Background Brugada syndrome (BrS) is a sudden death predisposing genetic condition characterized electrocardiographically by ST-segment elevation in the leads V1-V3. Given the prominent role of the transient outward current (Ito) in BrS pathogenesis, we hypothesized that rare gain-of-function mutations in KCND3 may serve as a pathogenic substrate for BrS. Methods Comprehensive mutational analysis of KCND3-encoded Kv4.3 (Ito) was conducted using PCR, DHPLC, and direct sequencing of DNA derived from 86 unrelated BrS1-8 genotype negative BrS patients. DNA from 780 healthy individuals was examined to assess allelic frequency for non-synonymous variants. Putative BrS-associated Kv4.3 mutations were engineered and co-expressed with wild-type KChIP2 in HEK293 cells. Wild-type and mutant Ito ion currents were recorded using whole cell patch clamp. Results Two BrS1-8 genotype-negative cases possessed novel Kv4.3 missense mutations. Both Kv4.3-L450F and Kv4.3-G600R were absent in 1560 reference alleles and involved residues highly conserved across species. Both Kv4.3-L450F and Kv4.3-G600R demonstrated a gain-of-function phenotype, increasing peak Ito current density by 146.2% (n=15, p<0.05) and 50.4% (n=15, p<0.05) respectively. Simulations employing a Luo-Rudy II AP model demonstrated the stable loss of the AP dome as a result of the increased Ito maximal conductance associated with the heterozygous expression of either L450F or G600R. Conclusions These findings provide the first molecular and functional evidence implicating novel KCND3 gain-of-function mutations in the pathogenesis and phenotypic expression of BrS, with the potential for a lethal arrhythmia being precipitated by a genetically enhanced Ito current gradient within the right ventricle were KCND3 expression is the highest.

Background A 2% to 5% background rate of rare SCN5A nonsynonymous single nucleotide variants (nsSNVs) among healthy individuals confounds clinical genetic testing. Therefore, the purpose of this study was to enhance interpretation of SCN5A nsSNVs for clinical genetic testing using estimated predictive values derived from protein-topology and 7 in silico tools. Methods and Results Seven in silico tools were used to assign pathogenic/benign status to nsSNVs from 2888 long-QT syndrome cases, 2111 Brugada syndrome cases, and 8975 controls. Estimated predictive values were determined for each tool across the entire SCN5A-encoded Nav1.5 channel as well as for specific topographical regions. In addition, the in silico tools were assessed for their ability to correlate with cellular electrophysiology studies. In long-QT syndrome, transmembrane segments S3−S5+S6 and the DIII/DIV linker region were associated with high probability of pathogenicity. For Brugada syndrome, only the transmembrane spanning domains had a high probability of pathogenicity. Although individual tools distinguished case- and control-derived SCN5A nsSNVs, the composite use of multiple tools resulted in the greatest enhancement of interpretation. The use of the composite score allowed for enhanced interpretation for nsSNVs outside of the topological regions that intrinsically had a high probability of pathogenicity, as well as within the transmembrane spanning domains for Brugada syndrome nsSNVs. Conclusions We have used a large case/control study to identify regions of Nav1.5 associated with a high probability of pathogenicity. Although topology alone would leave the variants outside these identified regions in genetic purgatory, the synergistic use of multiple in silico tools may help promote or demote a variant's pathogenic status.

Choroidal neovascularization (CNV) is an important characteristic of advanced wet age‐related macular degeneration (AMD) and leads to severe visual impairment among elderly patients. Previous studies have demonstrated that melatonin induces several biological effects related to antioxidation, anti‐inflammation, and anti‐angiogenesis. However, the role of melatonin in CNV, and its underlying mechanisms, has not been investigated thus far. In this study, we found that melatonin administration significantly reduced the scale and volume of CNV lesions, suppressed vascular leakage, and inhibited the capacity of vascular proliferation in the laser‐induced mouse CNV model. Additionally, the results also show that the melatonin‐treated retinal microglia in the laser‐induced mice exhibited enhanced expression of M1‐type markers, such as iNOS, CCL‐3, CCL‐5, and TNF‐α, as well as decreased production of M2‐type markers, such as Arg‐1, Fizz‐1, IL‐10, YM‐1, and CD206, indicating that melatonin switched the macrophage/microglia polarization from pro‐angiogenic M2 phenotype to anti‐angiogenic M1 phenotype. Furthermore, the RhoA/ROCK signaling pathway was activated during CNV formation, yet was suppressed after an intraperitoneal injection of melatonin. In conclusion, melatonin attenuated CNV, reduced vascular leakage, and inhibited vascular proliferation by switching the macrophage/microglia polarization from M2 phenotype to M1 phenotype via inhibition of RhoA/ROCK signaling pathway in CNV. This suggests that melatonin could be a novel agent for the treatment of AMD.

Loss of GGPPS from childhood mumps infection or deletion in mice results in constitutively activated MAPK and NF-kB signaling that induces spermatogonium apoptosis, macrophage invasion into seminiferous tubules, and sterility.