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 NaV1.5 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 NaV
1.5 currents in heterologous expression experiments, while WT β1 and β1B did. Incomplete penetrance, a well-recognized feature of the monogenic arrhythmia syndromes (12
), 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
), 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 NaV
1.5. 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 NaV
1.5 current and did not modulate the effect of the WT β1B protein. Furthermore, the voltage dependencies of activation and inactivation of NaV
1.5 coexpressed with p.Trp179X β1B were the same as those for NaV
1.5 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
), 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 NaV1.5 gating (shift in the voltage dependence of inactivation, in either the β1 or β1B background), supporting the idea that it associates with NaV1.5 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 NaV1.5 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
). 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 NaV
1.5 cell surface expression and/or altering gating (4
). 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
). A common feature in Brugada syndrome — consistent with reduced sodium current — is slowed conduction (28
). 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
1.5 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
), and conversely, in vitro electrophysiologic analysis of SCN5A
mutations linked to Brugada syndrome or isolated conduction disease consistently reveals loss of NaV
1.5 channel function (2
). Indeed, a single mutation segregating in a given family can lead to conduction disease in some family members and Brugada syndrome in others (6
). 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
The reported effects of β1 on NaV
1.5 channels are controversial (32
). Some groups have reported that β1 increases NaV
1.5 currents with or without affecting voltage dependence or channel kinetics, while others have reported no effect of β1 on NaV
1.5 current (20
). The β1B variant has to date only been studied in coexpression studies with the neuronal sodium channel NaV
1.2 (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 NaV
1.5 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 NaV
1.5 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 NaV
1.5 cell-surface expression and gating likely reside in the extracellular immunoglobulin domain. This is in line with previous studies of skeletal muscle (NaV
1.4 encoded by SCN4A
) and neuronal (NaV
1.2) 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
). 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 NaV
1.5 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
). 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 NaV
). 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
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
). Conversely, we have observed no neurological phenotype in our patients. Four SCN1B
mutations have been linked to epilepsy to date (47
), 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
). 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
) is also low in Brugada syndrome (12
). 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 NaV1.5 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.