Basic Mechanisms
The major focus of our network is the elucidation of new markers of SCD risk in human populations. In addition, each of the centers in this Leducq Network conducts studies to understand the basic mechanisms whereby genomic variants impact SCD risk. Examples are listed here.
Advanced molecular signatures in patients at high risk for SCD: The Nantes group has developed methods to perform transcriptional profiling for ion channel and related genes on endocardial biopsy material. Since SCN5A mutations are detected in only 20% of cases of Brugada Syndrome, these methods have been applied to identify alternate mechanisms that may modulate cardiac conduction and arrhythmogenesis. Accordingly, the Münster and Nantes groups collaborated in a study to perform transcriptional profiling on right ventricular septal endomyocardial biopsies from 10 unrelated probands with Brugada syndrome, 11 non-diseased organ donors, 7 heart transplant recipients, 10 patients with arrhythmogenic right ventricular cardiomyopathy and 9 patients with idiopathic right ventricular outflow tract tachycardia. Analysis of 77 transcripts encoding ion channel and transporter subunits revealed that Brugada Syndrome patients showed a pattern different from the other 4 groups. Specifically, 14 genes showed differential expression, including down-regulation of Nav1.5 (the protein encoded by SCN5A), Kv4.3 and Kir3.4, and upregulation of both ion channel genes (Nav2.1 and TWIK1) as well as genes involved in intracellular calcium homeostasis (RYR2 and NCX1). Moreover, the molecular profile of five Brugada patients with a mutation in SCN5A did not differ from that of six Brugada patients without an SCN5A mutation, indicating that multiple genetic mechanism can lead to a final arrhythmogenic phenotype, that can be assessed by transcriptional profiling.
Parallel studies in genetically-modified mice, conducted by the Nantes and Amsterdam centers, confirm that deletion of a single sodium channel allele confers a variable electrophysiologic phenotype, likely due to variability in the mechanisms that compensate for the initial genetic lesion. This ability to study human tissues and mouse models with tools developed within the network provides a unique opportunity to advance the science of identifying new molecular markers for SCD risk.
NOS1AP/CAPON signaling: Investigators at the Johns Hopkins site, in collaboration with investigators in Munich have used genome-wide association approaches combined with contemporary techniques in genetic epidemiology and biostatistics expertise, to identify a wholly unexpected locus modulating QT variability in the central German KORA cohort. A specific set of polymorphisms in NOS1AP (encoding Capon), a modulator of neuronal nitric oxide synthase, conferred a significant increase in QT interval in a normal population, and this result was replicated in two further populations. This work is important because it defines a new signaling pathway in cardiac electrogenesis, and NOS1AP polymorphisms were therefore included in the genotyping platforms deployed by the Network.
Because there had been no previous indication that the NOS1AP/Capon pathway had any impact at all on cardiac electrical function, workers at the Hopkins center undertook studies to determine how variants in this gene might act. Using adenoviral vectors containing a GFP marker, and the full NOS1AP sequence, guinea pig myocytes were found to express both a long and short form of Capon, and that cells over-expressing Capon displayed decreased ICa-L, increased IKs, no change in INa or IK1, and shortened action potentials. Collectively, these data support the hypothesis that variants in NOS1AP influence QTc and arrhythmia risk by modulating the function of specific ion channels.
Large-scale genomic variation in arrhythmia candidate genes: SNP interrogation is a widely-used approach in genomics, but may not detect larger genomic rearrangements increasingly well-recognized as modulators of human disease. To begin to probe the role of such variants, the Nantes group examined five Long QT Syndrome (LQTS) genes in 100 mutation-negative LQTS probands. A multiplex ligation-dependent probe amplification (MLPA) approach was used, and aberrant exon copy numbers were confirmed by Quantitative Multiplex PCR of Short Fluorescent Fragments (QMPSF). Array based CGH analysis was performed to further map genomic rearrangements, and family studies were performed to test segregation of the copy number variation. This study identified two large deletions in KCNH2 in 2 probands:
- A deletion spanning exon 4 to 15 and including 19 additional downstream genes (total ~650 Kb) was identified in a 20 year old woman with QTc = 554 msec. Family studies identified 3 additional affected individuals.
- A deletion was identified in a second proband with a QTc= 478 msec who experienced syncope after emotional stress. This deletion included exons 1-15, extended beyond KCNH2, and was ~145 Kb. No family study was performed since the proband was adopted.
New gene for cardiac arrhythmia: In a combined effort among the Amsterdam, Nantes and Nashville groups, we have identified mutations in SCN1B, encoding the cardiac Na-channel ß1-subunit, underlying the conduction disease and/or Brugada syndrome phenotype in 3 small kindreds. Two of these mutations were located in a newly-described alternately-processed transcript of this gene. Both the canonical and alternately-processed transcripts are expressed to a greater degree in Purkinje fiber and right ventricle than in left ventricle, consistent with the clinical syndromes. Coexpression of the mutant ß1-subunits with SCN5A showed loss of Na-channel function in heterologous expression system. These findings implicate SCN1B as a disease gene for human arrhythmia susceptibility.
Ankyrin-B variants are common in patients with arrhythmias: Mutations in the ankyrin-B gene (ANK2) cause LQT4, and have been described in kindreds with other arrhythmias. The frequency of ANK2 variants in large populations and molecular mechanisms underlying variability in the clinical phenotypes are not established. More importantly, there is no cellular explanation for the range of severity of cardiac phenotypes associated with specific ANK2 variants. In a screen of ANK2 coding exons in 445 patients with drug-induced or other arrhythmias, as well as 160 population controls, the Vanderbilt and Nantes groups identified 8 ANK2 variants. However, the clinical phenotypes associated with these variants vary strikingly, from no obvious phenotype to manifest LQTS and sudden death, suggesting that mutants confer a spectrum of cellular phenotypes. We tested this hypothesis by transfecting myocytes from wild-type or ankyrin knockout mice with mutant ankyrin constructs, and showed that variants generate variable in vitro phenotypes, including wild-type activity, simple loss-of-function, and dominant-negative action. These data indicate that varying degrees of ankyrin-B loss-of-function correlate with a range of severity of cardiac dysfunction.