DNA-pools targeted-sequencing as a robust cost-effective method to detect rare variants: Application to dilated cardiomyopathy genetic diagnosis.

Dilated cardiomyopathy (DCM) is a heart disease characterized by left ventricular dilatation and systolic dysfunction. In 30% of cases, pathogenic variants, essentially private to each patient, are identified in at least one of almost 50 reported genes. Thus, while costly, exons capture-based Next Generation Sequencing (NGS) of a targeted gene panel appears as the best strategy to genetically diagnose DCM. Here, we report a NGS strategy applied to pools of 8 DNAs from DCM patients and validate its robustness for rare variants detection at 4-fold reduced cost. Our pipeline uses Freebayes to detect variants with the expected 1/16 allele frequency. From the whole set of detected rare variants in 96 pools we set the variants quality parameters optimizing true positives calling. When compared to simplex DNA sequencing in a shared subset of 50 DNAs, 96% of SNVs/InsDel were accurately identified in pools. Extended to the 384 DNAs included in the study, we detected 100 variants (ACMG class 4 and 5), mostly in well-known morbid gene causing DCM such as TTN, MYH7, FLNC, and TNNT2. To conclude, we report an original pool-sequencing NGS method accurately detecting rare variants. This innovative approach is cost-effective for genetic diagnostic in rare diseases.

uAUG creating variants in the 5'UTR of ENG causing Hereditary Hemorrhagic Telangiectasia.

Hereditary Hemorrhagic Telangiectasia (HHT) is a rare, autosomal dominant, vascular disorder. About 80% of cases are caused by pathogenic variants in ACVRL1 (also known as ALK1) and ENG, with the remaining cases being unexplained. We identified two variants, c.-79C>T and c.-68G>A, in the 5'UTR of ENG in two unrelated patients. They create upstream AUGs at the origin of upstream overlapping open reading frames (uoORFs) ending at the same stop codon. To assess the pathogenicity of these variants, we performed functional assays based on the expression of wild-type and mutant constructs in human cells and evaluated their effect on ALK1 activity in a BMP-response element assay. This assay is mandatory for molecular diagnosis and has been so far only applied to coding ENG variants. These variants were associated with a decrease of protein levels in HeLa and HUVEC cells and a decreased ability to activate ALK1. We applied the same experiments on three additional uoORF-creating variants (c.-142A>T, c.-127C>T and c.-10C>T) located in the 5'UTR of ENG and previously reported in HHT patients. We found that all the analyzed variants alter protein levels and function. Additional experiments relying on an artificial deletion in our mutated constructs show that identified uAUGs could initiate the translation indicating that the associated effect is translation-dependent. Overall, we have identified two 5'UTR ENG variations in HHT patients and shed new light on the role of upstream ORFs on ENG regulation. Our findings contribute to the amelioration of molecular diagnosis in HHT.

Genome-wide association study of a semicontinuous trait: illustration of the impact of the modeling strategy through the study of Neutrophil Extracellular Traps levels.

Over the last years, there has been a considerable expansion of genome-wide association studies (GWAS) for discovering biological pathways underlying pathological conditions or disease biomarkers. These GWAS are often limited to binary or quantitative traits analyzed through linear or logistic models, respectively. In some situations, the distribution of the outcome may require more complex modeling, such as when the outcome exhibits a semicontinuous distribution characterized by an excess of zero values followed by a non-negative and right-skewed distribution. We here investigate three different modeling for semicontinuous data: Tobit, Negative Binomial and Compound Poisson-Gamma. Using both simulated data and a real GWAS on Neutrophil Extracellular Traps (NETs), an emerging biomarker in immuno-thrombosis, we demonstrate that Compound Poisson-Gamma was the most robust model with respect to low allele frequencies and outliers. This model further identified the MIR155HG locus as significantly (P = 1.4 × 10-8) associated with NETs plasma levels in a sample of 657 participants, a locus recently highlighted to be involved in NETs formation in mice. This work highlights the importance of the modeling strategy for GWAS of a semicontinuous outcome and suggests Compound Poisson-Gamma as an elegant but neglected alternative to Negative Binomial for modeling semicontinuous outcome in the context of genomic investigations.

Common and Rare 5'UTR Variants Altering Upstream Open Reading Frames in Cardiovascular Genomics.

High-throughput sequencing (HTS) technologies are revolutionizing the research and molecular diagnosis landscape by allowing the exploration of millions of nucleotide sequences at an unprecedented scale. These technologies are of particular interest in the identification of genetic variations contributing to the risk of rare (Mendelian) and common (multifactorial) human diseases. So far, they have led to numerous successes in identifying rare disease-causing mutations in coding regions, but few in non-coding regions that include introns, untranslated (UTR), and intergenic regions. One class of neglected non-coding variations is that of 5'UTR variants that alter upstream open reading frames (upORFs) of the coding sequence (CDS) of a natural protein coding transcript. Following a brief summary of the molecular bases of the origin and functions of upORFs, we will first review known 5'UTR variations altering upORFs and causing rare cardiovascular disorders (CVDs). We will then investigate whether upORF-affecting single nucleotide polymorphisms could be good candidates for explaining association signals detected in the context of genome-wide association studies for common complex CVDs.

Targeting Pdzrn3 maintains adult blood-brain barrier and central nervous system homeostasis.

Blood brain barrier (BBB) disruption is a critical component of the pathophysiology of cognitive impairment of vascular etiology (VCI) and associated with Alzheimer's disease (AD). The Wnt pathway plays a crucial role in BBB maintenance, but there is limited data on its role in cognitive pathologies. The E3 ubiquitin ligase PDZRN3 is a regulator of the Wnt pathway. In a murine model of VCI, overexpressing Pdzrn3 in endothelial cell (EC) exacerbated BBB hyperpermeability and accelerated cognitive decline. We extended these observations, in both VCI and AD models, showing that EC-specific depletion of Pdzrn3, reinforced the BBB, with a decrease in vascular permeability and a subsequent spare in cognitive decline. We found that in cerebral vessels, Pdzrn3 depletion protects against AD-induced Wnt target gene alterations and enhances endothelial tight junctional proteins. Our results provide evidence that Wnt signaling could be a molecular link regulating BBB integrity and cognitive decline under VCI and AD pathologies.

An artificial neural network approach integrating plasma proteomics and genetic data identifies PLXNA4 as a new susceptibility locus for pulmonary embolism.

Venous thromboembolism is the third common cardiovascular disease and is composed of two entities, deep vein thrombosis (DVT) and its potential fatal form, pulmonary embolism (PE). While PE is observed in ~ 40% of patients with documented DVT, there is limited biomarkers that can help identifying patients at high PE risk. To fill this need, we implemented a two hidden-layers artificial neural networks (ANN) on 376 antibodies and 19 biological traits measured in the plasma of 1388 DVT patients, with or without PE, of the MARTHA study. We used the LIME algorithm to obtain a linear approximate of the resulting ANN prediction model. As MARTHA patients were typed for genotyping DNA arrays, a genome wide association study (GWAS) was conducted on the LIME estimate. Detected single nucleotide polymorphisms (SNPs) were tested for association with PE risk in MARTHA. Main findings were replicated in the EOVT study composed of 143 PE patients and 196 DVT only patients. The derived ANN model for PE achieved an accuracy of 0.89 and 0.79 in our training and testing sets, respectively. A GWAS on the LIME approximate identified a strong statistical association peak (rs1424597: p = 5.3 × 10-7) at the PLXNA4 locus. Homozygote carriers for the rs1424597-A allele were then more frequently observed in PE than in DVT patients from the MARTHA (2% vs. 0.4%, p = 0.005) and the EOVT (3% vs. 0%, p = 0.013) studies. In a sample of 112 COVID-19 patients known to have endotheliopathy leading to acute lung injury and an increased risk of PE, decreased PLXNA4 levels were associated (p = 0.025) with worsened respiratory function. Using an original integrated proteomics and genetics strategy, we identified PLXNA4 as a new susceptibility gene for PE whose exact role now needs to be further elucidated.

A rare coding mutation in the MAST2 gene causes venous thrombosis in a French family with unexplained thrombophilia: The Breizh MAST2 Arg89Gln variant.

Rare variants outside the classical coagulation cascade might cause inherited thrombosis. We aimed to identify the variant(s) causing venous thromboembolism (VTE) in a family with multiple relatives affected with unprovoked VTE and no thrombophilia defects. We identified by whole exome sequencing an extremely rare Arg to Gln variant (Arg89Gln) in the Microtubule Associated Serine/Threonine Kinase 2 (MAST2) gene that segregates with VTE in the family. Free-tissue factor pathway inhibitor (f-TFPI) plasma levels were significantly decreased in affected family members compared to healthy relatives. Conversely, plasminogen activator inhibitor-1 (PAI-1) levels were significantly higher in affected members than in healthy relatives. RNA sequencing analysis of RNA interference experimental data conducted in endothelial cells revealed that, of the 13,387 detected expressed genes, 2,354 have their level of expression modified by MAST2 knockdown, including SERPINE1 coding for PAI-1 and TFPI. In HEK293 cells overexpressing the MAST2 Gln89 variant, TFPI and SERPINE1 promoter activities were respectively lower and higher than in cells overexpressing the MAST2 wild type. This study identifies a novel thrombophilia-causing Arg89Gln variant in the MAST2 gene that is here proposed as a new molecular player in the etiology of VTE by interfering with hemostatic balance of endothelial cells.

A novel rare c.-39C>T mutation in the PROS1 5'UTR causing PS deficiency by creating a new upstream translation initiation codon.

Autosomal dominant inherited Protein S deficiency (PSD) (MIM 612336) is a rare disorder caused by rare mutations, mainly located in the coding sequence of the structural PROS1 gene, and associated with an increased risk of venous thromboembolism. To identify the molecular defect underlying PSD observed in an extended French pedigree with seven PSD affected members in whom no candidate deleterious PROS1 mutation was detected by Sanger sequencing of PROS1 exons and their flanking intronic regions or via an multiplex ligation-dependent probe amplification (MLPA) approach, a whole genome sequencing strategy was adopted. This led to the identification of a never reported C to T substitution at c.-39 from the natural ATG codon of the PROS1 gene that completely segregates with PSD in the whole family. This substitution ACG→ATG creates a new start codon upstream of the main ATG. We experimentally demonstrated in HeLa cells that the variant generates a novel overlapping upstream open reading frame (uORF) and inhibits the translation of the wild-type PS. This work describes the first example of 5'UTR PROS1 mutation causing PSD through the creation of an uORF, a mutation that is not predicted to be deleterious by standard annotation softwares, and emphasizes the need for better exploration of such type of non-coding variations in clinical genomics.

Correction: Exome-wide association study reveals novel susceptibility genes to sporadic dilated cardiomyopathy.

[This corrects the article DOI: 10.1371/journal.pone.0172995.].

Whole blood levels of S1PR4 mRNA associated with cerebral vasospasm after aneurysmal subarachnoid hemorrhage.

OBJECTIVE: The authors sought to identify mRNA biomarkers of cerebral vasospasm in whole blood of patients suffering from aneurysmal subarachnoid hemorrhage (aSAH). METHODS: A prospective transcriptomic study for vasospasm was conducted in whole blood samples of 44 aSAH patients who developed (VSP+ group, n = 22) or did not develop (VSP- group, n = 22) vasospasm. Samples from all patients were profiled for 21,460 mRNA probes using the Illumina Human HT12v4.0 array. Differential statistical analysis was performed using a linear mixed model. RESULTS: Levels of sphingosine-1-phosphate receptor 4 (S1PR4) mRNA were significantly higher (p = 8.03 × 10-6) at presentation in patients who developed vasospasm after aSAH than in patients who did not. CONCLUSIONS: The results, which are consistent with findings of previous experimental investigations conducted in animal models, support the role of S1PR4 and its ligand, sphingosine-1-phosphate (S1P), in arterial-associated vasoconstriction, which suggests that S1PR4 could be used as a biomarker for cerebral vasospasm in aSAH patients.

Whole-Blood miRNA Sequencing Profiling for Vasospasm in Patients With Aneurysmal Subarachnoid Hemorrhage.

Background and Purpose- Arterial vasospasm is a well-known delayed complication of aneurysmal subarachnoid hemorrhage (aSAH). However, no validated biomarker exists to help clinicians discriminating patients with aSAH who will develop vasospasm (VSP+) and identifying those who then deserve aggressive preventive therapy. We hypothesized that whole-blood miRNAs could be a source of candidate biomarkers for vasospasm. Methods- Using a next-generation sequencing approach, we performed whole-blood miRNA profiling between VSP+patients with aSAH and patients who did not develop vasospasm (VSP-) in a prospective cohort of 32 patients. Profiling was performed on the admission day and 3 days before vasospasm. Results- Four hundred forty-two miRNAs were highly expressed in whole blood of patients with aSAH. Among them, hsa-miR-3177-3p demonstrated significant ( P=5.9×10-5; PBonferronicorrected=0.03) lower levels in VSP- compared with VSP+ patients. Looking for whole-blood mRNA correlates of hsa-miR-3177-3p, we observed some evidence that the decrease in hsa-miR-3177-3p levels after aSAH was associated with an increase in LDHA mRNA levels in VSP- ( P<10-3) but not in VSP+ ( P=0.66) patients. Conclusions- Whole-blood miRNA levels of hsa-miR-3177-3p could serve as a biomarker for vasospasm. Clinical Trial Registration- URL: https://www.clinicaltrials.gov . Unique identifier: NCT01779713.

Plasma levels of hsa-miR-152-3p are associated with diabetic nephropathy in patients with type 2 diabetes.

Background: MicroRNAs (miRNAs) are small non-coding RNAs participating in post-transcriptional regulation of genes. Their key role in modulating the susceptibility to human diseases is now widely recognized, in particular in the context of cardiometabolic disorders. The aim of the present study was to identify miRNAs associated with diabetic nephropathy (DN) in patients with type 2 diabetes (T2D). Methods: A next-generation sequencing-based miRNA profiling was performed in a case-control study for DN in plasma samples of 23 T2D patients with DN (cases) and 23 T2D without (controls). The main associations were confirmed using quantitative reverse transcription-polymerase chain reaction and tested for replication in an independent case-control collection of 100 T2D patients, 50 with DN and 50 without. Results: From the 381 known mature miRNAs that were found highly expressed in the discovery samples, we observed and replicated an association between increased plasma levels of hsa-miR-152-3p and DN (P = 4.03 × 10-4 in the combined samples). Hsa-miR-152-3p plasma levels were further found to be positively correlated (P = 0.003) to plasma osmolarity, a surrogate marker for solute carrier net activity, whose regulation is controlled by several genes including SLC5A3, one of the predicted targets of hsa-miR-152-3p. Conclusions: We observed strong evidence for the association of hsa-miR-152-3p plasma levels and DN in patients with T2D, confirming an association previously observed in patients with type 1 diabetes.

Exome-wide association study reveals novel susceptibility genes to sporadic dilated cardiomyopathy.

AIMS: Dilated cardiomyopathy (DCM) is an important cause of heart failure with a strong familial component. We performed an exome-wide array-based association study (EWAS) to assess the contribution of missense variants to sporadic DCM. METHODS AND RESULTS: 116,855 single nucleotide variants (SNVs) were analyzed in 2796 DCM patients and 6877 control subjects from 6 populations of European ancestry. We confirmed two previously identified associations with SNVs in BAG3 and ZBTB17 and discovered six novel DCM-associated loci (Q-value<0.01). The lead-SNVs at novel loci are common and located in TTN, SLC39A8, MLIP, FLNC, ALPK3 and FHOD3. In silico fine mapping identified HSPB7 as the most likely candidate at the ZBTB17 locus. Rare variant analysis (MAF<0.01) demonstrated significant association for TTN variants only (P = 0.0085). All candidate genes but one (SLC39A8) exhibit preferential expression in striated muscle tissues and mutations in TTN, BAG3, FLNC and FHOD3 are known to cause familial cardiomyopathy. We also investigated a panel of 48 known cardiomyopathy genes. Collectively, rare (n = 228, P = 0.0033) or common (n = 36, P = 0.019) variants with elevated in silico severity scores were associated with DCM, indicating that the spectrum of genes contributing to sporadic DCM extends beyond those identified here. CONCLUSION: We identified eight loci independently associated with sporadic DCM. The functions of the best candidate genes at these loci suggest that proteostasis regulation might play a role in DCM pathophysiology.

Role of lipid phosphate phosphatase 3 in human aortic endothelial cell function.

AIMS: Lipid phosphate phosphatase 3; type 2 phosphatidic acid phosphatase ß (LPP3; PPAP2B) is a transmembrane protein dephosphorylating and thereby terminating signalling of lipid substrates including lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P). Human LPP3 possesses a cell adhesion motif that allows interaction with integrins. A polymorphism (rs17114036) in PPAP2B is associated with coronary artery disease, which prompted us to investigate the possible role of LPP3 in human endothelial dysfunction, a condition promoting atherosclerosis. METHODS AND RESULTS: To study the role of LPP3 in endothelial cells we used human primary aortic endothelial cells (HAECs) in which LPP3 was silenced or overexpressed using either wild type or mutated cDNA constructs. LPP3 silencing in HAECs enhanced secretion of inflammatory cytokines, leucocyte adhesion, cell survival, and migration and impaired angiogenesis, whereas wild-type LPP3 overexpression reversed these effects and induced apoptosis. We also demonstrated that LPP3 expression was negatively correlated with vascular endothelial growth factor expression. Mutations in either the catalytic or the arginine-glycine-aspartate (RGD) domains impaired endothelial cell function and pharmacological inhibition of S1P or LPA restored it. LPA was not secreted in HAECs under silencing or overexpressing LPP3. However, the intra- and extra-cellular levels of S1P tended to be correlated with LPP3 expression, indicating that S1P is probably degraded by LPP3. CONCLUSIONS: We demonstrated that LPP3 is a negative regulator of inflammatory cytokines, leucocyte adhesion, cell survival, and migration in HAECs, suggesting a protective role of LPP3 against endothelial dysfunction in humans. Both the catalytic and the RGD functional domains were involved and S1P, but not LPA, might be the endogenous substrate of LPP3.

Preservation Analysis of Macrophage Gene Coexpression Between Human and Mouse Identifies PARK2 as a Genetically Controlled Master Regulator of Oxidative Phosphorylation in Humans.

Macrophages are key players involved in numerous pathophysiological pathways and an in-depth characterization of their gene regulatory networks can help in better understanding how their dysfunction may impact on human diseases. We here conducted a cross-species network analysis of macrophage gene expression data between human and mouse to identify conserved networks across both species, and assessed whether such networks could reveal new disease-associated regulatory mechanisms. From a sample of 684 individuals processed for genome-wide macrophage gene expression profiling, we identified 27 groups of coexpressed genes (modules). Six modules were found preserved (P < 10-4) in macrophages from 86 mice of the Hybrid Mouse Diversity Panel. One of these modules was significantly [false discovery rate (FDR) = 8.9 × 10-11] enriched for genes belonging to the oxidative phosphorylation (OXPHOS) pathway. This pathway was also found significantly (FDR < 10-4) enriched in susceptibility genes for Alzheimer, Parkinson, and Huntington diseases. We further conducted an expression quantitative trait loci analysis to identify SNP that could regulate macrophage OXPHOS gene expression in humans. This analysis identified the PARK2 rs192804963 as a trans-acting variant influencing (minimal P-value = 4.3 × 10-8) the expression of most OXPHOS genes in humans. Further experimental work demonstrated that PARK2 knockdown expression was associated with increased OXPHOS gene expression in THP1 human macrophages. This work provided strong new evidence that PARK2 participates to the regulatory networks associated with oxidative phosphorylation and suggested that PARK2 genetic variations could act as a trans regulator of OXPHOS gene macrophage expression in humans.

Removing Batch Effects from Longitudinal Gene Expression - Quantile Normalization Plus ComBat as Best Approach for Microarray Transcriptome Data.

Technical variation plays an important role in microarray-based gene expression studies, and batch effects explain a large proportion of this noise. It is therefore mandatory to eliminate technical variation while maintaining biological variability. Several strategies have been proposed for the removal of batch effects, although they have not been evaluated in large-scale longitudinal gene expression data. In this study, we aimed at identifying a suitable method for batch effect removal in a large study of microarray-based longitudinal gene expression. Monocytic gene expression was measured in 1092 participants of the Gutenberg Health Study at baseline and 5-year follow up. Replicates of selected samples were measured at both time points to identify technical variability. Deming regression, Passing-Bablok regression, linear mixed models, non-linear models as well as ReplicateRUV and ComBat were applied to eliminate batch effects between replicates. In a second step, quantile normalization prior to batch effect correction was performed for each method. Technical variation between batches was evaluated by principal component analysis. Associations between body mass index and transcriptomes were calculated before and after batch removal. Results from association analyses were compared to evaluate maintenance of biological variability. Quantile normalization, separately performed in each batch, combined with ComBat successfully reduced batch effects and maintained biological variability. ReplicateRUV performed perfectly in the replicate data subset of the study, but failed when applied to all samples. All other methods did not substantially reduce batch effects in the replicate data subset. Quantile normalization plus ComBat appears to be a valuable approach for batch correction in longitudinal gene expression data.

miR-322 regulates insulin signaling pathway and protects against metabolic syndrome-induced cardiac dysfunction in mice.

We identified murine miR-322, orthologous to human miR-424, as a new regulator of insulin receptor, IGF-1 receptor and sirtuin 4 mRNA in vitro and in vivo in the heart and found that miR-322/424 is highly expressed in the heart of mice. C57Bl/6N mice fed 10weeks of high fat diet (HFD) presented signs of cardiomyopathy and a stable miR-322 cardiac level while cardiac function was slightly affected in 11week-old ob/ob which overexpressed miR-322. We thus hypothesized that mmu-miR-322 could be protective against cardiac consequences of hyperinsulinemia and hyperlipidemia. We overexpressed or knocked-down mmu-miR-322 using AAV9 and monitored cardiac function in wild-type C57Bl/6N mice fed a control diet (CD) or a HFD and in ob/ob mice. The fractional shortening progressively declined while the left ventricle systolic diameter increased in HFD mice infected with an AAVcontrol or with an AAVsponge (decreasing miR-322 bioavailability) but also in ob/ob mice infected with AAVsponge. Similar observations were also found in CD-fed mice infected with AAVsponge. On the contrary over-expressing miR-322 with AAVmiR-322 was efficient in protecting the heart from HFD effects in C57Bl/6N mice. This cardioprotection could be associated with the regulation of identified targets IGF1R, INSR and CD1, a decrease in insulin signaling pathway and an enrichment of genes involved in mitochondrial function and fatty acid oxidation as demonstrated by transcriptome analysis. Altogether, these results emphasize miR-322 as a new potential therapeutic target against cardiac consequences of metabolic syndrome, which represents an increasing burden in the western countries.

Molecular Characterization of the NLRC4 Expression in Relation to Interleukin-18 Levels.

BACKGROUND: Interleukin-18 (IL-18) is a pleiotropic cytokine centrally involved in the cytokine cascade with complex immunomodulatory functions in innate and acquired immunity. Circulating IL-18 concentrations are associated with type 2 diabetes mellitus, cardiovascular events, and diverse inflammatory and autoimmune disorders. METHODS AND RESULTS: To identify causal variants affecting circulating IL-18 concentrations, we applied various omics and molecular biology approaches. By genome-wide association study, we confirmed association of IL-18 levels with a single nucleotide polymorphism in the untranslated exon 2 of the inflammasome component NLRC4 (NLR family, caspase recruitment domain-containing 4) gene on chromosome 2 (rs385076; P=2.4 × 10(-45)). Subsequent molecular analyses by gene expression analysis and reporter gene assays indicated an effect of rs385076 on NLRC4 expression and differential isoform usage by modulating binding of the transcription factor PU.1. CONCLUSIONS: Our study provides evidence for the functional causality of single nucleotide polymorphism rs385076 within the NLRC4 gene in relation to IL-18 activation.

Contribution of Rare and Common Genetic Variants to Plasma Lipid Levels and Carotid Stiffness and Geometry: A Substudy of the Paris Prospective Study 3.

BACKGROUND: We assess the contribution of common and rare putatively functional genetic variants (most of them coding) present on the Illumina exome Beadchip to the variability of plasma lipids and stiffness of the common carotid artery. METHODS AND RESULTS: Measurements were obtained from 2283 men and 1398 women, and after filtering and exclusion of monomorphic variants, 32,827 common (minor allele frequency >0.01) and 68,770 rare variants were analyzed. A large fraction of the heritability of plasma lipids is attributable to variants present on the array, especially for triglycerides (fraction of variance attributable to measured genotypes: V(G)/V(p)=31.4%, P<3.1×10(-11)) and high-density lipoprotein cholesterol (V(G)/V(p)=26.4%, P<4.2×10(-12)). Plasma lipids were associated with common variants located in known candidate genes, but no implication of rare variants could be established. Gene sets for plasma lipids, blood pressure, and coronary artery disease were defined on the basis of recent meta-analyses of genome-wide association studies. We observed a strong association between the plasma lipids gene set and plasma lipid variables, but none of the 3 genome-wide association studies gene sets was associated with the carotid parameters. Significant V(G)/V(p) ratios were observed for external (14.5%, P<2.7×10(-5)) and internal diameter (13.4%, P<4.3×10(-4)), stiffness (12.5%, P<8.0×10(-4)), intima-media thickness (10.6%, P<7.9×10(-4)), and wall cross-sectional area (13.2%, P<2.4×10(-5)). A significant association was observed between the common rs2903692 polymorphism of the CLEC16A gene and the internal diameter (P<4.3×10(-7)). CONCLUSIONS: These results suggest an involvement of CLEC16A, a gene that has been reported to be associated with immune disorders, in the modulation of carotid vasodilatation.

miR-421 and miR-30c inhibit SERPINE 1 gene expression in human endothelial cells.

In this work, we assessed whether SERPINE1 expression could be under the influence of microRNAs (miRNAs) predicted to bind the SERPINE1 3'UTR region. We specifically focused on the 3'UTR region harboring a common polymorphism, rs1050955, that have been found associated to SERPINE1 monocyte expression, and investigated whether the presence of different alleles at rs1050955 could modify the miRNAs binding efficiency and affect PAI-1 protein levels. We demonstrated that, in human umbilical vein endothelial cells, both miR-421 and miR-30c directly interacted with PAI-1 mRNA to inhibit the expression of the associated protein. However, these inhibitory mechanisms were independent on the allele present at the rs1050955 locus. We further showed that miR-421 levels correlated with PAI-1 activity in the plasma sample of 40 patients with venous thrombosis. Our results strongly suggest that the regulation of PAI-1 molecule could be under the influence of several miRNAs whose measurement in the plasma of patients could be envisaged as a biomarker for inflammatory and thrombotic disorders.