Daniela Ruggiero

BODY MASS INDEX IS DIRECTLY ASSOCIATED WITH BIOMARKERS OF ANGIOGENESIS AND INFLAMMATION IN CHILDREN AND ADOLESCENTS

OBJECTIVES Childhood obesity is associated with an increased risk of atherosclerosis, which can be mediated by an increase in angiogenesis and inflammation. The objective was to investigate the association between body mass index (BMI) and circulating biomarkers of angiogenesis, inflammation, and cardiac dysfunction in children and adolescents. METHODS The Genetic Park Study is a highly inclusive survey conducted in three isolated villages of southern Italy. One hundred fifty-one children and adolescents (age range 5-17 y, 45% male) were included and categorized as obese (BMI z-score ≥ 1.64, n = 38) or non-obese (n = 113). Metabolic and cardiovascular biomarkers included glucose, triacylglycerol, total cholesterol, high-density lipoprotein, vascular endothelial growth factor (VEGF), placental growth factor, soluble feline sarcoma virus (fms)-like tyrosine kinase-1, highly sensitive C reactive protein (hs-CRP), highly sensitive troponin T (hs-TnT), and N-terminal prohormone brain natriuretic peptide (NT-proBNP). RESULTS Obese subjects had higher levels of triacylglycerol (P = 0.03) and hs-CRP (P = 0.02) after adjustment for age and gender. Circulating levels of VEGF were directly associated with BMI z-score (r = 0.22, P = 0.007) and hs-CRP (r = 0.33, P < 0.001). BMI z-score was not associated with biomarkers of cardiac dysfunction (hs-TnT and NT-proBNP). CONCLUSION Increasing BMI was associated with plasma levels hs-CRP and VEGF, which are involved in the initiation and progression of atherosclerosis. The lack of association between BMI and markers of cardiac damage (hs-TnT) or ventricular volume overload (NT-proBNP) suggest that atherosclerotic risk may still at a preclinical stage in this population of obese but otherwise healthy young individuals. Collectively, this suite of biomarkers could provide mechanistic insights into the physiopathologic progression of cardiovascular risk associated with childhood obesity.

Meta-analysis of genome-wide association studies identifies common variants in CTNNA2 associated with Excitement-Seeking

The tendency to seek stimulating activities and intense sensations define excitement-seeking, a personality trait akin to some aspects of sensation-seeking. This trait is a central feature of extraversion and is a component of the multifaceted impulsivity construct. Those who score high on measures of excitement-seeking are more likely to smoke, use other drugs, gamble, drive recklessly, have unsafe/unprotected sex and engage in other risky behaviors of clinical and social relevance. To identify common genetic variants associated with the Excitement-Seeking scale of the Revised NEO Personality Inventory, we performed genome-wide association studies in six samples of European ancestry (N=7860), and combined the results in a meta-analysis. We identified a genome-wide significant association between the Excitement-Seeking scale and rs7600563 (P=2 × 10−8). This single-nucleotide polymorphism maps within the catenin cadherin-associated protein, alpha 2 (CTNNA2) gene, which encodes for a brain-expressed α-catenin critical for synaptic contact. The effect of rs7600563 was in the same direction in all six samples, but did not replicate in additional samples (N=5105). The results provide insight into the genetics of excitement-seeking and risk-taking, and are relevant to hyperactivity, substance use, antisocial and bipolar disorders.

Genetics of VEGF Serum Variation in Human Isolated Populations of Cilento: Importance of VEGF Polymorphisms

Vascular Endothelial Growth Factor (VEGF) is the main player in angiogenesis. Because of its crucial role in this process, the study of the genetic factors controlling VEGF variability may be of particular interest for many angiogenesis-associated diseases. Although some polymorphisms in the VEGF gene have been associated with a susceptibility to several disorders, no genome-wide search on VEGF serum levels has been reported so far. We carried out a genome-wide linkage analysis in three isolated populations and we detected a strong linkage between VEGF serum levels and the 6p21.1 VEGF region in all samples. A new locus on chromosome 3p26.3 significantly linked to VEGF serum levels was also detected in a combined population sample. A sequencing of the gene followed by an association study identified three common single nucleotide polymorphisms (SNPs) influencing VEGF serum levels in one population (Campora), two already reported in the literature (rs3025039, rs25648) and one new signal (rs3025020). A fourth SNP (rs41282644) was found to affect VEGF serum levels in another population (Cardile). All the identified SNPs contribute to the related population linkages (35% of the linkage explained in Campora and 15% in Cardile). Interestingly, none of the SNPs influencing VEGF serum levels in one population was found to be associated in the two other populations. These results allow us to exclude the hypothesis that the common variants located in the exons, intron-exon junctions, promoter and regulative regions of the VEGF gene may have a causal effect on the VEGF variation. The data support the alternative hypothesis of a multiple rare variant model, possibly consisting in distinct variants in different populations, influencing VEGF serum levels.

Angiogenesis and biomarkers of cardiovascular risk in adults with metabolic syndrome.

Abstract.  Siervo M, Ruggiero D, Sorice R, Nutile T, Aversano M, Stephan BCM, Ciullo M. (Address: Human Nutrition and Physiology, Department of Neuroscience, University “Federico II”, Faculty of Medicine, Napoli; Institute of Genetics and Biophysics “A. Buzzati‐Traverso”, CNR, Napoli, Italy; and Institute of Public Health, University of Cambridge, UK). Angiogenesis and biomarkers of cardiovascular risk in adults with metabolic syndrome. J Intern Med 2010; 268: 338–347.

Six Novel Loci Associated with Circulating VEGF Levels Identified by a Meta-analysis of Genome-Wide Association Studies

Vascular endothelial growth factor (VEGF) is an angiogenic and neurotrophic factor, secreted by endothelial cells, known to impact various physiological and disease processes from cancer to cardiovascular disease and to be pharmacologically modifiable. We sought to identify novel loci associated with circulating VEGF levels through a genome-wide association meta-analysis combining data from European-ancestry individuals and using a dense variant map from 1000 genomes imputation panel. Six discovery cohorts including 13,312 samples were analyzed, followed by in-silico and de-novo replication studies including an additional 2,800 individuals. A total of 10 genome-wide significant variants were identified at 7 loci. Four were novel loci (5q14.3, 10q21.3, 16q24.2 and 18q22.3) and the leading variants at these loci were rs114694170 (MEF2C, P = 6.79x10-13), rs74506613 (JMJD1C, P = 1.17x10-19), rs4782371 (ZFPM1, P = 1.59x10-9) and rs2639990 (ZADH2, P = 1.72x10-8), respectively. We also identified two new independent variants (rs34528081, VEGFA, P = 1.52x10-18; rs7043199, VLDLR-AS1, P = 5.12x10-14) at the 3 previously identified loci and strengthened the evidence for the four previously identified SNPs (rs6921438, LOC100132354, P = 7.39x10-1467; rs1740073, C6orf223, P = 2.34x10-17; rs6993770, ZFPM2, P = 2.44x10-60; rs2375981, KCNV2, P = 1.48x10-100). These variants collectively explained up to 52% of the VEGF phenotypic variance. We explored biological links between genes in the associated loci using Ingenuity Pathway Analysis that emphasized their roles in embryonic development and function. Gene set enrichment analysis identified the ERK5 pathway as enriched in genes containing VEGF associated variants. eQTL analysis showed, in three of the identified regions, variants acting as both cis and trans eQTLs for multiple genes. Most of these genes, as well as some of those in the associated loci, were involved in platelet biogenesis and functionality, suggesting the importance of this process in regulation of VEGF levels. This work also provided new insights into the involvement of genes implicated in various angiogenesis related pathologies in determining circulating VEGF levels. The understanding of the molecular mechanisms by which the identified genes affect circulating VEGF levels could be important in the development of novel VEGF-related therapies for such diseases.

Genetic and environmental factors influencing the Placental Growth Factor (PGF) variation in two populations.

Placental Growth Factor (PGF) is a key molecule in angiogenesis. Several studies have revealed an important role of PGF primarily in pathological conditions (e.g.: ischaemia, tumour formation, cardiovascular diseases and inflammatory processes) suggesting its use as a potential therapeutic agent. However, to date, no information is available regarding the genetics of PGF variability. Furthermore, even though the effect of environmental factors (e.g.: cigarette smoking) on angiogenesis has been explored, no data on the influence of these factors on PGF levels have been reported so far. Here we have first investigated PGF variability in two cohorts focusing on non-genetic risk factors: a study sample from two isolated villages in the Cilento region, South Italy (N = 871) and a replication sample from the general Danish population (N = 1,812). A significant difference in PGF mean levels was found between the two cohorts. However, in both samples, we observed a strong correlation of PGF levels with ageing and sex, men displaying PGF levels significantly higher than women. Interestingly, smoking was also found to influence the trait in the two populations, although differently. We have then focused on genetic risk factors. The association between five single nucleotide polymorphisms (SNPs) located in the PGF gene and the plasma levels of the protein was investigated. Two polymorphisms (rs11850328 and rs2268614) were associated with the PGF plasma levels in the Cilento sample and these associations were strongly replicated in the Danish sample. These results, for the first time, support the hypothesis of the presence of genetic and environmental factors influencing PGF plasma variability.

Genetic variants modulating CRIPTO serum levels identified by genome-wide association study in Cilento isolates.

Cripto, the founding member of the EGF-CFC genes, plays an essential role in embryo development and is involved in cancer progression. Cripto is a GPI-anchored protein that can interact with various components of multiple signaling pathways, such as TGF-β, Wnt and MAPK, driving different processes, among them epithelial-mesenchymal transition, cell proliferation, and stem cell renewal. Cripto protein can also be cleaved and released outside the cell in a soluble and still active form. Cripto is not significantly expressed in adult somatic tissues and its re-expression has been observed associated to pathological conditions, mainly cancer. Accordingly, CRIPTO has been detected at very low levels in the plasma of healthy volunteers, whereas its levels are significantly higher in patients with breast, colon or glioblastoma tumors. These data suggest that CRIPTO levels in human plasma or serum may have clinical significance. However, very little is known about the variability of serum levels of CRIPTO at a population level and the genetic contribution underlying this variability remains unknown. Here, we report the first genome-wide association study of CRIPTO serum levels in isolated populations (n = 1,054) from Cilento area in South Italy. The most associated SNPs (p-value<5*10-8) were all located on chromosome 3p22.1-3p21.3, in the CRIPTO gene region. Overall six CRIPTO associated loci were replicated in an independent sample (n = 535). Pathway analysis identified a main network including two other genes, besides CRIPTO, in the associated regions, involved in cell movement and proliferation. The replicated loci explain more than 87% of the CRIPTO variance, with 85% explained by the most associated SNP. Moreover, the functional analysis of the main associated locus identified a causal variant in the 5’UTR of CRIPTO gene which is able to strongly modulate CRIPTO expression through an AP-1-mediate transcriptional regulation.

A transnational collaborative network dedicated to the study and applications of the vascular endothelial growth factor-A in medical practice: the VEGF Consortium

Maria G. Stathopoulou, Ting Xie, Daniela Ruggiero, Jerome Chatelin, Marc Rancier, George Weryha, Mary Jo Kurth, Alex-Ander Aldasoro Arguinano, Vesna Gorenjak, Alexandros M. Petrelis, Georges Dagher, George Dedoussis, Panagiotis Deloukas, John Lamont, Janja Marc, Maurizio Simmaco, Ron H.N. van Schaik, Federico Innocenti, Jean-Louis Merlin, Jochen Schneider, Behrooz Ziad Alizadeh, Marina Ciullo, Sudha Seshadri and Sophie Visvikis-Siest*, The VEGF Consortium A transnational collaborative network dedicated to the study and applications of the vascular endothelial growth factor-A in medical practice: the VEGF Consortium