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

Ruggiero D, Nappo S, Nutile T, Sorice R, Talotta F, Giorgio E, Bellenguez C, Leutenegger AL, Liguori GL, Ciullo M - PLoS Genet. (2015)

Bottom Line: Overall six CRIPTO associated loci were replicated in an independent sample (n = 535).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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Genetics and Biophysics A. Buzzati-Traverso, CNR, Naples, Italy.

ABSTRACT
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.

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Transcriptional activity and schematic representation of reporter constructs.(A) 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 CRIPTO 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) NTERA2 cell line was transfected with firefly luciferase reporter constructs carrying 1,051bp of the CRIPTO 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.
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pgen.1004976.g003: Transcriptional activity and schematic representation of reporter constructs.(A) 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 CRIPTO 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) NTERA2 cell line was transfected with firefly luciferase reporter constructs carrying 1,051bp of the CRIPTO 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.

Mentions: The top 8 associated SNPs in the CRIPTO gene region were all included in a unique LD block (S3A–S3B Fig.). Among them the rs112481213 variant (LD with rs3806702, r2 = 0.99, p-value of association = 1.53*10–158), reported to be in a regulative region from ENCODE data (see the above paragraph), was also predicted to create an AP-1 binding site by bioinformatics analysis with MatInspector program and TRANSFAC database (S3C Fig.). To explore the possibility that this SNP, located in the 5’UTR of CRIPTO gene, might influence CRIPTO transcription we tested the SNP allele effect on the transcriptional activity in the NTERA2 teratocarcinoma cell line, expressing high levels of CRIPTO [38]. Two constructs, both containing a 1,051 bp region upstream the CRIPTO ATG start codon (including almost all the 5’UTR and also 342 bp upstream the transcription start site), but differing for the rs112481213 allele (-222A/luc and-222T/luc, respectively) were transfected in NTERA2 cells. Overall, 15 SNPs were included in that region. As one of those SNPs, the rs3806703 variant was in linkage disequilibrium with rs112481213 (r2 = 0.72), two additional constructs (-222T→A/luc, -222A→T/luc) were also produced by site-directed mutagenesis to discriminate the effect of each of the two variants on the transcription efficiency (Fig. 3A). The construct containing the rs112481213 A allele (-222A/luc) produced an about 5-fold increase (p-value = 9.2*10–6) of the luciferase activity compared to the construct containing the T allele (-222T/luc) indicating that rs112481213 SNP affects the transcription (Fig. 3B). Also, the luciferase activity was reported to high levels in the site-mutated-222T→A/luc construct demonstrating that the main effect on the transcription can be attributed to the rs112481213 variant. Interestingly, the activity produced by the-222T→A/luc is significantly higher than that observed for the-222A/luc (p-value = 2.9*10–2) and inverse results were obtained for-222A→T/luc and-222T/luc (p-value = 5.8*10–4), indicating that rs3806703 might also have an effect, although modest, on the transcription. A cooperative role of rs112481213 and rs3806703 was also statistically supported by an interaction model tested for the two variants (βrs112481213 = -1.18, SErs112481213 = 0.04, βrs3806703 = 0.27, SErs3806703 = 0.06, βinter = -0.15, SEinter = 0.03, p-valueinter = 7.08*10–8).


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

Ruggiero D, Nappo S, Nutile T, Sorice R, Talotta F, Giorgio E, Bellenguez C, Leutenegger AL, Liguori GL, Ciullo M - PLoS Genet. (2015)

Transcriptional activity and schematic representation of reporter constructs.(A) 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 CRIPTO 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) NTERA2 cell line was transfected with firefly luciferase reporter constructs carrying 1,051bp of the CRIPTO 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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4309561&req=5

pgen.1004976.g003: Transcriptional activity and schematic representation of reporter constructs.(A) 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 CRIPTO 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) NTERA2 cell line was transfected with firefly luciferase reporter constructs carrying 1,051bp of the CRIPTO 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.
Mentions: The top 8 associated SNPs in the CRIPTO gene region were all included in a unique LD block (S3A–S3B Fig.). Among them the rs112481213 variant (LD with rs3806702, r2 = 0.99, p-value of association = 1.53*10–158), reported to be in a regulative region from ENCODE data (see the above paragraph), was also predicted to create an AP-1 binding site by bioinformatics analysis with MatInspector program and TRANSFAC database (S3C Fig.). To explore the possibility that this SNP, located in the 5’UTR of CRIPTO gene, might influence CRIPTO transcription we tested the SNP allele effect on the transcriptional activity in the NTERA2 teratocarcinoma cell line, expressing high levels of CRIPTO [38]. Two constructs, both containing a 1,051 bp region upstream the CRIPTO ATG start codon (including almost all the 5’UTR and also 342 bp upstream the transcription start site), but differing for the rs112481213 allele (-222A/luc and-222T/luc, respectively) were transfected in NTERA2 cells. Overall, 15 SNPs were included in that region. As one of those SNPs, the rs3806703 variant was in linkage disequilibrium with rs112481213 (r2 = 0.72), two additional constructs (-222T→A/luc, -222A→T/luc) were also produced by site-directed mutagenesis to discriminate the effect of each of the two variants on the transcription efficiency (Fig. 3A). The construct containing the rs112481213 A allele (-222A/luc) produced an about 5-fold increase (p-value = 9.2*10–6) of the luciferase activity compared to the construct containing the T allele (-222T/luc) indicating that rs112481213 SNP affects the transcription (Fig. 3B). Also, the luciferase activity was reported to high levels in the site-mutated-222T→A/luc construct demonstrating that the main effect on the transcription can be attributed to the rs112481213 variant. Interestingly, the activity produced by the-222T→A/luc is significantly higher than that observed for the-222A/luc (p-value = 2.9*10–2) and inverse results were obtained for-222A→T/luc and-222T/luc (p-value = 5.8*10–4), indicating that rs3806703 might also have an effect, although modest, on the transcription. A cooperative role of rs112481213 and rs3806703 was also statistically supported by an interaction model tested for the two variants (βrs112481213 = -1.18, SErs112481213 = 0.04, βrs3806703 = 0.27, SErs3806703 = 0.06, βinter = -0.15, SEinter = 0.03, p-valueinter = 7.08*10–8).

Bottom Line: Overall six CRIPTO associated loci were replicated in an independent sample (n = 535).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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Genetics and Biophysics A. Buzzati-Traverso, CNR, Naples, Italy.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus