Data_Sheet_1_Mutational Landscapes and Phenotypic Spectrum of SWI/SNF-Related Intellectual Disability Disorders.DOCX

<p>Mutations in genes that encode proteins of the SWI/SNF complex, called BAF complex in mammals, cause a spectrum of disorders that ranges from syndromic intellectual disability to Coffin-Siris syndrome (CSS) to Nicolaides-Baraitser syndrome (NCBRS). While NCBRS is known to be a recognizable and restricted phenotype, caused by missense mutations in SMARCA2, the term CSS has been used lately for a more heterogeneous group of phenotypes that are caused by mutations in either of the genes ARID1B, ARID1A, ARID2, SMARCA4, SMARCB1, SMARCE1, SOX11, or DPF2. In this review, we summarize the current knowledge on the phenotypic traits and molecular causes of the above named conditions, consider the question whether a clinical distinction of the phenotypes is still adequate, and suggest the term “SWI/SNF-related intellectual disability disorders” (SSRIDDs). We will also outline important features to identify the ARID1B-related phenotype in the absence of classic CSS features, and discuss distinctive and overlapping features of the SSRIDD subtypes. Moreover, we will briefly review the function of the SWI/SNF complex in development and describe the mutational landscapes of the genes involved in SSRIDD.</p

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

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

<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

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

<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

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

Lrp4 binds the Bmp4 antagonist Gremlin1 <i>in vitro</i>.

<p>Lrp4 has been implicated in modulating the Bmp signaling pathway through binding of the Wnt and Bmp modulator Wise. Co-immunoprecipitation reveals Gremlin1 binding to Lrp4 <i>in vitro</i> (Panel A lane 4); we further confirmed the Lrp4 binding partners Wise, Dkk1 and SOST (Panel A, lanes 6, 10 and 12). The Wnt agonist R-spondin 2 did not interact with Lrp4 (Panel A lane 7 and 8). Transfection efficiency was confirmed by immunoblot analysis (Panel B).</p

Expression of branching regulators in Lrp4 mutants.

<p>Expression of c-Ret (a–f), GDNF (g–l) and Wnt11 (m–r) in E10.5 (a,b,g,h,m and n), E11.5 (c,d,i,j,o and p), and E12.5 (e,f,k,l,q,r) in wild-type (a,c,e,g,i,k,m,o, and q) and Lrp4 knockout (b,d,f,h,j,l,n,p and r) kidneys. C-Ret is expressed in the ureteric bud at basal levels in the Lrp4 knockout mice at E10.5 (a,b). At E11.5, the Lrp4 knockout ureteric bud fails to bifurcate or upregulate c-Ret expression at the tip of the ureteric bud (d) compared to wild-type embryos (c). At E12.5, the signal is greatly reduced in the knockout kidney (f). GDNF is expressed normally in the metanephric mesenchyme at both E10.5 and 11.5 in wild type and Lrp4 knockout animals (g–j). By E12.5, GDNF expression is completely lost from the Lrp4 knockout metanephric mesenchyme (k and l). Wnt11 is expressed normally at the tips of the ureteric bud at both E10.5 (m and n) and 11.5 (o and p) in Lrp4 mutants compared to wildtype. By E12.5 Wnt11 is absent from the ureteric bud of Lrp4 knockout animals (q and r). The Wolffian duct and ureteric bud are outlined by white dashed lines, mesenchyme (g, h, I, j) renal vesicles (e and q) and truncated ureteric bud (f, l, r) are indicated by black arrows.</p

Expression of Lrp4 in the developing kidney.

<p>At E10.5 Lrp4 is expressed throughout the Wolffian duct and the ureteric bud. (a). At E11.5, Lrp4 is expressed in the ureteric bud and the pre-tubular aggregates (b). At E12.5 and E 14.5, Lrp4 expression is maintained in the ureteric bud and the renal vesicles (c and d, respectively). The Wolffian duct and ureteric bud are outlined by dotted lines; the arrow points to the early ureteric bud in (a) or the renal vesicles in (c), respectively.</p