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

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

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

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

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

The rs112481213 variant alter the AP-1 binding site.

<p><b>(A)</b> Electrophoretic mobility shift assay (EMSA) for AP-1 binding in nuclear extracts (NEs) of PC-3 cell line. The binding activities of NEs (lanes 2–14) were analyzed by EMSA using biotin-labeled AP-1 probe (lanes 2–4), -222 T probe (lanes 5–9) and-222 A probe (lanes 10–14). Competition was performed with 50 and 200 fold molar excess of the cold probe (lanes 3–4, 6–7, 11–12). Cross competition was performed using the cold probe carrying the alternative allele (lane 8 and 13) and the cold scrambled probe (lane 9 and lane 14) at 200 fold molar excess. No binding was detectable for-222 T probe (lanes 5–9). The DNA-protein complex of-222 A probe (lane 10) can be eliminated by 50 or 200 fold molar excess of cold-222 A probe (lanes 11, 12) but not by 200 fold molar excess of cold-222 T probe and cold scrambled probe. <b>(B)</b> Specificity of AP-1 binding activity. Supershift assay was carried out for-222 A probe using specific antibodies for the AP-1 components c-Jun (lane 3), JunB (lane 4), JunD (lane 5), Fra-1 (lane 6) and Fra-2 (lane 8). A supershifted DNA-protein-antibody complex (indicated by the arrow) is observed (lanes 3, 5, 6, 8). Lane 7 shows that the specific complexes were eliminated by addition of 200 fold molar excess of cold-222 A probe. No supershifted complex was observed adding HA-probe antibody used as negative control (lane 9). The results are representative of three independent determinations.</p