Journal of 29 (2013) 71–76

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Journal of Arrhythmia

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Review Genetic basis of Brugada syndrome

Minoru Horie, MD, PhDn, Seiko Ohno, MD, PhD

Department of Cardiovascular and Respiratory Medicine, Shiga University of Medical Science, Seta-Tsukinowa-cho, Otsu, Shiga 520-2192, Japan article info abstract

Article history: Brugada syndrome (BrS) is associated with the familial sudden death syndrome, and more than 10 Received 11 December 2012 have been reported as causative for or modifiers of BrS. All mutations are related to functional changes Received in revised form of inward sodium or calcium currents or outward potassium currents. SCN5A was the first gene known to 11 January 2013 be associated with BrS; it encodes the a-subunit of the cardiac . Approximately 20% of BrS Accepted 15 January 2013 patients in genotyped cases were found to carry SCN5A mutations. The frequency for other BrS-associated Available online 21 March 2013 gene mutations is so low that genotype–phenotype correlations for these genes have not been studied to Keywords: thesameextentasSCN5A-related BrS. In some families with SCN5A mutations, the penetrance of the Brugada syndrome mutations is low, and pathophysiological changes in the right ventricular outflow tract were reported in Gene mutations patients with SCN5A mutations. Furthermore, the phenotypes of SCN5A-related BrS can overlap with other SCN5A phenotypes, including long QT and sick sinus syndrome, thereby suggesting that SCN5A mutations might be Overlap syndrome modifiers for BrS, but they do not direct cause BrS. Here, we summarize the genetic background of BrS, with a particular focus on recent progress in this field. & 2013 Japanese Heart Rhythm Society. Published by Elsevier B.V. All rights reserved.

Contents

1. Introduction ...... 72 2. SCN5A (BrS type 1 [BrS1]) ...... 72 3. GPD1L (BrS2) ...... 73 4. CACNA1C (BrS3), CACNB2 (BrS4), and CACNA2D (BrS9) ...... 73 5. SCN1B (BrS5) ...... 73 6. KCNE3 (BrS6) ...... 73 7. SCN3B (BrS7) ...... 73 8. KCNJ8 (BrS8)...... 73 9. KCND3 (BrS10) ...... 74 10. MOG1 (BrS11)...... 74 11. SLMAP (BrS12) ...... 74 12. SCN2B (BrS13) ...... 74 13. Other candidate genes ...... 74 13.1. KCNH2 ...... 74 13.2. KCNE5 (KCNE1L)...... 74 13.3. HCN4 ...... 74 14. The frequency of minor gene mutations in BrS ...... 75 15. Other types of mutations in BrS patients ...... 75 16. Conclusion ...... 75 Conflict of interest...... 75 Acknowledgments ...... 75 References ...... 75

n Corresponding author. Tel.: þ81 77 548 2213; fax: þ81 77 543 5839. E-mail address: [email protected] (M. Horie).

1880-4276/$ - see front matter & 2013 Japanese Heart Rhythm Society. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.joa.2013.01.008 72 M. Horie, S. Ohno / Journal of Arrhythmia 29 (2013) 71–76

1. Introduction number of families with a history of BrS that have been geno- typed. Presently, genetic testing is only diagnostic; however, once Brugada syndrome (hereafter referred to as BrS) is character- the proband is successfully genotyped, for example as a carrier of ized by a peculiar ST segment (J point) elevation in the right SCN5A mutation, the mutation-specific genetic test would be precordial leads of the electrocardiogram (ECG) as well as idio- highly recommended for family members because it can deter- pathic ventricular fibrillation, occurring particularly during sleep, mine the relatives that are at a potential risk for BrS and BrS- which often results in nocturnal sudden death. There is a definite related death. In this review, we summarize the genetic basis of male predominance in BrS-related morbidity, and men are 10- BrS, with a particular focus on recent reports. times more affected than women. Approximately 20% of the phenotype-positive probands have a family history of syncope or sudden death. These clinical features are partially similar to 2. SCN5A (BrS type 1 [BrS1]) those previously described as ‘‘Pokkuri’’ disease in Japan [1,2] and ‘‘Lai Tai’’ in Thailand [3]. Indeed, its prevalence is higher in Japan Voltage-gated sodium channels are essential for modulating the and Southeastern Asia, which may be partially related to an amplitude and upstroke velocity of the cardiac action potential, Asian-specific sequence in the promoter region of SCN5A [4]. which are parameters that are important determinants of impulse The disease was named after the report in 1992 by the Brugada propagation and conduction velocity through the myocardium. brothers [5], but several case reports on similar ECG findings and Functional analyses using patch-clamp techniques in a heterolo- clinical phenotypes from Italy and Japan were published previously gous expression system demonstrated that most of the SCN5A [1,2,6]. Some of these reports described an associated structural mutations had a loss-of-function effect, primarily manifested as a abnormality, especially in the right-side of the heart, suggesting the failure of intracellular trafficking to the cell membrane or mal- coexistence of subclinical arrhythmogenic right ventricular cardio- function of gating kinetics [12–15]. Surprisingly, SCN5A mutations myopathy [7]. Patients with BrS have been shown to carry SCN5A are associated with different phenotypes, in addition to BrS, even mutations, which raises the possibility that the specific structural among members of the affected family (Fig. 1). These phenotypes change is caused by SCN5A mutations [8,9]. include long QT syndrome (type 3) [16–21], progressive cardiac The inheritance pattern of BrS shows an autosomal dominant conduction defects [22–25], sick sinus syndrome [26–29], familial mode of transmission. Candidate gene analysis showed that muta- atrial fibrillation [30–32], early repolarization syndrome [33], tions in SCN5A were responsible for the phenotype of BrS in 3 families, dilated cardiomyopathy [34–36], and sudden infant death syn- and over 300 SCN5A mutations have been reported to date [10]. drome [37–39]. Very recently, a French research group reported an SCN5A encodes for the a subunit of the cardiac sodium SCN5A R222Q mutation in 3 unrelated families with multifocal channel, and SCN5A mutations are found in 11–28% of clinically ectopic Purkinje-related premature contractions (MEPPC) and diagnosed BrS patients [11]. Since 1998, genetic variants in over demonstrated that the mutation caused a gain-of-function of the 10 genes have been reported in patients with BrS. Table 1 lists cardiac sodium channel [40]. Even in a single patient, these these genes, the mutation locations, and the functional changes in phenotypes can overlap (so-called overlap syndrome). Character- each current induced by these mutations. In all istic clinical features differentiate the SCN5A-related BrS from other genotypes, either the decrease in inward sodium or calcium forms of BrS and can help predict the genetic diagnosis of BrS. The currents (INa or ICa) or the increase in outward potassium currents appearance of overlap phenotypes seems to be mutation-specific. is thought to be responsible for the BrS phenotype. For example, the SCN5A E1784K mutant has been linked to not BrS is, therefore, a very heterogeneous disease entity from the only BrS but also long QT and familial sick sinus syndrome [19]. perspective of its genetic background. In addition, it is notable Though they are the most studied among BrS-associated genes, that the genotypes of over two-thirds of BrS patients have not yet SCN5A genotype–BrS phenotype correlations remain controver- been determined despite extensive genetic testing, which limits sial. According to numerous functional assays, there is convincing the prognostic value of the genetic tests. Indeed, except for data for the hypothesis that the biophysical effects of SCN5A SCN5A-related BrS, the genotype–phenotype correlation of other mutations identified in BrS patients account for their phenotype. BrS-associated genetic changes is relatively not well studied. However, in extensive surveys of family members, SCN5A muta- The paucity of genetic studies of BrS is mainly due to the small tions were also identified in phenotype-negative individuals [41]. On the contrary, in a study on 13 BrS families containing Z5 Table 1 BrS patients [42], 8 individuals presented with typical type 1 BrS Brugada syndrome causative genes.

BrS type Gene name Function % of probands Reference BrS

BrS1 SCN5A 3p 21–23 INak 11–28% [12] ERS LQTS

BrS2 GPD1L 3p 24 INak Rare [44]

BrS3 CACNA1C 12p 13.3 ICak 6.6% [48]

BrS4 CACNB2 10p 12.33 ICak 4.8% [48] k FAF PCCD BrS5 SCN1B 19q 13.1 INa 1.1% [53] SCN5 BrS6 KCNE3 11q 13–14 Itom Rare [55]

BrS7 SCN3B 11q 23.3 INak Rare [57] m BrS8 KCNJ8 12p11.23 IKATP 2% [61] DCM SSS BrS9 CACNA2D1 7q21-22 ICak 1.8% [49]

BrS10 KCND3 1p13.3 Itom Rare [63]

BrS11 MOG1 17p13.1 INak Rare [65] SIDS MEPPC

BrS12 SLMAP 3p21.2-p14.3 INak Unknown [66]

BrS13 SCN2B 11q23 INak Unknown [67] Fig. 1. Scheme showing the multiple cardiac phenotypes that are associated with Other genes SCN5A mutations. DCM, dilated cardiomyopathy; ERS, early repolarization syn- drome; FAF, familial atrial fibrillation; LQTS, long QT syndrome (type 3); MEPPC, KCNH2 7q36.1 IKrm Unknown [64] multifocal ectopic Purkinje-related premature contractions; PCCD, progressive HCN4 15q24.1 Ifk Unknown [71] cardiac conduction defects; SIDS, sudden infant death syndrome; SSS, sick sinus KCNE5 Xq 22.3 Itom Unknown [70] syndrome. M. Horie, S. Ohno / Journal of Arrhythmia 29 (2013) 71–76 73 who lacked SCN5A mutations. Taken together, these data suggest investigation is essential to elucidate the pathophysiology of BrS that mutations in SCN5A alone do not directly cause but work as patients with LTCC mutations. modifiers to increase the likelihood of developing the BrS pheno- type in some BrS patients. 5. SCN1B (BrS5)

The sodium channel is composed of a pore-forming a subunit 3. GPD1L (BrS2) and auxiliary function-modifying b subunits [51].Inhumans,4b subunits have been identified [52].Amongthese,SCN1B encodes one In 2002, the second candidate of BrS was discovered on of the b subunits, b1. In 2008, Watanabe et al. [53] reported a novel chromosome 3p22-25 in a large Italian family by using linkage mutation, W179X, in SCN1B from a survey of 282 BrS patients. analysis [43]. The sodium channel genes located on that locus, The mutation was located in the transcriptional variant b1B, which SCN5A, SCN10A,andSCN12A, on chromosome 3 were excluded as had an alternative 30 sequence. ECG analysis of the proband with the candidates in the family. Therefore, London et al. [44] further SCN1B mutation showed an ST segment elevation, which is typical of searched and narrowed the BrS-associated region to 1 million bp BrS, and conduction abnormalities (prolonged PR interval of 220 ms on chromosome 3p24. Although 4 known genes were located on and left anterior hemiblock). In functional analysis, co-expression of this region, they were eliminated by single-strand conformational Nav1.5 and WT b1B increased the INa densities and evoked a polymorphism (SSCP) and/or direct sequencing. In the linkage negative shift in the activation and inactivation curves, whereas region, RNA from 2 genes of unknown function in the heart was these effects were diminished in cells co-expressing Nav1.5 and the identified using reverse-transcriptional polymerase chain reaction W179X b1B mutant. (PCR) and direct sequencing. Finally, a missense mutation was detected in one of the genes. The gene was GPD1L (NM_015141), and the mutation was a C/T base-pair change (C899T) leading to an 6. KCNE3 (BrS6) alanine-to-valine substitution at amino acid 280 (A280V). The co- expression of SCN5A and GPD1L-A280V, but not SCN5A and wild- KCNE3 is a homolog of KCNE1, which encodes MinK, and the type (WT) GPD1L, in human embryonic kidney (HEK) cells sig- encoded by KCNE3 is called MinK-related protein 2 nificantly changed the amplitude of INa. The data also showed that (Mirp2). Thus far, 5 KCNE genes have been identified and all the trafficking deficiency of Nav1.5 to the cell surface was the genes encode auxiliary b subunits, which modify many kinds of underlying mechanism responsible for decreased INa. Following potassium channels [54]. In a screening study of 105 BrS patients, this report, 3 novel GPD1L mutations, corresponding to E83K, an R99H Mirp2 mutation was found in a proband who was I124V, and R273C in GPD1L protein, were identified in 304 cases resuscitated from cardiac arrest [55]. In the analysis of the index of sudden infant death syndrome [45]. In the functional analyses, all family, all 4 mutation carriers showed ST segment elevation, mutant channels showed a reduction in the INa current. A study on whereas 3 non-carriers had normal ECG. In the normal condition, 80 Japanese BrS patients in Asia by Makiyama and his colleagues did Mirp2 decreased the transient outward potassium current (Ito). not identify any GPD1L mutations [46].TherelevanceofGPD1L However, the suppression of Ito by the R99H Mirp2 mutant was mutations in Asian BrS patients needs further study. smaller than that mediated by the WT protein, and the effect was observed even upon co-expression of the WT and R99H. These results showed that expression of the R99H mutant increased the 4. CACNA1C (BrS3), CACNB2 (BrS4), and CACNA2D (BrS9) Ito densities and caused BrS. Recently, another KCNE3 mutation, corresponding to T4A in Mrip2, was identified in a Japanese BrS The L-type (LTCC) in heart consists of 4 sub- patient [56]. This mutation also failed to suppress the Ito. units: a1, b2, a2, and d [47], encoded by CACNA1C, CACNB2, and CACNA2D, respectively, with the a2 and d subunits encoded by 7. SCN3B (BrS7) the gene CANA2D. In 2007, Antzelevitch et al. [48] reported 2 CACNA1C mutations and a CACNB2 mutation from a genotype SCN3B encodes the b3 subunit of the sodium channel. An survey of 82 BrS patients. The corresponding protein mutations SCN3B-mutation, corresponding to L10P in b3, was identified in a were A39V and G490R in CACNA1C and S481L in CACNB2. 64-year-old man [57]. The patient was asymptomatic and his ECG Carriers of all 3 mutations exhibited short QT intervals, lower showed coved-type ST elevation upon provocation by procaina- than 360 ms, with a coved-type ST elevation in ECG evaluations. mide. In a heterologous expression system, WT b3 increased the Therefore, BrS patients with LTCC mutations were considered to I densities, whereas co-expression of the WT b3 and L10P b3 have short QTc intervals. In their subsequent study, they identi- Na strongly decreased the I densities. The gating of the cardiac fied 15 and 5 mutation carriers in a cohort of 152 and 10 Na sodium channel also changed in cells with L10P b3 and the probands diagnosed with BrS and BrS with short QT intervals, inactivation curve of the mutant channel moved negatively, respectively [49]. Eight mutations were detected in CACNA1C,6in which caused the reduction in the total I . Furthermore, expres- CACNB2, and 3 in CACNA2D. We also screened 191 BrS and IVF Na sion of L10P b3 disturbed the normal trafficking of the cardiac patients who were negative for SCN5A mutations and identified 2 sodium channel to the membrane. More recently, another SCN3B CACNA1C mutations in 2 BrS patients [50]. mutation, corresponding to V110I in b3, was identified in 3 of 178 Functional analysis using patch-clamp methods was per- Japanese BrS patients [58]. Transfection of HEK 293-derived cells formed to address the impact of the 4 CACNA1C mutations with V110I b3 impaired the cytoplasmic trafficking of Nav1.5 and (A39V, G490R, V2014I, and E1829-Q1833dup) and 1 CACNB2 decreased the cell surface expression of Nav1.5. mutation (S481L) reported thus far [48,49]. Current densities in cells expressing any of the mutant LTCCs were decreased, and the expression of one of the mutants, A39V, impaired cell surface 8. KCNJ8 (BrS8) trafficking of Ca(v)1.2. The number of LTCC mutations identified in BrS patients and that of mutants whose channel functions have A KCNJ8 mutation, corresponding to an S422L mutation in been analyzed, are small compared to SCN5A mutations. Further the protein, was first identified in a 14-year-old girl with early 74 M. Horie, S. Ohno / Journal of Arrhythmia 29 (2013) 71–76 repolarization [59], who suffered frequent VF attacks. KCNJ8 co-expressing the WT protein. Moreover, membrane surface encodes Kir6.1, which has 2 transmembrane regions, and the KATP expression of Nav1.5 was impaired in cells harboring the channel is composed of a complex of Kir6.1 or Kir6.2 and the mutant SLMAP. sulfonylurea receptor (SUR) [60]. In a subsequent report, 87 BrS patients were genetically screened for changes in KCNJ8 and one asymptomatic BrS patient was identified with the S422L mutation 12. SCN2B (BrS13) [61]. A coved-type ST elevation in ECG evaluations of this patient was unmasked after flecainide administration. To confirm the More recently, another b subunit of the sodium channel, functional effect of the S422L mutation, glibenclamide-sensitive SCN2B, was reported to be a candidate gene involved in BrS KATP currents were recorded in cells co-expressing Kir6.1 and [67]. Three mutations, corresponding to R28Q, –Y69H and –P210L SUR2. The results showed that KATP current density of Kir6. in the b2 subunit, were identified in 4 patients from a survey of 1-S422L mutant channels was remarkably increased compared 269 BrS patients. In the functional analysis, I in cells with the to that of the Kir6.1-WT channels. Na mutant b2 were decreased compared to those with the WT protein, and all the mutants perturbed normal trafficking of 9. KCND3 (BrS10) Nav1.5 to the membrane.

KCND3 encodes Kv4.3, which is the a subunit of the that modulates I . The differences of I densities to to 13. Other candidate genes between the endocardium and epicardium, or the increase of Ito in the epicardium have been discussed as mechanisms underlying 13.1. KCNH2 BrS [62]. Therefore, KCND3 was expected to be a strong candidate gene, and indeed, 2 KCND3 mutation carriers were identified from KCNH2 is well known as the causative gene of long QT 86 BrS patients in whom no mutations were detected in other syndrome type 2, and it encodes Kv11.1, which is a constituent reported BrS genes [63]. The first patient with the Kv4.3-L450F of the I channel. In 2005, 2 KCNH2 mutations, corresponding to mutation was a 45-year-old man with a history of heart palpita- Kr G873S and N985S in Kv11.1, were detected in two asymptomatic tions at rest. His ECG showed slight ST elevation; therefore, he BrS patients [68]. Both mutant channels increased the I current. underwent a flecainide challenge test that induced a type 1 ECG Kr Subsequently, another KCNH2 mutation was identified in an pattern. Another patient with the Kv4.3-G600R mutation was a asymptomatic BrS patient with short QT interval and that mutant 22-year-old man with a history of heart palpitations and pre- channel also increased the I current [69]. The patients with syncope. He was discovered unconscious and unresponsive in Kr KCNH2 mutations showed atypical BrS phenotypes; therefore, bed. The 12-lead ECG examination conducted after hospitalization KCNH2 was considered a modifier that configures the ECG pattern revealed ST segment elevation in V1, V2, and V3. In the functional in various BrS types. analysis of the mutants in cells, co-expression of both Kv4.3 mutants with KChip2 increased the Ito densities, and the deacti- vation time constant of the Kv4.3-G600R channel was slower than 13.2. KCNE5 (KCNE1L) that of the WT. Computer simulations revealed that both Kv4.3 mutations caused a loss of the action potential dome of recon- KCNE5 is a member of the KCNE gene family and encodes an stituted right ventricular (RV) epicardial action potentials. auxiliary subunit of the potassium channel. Gender differences in BrS prevalence are well known, although the underlying reason is 10. MOG1 (BrS11) not fully elucidated. The KCNE5 gene is located on the X chromo- some. In 2009, 2 KCNE5 mutations, corresponding to Y81H and MOG1 is a recently identified protein that modifies the D92E-E93X on the protein, were identified in these BrS patients expression and trafficking of Nav1.5 [64]. A MOG1-E83D mutation [70]. Although KCNE5 modifies many kinds of potassium chan- was identified in a 40-year-old woman who was resuscitated nels, changes to the Ito were suspected to be affected by KCNE5 from cardiac arrest [65]. Her ECG showed ST elevation and an mutants to reproduce the phenotype of BrS. When co-expressed atypical right branch block just after the resuscitation. The ST with Kv4.3, which encodes the a subunit of Ito, the mutant KCNE5 elevation was normalized 24 h later. The functional analysis of channels increased the Ito densities. On the other hand, Ito MOG1-E83D by using patch-clamp methods showed decreased densities in cells co-expressing KCNE5-WT and mutants were almost the same as those expressing KCNE5-WT alone. This INa, and expression of the mutant in cells decreased the levels of Nav1.5 on the cell membrane. difference may explain the gender difference in BrS, such that an intact WT copy of KCNE5 on the second may provide protection from BrS in female patients. 11. SLMAP (BrS12)

Recently, 2 mutations in SLMAP were reported to cause BrS 13.3. HCN4 [66]. SLMAP encodes sarcolemmal membrane-associated protein

(SLMAP) that localizes at T-tubules. The functions of SLMAP have HCN4 encodes the If channel, also known as the pacemaker not been well elucidated. The mutations, V269I and E710A, were channel. In 2009, a splice mutation in HCN4 was identified in BrS identified in a 46-year-old and a 57-year-old male patient, patients [71], and a simulation model that integrated a ventri- respectively, and both of them experienced syncope and showed cular action potential of the If channel (Tusscher–Noble–Noble– spontaneous saddle-back (V269I) or coved-type (E710A) ST ele- Panfilov model) [72] showed that the If channel likely contributed vation. To elucidate the functional effect of SLMAP mutations on to the shortening of the action potential duration and the the cardiac INa, SLMAP-WT or mutants were co-expressed with prevention of early after-depolarization in bradycardia. These NaV1.5. The results showed that INa in cells co-expressing SLMAP- observations suggested that the If channel plays a preventive role V269I or E710A with NaV1.5 were significantly lower than that in triggering bradycardia-induced ventricular . M. Horie, S. Ohno / Journal of Arrhythmia 29 (2013) 71–76 75

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