The frequency of gene variant reclassification and its impact on clinical management in the inherited arrhythmia clinic.

BACKGROUND: Genetic testing in the inherited arrhythmia clinic informs risk stratification, clinical management, and family screening. Periodic review of variant classification is recommended as supporting evidence accrues over time. However, there is limited reporting of real-world data on the frequency and impact of variant reclassification. OBJECTIVE: The purpose of this study was to determine the burden of variant reclassification in our inherited arrhythmia clinic and the impact on clinical management. METHODS: Genetic testing reports for patients referred to our clinic from 2004-2020 were reviewed. Reported variants were reinvestigated using ClinVar, VarSome, and a literature review. Classification was updated using the American College of Medical Genetics and Genomics (ACMG) criteria and tested for association with arrhythmic events and modification of medical management. RESULTS: We identified 517 patients (median age 37 years) who underwent gene panel testing. A variant of uncertain significance (VUS) was reported for 94 patients (18.2%) and more commonly identified when using large gene panels (P <.001). A total of 28 of 87 unique VUSs (32.2%) were reclassified to pathogenic/likely pathogenic (n = 11) or benign/likely benign (n = 17). Of 138 originally reported pathogenic variants, 7 (5.1%) lacked support using ACMG criteria. Variant reclassification was not associated with arrhythmic events; however, it did impact genotype-specific counseling and future therapeutic options. CONCLUSION: In our large real-world patient cohort, we identify a clinically important proportion of both pathogenic variants and VUSs with evidence for reclassification. These findings highlight the need for informed pretest counseling, a regular structured review of variants reported in genetic testing, and the potential benefits to patients for supporting genotype-guided therapy.

Squaramides enhance NLRP3 inflammasome activation by lowering intracellular potassium.

The NLRP3 inflammasome is a component of the inflammatory response to infection and injury, orchestrating the maturation and release of the pro-inflammatory cytokines interleukin-1ß (IL-1ß), IL-18, and triggering pyroptotic cell death. Appropriate levels of NLRP3 activation are needed to avoid excessive tissue damage while ensuring host protection. Here we report a role for symmetrical diarylsquaramides as selective K+ efflux-dependent NLRP3 inflammasome enhancers. Treatment of macrophages with squaramides potentiated IL-1ß secretion and ASC speck formation in response to K+ efflux-dependent NLRP3 inflammasome activators without affecting priming, endosome cargo trafficking, or activation of other inflammasomes. The squaramides lowered intracellular K+ concentration which enabled cells to respond to a below-threshold dose of the inflammasome activator nigericin. Taken together these data further highlight the role of ion flux in inflammasome activation and squaramides as an interesting platform for therapeutic development in conditions where enhanced NLRP3 activity could be beneficial.

Design of Novel Series of Antimalarial PMX Inhibitors with Increased Half-Life via Molecular Property Optimization.

Plasmepsin X (PMX) has been identified as a multistage antimalarial target. PMX is a malarial aspartyl protease essential for merozoite egress from infected red blood cells and invasion of the host erythrocytes. Previously, we reported the identification of PMX inhibitors by structure-based optimization of a cyclic guanidine core. Preclinical assessment of UCB7362, which displayed both in vitro and in vivo antimalarial activity, revealed a suboptimal dose paradigm (once daily dosing of 50 mg for 7 days for treatment of uncomplicated malaria) relative to current standard of care (three-dose regime). We report here the efforts toward extending the half-life (t1/2) by reducing metabolic clearance and increasing volume of distribution (Vss). Our efforts culminated in the identification of a biaryl series, with an expected longer t1/2 in human than UCB7362 while maintaining a similar in vitro off-target hit rate.

Vimentin intermediate filaments provide structural stability to the mammalian Golgi complex.

The Golgi complex comprises a connected ribbon of stacked cisternal membranes localized to the perinuclear region in most vertebrate cells. The position and morphology of this organelle depends upon interactions with microtubules and the actin cytoskeleton. In contrast, we know relatively little about the relationship of the Golgi complex with intermediate filaments (IFs). In this study, we show that the Golgi is in close physical proximity to vimentin IFs in cultured mouse and human cells. We also show that the trans-Golgi network coiled-coil protein GORAB can physically associate with vimentin IFs. Loss of vimentin and/or GORAB had a modest effect upon Golgi structure at the steady state. The Golgi underwent more rapid disassembly upon chemical disruption with brefeldin A or nocodazole, and slower reassembly upon drug washout, in vimentin knockout cells. Moreover, loss of vimentin caused reduced Golgi ribbon integrity when cells were cultured on high-stiffness hydrogels, which was exacerbated by loss of GORAB. These results indicate that vimentin IFs contribute to the structural stability of the Golgi complex and suggest a role for GORAB in this process.

What Factors Are Associated With Stem Breakage in Distal Femoral Endoprosthetic Replacements Undertaken for Primary Bone Tumors?

BACKGROUND: The advantages of distal femoral replacement prostheses for reconstructions after tumors are well known; one such implant, the Global Modular Replacement System (GMRS), has been widely used since 2003. Although implant breakage has been reported, the frequency of this event has varied across different studies. QUESTIONS/PURPOSES: (1) What percentage of patients who underwent distal femur resection and replacement using the GMRS for primary bone tumors at one center experienced stem breakage? (2) At what timepoints did these breakages occur, and what factors were common among the stems that broke? METHODS: We performed a retrospective study of all patients who underwent distal femur resection and replacement using the GMRS for a diagnosis of primary bone sarcoma by the Queensland Bone and Soft-tissue Tumor service from 2003 to 2020 who had a minimum of 2 years of follow-up. Standard follow-up for primary bone sarcoma involves radiographic imaging of the femur at 6 weeks and 3 months postoperatively and yearly thereafter. From a chart review, we identified patients with femoral stem breakage. Patient and implant details were recorded and analyzed. A total of 116 patients had undergone a distal femoral replacement with the GMRS prosthesis for primary bone sarcoma; however, 6.9% (eight of 116 patients) died before completing the 2-year follow-up period and were excluded. Of the remaining 108 patients, 15% (16 patients) had died at the time of this review; however, given that they completed the 2-year follow-up period and did not experience stem breakage, they were included. Furthermore, 15% (16 patients) were considered lost to follow-up and excluded because they have not been seen in the past 5 years but were not known to have died or experienced stem breakage. This left 92 patients for analysis. RESULTS: Stem breakages were identified in 5.4% (five of 92) of patients. All stem breakages occurred in stem diameters 11 mm or less with a porous body construct; the percentage of patients with breakage in this group was 16% (five of 31). All patients with stem fracture demonstrated minimal ongrowth to the porous coated body. The median time to stem fracture was 10 years (range 2 to 12 years); however, two of the five stems broke within 3 years. CONCLUSION: We recommend the use of a larger-diameter GMRS cemented stem (> 11 mm), and either the line-to-line cementing method or an uncemented stem from an alternative company should be considered in order to achieve this larger stem in smaller canals. If a stem less than 12 mm in diameter must be used or there is evidence of minimal ongrowth, then close follow-up and prompt investigation of new symptoms should occur. LEVEL OF EVIDENCE: Level IV, therapeutic study.

PCYT2-regulated lipid biosynthesis is critical to muscle health and ageing.

Muscle degeneration is the most prevalent cause for frailty and dependency in inherited diseases and ageing. Elucidation of pathophysiological mechanisms, as well as effective treatments for muscle diseases, represents an important goal in improving human health. Here, we show that the lipid synthesis enzyme phosphatidylethanolamine cytidyltransferase (PCYT2/ECT) is critical to muscle health. Human deficiency in PCYT2 causes a severe disease with failure to thrive and progressive weakness. pcyt2-mutant zebrafish and muscle-specific Pcyt2-knockout mice recapitulate the participant phenotypes, with failure to thrive, progressive muscle weakness and accelerated ageing. Mechanistically, muscle Pcyt2 deficiency affects cellular bioenergetics and membrane lipid bilayer structure and stability. PCYT2 activity declines in ageing muscles of mice and humans, and adeno-associated virus-based delivery of PCYT2 ameliorates muscle weakness in Pcyt2-knockout and old mice, offering a therapy for individuals with a rare disease and muscle ageing. Thus, PCYT2 plays a fundamental and conserved role in vertebrate muscle health, linking PCYT2 and PCYT2-synthesized lipids to severe muscle dystrophy and ageing.

Disruptions in endocytic traffic contribute to the activation of the NLRP3 inflammasome.

Inflammation driven by the NLRP3 inflammasome is coordinated through multiple signaling pathways and is regulated by subcellular organelles. Here, we tested the hypothesis that NLRP3 senses disrupted endosome trafficking to trigger inflammasome formation and inflammatory cytokine secretion. NLRP3-activating stimuli disrupted endosome trafficking and triggered localization of NLRP3 to vesicles positive for endolysosomal markers and for the inositol lipid PI4P. Chemical disruption of endosome trafficking sensitized macrophages to the NLRP3 activator imiquimod, driving enhanced inflammasome activation and cytokine secretion. Together, these data suggest that NLRP3 can sense disruptions in the trafficking of endosomal cargoes, which may explain in part the spatial activation of the NLRP3 inflammasome. These data highlight mechanisms that could be exploited in the therapeutic targeting of NLRP3.

Immunofluorescence Microscopy of the Mammalian Golgi Apparatus.

Immunofluorescence is a technique that uses antibodies and fluorophores to label structures inside cells. The cells are normally fixed and permeabilized, and then structures are labelled using primary antibodies directly conjugated to fluorophores, or, more commonly, first with an antibody against an antigen of interest followed by a secondary antibody conjugated to a fluorophore that binds to the primary antibody. Fluorescence can be visualized using widefield, confocal, or super-resolution microscopy. Here we focus on labelling of the Golgi apparatus and show that different fixation and permeabilization conditions can significantly affect labelling of Golgi proteins and describe how to optimize fluorescent detection of Golgi proteins.

Discovery and Characterization of Potent, Efficacious, Orally Available Antimalarial Plasmepsin X Inhibitors and Preclinical Safety Assessment of UCB7362.

Plasmepsin X (PMX) is an essential aspartyl protease controlling malaria parasite egress and invasion of erythrocytes, development of functional liver merozoites (prophylactic activity), and blocking transmission to mosquitoes, making it a potential multistage drug target. We report the optimization of an aspartyl protease binding scaffold and the discovery of potent, orally active PMX inhibitors with in vivo antimalarial efficacy. Incorporation of safety evaluation early in the characterization of PMX inhibitors precluded compounds with a long human half-life (t1/2) to be developed. Optimization focused on improving the off-target safety profile led to the identification of UCB7362 that had an improved in vitro and in vivo safety profile but a shorter predicted human t1/2. UCB7362 is estimated to achieve 9 log 10 unit reduction in asexual blood-stage parasites with once-daily dosing of 50 mg for 7 days. This work demonstrates the potential to deliver PMX inhibitors with in vivo efficacy to treat malaria.

ECG-I phenotyping of persistent AF based on driver burden and distribution to predict response to pulmonary vein isolation (PHENOTYPE-AF).

BACKGROUND: This prospective trial sought to phenotype persistent atrial fibrillation (AF) based on AF mechanisms using electrocardiographic imaging (ECGI) mapping to determine whether this would predict long-term freedom from arrhythmia after pulmonary vein isolation (PVI). METHODS: Patients with persistent AF of <2 years duration underwent cryoballoon PVI. ECGI mapping was performed before PVI to determine potential drivers (PDs) defined as rotational activations completing ≥1.5 revolutions or focal activations. The coprimary endpoint was the association between (1) PD burden (defined as the number of PD occurrences) and (2) PD distribution (defined as the number of segments on an 18-segment model of the atria harboring PDs) with freedom from arrhythmia at 1-year follow up. RESULTS: Of 100 patients, 97 completed follow up and 52 (53.6%) remained in sinus rhythm off antiarrhythmic drugs. Neither PD burden nor PD distribution predicted freedom from arrhythmia (hazard ratio [HR]: 1.01, 95% confidence interval [CI]: 0.99-1.03, p = .164; and HR: 1.04, 95% CI: 0.91-1.17, p = .591, respectively). Otherwise, the burden of rotational PDs, rotational stability, and the burden of PDs occurring at the pulmonary veins and posterior wall all failed to predict arrhythmia recurrence (all p > .10). CONCLUSIONS: AF mechanisms as determined using ECGI mapping do not predict outcomes after PVI for persistent AF. Further studies using different methodologies to characterize AF mechanisms are warranted (NCT03394404).

Assessment of endocytic traffic and Ocrl function in the developing zebrafish neuroepithelium.

Endocytosis allows cells to internalise a wide range of molecules from their environment and to maintain their plasma membrane composition. It is vital during development and for maintenance of tissue homeostasis. The ability to visualise endocytosis in vivo requires suitable assays to monitor the process. Here, we describe imaging-based assays to visualise endocytosis in the neuroepithelium of living zebrafish embryos. Injection of fluorescent tracers into the brain ventricles followed by live imaging was used to study fluid-phase or receptor-mediated endocytosis, for which we used receptor-associated protein (RAP, encoded by Lrpap1) as a ligand for low-density lipoprotein receptor-related protein (LRP) receptors. Using dual-colour imaging combined with expression of endocytic markers, it is possible to track the progression of endocytosed tracers and to monitor trafficking dynamics. Using these assays, we reveal a role for the Lowe syndrome protein Ocrl in endocytic trafficking within the neuroepithelium. We also found that the RAP-binding receptor Lrp2 (encoded by lrp2a) appears to contribute only partially to neuroepithelial RAP endocytosis. Altogether, our results provide a basis to track endocytosis within the neuroepithelium in vivo and support a role for Ocrl in this process. This article has an associated First Person interview with the first author of the paper.

The inositol 5-phosphatase INPP5B regulates B cell receptor clustering and signaling.

Upon antigen binding, the B cell receptor (BCR) undergoes clustering to form a signalosome that propagates downstream signaling required for normal B cell development and physiology. BCR clustering is dependent on remodeling of the cortical actin network, but the mechanisms that regulate actin remodeling in this context remain poorly defined. In this study, we identify the inositol 5-phosphatase INPP5B as a key regulator of actin remodeling, BCR clustering, and downstream signaling in antigen-stimulated B cells. INPP5B acts via dephosphorylation of the inositol lipid PI(4,5)P2 that in turn is necessary for actin disassembly, BCR mobilization, and cell spreading on immobilized surface antigen. These effects can be explained by increased actin severing by cofilin and loss of actin linking to the plasma membrane by ezrin, both of which are sensitive to INPP5B-dependent PI(4,5)P2 hydrolysis. INPP5B is therefore a new player in BCR signaling and may represent an attractive target for treatment of B cell malignancies caused by aberrant BCR signaling.

Transseptal puncture for left atrial ablation: Risk factors for cardiac tamponade and a proposed causative classification system.

AIMS: Cardiac tamponade is a high morbidity complication of transseptal puncture (TSP). We examined the associations of TSP-related cardiac tamponade (TRCT) for all patients undergoing left atrial ablation at our center from 2016 to 2020. METHODS AND RESULTS: Patient and procedural variables were extracted retrospectively. Cases of cardiac tamponade were scrutinized to adjudicate TSP culpability. Adjusted multivariate analysis examined predictors of TRCT. A total of 3239 consecutive TSPs were performed; cardiac tamponade occurred in 51 patients (incidence: 1.6%) and was adjudicated as TSP-related in 35 (incidence: 1.1%; 68.6% of all tamponades). Patients of above-median age [odds ratio (OR): 2.4 (1.19-4.2), p = .006] and those undergoing re-do procedures [OR: 1.95 (1.29-3.43, p = .042] were at higher risk of TRCT. Of the operator-dependent variables, choice of transseptal needle (Endrys vs. Brockenbrough, p > .1) or puncture sheath (Swartz vs. Mullins vs. Agilis vs. Vizigo vs. Cryosheath, all p > .1) did not predict TRCT. Adjusting for operator, equipment and demographics, failure to cross the septum first pass increased TRCT risk [OR: 4.42 (2.45-8.2), p = .001], whilst top quartile operator experience [OR: 0.4 (0.17-0.85), p = .002], transoesophageal echocardiogram [TOE prevalence: 26%, OR: 0.51 (0.11-0.94), p = .023], and use of the SafeSept transseptal guidewire [OR: 0.22 (0.08-0.62), p = .001] reduced TRCT risk. An increase in transseptal guidewire use over time (2016: 15.6%, 2020: 60.2%) correlated with an annual reduction in TRCT (R2 = 0.72, p < .001) and was associated with a relative risk reduction of 70%. CONCLUSIONS: During left atrial ablation, the risk of TRCT was reduced by operator experience, TOE-guidance, and use of a transseptal guidewire, and was increased by patient age, re-do procedures, and failure to cross the septum first pass.

A novel nanoluciferase transgenic reporter measures proteinuria in zebrafish.

The zebrafish is an important animal system for modeling human diseases. This includes kidney dysfunction as the embryonic kidney (pronephros) shares considerable molecular and morphological homology with the human nephron. A key clinical indicator of kidney disease is proteinuria, but a high-throughput readout of proteinuria in the zebrafish is currently lacking. To remedy this, we used the Tol2 transposon system to generate a transgenic zebrafish line that uses the fabp10a liver-specific promoter to over-express a nanoluciferase molecule fused with the D3 domain of Receptor-Associated Protein (a type of molecular chaperone) which we term NL-D3. Using a luminometer, we quantified proteinuria in NL-D3 zebrafish larvae by measuring the intensity of luminescence in the embryo medium. In the healthy state, NL-D3 is not excreted, but when embryos were treated with chemicals that affected either proximal tubular reabsorption (cisplatin, gentamicin) or glomerular filtration (angiotensin II, Hanks Balanced Salt Solution, Bovine Serum Albumin), NL-D3 is detected in fish medium. Similarly, depletion of several gene products associated with kidney disease (nphs1, nphs2, lrp2a, ocrl, col4a3, and col4a4) also induced NL-D3 proteinuria. Treating col4a4 depleted zebrafish larvae (a model of Alport syndrome) with captopril reduced proteinuria in this system. Thus, our findings validate the use of the NL-D3 transgenic zebrafish as a robust and quantifiable proteinuria reporter. Hence, given the feasibility of high-throughput assays in zebrafish, this novel reporter will permit screening for drugs that ameliorate proteinuria, thereby prioritizing candidates for further translational studies.

Pacsin2 is required for endocytosis in the zebrafish pronephric tubule.

Endocytosis mediates the cellular uptake of numerous molecules from the extracellular space and is a fundamentally important process. In the renal proximal tubule, the scavenger receptor megalin and its co-receptor cubilin mediate endocytosis of low molecular weight proteins from the renal filtrate. However, the extent to which megalin endocytosis relies on different components of the trafficking machinery remains relatively poorly defined in vivo. In this study, we identify a functional requirement for the F-BAR protein pacsin2 in endocytosis in the renal proximal tubule of zebrafish larvae. Pacsin2 is expressed throughout development and in all zebrafish tissues, similar to the mammalian orthologue. Within renal tubular epithelial cells, pacsin2 is enriched at the apical pole where it is localised to endocytic structures. Loss of pacsin2 results in reduced endocytosis within the proximal tubule, which is accompanied by a reduction in the abundance of megalin and endocytic organelles. Our results indicate that pacsin2 is required for efficient endocytosis in the proximal tubule, where it likely cooperates with other trafficking machinery to maintain endocytic uptake and recycling of megalin.

Assessing Noninvasive Delineation of Low-Voltage Zones Using ECG Imaging in Patients With Structural Heart Disease.

OBJECTIVES: This study sought to assess the association between electrocardiographic imaging (ECGI) parameters and voltage from simultaneous electroanatomic mapping (EAM). BACKGROUND: ECGI offers noninvasive assessment of electrophysiologic features relevant for mapping ventricular arrhythmia and its substrate, but the accuracy of ECGI in the delineation of scar is unclear. METHODS: Sixteen patients with structural heart disease underwent simultaneous ECGI (CardioInsight, Medtronic) and contact EAM (CARTO, Biosense-Webster) during ventricular tachycardia catheter ablation, with 7 mapped epicardially. ECGI and EAM geometries were coregistered using anatomic landmarks. ECGI points were paired to the closest site on the EAM within 10 mm. The association between EAM voltage and ECGI features from reconstructed epicardial unipolar electrograms was assessed by mixed-effects regression models. The classification of low-voltage regions was performed using receiver-operating characteristic analysis. RESULTS: A total of 9,541 ECGI points (median: 596; interquartile range: 377-737 across patients) were paired to an EAM site. Epicardial EAM voltage was associated with ECGI features of signal fractionation and local repolarization dispersion (N = 7; P < 0.05), but they poorly classified sites with bipolar voltage of <1.5 mV or <0.5 mV thresholds (median area under the curve across patients: 0.50-0.62). No association was found between bipolar EAM voltage and low-amplitude reconstructed epicardial unipolar electrograms or ECGI-derived bipolar electrograms. Similar results were found in the combined cohort (n = 16), including endocardial EAM voltage compared to epicardial ECGI features (n = 9). CONCLUSIONS: Despite a statistically significant association between ECGI features and EAM voltage, the accuracy of the delineation of low-voltage zones was modest. This may limit ECGI use for pr-procedural substrate analysis in ventricular tachycardia ablation, but it could provide value in risk assessment for ventricular arrhythmias.

Regenerative matching axial vascularisation of absorbable 3D-printed scaffold for large bone defects: A first in human series.

BACKGROUND: We describe the first clinical series of a novel bone replacement technique based on regenerative matching axial vascularisation (RMAV). This was used in four cases: a tibial defect after treatment of osteomyelitis; a calvarial defect after trauma and failed titanium cranioplasty; a paediatric tibial defect after neoadjuvant chemotherapy and resection of Ewing sarcoma; and a paediatric mandibular deficiency resulting from congenital hemifacial microsomia. METHOD: All patients underwent reconstruction with three-dimensional (3D)-printed medical-grade polycaprolactone and tricalcium phosphate (mPCL-TCP) scaffolds wrapped in vascularised free corticoperiosteal flaps. OUTCOME: Functional volumes of load-sharing regenerate bone have formed in all cases after a moderate duration of follow-up. At 36 cm, case 1 remains the longest segment of load bearing bone ever successfully reconstructed. This technique offers an alternative to existing methods of large volume bone defect reconstruction that may be safe, reliable, and give predictable outcomes in challenging situations. It achieves this by using a bioresorbable scaffold to support and direct the growth of regenerate bone, driven by RMAV. CONCLUSION: This technique may facilitate the reconstruction of bone defects previously thought unreconstructable, reduce the risk of long-term implant-related complications and achieve these outcomes in a hostile environment. These potential benefits must now be formally tested in prospective clinical trials.