SCREENING DEI GENI ¿PACEMAKER¿ IN PAZIENTI CON EPILESSIA IDIOPATICA GENERALIZZATA: IDENTIFICAZIONE DI UNA MUTAZIONE RECESSIVA NEL CANALE HHCN2

It is well established that several forms of inheritable idiopathic epilepsy sindrome are ion channelopathies, that is pathologies associated with dysfunctional ion channels, even if the functional link between channel dysfunction and clinical phenotype is often unresolved. Hyperpolarization-activated, Cyclic-Nucleotide gated (HCN) channels are a class of voltage- and cAMP-dependent channels. They mediate the hyperpolarization-activated Ih current, which control synaptic integration and intrinsic excitability in various brain areas. Ih is pathologically altered after experimentally-induced seizures and has been proposed to have a role in different forms of epileptogenesis. Hcn1 and Hcn2 genes variants have been identified in patients with febrile seizure or GEFS+. While existing data therefore clearly show a link between HCN channel dysfunction and epileptogenesis, no specific mutation-induced HCN channel modification has so far been correlated functionally with increased neuronal excitability. To investigate this we used a candidate gene approach and screened a panel of idiopathic generalized epilepsy patients and related families for mutations in the Hcn1 and Hcn2 genes. We found a form of sporadic IGE associated with a recessive point mutation in the gene coding for the HCN2 channel. The protein mutation E515K is located in the C-linker region (exon 5 of the Hcn2 gene). Functional analysis revealed that homomeric mutant, but not heteromeric wild-type/E515K channels, have a negative shift in the activation kinetics. Furthermore, the time-constant curve for homomeric E515K channels was also shifted to the negative direction. Moreover, omomeric mutant, but not heteromeric wild type/mutant channels, showed a lowering of the threshold of action potential firing and a strongly increased cell excitability and firing frequency when compared to wild-type channels. In conclusion, our results show that the homozygous E515K mutation in human HCN2 channels is a loss-of-function mutation causing a large negative shift of the activation curve and slowing of activation, and a consequent strong reduction of Ih availability near resting voltages. These changes cause a substantial increase of neuronal excitability, a condition predisposing to epileptogenesis, and are associated with a recessive type of inheritance compatible with the idiopathic generalized epilepsy of the proband in the family pedigree.