current observed in each trace. In this example, peak current for WT was 4.4 nA and for M1766L was 0.29 nA. (B) Summary data for late INa expressed as the ratio of late INa to peak INa under different culture conditions. Late INa was measured as the mean current between 650 and 750 ms after the start of the depolarization. Under each condition, the difference in the late current amplitude between the M1766L and the WT was significant (*P<0.001). From left to right, the bars represent mean data for n = 16, 16, 5, 11, 6, and 3 experiments under the culture conditions indicated, bars represent S.E.M.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
Clinically, this infant presented with self-terminating episodes of TdP and marked QT interval prolongation consistent with LQT3. The increased late INa found for M1766L (Fig. 6) is characteristic of the LQT3 molecular phenotype [18]. In comparison to individuals with LQT1 and LQT2, clinical events in LQT3 tend to occur at rest or during sleep, occur later in life, be more lethal, and tend to be less responsive to β adrenergic blocker therapy [19]. This infant had a nearly fatal arrhythmic event early in life, with activity, and with apparent protection provided by beta-blockers and mexiletine.

Mutations in SCN5A with late INa typical of LQT3, however, have recently been diagnosed in two young children [20,21] and in two documented cases of sudden infant death syndrome [9]. This would represent a fifth case of symptomatic LQT3 in an infant. What is different about these mutations?

In one of the most studied LQT3 mutations ({Delta}KPQ), the late INa decays and recovers slowly [13]. This can lead to accumulation of inactivation and decreased late INa at higher rates of stimulation [22]. Thus, at faster heart rates, late INa would be reduced, the action potential plateau abbreviated, and the QT interval would shorten, accounting for the enhanced heart rate adaptation of the QT interval found in most LQT3 patients [23]. However, in the M1766L mutation and the SIDS mutations A997S and R1826H, the late current did not decay suggesting that mutations lacking the mechanism for enhanced QT-rate adaptation may represent a more lethal phenotype [9]. This might also explain this infant's apparent response to β blockers and to his sudden collapse during intense sympathetic stimulation. Of the two other LQT3 mutations, late INa for S941N showed decay in oocytes [20] and no late INa was reported for A1330P [21] indicating that other mechanisms may also account for early lethality.

In addition, the M1766L mutant also showed a dramatic decrease in INa density when expressed in HEK cells. Such a ‘loss-of-function’ is more typical of Brugada syndrome [24]. There was no ECG evidence of the right bundle branch block morphology and ST elevation of Brugada syndrome seen in this infant. ‘Loss-of-function’ has also been described in an SCN5A mutation in idiopathic ventricular fibrillation without Brugada criteria [25]. ‘Overlap’ SCN5A mutations with characteristics of both syndromes have been reported [26,27]. The novel M1766L mutation reported here might represent another example of an overlap syndrome with ‘gain-of-function’ for late INa and ‘loss-of-function’ for peak INa.

At first glance, however, it is difficult to implicate both mechanisms in the case study. If the mutant channel does not express well, then how is late INa generated to account for the prolonged QT interval? Although oocytes [28] and mammalian cell expression systems [29] have been used experimentally to characterize expression defects in SCN5A mutations for Brugada syndrome as well as HERG mutations [15] for LQT2, it is uncertain whether or not the effects would necessarily be the same in the environment of the myocardial cell. Alternatively, expression defects to cause Brugada syndrome might involve regional differences in INa expression [4]. The mechanisms controlling INa expression in the HEK cell as well as the myocyte are not well-characterized. The expression defect seen when expressing M1766L in HEK cells might suggest a trafficking defect that might occur only under certain conditions, regions, or times in the myocardium. This would allow for both loss-of-function and gain-of-function to play a role in the pathogenesis of the arrhythmia in this patient at different times, or at the same time and in different tissues, by introducing increased electrophysiological heterogeneity. TdP is speculated to be triggered by a prolonged QT interval and early afterdepolarizations [30], but perhaps maintained by reentrant mechanisms which are favored by heterogeneity of repolarization [31]. It is interesting to speculate that the infant's response to mexiletine may have improved his condition by simultaneously upregulating peak INa density and restoring homogeneity and at the same time reducing the trigger by blocking late INa [32]. Peak INa may be mostly unblocked at normal rates because of the use-dependent block properties and rapid off kinetics of mexiletine. The gastrointestinal illness preceding his death may have decreased his intake or absorption of mexiletine and contributed to his demise. This rescue of INa density by mexiletine is a novel potential antiarrhythmic drug mechanism.

In conclusion, the M1766L mutation shows both ‘gain-of-function’ in late INa as in other LQT3 mutations, and ‘loss-of-function’ in peak INa density. Moreover, the defect in INa density could be ‘rescued’ by the antiarrhythmic drug mexiletine, suggesting a novel antiarrhythmic mechanism that may have played a role in this infant's clinical course.

Time for primary review 27 days.


   Notes
 
1 C.R.V. and M.J.A. are co-equal first authors. Back


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

  1. Goldin A.L., Barchi R.L., Caldwell J.H., et al. Nomenclature of voltage-gated sodium channels. Neuron (2000) 28:365–368.[CrossRef][Web of Science][Medline]
  2. Ackerman M.J. The long QT syndrome: ion channel diseases of the heart. Mayo Clin Proc (1998) 73:250–269.[Abstract]
  3. Splawski I., Shen J., Timothy K.W., et al. Spectrum of mutations in long-QT syndrome genes. KVLQT1, HERG, SCN5A, KCNE1, and KCNE2. Circulation (2000) 102:1178–1185.[Abstract/Free Full Text]
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C. A. Remme and A. A.M. Wilde
SCN5A overlap syndromes: no end to disease complexity?
Europace, November 1, 2008; 10(11): 1253 - 1255.
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