Sodium channel β1 subunit mutations associated with Brugada syndrome and cardiac conduction disease in humans

Brugada syndrome is a genetic disease associated with sudden cardiac death that is characterized by ventricular fibrillation and right precordial ST segment elevation on ECG. Loss-of-function mutations in SCN5A, which encodes the predominant cardiac sodium channel α subunit Na_V1.5, can cause Brugada syndrome and cardiac conduction disease. However, SCN5A mutations are not detected in the majority of patients with these syndromes, suggesting that other genes can cause or modify presentation of these disorders. Here, we investigated SCN1B, which encodes the function-modifying sodium channel β1 subunit, in 282 probands with Brugada syndrome and in 44 patients with conduction disease, none of whom had SCN5A mutations. We identified 3 mutations segregating with arrhythmia in 3 kindreds. Two of these mutations were located in a newly described alternately processed transcript, β1B. Both the canonical and alternately processed transcripts were expressed in the human heart and were expressed to a greater degree in Purkinje fibers than in heart muscle, consistent with the clinical presentation of conduction disease. Sodium current was lower when Na_V1.5 was coexpressed with mutant β1 or β1B subunits than when it was coexpressed with WT subunits. These findings implicate SCN1B as a disease gene for human arrhythmia susceptibility.

Voltage-gated sodium channels are critical for the generation and propagation of the cardiac action potential, and mutations in SCN5A, the gene encoding the major pore-forming sodium channel α subunit in the heart (Na_V1.5), cause multiple cardiac arrhythmia syndromes (1–4). Mutations producing enhanced inward current during the course of the action potential plateau, often as a consequence of destabilized fast inactivation of the channel, cause long QT syndrome type 3 (LQT3; OMIM 603830) (1). On the other hand, a reduction in sodium current leads to cardiac conduction disease, which may be progressive (OMIM 113900) (2, 3), and Brugada syndrome (OMIM 601144), characterized by ST segment elevation in the right precordial leads (V1 to V3) of the 12-lead ECG and episodes of ventricular fibrillation (4). Multiple mechanisms have been described that reduce sodium current in these syndromes, including altered gating of the channel or reduced cell-surface expression (5). In addition, mutations in SCN5A may manifest with an overlap of these different phenotypes (6–10). However, mutations in SCN5A are found in fewer than 30% of patients with Brugada syndrome, indicating involvement of other genes (11). A mutation in the glycerol-3-phosphate dehydrogenase 1–like gene (GPD1L) has recently been reported in a large kindred with Brugada syndrome (12); however, GPD1L mutations are rare in Brugada syndrome (13). Antzelevitch et al. have recently reported mutations in the gene encoding the L-type calcium channel (CACNA1C) or its β2b subunit (CACNB2b) in Brugada syndrome patients with unusually short QT intervals (14), but the frequency of these defects as a cause for more-typical Brugada syndrome is unknown. SCN5A mutations are also not identified in the majority of patients with cardiac conduction disease (15).

Sodium channels are multisubunit protein complexes composed not only of pore-forming α subunits but also of multiple other protein partners including auxiliary function-modifying β subunits (16, 17). In humans, 4 sodium channel β subunits (β1 to β4, encoded by SCN1B to SCN4B) have been identified, and they share a common predicted protein topology: a large extracellular N-terminal domain (including an immunoglobulin-like domain), a single transmembrane segment, and an intracellular C-terminal domain (16). Functions attributed to β subunits include an increase in sodium channel expression at the cell surface, modulation of channel gating and voltage dependence, and a role in cell adhesion and recruitment of cytosolic proteins such as ankyrin-G (16).

The β1 transcript arises from splicing of exons 1–5 of the SCN1B gene (Figure 1, A and B). More recently, a second transcript has been described, arising from splicing of exons 1–3 with retention of a segment of intron 3 (termed exon 3A), leading to an alternate 3ι sequence (Figure 1, A and B) (18, 19). This latter transcript encodes the β1B subunit, which, in spite of the different 3ι sequence, has a predicted protein topology similar to that of β1 (Figure 1C) (19). The β1B subunit has been shown to increase a neuronal sodium current (Na_V1.2) (19), but its effects on Na_V1.5 current have not yet been investigated, although β1 and β1B are both expressed in heart (19, 20).

Figure 1

Structure of β1 and β1B subunits. (A) Genomic structure of SCN1B. (B) Extension of exon 3 (c.208–458) into intron 3 creates a novel 3′ end of the transcript (exon 3A, c.208–978) and generates an alternate transcript encoding β1B. The gray region indicates the unique sequence of exon 3A. (C) Predicted topology of β1 and β1B. The β1B protein has unique juxtamembrane, transmembrane, and intracellular domains. The arrow indicates the initial amino acid of the β1B-specific segment. Circles indicate the locations of the mutations.

Since loss-of-function Na_V1.5 mutations cause conduction disease and Brugada syndrome, one could envision that mutations in sodium channel β subunits could also underlie these disorders by decreasing sodium current. Therefore, we tested the hypothesis that mutations in SCN1B coding sequences, for either β1 or β1B, underlie cases of conduction disease and Brugada syndrome. We identified 3 mutations segregating with arrhythmia in 3 kindreds, and 2 of the mutations were located in the newly described β1B transcript. Both β1 and β1B transcripts were expressed in the human heart and were abundant in Purkinje fibers that play a critical role in electric pulse conduction in heart. Electrophysiologic study of heterologously expressed sodium channels revealed loss of sodium current with mutant subunits.

Mutation analysis and clinical data. We screened 282 probands with Brugada syndrome and 44 with conduction disease for mutations in exons 1–5 of SCN1B encoding the β1 subunit and in exon 3A retained in the β1B transcript (Figure 1, A and B). SCN5A coding region mutations had been previously excluded in all 326 subjects. Three variants were identified in probands and family members (Figure 2A). These variants were absent in 1,404 population controls (see Methods).

Figure 2

SCN1B mutations found in patients with Brugada syndrome and conduction disease. (A) Pedigrees and phenotypes of the families affected by Brugada syndrome and/or conduction disease. Individuals carrying the mutation are indicated (+). Individuals who tested negative for the mutation are indicated (–). Individuals I-1 from family 1; and I-1, I-2, and II-3 from family 2 did not undergo genetic testing. Arrows indicate probands. (B) The c.259G→C mutation in SCN1B resulting in p.Glu87Gln found in family 1. (C) Alignment of β1 across species showing the high conservation of Glu87. (D) The c.536G→A (middle) and c.537G→A (right) mutations in exon 3A of β1B, both resulting in p.Trp179X found in families 2 and 3, respectively. (E) Twelve-lead ECG from the proband of family 2 (II-4). The arrowheads indicate ST-segment elevation typical of Brugada syndrome.

A missense mutation, c.259G→C (Figure 2B) in exon 3, resulting in p.Glu87Gln within the extracellular immunoglobulin loop of the protein (Figure 1C) was identified in a Turkish kindred affected by conduction disease (family 1; Figure 2A). Alignment of the β1 subunit amino acid sequence from multiple species demonstrated that Glu87 is highly conserved, supporting the importance of glutamate at this position (Figure 2C). The proband was a 50-year-old white Turkish female (II-1) who presented with palpitations and dizziness. Physical examination and echocardiography were normal, and her ECG showed complete left bundle branch block. A clinical electrophysiological study revealed a prolonged His-ventricle interval of 80 ms and inducible atrioventricular nodal reentrant tachycardia; complete atrioventricular block occurred following atrial programmed stimulation and during induced tachycardia. A dual-chamber pacemaker was implanted with resolution of symptoms. The same mutation was found in her brother (II-3), who had bifascicular block (right bundle branch block and left anterior hemiblock), and her mother (I-2), who had a normal ECG. There was no family history of syncope, sudden cardiac death, or epilepsy.

A nonsense mutation, c.536G→A in exon 3A (Figure 1B and Figure 2D), was identified in a French kindred affected with Brugada syndrome and conduction disease (family 2; Figure 2A). This mutation results in p.Trp179X and is predicted to generate a prematurely truncated protein lacking the membrane-spanning segment and intracellular portion of the protein (Figure 1C). The proband was a 53-year-old white male (II-4) who presented with chest pain. Physical examination, echocardiography, and coronary angiography were normal. His ECG showed ST segment elevation typical of Brugada syndrome and conduction abnormalities (prolonged PR interval of 220 ms and left anterior hemiblock; Figure 2E) (21). Ventricular fibrillation was induced by programmed electrical stimulation in basal state (in the absence of drugs). The same mutation was detected in his brother (II-1), nephew (III-1), and sister (II-2). The brother had no palpitations or history of syncope. His baseline ECG showed left anterior hemiblock and minor ST segment elevation suggestive of Brugada syndrome at baseline (type II saddleback abnormality; ref. 21); with flecainide challenge, the ST segment elevation was further exaggerated but did not meet criteria for a diagnostic (type I) pattern. The nephew had right bundle branch block and type II Brugada syndrome ECG after flecainide challenge, and the sister had a normal ECG and a negative flecainide test. There was no family history of tachyarrhythmias, syncope, sudden cardiac death, or epilepsy.

A different nonsense mutation, c.537G→A in exon 3A (Figure 2D), resulting in p.Trp179X, affecting the same codon as in family 2, was identified in a Dutch kindred (family 3; Figure 2A). The proband was a 17-year-old white female (II-1). Physical examination and echocardiography were normal, and a flecainide test for Brugada syndrome was negative. Her ECG showed right bundle branch block and prolonged PR interval of 196 ms (normal upper limit in teenagers, 180 ms) (22). The same mutation was found in her father (I-1), with normal ECG and negative flecainide test. The family history was negative for syncope, sudden cardiac death, or epilepsy.

β1 and β1B transcript expression. To confirm and extend previous reports that β1B is expressed in brain, heart, skeletal muscle, and other organs (19), we used quantitative real-time PCR in nondiseased human heart. Both β1 and β1B transcripts were detected in right and left ventricles and in Purkinje fibers (Figure 3). The β1 transcript level was higher in Purkinje fibers (which make up the conduction system in the ventricle) than left- (2.4-fold; P < 0.05) and right-ventricular (1.6-fold; P = NS) free walls. β1B transcript levels showed an even greater difference: Purkinje fibers versus left- (4.8-fold; P < 0.001) and right-ventricular (3.7-fold; P < 0.001) free walls. Levels of both transcripts were also slightly (but not statistically significantly) higher in right- versus left-ventricular free wall (1.5-fold and 1.3-fold for β1 and β1B transcripts, respectively).

Figure 3

Expression profile of β1 and β1B transcripts in nondiseased human ventricular tissue as determined by quantitative real-time PCR. Relative expression levels of the β1 and β1B subunits are presented, normalized to those of HPRT1 in LV (circles), RV (squares), and Purkinje fibers (triangles). Tissues for each group were collected from 6 human donors (nondiseased hearts, n = 6). Data points indicate the average of 2 measurements in each tissue sample. Larger symbols and error bars indicate median ± median absolute deviation for all samples.

Cellular electrophysiology. The effects of mutant and WT β1 and β1B variants on Na_V1.5 sodium current were assessed using the whole-cell patch-clamp technique in transfected CHO cells. As described in Methods, bicistronic expression vectors encoding a reporter (GFP or DsRed) with or without β subunits were cotransfected with expression vector encoding Na_V1.5. Currents were compared in cells transfected with SCN5A alone or SCN5A plus WT, mutant, or both β subunits.

Figure 4A shows representative current traces in cells expressing Na_V1.5 alone and Na_V1.5 plus WT or mutant β1B (p.Trp179X β1B) or their combination; current densities at –30 mV are summarized in Figure 4B. Coexpression of Na_V1.5 with WT β1B significantly increased sodium current density over Na_V1.5 alone, by 69%, while currents recorded with p.Trp179X β1B coexpression were no different from Na_V1.5 alone. Similarly, while coexpression of WT subunit with Na_V1.5 shifted the voltage dependence of both activation and inactivation to more negative potentials compared with those with Na_V1.5 alone, no such shift was observed with the mutant (Figure 4C and Table 1). This result indicates that while WT β1B modulates Na_V1.5 gating (in a fashion similar to WT β1; see below), the mutant exerts no such effect. Coexpression of WT or mutant β1B with Na_V1.5 did not alter recovery from inactivation (Figure 4D and Table 1).

Figure 4

Electrophysiological characteristics of the p. Trp179X β1B mutant. (A) Representative traces of sodium current demonstrating an increase in sodium current with WT but not mutant subunit. (B) Sodium current density at –30 mV for Na_V1.5 alone (n = 29), Na_V1.5 coexpressed with WT β1B (n = 28), Na_V1.5 coexpressed with p.Trp179X β1B (n = 18), Na_V1.5 coexpressed with WT β1B plus p.Trp179X β1B (1 μg for each; n = 14), and Na_V1.5 coexpressed with WT β1B plus p.Trp179X β1B (0.5 μg for each; n = 10). (C) Voltage dependence of activation and inactivation. Filled circles, open circles, and squares indicate Na_V1.5 alone, Na_V1.5 coexpressed with WT β1B, and Na_V1.5 coexpressed with p.Trp179X β1B, respectively. The pulse protocol used to study the voltage dependence of inactivation is shown in the inset. (D) Recovery from inactivation. Biophysical properties are provided in Table 1.

Table 1

Biophysical parameters of WT and mutant β1B

To examine whether expression of the mutant influences the effect of WT β1B on Na_V1.5 current (e.g., to produce a dominant negative action), cells were transfected with Na_V1.5 and varying amounts of WT and p.Trp179X β1B. Figure 4B shows that the sodium current increase over Na_V1.5 alone recorded with transfection of 1 μg of both β1B subunit constructs was identical to the increase with that of 1 μg of WT β1B. In addition, the increase in sodium current recorded with transfection of 0.5 μg of both β1B subunit constructs was 51% of that with 1 μg of β1B alone. These data indicate that p.Trp179X β1B does not exert a dominant negative effect on WT β1B function and further support the finding that the mutant, unlike WT, does not affect sodium channel function.

Figure 5A shows representative current traces of Na_V1.5 and Na_V1.5 coexpressed with WT and/or mutant β1 (p.Glu87Gln β1); current densities are summarized in Figure 5B. Coexpression of Na_V1.5 with WT β1 significantly increased sodium current density at –30 mV, by 76%, while coexpression with mutant β1 (p.Glu87Gln β1) did not increase the sodium current. The increase in sodium current recorded with coexpression of Na_V1.5 and 1 μg of both WT and p.Glu87Gln β1 (+20%) was markedly smaller than the increase with coexpression of Na_V1.5 with 1 μg WT β1 alone (+76%), indicating that this mutant exerts a dominant negative effect on WT β1 function. Figure 5C shows that WT β1 produced negative shifts in the voltage dependence of Na_V1.5 activation and inactivation similar to those observed with WT β1B. p.Glu87Gln β1 shifted the voltage dependence of inactivation to negative potentials (similar to WT β1) but did not alter the voltage dependence of activation (Table 2). Coexpression of WT or mutant β1 with Na_V1.5 did not alter recovery from inactivation (Figure 5D and Table 2).

Figure 5

Electrophysiological characteristics of the p.Glu87Gln mutant. (A) Representative traces of sodium current. (B) Current density at –30 mV for Na_V1.5 alone (n = 13), Na_V1.5 coexpressed with WT β1 (n = 17), Na_V1.5 coexpressed with p.Glu87Gln β1 (n = 18), and Na_V1.5 coexpressed with WT β1 plus p.Glu87Gln β1 (n = 15). (C) Voltage dependence of activation and inactivation. Filled circles, open circles, and squares indicate Na_V1.5 alone, Na_V1.5 coexpressed with WT β1, and Na_V1.5 coexpressed with p.Glu87Gln β1, respectively. (D) Recovery from inactivation. Biophysical properties are provided in Table 2.

Table 2

Biophysical parameters of WT and mutant β1

Since Glu87 is located in a region of the protein common to both β1 and β1B, we also studied the effects of p.Glu87Gln β1B on Na_V1.5 current properties (Supplemental Figure 1 and Supplemental Table 1; supplemental material available online with this article; doi: 10.1172/JCI33891DS1). While WT β1B increased Na_V1.5 current by 69% (Figure 4), p.Glu87Gln β1B did not increase the sodium current compared with Na_V1.5 alone. Similarly, WT β1B produced a negative shift in voltage dependence of both activation and inactivation (Table 1), while p.Glu87Gln β1B shifted only the voltage dependence of inactivation compared with Na_V1.5 alone. As with the other β subunit constructs studied, there was no change in recovery from inactivation. Thus, the effects of p.Glu87Gln were comparable in the β1 and the β1B backbones.

Since Glu87 is located in a region of the protein common to both β1 and β1B, we also studied the effects of p.Glu87Gln β1B on NaV1.5 current properties (Supplemental Figure 1 and Supplemental Table 1). While WT β1B increased NaV1.5 current by 69% (Figure 4), p.Glu87Gln β1B did not increase the sodium current compared with NaV1.5 alone. Similarly, WT β1B produced a negative shift in voltage dependence of both activation and inactivation (Table 1), while p.Glu87Gln β1B shifted only the voltage dependence of inactivation compared with NaV1.5 alone. As with the other β subunit constructs studied, there was no change in recovery from inactivation. Thus, the effects of p.Glu87Gln were comparable in the β1 and the β1B backbones.

In this study, we provide what we believe to be the first report of mutations in SCN1B sequences encoding the β1 and β1B transcript variants in patients with conduction disease and/or Brugada syndrome. Further, we provide new data indicating that β1 and β1B transcripts in the heart vary by region; greater expression in Purkinje fibers is consistent with the conduction system phenotype we describe in mutation carrier patients. Finally, we demonstrate that the β1 and β1B variants modulate function of the major cardiac sodium channel α subunit Na_V1.5 and that the identified SCN1B mutations blunt or inhibit this effect.

The 3 mutations were identified in 3 probands with conduction disease and/or Brugada syndrome as well as in other family members with or without these arrhythmia phenotypes. Formal linkage analysis was not possible because the families are too small and penetrance is incomplete. Thus, evidence in support of disease causality of these mutations (beyond their identification in subjects with clinical phenotypes) includes the findings that both β1 and β1B transcripts are expressed in heart and that the mutant subunits (p.Glu87Gln β1, p.Glu87Gln β1B, and p.Trp179X β1B) did not increase Na_V1.5 currents in heterologous expression experiments, while WT β1 and β1B did. Incomplete penetrance, a well-recognized feature of the monogenic arrhythmia syndromes (12, 23), was observed. For SCN5A mutations linked to Brugada syndrome, penetrance as low as 12.5% has been described (24). A role for sex, age, and genetic modifiers (e.g., common polymorphisms) is suspected (5, 25, 26), but the mechanisms for this common clinical finding remain poorly understood.

Two types of mutations were identified. The c.536G→A and c.537G→A mutations in exon 3A both result in a stop codon at position 179, predicted to generate a β1B protein lacking the transmembrane and cytoplasmic domains and thus unable to integrate into the sarcolemma and to associate with Na_V1.5. Thus, the a priori assumption is that a mutation such as this will cause disease by simple haploinsufficiency. The electrophysiologic data support this idea, since coexpression of p.Trp179X β1B failed to increase Na_V1.5 current and did not modulate the effect of the WT β1B protein. Furthermore, the voltage dependencies of activation and inactivation of Na_V1.5 coexpressed with p.Trp179X β1B were the same as those for Na_V1.5 alone, in contrast to the shifts observed with WT β1B. While Scn1b-knockout mice display clear ECG changes (27), studies with young (17- to 18-day-old) heterozygotes identified no difference from WT. Since age-related changes in conduction are a recognized feature of cardiac conduction disease and conduction delay is one of the proposed mechanisms of Brugada syndrome (2, 28), aging may be important for the β subunit–mediated phenotype.

On the other hand, the c.259G→C mutation leads to an amino acid substitution (p.Glu87Gln) within the extracellular domain of the protein. The electrophysiological data demonstrate that the mutant subunit did modulate Na_V1.5 gating (shift in the voltage dependence of inactivation, in either the β1 or β1B background), supporting the idea that it associates with Na_V1.5 at the cell surface. In addition, in contrast to the p.Trp179X β1B, p.Glu87Gln did exert a dominant negative effect on the WT subunit. Thus, the 3 mutations lead to a decrease in Na_V1.5 current through somewhat different mechanisms. This reduction of current is consistent with the conduction disease and Brugada syndrome phenotypes of the patients.

Normal impulse propagation in the atria, ventricles, and Purkinje network is critically dependent on normal sodium channel function. Dysfunction of the sodium channel leads to conduction delay, and loss-of-function mutations in SCN5A have been described in isolated conduction disease unassociated with structural heart disease (2, 3). Thus, our finding of SCN1B mutations associated with reduced sodium current in patients with conduction disease is consistent with previous studies of the mechanism of this disorder. The preferential expression of the β1 and β1B transcripts in human Purkinje fibers further supports the prominent conduction delay seen as part of the clinical phenotypes.

Loss-of-function mutations in SCN5A were the first reported cause of the Brugada syndrome (4). These mutations reduce sodium current by reducing Na_V1.5 cell surface expression and/or altering gating (4, 5, 29). A common view is that in epicardial cells, this reduction in sodium current produces marked action potential shortening, attributed to an “unopposed” early transient outward potassium current. By contrast, reduction of sodium current in endocardial cells is thought to produce only modest action potential shortening. The resultant increased heterogeneity of repolarization predisposes to rapid reentry, resulting in ventricular fibrillation (4, 30). A common feature in Brugada syndrome — consistent with reduced sodium current — is slowed conduction (28, 31). Indeed, an alternate proposed mechanism suggests that the characteristic right-precordial ST-segment elevation on the ECG and initiation of arrhythmias is attributable primarily to right-ventricular outflow tract conduction delay (28). The trend to higher expression levels of β1B in right ventricle may thus contribute to the Brugada syndrome phenotype.

This idea is further supported by functional studies in a single large kindred in which a GPD1L mutation was linked to Brugada syndrome: coexpression of mutant GPD1L with Na_V1.5 was reported to decrease sodium current, consistent with the observation that loss-of-function mutations in SCN5A cause Brugada syndrome (12). In principle, reduction in L-type calcium current might also produce differential effects in epicardial and endocardial sites and thus cause Brugada syndrome; rare kindreds with this mechanism have now been described (14).

Conduction disease was observed in families 1 and 3, while in family 2, mutation carriers presented either solely with conduction disease or conduction disease in combination with ECGs typical of Brugada syndrome. This phenomenon of overlapping clinical phenotypes is common in individuals with SCN5A mutations leading to loss of sodium channel function (6, 7), and conversely, in vitro electrophysiologic analysis of SCN5A mutations linked to Brugada syndrome or isolated conduction disease consistently reveals loss of Na_V1.5 channel function (2, 4). Indeed, a single mutation segregating in a given family can lead to conduction disease in some family members and Brugada syndrome in others (6, 7). What determines the ultimate phenotype — Brugada syndrome versus isolated conduction disease — is unknown. Sex, age, and genetic modifiers (e.g., common polymorphisms) have been proposed as modulators of the clinical phenotypes (5, 25, 26).

The reported effects of β1 on Na_V1.5 channels are controversial (32). Some groups have reported that β1 increases Na_V1.5 currents with or without affecting voltage dependence or channel kinetics, while others have reported no effect of β1 on Na_V1.5 current (20, 33–37). The β1B variant has to date only been studied in coexpression studies with the neuronal sodium channel Na_V1.2 (encoded by SCN2A), where it was shown to increase sodium current and cause a small negative shift in voltage dependence of activation (19). In our experiments, WT β1 and β1B had similar effects on Na_V1.5 current: both increased sodium currents and led to hyperpolarizing (negative) shifts in voltage dependence of activation and inactivation.

Not only were the effects of the WT β subunits on Na_V1.5 current similar, but the effects of the p.Glu87Gln mutation in the β1 background (p.Glu87Gln β1) were also similar to those in the β1B background (p.Glu87Gln β1B). Although the β1 and β1B variants share the same topology (an N-terminal extracellular immunoglobulin domain, a transmembrane domain, and a C-terminal cytoplasmic domain), their sequence identity is limited to the extracellular immunoglobulin domain; the C-terminal half of β1B, residues 150–268, has only approximately 17% amino acid sequence identity with β1 (19). Taken together, the data suggest that the molecular determinants of β1 and β1B modulation of Na_V1.5 cell-surface expression and gating likely reside in the extracellular immunoglobulin domain. This is in line with previous studies of skeletal muscle (Na_V1.4 encoded by SCN4A) and neuronal (Na_V1.2) sodium channel α subunits that have shown that deletion of the intracellular domain of the β1 subunit has no effect on its modulation of α subunit function, whereas deletions within the extracellular domain block modulation (38–40). Alternatively, specific residues may not be as important as preservation of overall structural motifs, as suggested by the data of Zimmer and Benndorf, who reported that the β1 subunit modulates Na_V1.5 via the membrane anchor plus additional intracellular or extracellular regions (41).

In addition to modulation of sodium channel α subunit expression and function, other roles have been suggested for β subunits: these include acting as adhesion molecules or as participants in signal transduction (16, 32). The different transmembrane and C-terminal domains of β1 and β1B might therefore lead to participation in different signaling pathways. For instance, phosphorylation of the tyrosine at position 181 of the β1 C terminus regulates its interaction with ankyrin-G (42), which is thought to be critical for ankyrin-G localization within cardiomyocytes (intercalated discs versus T tubules). β1B lacks this tyrosine in its C-terminal domain, so a role for β1B as a modulator of this function seems less likely.

Another mechanism regulating function of β subunits is the potential for cleavage by β-site amyloid precursor protein–cleaving enzyme (BACE1) and γ-secretase, resulting in the release of the N- and C-terminal fragments (43). The processed C-terminal fragment of β2 and β4 has been reported to be associated with cell adhesion, migration, and morphogenesis in neuronal cells as well as regulation of the expression level of the neuronal sodium channel Na_V1.1 (44–46). Thus, p.Trp179X β1B may result in absence of functions depending on the generation of a β subunit C-terminal fragment by BACE1. However, a role for BACE1 cleavage has not been studied in either human β1 subunit or cardiomyocytes, and the cleavage site located at the common juxtamembrane domain in β1 and β1B is not conserved between human and mice (19, 43).

Mutations in SCN1B have been previously reported in generalized epilepsy with febrile seizures plus (47), and β1-null mice exhibit a severe seizure disorder and die at approximately 3 weeks of age (48). In addition, these mice exhibit bradycardia and prolonged rate-corrected QT intervals (27). These changes suggest that β1 plays an important role in the murine heart, although it is possible that the changes are a consequence of the severe overall developmental phenotype in this model (48). To our knowledge, defects in cardiac function have not been investigated in SCN1B mutation carriers presenting with epilepsy (32, 49). Conversely, we have observed no neurological phenotype in our patients. Four SCN1B mutations have been linked to epilepsy to date (47, 49, 50), all of which localize to the extracellular immunoglobulin-like fold of the protein, as does the p.Glu87Gln mutation reported here. One additional possible link between the cardiac and neurological phenotypes associated with β1 mutations is the syndrome of sudden unexpected death in epilepsy (SUDEP) (51), where a role for cardiac bradyarrhythmias has been proposed (52).

To date, SCN5A mutations are the most common cause identified in cases of Brugada syndrome, and SCN5A is the only identified causative gene in conduction disease (2, 11). However, SCN5A mutations are not identified in the majority of patients, and it has been reported that the frequency of mutations in other implicated genes (CACNA1C, CACNB2b, GPD1L) is also low in Brugada syndrome (12–14). In this study, SCN1B mutations were identified in less than 1% of probands with Brugada syndrome and less than 5% of probands with conduction disease and thus account for a small subset of these inherited arrhythmic syndromes.

A conventional heterologous mammalian expression system was used for functional assessment of the mutations. The environment in this approach is different from that in native cardiomyocytes, and other proteins known to associate with the sodium channel complex, such as other β subunits, are generally absent. Despite these limitations, the in vitro characteristics of the mutations were concordant with the phenotype observed in the patients, which, in combination with the genetic data presented, supports the hypothesis of a causal relationship between the mutations and disease.

In summary, we have for the first time to our knowledge identified SCN1B mutations in families with conduction disease and Brugada syndrome. We have shown expression of the β1 subunit transcript and the alternate β1B subunit transcript variant in human heart and demonstrated reduced Na_V1.5 sodium current as a result of loss or altered β subunit modulation of Nav1.5 current. These findings implicate SCN1B as a disease gene for human arrhythmia susceptibility.

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We thank Leander Beekman, Peter van Tintelen, Arie O. Verkerk, Carol Ann Remme, Alfred George Jr., Katherine Murray, Sabina Kupershmidt, Kai Liu, Sameer Chopra, Nathalie Gaborit, Satoru Komura, Mahmut Akyol, and Moritz Sinner for their contributions to performing and/or analyzing this work and for helpful discussions. This work was supported by grants from the NIH (HL46681 and HL65962 to D.M. Roden), a Fondation Leducq Trans-Atlantic Network of Excellence grant (05 CVD 01, Preventing Sudden Death, to D. Escande, J.-J. Schott, A.M. Wilde, S. Kääb, and D.M. Roden), Netherlands Heart Foundation grant 2003T302 (to A.A.M. Wilde), the Interuniversity Cardiology Institute of The Netherlands (project 27, to A.A.M. Wilde), Agence Nationale de la Recherche grant 05-MRAR-028 (to J.-J. Schott), Agence Nationale de la Recherche grants 05-MRAR-028 and 06-MRAR-022 (to J.-J. Schott), German Federal Ministry of Education and Research (BMBF) grants 01GI0204, 01GS0499, 01GI0204, and 01GR0103 (to S. Kääb, A. Pfeufer, and H.-E. Wichmann), and a Sumitomo Life Social Foundation grant (to H. Watanabe). The KORA platform is funded by the BMBF and by the State of Bavaria. Resequencing of Coriell samples was performed by the J. Craig Venter Institute through the NHLBI Resequencing and Genotyping Program. We also thank Andras Varro (University of Szeged) for providing the human tissues. C.R. Bezzina is an Established Investigator of The Netherlands Heart Foundation (grant 2005/T024).

Address correspondence to: Connie R. Bezzina, Heart Failure Research Center, Department of Experimental Cardiology, L2-108-1, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands. Phone: 31-20-5665403; Fax: 31-20-6976177; E-mail: C.R.Bezzina@amc.uva.nl.

Conflict of interest: The authors have declared that no conflict of interest exists.

Reference information: J. Clin. Invest.118:2260–2268 (2008). doi:10.1172/JCI33891

Hiroshi Watanabe, Tamara T. Koopmann, and Solena Le Scouarnec contributed equally to this work.

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