The HCN4 gene encodes a subunit of the hyperpolarization‐activated cyclic nucleotide‐gated channel, type 4 that 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. Here, we report the in vitro functional characterization of four rare variants of uncertain significance (VUS) in HCN4, identified through testing a cohort of 296 sudden unexpected natural deaths. The variants are 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. We also identified a likely benign variant, p. P1063T, which has a high minor allele frequency in the gnomAD, which is utilized here 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, we reclassified three rare HCN4 VUS to likely benign variants, eliminating the necessity for costly and time‐consuming further study. Our studies also provide a new lead to investigate 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.

Background: Molecular testing of the deceased (Molecular Autopsy) is an overlooked area in the United States healthcare system and is not covered by medical insurance, leading to ineffective care for surviving families of thousands of sudden unexpected natural deaths each year. We demonstrated the precision management of surviving family members through the discovery of a novel de novo pathogenic variant in a decedent. Methods: Forensic investigation and molecular autopsy were performed on an 18year-old female who died suddenly and unexpectedly. Co-segregation family study