PGF levels according to sex in the Cilento and Denmark samples.

<p>Mean levels of the log-transformed PGF and the standard error are reported for each gender and population sample. A univariate analysis of variance including the log-PGF as a dependent variable, sex and village as fixed factors and age, menstruation, smoking and “disease status” as covariates was performed. The corresponding p-value for the PGF level difference between the population samples is shown in the left corner of the plot.</p

Association results between the SNPs in the <i>PGF</i> gene and the protein levels according to the best fitting models for the Cilento and Denmark samples.

<p>Association for the five <i>PGF</i> SNPs and the protein levels is reported for Cilento; only the SNPs associated in Cilento were tested in Denmark. Statistical associations are all adjusted for age, sex, menstruation, smoking, sex/smoking interaction (only for Cilento) and “disease status”.</p>*<p>Minor allele referred to the reverse strand according to <i>PGF</i> position.</p>†<p>The CEU sample was chosen as reference population of the HapMap data.</p>‡<p>Test corrected for relatedness between individuals. Multiple testing p-value threshold = 0.0127. Significant results are given in bold.</p>§<p>P-value from meta-analysis (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042537#s2" target="_blank">Material and Methods</a> section for details).</p

PGF level according to smoking and sex in Cilento and Denmark.

<p>Mean levels of the log-transformed PGF and the standard error are reported. A linear regression model includes the log-PGF as a dependent variable, age, sex, menstruation, smoking, sex/smoking interaction, and “disease status” as independent variables for the Cilento sample (A) and age, sex, menstruation, smoking, and “disease status” as regressors for the Denmark sample (B). The p-values for sex, smoking and sex/smoking interaction are shown.</p

Genetic and Environmental Factors Influencing the Placental Growth Factor (PGF) Variation in Two Populations

<div><p>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 <em>PGF</em> 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.</p> </div

Cumulative effect of the environmental and genetic factors on the PGF levels.

<p>Mean PGF levels (right vertical axis) are shown as solid black dots connected by solid lines for categories of the cumulative risk score. The standard error is reported as error bar. The shaded bars show the distribution of the cumulative risk score in the whole population (left vertical axis) in the Cilento (A) and Denmark (B) sample.</p

The baseline characteristics of the samples.

<p>The mean and the standard deviation (SD) are reported for the age, the median and the interquartile range (IQR) for the PGF levels.</p>*<p>Plasma levels for Cilento and serum levels for Denmark.</p

Genetic Variants Modulating CRIPTO Serum Levels Identified by Genome-Wide Association Study in Cilento Isolates

<div><p><i>Cripto</i>, 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. <i>Cripto</i> 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 <i>CRIPTO</i> 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 <i>CRIPTO</i>, 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 <i>CRIPTO</i> gene which is able to strongly modulate <i>CRIPTO</i> expression through an AP-1-mediate transcriptional regulation.</p></div

Functional analysis of the associated genes.

<p>The network was algorithmically constructed by Ingenuity Pathway Analysis (IPA) software on the basis of the functional and biological connectivity of genes. The network highlights the interconnections of 3 loci (marked in grey) identified from serum CRIPTO levels GWAS with a p-value = 1*10^–6. <i>CRIPTO</i> gene is reported as <i>TDGF1</i>. Lines between genes represent known interactions and the nodes are displayed using various shapes that represent the functional class of the gene product (legend).</p

Transcriptional activity and schematic representation of reporter constructs.

<p><b>(A)</b> The two constructs (222A/luc and-222T/luc) contain a 1,051 bp region including the 5’UTR and 342 bp upstream the transcription start site of <i>CRIPTO</i> and differ for the rs112481213 allele. The-222A→T/luc and 222T→A/luc constructs derive from a site-directed mutagenesis at the rs112481213 SNP position. <b>(B)</b> NTERA2 cell line was transfected with firefly luciferase reporter constructs carrying 1,051bp of the <i>CRIPTO</i> gene with either the A allele or the T allele at rs112481213. The rs112481213 A allele produced a 5-fold increase in luciferase activity compared to the T allele. Site-mutated constructs at the rs112481213 allowed to discriminate the effect of rs112481213 and rs3806703 on the transcription. Data are presented as fold-induction compared with promoter-less vector (pGL3-BV). Data are shown as mean±SD from five experiments.</p

Manhattan plot of genome-wide association results in discovery analysis.

<p>Truncated Manhattan Plot showing -log₁₀(p-values) for all SNPs of the CRIPTO discovery GWAS ordered by their chromosomal position. The dashed horizontal line represents the threshold for genome-wide significance (p-value<5*10^-8). SNPs associated at the genome-wide significance level are all located on chromosome 3 (lowest p-value = 1.03*10^–159).</p