E-cadherin is a key player in gastric cancer (GC) and germline alterations of CDH1, its encoding gene, are responsible for Hereditary Diffuse Gastric Cancer (HDGC) syndrome. This study aimed at elucidating the role of genetic variants and DNA methylation of CDH1 promoter and enhancers in the regulation of gene expression. For this purpose, we analyzed genetic variants of the CDH1 gene through Next-Generation Sequencing (NGS) in a series of GC cell lines (NCI-N87, KATO-III, SNU-1, SNU-5, GK2, AKG, KKP) and the corresponding CDH1 expression levels. By bisulfite genomic sequencing, we analyzed the methylation status of CDH1 regulatory regions in 8 GC cell lines, in a series of 13 sporadic GC tissues and in a group of 20 HDGC CDH1-negative patients and 6 healthy controls. The NGS analysis on CDH1 coding and regulatory regions detected genetic alterations in 3 out of 5 GC cell lines lacking functional E-cadherin. CDH1 regulatory regions showed different methylation patterns in patients and controls, GC cell lines and GC tissues, expressing different E-cadherin levels. Our results showed that alterations in terms of genetic variants and DNA methylation patterns of both promoter and enhancers are associated with CDH1 expression levels and have a role in its regulation.

Background: Diffuse gastric cancer (DGC) is the most common manifestation in germline CTNNA1 variant carriers, with one study estimating a 49–57% lifetime risk by age 80. Knowledge on CTNNA1 -associated hereditary diffuse gastric cancer (HDGC), loss-of-function mechanisms, variant-type causality, disease spectrum and cancer risks remains scarce. Objective: Explore CTNNA1 genotype–phenotype associations to improve genetic testing criteria, surveillance and risk-reduction recommendations for carriers. Design: Using molecular, clinical and population data from 1308 individuals from 351 CTNNA1- variant carrier families and 37 428 non-carriers from European and American ancestries, we analysed genotype–phenotype associations with multivariable logistic regression. With CRISPR/Cas9 CTNNA1 -knockout gastric cancer (GC) cells and CTNNA1 -humanised Drosophila , we assessed CTNNA1 -associated loss-of-function mechanisms. Results: CTNNA1 -truncating transcripts are degraded by nonsense-mediated mRNA decay (NMD), and DGCs from germline CTNNA1 -truncating carriers lose αE-catenin. These transcripts are non-functional in Drosophila , in contrast to non-truncating transcripts. DGC risk is eightfold higher in truncating, compared with non-truncating carriers. The risk of GC and lobular breast cancer (LBC) development in CTNNA1 -truncating variant carriers is fivefold and eightfold lower than in CDH1 pathogenic/likely pathogenic variant carriers, respectively. Compared with wild-type individuals, GC risk is 7-fold higher in CTNNA1 -truncating and 38-fold higher in CDH1 -truncating variant carriers. LBC is recurrent among CTNNA1 -truncating carriers, some lacking HDGC criteria. Simplification of previous criteria for CTNNA1 genetic testing produced the ‘Porto’ criteria, which increased CTNNA1 -carrier families’ pick-up rate by 9%, without performance loss compared with the HDGC 2020 clinical guidelines. Macular dystrophy patterned-2 was positively associated with non-truncating variants, specifically in the αE-catenin M-fragment. Conclusion: We provide compelling evidence supporting that CTNNA1 -truncating variants positively associate with DGC and LBC, and NMD as the pathophysiological mechanism leading to CTNNA1 downregulation. We demonstrate that compared with CDH1 , CTNNA1 is a moderate penetrance HDGC gene. This new knowledge is essential to define surveillance and/or prophylactic measures for CTNNA1 -carrier individuals and families.

Cadherins are cell–cell adhesion molecules, fundamental for cell architecture and polarity. E-cadherin to P-cadherin switch can rescue adherens junctions in epithelial tumours. Herein, we disclose a mechanism for E-cadherin to P-cadherin switch in gastric cancers. CDH1 and CDH3 mRNA expression was obtained from 42 gastric tumours’ RNA-seq data. CRISPR-Cas9 was used to knock out CDH1 and a putative regulatory element. CDH1-depleted and parental cells were submitted to proteomics and enrichment GO terms analysis; ATAC-seq/4C-seq with a CDH1 promoter viewpoint to assess chromatin accessibility and conformation; and RT-PCR/flow cytometry to assess CDH1/E-cadherin and CDH3/P-cadherin expression. In 42% of gastric tumours analysed, CDH1 to CDH3 switch was observed. CDH1 knockout triggered CDH1/E-cadherin complete loss and CDH3/P-cadherin expression increase at plasma membrane. This switch, likely rescuing adherens junctions, increased cell migration/proliferation, commonly observed in aggressive tumours. E- to P-cadherin switch accompanied increased CDH1 promoter interactions with CDH3–eQTL, absent in normal stomach and parental cells. CDH3–eQTL deletion promotes CDH3/CDH1 reduced expression. These data provide evidence that loss of CDH1/E-cadherin expression alters the CDH3 locus chromatin conformation, allowing a CDH1 promoter interaction with a CDH3-eQTL, and promoting CDH3/P-cadherin expression. These data highlight a novel mechanism triggering E- to P-cadherin switch in gastric cancer.

PURPOSE: Hereditary cancers represent 5%-10% of all cancers, typically characterized by familial aggregation, early onset, and/or multiple primary tumors. Isolated cases with extreme early-onset or multiple unrelated cancers are rare and frequently underdiagnosed. This study aimed to improve genetic diagnostic yield in unresolved patients with strong clinical suspicion of hereditary cancer. Inclusion criteria were (1) ≥4 primary tumors in different organs (or ≥3 if two are rare), (2) adult-type cancers at ≤25 years, or (3) profuse gastrointestinal adenomatous polyposis before age 50 years or profuse polyposis of unknown type before age 30 years. METHODS: Germline DNA from 98 patients selected through ERN-GENTURIS underwent short-read whole-genome sequencing (WGS). Ninety had received negative results from standard genetic testing, whereas eight had not been tested. Variant analysis was performed using the RD-Connect GPAP and Solve-RD frameworks. RESULTS: Pathogenic or likely pathogenic variants in phenotype-related high-penetrance genes were found in 6% of patients, including APC and TP53 mosaicisms and germline variants in TP53 , BAP1 , BARD1 , and MBD4 (homozygous). Three patients carried pathogenic variants in hereditary cancer genes unrelated to their phenotype, and a heterozygous ERCC3 pathogenic variant was detected in a young patient with breast cancer. Suggestive variants of uncertain significance were identified, including a chromosome 3 inversion affecting VHL and MLH1 in a patient with renal and gastric cancers. Potentially regulatory noncoding variants in phenotype-associated genes were observed in 15% of patients (22 variants). CONCLUSION: Comprehensive resequencing of patients suspected of hereditary cancer increased the diagnostic yield by 6%. Detected pathogenic variants involved phenotype-related genes not previously analyzed or missed because of mosaicism. WGS further enables identification of novel gene associations and structural, deep intronic, or regulatory variants.

Missing heritability in hereditary diffuse gastric cancer (HDGC) ranges from 60 to 90%. These HDGC-like families, despite complying with HDGC clinical criteria, lack CDH1 and CTNNA1 actionable germline variants, and are not offered HDGC-targeted life-saving disease prevention measures. Herein, we explored novel HDGC predisposition mechanisms affecting the CDH1 -regulatory network. We called single-nucleotide (SNV) and copy-number variants (CNV) from 19 HDGC-like probands from whole-genome sequencing data and performed gene-ontology analysis. Chromatin enhancer marks and CDH1 promoter interactions were evaluated in normal stomach by ChIP-seq, ATAC-seq and 4C-seq, variant causality was assessed by RT-PCR, immunohistochemistry and microsatellite instability (MSI) analysis in tumours. Functional analysis was performed using CRISPR-Cas9, RT-PCR and flow cytometry in cell lines, and enhancer assays using mouse embryos. Within the CDH1 topologically associating domain (TAD), we found two deletions in Family F4 and F9. F4 carried a heterozygous CDH3 20kb-CNV triggering CDH1 mRNA/protein loss in homozygosity by CRISPR-Cas9 editing, similarly to a CDH1 coding deletion. This 20kb sequence encloses two hypomorphic tissue-specific regulatory elements (REs), each contributing 50% to CDH1 expression regulation. F9 carried a heterozygous 39bp-intergenic CNV downstream of CDH1 , triggering CDH1 mRNA/protein loss by CRISPR-Cas9. F15, presenting gastric but not colorectal cancer, carried an MLH1 heterozygous 2.7Kb germline CNV overlapping a stomach-specific RE found by ChIP-seq. The gastric tumour of mixed histology displayed Microsatellite instability (MSI), reduced MLH1 mRNA and protein, and reduced CDH1 and E-cadherin protein. CRISPR-Cas9 clones mimicking the MLH1 heterozygous CNV, triggered loss of MLH1 and CDH1 /E-cadherin mRNA and protein, similar to a coding deletion. Beyond the CDH1 TAD and tumour risk syndrome genes, multiple deletions of stomach accessible chromatin sequences were found in particularly young-affected individuals from additional 6 families. This oligogenic pattern impaired specifically mucin genes and multiple immune-related pathways. Herein, we pinpointed novel mechanisms behind HDGC predisposition. One involves deletions of CDH1 -REs in the TAD or stomach-specific CDH1 -REs in the MLH1 locus. The second involves multiple deletions of stomach REs affecting mucin and immune-related genes, favouring a gastric immune-deficient phenotype. Altogether, by combining stomach-specific chromatin accessibility and promoter interactions with whole genome sequencing, we solved the missing heritability in 47% of HDGC-like families within our cohort.