Combined loss of CDH1 and downstream regulatory sequences drive early-onset diffuse gastric cancer and increase penetrance of hereditary diffuse gastric cancer

Abstract

Background: Germline CDH1 pathogenic or likely pathogenic variants cause hereditary diffuse gastric cancer (HDGC). Once a genetic cause is identified, stomachs' and breasts' surveillance and/or prophylactic surgery is offered to asymptomatic CDH1 carriers, which is life-saving. Herein, we characterized an inherited mechanism responsible for extremely early-onset gastric cancer and atypical HDGC high penetrance.

Methods: Whole-exome sequencing (WES) re-analysis was performed in an unsolved HDGC family. Accessible chromatin and CDH1 promoter interactors were evaluated in normal stomach by ATAC-seq and 4C-seq, and functional analysis was performed using CRISPR-Cas9, RNA-seq and pathway analysis.

Results: We identified a germline heterozygous 23 Kb CDH1-TANGO6 deletion in a family with eight diffuse gastric cancers, six before age 30. Atypical HDGC high penetrance and young cancer-onset argued towards a role for the deleted region downstream of CDH1, which we proved to present accessible chromatin, and CDH1 promoter interactors in normal stomach. CRISPR-Cas9 edited cells mimicking the CDH1-TANGO6 deletion display the strongest CDH1 mRNA downregulation, more impacted adhesion-associated, type-I interferon immune-associated and oncogenic signalling pathways, compared to wild-type or CDH1-deleted cells. This finding solved an 18-year family odyssey and engaged carrier family members in a cancer prevention pathway of care.

Conclusion: In this work, we demonstrated that regulatory elements lying down-stream of CDH1 are part of a chromatin network that control CDH1 expression and influence cell transcriptome and associated signalling pathways, likely explaining high disease penetrance and very young cancer-onset. This study highlights the importance of incorporating scientific-technological updates and clinical guidelines in routine diagnosis, given their impact in timely genetic diagnosis and disease prevention.

Keywords: CDH1; Copy number variants; Deletion; Hereditary diffuse gastric cancer; Regulatory elements; Type-I interferon immune response.

Fig. 1

Diagnostic odyssey, pedigree, variant calling details and cancer histology of a HDGC family carrying a causative CDH1 CNV. A Current family pedigree. Full black symbols: individuals with confirmed DGC; Red outline: negative for SNVs (Sanger sequencing); green outline: negative for SNVs (WES); orange outline: positive for CNVs (WES); blue outline: MLPA-positive for the CDH1 deletion; traced orange outline: negative for CNVs (WES); traced blue outline: MLPA-negative for the CDH1 deletion. All family members, submitted to multiple genetic tests herein described, signed informed consents. The research work has been approved by the Ethics Committee of Centro Hospitalar Universitário São João, in Porto, Portugal, in the frame of the Solve-RD project with the reference CHUSJ_445/2020; B Timeline of events and diagnostic odyssey of the family; C CDH1 CNV found by WES, encompassing part of the CDH1 and TANGO6 genes (represented in green) and IGV coverage; Green text represents first and last edited exons; D Detected CDH1 CNV in ClinCNV [31], ExomeDepth [30] and Manta [32]; E MLPA analysis performed using SALSA MLPA-Probemix P083 CDH1 (MRC Holland) in patient III-3; Ratio above blue line indicates increased copy number, whilst ratio below red line indicates reduced copy number; Blue highlight represents CDH1 probes and 2 neighbour genes; Grey highlight represents reference probes; F Haematoxylin and eosin staining of DGC depicting signet-ring cells (arrow heads) (100 × magnification) from the proband III-5

Fig. 2

CDH1 and TANGO6 regulatory network and characterization of CRISPR-cas9-edited clones. A CDH1/TANGO6 potential enhancers depicted in GeneHancer, accessible chromatin and CDH1 promotor interactors in normal stomach tissue; B Sanger sequencing genotyping of the CNV breakpoints, and deletion coordinates in CDH1 and CDH1-TANGO6 CRISPR-cas9-edited clones; C mRNA genotyping of CDH1 and CDH1-TANGO6-edited clones resourcing to a probe located in the deleted CDH1 region; D CDH1 mRNA expression of CDH1- and CDH1-TANGO6-edited clones resourcing to a probe located in exons 6–7 (Hs.PT.58.3324071, TaqMan); E E-cadherin protein expression of CDH1- and CDH1-TANGO6-edited clones measured by flow cytometry (monoclonal antibody HECD-1, Invitrogen, 1:100 dilution; and mouse Alexa Fluor 647, Invitrogen, 1:500 dilution); F RNA-seq expression read counts of CDH1 and TANGO6 genes. Data are represented as the mean ± SEM, MFI: median fluorescence intensity. Experiments depicted in panels C–E were performed in triplicates and differences considered statistically significant if p value < 0.05 in a t-test (details in Materials and Methods)

Fig. 3

Genome-wide transcriptome regulation of CDH1 CNV and CDH1-TANGO6 CNV. A) Volcano-plot illustrating CDH1 CNV differentially expressed genes with CDH1 WT; B Volcano-plot illustrating CDH1-TANGO6 CNV differentially expressed genes with CDH1 WT; C Volcano-plot illustrating CDH1-TANGO6 CNV differentially expressed genes with CDH1 CNV; D Heatmap illustrating genome-wide differentially expressed genes; E Selected genome-wide transcriptomic misregulated pathways of CDH1-TANGO6 CNV and CDH1 CNV. Grey scale represents q value and red–green scale represents z-score (red for pathways with mainly downregulated genes, white for equally downregulated and upregulated genes and green for pathways with mainly upregulated genes); F Response to interferon, TGF-β and Wnt signalling pathways misregulated in CDH1-TANGO6 CNV vs CDH1 CNV, created with BioRender.com. Red–green scale represents down- to upregulated genes and blue represents normal expressed genes