This article reports the in vitro functional characterization of four rare variants of uncertain significance (VUS) in the HCN4 gene, identified through testing a cohort of 296 sudden unexpected natural deaths.
The HCN4 gene encodes a subunit of the hyperpolarization-activated cyclic nucleotide-gated channel type 4, which is essential for the proper generation of pacemaker potentials in the sinoatrial node. The HCN4 gene is often present in targeted genetic testing panels for various cardiac conduction system disorders and there are several reports of HCN4 variants associated with conduction disorders. The current study found that the variants in the studied cases of natural deaths were all missense alterations, leading to single amino acid changes: p.E66Q in the N-terminus, p.D546N in the C-linker domain, and both p.S935Y and p.R1044Q in the C-terminus distal to the CNBD. The study also identified a likely benign variant, p. P1063T, which has a high minor allele frequency in the gnomAD, which was used as a negative control. Three of the HCN4 VUS (p.E66Q, p.S935Y, and p.R1044Q) had electrophysiological characteristics similar to the wild-type channel, suggesting that these variants are benign. In contrast, the p.D546N variant in the C-linker domain exhibited a larger current density, slower activation, and was unresponsive to cyclic adenosine monophosphate (cAMP) compared to wild-type. With functional assays, the study reclassified three rare HCN4 VUS to likely benign variants, eliminating the necessity for costly and time-consuming further study. The study also provided a new lead to investigate regarding how a VUS located in the C-linker connecting the pore to the cAMP binding domain may affect the channel open state probability and cAMP response. (publisher abstract modified)