Crystal structure of the nonclassical cadherin-17 N-terminus and implications for its adhesive binding mechanism.

The cadherin superfamily of calcium-dependent cell-adhesion proteins has over 100 members in the human genome. All members of the superfamily feature at least a pair of extracellular cadherin (EC) repeats with calcium-binding sites in the EC linker region. The EC repeats across family members form distinct complexes that mediate cellular adhesion. For instance, classical cadherins (five EC repeats) strand-swap their N-termini and exchange tryptophan residues in EC1, while the clustered protocadherins (six EC repeats) use an extended antiparallel `forearm handshake' involving repeats EC1-EC4. The 7D-cadherins, cadherin-16 (CDH16) and cadherin-17 (CDH17), are the most similar to classical cadherins and have seven EC repeats, two of which are likely to have arisen from gene duplication of EC1-2 from a classical ancestor. However, CDH16 and CDH17 lack the EC1 tryptophan residue used by classical cadherins to mediate adhesion. The structure of human CDH17 EC1-2 presented here reveals features that are not seen in classical cadherins and that are incompatible with the EC1 strand-swap mechanism for adhesion. Analyses of crystal contacts, predicted glycosylation and disease-related mutations are presented along with sequence alignments suggesting that the novel features in the CDH17 EC1-2 structure are well conserved. These results hint at distinct adhesive properties for 7D-cadherins.

Identification and Clinical Associations of 3 Forms of Circulating T-cadherin in Human Serum.

CONTEXT: T-cadherin (T-cad) is a glycosylphosphatidylinositol (GPI)-anchored cadherin that mediates adiponectin to induce exosome biogenesis and secretion, protect cardiovascular tissues, promote muscle regeneration, and stimulate therapeutic heart protection by transplanted mesenchymal stem cells. CDH13, the gene locus of T-cad, affects plasma adiponectin levels most strongly, in addition to affecting cardiovascular disease risk and glucose homeostasis. Recently, it has been suggested that T-cad exists in human serum, although the details are still unclear. OBJECTIVE: To validate the existence of T-cad forms in human serum and investigate the association with clinical parameters of type 2 diabetes patients. METHODS: Using newly developed monoclonal antibodies against T-cad, pooled human serum was analyzed, and novel T-cad enzyme-linked immunosorbent assays (ELISAs) were developed. The serum T-cad concentrations of 183 Japanese type 2 diabetes patients were measured in a cross-sectional observational study. The main outcome measure was the existence of soluble T-cad in human serum. RESULTS: There were 3 forms of soluble T-cad: a 130-kDa form with a prodomain, a 100-kDa mature form, and a 30-kDa prodomain in human serum. Using newly developed ELISAs to measure them simultaneously, we found that the 130-kDa form of T-cad positively correlated with plasma adiponectin (r = 0.28, P < .001), although a physiological interaction with adiponectin was not observed in serum. The unique 30-kDa prodomain was associated with several clinical parameters in diabetes patients. CONCLUSION: We identified 3 novel forms of soluble T-cad. Their importance as disease markers and/or biomarkers of adiponectin function and the possible bioactivity of the respective molecules require further investigation.

An electrochemical biosensor for the detection of epithelial-mesenchymal transition.

Epithelial-mesenchymal transition (EMT) is critically involved in a variety of biological processes. Electrochemical sensing offers potential to develop more effective technology for EMT detection. In this study, by using the unique performance of quantum dot (QD)-nanocomposite materials, we establish an electrochemical biosensor that can specifically detect the change of E-cadherin and analyze different stages of EMT. The signal for EMT is largely magnified due to the transmission of molecular information to the electronic device. In addition, differential pulse voltammetry reveals that the response of the electrochemical signals is rapid and sensitive, due to the synergistic effect of QDs and carbon nanotube-gold nanoparticles. Our study thus suggests that electrochemical sensing is an effective technology for detecting EMT and may have broad applications in analyzing various cell type transitions.

Electrochemical immunosensor for CDH22 biomarker based on benzaldehyde substituted poly(phosphazene) modified disposable ITO electrode: A new fabrication strategy for biosensors.

A novel label-free impedimetric immunosensor was fabricated for rapid, selective and sensitive detection and quantification of Cadherin-like protein 22 (CDH22), a cancer marker, in human serum by using easy and quickly prepared disposable ITO immunoelectrode. cancer marker, in human serum by using easy and quickly prepared disposable ITO immunoelectrode. The biosensing approach implied the use of ITO electrode coated with poly(phosphazene) polymer including benzaldehyde groups attached with CDH22 antibody and CDH22 antigens. Benzaldehyde side groups containing poly(phosphazene) film coated disposable ITO electrode were utilized as an immunosensing platform and anti-CDH22 antibodies bound to aldehyde groups of benzaldehyde substituted poly(phosphazene) (P-PHP) covalently. The immunosensor modification steps and affinity interaction between anti-CDH22 antibodies and CDH22 antigens were observed by EIS and CV in the presence of the redox couple. Furthermore, antibody immobilization was followed via FTIR and Raman spectroscopy. The morphological analyses of the suggested immunosensor during the fabrication steps were carried out with SEM and AFM monitoring. All the experimental parameters affecting the construction of the immunoelectrodes were optimized. The fabricated immunosensor exhibited an excellent working performance with a wide detection linear range (0.015-2.9 pg/mL) and low limit of detection (4.4 fg/mL). Moreover, the proposed immunosensor had great reproducibility, repeatability and long-term stability. Additionally, the fabricated immunosensor was successfully used in the quantification of CDH22 in human serum without any pretreatment.

Measuring Force-Induced Dissociation Kinetics of Protein Complexes Using Single-Molecule Atomic Force Microscopy.

Proteins respond to mechanical force by undergoing conformational changes and altering the kinetics of their interactions. However, the biophysical relationship between mechanical force and the lifetime of protein complexes is not completely understood. In this chapter, we provide a step-by-step tutorial on characterizing the force-dependent regulation of protein interactions using in vitro and in vivo single-molecule force clamp measurements with an atomic force microscope (AFM). While we focus on the force-induced dissociation of E-cadherins, a critical cell-cell adhesion protein, the approaches described here can be readily adapted to study other protein complexes. We begin this chapter by providing a brief overview of theoretical models that describe force-dependent kinetics of biomolecular interactions. Next, we present step-by-step methods for measuring the response of single receptor-ligand bonds to tensile force in vitro. Finally, we describe methods for quantifying the mechanical response of single protein complexes on the surface of living cells. We describe general protocols for conducting such measurements, including sample preparation, AFM force clamp measurements, and data analysis. We also highlight critical limitations in current technologies and discuss solutions to these challenges.

Downregulation of MicroRNA-320d predicts poor overall survival and promotes the growth and invasive abilities in glioma.

Previous studies have demonstrated that miRNAs play an important role in tumor development and progression. The role of miR-320d has been studied in several cancers except for glioma. The aim of the study was to investigate the expression levels, biological function, and mechanism of miR-320d in glioma. The expression levels of miR-320d were detected in glioma tissues and cell lines (U87 and U251) by RT-qPCR. Cell proliferation, colony formation, apoptosis, cell cycle, and transwell assays were performed in glioma cell lines transfected with miR-320d mimics or controls to evaluate the effects of miR-320d in vitro. The expression levels of invasive-related proteins were determined by Western blot analysis. Results showed that the expression of miR-320d was significantly decreased in glioma tissues and cell lines. Overexpression of miR-320d could significantly suppress cell growth, migration and invasion, and induced cell apoptosis as well as cell cycle at G0/G1 arrest in U87 and U251 cell lines. Additionally, expression levels of MMP-2, MMP-9, N-cadherin, and integrin-ß1 reduced, while E-cadherin increased in miR-320d mimic group. Overall, this study is the first to demonstrate that miR-320d may serve as an independent prognostic factor, indicating that miR-320d is a biomarker for prognosis and therapy in glioma.

Crystal Structure of Human E-Cadherin-EC1EC2 in Complex with a Peptidomimetic Competitive Inhibitor of Cadherin Homophilic Interaction.

Cadherins are transmembrane cell adhesion proteins whose aberrant expression often correlates with cancer development and proliferation. We report the crystal structure of an E-cadherin extracellular fragment in complex with a peptidomimetic compound that was previously shown to partially inhibit cadherin homophilic adhesion. The structure reveals an unexpected binding mode and allows the identification of a druggable cadherin interface, thus paving the way to a future structure-guided design of cell adhesion inhibitors against cadherin-expressing solid tumors.

Rapid and Sensitive Detection of sFAT-1 Transgenic Pigs by Visual Loop-Mediated Isothermal Amplification.

Genetically modified (GM) livestock have the potential to contribute to improving the environment and human health, with consumption of fewer resources and reduced waste production. However, the transgene process also poses risks. The safety assessment and control of transgenic animal products have drawn wide attention, and the relevant regulations and technology are being developed. Quick testing technology plays a significant role in on-site and customs sampling. Nowadays, loop-mediated isothermal amplification (LAMP) was widely applied in nucleic acid analysis because of its simplicity, rapidity, high efficiency and specificity. In this study, a specific, sensitive detection system for detecting sFAT-1 transgenic pigs was designed. A set of six primers including two loop primers was designed for the target sequence. The DNA samples were amplified in less than 1 h at the optimized temperature and detecting by both Nephelometer LA-320c and unaided eyes directly adding calcein. The detection limit of sFAT-1 LAMP was as low as 1.26 ng/µL. Furthermore, blind tests of transgenic and non-transgenic DNA samples were all correctly detected. Hence, the results in this study demonstrated that LAMP is a very useful tool for transgenic detection.

Spatio-temporally restricted expression of cell adhesion molecules during chicken embryonic development.

Differential cell adhesive properties are known to regulate important developmental events like cell sorting and cell migration. Cadherins and protocadherins are known to mediate these cellular properties. Though a large number of such molecules have been predicted, their characterization in terms of interactive properties and cellular roles is far from being comprehensive. To narrow down the tissue context and collect correlative evidence for tissue specific roles of these molecules, we have carried out whole-mount in situ hybridization based RNA expression study for seven cadherins and four protocadherins. In developing chicken embryos (HH stages 18, 22, 26 and 28) cadherins and protocadherins are expressed in tissue restricted manner. This expression study elucidates precise expression domains of cell adhesion molecules in the context of developing embryos. These expression domains provide spatio-temporal context in which the function of these genes can be further explored.

Common protein biomarkers assessed by reverse phase protein arrays show considerable intratumoral heterogeneity in breast cancer tissues.

Proteins are used as prognostic and predictive biomarkers in breast cancer. However, the variability of protein expression within the same tumor is not well studied. The aim of this study was to assess intratumoral heterogeneity in protein expression levels by reverse-phase-protein-arrays (RPPA) (i) within primary breast cancers and (ii) between axillary lymph node metastases from the same patient. Protein was extracted from 106 paraffin-embedded samples from 15 large (≥3 cm) primary invasive breast cancers, including different zones within the primary tumor (peripheral, intermediate, central) as well as 2-5 axillary lymph node metastases in 8 cases. Expression of 35 proteins including 15 phosphorylated proteins representing the HER2, EGFR, and uPA/PAI-1 signaling pathways was assessed using reverse-phase-protein-arrays. All 35 proteins showed considerable intratumoral heterogeneity within primary breast cancers with a mean coefficient of variation (CV) of 31% (range 22-43%). There were no significant differences between phosphorylated (CV 32%) and non-phosphorylated proteins (CV 31%) and in the extent of intratumoral heterogeneity within a defined tumor zone (CV 28%, range 18-38%) or between different tumor zones (CV 24%, range 17-38%). Lymph node metastases from the same patient showed a similar heterogeneity in protein expression (CV 27%, range 18-34%). In comparison, the variation amongst different patients was higher in primary tumors (CV 51%, range 29-98%) and lymph node metastases (CV 65%, range 40-146%). Several proteins showed significant differential expression between different tumor stages, grades, histological subtypes and hormone receptor status. Commonly used protein biomarkers of breast cancer, including proteins from HER2, uPA/PAI-1 and EGFR signaling pathways showed higher than previously reported intratumoral heterogeneity of expression levels both within primary breast cancers and between lymph node metastases from the same patient. Assessment of proteins as diagnostic or prognostic markers may require tumor sampling in several distinct locations to avoid sampling bias.

Protein-protein interaction techniques: dissect PCP signaling in Xenopus.

The planar cell polarity (PCP) pathway is a ß-catenin-independent branch of the Wnt signaling cascade. In vertebrate embryos PCP signaling regulates morphogenetic events including convergent extension (CE) movements during gastrualtion. Xenopus embryo has been established as an excellent model system to dissect PCP signaling in vertebrates because morphogenetic cell behaviors including CE can easily be monitored in vivo. Xenopus Paraxial protocadherin (xPAPC) is a transmembrane protein which serves as a link between patterning factors in the Spemann's organizer and regulators of the morphogenetic movements. xPAPC regulates morphogenesis in part by modulating cell adhesion and PCP signaling. Here two methods, GST pull-down assay and yeast two-hybrid assay, are described for the identification of xPAPC interacting proteins to elucidate the mechanism by which xPAPC regulates PCP signaling.

Stability studies of extracellular domain two of neural-cadherin.

Neural- (NCAD) and epithelial- (ECAD) cadherin are calcium-dependent cell-adhesive molecules, and are localized at excitatory and inhibitory synapses respectively. They play an important role in synaptogenesis, synapse maintenance and plasticity. The extracellular region plays a critical role in cadherin-mediated cell adhesion, and has five tandemly repeated ectodomains (EC1-EC5). Calcium binding is required for dimer formation between first two N-terminal domains (EC1-EC2). Despite similarity in the primary structure, the extracellular domains of NCAD and ECAD have different intrinsic stability, dimerization affinity and kinetics of disassembly. To investigate the origin of these differences, we are characterizing the modular domains individually. Here, we report studies of NCAD2, EC2 of NCAD. This domain is important for calcium binding and is the physical linkage between the dimerization interface in EC1 and the membrane proximal modular domains. Thermal-denaturation studies show that NCAD2 is less stable than ECAD2 and less influenced by the adjoining 7-residue, N- and C-terminal linker segments. In addition the NCAD2 constructs are less influenced by added salt. This difference is likely due to variation in the overall number and distribution of charges on these anionic proteins. Our studies indicate that despite their sequence similarity and apparently passive role in adhesive dimer formation, EC2 of E- and N-cadherins are distinctly different and may contribute to the differences in energetics and kinetics of dimerization.

Characterization of multiple stable conformers of the EC5 domain of E-cadherin and the interaction of EC5 with E-cadherin peptides.

The objectives of this work were to express the EC5 domain of E-cadherin and determine its structural characteristics as well as to evaluate the binding properties of HAV and BLG4 peptides to EC5 using spectroscopic methods. Homophilic interactions of E-cadherins are responsible for cell-cell adhesion in the adherens junctions of the biological barriers (i.e. intestinal mucosa and blood-brain barriers). The EC5 domain of E-cadherin has an important role in T-cell adhesion to intestinal mucosa via alpha(E)beta(7) integrin-E-cadherin interactions. In this study, the expressed EC5 has a high thermal stability (T(m) = 64.3 degrees C); it also has two stable conformations at room temperature, which convert to one conformation at approximately 54.5 degrees C. NMR and FTIR showed that HAV and BLG4 peptides bind to EC5. HSQC-NMR showed that either Asn or Gln of EC5 was involved in the interactions with HAV and BLG4 peptides. EC5 underwent a conformational change upon interaction with the HAV and BLG4 peptides. Finally, the binding properties of both peptides were modeled by docking experiments, and the results suggest that Asn-46 and Asn-75 of EC5 could be involved during the interaction with the peptides and that the Ser and Trp residues of the HAV and BLG4 peptides, respectively, were important for binding to EC5.

Structure of natural killer cell receptor KLRG1 bound to E-cadherin reveals basis for MHC-independent missing self recognition.

The cytolytic activity of natural killer (NK) cells is regulated by inhibitory receptors that detect the absence of self molecules on target cells. Structural studies of missing self recognition have focused on NK receptors that bind MHC. However, NK cells also possess inhibitory receptors specific for non-MHC ligands, notably cadherins, which are downregulated in metastatic tumors. We determined the structure of killer cell lectin-like receptor G1 (KLRG1) in complex with E-cadherin. KLRG1 mediates missing self recognition by binding to a highly conserved site on classical cadherins, enabling it to monitor expression of several cadherins (E-, N-, and R-) on target cells. This site overlaps the site responsible for cell-cell adhesion but is distinct from the integrin alpha(E)beta(7) binding site. We propose that E-cadherin may coengage KLRG1 and alpha(E)beta(7) and that KLRG1 overcomes its exceptionally weak affinity for cadherins through multipoint attachment to target cells, resulting in inhibitory signaling.

Expression study of cadherin7 and cadherin20 in the embryonic and adult rat central nervous system.

BACKGROUND: Vertebrate classic cadherins are divided into type I and type II subtypes, which are individually expressed in brain subdivisions (e.g., prosomeres, rhombomeres, and progenitor domains) and in specific neuronal circuits in region-specific manners. We reported previously the expression of cadherin19 (cad19) in Schwann cell precursors. Cad19 is a type II classic cadherin closely clustered on a chromosome with cad7 and cad20. The expression patterns of cad7 and cad20 have been reported previously in chick embryo but not in the developing and adult central nervous system of mammals. In this study, we identified rat cad7 and cad20 and analyzed their expression patterns in embryonic and adult rat brains. RESULTS: Rat cad7 protein showed 92% similarity to chick cad7, while rat cad20 protein had 76% similarity to Xenopus F-cadherin. Rat cad7 mRNA was initially expressed in the anterior neural plate including presumptive forebrain and midbrain regions, and then accumulated in cells of the dorsal neural tube and in rhombomere boundary cells of the hindbrain. Expression of rat cad20 mRNA was specifically localized in the anterior neural region and rhombomere 2 in the early neural plate, and later in longitudinally defined ventral cells of the hindbrain. The expression boundaries of cad7 and cad20 corresponded to those of region-specific transcription factors such as Six3, Irx3 and Otx2 in the neural plate, and Dbx2 and Gsh1 in the hindbrain. At later stages, the expression of cad7 and cad20 disappeared from neuroepithelial cells in the hindbrain, and was almost restricted to postmitotic cells, e.g. somatic motor neurons and precerebellar neurons. These results emphasized the diversity of cad7 and cad20 expression patterns in different vertebrate species, i.e. birds and rodents. CONCLUSION: Taken together, our findings suggest that the expression of cad7 and cad20 demarcates the compartments, boundaries, progenitor domains, specific nuclei and specific neural circuits during mammalian brain development.

Anopheles gambiae cadherin AgCad1 binds the Cry4Ba toxin of Bacillus thuringiensis israelensis and a fragment of AgCad1 synergizes toxicity.

A midgut cadherin AgCad1 cDNA was cloned from Anopheles gambiae larvae and analyzed for its possible role as a receptor for the Cry4Ba toxin of Bacillus thuringiensis strain israelensis. The AgCad1 cadherin encodes a putative 1735-residue protein organized into an extracellular region of 11 cadherin repeats (CR) and a membrane-proximal extracellular domain (MPED). AgCad1 mRNA was detected in midgut of larvae by polymerase chain reaction (PCR). The AgCad1 protein was localized, by immunochemistry of sectioned larvae, predominately to the microvilli in posterior midgut. The localization of Cry4Ba binding was determined by the same technique, and toxin bound microvilli in posterior midgut. The AgCad1 protein was present in brush border membrane fractions prepared from larvae, and Cry4Ba toxin bound the same-sized protein on blots of those fractions. The AgCad1 protein was expressed transiently in Drosophila melanogaster Schneider 2 (S2) cells. 125I-Cry4Ba toxin bound AgCad1 from S2 cells in a competitive manner. Cry4Ba bound to beads extracted 200 kDa AgCad1 and a 29 kDa fragment of AgCad1 from S2 cells. A peptide containing the AgCad1 region proximal to the cell (CR11-MPED) was expressed in Escherichia coli. Although Cry4Ba showed limited binding to CR11-MPED, the peptide synergized the toxicity of Cry4Ba to larvae. AgCad1 in the larval brush border is a binding protein for Cry4Ba toxin. On the basis of binding results and CR11-MPED synergism of Cry4Ba toxicity, AgCad1 is probably a Cry4Ba receptor.

Molecular cloning and expression analysis of three cadherin-8 isoforms in the embryonic chicken brain.

Three types of full-length cDNAs encoding chicken cadherin-8 (Cdh8) were identified and their expression in the embryonic chicken brain was investigated. The longest type of cDNA is closely similar to that of other classic cadherins, and the predicted protein shows a high similarity to rat and human Cdh8. The second type of cDNA is considerably shorter. The deduced protein lacks the cytoplasmic tail and the transmembrane domain, and contains a truncated fifth cadherin repeat in the extracellular domain (EC5; 68 amino acids), suggesting that it is a soluble isoform. The third type resembles the second one but is even shorter (only 30 amino acids in EC5). All types contain unique short sequences at their C terminus. Genomic analysis demonstrated that all of the three Cdh8 isoforms are located on chromosome 11, and consist of 12, 10, and 9 exons, respectively. Semi-quantitative RT-PCR with type-specific primers showed that the transcription of the three Cdh8 isoforms was temporally and spatially regulated in different parts of the embryonic chicken brain. The distinct regulation of gene expression suggested that the three isoforms likely play different roles during brain development. Northern blot analysis revealed that the transcription of the long isoform was much stronger than that of the two shorter ones. In situ hybridization showed that the long isoform of Cdh8 is expressed by a specific subset of brain nuclei, regions and layers in all major parts of the brain.

Isolation of high quality protein samples from punches of formalin fixed and paraffin embedded tissue blocks.

In general, it is believed that the extraction of proteins from formalin-fixed paraffin embedded samples is not feasible. However, recently a new technique was developed, presenting the extraction of non-degraded, full length proteins from formalin fixed tissues, usable for western blotting and protein arrays. In the study presented here, we applied this technique to punch biopsies of formalin fixed tissues embedded in paraffin to reduce heterogeneity of the tissue represented in sections, and to ensure analysing mainly defined cellular material. Successful extraction was achieved even from very small samples (0.7 mm(3)). Additionally, we were able to detect highly glycosylated proteins and protein modification, such as phosphorylation. Interestingly, with this technique it is feasible to extract high quality proteins from 14 year old samples. In summary, the new technique makes a great pool of material now usable for molecular analysis with high throughput tools.

Mint3/X11gamma is an ADP-ribosylation factor-dependent adaptor that regulates the traffic of the Alzheimer's Precursor protein from the trans-Golgi network.

Beta-amyloid peptides (Abeta) are the major component of plaques in brains of Alzheimer's patients, and are they derived from the proteolytic processing of the beta-amyloid precursor protein (APP). The movement of APP between organelles is highly regulated, and it is tightly connected to its processing by secretases. We proposed previously that transport of APP within the cell is mediated in part through its sorting into Mint/X11-containing carriers. To test our hypothesis, we purified APP-containing vesicles from human neuroblastoma SH-SY5Y cells, and we showed that Mint2/3 are specifically enriched and that Mint3 and APP are present in the same vesicles. Increasing cellular APP levels increased the amounts of both APP and Mint3 in purified vesicles. Additional evidence supporting an obligate role for Mint3 in traffic of APP from the trans-Golgi network to the plasma membrane include the observations that depletion of Mint3 by small interference RNA (siRNA) or mutation of the Mint binding domain of APP changes the export route of APP from the basolateral to the endosomal/lysosomal sorting route. Finally, we show that increased expression of Mint3 decreased and siRNA-mediated knockdowns increased the secretion of the neurotoxic beta-amyloid peptide, Abeta(1-40). Together, our data implicate Mint3 activity as a critical determinant of post-Golgi APP traffic.

Endogastric capsule for E-cadherin gene (CDH1) promoter hypermethylation assessment in DNA from gastric juice of diffuse gastric cancer patients.

BACKGROUND: We investigated whether an endogastric capsule (EC) may be a valuable tool for collecting DNA from exfoliated cells from the gastric mucosa and for carrying out an analysis of promoter methylation status of the E-cadherin (CDH1) gene in poorly differentiated, diffuse gastric cancer (DGC). MATERIAL AND METHODS: Consecutive patients with a confirmed diagnosis of poorly differentiated DGC underwent collection of gastric juice by EC. Subjects without cancer and premalignant lesions were also accrued as controls. The samples of gastric juice were processed for DNA isolation and amplification. Then they were used for analysis of CDH1 promoter hypermethylation. RESULTS: The procedure successfully allowed the analysis of CDH1 promoter hypermethylation in 20 patients and 14 controls. This pilot study showed feasibility of the procedure and a significantly different CDH1 promoter hypermethylation status between DGC patients and controls was detected. CONCLUSIONS: The EC may represent an innovative and noninvasive tool for the analysis of a specific epigenetic change in DGC patients. Our findings deserve additional studies as this method may represent a cost-effective tool for early detection of sporadic as well as hereditary DGC in CDH1 germline mutations carriers.