ClinicalGeneticAn international compendium of mutations in the SCN5A-encoded cardiac sodium channel in patients referred for Brugada syndrome genetic testing
Introduction
Brugada syndrome (BrS) is a rare heritable arrhythmia syndrome characterized by an electrocardiographic (ECG) pattern consisting of coved-type ST-segment elevation in the right precordial leads V1 through V3 (often referred to as a type-1 Brugada ECG pattern) and an increased risk for sudden cardiac death (SCD).1, 2 The penetrance and expressivity of this autosomal-dominant disorder is highly variable, ranging from a lifelong asymptomatic course to SCD during the first year of life. The syndrome is thought to account for up to 4% of all SCDs and 20% of unexplained sudden death in the setting of a structurally normal heart;3 however, some patients display a more benign course. BrS is generally considered a disorder involving young male adults, with arrhythmogenic manifestation first occurring at an average age of 40 years, with sudden death typically occurring during sleep.4 However, BrS has also been demonstrated in children and infants as young as 2 days old and may serve as a pathogenic basis for some cases of sudden infant death syndrome.3
Since the disorder's sentinel clinical and ECG description in 1992 by Drs. Pedro and Josep Brugada,5SCN5A-encoded cardiac sodium channel loss-of-function mutations have been shown to confer the pathogenic basis for an estimated 15% to 30% of BrS, currently representing the most common BrS genotype and classified as Brugada syndrome type 1 (BrS1).6, 7, 8 Loss-of-function mutations in SCN5A reduce the overall available sodium current (INa) through either impaired intracellular trafficking of the ion channel to the plasma membrane, thereby reducing membrane surface channel expression, or through altered gating properties of the channel. Gain-of-function SCN5A mutations cause a clinically and mechanistically distinct arrhythmia syndrome, long-QT syndrome type 3 (LQT3). Interestingly, some identical SCN5A mutations may provide either a loss-of-function BrS1-phenotype or a gain-of-function LQT3-phenotype, depending on the individual host. In fact, LQT3/BrS/conduction-disorder SCN5A overlap syndromes do exist within single large families.9, 10
After a decade of genetic testing by research laboratories worldwide, BrS genetic testing has made the transition from discovery to translation to clinical implementation with the availability of clinical BrS1 genetic testing (since 2004 in North America and even earlier in Europe), which provides comprehensive open-reading frame and canonical splice site mutational analysis of SCN5A. However, it must be recognized that nearly 2% of healthy Caucasians and 5% of healthy nonwhite subjects also host rare missense SCN5A variants, leading to a potential conundrum in the interpretation of the genetic test results.11 Distinguishing pathogenic mutations from rare harmless genetic variants is of critical importance in the interpretation of genetic testing and the management of genotype-positive BrS patients.
Presently, there are over 100 BrS1-associated mutations publicly available (http://www.fsm.it/cardmoc). We sought to assemble an international compendium of putative BrS1-associated mutations through a retrospective analysis of BrS genomic databases from 9 reference centers throughout the world (5 Europe, 3 United States, 1 Japan) that have each genotyped >100 unrelated cases of clinically suspected BrS. Such a compendium may illuminate further key structure–function properties and provide a foundational building block for the development of algorithms to assist in distinguishing pathogenic mutations from similarly rare but otherwise innocuous ones.
Section snippets
Study population
A retrospective analysis of BrS databases from 9 centers throughout the world that have each genotyped >100 unrelated cases of clinically suspected BrS was performed. In total, 2,111 unrelated patients (78% male, mean age 39 ± 15 years) were referred for SCN5A genetic testing (Table 1). For the purpose of this compendium of identified mutations, only minimal demographic information for each center's cohort, such as the average age and range of age at diagnosis and the number of male and female
Mutational analysis
Patient genomic DNA was analyzed for mutations in all 27 translated exons, including splice sites and adjacent regions, of the SCN5A-encoded cardiac sodium channel NaV1.5 using a combination of polymerase chain reaction (PCR), either denaturing high-performance liquid chromatography or single-stranded conformation polymorphism and DNA sequencing.12 In addition, frequency, location, and mutation type of SCN5A genetic variation found among 1,300 ostensibly healthy volunteers,11, 13 including 649
Mutation nomenclature
All possible BrS1-associated mutations were denoted using the accepted Human Genome Variation Society's guidelines for nomenclature.14 The nucleotide and amino acid designations were based on the SCN5A transcript NM_198056.2. For example, the missense mutation E1784K would indicate the wild-type amino acid (E = glutamic acid) at position 1784 is replaced by lysine (K). Frameshift mutations resulting from nucleotide insertions or deletions were annotated using the F861WfsX90 format, which
Defining terminology: variant versus mutation
For the purposes of this compendium, a variant will be defined as any change to the wild-type sequence, whether it is in case or control subjects. Mutations will be identified as rare, case-only (absent in the 1,300+ healthy volunteers) variants that are possibly pathogenic. Variants identified with a minor allele frequency (MAF) >0.5% among the 1,300 healthy control subjects will be termed common polymorphisms. If the MAF is <0.5%, these variants will be termed uncommon/rare polymorphisms.
Defining a variant as a possible BrS1-causative mutation
To be considered as a possible BrS1-causing mutation, the variant must disrupt either the open reading frame (i.e., missense, nonsense, insertion/deletion, or frameshift mutations) or the splice site (polypyrimidine tract, splice acceptor, or splice donor recognition sequences). In addition to the exonic splice sites described above, the acceptor splice site was defined as the 3 intronic nucleotides preceding an exon (designated as IVS-1, -2, or -3) and the donor splice site as the first 5
Results
Overall, 2,111 unrelated patients (78% male, average age at testing 39 ± 15 years) were referred for BrS genetic testing across 9 testing centers (Table 1). As expected, rare SCN5A missense mutations were far more common among BrS cases (438/2,111, 21%) than similarly rare genetic variants were among control subjects (43/1,300 [11/649, 1.7% white subjects and 31/651, 4.8% nonwhite subjects], P <10−55). The yield differed significantly across centers (P = .0017; chi2 = 24.7, degrees of freedom
Discussion
Since the first report by Chen et al26 in 1998, a little over 100 unique SCN5A mutations have been implicated as possibly causative for BrS1. Previous small cohort studies have indicated that the prevalence of SCN5A mutations in BrS is roughly 15% to 20%, and possibly as high as 40% in cases of familial BrS. In 2000, Priori et al6 reported a 15% yield with respect to SCN5A mutations among 52 unrelated patients. In 2002, these investigators extended their analysis to 130 probands (20% with a
Study limitations
For the purpose of this compendium of identified mutations, only minimal demographic information from each center's cohort was made available because the focus was on the prevalence, spectrum, and localization of SCN5A mutations among suspected cases of BrS rather than an attempt to establish any particular genotype–phenotype correlates. Nevertheless, there is significant clinical value due to the data in aggregate. For example, the rare missense mutations seen numerous times among these cases
Conclusion
Since the sentinel discovery of BrS as a cardiac channelopathy in 1998, our genomic understanding of this potentially lethal disorder has matured from a phase of discovery to one of translational medicine. This international consortium of BrS genetic testing centers has tripled the catalog of possible BrS1-associated mutations with the addition of 200 new mutations to the public domain and has provided a template to draw upon for further genetic testing interpretation and biological inquiry.
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Support for data analysis for this project was provided by the Mayo Clinic Windland Smith Rice Comprehensive Sudden Cardiac Death Program (Dr. Ackerman), grant HL47678 from the National Institutes of Health (Dr. Antzelevitch), New York State and Florida Free and Accepted Masons, the GIS Institut des Maladies Rares, the AFM (ANR-06-MRAR-022, PG, Dr. Schott), The Health Sciences Research Grants (H18, Research on Human Genome, 002) and the Research Grant for the Cardiovascular Diseases (21C-8) from the Ministry of Health, Labor, and Welfare of Japan (Dr. Shimizu), The Fondation Leducq Trans-Atlantic Network of Excellence Grant (05 CVD 01, Preventing Sudden Death, Dr. Schott), ANR grant ANR/-05-MRAR-028-01 (Dr. Schott), grant from the Fondation pour la recherche Medicale (Dr. Schott), FIS-ISCiii (Dr. Brugada), CNIC (Dr. Brugada), Ramon Brugada Sr. Foundation (Dr. Brugada), Leducq Foundation, grant 05 CVD, Alliance against Sudden Cardiac death (Drs. Wilde, Schott, and Schulze-Bahr), and Deutsche Forschungsgemeinschaft (Dr. Schulze-Bahr).
All mutational analyses performed in this study were conducted at individual centers.
Dr. Ackerman is a consultant for PGxHealth. Intellectual property derived from Dr. Ackerman's research program resulted in license agreements in 2004 between Mayo Clinic Health Solutions (formerly Mayo Medical Ventures) and PGxHealth (formerly Genaissance Pharmaceuticals).
Mr. Kapplinger and Mr. Tester contributed equally to this work.