Most pathogenic HCN1 mutations identified so far in patients with epilepsy or epileptic encephalopathies are missense mutations i.e. single nucleotide substitutions that result in the replacement of an amino acid by another one. Mutations causing severe phenotypes are mainly located in the transmembrane domains or the domains important for the channel function. Mutations associated with milder epilepsies are usually located outside transmembrane domains in the N- or C-terminal parts of the channel.
The precise mechanisms by which the mutations lead to epilepsy is not completely elucidated so far, but all HCN1 mutations studied have been shown to alter the biophysical properties of the channel. Different impacts of the mutations on HCN1 homomeric channels have been observed ranging from complete loss-of-function to significant shifts in activation kinetics and/or voltage dependence (gains-of-function). It is very likely that many variants associated with apparent loss-of-function in fact lead to dominant negative effects on normal HCN1 subunit expressed from the normal remaining allele.
Other mutations types, including a nonsense variant and intragenic deletions encompassing the exon encoding the pore of the channel, have been reported in patients with intellectual disability or autism spectrum disorders without seizures but the contribution of these variants to the patients’ phenotype is still unclear.
Identification of a HCN1 mutation can be achieved by different methods: targeted analysis of the HCN1 gene by Sanger sequencing or next-generation sequencing; gene panel sequencing including HCN1, exome or genome sequencing. Intragenic deletions have to be searched for by appropriate methods like microarrays analysis or any method allowing to assess the copy number of each exon.
Interpretation of a novel HCN1 variant should be performed according to the ACMG criteria, taking into account the inheritance of the mutation (de novo/inherited), the segregation of the mutation with the disorder in the family (for inherited variants ), the location of the mutation on the protein and conservation of the amino acid altered in othologs and paralogs. A functional test of the variant on the channel properties can also be required to conclude that the variant is pathogenic. Although HCN1 has a probability of being loss-of-function intolerant (pLI) score of 1, truncating variants should be interpreted with caution, as they have been described in a few healthy individuals.