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Supplementary Methods
Lynch syndrome genetic profiling and molecular characterization of defects in the DNA
mismatch repair genes and miR-155 expression levels in resected specimens by means of
qPCR assay.
Lynch syndrome genetic profiling and molecular characterization of defects in the DNA mismatch
repair genes
Molecular pathology testing for microsatellite instability (MIN) alterations typical of Lynch
syndrome was performed on tissue samples from the index patient with PVAC, her brother with
colorectal cancer and her nephew with rectal polyps. In details, MIN was determined by the
mobility shift of PCR products using Bethesda panel microsatellite (BAT25, BAT26, D5S346,
D17S250 and D2S123) in formalin-fixed-paraffin-embedded (FFPE) tissue blocks, containing
either tumour or non-neoplastic epithelial areas which were sectioned at 10 μm, harvested by
manual dissection and placed in 50 μL DNA digestion buffer. Tumours showing instability in four
or more markers were classified as high microsatellite instability (MSI-H), those showing it in two
marker as low MSI (MSI-L), and those showing no instability as microsatellite-stable (MSS), (1).
MSI-H was revealed in tumours from all the 3 patients by using 5 mononucleotide markers.
Mismatch repair proteins were evaluated by immunohistochemistry (IHC) on tissue
specimens from the PVAC of the index case and from colorectal cancer of her brother. IHC
evaluation of MSH2, MLH1, MSH6, MSH3 and PMS2 proteins was performed by using FFPE
tissue blocks containing tumour and adjacent normal epithelial areas, as previously reported (2).
The antibodies used for these studies were anti-hMSH2 NA26 (Oncogene Research, Darmstadt,
Germany); anti-hMSH6 G70220 (Transduction Laboratories, Basel, Switzerland); anti-hMLH1
13271A (PharMingen, Basel, Switzerland); anti-hPMS2 65861A (PharMingen, Basel, Switzerland).
MLH1/PMS2 proteins deficiency was observed in both tumour tissues.
Since there was a DNA repair defect, mutational analysis of mismatch repair genes was
recommended. Thus, in all the 3 subjects with MSI-H tumours, the entire coding regions of MLH1
gene, including all splice junctions and adjacent intronic regions, were sequenced. Mutational
analysis was performed on genomic DNA extracted from the peripheral blood using a standard
procedure, and amplified by PCR using primers located in the flanking introns approximately 50
base pairs from the respective intron/exon borders (3). Forward and reverse PCR amplified products
were sequenced using the automated ABI 310 DNA Sequencer (Applied Biosystems, Foster City,
CA, USA). To exclude deletion or insertion in MLH1 and/or PMS2 gene, we performed the
Multiplex Ligation-dependent Probe Amplification (MLPA) assay , by using using three different
SALSA MLPA KIT (P003, P008 and P248 kits, MCR-Holland, Amsterdam, The Netherlands),
according to the protocol supplied with the kit. PCR products were analyzed by using an ABI 310
genetic analyzer (Applied Biosystems, Foster City, CA, USA). However, neither mutations in
MLH1 gene nor large deletions or amplifications in the MLH1/PMS2 genes was found in these
subjects.
miR-155 expression levels in biopsies specimens by using qPCR assay
The miRNA fraction was extracted from fresh biopsie of PVAC for index case, of colon for
her brother and of rectal polyps for her nephew by using Qiazol Lysis Reagent (Qiagen, Hilden,
Germany), according to manufacturer’s instructions. Then, cDNA was synthesized by using genespecific primer according to the TaqMan MicroRNA Assay protocol (Applied Biosystems, Foster
City, CA, USA), as previously reported (4). Quantitative PCR was performed using an Applied
Biosystems 7700 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). The Ct
data was determinate using default threshold settings. In each tissue sample, miRNA relative
expression level of miR-155 in tumour compared to normal tissue was obtained using the 2-Ct
method of relative quantification, after normalizing for the expression of the endogenous RNU6B
(5).
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H, Neuweiler J, Riehle HM, Cattaruzza MS, Heinimann K, Schär P, Jiricny J, Marra G.
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