Abstract 12815: High Throughput Variant Identification in the S3-S5 Helices of KCNH2 (hERG)

Introduction: LQTS is a multi-factorial cardiac repolarization disorder characterized by a prolonged corrected QT interval (QTc) that predisposes individuals to sudden death. Variants in KCNH2 account for ~30% of LQTS cases. Variants in a ‘hotspot’ domain of KCNH2 are of concern, as variant location in KCNH2 is strongly associated with increased risk of cardiac events. Here, we determined the trafficking phenotype of all possible variants in the S3 and S4 helices (Voltage sensor) and S5 helix and p-loop (pore domain), both ‘hotspot’ domains, of KCNH2 . Method: We used a HA tagged KCNH2 construct containing mCherry to enable massively parallel characterization of channel trafficking of missense KCNH2 variants. We studied a total of 3315/3381 possible variants for residues 477-637. Results: Our preliminary data revealed a heterogenous distribution of trafficking defects (range: 0-157% of WT). Some regions (e.g. 562-566) were largely intolerant to substitution (trafficking scores ~0% for all variants) whereas other regions (e.g. 509-521) were very permissive of substitution (trafficking ranged from 78-146% of WT). For 51 clinically identified variants, we compared trafficking results with peak tail current density data and found that 4/6 were correctly classified as functionally normal and 42/44 correctly classified as loss of function. Conclusion: Massively parallel trafficking assay can identify KCNH2 defects and illuminate important channel biophysics. Massively parallel trafficking assays will enable prospective and accurate variant assessment for any missense variant in KCNH2 and subsequently other proteins where misfolding is a major loss-of-function mechanism.