A Gene-first Strategy in the Early-identification of Inherited Arrhythmia Syndromes

Grants and Contracts Details

Description

Every year congenital long QT syndrome (LQTS) kills thousands. This is not because LQTS is difficult to treat, but it is difficult to diagnose early before a deadly arrhythmia occurs.1-3 Ideally genetic screening could facilitate the early identification and prophylactic treatment of LQTS, but unfortunately genetic screening often identifies novel, rare variants of uncertain physiological significance (VUS). The frequency of these VUS far outpaces the true incidence of the disease and the ability to perform functional screens as they are discovered. The Geisinger MyCode® Biobank has 31,000 Whole Exome Sequences (WES) directly linked to the patients’ electronic health records (EHR). This allows Geisinger to uncover new relationships between genetics and cardiovascular disease. One goal of the MyCode® Initiative is to make early diagnoses of chronic or serious conditions. To facilitate the identification of LQTS patients from the tens of thousands of patients in the MyCode® Biobank, we propose a strategy to study candidate LQTS mutations similar to industry-based techniques to predict drug safety. Specifically, we will adopt the Comprehensive In Vitro Proarrhythmia Assay (CiPA) being developed by the Cardiac Safety Research Consortium, Health and Environmental Sciences Institute, and US Food and Drug Administration. Objective 1. Determine the functional significance of the MyCode KCNH2 variants in vivo. Zebrafish express the molecularly and functionally similar KCNH2 orthologue kcnh6. Zebrafish embryos injected with antisense kcnh6 morpholino (MO) display measurable repolarization abnormalities. Co-injection of MO and human KCNH2 cRNA restores normal repolarization, but co-injection MO and LQT2-linked mutant cRNA does not. We will use this assay to determine if any of the MyCode variants cause a LOF in vivo. If additional MyCode variant cause a LOF in vivo, we will examine the corresponding EHRs to test for possible indicators of LQTS. Objective 2. Develop a comprehensive mutagenic KCNH2 library. We will use an innovative strategy that combines mutagenic PCR, next generation sequencing and combinatorial pooling to create comprehensive, sequence-indexed library of randomly mutated KCNH2 constructs. A proof-of-principle study has already generated a large number of KCNH2 variants, including many reported in ClinVar. Efforts will be expanded to generate a saturated mutagenic KCNH2 library for future off the shelf testing. Objective 3: Identify candidate LQT2-causing mutations from ClinVar. We will use in vitro, in silico, and in vivo testing to identify LOF KCNH2 variants reported in ClinVar. In vitro: We will use Western blot to screen hundreds of VUS generated in Aim 2. Suspected trafficking-deficient KCNH2 mutations will be confirmed using voltage-clamping. We will also use voltage-clamping to assess the functional properties of trafficking-competent Kv11.1 channels. In silico: Predict how mutagenic changes in Kv11.1 channel function impact the ventricular action potential (AP). We will mimic changes in Kv11.1 channel function by modifying a Markovian model for IKr in computational simulations of the human ventricular AP. KCNH2 variants that predict a 12% prolongation in the AP duration (a surrogate for the QT interval) will be considered candidate LQT2-causing mutations. In vivo: Co-injection of antisense kcnh6 MO and WT human KCNH2 cRNA restores normal repolarization and prevents AV block/asystole. We will test if co-injecting kcnh6 MO and candidate LQT2-causing mutant cRNA fails to restore cardiac repolarization.
StatusFinished
Effective start/end date5/1/174/30/18

Funding

  • American Heart Association: $250,000.00

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