Neves, Raquel

Abstract Aims Calcium release channel deficiency syndrome (CRCDS) results from loss-of-function (LOF) variants in the RYR2-encoded type 2 ryanodine receptor (RyR2), predisposing patients to sudden cardiac arrest/death (SCA/SCD) without abnormalities on a stress electrocardiogram (ECG). Undetected CRCDS may underlie idiopathic ventricular fibrillation (IVF) and sudden unexplained death in the young (SUDY). We aimed to determine the prevalence of potential CRCDS-causative RYR2 variants in IVF and SUDY. Methods and results We reviewed clinical evaluation and RYR2 genetic analysis of 169 IVF patients and 279 SUDY victims. Only ultra-rare (<0.005% in gnomAD) nonsynonymous RYR2 variants were considered potentially pathogenic. Among IVF patients, 6/169 (3%) overall—and 6/67 (9%) with exertion-related SCA—harboured an RYR2 variant and represent potential CRCDS cases. All exhibited normal resting and stress ECGs. Genetic analysis revealed six distinct RYR2 variants, two previously characterized as LOF. In SUDY, 31/279 victims (11%) had a RYR2 variant (30 unique variants), predominantly observed in exertion-related SCD 20/83 (24%) vs. rest-related 11/196 (6%). Of the 14 SUDY victims with functionally characterized RYR2 variants, five (2% of total cohort) had a LOF variant; among the 56 exertion-related SUDY cases, four (7%) had a LOF variant. Conclusion CRCDS may account for 3% of IVF overall and 9% of exertion-related SCA in IVF. Ultra-rare RYR2 variants may underlie up to 11% of SUDY, with 65% of RYR2-positive cases occurring during exertion. LOF-RYR2 variants may contribute to ≥7% of exercise-associated SUDY. Accurate identification of the underlying ryanodinopathy is essential for clinical management of affected patients.

Background: Current guidelines recommend prophylactic beta-blocker therapy for all genotype-positive newborns with long QT syndrome (LQTS), despite the potential for QT normalization in the first few weeks of life and the rarity of cardiac events during infancy. We previously reported our early experience with intentional non-therapy (INT) for low-risk adolescents and adults. Objective: To evaluate our single-center experience regarding INT for infants with genotype-positive LQTS identified following family screening/cascade testing. Methods: We retrospectively reviewed all infants diagnosed with genotype-positive LQTS between 2015 and 2024. Only LQTS patients with clinical and ECG data within the first 2 years of life and follow-up were included. INT was defined as adherence to precautionary QT-preventative measures and structured periodic follow-up rather than treating with prophylactic drugs, devices, or denervation. Candidacy for INT was based on absence of symptoms, down-trending QT during first few weeks of life, family agreement with the strategy, and frequent surveillance (e.g., ECG and Holter every 6 months). Prophylactic drug therapy was initiated if a surveillance QTc exceeded 470ms on 2 consecutive ECGs or at family request. Results: Of 94 infants, 38 [40%; 19 females (50%), mean age at diagnosis 0.3±0.4 years] were managed with INT (14 LQT1 (36%), 15 LQT2 (39%), 8 LQT3 (21%)). Genetic testing and ECG screening were performed because of a family history in all 38 infants including 11 (29%) with a family history of SCA/SCD. None had fetal bradycardia reported or any cardiac symptoms prior to diagnosis. Mean neonatal QTc during the first week of life was 477±41ms and 442±6ms at 3 months of age. No cardiac events were reported during a mean follow-up of 3.9±2.3 years. Management was escalated from INT to prophylactic pharmacologic therapy in 8 infants (21%) with nadolol in 4, propranolol in 3, and LQT3 mutation-specific therapy with flecainide in 1 based on 2 consecutive QTc values>470ms, at a mean age of 4.3±2 years (mean QTc at treatment initiation 485±13ms). Conclusions: In selected LQTS infants with favorable down-trending QT and access to close follow-up, INT rather than universal prophylactic drug therapy can be considered. This approach may offer an alternative strategy in infancy, avoiding unnecessary early exposure to medication-related side effects and reinforcing individualized genotype- and phenotype-directed tailoring of therapeutic options.

Background: Congenital long QT syndrome (LQTS) is characterized by a prolongation of the heart rate-corrected QT interval (QTc) and an increased risk of syncope, seizure and sudden cardiac arrest/death (SCA/SCD). Genome-wide association studies have identified numerous QT interval-influencing loci of small effect sizes. Herein, we sought to investigate whether a polygenic background for the QT interval contributes to the phenotypic heterogeneity in patients with genotype-positive LQTS. Methods: We used summary statistics from a recent genome-wide meta-analysis for QT interval (n = 117,532) to derive 160 different polygenic scores for QT interval (PGS QT ) using LDpred2-grid, each comprising ~1.4 million common variants with varying assumptions of the underlying genetic architecture. Next, we applied these PGS QT to predict the QTc value in a genetically unrelated, European ancestry subset of the ‘All of Us’ cohort with available electrocardiogram and genetic data (n=6,404), where the best performing PGS QT was determined based on explained variance. Next, using 225 patients with genetically confirmed LQTS who were evaluated in the Mayo Clinic Windland Smith Rice Genetic Heart Rhythm Clinic, we tested the effect of the best performing PGS QT on QTc duration and LQTS-associated cardiac events (defined as a composite of syncope, seizures, sustained ventricular arrhythmia, appropriate ICD shocks, and SCA/SCD) using Bayesian regression models. Results: Overall, the best performing PGS QT explained 11.8% of the variance in the QTc in the ‘All of Us’ cohort. Among patients with genotype-positive LQTS (n [%]; KCNQ1 125 [55.6], KCHN2 76 [33.8], SCN5A 21 [9.3], Other 3 [1.3]), those with higher PGS QT had a higher QTc (predicted mean QTc [95% uncertainty interval], 95 th percentile vs 50 th percentile for PGS QT , 478 ms [464-491] vs 459 ms [449-468]). In comparison to the median PGS, those LQTS patients with a PGS QT >95 th percentile were more than twice as likely to have experienced a LQTS-associated cardiac event (Odds ratio [95% UI], 95 th percentile vs 50 th percentile for PGS QT ; 2.16 [1.02-5.41]), ( Figure ). Conclusions: Individuals with genotype-positive LQTS who have high polygenic susceptibility for QTc prolongation are more likely to phenotypically exhibit a higher QTc value and experience potentially life-threatening LQTS-associated cardiac events. Accounting for polygenic background may refine risk stratification strategies for patients with genotype-positive LQTS

Background Disease‐causative variants in LMNA ‐encoded lamin A/C cause a genetic cardiomyopathy characterized by atrioventricular block, atrial fibrillation, ventricular arrhythmias, and systolic dysfunction. The influence of LMNA variant type/localization on late gadolinium enhancement (LGE) patterns and clinical outcomes remains unclear. Methods Retrospective analysis of 822 genotype‐positive patients with arrhythmogenic/dilated cardiomyopathy was used to identify those with disease‐causative variants in LMNA . Data on LGE distribution and prevalence of advanced heart failure, thromboembolic and sudden cardiac death/major ventricular arrhythmia events were extracted from the electronic record and analyzed by variant type/localization. Results Among the 72/116 (62%) LMNA variant‐positive patients with cardiac magnetic resonance imaging data, LGE was observed in 40/72 (56%) cases. Most exhibited a nonischemic, midmyocardial or subepicardial pattern (73%), and 6/40 (15%) showed a unique “pseudo‐infarct” transmural pattern, predominantly affecting the apical segments. All 6 patients with this distinct LGE pattern harbored C‐terminal IgD (immunoglobulin‐like domain) variants (p.Arg471His or p.Arg541His). In patients with clinically manifest disease (76/116), those with IgD‐localizing variants had a lower prevalence of atrioventricular block (25% versus 72%, P =0.002) and atrial fibrillation (50% versus 81%, P =0.019) but higher rates of thromboembolic events (42% versus 16%, P =0.038). During a median follow‐up of 37 months, IgD variant presence independently predicted sudden cardiac death/major ventricular arrhythmia (hazard ratio, 2.391 [95% CI, 1.046–5.464]; P =0.039). Conclusions LMNA missense variants localizing to IgD present a distinct apical pseudo‐infarct LGE pattern associated with increased risk of ventricular arrhythmias and thromboembolic events but reduced atrioventricular block and atrial fibrillation. Multicenter studies are warranted to develop variant‐specific risk‐stratification strategies in cardiac laminopathy.

Background: LQTS is a genetic heart disease associated with an increased risk of sudden death. Despite advances in genetic testing, a small subset of patients still lacks identifiable genetic markers. Consequently, the true prevalence and clinical phenotype of genotype negative remains to be determined. Objective: To determine the prevalence and clinical characteristics of patients with a clinical diagnosis of LQTS but a negative genetic test. Methods: A retrospective review was conducted on patients evaluated and treated at a tertiary hospital between 2000 and 2024, focusing on those with a clinical diagnosis of LQTS, but a negative genetic test for all LQTS-associated genes, with available follow-up of at least 1 year. Results: After exclusion for follow-up and lack of genetic testing, the study cohort consisted of 1834 patients with LQTS including 1708 (93%) with a (likely) pathogenic (LP/P), LQTS-causative variant. Also, in 95 patients (5%), their variant of uncertain significance was clinically upgraded to a LP/P-variant, leaving only 31 patients classified as phenotype positive but genotype negative (1.6%). Of these, 16/31 underwent next-generation genetic sequencing, revealing the genetic cause in 6/16 cases (37%; 0.3% of all patients). For the remaining 25 genotype negative LQTS patients (1.3%), the mean age at diagnosis was 26 ± 15 years, with 19 (76%) being female, and an average initial QTc of 497 ± 41 ms. In total, 11 patients (44%) experienced a cardiac event before diagnosis [1 sudden cardiac arrest (SCA), 1 Torsades, 1 VT, 8 syncopes] and 1 patient had his first cardiac event during follow-up; 7/12 patients had more than one subsequent event. Twenty patients were proband (80%) and 6 had a family history of SCA. Eleven patients received an ICD (44%), left cardiac sympathetic denervation was performed in 4 (16%), and 2 patients experienced arrhythmia-related deaths (8%). The mean follow-up since diagnosis was 7 ± 5 years. Conclusion: With a prevalence of genotype-negative LQTS of 2%, the number of these patients is much lower than previously suggested, underscoring the advancements in identifying new genetic markers and the role of phenotype-enhanced variant adjudication and next-generation sequencing. Additionally, significant disease severity was observed in the remaining patients, emphasizing the pivotal role of genotype-phenotype correlations and risk stratification in managing and counseling patients lacking identified LQTS-causing mutations.

Background: Arrhythmogenic cardiomyopathy (ACM) is a genetic heart disease characterized by proclivity for ventricular arrhythmias and systolic dysfunction. Rarely, sudden cardiac arrest (SCA) is the presenting event in ACM. These individuals may have concealed disease with no appreciable structural manifestations on imaging. Aims: To determine the prevalence, clinical features, and outcomes of patients with genotype-positive ACM who presented with a sentinel SCA. Methods: In this retrospective study, 445 genotype-positive ACM patients evaluated from 1/2015-4/2024 were analyzed to identify those whose initial presentation was SCA. Pertinent demographic, clinical, genetic, and outcome data were extracted from the electronic medical record. Structural manifestations were defined by having one of the following on baseline TTE: EF < 50%, LVEDV >56 mL (male) or >51 mL (female), or regional RV akinesia, dyskinesia, or aneurysm. Categorical variables are presented as number (%), and continuous variables as median (IQR). Statistical analysis was done using Fisher’s exact test (categorical), t-test (continuous), and Logrank test (survival). Patients were censored at time of transplant, death, or last followup. Results: Overall, 14/445 (3%) ACM patients presented with sentinel SCA [71% male; median age at SCA 39 (IQR 30-46)]. The most common genetic substrates were DSP -encoded desmoplakin (n=4; 29%), LMNA -encoded lamin A/C (n=3; 21%), and PKP2 -encoded plakophilin-2 (n=3; 21%). During a median followup of 8.4 years, 12 (86%) experienced appropriate ICD shocks, 8 (57%) required catheter ablation for ventricular arrhythmias, 6 (42%) underwent transplant, and 2 (14%) died of cardiac causes. Notably, 5 (35%) patients did not have evidence of structural disease during baseline evaluation. Compared to those with structural disease, this group was younger [median age 28 (IQR 21-29) vs 45 (IQR 41-48), p = 0.002] and less likely to undergo transplant [0 (0%) vs 6 (42%), p = 0.03]. Conclusions: Sentinel SCA is a rare presentation in ACM and may precede overt structural cardiac changes in a subset of younger patients. This population remains at increased risk for recurrent ventricular arrhythmias and often require advanced therapies.

Background: Although exercise stress testing (EST) has shown to unmask latent electrical substrates in PKP2 -mediated arrhythmogenic cardiomyopathy (ACM), the prognostic role of EST-induced ventricular arrhythmia (VA) patterns in PKP2-ACM patients is unclear. Objective: To examine the exercise-induced VA patterns observed during EST in PKP2-ACM patients and explore their correlation with major VA outcomes. Methods: We retrospectively analyzed 621 patients with either genotype-positive ACM or genotype-positive dilated cardiomyopathy evaluated between 01/2015 and 04/2024 to identify those with a disease-causative variant in PKP2 (N = 108). After exclusion of 38 PKP2-ACM patients without EST data, demographic and electrocardiographic data was extracted from the electronic medical record. The arrhythmic burden [premature ventricular contractions (PVCs), PVCs in bigeminy, PVC couplets, non-sustained VT (NSVT), sustained VT, ventricular fibrillation (VF), and PVCs/minute] during each EST phase (baseline, peak, active and passive recovery) were reviewed and correlated with major VA outcomes [sudden cardiac arrest (SCA), sustained VT, and appropriate implantable cardioverter-defibrillator (ICD) shocks]. Results: A total of 186 exercise tests from 70/108 (65%) PKP2-ACM patients (mean age 36 ± 20 years, 40 % male) were analyzed. The presence and burden of PVCs occurring singly, bigeminy, couplets. and NSVT were more prevalent during the recovery phase, specifically the passive recovery phase ( Table ). PKP2-ACM patients with structural disease (64%) were more likely to have an increased VA burden during recovery than those without structural disease [28/45 (62%) vs 4/25 (16%); p<0.001]. Interestingly, the presence of an increased arrhythmic burden (worsening PVCs/min or complex PVCs) during the recovery was associated with major VA over a median follow-up of 47 months (HR 8.576 [1.015-72.449]; p= 0.019). Conclusion: Increased arrhythmic burden during the recovery phase (late > early) is associated with an increased risk of major VAs in PKP2-ACM. Further studies are needed to understand if this observation extends to other ACM genotypes and whether EST-induced VA patterns can be used to assess therapeutic efficacy in PKP2-ACM.

Background: We identified a novel, ultra-rare missense variant (p.R534S) in DENND3 as a potential disease-susceptibility variant in a multi-generational pedigree of familial idiopathic ventricular fibrillation (IVF). DENND3 is a guanine nucleotide exchange factor and activator for small GTPase Rab proteins involved in endosome and ion channel membrane trafficking. However, the role of DENND3 in the heart and its potential involvement in cardiac arrhythmias are unknown. Objective: To functionally characterize DENND3-p.R534S and determine its impact on channel trafficking. Methods: Heterologous co-expression studies in TSA201 cells were performed with DENND3-WT or DENND3-p.R534S and ion channels: KCNQ1 (Kv7.1, IKs), KCNH2 (Kv11.1, IKr), SCN5A (Nav1.5, INa), or CACNA1C (Cav1.2, ICa) as well as Rab proteins (Rab4, Rab5, Rab6, Rab7, Rab11, Rab12). Patient-specific DENND3-p.R534S iPSC-derived cardiomyocytes (iPSC-CMs) and isogenic controls (IC) were created. Localization and expression of ion channel and Rab proteins was assessed using immunofluorescence (IF) by super resolution AiryScan microscopy. GTPase-Glo assays measured Rab protein activity. Electrophysiology studies using patch-clamp and FluoVolt were performed on iPSC-CM models. Results: IF imaging showed significant increases in membrane expression of KCNQ1 (11714±157to 21929±378, p<0.05), KCNH2 (11378±160 to 31965±544, p<0.05), , SCN5A (10339±8 to 11225±23, p<0.05), and CACNA1C (11879±83 to 13749±138, p<0.05) in DENND3-p.R534S compared to DENND3-WT expressing TSA201 cells. Rab5 distribution was altered significantly by the DENND3-p.R534S variant, with increased dispersion away from the nucleus (0.23±0.01 vs. 0.17±0.01, p<0.05) and increased proximity to the plasma membrane (0.16±0.02 vs. 0.23±0.01, p<0.05). Additionally, Rab5 activity was enhanced significantly (57% increase) by p.R534S versus WT (p<0.05). iPSC-CMs revealed similar increases in channel membrane accumulation and irregular arrhythmic activity including frequent action potential duration alternans and afterdepolarizations in 78% of p.R534S iPSC-CMs versus none in IC cells (p<0.05). Conclusion: DENND3 is a novel ion channel trafficking protein in the heart. The DENND3-p.R534S variant disrupts cardiac ion channel trafficking. These findings highlight DENND3’s role in cardiac ion channel distribution, cellular function, and potential association with arrhythmogenic risk.

Background: Cardiac channelopathies are a heterogenous group of genetic heart diseases (GHDs) that affect cardiac ion channel function. The most prevalent among these are long QT syndrome (LQTS) and catecholaminergic polymorphic ventricular tachycardia (CPVT) which predispose patients to potentially life threatening ventricular arrhythmias. Accurate and timely diagnosis is important for the management and risk stratification of the index case and identification of potentially at-risk relatives. Unfortunately, despite rising awareness of these GHDs, critical warning signs are overlooked. Objective: To describe the phenotypes of patients who present with a delayed or missed diagnosis of LQTS or CPVT. Methods: EMR data was reviewed for all patients evaluated and treated at Mayo Clinic between 2000-2023 who arrived with a mis- or delayed diagnosis of LQTS or CPVT. Data was abstracted for patient demographics, clinical characteristics, family history of SCD, and cardiac and genetic test results. Results: Overall, 47/2119 (2%) patients [33 (70%) female, mean age at correct diagnosis 26±18 years] diagnosed with LQTS (30; 2% of LQTS cohort) or CPVT (17; 7% of CPVT cohort) arrived with an outside misdiagnosis (37, 79%) or underdiagnosis (10, 21%). The most common misdiagnosis was epilepsy (20; 43%) or vasovagal syncope (14; 30%) and 3 CPVT patients (6%) were misdiagnosed with LQTS. Of the 10 underdiagnosed patients, 4 (40%) had QT prolongation, 4 (40%) family history of SCD, 3 (30%) bilateral deafness, and 1 (10%) fetal bradycardia and syndactyly. Overall, 40/47 (85%) were symptomatic (syncope: 29 [73%], seizures: 21 [53%], SCA: 10 [25%]) prior to diagnosis. Of note, 34/40 (85%) had symptoms with exertion, 5 (13%) at rest, and trigger was unknown in 1 (2%). Of the 7 asymptomatic patients (15%), 4 (57%) had QT prolongation and 4 (57%) had family history of SCD. In total, 20/47 patients (43%) had positive family history of SCD under 45 years of age. Overall, patients experienced a median delay in diagnosis of 8 years during which a median of 4 events (range: 1-150) occurred. As a result, an additional 29 family members of these patients received a delay in their diagnosis. Conclusions: Recognizing the signs and symptoms of patients with GHDs is important to reduce the ongoing burden of misdiagnosis or delayed diagnosis in LQTS and CPVT. The most important factors for suspicion of an inherited channelopathy are symptoms during exertion and family history of SCD.

Background: Long QT syndrome (LQTS) is a potentially lethal cardiac channelopathy. Among women with LQT2, the post-partum period has been considered high risk for cardiac events. However, whether this risk persists after establishing their diagnosis and implementing their LQT2-directed treatment program remains to be determined. Objective: To describe the management and outcomes of LQT2 women during the 9 months post-partum period. Methods: A retrospective analysis of 1869 patients with LQTS treated and evaluated at a tertiary center specializing in Genetic Heart Disease from January 2000 to November 2023 was performed to identify women with diagnosed and treated LQT2 who had a pregnancy during follow-up. Data were abstracted for patient demographics, clinical characteristics, symptomatic status, and treatment plans before and after pregnancy. Results: Overall, 30 pregnancies occurred in 22 women with LQT2. Their average QTc was 489 ± 34 ms with 7 patients (32%) having a resting QTc > 500 ms. Prior to their first post-partum period, 5/22 (23%) were symptomatic with 2 (9%) experiencing a LQT2-triggered sudden cardiac arrest (SCA). Before their post-partum period, their LQT2-directed therapy comprised preventative measures only in 7 (23%), drug therapy in 16 (53%), combination therapy in 7 (23%), and 10 women (43%) had an implantable cardioverter defibrillator (ICD). Pre-emptive treatment intensification was done for 24/30 post-partum periods. Only a single VF-terminating ICD therapy occurred in 1 (3%) of the 30 post-partum periods involving a 21-year-old with p.Lys610Asn-KCNH2 variant, QTc = 490 ms, and a pre-diagnosis presentation of seizures. Conclusion: Although the post-partum period is regarded as a ‘high risk’ window of time for women with LQT2, the risk of a LQT2-triggered cardiac event after diagnosis and implementation of contemporary therapies is very low. This designation of “high risk” among correctly diagnosed and treated women is misleading and generates inappropriate and unnecessary anxiety.