CDH1 Germline Variants in a Tunisian Cohort with Hereditary Diffuse Gastric Carcinoma

Abstract

Mutational screening of the CDH1 gene is a standard treatment for patients who fulfill Hereditary Diffuse Gastric Cancer (HDGC) testing criteria. In this framework, the classification of variants found in this gene is a crucial step for the clinical management of patients at high risk for HDGC. The aim of our study was to identify CDH1 as well as CTNNA1 mutational profiles predisposing to HDGC in Tunisia. Thirty-four cases were included for this purpose. We performed Sanger sequencing for the entire coding region of both genes and MLPA (Multiplex Ligation Probe Amplification) assays to investigate large rearrangements of the CDH1 gene. As a result, three cases, all with the HDGC inclusion criteria (8.82% of the entire cohort), carried pathogenic and likely pathogenic variants of the CDH1 gene. These variants involve a novel splicing alteration, a missense c.2281G > A detected by Sanger sequencing, and a large rearrangement detected by MLPA. No pathogenic CTNNA1 variants were found. The large rearrangement is clearly pathogenic, implicating a large deletion of two exons. The novel splicing variant creates a cryptic site. The missense variant is a VUS (Variant with Uncertain Significance). With ACMG (American College of Medical Genetics and Genomics) classification and the evidence available, we thus suggest a revision of its status to likely pathogenic. Further functional studies or cosegregation analysis should be performed to confirm its pathogenicity. In addition, molecular exploration will be needed to understand the etiology of the other CDH1- and CTNNA1-negative cases fulfilling the HDGC inclusion criteria.

Keywords: CDH1; CTNNA1; Tunisian patients; germline variants; hereditary diffuse gastric cancer; large rearrangements.

Figure 1

Family history of index cases carrying selected variants. (A) Family history of “JI-014” harboring the novel Indel variant c.1563 + 3_1563 + 4delinsGT located in intron 10, predicted to be probably pathogenic. (B) Family history of “JI-007” harboring the missense variant c.2281 G > A at exon 14 of the CDH1 gene, classified as a VUS in the ClinVar database. (C) Familial history of “JI-020” carrying the large deletion of two exons (one and two) identified by MLPA assay.

Figure 2

Indel c.1565 + 3_1565 + 4delinsGT effect for the index case JI-014, as shown by Alamut Visual Interactive Biosoftware covering several in silico prediction tools, such as Splice Site Finder-like, MaxEntScan, NNSPLICE, and GeneSplicer.

Figure 3

E-cadherin expression status in tumor gastric tissue. (A) H and E staining of JI-014 tumor tissue (X100). (B) E-cadherin immunostaining expression in gastric tumor tissue (X100). Black arrow shows normal membranous E-cadherin staining in crypt and glandular cells. (C) Loss of membranous E-cadherin expression in tumor cells (X200). (D) Red arrow shows a loss/reduction of E-cadherin expression in tumor cells and residual glands (X400).

Figure 4

Detection of CDH1 exon deletions by MLPA assay. (Blue) Control probes, (Red) Index cases harboring exon deletions (CDH1 Exons 1 and 2).

Figure 5

In silico analysis of p.G761R effect. (A) Schematic representation of the E-cadherin/p120 complex that includes the JMD core and the position of the mutation. (B) Co-crystal structure of the JMD core with p120 ARM domain showing the position of the mutated residue (light orange). (C) Interaction of G761 and R761 with the nearby amino acids in the WT form and the mutant form, respectively. (D) Cumulative likelihood of occurrence as a function of the backbone RMSD of the JMD core. All the structures of the ensembles were first fitted to the bound conformation of the JMD core prior to the calculation of the RMSD. (E) Root Mean Square Fluctuation (RMSF) profiles of the JMD core residues calculated for the WT and mutant forms.

Figure 6

E-cadherin/β-catenin signaling pathway alteration in the presence of p.G761R (inspired from [43]).