Additional file 3: of An improved sequencing-based strategy to estimate locus-specific DNA methylation

Validation of the plasmid DNA standards. (PDF 277 kb

Additional file 6: of An improved sequencing-based strategy to estimate locus-specific DNA methylation

Inverse association between miR-200c/miR-141 locus methylation and miR-200c expression. (PDF 1483 kb

Additional file 5: of An improved sequencing-based strategy to estimate locus-specific DNA methylation

miR-200c/miR-141 locus methylation of MDA-MB-231 breast cancer cell line determined by BSP and NBSP performed with forward and reverse primers. (PDF 455 kb

Sequencing chromatograms showing the novel <i>CDH1</i> germline mutations.

<p>(A) ID 5, new intronic mutation close to exon 1 (IVS1 c.48+7C>T) found in one patient affected by AMAG (S121). (B) ID 13, silent mutation (c.1416C>T) with conserved Ala residue at position 472 in <i>CDH1</i> exon 10 (p.T472T) found in one GC (S4). (C) ID 18, the silent mutation (c.2073C>T) with conserved Ala residue at position 691 in <i>CDH1</i> exon 13 (p.A691A) found in one GC (S48).</p

Clinicopathological characteristics of patients with gastric cancer.

<p>Clinicopathological characteristics of patients with gastric cancer.</p

Evaluation of potential effects on splicing on <i>CDH1</i> gene of the intronic variants found.

<p>Total mRNA was extracted from PBMCs of each represented sample and retrotrascribed to single strand cDNA to amplify: (A) Exons 1–5 of about 768 bp. In the second lane AMAG (S121) patient carrying the ID 5 mutation (IVS1 c.48+7C>T); in the third lane FDR (S97) carrying the ID 9 mutation (IVS4 c.532-18C>T); (B) Exons 10–13 of about 686 bp. GC (S10), GC (S46) and BD (S190) carrying the ID 17 mutation (IVS12 c.1937-13T>C). Yellow arrows evidence a smaller band corresponding to aberrant transcripts lacking the exon 11; (C) β-actin was used as internal amplification control. MW: 1 Kb DNA ladder. PCR products were run in a 4% agarose and gel stained with SYBR Green dyef</p

Hematoxylin and eosin stain gastric sections and immunohistochemical staining for E-cadherin and β-catenin.

<p>(A) Hematoxylin and eosin staining in normal gastric tissue. (B) Hematoxylin and eosin staining in the tumor tissue of GC S10 with signet ring cell carcinoma: signet ring cells are highlighted by black arrows. (C) Hematoxylin and eosin staining evidence the diffuse histotype of GC S46. (D) E-cadherin staining in normal gastric tissue. (E) Reduction of E-cadherin staining in the signet ring cells of GC S10 respect to adjacent normal cells; signet ring cells are highlighted by black arrows. (F) Loss of E-cadherin expression in the diffuse tumor cells of GC S46 compared to normal tissue (on the right side of the photomicrograph). (G) β-catenin staining in normal gastric tissue. (H) Weakly β-catenin staining in signet ring cells (black arrows) of GC S10. (I) Loss of β-catenin staining in the tumor tissue of GC S46 compared to normal tissue (on the right side of the photomicrograph). All the photomicrographs were taken at 400× magnification.</p

Graphic presentation of relative E-cadherin quantification by quantitative real time RT-PCR.

<p>Relative quantification of E-cadherin mRNA levels in EBV immortalized lymphocytes (LCL). LCLs were generated from the seeding of 2,5×10⁶ PBMCs immortalized by B.95.8 EBV and cultured in suspension. About 8 million of cells were harvested for each sample after immortalization. Patients tested were: GC S10 harbouring the ID 17 variant (IVS12 c.1937-13T>C), FDR S97 carrying the ID 9 (IVS4 c.532-18C>T), BD S190 with ID 17 and BD S189 without any <i>CDH1</i> mutation. S189 was considered as the reference (value of 1) Results are representative of three independent experiments. E-cadherin expression level was normalized normalized to β-actin Data are represented as means ± SD. *, p<0.05, **, p<0.01.</p

Analysis of <i>CDH1</i> germline mutations found in 190 individuals.

<p><b>Legend:</b> ID = Variant code; GenReg = Gene Region; (c) = nucleotide code position; (p) = protein code position; BDs, Blood Donors (n = 52); Freq = Frequency; rs = refSNPcluster; Ref = Reference; Ex = Exon; UTR = Untranslated; d/i = deletion/insertion; Intr = Intronic; I = Intestinal, D = Diffuse, M = Mixed, Ind = Indeterminate, histotypes; Polym. = Polymorphic; mut = mutation. Other abbreviations are described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077035#pone-0077035-t002" target="_blank">Table 2</a> Abbreviations list.</p><p>#, in the column are indicated the number of subjectss having the ID mutation.</p><p>##, Frequency of variants was calculated in 111 healthy subjects (52BDs+59FDRs). 16 FDRs were first degree relatives of our GC series, when one of the variant was present in GC and in its related FDR case, we excluded the FDR individual from the frequency calculation.</p>*<p>: new mutation frequency was evaluated by extending BD subjects analysis from 52 to 108 individuals.</p>**<p>: novel mutation with uncertain pathogenetic effect that we have previously reported <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077035#pone.0077035-Garziera1" target="_blank">[26]</a>.</p>°<p>: variant with frequencies in NHLBI exome sequencing project (ESP).</p>°°<p>: variant with frequencies in NHLBI exome sequencing project (ESP) and 1000 Genomes project.</p>°°°<p>: variant with frequencies in 1000 Genomes project.</p

Structural modelling of the extracellular domains of the E-cadherin protein.

<p>In A–B PyMOL and Coot softwares representations of EC1–EC2 protomers based on human sequence (PDB code: 2O72); in C–F, EC3 and EC4 protomers based on murine E-cadherin (PDB code: 3Q2W) by Coot software. (A) Cartoon representation highlights A144 (<i>yellow</i>) position: A144 is near to calcium sites (<i>purple</i>) and in proximity of the dimerization interface between EC1 (<i>blue</i>)-EC2 (<i>green</i>) domains. (B) Structural representation of A144T substitution in EC2 domain. Position of AT144 is spotlighted in yellow. Threonine in position 144 is quite dramatic for local structure because it interacts with two Aspartic acid residues (<i>green</i>) directly involved in calcium sites, and these bond lengths are particularly stressed being less than 3 Å. (C) The T316 (<i>yellow</i>) is <i>O</i>-glycosilated (<i>blue</i>) and present on the surface of EC3 domain. (D) The hydrophobic lateral chain of I316 (<i>yellow</i>) cannot be <i>O</i>-glycosilated and promotes protein-protein interactions. (E) The methylenic side chain of A438 (<i>yellow</i>) allows conformational freedom on the surface of EC4 domain. (F) T438 (<i>yellow</i>) substitution is not dramatic for local structure but it could represent a potential site for post-translational modifications. Calcium ions are highlighted (<i>green</i>).</p