Risultati per: Percorsi personalizzati contro il cancro legati al gene BRCA

Circulation, Volume 150, Issue Suppl_1, Page A4145948-A4145948, November 12, 2024. Background:Mutations in desmoplakin gene (DSP) are associated with arrhythmogenic cardiomyopathy (ARVC). Myosin-binding protein C gene (MYBPC3) is a sarcomere gene and is associated with hypertrophic cardiomyopathy (HCM). The expression of both of these genes has only been reported twice in the literature. It remains unclear whether the expression of both mutation carries a more severe phenotype than carrying on of the mutations only.Case Description:A 21-year-old female with history of sustained ventricular fibrillation (VT) at age 18, VT arrest at age 20,dilated cardiomyopathy with LVEF 30-35% and subcutaneous ICD, who was admitted after multiple ICD defibrillations. Device interrogation demonstrated 23 monomorphic VT episodes treated with 44 shocks by the ICD. Coronary angiogram demonstrated non-obstructive coronary arteries. Patient underwent VT ablation, complicated by incessant VT from LV summit which was eliminated during procedure. Cardiac MRI demonstrated RVEF 33%, regional dyskinesis in the sub-tricuspid area and RV outflow tract. cMRI also demonstrated subepicardial late gadolinium enhancement at the level of the mid antero/infero lateral walls with extension into the apicolateral wall and mid inferoseptal territory. These findings heightened suspicion for left ventricle fatty infiltration. However, endomyocardial biopsy negative for inflammation or granuloma, no fibrosis, negative iron and amyloid staining. Left-dominant arrhythmogenic cardiomyopathy was diagnosed based on cMRI findings, LV systolic dysfunction, and arrhythmia of LV origin. Subcutaneous ICD was explanted and replaced with a dual chamber ICD. Patient was discharged on Mexiletine and GDMT for HFrEF. Genetics testing later revealed rare co-occurance of two genetic mutations including MYBPC3 and DSP.Discussion:The presentation of both DSP and MYBPC3 mutations in patients diagnosed with ARVC has only been reported twice in the literature and a is an extremely rare phenomenon. There is currently no sufficient understanding of the genotype-phenotype correlation of DSP and MYBPC3 mutations in the clinical expression of their associated inherited cardiac diseases. Therefore, more research need to investigate this phenomena and to study whether the co-presence of both of these two mutations is associated with a more severe phenotypic expression of HCM and ARVC.

Circulation, Volume 150, Issue Suppl_1, Page A4132120-A4132120, November 12, 2024. Epidemiological studies indicate that individuals with Down syndrome (DS) have an increased risk of leukemia and neuronal diseases but a significantly reduced incidence of most solid tumors and advanced vascular dysfunction. This suggests that one or a combination of trisomy genes on human chromosome 21 (HSA21) or murine chromosome 16 (Mmu16) may be responsible for protecting against vasculopathy. Our previous research has shown that theDown syndrome critical region (Dscr)-1gene, located on HSA21 orMmu16, encodes a feedback modulator of calcineurin-NFAT signaling in endothelial cells (ECs). Null mutation or overexpression of Dscr-1 has been found to increase or prevent septic mortality, angiogenesis, liver steatosis, and susceptibility of tumor metastasizing to the lung, respectively, demonstrating its significant role in these processes.In a unique approach, we crossed DS model mice withApoE-null mice and fed them a high-fat diet (HFD) for 12 weeks to survey the effects of DS-related genes on atherosclerosis. The results were striking, with HFD-mediated increased LDL and triglyceride levels inApoE-null mice significantly reduced in the combined DS plusApoE-null mice. This innovative method allowed us to observe a remarkable (~35%) reduction in ApoE-null mediated atheroma formation in the DS model. This finding was also replicated in EC-specific DSCR-1 transgenic (DSCR-1ECTg) mice.Furthermore, to test the effect of HSA21, we have collected DS patient-derived trisomy and disomy iPS cells and executed them for the chromatin conformation capture analysis and thein vitroECs differentiation experiments. DS-derived trisomy iPS cells already changed the whole chromatin structure, typically within the X chromosome. Moreover, the trisomy iPS-derived ECs revealed less proliferation compared to the disomy. RNA-seqs indicated trisomy iPS derived ECs dramatically (21-fold) increasedDSCR-1, but some HSA21 genes, Alzheimer’s relatedAppandBace2, e.g., were inversely reduced the expression.Our comprehensive studies not only shed new light on the mechanisms of atherosclerosis in vascular pathology but also offer potential therapeutic strategies. The discovery of theDscr-1gene on the DS chromosome, which could be a powerful tool in combating lifestyle-related vascular diseases, underscores the significance of our research.

Circulation, Volume 150, Issue Suppl_1, Page A4133040-A4133040, November 12, 2024. Introduction:Heart failure (HF) is associated with high mortality and morbidity, and there are limited therapeutic options available to improve cardiac muscle function. Recent studies suggest that the impairment of cardiomyocyte T-tubule microdomains is a key factor in HF pathophysiology. AAV9-cBIN1 gene therapy effectively addresses this impairment. However, the kinetics and biodistribution of AAV9-cBIN1 have not been explored, limiting the knowledge of the transcription efficiency and organ specificity of cBIN1 gene therapy when delivered systemically.Aim:This study aims to elucidate the kinetics and biodistribution of AAV9-cBIN1 in a murine model.Method:Adult male C57BL/6J mice (n=20) were administered a single retro-orbital injection of AAV9-CMV-cBIN1-V5 at a dose (2×10^12 vg/kg) known to effectively transduce the majority of cardiomyocytes. Mice were sacrificed at pre-determined time points: prior to injection (Day 0, n=2) and post-injection at Days 3, 7, 14, 28, 56, and 84 (n=3/group). Tissues were harvested from the heart, lung, liver, kidney, skeletal muscle, and spleen. Genomic DNA was extracted and analyzed using qPCR to determine vector genome (vg) copies. For transcriptional analysis of the AAV9-transduced exogenous gene, mRNA was extracted and converted to cDNA for qRT-PCR quantification ofcBin1-V5, normalized to the housekeeping geneHprt1.Results:All mice survived to their designated sacrifice times. Kinetic analysis demonstrated a time-dependent expression of the exogenouscBin1-V5gene, with peak expression observed at 4 weeks in both the heart and liver, with expression declining in the liver after this peak (Figure 1A). Genomic DNA analysis showed a gradual increase and plateau of viral genome distribution in the heart between 2-4 weeks, while in the liver, distribution peaked sharply between Days 3-7 and then decreased. When correcting the gene transcription ofcBin1-V5(ΔCq ofV5/Hprt1) for vector DNA vg copies in the tissue, the transcription efficiency in the heart was higher than in the liver (Figure 1B). Outside of the liver and heart,cBin1-V5mRNA expression was undetectable in all other tissues by Day 56.Conclusion:In adult male mice, a single systemic administration of AAV9-cBIN1 results in robust transduction and higher transcription efficiency in the heart compared to the liver. AAV9-cBIN1 holds promise for targeted gene therapy in cardiac tissues, with minimal off-target expression in other organs.

Circulation, Volume 150, Issue Suppl_1, Page A4147026-A4147026, November 12, 2024. Introduction:Lipoprotein a (Lp(a)) levels are an often unmeasured predictor of future risk of cardiovascular disease. Unlike LDL, HDL, and triglycerides, Lp(a) levels are believed to be genetically determined; they are not altered by lipid-lowering medications or dietary changes, and are consistent over a lifetime, making them a potential early-life predictor of adult morbidity. However, genetic predictors of Lp(a) are challenging to design. Repeats of the kringle IV subtype 2 domain (KIV-2) contribute significantly but are extremely challenging to quantify via next-generation sequencing; this variability is particularly impactful in non-European ancestries.Methods:We analyzed ~110K individuals with paired clinical and exome sequencing data from six medical centers. We developed a coverage-based method to determine KIV-2 repeat counts. We validated that these counts associated with clinical Lp(a) measurements derived from the course of care (n = 2039 individuals). We calculated an expected Lp(a) in units of nmol/L using a genetic risk score (GRS) based on 43 variants in the Lp(a) gene. We identified instances of CAD via ICD9CM, ICD10CM and SNOMED terms present in the medical record.Results:Adding the number of KIV-2 repeats to the GRS substantially improved prediction of clinical Lp(a) measurements, for example improving the r-squared for non-Europeans five-fold (from 0.05 to 0.22). Individuals with fewer than 15 estimated KIV-2 repeats and a high GRS-predicted Lp(a) (high risk group) have a mean measured Lp(a) of that is 10-fold that of those in the lowest category of GRS-predicted Lp(a) (low risk group). In the full cohort, most of whom had not been measured for Lp(a), we find that relative to the low risk group, the high risk individuals are 1.6x more likely to have CAD.Conclusion:Lp(a) is a potentially useful biomarker with consistency over a lifetime and implications for future cardiovascular morbidity. We developed a method for estimating the number of KIV-2 repeats, a challenging component of genetic prediction of Lp(a). This estimate can be used in combination with a GRS to predict Lp(a), especially in individuals with non-European ancestry where GRS-based estimates fall short. The final prediction is associated with increased risk of CAD.

Circulation, Volume 150, Issue Suppl_1, Page A4145103-A4145103, November 12, 2024. Dilated cardiomyopathy (DCM) is a clinical condition with tremendous diversity of etiology. In familial forms of DCM, there has been a compelling evidence of immense genetic heterogeneity that implicate various mechanisms. Recently, biallelic loss-of-function (LOF) variants in the nebulin-related-anchoring protein (NRAP) gene have been reported in an ultra-rare form of DCM inherited recessively.In this study, we sought to describe the clinical and genetic spectrum associated withNRAPvariants in our highly consanguineous population. Genetic analysis with whole exome sequencing, conducted on consecutively recruited cases of DCM, identified 13 consanguineous families with 6 different LOF variants inNRAP. Segregation analysis detected 25 homozygous and 25 heterozygous individuals. Interestingly, only 18 of the 25 homozygous cases were symptomatic with a remarkable variability of age of onset (9 months to 47 years, median 5 years), of whom 9 cases died with a median age of death of 3 years (range: 9 months to 13 years). None of the heterozygous individuals was clinically symptomatic. Of note, 3 homozygous cases underwent heart transplantation at the ages of 8, 11, and 14 years who are alive with a follow up of 6 years, 3 years, and 3 months respectively.Our work illustrates a notable clinical variability of DCM associated withNRAPLOF variants. This observation suggests an underling complex mechanism ofNRAP-related DCM that deviates from simple mendelian inheritance and plausibly implicates modifiers. The reduced penetrance and variable expression underscore the need of exercising precaution when counseling individuals and families found to haveNRAPvariants.

Circulation, Volume 150, Issue Suppl_1, Page A4143995-A4143995, November 12, 2024. Background:Genetic cardiomyopathies (CM) are increasingly diagnosed among patients with ventricular arrhythmias (VA). There is increasing recognition of the regional nature of potential arrhythmic substrate in genetic CM, including involvement of the basal left ventricular (LV) septum and LV summit. Whether anatomically constrained ventricular arrhythmias predict genetic CM is not known.Research question:Are LV summit arrhythmias associated with pathogenic/likely pathogenic (P/LP) gene variants?Aims:To examine whether LV summit VA is associated with P/LP gene variants in patients referred for ventricular tachycardia (VT) or premature ventricular complex (PVC) ablation.Methods:The cohort included patients with arrhythmia and cardiomyopathy panel genetic testing (Invitae) between 2018 and 2024 who also underwent VT/PVC ablation at the University of Washington. VA location (summit vs. non-summit) was defined using electroanatomic voltage and activation mapping. The prevalence of P/LP gene variants in those with and without LV summit VA were compared using Fisher’s exact testing. Logistic regression was used to examine the association of LV summit VA with P/LP gene variants with multivariable adjustment for anti-rhythm drugs and standard risk factors including age, sex, and LV ejection fraction (LVEF).Results:The study included 57 patients (mean age 55±16 years, 82% male, LVEF 44±13%, n=22/35 PVC/VT ablation). LV summit VA was identified in 10 patients and P/LP gene variants were found in 22 patients. The prevalence of P/LP genetic variants was significantly higher in LV summit vs. non-summit patients (70% vs. 32%, p=0.04). Those with LV summit arrhythmias had a 7-fold higher odds of P/LP genetic variants compared to non-LV summit arrhythmias (adj OR [95% CI]: 6.6 [1.1-39.6]. P/LP variants in LV summit VA individuals were either TTN (n=3 [2 truncating variant, 1 splice site]) or LMNA (n=4 [3 missense, 1 deletion]).Conclusion:LV summit VA are associated with P/LP genetic variants with known tropism for basal septal pathology. This preliminary work supports efforts to expand the paradigm that anatomically-specific VA may represent the electrical manifestation of genetic CM.

Circulation, Volume 150, Issue Suppl_1, Page A4117767-A4117767, November 12, 2024. Introduction:Hypertrophic Cardiomyopathy (HCM) is an inherited heart disease and the pathogenesis of HCM involves genetic mutations, hemodynamic stress, and metabolic factors, with myocardial fibrosis playing a crucial role in severe clinical events. IL-33/ST2 signaling pathway known for its roles in immune response and tissue repair, participates in cardiac protection and anti-cardiac fibrosis in heart failure. The role of ST2 in HCM remains unclear, and IL-33/ST2 pathway and broader inflammatory responses may be critical in HCM.Hypothesis:ST2 gene downregulation and dysregulation of inflammatory pathways represent a novel mechanism in HCM.Methods:We re-analyzed the raw whole transcriptome sequencing data using a comprehensive bioinformatics pipeline from 9 GEO datasets. After removing low-quality samples, myocardial tissue samples from 109 HCM patients and 210 non-HCM controls were included. HTSeq-count was used to obtain the read count of each gene. Differential expression analyses were conducted using DESeq2. Spearman correlation analysis was performed for ST2 with all other protein-coding genes. Gene set enrichment analysis (GSEA) was performed to explore the biological significance of ST2 related genes. CIBERSORTx was used to estimate the immune cell composition of heart tissues. Additionally, network analyses and statistical correlations were conducted.Results:Our analysis identified significant downregulation of the ST2 gene in HCM patients compared to controls (log2FC = -5.0, p = 2.7E-147). In total, 5,180 upregulated and 741 downregulated genes were detected. Additionally, 208 genes were significantly correlated with the expression of ST2. GSEA based on the correlation coefficients revealed a significant increase in immune response, inflammation, and IFN-γ pathway activity, along with a decrease in the expression of genes related to sarcomeric function, cardiac morphogenesis, and metabolism in HCM. Notably, the significant inverse correlation between ST2 expression and regulatory T cells (r = -0.34) and a positive correlation with neutrophils (r = 0.39) suggest that ST2 may modulate immune cell populations in HCM. Pathway analysis indicated ST2’s central role in networks involving inflammatory and fibrotic responses.Conclusion:The interplay between ST2 and inflammatory pathways may drive myocardial remodeling and fibrosis in HCM. This study paves a new way for investigating pathogenic mechanisms and therapeutic strategies for HCM.

Circulation, Volume 150, Issue Suppl_1, Page A4142380-A4142380, November 12, 2024. Background:Heart failure with preserved ejection fraction (HFpEF) continues to be poorly understood at the molecular level. While previous studies have identified gene expression signatures unique to HFpEF, genes associated with clinical decompensation have not been determined. Here, we performed exploratory analysis of myocardial RNA-seq data to identify genes associated with event-free survival in HFpEF.Methods:We analyzed previously published RNA sequencing data of right ventricular septal endomyocardial biopsies from HFpEF patients (n=41) with paired clinical, echocardiographic, and outcome data (including mortality and heart failure hospitalizations). We constructed random survival forests with forward stepwise regularization (the “variable hunting” method) to determine genes associated with time to first event using a combined end-point of heart failure hospitalization or all-cause death. Selection of candidate forest variables was tested with both random and weighted sampling methods.Results:We identified 33 genes that are predictive of survival in HFpEF (Figure 1A). This set includes genes previously implicated in heart failure, includingADAMTSL2,ADRB1,BMP6, andMETRNL. Patients with HFpEF were categorized into 3 groups (Gene Group) based on their expression of this survival gene signature. Gene Group 1 had the worst survival compared to Gene Groups 2 and 3 (log rank p values 7.85e-5 for Gene Group 1 vs 2, 1.16e-5 for Gene Group 1 vs 3, adjusted for age, sex, and renal function, Figure 1B). Survival was not significantly different between Gene Groups 2 and 3 (adjusted log-rank p value 0.17). Survival forests constructed using these genes outperform those constructed from clinical and hemodynamic parameters alone (out-of-bag C-index 0.894 vs 0.545). Moreover, in survival forests constructed from both gene data and hemodynamic measurements, individual survival genes consistently showed higher variable importance than clinical/hemodynamic parameters (Figure 1C).Conclusions:Random survival forests identify myocardial gene signatures that may better model HFpEF prognosis than clinical measurements. Further studies are warranted to validate findings in independent cohorts.

Circulation, Ahead of Print. Cardiovascular disease remains the foremost cause of morbidity and mortality globally, affecting millions of individuals. Recent discoveries illuminate the substantial role of genetics in cardiovascular disease pathogenesis, encompassing both monogenic and polygenic mechanisms and identifying tangible targets for gene therapies. Innovative strategies have emerged to rectify pathogenic variants that cause monogenic disorders such as hypertrophic, dilated, and arrhythmogenic cardiomyopathies and hypercholesterolemia. These include delivery of exogenous genes to supplement insufficient protein levels caused by pathogenic variants or genome editing to correct, delete, or modify mutant sequences to restore protein function. However, effective delivery of gene therapy to specified cells presents formidable challenges. Viral vectors, notably adeno-associated viruses and nonviral vectors such as lipid and engineered nanoparticles, offer distinct advantages and limitations. Additional risks and obstacles remain, including treatment durability, tissue-specific targeting, vector-associated adverse events, and off-target effects. Addressing these challenges is an ongoing imperative; several clinical gene therapy trials are underway, and many more first-in-human studies are anticipated. This science advisory reviews core concepts of gene therapy, key obstacles, patient risks, and ongoing research endeavors to enable clinicians to understand the complex landscape of this emerging therapy and its remarkable therapeutic potential to benefit cardiovascular disease.

Circulation, Volume 150, Issue Suppl_1, Page A4143866-A4143866, November 12, 2024. Background:S100A6 is a small calcium-binding protein that is important in managing calcium storage and myocyte contractility. This protein has a low basal cardiac expression and is upregulated following Myocardial Infarction (MI). Previous experiments have demonstrated that S100A6 gene transfer improves left ventricular function after acute ischemia/reperfusion injury.Hypothesis: Delivery of S100A6 by Ultrasound-Targeted Microbubble Destruction (UTMD) after established MI model results in improved left ventricular function and prevention of adverse ventricular remodeling.Aim:Assess the impact of UTMD delivery of S100A6 on cardiac function following experimental MI.Methods:MI was induced through permanent left anterior descending (LAD) coronary artery ligation in 8-week-Spragues Dawley (SD) male rats on day 0. On day 28, by using UTMD methods, we delivered microbubbles (1×109) coupled with either 200 μg of human S100A6 mini-circle DNA or empty minicircles to the left ventricle (LV), while control animals received no therapy. The three groups were monitored weekly for four weeks post-gene delivery using serial echocardiography to follow LV function, followed by tissue collection from various regions of the myocardium.Results:At day 28 post-MI, all groups showed reduced LV ejection fraction (LVEF) and fractional shortening (FS), with LVEF and FS values of 39.47±1.49% and 19.75±2.38%, respectively. Meanwhile, healthy animals showed LVEF and FS values of (63.75±2.38% and 36.20±1.29% (p

Circulation, Volume 150, Issue Suppl_1, Page A4135301-A4135301, November 12, 2024. Background:We have previously demonstrated that diabetes mellitus (DM) causes cardiac inflammation leading to heart failure with preserved ejection fraction (HFpEF). Arachidonate 5-lipoxygenase (5-LOX), encoded by the ALOX5 gene, is an enzyme that is highly expressed in leukocytes. 5-LOX synthesizes specialized pro-resolving mediators (SPMs) including Resolvin D1 (RvD1), which reduce inflammation.Hypothesis:We hypothesize that cardiac-specific ALOX5 upregulation could prevent diabetes-associated HFpEF without inducing systemic immunosuppression.Methods:DM was induced by feeding mice with a high fat diet (HFD, 60% fat by kcal) starting from 6 weeks of age for 22-24 weeks. At 24 weeks of age, DM mice were treated with RvD1, by intraperitoneal injection at 100 ng/mouse for 2 weeks. To specifically upregulate 5-LOX in the heart, another cohort of DM mice were injected with either AAV9-plain or AAV9-ALOX5viral vectors under the control of the α-myosin heavy chain (MHC) at 22 weeks old. At 28 weeks, we performed echocardiographic measurement of diastolic function and heart extraction.Results:HFD-induced DM mice showed lower cardiac 5-LOX expression (0.72 ± 0.05 a.u. vs. 1.09 ± 0.12, p < 0.05) and RvD1 levels (1 ± 0.08 vs. 1.9 ± 0.14 fold change over DM mice, p

Circulation, Volume 150, Issue Suppl_1, Page A4143876-A4143876, November 12, 2024. Background:Traditional combustible cigarette (combustible) use induces endothelial dysfunction accelerating cardiovascular disease progression. Young adults frequently use electronic cigarettes (e-cig) that is linked to impaired endothelial cell (EC) function. We aimed to use EC gene expression analysis to explore altered pathways across patterns of tobacco product use.Methods:Healthy adults aged 18-45 who regularly use e-cig (sole or dual), combustible, and non-use controls were with forearm vein EC biopsy and magnetic bead purification. EC total RNA was isolated and profiled on a NextSeq sequencer. Samples with transcript integrity number (TIN) < 10 were removed, genes were filtered with edgeR::filterByExpr, and analyzed with DESeq2. Differentially expressed genes (DEGs) comparing each use group to the non-use group were defined as log2fold change >1 and FDR p1 and p

Circulation, Volume 150, Issue Suppl_1, Page A4147345-A4147345, November 12, 2024. Introduction:Limited knowledge exists regarding the influence of sex on rejection-related gene-expression profiling using the Molecular Microscope (MMDx).Methods:We prospectively included all heart transplant recipients who underwent endomyocardial biopsy for clinical indication from November 2022 to May 2024 at New York Presbyterian. During the biopsy procedure, four samples were sent for histologic evaluation, and two for molecular analysis. Donor-derived cell-free DNA levels (dd-cfDNA%), peripheral gene-expression profiling (pGEP) scores, MMDx rejection scores, and rejection rates on histology and MMDx were compared based on sex.Results:A total of 414 samples were included, 130 (31.4%) were from female recipients. dd-cfDNA levels were higher in females compared to male recipients (0.30%[0.15-0.80] vs.0.22%[0.11-0.45]; p=0.02). Despite similar rates of pGEP scores (32 [28-34] vs. 32 [29-35]; p=0.70) or pGEP positivity (14.6% vs 17.0%; p=0.18), there was a trend towards a higher prevalence of donor-specific antibodies in female recipients (51.5% vs. 44.4%; p=0.20). Overall, rejection rates on MMDx (30.8% vs. 19.1%; p=0.01) and MMDx rejection scores (0.40[0.17-0.63] vs.0.27[0.11-0.55]; p=0.005) were higher in female recipients (Figure 1), primarily due to increased rates of antibody-mediated rejection (ABMR) diagnosis in this group (21.5%vs.10.8%; p=0.003). No differences in rejection rates were noted when assessed by histology (6.9% vs 8.0%; p=0.37).Conclusion:In a cohort of heart transplant recipients biopsied due to suspected rejection, female recipients were associated with increased MMDX-rejection scores and higher rejection rates, primarily driven by a twofold higher frequency of ABMR detection by MMDx compared to males.

Circulation, Volume 150, Issue Suppl_1, Page A4144882-A4144882, November 12, 2024. Objective:To elucidate the molecular mechanisms by which CD73 variants contribute to inflammation and atherosclerosis in humans.Approach and Results:To explore this knowledge gap, we conducted phenotype-wide association analysis (PheWAS) in a cohort of 25,000 patients to identify ASCVD-associated variants in theNT5E(CD73) gene in a non-biased manner. PheWAS analysis of coding variants identified that the rs200648774 single nucleotide polymorphisms in ecto-5’-nucleotidase (NT5E; CD73) that was associated with atherosclerosis (ICD-9; 440.x codes) in this cohort. The SNP rs200648774 (C– >T; MAF 0.000188) results in an Arginine (R) to Cysteine (C) substitution at amino acid 354 within the active site of CD73 (OR: 10.18, SE: 1.1; P=0.035). This Arginine residue is evolutionarily conserved across species. The variant protein is expressed but displays a loss of function (LOF; CD73 Cys354 variant). To examinethe impact of the CD73 Cys354 variant, we generated a murine model usingEfficientadditions withssDNAinserts-CRISPR (Easi-CRISPR) gene editing. CD73-Arg354/Arg354 (cd73wt/wt) and CD73-Cys354/Cys354 (cd73Δ/Δ) mice in anLdlr-/-background were placed on HFD for 12 weeks then sacrificed for quantification of aortic root atherosclerosis. Representative images of oil red O staining of aortic valve from CD73-Arg354/Arg354-Ldlr-/-(cd73wt/wt,Ldlr-/-) mice (Fig. A; Bar= 100 um) and CD73-Cys354/Cys354Ldlr-/-(cd73Δ/Δ,Ldlr-/-) mice (Fig. B; Bar= 100 um). Quantified area of atherosclerosis in CD73-Cys354/Cys354 (cd73Δ/Δ) aortic roots were significantly higher in CD73-Cys354/Cys354 (cd73Δ/Δ) than in CD73-Arg354/Arg354 (cd73wt/wt) (Fig. C; p=0.0096). The Percentage of Oil Red O staining in aortic root atherosclerotic lesions was also significantly higher in CD73-Cys354/Cys354 (cd73Δ/Δ) aortic roots compared to CD73-Arg354/Arg354 (cd73wt/wt) aortic roots. Together these data demonstrate that the rs200648774 single nucleotide polymorphisms resulting in cysteine substitution for arginine in the active site of CD73 results in a loss of CD73 function that also associates withincreased atherosclerosis in genetically susceptible mice.Conclusion:The LOF rs200648774 single nucleotide polymorphisms associates with atherosclerosis in humans and generation of the CD73-Cys354/Cys354 polymorphism inLdlr-/-mice results in increased HFD induced atherosclerosis

Circulation, Volume 150, Issue Suppl_1, Page A4137902-A4137902, November 12, 2024. Background:The mechanism of hemodialysis arteriovenous fistula (AVF) vein intima hyperplasia (IH) may be specific to its layers (intima, media, and adventitia) but is poorly understood. To address this gap, we studied layer-specific transcriptomics related to IH in veins of two-stage AVF creation.Methods:Vein samples were collected during the initial creation of brachial artery-basilic vein AVF (1st stage) and vein transposition surgery 6 weeks later (2nd stage) and were sectioned in OCT. Tissues from each layer were separately collected by laser microdissection. Following Takara’s SMARTer Stranded Total RNA-Seq Pico Input protocol, libraries were constructed and sequenced in NovaSeq. The sequencing reads were processed by fastp, aligned to genome GENCODE 45 by STAR and gene-level counts were estimated by RSEM. Intima was assessed by histology. Layer-specific differentially expressed genes in pre-access or AVF veins associated with IH in AVF veins were detected by edgeR and further identified the enriched pathways and predicted functions using Qiagen IPA.Results:44 veins were collected from 22 patients. 13,522 protein-coding genes that had a raw count of at least 10 in more than 70% of samples were included in analysis. 10 AVF veins had severe IH. Among the layers of pre-access vein, intima had the largest number of genes significantly associated with severe AVF IH and most or all these genes were upregulated (35/4, 26/2, 5/0 for the number of up/downregulated genes in intima, media, and adventitia of pre-access vein, respectively); cellular movement and immune cell trafficking were more strongly activated in intima of pre-access vein with severe AVF IH. No genes in any layer of AVF vein were significantly associated with severe AVF IH. From 1stto 2ndstage, more genes significantly changed expression in intima with severe AVF IH than intima without severe AVF IH (197/150 vs 144/93 for up/downregulated genes), while media (20/4 vs 149/31) and adventitia (99/27 vs 196/70) were the opposite; TGFB1, angiotensinogen, and TNF were more strongly activated in intima, while cell survival and migration were diminished in media and adventitia with severe AVF IH, respectively.Conclusion:Intima of pre-access vein has more genes associated with severe AVF IH. The layer-specific gene expression changes during AVF development and predicted functions are associated with AVF IH. These data can be used to develop layer-specific therapies to reduce AVF IH.

Circulation, Volume 150, Issue Suppl_1, Page A4147419-A4147419, November 12, 2024. Introduction:Nav1.5, the predominant Na+channel in cardiac myocytes, is encoded bySCN5Aand is essential for depolarization and impulse propagation in the heart. Loss of function mutations of Nav1.5 can lead to tachyarrhythmias, conduction disease and dilated cardiomyopathy by altering channel expression, kinetic properties, and/or membrane trafficking. We previously reported that amino acid K1479 of Nav1.5 is acetylated, that K1479 acetylation or mutation to the acetylation mimic glutamine (K1479Q) decreases inward Na+current (INa) in heterologous expression systems by decreasing membrane trafficking, that cardiac myocytes from mice lacking the deacetylase Sirt1 (Sirt1-/-) are hyperacetylated and have less INa, and that Sirt1-/-mice have conduction disease and arrhythmias. Here, we studied the effect of the K1479Q mutation that mimics acetylationin-vivo.Methods:K1479Q knock-in mice were engineered via CRISPR/Cas9 on a C57BL6 background and backcrossed 4 times with C57BL6 wild type (WT) mice. Nav1.5WT/Qheterozygous (het) mice were mated, and male (M) and female (F) gene-targeted offspring and littermate controls were studied by electrocardiograms (EKGs) and echocardiograms (Echos) at 3 and 6 months of age. Data is shown as mean±SEM.Results:The K1479Q mutation was homozygous (hom) embryonic lethal (Nav1.5WT/Qx Nav1.5WT/Qcrosses: WT, n=23; Het, n=49; Hom, n=0). On EKG (WT: n = 5M, 3F; Het: n = 6M, 5F), Nav1.5WT/Qmice had a prolonged PR interval at 3 months (42±1 vs. 36±1 ms, p=0.0003) and at 6 months (46±1 vs. 39±1 ms, p=