Figure 1.
Identified FGFR2 mutation in tumor tissue.
The upper sequence chromatogram shows the heterozygous missense mutation in exon 14 in FGFR2, c.1980G>C (p.K660N), found in somatic tumor breast tissue of patient BC80. The middle and lower chromatograms illustrate the normal sequence in non-tumor breast tissue and blood-derived DNA of the same patient.
Figure 2.
Location and conservation of FGFR2 mutations. A) Schematic model of FGFR2 with bound ligand (FGF).
The locations of the novel p.K660N and p.R203C mutations are marked by red dots. TK1/2: tyrosine kinase domains 1 and 2; D1–3: immunoglobulin-like domains 1–3. B) Conservation of FGFR2 mutations. Arrows indicate localization of mutations. Above CLUSTALW alignment of vertebrate FGFR2s and human FGFRs. Below: ConSeq prediction. Amino acid conservation grade is colour-coded. The predicted status of each residue, buried (b) or exposed (e), is marked below the amino acid sequence. Slowly evolving and exposed residues are predicted to be functional (f), whereas slowly evolving and buried residues are predicted to be structurally important (s).
Figure 3.
Representative MLPA chromatogram and quantification with SequencePilot software. The probe mix contained 34 probes, 6 hybridizing to the FGF10 gene, 8 to the FGFR2 gene, and 20 controls hybridizing to single-copy genes located on other chromosomes. Peak areas lower than 75% of the controls are indicative for a deletion, peak area higher than 125% of the controls for a duplication.
Figure 4.
FGFR2 mRNA isoform expression analysis in tumor tissue of three sporadic breast cancer patients. A) Representative electropherogram of GeneScan analysis.
Isoforms FGFR2-IIIb and FGFR2-IIIc differ in exon 8, resulting in a variation of 3 bp in length of mature mRNA. A PCR fragment of 297 bp for FGFR2-IIIb or 294 bp for FGFR2-IIIc cDNA spanning exon 8 of both isoforms was amplified by PCR using a fluorescently-labeled primer pair located in exons 7 and 9, which are common in both isoforms. Fragment analysis showing a single sharp peak. B) Representative sequence electropherogram showing the expression of IIIb isoform.
Figure 5.
Activating FGFR2 breast cancer mutations.
A) Representative Western blot showing increased tyrosine kinase activities of FGFR2-IIIb breast cancer mutants compared to tyrosine kinase activities of wt FGFR2-IIIb (WT), of a kinase defective (KD) FGFR2-IIIb mutant, and of a Pfeiffer syndrome gain-of-function mutant (K642R, previously named K641R according to another reference sequence) as controls. HEK293 cells were transiently transfected using lipofectamine with pRK5 vectors containing cDNA coding for wt FGFR2-IIIb or FGFR2-IIIb variants containing the indicated amino acid substitution. Untransfected cells served as negative control (−). Lysates from cells were subjected to immunoprecipitation with anti-FGFR2 antibodies (Bek(C-17)) followed by SDS-PAGE and immunoblotting (IB) with anti-FGFR2 or anti-p-Tyr antibodies (PY99). B) Representative Western blots showing increased substrate phosphorylation by FGFR2-IIIb breast cancer mutants compared to substrate phosphorylation by wt FGFR2-IIIb (WT), a kinase defective (KD) FGFR2-IIIb mutant, and a Pfeiffer syndrome gain-of-function mutant (K642R, previously named K641R according to another reference sequence) as controls. For transient transfection calcium phosphate was used. Untransfected cells served as negative control (−). HEK293 cells expressing FGFR2-IIIb as well as untransfected cells were stimulated with FGF1. Lysates of unstimulated or FGF1-stimulated cells were subjected to SDS-PAGE followed by immunoblotting with anti-p-FRS2, anti-p-MEK1/2, anti-p-STAT3 and anti-ß-Actin as loading control.