Supplementary Materials and Methods
Histology and immunohistochemistry
Sections from formalin-fixed, paraffin-embedded specimens were stained with hematoxylin/eosin.
The antibodies used for immunohistochemistry were: CD117 and S100 (rabbit polyclonal), and
vimentin (mouse monoclonal) (DAKO, Glostrup, Denmark, 1:400, 1:800 and 1:200, respectively),
DOG1 (Spring Bioscience, Pleasanton, CA, rabbit polyclonal, 1:100), CD34 (Novocastra,
Newcastle, UK, 1:50) and PDGFRA (sc-338) (Santa Cruz, Santa Cruz, CA, rabbit polyclonal,
1:400). Antigen retrieval was performed for DOG1, S100, vimentin and PDGFRA (10 minutes in
0.01 M citrate buffer, pH 6, 750 W microwave). Specific pre-immune sera or isotype-specific
unrelated primary antibodies were used for the controls. Hydrogen peroxide, serum biotinylated
immunoglobulins, and avidin-biotin complexes were used following the manufacturer's instructions;
after color induction with diaminobenzidine solution (Dakopatts, Glostrup, Denmark), slides were
counterstained with hematoxylin.
Sequence analysis in KIT and PDGFRA genes
Informed written consent was obtained from all tested individuals. DNA was obtained from paraffinembedded tissues or buccal swab samples (the latter performed in all patients tested except
patient II-2 and II-3, where formalin-fixed, paraffine embedded tissues were available first). Slides
were cut from paraffin-embedded tissues of patients II-2, II-3, III-4 and III-5, treated twice with
xylene and washed with ethanol; pathologic areas (nearly 100% disease-specific tissue) were
macro-dissected on slides; mutational analysis was also performed in normal tissue
(gastrointestinal wall layers, or lymph nodes). DNA was extracted using the QIAamp tissue mini kit
(Qiagen, Hilden, Germany). KIT (exons 9, 11 13, and 17) and PDGFRA (exons 12, 14, and 18)
genes were amplified using the same primers and PCR conditions described elsewhere(1-3).
Briefly, DNA (100-200 ng) was amplified in a mixture containing 1xPCR buffer [20 mM TRIS (pH
8.3), 50 mMKCl, 1.5 mM MgCl2], dNTPs (200 mM each), primers (20 pM each), and 0.5 U Taq
polymerase platinum (Invitrogen, Milan, Italy) in a 25 l final volume. PCR conditions were: 8-min
initial denaturation at 95°C, then 35 cycles at 95°C for 40s, 55°C for 40s, and 72°C for 40s. After
visualization onto agarose gel, PCR products were treated with ExoSAP-IT (USB Corp, Cleveland,
OH) following the manufacturer's protocol, amplified with BigDye Terminator version 3.1 cycle
sequencing kit (Applied Biosystems) using forward and reverse primers, and sequenced with an
ABI PRISM 3100-Avant Genetic Analyzer (Applied Biosystems). Water was the negative control.
Bioinformatics analysis
The P653L PDGFRA missense mutation was predicted with 4 different computational tools: “SIFT”,
a predictor based on the degree of conservation of amino-acid residues in sequence alignments
derived from closely related sequences(4); “PolyPhen-2”, a tool that predicts possible impact of a
substitution of an amino-acid on the structure and function of a human protein using physical and
comparative considerations(5); “SNPs&GO”, a predictor of human disease-related mutations in
proteins that considers information from evolutionary informations, gene ontology terms and
protein sequence(6); PROVEAN, an alignment-based score which measures the change in
sequence similarity of a query sequence to a protein sequence homolog before and after the
introduction of an amino acid variation to the query sequence(7).
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