Integrative genomic analyses identify candidate causal genes for calcific aortic valve stenosis involving tissue-specific regulation.

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.

Enhancer promoter interactome and Mendelian randomization identify network of druggable vascular genes in coronary artery disease.

Coronary artery disease (CAD) is a multifactorial disorder, which is partly heritable. Herein, we implemented a mapping of CAD-associated candidate genes by using genome-wide enhancer-promoter conformation (H3K27ac-HiChIP) and expression quantitative trait loci (eQTL). Enhancer-promoter anchor loops from human coronary artery smooth muscle cells (HCASMC) explained 22% of the heritability for CAD. 3D enhancer-promoter genome mapping of CAD-genes in HCASMC was enriched in vascular eQTL genes. By using colocalization and Mendelian randomization analyses, we identified 58 causal candidate vascular genes including some druggable targets (MAP3K11, CAMK1D, PDGFD, IPO9 and CETP). A network analysis of causal candidate genes was enriched in TGF beta and MAPK pathways. The pharmacologic inhibition of causal candidate gene MAP3K11 in vascular SMC reduced the expression of athero-relevant genes and lowered cell migration, a cardinal process in CAD. Genes connected to enhancers are enriched in vascular eQTL and druggable genes causally associated with CAD.

Genome-wide chromatin contacts of super-enhancer-associated lncRNA identify LINC01013 as a regulator of fibrosis in the aortic valve.

Calcific aortic valve disease (CAVD) is characterized by a fibrocalcific process. The regulatory mechanisms that drive the fibrotic response in the aortic valve (AV) are poorly understood. Long noncoding RNAs derived from super-enhancers (lncRNA-SE) control gene expression and cell fate. Herein, multidimensional profiling including chromatin immunoprecipitation and sequencing, transposase-accessible chromatin sequencing, genome-wide 3D chromatin contacts of enhancer-promoter identified LINC01013 as an overexpressed lncRNA-SE during CAVD. LINC01013 is within a loop anchor, which has contact with the promoter of CCN2 (CTGF) located at ~180 kb upstream. Investigation showed that LINC01013 acts as a decoy factor for the negative transcription elongation factor E (NELF-E), whereby it controls the expression of CCN2. LINC01013-CCN2 is part of a transforming growth factor beta 1 (TGFB1) network and exerts a control over fibrogenesis. These findings illustrate a novel mechanism whereby a dysregulated lncRNA-SE controls, through a looping process, the expression of CCN2 and fibrogenesis of the AV.

Enhancer-associated aortic valve stenosis risk locus 1p21.2 alters NFATC2 binding site and promotes fibrogenesis.

Genome-wide association studies for calcific aortic valve stenosis (CAVS) previously reported strong signal for noncoding variants at 1p21.2. Previous study using Mendelian randomization suggested that the locus controls the expression of PALMD encoding Palmdelphin (PALMD). However, the molecular regulation at the locus and the impact of PALMD on the biology of the aortic valve is presently unknown. 3D genetic mapping and CRISPR activation identified rs6702619 as being located in a distant-acting enhancer, which controls the expression of PALMD. DNA-binding assay showed that the risk variant modified the DNA shape, which prevented the recruitment of NFATC2 and lowered the expression of PALMD. In co-expression network analysis, a module encompassing PALMD was enriched in actin-based process. Mass spectrometry and functional assessment showed that PALMD is a regulator of actin polymerization. In turn, lower level of PALMD promoted the activation of myocardin-related transcription factor and fibrosis, a key pathobiological process underpinning CAVS.

System Genetics Including Causal Inference Identify Immune Targets for Coronary Artery Disease and the Lifespan.

BACKGROUND: Randomized clinical trials indicate that the immune response plays a significant role in coronary artery disease (CAD), a disorder impacting the lifespan potential. However, the identification of targets critical to the immune response in atheroma is still hampered by a lack of solid inference. METHODS: Herein, we implemented a system genetics approach to identify causally associated immune targets implicated in atheroma. We leveraged genome-wide association studies to perform mapping and Mendelian randomization to assess causal associations between gene expression in blood cells with CAD and the lifespan. Expressed genes (eGenes) were prioritized in network and in single-cell expression derived from plaque immune cells. RESULTS: Among 840 CAD-associated blood eGenes, 37 were predicted causally associated with CAD and 6 were also associated with the parental lifespan in Mendelian randomization. In multivariable Mendelian randomization, the impact of eGenes on the lifespan potential was mediated by the CAD risk. Predicted causal eGenes were central in network. FLT1 and CCR5 were identified as targets of approved drugs, whereas 22 eGenes were deemed tractable for the development of small molecules and antibodies. Analyses of plaque immune single-cell expression identified predicted causal eGenes enriched in macrophages (GPX1, C4orf3) and involved in ligand-receptor interactions (CCR5). CONCLUSIONS: We identified 37 blood eGenes predicted causally associated with CAD. The predicted expression for 6 eGenes impacted the lifespan potential through the risk of CAD. Prioritization based on network, annotations, and single-cell expression identified targets deemed tractable for the development of drugs and for drug repurposing.

Single-cell expression and Mendelian randomization analyses identify blood genes associated with lifespan and chronic diseases.

The human lifespan is a heritable trait, which is intricately linked to the development of disorders. Here, we show that genetic associations for the parental lifespan are enriched in open chromatin of blood cells. By using blood expression quantitative trait loci (eQTL) derived from 31,684 samples, we identified for the lifespan 125 cis- and 559 trans-regulated expressed genes (eGenes) enriched in adaptive and innate responses. Analysis of blood single-cell expression data showed that eGenes were enriched in dendritic cells (DCs) and the modelling of cell ligand-receptor interactions predicted crosstalk between DCs and a cluster of monocytes with a signature of cytotoxicity. In two-sample Mendelian randomization (MR), we identified 16 blood cis-eGenes causally associated with the lifespan. In MR, the majority of cis-eGene-disorder association pairs had concordant effects with the lifespan. The present work underlined that the lifespan is linked with the immune response and identifies eGenes associated with the lifespan and disorders.

Enhancer-mediated enrichment of interacting JMJD3-DDX21 to ENPP2 locus prevents R-loop formation and promotes transcription.

ENPP2, which encodes for the enzyme autotaxin (ATX), is overexpressed during chronic inflammatory diseases and various cancers. However, the molecular mechanism involved in the ENPP2 transcription remains elusive. Here, in HEK 293T cells, we demonstrated that lipopolysaccharide (LPS) increased the transcription process at ENPP2 locus through a NF-кB pathway and a reduction of H3K27me3 level, a histone repressive mark, by the demethylase UTX. Simultaneously, the H3K27me3 demethylase JMJD3/KDM6B was recruited to the transcription start site (TSS), within the gene body and controlled the expression of ENPP2 in a non-enzymatic manner. Mass spectrometry data revealed a novel interaction for JMJD3 with DDX21, a RNA helicase that unwinds R-loops created by nascent transcript and DNA template. Upon LPS treatment, JMJD3 is necessary for DDX21 recruitment at ENPP2 locus allowing the resolution of aberrant R-loops. CRISPR-Cas9-mediated deletion of a distant-acting enhancer decreased the expression of ENPP2 and lowered the recruitment of JMJD3-DDX21 complex at TSS and its progression through the gene body. Taken together, these findings revealed that enhancer-mediated enrichment of novel JMJD3-DDX21 interaction at ENPP2 locus is necessary for nascent transcript synthesis via the resolution of aberrant R-loops formation in response to inflammatory stimulus.

DNA methylation of a PLPP3 MIR transposon-based enhancer promotes an osteogenic programme in calcific aortic valve disease.

Aims: Calcific aortic valve disease (CAVD) is characterized by the osteogenic transition of valve interstitial cells (VICs). In CAVD, lysophosphatidic acid (LysoPA), a lipid mediator with potent osteogenic activity, is produced in the aortic valve (AV) and is degraded by membrane-associated phospholipid phosphatases (PLPPs). We thus hypothesized that a dysregulation of PLPPs could participate to the osteogenic reprograming of VICs during CAVD. Methods and results: The expression of PLPPs was examined in human control and mineralized AVs and comprehensive analyses were performed to document the gene regulation and impact of PLPPs on the osteogenic transition of VICs. We found that PLPP3 gene and enzymatic activity were downregulated in mineralized AVs. Multidimensional gene profiling in 21 human AVs showed that expression of PLPP3 was inversely correlated with the level of 5-methylcytosine (5meC) located in an intronic mammalian interspersed repeat (MIR) element. Bisulphite pyrosequencing in a larger series of 67 AVs confirmed that 5meC in intron 1 was increased by 2.2-fold in CAVD compared with control AVs. In isolated cells, epigenome editing with clustered regularly interspersed short palindromic repeats-Cas9 system containing a deficient Cas9 fused with DNA methyltransferase (dCas9-DNMT) was used to increase 5meC in the intronic enhancer and showed that it reduced significantly the expression of PLPP3. Knockdown experiments showed that lower expression of PLPP3 in VICs promotes an osteogenic programme. Conclusions: DNA methylation of a MIR-based enhancer downregulates the expression of PLPP3 and promotes the mineralization of the AV.

OxLDL-derived lysophosphatidic acid promotes the progression of aortic valve stenosis through a LPAR1-RhoA-NF-κB pathway.

AIMS: Oxidatively modified lipoproteins may promote the development/progression of calcific aortic valve stenosis (CAVS). Oxidative transformation of low-density lipoprotein (OxLDL) generates lysophosphatidic acid (LPA), a lipid mediator that accumulates in mineralized aortic valves. LPA activates at least six different G protein-coupled receptors, which may play a role in the pathophysiology of CAVS. We hypothesized that LPA derived from OxLDL may promote a NF-κB signature that drives osteogenesis in the aortic valve. METHODS AND RESULTS: The role of OxLDL-LPA was examined in isolated valve interstitial cells (VICs) and the molecular pathway was validated in human explanted aortic valves and in a mouse model of CAVS. We found that OxLDL-LPA promoted the mineralization and osteogenic transition of VICs through LPAR1 and the activation of a RhoA-NF-κB pathway. Specifically, we identified that RhoA/ROCK activated IκB kinase alpha, which promoted the phosphorylation of p65 on serine 536 (p65 pS536). p65 pS536 was recruited to the BMP2 promoter and directed an osteogenic program not responsive to the control exerted by the inhibitor of kappa B. In LDLR-/-/ApoB100/100/IGFII transgenic mice (IGFII), which develop CAVS under a high-fat and high-sucrose diet the administration of Ki16425, a Lpar1 blocker, reduced by three-fold the progression rate of CAVS and also decreased the osteogenic activity as measured with a near-infrared fluorescent probe that recognizes hydroxyapatite of calcium. CONCLUSIONS: OxLDL-LPA promotes an osteogenic program in the aortic valve through a LPAR1-RhoA/ROCK-p65 pS536 pathway. LPAR1 may represent a suitable target to prevent the progression of CAVS.