J. Peter van Tintelen

Complex diseases such as progressive cardiomyopathies are often insufficiently recapitulated in small animals or in vitro models. Large animal species such as pigs provide a valuable alternative, but constitutive genetic manipulation has not yet been applied to pigs in an effective manner, mainly due to biological and logistical limitations. Here we describe the generation of a humanized pig model for phospholamban-mediated cardiomyopathy and compare different methods for activating a pathogenic R14del mutation by Cre-mediated recombination. Both Cre treatment of pig primary cells before somatic cell nuclear transfer as well as microinjection of Cre-encoding mRNA into zygotes were similarly efficient in delivering piglets with an activated R14del mutation. Alternatively, administration of Cre-encoding adeno-associated virus into piglets was sufficient, albeit to a varying extent. Together, we describe a highly effective process to establish complex inducible genetic traits in pig and demonstrate that the lack of Cre-driver lines can be compensated by various interventions during reproduction or postnatally.

BACKGROUND: Current guidelines recommend regular screening for first-degree relatives of gene-elusive arrhythmogenic right ventricular cardiomyopathy (ARVC) patients using a similar regimen as for genotype-positive/phenotype-negative relatives. However, the multifactorial nature of gene-elusive ARVC may necessitate a different approach. This study aimed to determine the yield of cardiac screening in first-degree relatives of ARVC probands without a validated genetic cause. METHODS: We included all first-degree relatives of probands who (1) met the 2010 Task Force Criteria, (2) underwent next-generation sequencing that included all genes with at least moderate evidence for ARVC causation per Clinical Genome Resource appraisal (validated ARVC genes), and (3) had no pathogenic/likely pathogenic (P/LP) variants identified in these genes. The primary and secondary end points were definite ARVC by the 2010 Task Force Criteria and ventricular arrhythmia, respectively. RESULTS: We included 44 relatives (39.0 [22.3–45.8] years; 36% male) from 24 families. In 4 (17%) families, a P/LP variant was identified in a different cardiomyopathy/arrhythmia gene ( SCN5A , LMNA , CDH2 , FLNC ). Overall, 10 (23%) relatives had definite ARVC at baseline evaluation. Of the 20 relatives without definite ARVC who had follow-up available, 8/20 (40%) relatives progressed to definite ARVC during 9.0 (5.8–14.4) years of follow-up. No statistical difference in the yield of baseline screening or serial evaluation between relatives from families with a P/LP variant and relatives from families without a P/LP variant was observed. Of the 27 relatives who had follow-up available, ventricular arrhythmia was observed in 2/27 (7%) relatives and occurred 6.3 and 13.8 years after definite ARVC diagnosis. Both of those relatives were from families without a P/LP variant. CONCLUSIONS: These findings highlight the importance of managing first-degree relatives of ARVC probands without a validated genetic cause similarly to genotype-positive ARVC relatives. Furthermore, using a broad cardiomyopathy and arrhythmia gene panel in ARVC probands, rather than limiting testing to validated ARVC genes alone, is warranted.

Studying the global distribution of the pathogenic variant c.40_42delAGA;p.(Arg14del) in the phospholamban (PLN) gene is highly important for raising awareness among healthcare providers and may help uncover factors contributing to variability in the development of associated cardiomyopathy phenotypes. PLN p.(Arg14del)-positive individuals were identified through a PubMed literature search, our clinical and research networks, and ClinVar. Additionally, population prevalences were determined using publicly available genetic databases. Furthermore, haplotype analysis was conducted using haplotype markers or whole genome sequencing data to assess whether newly identified cases across different continents share common ancestry. The PLN p.(Arg14del) variant was identified in 21 countries across four continents. Haplotype marker analysis suggest that most analyzed individuals, except those from Greece, shared at least part of a common haplotype. The PLN p.(Arg14del) variant is present in at least 2000 carriers globally. While the majority share at least part of a common haplotype, suggesting a common founder, data suggest an independent mutational event in Greek patients.

Digital interventions are potentially promising to improve accessibility and efficiency of genetic counseling services. However, current literature on user perspectives toward digital tools for cascade testing is limited. Therefore, this focus‐group study aimed to gain insights into the attitude and perspectives of probands, at‐risk relatives (ARR), and genetic healthcare professionals (HCP) toward digital innovations for assistance with both pretest and posttest counseling and cascade genetic testing in cardiogenetics. We conducted seven online focus groups, which were transcribed and thematically analyzed. In total, 37 individuals participated (10 probands, 11 ARR and 16 HCP). Thematic analysis of focus group transcripts showed a first theme of (1) acceptability of digital tools. Other identified themes were defined as “domains,” where digital tools impact traditional, in‐person clinical genetic care, being (2) family communication, (3) decision‐making, (4) care relations, and (5) the genetic care system. Participants expressed a predominantly positive attitude toward the digitization of (parts of) the predictive genetic counseling journey in cardiogenetics under the condition that access to human contact is preserved. In the clinical setting of predictive counseling, efforts should be made to ensure access to genetic services for all ARR and to protect in‐person involvement of HCP.

Dipeptidyl aminopeptidase–like protein 6 (DPP6) is a subunit of the K _v 4 channels that carry the transient-outward current ( I _to ) in cardiac Purkinje cells (PCs) and ventricular myocytes (VMs). DPP6 genetic variants have been linked to severe arrhythmia syndromes. Given the influence of other I _to subunits on the Na _v 1.5-mediated cardiac sodium current ( I _Na ), we examined whether DPP6 regulates both I _to and I _Na . We explored the impact of the DPP6 missense variants c.821G>A and c.637C>T, segregating in families with long-QT syndrome (LQTS), and c.2252C>T and c.1578G>C, associated with J-wave syndromes (JWSs) and unexplained syncope. In human and mouse heart slices, DPP6 localized within 40 nanometers of Na _v 1.5. Functionally, DPP6 reduced I _Na and increased I _to density in transfected Chinese hamster ovary cells. DPP6 variants linked to LQTS and JWSs led to a hypo- and hyperinhibition of I _Na , respectively. Conversely, I _to was increased by the JWS variants and decreased by the LQTS variants coexpressed with PC (but not VM) I _to subunits. These findings were validated in human induced pluripotent stem cell–derived cardiomyocytes. In silico modeling of I _Na and I _to data into PC and VM single-cell action potentials, subsequently integrated in two-dimensional tissue simulations, produced steep repolarization gradients for LQTS-c.821G>A versus slowed conduction for JWS-c.2252C>T. Noninvasive electrocardiographic imaging, used for advanced clinical phenotyping, showed dispersed and prolonged repolarization in the DPP6 c.821G>A index patient versus right ventricular outflow tract delayed activation of a DPP6 c.2252C>T carrier. In conclusion, DPP6 variants play an important role in the mutually antagonistic regulation of I _Na and I _to , contributing to cardiac electrophysiology and arrhythmogenesis.

Background: Pathogenic desmin (DES) variants have been implicated in early-onset atrial disease, yet the mechanisms by which desmin dysfunction alters atrial structure and function remain unclear. Desmin anchors the cytoskeleton to the nuclear envelope (NE) through the linker of nucleoskeleton and cytoskeleton (LINC) complex, suggesting that defects in this network may drive atrial cardiomyopathy. Methods: Human desmin wild-type (WT) and the pathogenic variants p.S13F, p.N342D, and p.R454W were stably expressed in HL-1 atrial cardiomyocytes. Desmin organization, nuclear morphology, LINC-complex integrity (nesprin-3, lamin A/C), and DNA leakage, assessed by cyclic GMP?AMP synthase (cGAS), were analyzed by confocal microscopy. Action potential duration (APD) and calcium transients (CaT) were measured optically. Human myocardium samples from DES variant carriers were analyzed for validation. Data-independent acquisition (DIA) mass spectrometry profiled atrial proteomes from desmin-network variant carriers. The heat-shock proteins (HSPs) inducer geranylgeranylacetone (GGA) was evaluated for rescue effects. Results: p.N342D caused severe filament-assembly defects with prominent perinuclear aggregates, whereas p.S13F showed mixed phenotypes with frequent perinuclear aggregates, and p.R454W largely preserved filamentous networks. p.N342D and p.S13F induced nuclear deformation with disrupted nesprin-3 and lamin A/C distribution. In p.N342D and p.S13F, desmin aggregates drove focal lamin A/C accumulation, nuclear envelope (NE) rupture, DNA leakage, and increased cGAS activation. DES variants significantly shortened APD20/90 and reduced CaT amplitude, indicating pro-arrhythmic electrical remodeling. Atrial proteomics revealed a desmin-network?specific signature enriched for cytoskeletal, NE, intermediate filament, and chaperone pathways, consistent with the structural injury observed in vitro. GGA prevented desmin aggregation and nuclear morphology changes, and mitigated APD shortening in p.N342D-expressing cardiomyocytes. Human myocardium from DES variant carriers showed concordant desmin aggregation and polarized lamin A/C distribution. Conclusions: DES variants induce a desmin-dependent atrial cardiomyopathy characterized by cytoskeletal disorganization, disruption of LINC-complex, NE rupture with DNA leakage, and pro-arrhythmic electrophysiological remodeling. These findings provide mechanistic insight into how desmin-network variants promote atrial disease. HSPs induction by GGA partially restores structural and functional integrity, identifying a potential therapeutic approach for desmin-related atrial cardiomyopathy.

Genetic testing has become an integral part of the diagnostic workup of patients with dilated cardiomyopathy (DCM). While the initial goal of genetic testing was to identify family members at risk, recent advances have now extended their relevance to clinical decision-making. Our knowledge of the genetic architecture of DCM has expanded significantly, promoting a shift from the monogenic dogma towards a broader polygenic spectrum. However, current genetic testing strategies still primarily rely on the model of monogenic inheritance with an incomplete penetrance. Large studies have shown a yield varying from 8% to 36% of genetic testing in patients with DCM, depending on aetiology or family history. Genetic testing is generally warranted for every patient with DCM where genetic results could have an impact on risk stratification, the prognosis or the treatment of the patient, or its family members with an opportunity for reassurance or early disease detection. There are various strategies for genetic testing including broad multigene panels, or more targeted panels limited to specific disease-associated genes. Identified variants are classified by genetic laboratories, where pathogenic or likely pathogenic variants often have actionable clinical implications. It is crucial to interpret these variants in the context of the individual patient considering the phenotype and other contributing factors. When the genetic results are consistent with the patients’ broader phenotype, potential clinical implications may include decision for device therapy, recommendations for family screening, and reproductive options. A comprehensive approach to integrate genetic testing in the clinical care of patients with DCM is proposed.

BACKGROUND: The arrhythmogenic right ventricular cardiomyopathy (ARVC) risk calculator estimates the risk of incident sustained ventricular arrhythmia (VA) and performs well in ARVC populations meeting 2010 Task Force Criteria. However, the calculator includes no measure of left ventricular (LV) structure and function, while late gadolinium enhancement (LGE) on cardiac magnetic resonance shows promise in arrhythmic risk prediction. This study aims to evaluate whether LV LGE on cardiac magnetic resonance can further refine ARVC VA risk stratification. METHODS: Patients with definite ARVC, no prior sustained VA, and contrast-enhanced cardiac magnetic resonance at baseline were followed at 17 centers. Survival analyses were performed to assess LV LGE effect on VA prediction, and its incremental prognostic value on the risk calculator was evaluated using Cox proportional hazard models. The presence of high-risk LGE, defined as LV epicardial, transmural, or combined septal and free-wall LGE, was studied as a sensitivity analysis. RESULTS: Of 385 patients (39.6±15.4 years, 39.7% male, 54.0% probands), 132 (34.3%) had LV LGE on cardiac magnetic resonance, with 98 (25.5%) having a high-risk pattern. Over 3.1 [1.2–5.8] years of follow-up, 67 (17.4%) patients experienced VA. In univariable analysis, both LV LGE (hazard ratio, 1.82; P =0.014) and high-risk LV LGE (hazard ratio, 1.85; P =0.017) were associated with higher risk for VA. However, after adjusting for the ARVC calculator–estimated risk, the presence of neither LV LGE ( P =0.85) nor high-risk LV LGE ( P =0.87) independently predicted sustained VA. (graphic abstract) CONCLUSIONS: While associated with the risk of VA in ARVC, LV LGE did not provide incremental prognostic value for incident VA risk prediction compared with the ARVC risk calculator.

BACKGROUND: Inflammatory, myocarditis-like episodes precede and are associated with higher risk of sustained ventricular arrhythmias and heart failure in patients with pathogenic or likely pathogenic desmoplakin (DSP) variants. Whether the recurrence and treatment of myocarditis-like episodes influence the outcomes in this population is unknown. This study aimed to assess the prognostic impact of the recurrence and treatment of myocarditis-like episodes in patients with pathogenic or likely pathogenic DSP variants. METHODS: The study was designed as an observational cohort study using all patients with pathogenic or likely pathogenic DSP variants in the DSP -ERADOS Network ( Desmopakin Specific Effort for a Rare Disease Outcome Study) enrolled at 31 institutions up until April 30, 2024. Survival from ventricular arrhythmia and heart failure end points and recurrent myocarditis-like episodes was evaluated for both first and recurrent documented myocarditis-like episodes and the impact of immunosuppressive treatment on outcomes assessed. RESULTS: A total of 1014 patients with pathogenic or likely pathogenic DSP variants (62.4% female; mean age, 40.4±17.4 years; 47.1% probands) were included. Among them, 177 individuals (17.5%) experienced ≥1 documented myocarditis-like episode, with 63 of them (35.6%) receiving immunosuppressive treatment. Immunosuppressive treatment of the first documented myocarditis-like episode was associated with reduced risk of ventricular arrhythmia and heart failure outcomes (hazard ratio, 0.18 [95% CI, 0.07–0.45]; P P =0.001, respectively) over a median follow-up of 6.4 years (95% CI, 4.0–10.5). This risk was comparable to that of patients with no history of myocarditis-like episode but did not correlate with a reduction in recurrent episodes after completion of initial immunosuppression (hazard ratio, 0.85 [95% CI, 0.47–1.54]; P =0.599). In contrast, nonsteroidal anti-inflammatory drugs or colchicine treatment did not influence outcomes. CONCLUSIONS: Immunosuppressive treatment of myocarditis-like episodes in DSP patients was associated with improved combined ventricular arrhythmia or heart failure outcomes, supporting future prospective evaluation in DSP cohorts.

Complex diseases such as progressive cardiomyopathies are often insufficiently reflected in small animal and in vitro models. Large animal species such as pigs provide a valuable alternative, but constitutive genetic manipulation (cGM) has not yet been applied to pig in an effective manner, mainly due to biological and logistic limitations. Here, we describe the generation of a novel humanized model for PLN-mediated cardiomyopathy and compare different methods of activating a pathogenic R14del mutation by Cre-mediated recombination. Both, the Cre- treatment of pig primary cells before somatic cell nuclear transfer as well as the micro-injection of Cre-encoding mRNA into zygotes, were similarly efficient in delivering piglets with an activated R14del mutation. Alternatively, administration of Cre-encoding AAV into piglets was sufficient in cGM, albeit at to a varying extent. Together, we here describe a highly effective process to establish complex cGM traits in pig and demonstrate that the lack of Cre-driver lines can be compensated by various interventations during reproduction or post-natally.