Efficient in vivo genome editing prevents hypertrophic cardiomyopathy in mice.

Dominant missense pathogenic variants in cardiac myosin heavy chain cause hypertrophic cardiomyopathy (HCM), a currently incurable disorder that increases risk for stroke, heart failure and sudden cardiac death. In this study, we assessed two different genetic therapies-an adenine base editor (ABE8e) and a potent Cas9 nuclease delivered by AAV9-to prevent disease in mice carrying the heterozygous HCM pathogenic variant myosin R403Q. One dose of dual-AAV9 vectors, each carrying one half of RNA-guided ABE8e, corrected the pathogenic variant in ≥70% of ventricular cardiomyocytes and maintained durable, normal cardiac structure and function. An additional dose provided more editing in the atria but also increased bystander editing. AAV9 delivery of RNA-guided Cas9 nuclease effectively inactivated the pathogenic allele, albeit with dose-dependent toxicities, necessitating a narrow therapeutic window to maintain health. These preclinical studies demonstrate considerable potential for single-dose genetic therapies to correct or silence pathogenic variants and prevent the development of HCM.

Plakophilin-2 truncating variants impair cardiac contractility by disrupting sarcomere stability and organization.

Progressive loss of cardiac systolic function in arrhythmogenic cardiomyopathy (ACM) has recently gained attention as an important clinical consideration in managing the disease. However, the mechanisms leading to reduction in cardiac contractility are poorly defined. Here, we use CRISPR gene editing to generate human induced pluripotent stem cells (iPSCs) that harbor plakophilin-2 truncating variants (PKP2tv), the most prevalent ACM-linked mutations. The PKP2tv iPSC­derived cardiomyocytes are shown to have aberrant action potentials and reduced systolic function in cardiac microtissues, recapitulating both the electrical and mechanical pathologies reported in ACM. By combining cell micropatterning with traction force microscopy and live imaging, we found that PKP2tvs impair cardiac tissue contractility by destabilizing cell-cell junctions and in turn disrupting sarcomere stability and organization. These findings highlight the interplay between cell-cell adhesions and sarcomeres required for stabilizing cardiomyocyte structure and function and suggest fundamental pathogenic mechanisms that may be shared among different types of cardiomyopathies.

Predictors of cardiovascular events after one year of molecular screening for Familial hypercholesterolemia.

BACKGROUND AND AIMS: This study reports the first year follow-up of individuals enrolled in Brazil's genetic cascade screening program for Familial Hypercholesterolemia (FH), Hipercol Brasil. Predictors for the occurrence of cardiovascular (CV) events in individuals screened for FH were studied. METHODS: This is an open prospective cohort of individuals who were included in a cascade genetic screening program for FH. The first prospective follow-up was carried out one year after patients received their genetic test result. Individuals included in this study were index cases (probands) and relatives with identified (M +) or not genetic mutations (M -). Logistic regression analysis was performed to determine predictive variables for the occurrence of CV events censored at one-year of follow-up. RESULTS: A total of 818 subjects were included, 47 first CV events were ascertained, with 14 (29.7%) being fatal. For index cases, the only factor independently associated with increased risk of CV events was the presence of corneal arcus (OR: 9.39; 95% CI: 2.46-35.82). There was an inverse association of CV events with higher HDL-cholesterol levels (OR: 0.95; 95% CI: 0.90-0.99). For M+ relatives, risk factors associated with increased CV events risk were diabetes mellitus (OR: 7.97; 95% CI: 2.07-30.66) and tobacco consumption (OR: 3.70; 95% CI: 1.09-12.50). CONCLUSIONS: A high one-year incidence of CV events was found in this cascade-screening cohort. Predictors of events differed between index cases and relatives and can be useful for the development of preventive efforts in this highly susceptible group of individuals.

Peripheral vascular reactivity and serum BDNF responses to aerobic training are impaired by the BDNF Val66Met polymorphism.

Besides neuronal plasticity, the neurotrophin brain-derived neurotrophic factor (BDNF) is also important in vascular function. The BDNF has been associated with angiogenesis through its specific receptor tropomyosin-related kinase B (TrkB). Additionally, Val66Met polymorphism decreases activity-induced BDNF. Since BDNF and TrkB are expressed in vascular endothelial cells and aerobic exercise training can increase serum BDNF, this study aimed to test the hypotheses: 1) Serum BDNF levels modulate peripheral blood flow; 2) The Val66Met BDNF polymorphism impairs exercise training-induced vasodilation. We genotyped 304 healthy male volunteers (Val66Val, n = 221; Val66Met, n = 83) who underwent intense aerobic exercise training on a running track three times/wk for 4 mo. We evaluated pre- and post-exercise training serum BDNF and proBDNF concentration, heart rate (HR), mean blood pressure (MBP), forearm blood flow (FBF), and forearm vascular resistance (FVR). In the pre-exercise training, BDNF, proBDNF, BDNF/proBDNF ratio, FBF, and FVR were similar between genotypes. After exercise training, functional capacity (V̇o2 peak) increased and HR decreased similarly in both groups. Val66Val, but not Val66Met, increased BDNF (interaction, P = 0.04) and BDNF/proBDNF ratio (interaction, P < 0.001). Interestingly, FBF (interaction, P = 0.04) and the FVR (interaction, P = 0.01) responses during handgrip exercise (HG) improved in Val66Val compared with Val66Met, even with similar responses of HR and MBP. There were association between BDNF/proBDNF ratio and FBF (r = 0.64, P < 0.001) and FVR (r = -0.58, P < 0.001) during HG exercise. These results show that peripheral vascular reactivity and serum BDNF responses to exercise training are impaired by the BDNF Val66Met polymorphism and such responsiveness is associated with serum BDNF concentrations in healthy subjects.

A Variant Detection Pipeline for Inherited Cardiomyopathy-Associated Genes Using Next-Generation Sequencing.

In inherited cardiomyopathies, genetic testing is recognized as an enriching procedure in the diagnostic closure of a cardiac condition. Many genetic mutations have been described as pathogenically related to cardiomyopathies, turning next-generation sequencing into an extremely reliable scenario. Here we describe the validation process of a pipeline constructed with a target panel of 74 cardiomyopathy-related genes sequenced using a next-generation sequencing system. Fifty-two samples from a hypertrophic cardiomyopathy casuistic with previous molecular diagnostics (Sanger-sequenced for MYH7, MYBCP3, and TNNT2; 19 positives and 33 negatives) were processed in parallel with a HapMap reference sample (NA12878) applied for a complete panel assessment. Sequencing coverage values were satisfactory, with a mean of 250× (95% CI, 226.03-273.91) and 95.2% of target bases with a coverage of ≥10×. With a total of 567 variants, variant call sensitivity was tested in five scenarios of coverage and variant allele frequency cutoffs. Maximum achieved sensitivity was 96.7% for single-nucleotide variants and 28.5% for indels, and positive predictive values remained above 0.959 during the whole process. Inter- and intra-assay reproducibility values were 89.5% and 87.3%, respectively. After a careful assessment of analytical performance, we infer that the assay presents potential feasibility for application in diagnostic routines, with minimal time requirements and a simple bioinformatics structure.

Familial hypercholesterolemia in Brazil: cascade screening program, clinical and genetic aspects.

BACKGROUND: There is little knowledge about familial hypercholesterolemia in Brazil. This study presents the first results of genetic cascade screening performed in the city of Sao Paulo. MATERIAL AND METHODS: Two-hundred and forty-eight suspected index cases were initially included. DNA was extracted from peripheral blood and the complete coding sequence of low-density lipoprotein receptor, exon 7 of proprotein convertase subtilisin/kexin type 9 gene and part of exon 26 of apolipoprotein B genes were sequenced. Multiplex Ligation-dependent Probe Amplification was performed on cases where a causal mutation was not identified through sequencing. After the identification of a causal mutation screening in first-degree relatives was pursued. RESULTS: From 248 index cases, a mutation was found in 125 individuals (50.4%). 394 relatives were included in the cascade screening program and a mutation was identified in 59.4%. Seventy different causal mutations in the low-density lipoprotein receptor gene (97.2%) and 2 in the apolipoprotein B gene (2.8%) were found. No mutations were encountered in the proprotein convertase subtilisin/kexin type 9 gene. Mutations in exons 14 and 4 were the most prevalent and, 10 cases of true homozygotes (8 index cases and 2 relatives) and 1 compound heterozygote were identified. The most frequent mutation found was of Lebanese origin, the p.(Cys681*) mutation in exon 14 (8.5%). CONCLUSION: Genetic familial hypercholesterolemia cascade screening is feasible in Brazil and leads to identification of a mutation in approximately half of the index cases with higher rates of success in their relatives.