Identification of the molecular basis of the lacrimo-auriculo-dento-digital (LADD) syndrome

Lacrimo-auriculo-dento-digital (LADD) syndrome, also known as Levy-Hollister syndrome, is a rare autosomal dominant developmental disorder, mainly characterized by abnormalities of the lacrimal system and salivary glands, ears and hearing, teeth and distal limb development. Besides these cardinal features, facial dysmorphism and malformations of the kidney and the respiratory system have been reported. In this study, the LADD1 locus was mapped to chromosome 10q26 by genome wide linkage analysis using the Affymetrix GeneChip 10K array in three large LADD families. In all three LADD families and in one sporadic case, heterozygous missense mutations were found in exon 16 of the gene encoding the fibroblast-growth-factor-receptor 2 (FGFR2). After exclusion of the FGFR2 locus by haplotype analysis in two additional LADD families, one missense mutation was identified in FGFR3 and one mutation was found in the fibroblast-growth-factor 10 (FGF10), a known ligand of FGFR2 [Rohmann et al., 2006]. The functional properties of FGF10 LADD and FGFR2 LADD mutants were analyzed and compared to the activities of their normal counterparts. Protein expression in BL21 cells and binding studies showed that each of the three analyzed FGF10 mutations demonstrated severely impaired activity by different mechanisms. Transient and stable expression studies exhibited that the FGFR2 mutations possess a reduced autophosphorylation and a weaker tyrosine kinase activity. Mutations also lead to diminished phosphorylation activity in FGFR2-mediated substrates (e. g. FRS2 and Shc) and to a decreased downstream signaling pathway, as shown by MAPK activity. While tested FGF10 LADD mutations caused haploinsufficiency, the FGFR2 LADD mutants could exert a dominant-negative effect on normal FGFR2 protein [Shams and Rohmann et al., 2007]. An in vitro kinase assay and crystallization of both, FGFR2 WT and the p.A628T missense mutation in the catalytic part of the tyrosine kinase domain, demonstrated that the A628T LADD mutation disrupts the catalytic activity due to conformational changes, leading to LADD syndrome. In addition, the newly described crystal structure of FGFR2 in comparison to FGFR1 revealed that the FGFR2 utilizes a less stringent mode of autoinhibition [Lew, Bae and Rohmann et al., 2007]

Activating Somatic <em>FGFR2</em> Mutations in Breast Cancer

<div><p>It is known that <i>FGFR2</i> gene variations confer a risk for breast cancer. FGFR2 and FGF10, the main ligand of FGFR2, are both overexpressed in 5–10% of breast tumors. In our study, we sequenced the most important coding regions of <i>FGFR2</i> in somatic tumor tissue of 140 sporadic breast cancer patients and performed MLPA analysis to detect copy number variations in <i>FGFR2</i> and <i>FGF10</i>. We identified one somatic heterozygous missense mutation, p.K660N (c.1980G>C), within the tyrosine kinase domain of FGFR2 in tumor tissue of a sporadic breast cancer patient, which is likely mediated by the FGFR2-IIIb isoform. The presence of wild type and mutated alleles in equal quantities suggests that the mutation has driven clonal amplification of mutant cells. We have analyzed the tyrosine kinase activity of p.K660N and another recently described somatic breast cancer mutation in FGFR2, p.R203C, after expression in HEK293 cells and demonstrated that the intrinsic tyrosine kinase activity of both mutant proteins is strongly increased resulting in elevated phosphorylation and activity of downstream effectors. To our knowledge, this is the first report of functional analysis of somatic breast cancer mutations in FGFR2 providing evidence for the activating nature of FGFR2-mediated signalling in the pathogenesis of breast cancer.</p> </div

<i>FGFR2</i> mRNA isoform expression analysis in tumor tissue of three sporadic breast cancer patients. A) Representative electropherogram of GeneScan analysis.

<p>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 <i>FGFR2</i>-IIIb or 294 bp for <i>FGFR2</i>-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>B) Representative sequence electropherogram showing the expression of IIIb isoform.</b></p

<i>FGFR2/FGF10</i> MLPA analysis.

<p>Representative MLPA chromatogram and quantification with SequencePilot software. The probe mix contained 34 probes, 6 hybridizing to the <i>FGF10</i> gene, 8 to the <i>FGFR2</i> 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.</p

Lacrimo-Auriculo-Dento-Digital Syndrome Is Caused by Reduced Activity of the Fibroblast Growth Factor 10 (FGF10)-FGF Receptor 2 Signaling Pathway▿

Lacrimo-auriculo-dento-digital (LADD) syndrome is characterized by abnormalities in lacrimal and salivary glands, in teeth, and in the distal limbs. Genetic studies have implicated heterozygous mutations in fibroblast growth factor 10 (FGF10) and in FGF receptor 2 (FGFR2) in LADD syndrome. However, it is not clear whether LADD syndrome mutations (LADD mutations) are gain- or loss-of-function mutations. In order to reveal the molecular mechanism underlying LADD syndrome, we have compared the biological properties of FGF10 LADD and FGFR2 LADD mutants to the activities of their normal counterparts. These experiments show that the biological activities of three different FGF10 LADD mutants are severely impaired by different mechanisms. Moreover, haploinsufficiency caused by defective FGF10 mutants leads to LADD syndrome. We also demonstrate that the tyrosine kinase activities of FGFR2 LADD mutants expressed in transfected cells are strongly compromised. Since tyrosine kinase activity is stimulated by ligand-induced receptor dimerization, FGFR2 LADD mutants may also exert a dominant inhibitory effect on signaling via wild-type FGFR2 expressed in the same cell. These experiments underscore the importance of signal strength in mediating biological responses and that relatively small changes in receptor signaling may influence the outcome of developmental processes in cells or organs that do not possess redundant signaling pathway

Identified <i>FGFR2</i> mutation in tumor tissue.

<p>The upper sequence chromatogram shows the heterozygous missense mutation in exon 14 in <i>FGFR2</i>, 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.</p

Activating FGFR2 breast cancer mutations.

<p><b>A)</b> 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>B</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.</p

Location and conservation of FGFR2 mutations. A) Schematic model of FGFR2 with bound ligand (FGF).

<p>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>B) Conservation of FGFR2 mutations.</b> Arrows indicate localization of mutations. <i>Above</i> CLUSTALW alignment of vertebrate FGFR2s and human FGFRs. <i>Below</i>: 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).</p

Mutations in different components of FGF signaling in LADD syndrome.

Contains fulltext : 50020.pdf (publisher's version ) (Closed access)Lacrimo-auriculo-dento-digital (LADD) syndrome is characterized by lacrimal duct aplasia, malformed ears and deafness, small teeth and digital anomalies. We identified heterozygous mutations in the tyrosine kinase domains of the genes encoding fibroblast growth factor receptors 2 and 3 (FGFR2, FGFR3) in LADD families, and in one further LADD family, we detected a mutation in the gene encoding fibroblast growth factor 10 (FGF10), a known FGFR ligand. These findings increase the spectrum of anomalies associated with abnormal FGF signaling