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Germline variants in the SEMA4A gene predispose to familial colorectal cancer type X.

Schulz E, Klampfl P, Holzapfel S, Janecke AR, Ulz P, Renner W, Kashofer K, Nojima S, Leitner A, Zebisch A, Wölfler A, Hofer S, Gerger A, Lax S, Beham-Schmid C, Steinke V, Heitzer E, Geigl JB, Windpassinger C, Hoefler G, Speicher MR, Boland CR, Kumanogoh A, Sill H - Nat Commun (2014)

Bottom Line: Here we identify the SEMA4A p.Val78Met germline mutation in an Austrian kindred with FCCTX, using an integrative genomics strategy.In a cohort of 53 patients with FCCTX, we depict two further SEMA4A mutations, p.Gly484Ala and p.Ser326Phe and the single-nucleotide polymorphism (SNP) p.Pro682Ser.Our study shows previously unidentified germline variants in SEMA4A predisposing to FCCTX, which has implications for surveillance strategies of patients and their families.

View Article: PubMed Central - PubMed

Affiliation: Division of Hematology, Department of Internal Medicine, Medical University of Graz, A-8036 Graz, Austria.

ABSTRACT
Familial colorectal cancer type X (FCCTX) is characterized by clinical features of hereditary non-polyposis colorectal cancer with a yet undefined genetic background. Here we identify the SEMA4A p.Val78Met germline mutation in an Austrian kindred with FCCTX, using an integrative genomics strategy. Compared with wild-type protein, SEMA4A(V78M) demonstrates significantly increased MAPK/Erk and PI3K/Akt signalling as well as cell cycle progression of SEMA4A-deficient HCT-116 colorectal cancer cells. In a cohort of 53 patients with FCCTX, we depict two further SEMA4A mutations, p.Gly484Ala and p.Ser326Phe and the single-nucleotide polymorphism (SNP) p.Pro682Ser. This SNP is highly associated with the FCCTX phenotype exhibiting increased risk for colorectal cancer (OR 6.79, 95% CI 2.63 to 17.52). Our study shows previously unidentified germline variants in SEMA4A predisposing to FCCTX, which has implications for surveillance strategies of patients and their families.

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SEMA4AV78M shows normal surface expression and leads to cell cycle changes in HCT-116 cells.(a) ARPE-19 cells were transfected with the plasmid constructs expressing Sema4AWT-EGFP or Sema4AV78M-EGFP proteins, incubated for 48 h and stained with phalloidin. Green, Sema4A-EGFP; red, phalloidin (actin). Representative images obtained by confocal microscopy are shown. The size of the scale bar is 20 μm. (b,c) Representative density plots and statistical analysis of GFP-positive SEMA4A-transfected HCT-116 cells stained by 7-AAD and APC anti-BrdU antibodies for cell cycle analysis. Cells were analysed 48 h after transfection. Significantly, more SEMA4AV78M than SEMA4AWT-transfected cells are in S phase and significantly less in G2/M phase, respectively (mean±s.e.m.; n=3 per group; two-tailed paired Student's t-test; *P<0.05 compared with WT). Cell cycle phase: Sub-G1 (R1), G1/G0 (R2), S (R3), G2/M (R4). (d,e) Representative immunoblots and statistical analysis of SEMA4A-transfected HCT-116 cells (whole-cell lysates) lyzed 48 h after transfection. SEMA4AV78M-transfected cells show increased phosphorylation of Akt and Erk (mean±s.e.m.; n=6 per group; two-tailed paired Student's t-test; *P<0.05 compared with WT). (p-)GSK3β and (active) β-catenin proteins were blotted on a separate membrane in this experiment. No effects on GSK3β and β-catenin phosphorylation were seen in repeated experiments.
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f4: SEMA4AV78M shows normal surface expression and leads to cell cycle changes in HCT-116 cells.(a) ARPE-19 cells were transfected with the plasmid constructs expressing Sema4AWT-EGFP or Sema4AV78M-EGFP proteins, incubated for 48 h and stained with phalloidin. Green, Sema4A-EGFP; red, phalloidin (actin). Representative images obtained by confocal microscopy are shown. The size of the scale bar is 20 μm. (b,c) Representative density plots and statistical analysis of GFP-positive SEMA4A-transfected HCT-116 cells stained by 7-AAD and APC anti-BrdU antibodies for cell cycle analysis. Cells were analysed 48 h after transfection. Significantly, more SEMA4AV78M than SEMA4AWT-transfected cells are in S phase and significantly less in G2/M phase, respectively (mean±s.e.m.; n=3 per group; two-tailed paired Student's t-test; *P<0.05 compared with WT). Cell cycle phase: Sub-G1 (R1), G1/G0 (R2), S (R3), G2/M (R4). (d,e) Representative immunoblots and statistical analysis of SEMA4A-transfected HCT-116 cells (whole-cell lysates) lyzed 48 h after transfection. SEMA4AV78M-transfected cells show increased phosphorylation of Akt and Erk (mean±s.e.m.; n=6 per group; two-tailed paired Student's t-test; *P<0.05 compared with WT). (p-)GSK3β and (active) β-catenin proteins were blotted on a separate membrane in this experiment. No effects on GSK3β and β-catenin phosphorylation were seen in repeated experiments.

Mentions: Compound heterozygous germline mutations in SEMA4A have been reported in patients with retinal degenerative diseases and studies in knock-in mice showed that one of these mutations (F350C) leads to an abnormal Sema4A localization in retinal pigment epithelial cells1718. A three-dimensional protein model of human SEMA4A predicts that Val78 has no spatial relationship to residues associated with retinal disorders (Fig. 2b). In agreement with this prediction and the family's history lacking apparent ocular manifestations, the expression of a fusion gene composed of Sema4AV78M and carboxyl-terminal green fluorescent protein (GFP) in human retinal ARPE-19 cells showed normal GFP signal distribution (Fig. 4a).


Germline variants in the SEMA4A gene predispose to familial colorectal cancer type X.

Schulz E, Klampfl P, Holzapfel S, Janecke AR, Ulz P, Renner W, Kashofer K, Nojima S, Leitner A, Zebisch A, Wölfler A, Hofer S, Gerger A, Lax S, Beham-Schmid C, Steinke V, Heitzer E, Geigl JB, Windpassinger C, Hoefler G, Speicher MR, Boland CR, Kumanogoh A, Sill H - Nat Commun (2014)

SEMA4AV78M shows normal surface expression and leads to cell cycle changes in HCT-116 cells.(a) ARPE-19 cells were transfected with the plasmid constructs expressing Sema4AWT-EGFP or Sema4AV78M-EGFP proteins, incubated for 48 h and stained with phalloidin. Green, Sema4A-EGFP; red, phalloidin (actin). Representative images obtained by confocal microscopy are shown. The size of the scale bar is 20 μm. (b,c) Representative density plots and statistical analysis of GFP-positive SEMA4A-transfected HCT-116 cells stained by 7-AAD and APC anti-BrdU antibodies for cell cycle analysis. Cells were analysed 48 h after transfection. Significantly, more SEMA4AV78M than SEMA4AWT-transfected cells are in S phase and significantly less in G2/M phase, respectively (mean±s.e.m.; n=3 per group; two-tailed paired Student's t-test; *P<0.05 compared with WT). Cell cycle phase: Sub-G1 (R1), G1/G0 (R2), S (R3), G2/M (R4). (d,e) Representative immunoblots and statistical analysis of SEMA4A-transfected HCT-116 cells (whole-cell lysates) lyzed 48 h after transfection. SEMA4AV78M-transfected cells show increased phosphorylation of Akt and Erk (mean±s.e.m.; n=6 per group; two-tailed paired Student's t-test; *P<0.05 compared with WT). (p-)GSK3β and (active) β-catenin proteins were blotted on a separate membrane in this experiment. No effects on GSK3β and β-catenin phosphorylation were seen in repeated experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4214414&req=5

f4: SEMA4AV78M shows normal surface expression and leads to cell cycle changes in HCT-116 cells.(a) ARPE-19 cells were transfected with the plasmid constructs expressing Sema4AWT-EGFP or Sema4AV78M-EGFP proteins, incubated for 48 h and stained with phalloidin. Green, Sema4A-EGFP; red, phalloidin (actin). Representative images obtained by confocal microscopy are shown. The size of the scale bar is 20 μm. (b,c) Representative density plots and statistical analysis of GFP-positive SEMA4A-transfected HCT-116 cells stained by 7-AAD and APC anti-BrdU antibodies for cell cycle analysis. Cells were analysed 48 h after transfection. Significantly, more SEMA4AV78M than SEMA4AWT-transfected cells are in S phase and significantly less in G2/M phase, respectively (mean±s.e.m.; n=3 per group; two-tailed paired Student's t-test; *P<0.05 compared with WT). Cell cycle phase: Sub-G1 (R1), G1/G0 (R2), S (R3), G2/M (R4). (d,e) Representative immunoblots and statistical analysis of SEMA4A-transfected HCT-116 cells (whole-cell lysates) lyzed 48 h after transfection. SEMA4AV78M-transfected cells show increased phosphorylation of Akt and Erk (mean±s.e.m.; n=6 per group; two-tailed paired Student's t-test; *P<0.05 compared with WT). (p-)GSK3β and (active) β-catenin proteins were blotted on a separate membrane in this experiment. No effects on GSK3β and β-catenin phosphorylation were seen in repeated experiments.
Mentions: Compound heterozygous germline mutations in SEMA4A have been reported in patients with retinal degenerative diseases and studies in knock-in mice showed that one of these mutations (F350C) leads to an abnormal Sema4A localization in retinal pigment epithelial cells1718. A three-dimensional protein model of human SEMA4A predicts that Val78 has no spatial relationship to residues associated with retinal disorders (Fig. 2b). In agreement with this prediction and the family's history lacking apparent ocular manifestations, the expression of a fusion gene composed of Sema4AV78M and carboxyl-terminal green fluorescent protein (GFP) in human retinal ARPE-19 cells showed normal GFP signal distribution (Fig. 4a).

Bottom Line: Here we identify the SEMA4A p.Val78Met germline mutation in an Austrian kindred with FCCTX, using an integrative genomics strategy.In a cohort of 53 patients with FCCTX, we depict two further SEMA4A mutations, p.Gly484Ala and p.Ser326Phe and the single-nucleotide polymorphism (SNP) p.Pro682Ser.Our study shows previously unidentified germline variants in SEMA4A predisposing to FCCTX, which has implications for surveillance strategies of patients and their families.

View Article: PubMed Central - PubMed

Affiliation: Division of Hematology, Department of Internal Medicine, Medical University of Graz, A-8036 Graz, Austria.

ABSTRACT
Familial colorectal cancer type X (FCCTX) is characterized by clinical features of hereditary non-polyposis colorectal cancer with a yet undefined genetic background. Here we identify the SEMA4A p.Val78Met germline mutation in an Austrian kindred with FCCTX, using an integrative genomics strategy. Compared with wild-type protein, SEMA4A(V78M) demonstrates significantly increased MAPK/Erk and PI3K/Akt signalling as well as cell cycle progression of SEMA4A-deficient HCT-116 colorectal cancer cells. In a cohort of 53 patients with FCCTX, we depict two further SEMA4A mutations, p.Gly484Ala and p.Ser326Phe and the single-nucleotide polymorphism (SNP) p.Pro682Ser. This SNP is highly associated with the FCCTX phenotype exhibiting increased risk for colorectal cancer (OR 6.79, 95% CI 2.63 to 17.52). Our study shows previously unidentified germline variants in SEMA4A predisposing to FCCTX, which has implications for surveillance strategies of patients and their families.

Show MeSH
Related in: MedlinePlus