The Spectrum of SCN5A Gene Mutations in Spanish Brugada Syndrome Patients

INTRODUCTION

Brugada syndrome is a cardiac arrhythmia characterized by right bundle branch block and ST elevation in the right precordial leads of the electrocardiogram (ECG) in the absence of structural heart disease.¹ In many cases it is difficult to distinguish between Brugada syndrome and other arrhythmias, although it can be unmasked with sodium channel blockers.² The syndrome is associated with an increased risk of sudden death, sometimes during infancy. It can be diagnosed from a few days after birth until adulthood and the mean age of sudden death is around 40 years. The prevalence is around 5 cases per 10 000 inhabitants, although the ECG may appear normal in many individuals with the syndrome, making the true frequency difficult to determine. Brugada syndrome may be responsible for around 20% of sudden deaths in men younger than 40 years with structurally normal hearts.

Many patients have a family history of the disease, which follows an autosomal dominant pattern of inheritance. In 25% of patients, mutations are present in the SCN5A gene, which encodes an alpha subunit of the cardiac sodium channel.^3-5 More than 100 different SCN5A mutations have been described in patients with Brugada syndrome, as well as in some cases of type-3 long-QT syndrome (LQT3), Lev-Lenegre syndrome, and atrial fibrillation.⁶ All of these mutations cause a reduction in sodium channel function. Recent reports have suggested that Brugada syndrome may also be associated with mutations in other genes, such as glycerol-3-phosphate dehydrogenase (GPD1L) and the alfa-1 and beta subunits of the L-type cardiac calcium channel (CACNA1C and CACNB2b).⁷ It remains unclear what proportion of patients without mutations in SCN5A carry mutations in one of these other genes.

The aim of this study was to characterize the array of SCN5A mutations in a group of Spanish patients and define the phenotype associated with the mutations identified.

METHODS

Patients

The study included 25 patients diagnosed by specialists from the Department of Cardiology at Hospital Universitario Central de Asturias. Diagnosis was based on the presence of a type-1 ECG with ST-segment elevation followed by a negative T wave. Patients with type-1 or -2 ECG were diagnosed with Brugada syndrome if they had a type-1 pattern in more than 1 of the right precordial leads (V1-V3) after administration of flecainide. In addition, diagnostic workup included an electrophysiology study to assess inducibility of ventricular arrhythmias and measure conduction times. Table 1 shows the main characteristics of the patient population.

Genetic Study

Genomic DNA was obtained by extraction from leukocytes in 10 mL of peripheral blood. Polymerase chain reaction (PCR) was used to amplify the 27 coding exons from SCN5A with primers designed using the flanking intronic regions. Each fragment was purified and sequenced using BigDye chemistry in an ABI3130 sequencer (Applied Biosystems, Foster City, California, USA). The sequence for each patient was compared with the SCN5A sequence in the Ensembl sequence database (reference SCN5A ENSG00000183873; www.ensembl.org). Further information on the primers and PCR conditions can be obtained from the corresponding author.

Any sequence variant that had not been described previously was analyzed in 200 healthy individuals (ages, 20-70 years) recruited through the Principality of Asturias blood bank. None of these control subjects had symptoms of heart disease, although ECG was not performed to rule out arrhythmias. Each fragment in which a possible mutation had been identified was amplified in the patient and the 200 controls and the genotype defined by single-strand conformational polymorphism (SSCP). SSCP was also used to define the genotype of the H558R polymorphism in patients and controls.

In the cases in which a mutation in SCN5A was observed, all family members were offered a diagnostic workup including baseline ECG and screening for the mutation. Family members who were carriers of the mutation but had a normal ECG were studied further using the flecainide test. All individuals included in the study (patients, family members, and controls) provided signed informed consent to the genetic study.

RESULTS

Of the 25 patients included in the study, 18 (64%) had a type-1 baseline ECG and 7 (36%) had type 2 or 3 ECGs that were converted to type 1 in the flecainide test (Table 1). In those patients, we found 16 nucleotide variants of SCN5A, and only 4 of those were not also found in at least 1 of the corresponding 200 controls. None of the 4 mutations had been described previously in patients with Brugada syndrome (Table 2). The Ala2>Tre, Ala735>Tre, and Val1340>Ile mutations affected conserved amino acids. The 4 patients were homozygous for histidine 558; consequently, the effect of this polymorphism on the phenotype could not be assessed. Below we describe the main characteristics of the patients and their family members.

Ala2Thr (c.4G>A) Mutation

The patient was a 52-year-old man with a family history of Brugada syndrome diagnosed during routine screening. He had a type-2 ECG, which was converted to type 1 by flecainide. The electrophysiology study was negative. His mother (75-year-old) was a carrier of the mutation but was asymptomatic and had a normal ECG both at baseline and following flecainide challenge.

Ala735Thr (c.2203G>A) Mutation

The patient was a 60-year-old woman with recurring palpitations and no family history of sudden death. She had a type-1 baseline ECG and the electrophysiology study was normal. The patient, a sister aged 64 years, and 2 daughters aged 29 and 28 years were carriers of the mutation. Her sister and younger daughter had a type-2 baseline ECG that was converted to type 1 following flecainide challenge. The elder daughter had a normal ECG both at baseline and following flecainide challenge.

IVS18-1G>A (c.3390-1G>A) Mutation

Male patient diagnosed at 40 years of age with a type-1 baseline ECG and a positive electrophysiology study. The patient's mother and a son had died suddenly at 52 and 20 years of age, respectively. We found a mutation in the last nucleotide of intron 18 (G>A), and analysis using a computer program (http://www.fruitfly.org/seq_tools/other. html) showed that the mutation would affect RNA processing, eliminating exon 19 of the mRNA. This would represent a splicing mutation, and the patient's 30-year-old daughter was also a carrier. The daughter had a normal baseline ECG that was converted to type 1 in the flecainide test.

Val 1344Ile (c.4018G>A) Mutation

The index case was a 55-year-old man with a type-1 ECG whose brother had died suddenly at age 45.

The electrophysiology study was negative. The patient and 4 family members were carriers of the mutation: mother (76 years), sister (52 years), and 2 sons (32 and 36 years). All had a normal ECG both at baseline and in the flecainide test.

DISCUSSION

In our study, 16% of patients carried mutations in SCN5A, a rate that is similar to previously reported results.^5,8 As in other studies, we analyzed coding exons and some intronic bases. Consequently, we cannot rule out the possibility that mutations were present in other regions of the gene (such as the promoter or intronic sequences). Our study only analyzed SCN5A, but other genes have recently been described that could also be mutated (although less frequently) in patients with Brugada syndrome.^7,9 Patients with mutations in these genes would have a shorter QT interval and a new clinical entity has therefore been proposed combining Brugada syndrome and short-QT syndrome.

The 4 mutations identified in our patients had not been described previously (see the Biobase:HGMD database, available from www.biobase-international. com). This suggests that direct analysis would not be effective for the identification of mutations and in most cases sequencing of SCN5A would be necessary. Although we have not undertaken studies to assess the functional consequences of the mutations identified here, it can be expected that they would lead to malfunction of the sodium channel and cardiac conduction abnormalities.

Only 2 of the patients (50%) had a family history of Brugada syndrome, and among the family members who were carriers of the mutation, many were asymptomatic and had normal ECG results. In the largest patient series that has been published (130 patients), Priori et al⁵ found that 23% of family members who also carried the mutation did not have symptoms of the disease. Only the coexistence of syncope and ST-segment elevation in the ECG would be an indicator of poor prognosis in these patients, since the mutations are not associated worse prognosis. Those authors concluded that genetic data would serve as a diagnostic criterion but would be of little use in predicting the risk of adverse events.

Finally, some polymorphisms in SCN5A (such as H558R) could also be phenotypic modifiers in patients with mutations in this gene. The genotype of these polymorphisms may explain clinical or ECG differences among carriers of the mutation, even among family members.¹⁰ Since all of the carriers of mutations in SCN5A were homozygotes for histidine 558, we were unable to draw conclusions regarding the phenotypic effect of this polymorphism.

In conclusion, the frequency of SCN5A mutations in our patients was similar to that described in other populations. The existence of family members who were carriers of the mutation but remained asymptomatic and had a normal ECG indicates that genetic studies are of limited usefulness for determining risk but remain helpful for diagnosis.

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Study funded by grants from the Spanish Health Research Fund and European Regional Development Fund (project FIS-06/0214; principal investigator, Eliecer Coto García) and from the Red de Investigación Renal-REDINREN (RD06/0016) of the Carlos III Health Institute.

Correspondence: Dr. E. Coto.
Laboratorio de Genética Molecular. Hospital Universitario Central de Asturias (Maternidad).
33006 Oviedo. Asturias. Spain.
E-mail: [e-mail]

Manuscript received May 8, 2009.
Accepted for publication July 31, 2009.