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Background In the past decade, we and others have reported three families with rare genetic variants in TNNI3K, encoding the cardiac-specific troponin-I interacting kinase (TNNI3K), co-segregating with a mixed, but highly penetrant, cardiac phenotype that features predominant atrial/junctional tachycardia occurring in combination with cardiac conduction disease and dilated cardiomyopathy. We demonstrated that while the p.Thr539Ala and p.Gly526Asp TNNI3K variants had decreased auto-phosphorylation activity the p.Glu768Lys variant, present in 3 independent families, leads to increased auto-phosphorylation levels, in line with the finding that increased levels of Tnni3k expression are associated with slower atrial-ventricular conduction in mice. Objective Identifying new genetic variants in the TNNI3K gene associated with cardiac disease and assessing their impact on TNNI3K auto-phosphorylation levels. Methods Through next generation sequencing of a panel of genes associated with cardiac disease we assessed TNNI3K in patients with cardiac arrhythmias and cardiomyopathies. All variants identified were assessed in vitro for effects on auto-phosphorylation. Briefly, wild-type and mutant TNNI3K constructs were transfected into HEK293 cells, protein was extracted after 48 hours and analyzed with anti-flag and anti-phospho-tyrosine antibodies on Western blot. Results We identified 7 novel and rare variants in TNNI3K in 11 additional probands, with predominantly cardiac conduction disease, with or without dilated cardiomyopathy, and atrial-ventricular-re-entry-tachycardia (AVNRT). Of these, multiple variants were found to have aberrant auto-phosphorylation including almost absent auto-phosphorylation capacity for one (TNNI3K-p.Val510Leu). All three-independent wild type TNNI3K transfected HEK293 cell lysates showed similar phosphorylated TNNI3K levels and the kinase-dead negative control demonstrated no phosphorylation activity. Conclusion We here present 7 novel genetic variants in TNNI3K in patients with a remarkable overlap in cardiac phenotype consisting mainly of AVNRT and cardiac conduction disease. We further show that some of these variants alter the auto-phosphorylation of TNNI3K. These results indicate a more prevalent role of variants in TNNI3K in human cardiac disease and a possible in vitro functional assay to assess the pathogenicity of such variants. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): The Dutch Research Council (NWO Talent Scheme VIDI-91718361)

Background Left ventricular non-compaction (LVNC) is a condition characterised by trabeculations in the myocardial wall and is the subject of considerable conjecture as to whether it represents a distinct pathology or a secondary phenotype associated with other cardiac diseases, particularly cardiomyopathies. Purpose To investigate the genetic architecture of LVNC by identifying genes and variant classes robustly associated with disease and comparing these to other genetically characterised cardiomyopathies. Methods We performed rare variant association analysis using six different LVNC cohorts comprising 840 cases together with 125,748 gnomAD population controls and compared results to similar analyses with dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM) cases. Results We observed substantial overlap in genes and variant classes enriched in LVNC and DCM/HCM, indicating that in many cases LVNC belongs to a spectrum of more established cardiomyopathies, with non-compaction representing a phenotypic variation in patients with DCM- or HCM-causing variants. In contrast, five variant classes were uniquely enriched in LVNC cases, of which truncating variants in MYH7, ACTN2 and PRDM16 may represent a distinct LVNC aetiology. MYH7 truncating variants are generally considered as non-pathogenic but were detected in 2% of LVNC cases compared to 0.1% of controls, including a cluster of variants around a single splice region. Additionally, structural variants (exon deletions) in RYR2 and missense variants in the transmembrane region of HCN4 were enriched in LVNC cases, confirming prior reports regarding the association of these variant classes with combined LVNC and arrhythmia phenotypes. Conclusions We demonstrated that genetic association analysis can clarify the relationship between LVNC and established cardiomyopathies, highlighted substantial overlap with DCM/HCM but also identified variant classes associated with distinct LVNC and with joint LVNC/arrhythmia phenotypes. These results underline the complex genetic landscape of LVNC and inform how genetic testing in LVNC cases should be pursued and interpreted. Cardiomyopathy rare variant frequencies Funding Acknowledgement Type of funding source: None

Aims Non-cardiac drugs that impair cardiac repolarization (electrocardiographic QT prolongation) are associated with an increased sudden cardiac arrest (SCA) risk. Emerging evidence suggests that non-cardiac drugs that impair cardiac depolarization and excitability (electrocardiographic QRS prolongation) also increase the risk for SCA. Nortriptyline, which blocks the SCN5A -encoded cardiac sodium channel, may exemplify such drugs. We aimed to study whether nortriptyline increases the risk for SCA, and to establish the underlying mechanisms. Methods and results We studied QRS durations during rest/exercise in an index patient who experienced ventricular tachycardia during exercise while using nortriptyline, and compared them with those of 55 controls with/without nortriptyline and 24 controls with Brugada syndrome (BrS) without nortriptyline, who carried an SCN5A mutation. We performed molecular-genetic (exon-trapping) and functional (patch-clamp) experiments to unravel the mechanisms of QRS prolongation by nortriptyline and the SCN5A mutation found in the index patient. We conducted a prospective community-based study among 944 victims of ECG-documented SCA and 4354-matched controls to determine the risk for SCA associated with nortriptyline use. Multiple mechanisms may act in concert to increase the risk for SCA during nortriptyline use. Pharmacological (nortriptyline), genetic (loss-of-function SCN5A mutation), and/or functional (sodium channel inactivation at fast heart rates) factors conspire to reduce the cardiac sodium current and increase the risk for SCA. Nortriptyline use in the community was associated with a 4.5-fold increase in the risk for SCA [adjusted OR: 4.5 (95% CI: 1.1–19.5)], particularly when other sodium channel-blocking factors were present. Conclusions Nortriptyline increases the risk for SCA in the general population, particularly in the presence of genetic and/or non-genetic factors that decrease cardiac excitability by blocking the cardiac sodium channel.