There has been increased interest in a possible association between epilepsy channelopathies and cardiac arrhythmias, such as long QT syndrome (LQTS). We report a kindred that features LQTS, idiopathic epilepsy, and increased risk of sudden death. Genetic study showed a previously unreported heterozygous point mutation (c.246T>C) in the KCNH2 gene. Functional studies showed that the mutation induces severe loss of function. This observation provides further evidence for a possible link between idiopathic epilepsy and LQTS.
Sudden unexpected death in epilepsy (SUDEP) is a catastrophic complication of human epilepsy, with an incidence of 6.3–9.3 per 1,000 person years in patients who enter epilepsy surgery programs (Tomson et al., 2008; Surges et al., 2010). However, mechanisms and methods to prevent SUDEP are still largely unknown. A leading hypothesis suggests a dysfunction of excitability that could underlie both epilepsy and cardiac arrhythmias, leading to death (Nashef et al., 2007; Tomson et al., 2008; Surges et al., 2009).
In recent years there has been increased interest in a possible association between epilepsy channelopathies and cardiac arrhythmias, such as long QT syndrome (LQTS; Heron et al., 2010). A “seizure phenotype” was recorded in about 30% of unrelated patients from two independent cohorts with genetically confirmed LQT2 (KCNH2 mutations) (Johnson et al., 2009). A recent postmortem study identified mutations in KCNH2 or SCN5A genes, all previously associated with LQTS, in 6 (13%) of 68 patients with SUDEP (Tu et al., 2011). Moreover, mutations in KCNH2, encoding the potassium channel hERG-1 (human ether-à-go-go-related gene-1) that generates the rapid component of cardiac delayed rectifier potassium current, have been reported recently in patients with recurrent seizures and prolonged QTc interval (Glasscock et al., 2010; Anderson et al., 2012). In addition, mouse lines bearing dominant mutations in the Kv1.1 Shaker-like potassium channel, associated with LQTS type 1 in humans, exhibit severe epilepsy and premature death (Heron et al., 2010; Anderson et al., 2012; Zamorano-León et al., 2012; Parisi et al., 2013). We report a kindred featuring LQTS, idiopathic epilepsy, and increased risk of sudden death carrying a loss-of-function mutation of KCNH2.
Patients' evaluation included clinical history, routine blood workup, electroencephalography (EEG) recordings, neuroimaging, and treatment. Seizures and epilepsy were defined according to international recommendations. A peripheral blood sample was obtained to extract genomic DNA. The Ethics Committee of ‘Federico II’ University, Napoli, Italy, approved the study, and written informed consent was signed by the participants.
Two 18-year-old dizygotic twin sisters (III:1, III:2; Fig. 1A) were referred for myoclonic jerks at upper limbs, and occasional (about 2–3 episodes/year) tonic–clonic seizures started from age 13 and 15 years. No specific precipitating (e.g., visual, auditory stimuli) factors were reported. Interictal EEG showed normal background activity and generalized spike-wave and spike-slow wave complexes, increased by sleep (Fig. 1B). Brain magnetic resonance imaging (MRI) and neurologic examination were unremarkable. The patients became seizure-free with levetiracetam (1,500 mg/day) started from age 16 years. Surface electrocardiography (ECG) revealed marked QT prolongation (QTc 550 msec) and abnormal T-wave (Fig. 1C). Echocardiography and multislice computed tomography (CT) were normal. Both patients refused treatment for cardiac arrhythmia. They patients come from a socially disadvantaged background, and tracking of medical history was possible only with the help of a caregiver. Their mother (individual II:4) and grandmother (individual I:1) had LQTS and repeated episodes of loss of consciousness. No clinical documentation was available for these subjects who died suddenly between the age of 25 and 30 years.
At age 18 years, individual III:1 was found dead lying prone on her bed in the morning without any apparent signs of convulsion (blood, incontinence, morsus, drooling). Autopsy excluded any pharmacologic/toxicologic or anatomic cause of death. At the examination, no external lesion was present and body cavities appeared normal. No gross or brain microscopic abnormalities were evident. The heart weight was 280 g and external examination was negative. Mild pulmonary congestion and edema were observed. In the following weeks, her sister (III:2) finally underwent implantation of cardioverter-defibrillator.
Molecular genetics and functional study
Due to clear association of idiopathic epilepsy, LQTS, and sudden death we performed direct sequence analysis of KCNH2, SCN5A, and KCNQ1 from genomic DNA of individuals III:1 and III:2. Genetic analysis failed to find mutation in SCN5A and KCNQ1 but revealed a novel nonsynonymous heterozygous missense mutation (c.246T>C) in exon 2 of the KCNH2 gene (Fig. 1D), affecting the isoleucine at position 82 (p.I82T), localized at the N-terminus of the protein in the PAS (Per-Arnt-Sim) domain, in both patients. The variant, evolutionarily conserved in many species (http://www.uniprot.org/), was absent in the father and was not found in 600 control chromosomes of European descent. In addition, it was not reported in the 1000 Genomes Project (http://browser.1000genomes.org/) and in the Exome Sequencing Project (http://evs.gs.washington.edu/EVS/) database. Search through the in silico Condel platform (CONsensus DELeteriousness score of missense SNVs data base) (http://bg.upf.edu/condel/home) predicted pathogenic effect of this change.
The mutation I82T was introduced by standard polymerase chain reaction (PCR) mutagenesis into the hERG-1 DNA clone (cDNA) in the pCDNA3.1 plasmid. We transfected tsA-201 cells with wild-type (WT) or I82T hERG cDNA and the plasmid pEYFP-N1, which expresses yellow fluorescent protein (Cestèle et al., 2008) and recorded hERG currents with the whole-cell configuration of the patch clamp technique (Smith et al., 1996). We initially used recording solutions with physiologic extracellular K+ concentration (5 mm) observing robust hERG currents in all the cells expressing WT channels (n = 7; Fig. 2A), but small currents and only in a minority of cells expressing I82T (2 over 11 cells recorded; Fig. 2B), which did not allow a straightforward study of biophysical properties. To increase inward K+ current amplitude, we did further experiments using 35 mm extracellular K+, which elicited larger currents for WT (Fig. 2C) and more robust currents in 7 of the 11 recorded cells expressing I82T (Fig. 2D). The comparison of the maximal current density confirmed that I82T showed a severe reduction with both 5 and 35 mm extracellular K+ (Fig. 2E). Analysis of the voltage dependence of activation did not show differences. However, deactivation kinetics at −110 mV was faster for I82T (τ = 113 ± 14 msec for WT, 21 ± 3 msec for I82T; p < 0.01), similar to most of LQT2 mutations within the PAS domain, and the inactivation curve showed a positive shift of 32.5 mV. Overall, the striking reduction in current density was the main effect of the mutation.
This family showed the association of idiopathic epilepsy, LQTS, and sudden death. LQTS may be easily misdiagnosed with epilepsy and treated with anticonvulsants. However, our patients showed clear electroclinical features that indicated the diagnosis of idiopathic generalized epilepsy, and seizures never occurred after sound stimuli, the typically known triggers for arrhythmias in LQTS2 (Keller et al., 2009). The pathogenicity of the KCNH2 mutation in our patients was suggested by the highly aggressive phenotype in mutated individuals, the evolutionary conservation of the Iso82 residue, and its absence in healthy controls. Moreover, functional studies revealed a severe reduction in current density and modifications of gating properties. The overall effect was a severe loss of function.
KCNH2, the target of LQT2 mutations, encodes the α-subunit of the hERG-1 voltage-gated K+ channel expressed in several tissues such as heart and brain.11 Hundreds of LQT2-associated mutations have been described and most cause a loss of function by disrupting subunit folding, assembly, or trafficking of the channel to the cell surface. Genotype–phenotype correlations suggest that mutations localized in the pore-forming domain (S5-loop-S6) and the N-terminus region (in particular in the PAG domain) have a major functional effect (Shimizu et al., 2009), as confirmed by results of our functional study.
hERG channels, encoded by KCNH2, can regulate neuronal firing and are particularly active in astrocytes: perturbations of this channel may confer susceptibility for recurrent seizure activity (Shimizu et al., 2009). Therefore, our observations are consistent with the possibility that KCNH2 mutations may confer susceptibility for recurrent seizure activity, supporting the emerging concept of a genetically determined cardiocerebral channelopathy (Heron et al., 2010; Anderson et al., 2012; Zamorano-León et al., 2012; Parisi et al., 2013). The absence of an epilepsy phenotype in some carriers is consistent with the wide phenotype heterogeneity of LQTS2, which is influenced by the individual genetic background. Nevertheless, this observation further supports the suggestion of a pathogenic link between idiopathic epilepsy and LQTS.
This work was partly supported by the Cardiovascular Genetics Center, University of Girona, Spain, by ‘University of Genova (Fondi di Ateneo to P.S.), ‘Università Cattolica del Sacro Cuore’, (Fondi di Ateneo Linea D1 to A.O.), Fondazione Telethon (grant no. GGP10186 to A.O), and ‘LabEx ICST – France’ (to M.M.). We wish also to thank Dr. Amedeo Bianchi, Dr. Angela La Neve, and the Collaborative Group on SUDEP of Italian League Against Epilepsy (LICE) for the great support.
No financial support has been received for this study. There is no financial relationship to disclose for all the authors.
The authors have no conflicts of interests. We confirm that we have read the Journal's position on issues involved in ethical publication and affirm that this report is consistent with those guidelines.