BACKGROUND AND AIMS:The mechanisms underlying sex differences in calcific aortic valve stenosis (CAVS) are poorly understood. We aimed to uncover sex-specific gene expression signatures of CAVS, including genes located on sexual chromosomes. METHODS:We performed mRNA sequencing from the explanted aortic valves of 500 individuals from our institute who underwent aortic valve replacement (n = 440) or a heart transplant (n = 60). Differential gene expression analyses were performed according to biological sex (n = 175 women; n = 311 men), comparing individuals with severe forms of CAVS to those with no significant CAVS. Interaction analyses were used to identify genes exhibiting sex-specific variations in differential expression magnitude. We prioritized differentially expressed genes (DEGs) known to escape X chromosome inactivation (XCI) and with higher gene expression in women. RESULTS:When comparing valves with severe CAVS to valves with no significant CAVS, we identified 2556 DEGs on the autosomes in female samples, and 2042 DEGs in male samples (|FC|≥2, FDR<5%). The interaction analysis revealed 114 genes with significantly different changes between sexes (FDR<5%). We prioritized 39 genes specifically dysregulated in women, primarily involved in extracellular matrix organization and cell adhesion, such as PRSS2 and MMP12. In contrast, 24 genes were more strongly dysregulated in men, including a mineralization network involving DMP1 and MEPE. Genes known to escape XCI, like PRKX and GYG2, showed evidence of sex-specific dysregulation. CONCLUSIONS:A mineralization-associated network comprising DMP1 and MEPE was specifically dysregulated in CAVS in men while genes escaping XCI were more strongly associated with disease in women.

Abstract Aortic valve disease is common, yet its regulatory mechanisms remain poorly understood. We performed multi-omic profiling of human aortic valve interstitial cells (HAVICs), identifying 11,891 allele-specific chromatin accessibility QTLs (as-caQTLs), 48% novel to this cell type. These variants were enriched in active enhancers, disrupted transcription factor (TF) motifs, particularly AP-1, TEAD and GATA families, and were validated by allele-specific TF binding assays. A fine-tuned deep DNA sequence model prioritized common and rare variants at risk loci predicted to impact chromatin accessibility. Single-cell CRISPRi perturbation of 247 variants identified cis-target genes at 55 as-caQTL elements, including loci without eQTLs. We demonstrate that common regulatory variants controlling elastin and fibrillin impact the development of the aortic valve apparatus. We provide genetic evidence and a mechanistic framework for the contribution of a reduced aortic root size to CAVD risk. Perturbations identified core cell programs led by upstream regulators AHNAK , PDIA6 , and RNFT1 converging on extracellular matrix production and iron transport.

The influence of germline genetics on the molecular transition from lung to tumor and different transcriptional patterns of tumors remains underexplored. Additionally, little is known about the genetic contributions to lung cancer relapse and mortality following surgery. The transcriptional transition from non-affected lung tissue to tumor was investigated using an extended eQTL approach. Associations between variants and expression differences in tumors relative to matched surrounding lung tissue were examined in 515 surgical cases of lung adenocarcinoma. We also used Cox regression in a genome-wide association study to identify variants associated with survival (n = 3472) and relapse (n = 3369) in patients with NSCLC. The number of significant eQTL was approximately threefold lower in tumor than in lung tissue. Variants that showed differential cis-regulatory control on expression between lung and tumor tissues were more frequently located in bivalent or distal regulatory elements. Those variants altered the transcription factor binding landscape and the expression of genes that are differentially expressed in tumors. Co-expression modules derived from WGCNA identified module-QTLs and putative regulatory signaling genes (CDK15, CD79B, CCDC80, and SCARA5) involved in immunity and mesenchymal cell fate. Differential eQTLs for three genes (NRG1, SECISBP2L, and JAML) colocalized with GWAS-nominated lung cancer risk loci. Prognostic lung cancer loci identified 6p22.3-MBOAT1 and 16p13.3-RBFOX1. The lung-to-tumor transcriptomic transition is characterized by an attenuated contribution of germline variants. Differential eQTLs inform differences in gene expression between lung and tumor tissues, implicate putative cancer-related genes and pathways, and identify target genes underlying GWAS loci.

CONTEXT:Genome-wide association studies (GWAS) have identified dozens of genetic loci linked with metabolic dysfunction-associated steatotic liver disease (MASLD). OBJECTIVE:To identify liver-expressed genes that may represent therapeutic candidates for MASLD. METHODS:We conducted a new GWAS meta-analysis including 16 532 cases and 1 240 188 controls. We also generated RNA sequencing data of liver samples and genome-wide genotyping of 504 individuals of the Quebec Obesity Biobank. RESULTS:Using mendelian randomization (MR) and genetic colocalization, we confirm the implication of genes previously linked with MASLD and identified novel ones including AKNA (AT-hook transcription factor), EPHA2 (EPH receptor A2), CHEK2 (encoding checkpoint kinase 2), and PCCB (propionyl-CoA carboxylase subunit β). More specifically, we found a strong and positive effect of long noncoding (lnc)RNA TRIB1AL on MASLD. The lead genetic variant was not linked with expression levels of the nearby protein-coding gene TRIB1 (Tribbles pseudokinase 1). In UK Biobank participants with whole-exome sequencing data available, rare loss-of-function variants in TRIB1 were not associated with liver fat accumulation or plasma triglyceride levels, suggesting that the lncRNA TRIB1AL may carry cardiometabolic effects independently of TRIB1. Targeted- and phenome-wide MR also identified lower liver-expressed TRIB1AL as being associated with reduced liver fat accumulation, lower plasma lipoprotein-lipid levels, and decreased atherosclerotic cardiovascular disease risk. CONCLUSION:These results open the door to liver-targeted therapeutics silencing of the noncoding genome for the prevention and treatment of MASLD and cardiometabolic diseases.

BACKGROUND:Bicuspid aortic valve (BAV) is a frequent congenital heart defect with a high heritability. Despite this, only a limited number of genes have been associated with the disease, and the molecular mechanisms remain unexplained in most cases. This study aimed to further understand the genetic architecture of BAV. METHODS:A genome-wide association study meta-analysis including 9631 cases among 65 677 participants was performed. Genes were prioritized using transcriptomic analyses based on RNA sequencing in relevant tissues, including human fetal and adult aortic valves. The impact of the knockdown or knockout of 4 candidate genes on cardiac development was verified in zebrafish. A polygenic risk score was developed, its association with BAV was evaluated in an independent cohort, and its association with a wide range of phenotypes (n=976) was evaluated in UK Biobank (n=355 618 individuals). RESULTS:Thirty-six genomic loci were identified, including 32 that were not described previously. Among the prioritized genes, KANK2 and ERBB4 were identified as potentially causal through transcriptomic analyses, colocalization, and Mendelian randomization based on gene expression in human aortic valves (n=484), whereas PRDM6 and STRN were prioritized using similar analyses from aortic (n=326) and left ventricular tissues (n=326), respectively. Targeting 4 candidate genes (WNT4, LEF1, STRN, and KANK2) in zebrafish led to disruption in cardiac development. A polygenic risk score was associated with an odds ratio of 2.07 (95% CI, 1.90-2.25; P=5.43×10-62) per SD for BAV and significantly associated with thoracic aortic aneurysm and atrial fibrillation in UK Biobank. CONCLUSIONS:This study supports a significant polygenic contribution to BAV, where the combination of multiple common variants in genes involved in heart morphogenesis disrupts aortic valve development.

Background Lp(a) (lipoprotein(a)) is an independent risk factor for calcific aortic valve stenosis (CAVS). Whether patients with CAVS and high Lp(a) levels are at higher risk of valvular or cardiovascular events is unknown. The aim of this study is to determine whether higher Lp(a) levels are associated with valvular and cardiovascular outcomes in patients with CAVS. Methods and Results We identified 1962 patients from the UK Biobank with an electronic health record or self‐reported CAVS diagnosis but who did not previously undergo aortic valve replacement (AVR) and had a minimal follow‐up time of 2.5 years. Cox proportional hazard regression was used to evaluate the effect of Lp(a) on AVR, AVR or cardiac death, and valvular or cardiovascular events (AVR, cardiac death, myocardial infarction, stroke, heart failure, or coronary artery bypass grafting). The maximal follow‐up time was set to 5 years. During the follow‐up, 198 patients underwent AVR, 260 had AVR or cardiac death, and 435 had at least 1 valvular or cardiovascular event. Patients with Lp(a) levels ≥125 versus <125 nmol/L were at higher risk of AVR (hazard ratio [HR], 1.58 [95% CI, 1.17–2.12]), AVR or cardiac death (HR, 1.43 [95% CI, 1.10–1.86]), and cardiovascular or valvular events (HR, 1.36 [95% CI, 1.11–1.68]). Point estimates were comparable in men versus women, younger versus older patients, and in patients with higher versus lower plasma C‐reactive protein levels. Conclusions In patients with CAVS, Lp(a) levels predicted a higher risk of valvular and cardiovascular outcomes. The impact of Lp(a)‐lowering therapies on valvular and cardiovascular health should be assessed in a long‐term randomized clinical trial.

There is currently no medical therapy for calcific aortic valve stenosis. We aimed to identify transcriptomic signatures of the disease severity. We performed mRNA sequencing from explanted human aortic valves of 500 individuals who underwent aortic valve replacement (n = 440) or a heart transplant (n = 60). We performed differential gene expression analyses according to hemodynamic severity, valve morphology, calcification grade, and age of onset, and we estimated immune cell proportions. We identified immune response, inflammation, and adipocyte metabolism as predominant pathways associated with calcific aortic valve stenosis severity, offering new perspectives for the development of pharmacological treatments.

Calcific aortic valve stenosis (CAVS) is the most frequent heart valve disease. Elucidating specific gene expression patterns in the aortic valve could provide new insights for understanding disease pathophysiology. We used local RNA sequencing data from 500 explanted human aortic valves to identify aortic valve-specific genes and compared their expression according to disease status and CAVS severity. We identified 100 specific protein-coding genes in the aortic valve compared to 45 other tissues from the Genotype-Tissue Expression (GTEx) project. Among them, 38 were differentially expressed in CAVS. Ten had a gradient of expression between severity levels and were central in a protein-protein interaction network, most of which were involved in extracellular matrix regulation or inflammation. Among the aortic valve-specific genes, four of the corresponding proteins had a significantly different plasma level in individuals with CAVS. These findings represent a robust foundation for the development of specific biomarkers and therapies for CAVS.

Background:Valvular lesions in calcific aortic valve stenosis are sex-specific: female patients reach a similar level of severity as male patients but with less valvular calcification and more valvular fibrosis. We thus aim to assess the transcriptome of stenotic aortic valves according to patients' sex. Methods:A total of 300 valves were collected, and genomewide gene expression was quantified using a microarray on 240. Among these, 62 female patients were matched with 62 male patients, for age (within 2 years), body mass index (within 2 kg/m2), arterial pressure (within 10/5 mm Hg), diabetes (exact), hypertension (exact), and calcific aortic valve stenosis severity. Among the 60 remaining valves, 16 female and 16 male patients were similarly matched for real-time quantitative polymerase chain reaction analysis. Results:Clinical and echocardiographic characteristics of the patients were comparable between female and male patients, except for the incidence of coronary artery disease and body surface area (greater in male patients). A total of 190 genes were regulated differently in female vs male patients-132 on autosomes, and 58 on sexual chromosomes. Differences were found in inflammation and lipid metabolism-associated genes. Genes linked to intensified fibrosis processes (eg, TGFβ2, KIF1A, FRAS1) were overexpressed in female vs male patients. Genes associated with increased calcification were overexpressed in both male (CPAMD8, STC2) and female (RCN2, TPD52L1) patients. Genes involved in apoptosis (CES4, SFRP4, TGFB2) were overexpressed in female vs male patients. Only KIF1A was validated by real-time quantitative polymerase chain reaction analyses. Conclusions:This study provides evidence that sex may influence aortic valve gene expression through different mechanisms in female vs male individuals.

Genome-wide association studies (GWAS) have identified dozens of genetic loci linked with metabolic dysfunction-associated steatotic liver disease (MASLD). To identify liver-expressed genes that may represent therapeutic candidates for MASLD, we conducted a new GWAS meta-analysis including 16,532 cases and 1,240,188 controls, as well as RNA sequencing of liver samples and genome-wide genotyping of 504 individuals of the Quebec Obesity Biobank. Using Mendelian randomization (MR) and genetic colocalization, we confirm the implication of genes previously linked with MASLD and identified novel ones including AKNA (AT-hook transcription factor), EPHA2 (EPH receptor A2), CHEK2 (encoding Checkpoint kinase 2) and PCCB (Propionyl-CoA carboxylase subunit beta). More specifically, we found a strong and positive effect of the long non-coding RNA TRIB1AL on MASLD. The lead genetic variant was not linked with expression levels of the nearby protein-coding gene TRIB1 (Tribbles Pseudokinase 1). In participants of the UK Biobank with whole exome sequencing data available, rare loss-of-function variants in TRIB1 were not associated with liver fat accumulation or plasma triglyceride levels, suggesting that the long non-coding RNA TRIB1AL may carry cardiometabolic effects independently of TRIB1. Targeted- and phenome-wide MR also identified lower liver-expressed TRIB1AL as being associated with reduced liver fat accumulation, lower plasma lipoprotein-lipid levels, decreased atherosclerotic cardiovascular disease risk, and increased human lifespan. These results open the door to liver-targeted therapeutics silencing of the non-coding genome for the prevention and treatment of MASLD and cardiometabolic diseases. ### Competing Interest Statement BJA is a consultant for Novartis, Eli Lilly, and Silence Therapeutics and has received research contracts from Pfizer, Eli Lilly and Silence Therapeutics. AT receives research funding from Johnson & Johnson, Medtronic, and G.I. windows for studies related to bariatric surgery as well as consulting fees from Bausch Health and Novo Nordisk. The remaining authors disclose no conflicts. ### Funding Statement This study was supported by the Canadian Institutes of Health Research (CIHR), the Fondation IUCPQ as well as Silence Therapeutics. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish results. ÉG and LJR hold a Doctoral Research Award from the CIHR. JB holds a Masters Research Award from the Fonds de recherche du Quebec: Sante (FRQS). EG holds a Doctoral Research Award from the FRQS. YB holds a Canada Research Chair in Genomics of Heart and Lung Diseases. P.M. is recipient of the Joseph C. Edwards Foundation granted to Universite Laval. BJA holds a Senior Scholar Award from the FRQS. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Patients of the Quebec Obesity Biobank provided informed consent to participate to this institutional biobank. The study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Review Board (Ethics Committee) of Institut universitaire de cardiologie et de pneumologie de Quebec-Universite Laval (IUCPQ-UL) (approval number 2021-3656; date of approval: June 17th 2021). UK Biobank received approval from the British National Health Service, North West - Haydock Research Ethics Committee (16/NW/0274). The analysis in the UK Biobank were performed using data application number 25205. All genotype and phenotype data were collected according to an informed consent obtained at the baseline assessment from all participants. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors.

Lamins A/C, coded by LMNA gene, are crucial for nuclear architecture preservation. Pathogenic LMNA variants cause a wide range of inherited diseases called "laminopathies". A subgroup is referred to "progeroid syndromes" characterized by premature aging and other manifestations including cardiac valve abnormalities. Atypical phenotypes, generally less severe, have also been reported. We report the case of a 26-year-old male with calcific tricuspid aortic and mitral valve diseases. His father was diagnosed with severe aortic valve stenosis and mitral annulus calcification at the age of 38. The goal of this study was to identify the putative variant causing this non-syndromic multivalvular disease. Known disease-causing variants in NOTCH1, FLNA, and DCHS1 were first excluded by Sanger sequencing. Whole-exome sequencing was then performed in five family members. A LMNA variant (p.Glu262Val) was identified with in silico evidences of pathogenicity (CADD [combined annotation dependent depletion] = 33). Cells transfected with the cDNA construct harboring p.Glu262Val were characterized by abnormal nuclear morphology. Along with a literature review, the variant was classified as likely pathogenic. Elucidating the mechanism by which LMNA p.Glu262Val specifically affects cardiac heart valves is likely to provide insight about the pathogenesis of Mendelian forms of valvular heart diseases and may help guide the development of therapies.

Aims RNA interference therapies targeting liver expression of the gene proprotein convertase subtilisin/kexin type 9 (PCSK9) lower LDL-cholesterol (LDL-C) and apolipoprotein B (apoB) levels. As opposed to monoclonal antibodies, which neutralise PCSK9 circulating protein, their effect on atherosclerotic cardiovascular disease (ASCVD) outcomes is unknown. We used genetic variants in the PCSK9 locus influencing PCSK9 function or gene expression in the liver to determine whether antibodies against PCSK9 and RNA interference therapies could have comparable effects on ASCVD.Methods and results We performed genome-wide genotyping and RNA sequencing of 504 human liver sample and identified a genetic variant (rs472495) explaining 5.6% of liver PCSK9 gene expression to mimic lifelong RNA interference of PCSK9. We used the PCSK9 R46L variant, known to alter PCSK9 function, to model antibody-based PCSK9 inhibition. For each standard deviation decrease in apoB levels, both variants were similarly associated with coronary artery disease risk: (odds ratio [OR] = 0.40, 95% confidence interval [CI]: 0.31-0.51, P = 3.7e-13 for rs472495 which affects liver PCSK9 expression) and (OR = 0.48, 95% CI: 0.43-0.55, P = 1.3e-28 for R46L which affects protein levels). Comparable effects of these two genetic inhibition approaches were observed for aortic stenosis, heart failure, ischemic stroke, Type 2 diabetes and glycemic traits as well as non-alcoholic fatty liver disease and liver enzymes.Conclusion For a given reduction in apoB levels, genetically predicted reductions in PCSK9 function (mimicking PCSK9 neutralizing antibodies) and liver PCSK9 gene expression levels (mimicking PCSK9 RNA interference) were comparably associated with a lower risk of coronary artery disease. These genetic data suggest that LDL-C/apoB reductions may provide cardiovascular benefits, regardless of how PCSK9 function is inhibited.

BACKGROUND:Polygenic risk scores (PRS) could help to identify individuals with a high genetic risk profile for coronary artery disease (CAD). We aimed to evaluate the association between previously reported PRS and myocardial infarction (MI) as well as the extent and recurrence of coronary artery lesions. METHODS:We validated previously reported CAD-PRS and 6 cardiovascular (CV) risk factors PRS (systolic blood pressure [SBP], type 2 diabetes [T2D], body-mass index [BMI], low-density lipoprotein cholesterol [LDL], triglycerides [TG], and lipoprotein-[a][Lp(a)]) in individuals of European ancestry from two Canadian population-based cohorts, the Canadian Longitudinal Study on Aging (CLSA, N = 24,599) and CARTaGENE (N = 26,806). Using a stepwise model, we determined an optimal combination of PRS to identify MI. We tested the selected PRS for association with the severity and recurrence of atherosclerotic CAD evaluated by coronary angiography in patients undergoing cardiac surgery (QUEBEC-ANGIO, N = 4108). RESULTS:We show that the CAD-PRS most strongly associated with MI has odds ratios per standard deviation increment of 1.75 [1.64-1.86] (P = 1.57E-70) in CLSA and 1.87 [1.73-2.03] (P = 3.06E-53) in CARTaGENE. In CLSA, the optimal model includes CAD-PRS, SBP-PRS, BMI-PRS, LDL-PRS, TG-PRS and Lp(a)-PRS. Adding these PRS increases modestly yet significantly the discriminative capacity when compared to traditional risk factors (difference of AUC = 0.025 [0.019-0.031] in CLSA, 0.018 [0.012-0.024] in CARTaGENE). In QUEBEC-ANGIO, the CAD-PRS is gradually and significantly associated with the extent and recurrence of CAD. CONCLUSIONS:Screening multiple validated PRS may significantly improve genetic risk estimation of MI as well as the extent and recurrence of coronary artery lesions.

Aortic stenosis (AS) is a common valvular heart disease and has no pharmacological therapies. We performed a multi-ancestry genome-wide association meta-analysis of 86,864 AS cases among 2,853,408 individuals, discovering 241 autosomal independent risk loci and 3 X chromosome risk loci. We additionally performed sex-stratified and ancestry-stratified genome-wide association studies (GWASs), identifying an additional 5 sex-specific risk loci, 11 risk loci in European ancestry individuals and 1 risk locus in African ancestry individuals. We also performed a transcriptome-wide association study using expression quantitative trait loci from human aortic valves, discovering 54 new genes for which genetically predicted expression influences the risk of AS. We then generated a new polygenic risk score for AS. Finally, we performed gene silencing experiments targeting biologically relevant genes identified by our GWAS. Silencing of CMKLR1 and LTBP4 in human valvular interstitial cells substantially decreased mineralization, implicating a role for polyunsaturated fatty acids and transforming growth factor β signaling in AS.

Excess liver fat (LF) is associated with low cardiorespiratory fitness (CRF), low physical activity, and a deteriorated cardiometabolic health profile including increased visceral adipose tissue (VAT). Whether the association between LF and CRF is mediated by visceral adiposity is unknown. We studied the contribution of VAT to the relationship between CRF and LF in asymptomatic women and men. The sample included 320 participants (43% women) who underwent LF quantification by magnetic resonance spectroscopy. VAT was measured by magnetic resonance imaging, CRF using maximal cardiorespiratory exercise testing, and moderate-to-vigorous intensity physical activity (MVPA) using a 3-day journal. Mean age was 50.3 ± 8.6 years, waist circumference was 89.3 ± 11.4 cm, and LF content was 4.3 ± 5.7%. LF was inversely correlated with CRF (p < 0.0001), MVPA (p < 0.05) and cardiometabolic health score (p < 0.0001), and positively related with VAT (p < 0.0001) in both sexes. Significantly higher levels of VAT (p < 0.0001) and subcutaneous adipose tissue (p < 0.0001) and a worsening cardiometabolic health score (p < 0.05) and CRF (p = 0.0001) were found across increasing sex-specific tertiles of LF levels. Lower levels of LF (p < 0.01) and VAT (p < 0.0001) and a higher cardiometabolic health score (p < 0.0001) and MVPA (p < 0.05) were noted across increasing sex-specific CRF tertiles. Multivariable regression analyses showed that visceral adiposity explained the majority of the variance in LF in both sexes (p < 0.0001). Finally, serial mediation analyses revealed that VAT but not body fat percentage was a mediator in the relationship between CRF and LF in both sexes. Thus, visceral adiposity appears to be an important mediator in the relationship between CRF and LF, even after controlling for total adiposity.

There is currently no medical therapy to prevent calcific aortic valve stenosis (CAVS). Multi-omics approaches could lead to the identification of novel molecular targets. Here, we perform a genome-wide association study (GWAS) meta-analysis including 14,819 cases among 941,863 participants of European ancestry. We report 32 genomic loci, among which 20 are novel. RNA sequencing of 500 human aortic valves highlights an enrichment in expression regulation at these loci and prioritizes candidate causal genes. Homozygous genotype for a risk variant near TWIST1 , a gene involved in endothelial-mesenchymal transition, has a profound impact on aortic valve transcriptomics. We identify five genes outside of GWAS loci by combining a transcriptome-wide association study, colocalization, and Mendelian randomization analyses. Using cross-phenotype and phenome-wide approaches, we highlight the role of circulating lipoproteins, blood pressure and inflammation in the disease process. Our findings pave the way for the development of novel therapies for CAVS.