Abstracts

Rationale: Hyperpolarization-activated cyclic nucleotide-gated (HCN) channel plays critical roles in regulating rest membrane potential and integrating synaptic transmission. Recent studies have shown HCN1 mutation is related to early infant epileptic encephalopathy. However, mechanisms of the epilepsy caused by HCN1 still remains to be elucidated since only a few of pathogenic variants of HCN1 were reported. Thus, we aim to identify novel pathogenic variants of HCN1, to expand the phenotypic spectrum and to provide their functional analysis. Methods: Likely pathogenic variants in HCN1 were detected using next-generation sequencing and confirmed by bidirectional sanger sequencing. The mutations were engineered into human HCN1 cDNA in pcDNA3 vector. Plasmids of either HCN1 or mutations were co-transfected with GFP-containing plasmids in Human embryonic kidney cells (HEK293) for whole-cell voltage clamp experiments. Results: We identified three novel de novo HCN1 variants (p.E240G, p.A395G and p.I380F) in sporadic patients with epileptic encephalopathy (figure 1). Patients with these variants presented with different phenotypes including genetic generalized epilepsy with febrile seizure plus, infantile epileptic encephalopathy and malignant migrating partial seizure. Functional experiments showed that the p.E240G, p.A395G and p.I380F variants caused a -38 mV, -30 mV and -10.8 mV shifts in activation curve (n=14-16), respectively, compared to wild-type HCN1 channel. The p.A395G and p.I380F variants presented decreased current densities and slower activation kinetics at tested voltages, while the p.E240G variant had no effect on current densities or activation kinetics. The above results indicated p.A395G and p.I380F variants result in a strong loss-of-function effect, while the p.E240G variant presented a mild loss-of-function effect. Additionally, the co-expression experiments revealed that the p.A395G variant produced a significant dominant-negative loss-of-function effect on HCN1 channels due to a hyperpolarizing shift of activation curves and a decreased current density, and the p.I380F variant led to a slight dominant-negative loss-of-function effect by hyperpolarizing the activation curve without affecting the current density. In contrast to the p.A395G and p.I380F variants, we failed to detect any dominant-negative effect of the p.E240G variant on HCN1 wild-type channels. Conclusions: We have detected novel pathogenic mutations in HCN1, whose functional analyses indicate that the severity of HCN1 channel dysfunction may account for clinical phenotypic variability. Therefore, the functional changes of HCN1 mutations should be focused in order to seek for effective therapeutic strategies. Funding: National Natural Science Foundation of China No.81771409