CDH1 somatic variants
CDH1 mutations have been reported with frequencies ranging from 4.5 to 50% in diffuse tumors [
4,
5]. In our study, a total of 13 validated variants (including polymorphisms) in the
CDH1 gene were observed; however, we have not evidence to consider any of them as pathogenic.
The c.-137C > A variant found was previously reported for the Human Longevity Project (rs1046078040) [
13] but frequency and functionality data is lacking. In silico analysis suggested that this variant can affect the expression of E-cadherin protein, because two new binding sites for the transcription factors NFI/CTF and C/EBPbeta are created, as well the binding site for the transcription factor AhR: Arnt destroyed. Immunohistochemistry confirm the low expression of E-cadherin in the carrier of the c.-137C > A variant. Dysregulation in the expression of the
CDH1 gene can be explained by the modification of the binding sites for transcription factors, the NFI transcription factor play an important role during normal development, it functions by regulating cell proliferation and differentiation via the transcriptional control of their target genes, also it has been implicated in cancer, evidences suggests a converging role in development and cancer, with both oncogenic and tumor suppressor potential, depending on the carcinoma type and its tissue origin (Chen et al., 2017 [
14]). On the other hand, C/EBPbeta activates transcription of genes with a specific role in the nervous system, cytokines and transporter proteins that confer multidrug resistance as ABCC2 and ABCB1. C/EPBbeta is overexpressed in gastric tumors, with notable differences between histotypes, MGC (50%), IGC (46.2%) and DGC (4.4%) [
15]). Regarding to the AhR, it have a role in TCDD toxicity, but also is involved in tumorigenesis and is found at elevated levels in aggressive tumors and tumor cell lines [
16], however with the variant the binding site for this transcription factor is destroyed.
Regarding the variants c.1138-92delA, c.1138-75insA, and c.1221insC detected by NGS but not confirmed by capillary sequencing, it is important to highlight the need to confirm NGS findings as false positives can occur, particularly in repeated mononucleotides regions [
17]; another possibility is that, due to NGS higher sensitivity, such variants could be detected even if is present in a minority of DNA molecules within the tumor. Regarding the c.1221insC variant, interestingly, a mutation affecting neighbor nucleotides, c.1220delC (p.P407Qfs10), was reported in three members of a Spanish family with hereditary DGC [
18]; both, the c.1221insC variant and the c.1220delC mutation are predicted to result in a premature stop codon, which would lead to mRNA degradation and therefore to reduced E-cadherin expression and carcinogenesis, however c.1221insC was not confirmed through Sanger sequencing method.
Regarding known variants c.-197A > C, c.-176C > T, c.2164 + 17dupA, and c.2439 + 52G > A, in silico analysis showed that those located within the promoter can lead to changes in the binding site of some transcription factors. Recently was reported that the variant c.-197A > C (also denominated -73A > C) may lead to allele-specific repressions of
CDH1 gene, the allele C was related to lesser methylation, higher transcription levels and longer survival [
19], in our study observed three carriers for this allele, only one of them had normal expression of E-cadherin protein and CDH1 no methylated. For the c.-176C > T variant, no information published about their impact or functionality on CDH1 gene expression. The variants c.2164 + 17dupA and c.2439 + 52G > A, alters exonic splicing sites (ESS and ESE), the c.2164 + 17dupA is a common non-coding variant observed in 13/20 patients in our study, it is considered as benign by ClinVar, also, it was found in patients with breast cancer but was considered as no pathogenic [
20]). On the other hand, the c.2439 + 52G > A variant was observed in our study only in one patient, the clinical significance information is not yet reported in ClinVar, so in both cases the functional role need be clarified in future research.
For the known variants c.-285C > A (rs16260), c.48 + 6C > T (rs3743674), c.531 + 10G > C (rs33963999), c.1680G > A (rs35741240), c.1937-13 T > C (rs2276330), c.2076C > T (rs1801552), c.2164 + 17dupA (rs35686369), c.2253C > A (rs33964119), and c.54C > A (rs1801026), ClinVar considers them all as variants with clinical significance “benign” except for c.-285C > A that consider it as “risk factor” for prostate cancer. On the other hand, the Variant Effect Predictor tool predicts a “low” impact for the variants c.48 + 6C > T, c.1680G > A, c.2076C > T, and c.2253C > A, while the variants c.-285C > A, c.531 + 10G > C, c.1937-13 T > C, and c.54C > A are considered with an impact “modifier”. Additionally, some of these variants (rs35741240, rs1801552, rs33964119) are located in exonic regions but they are synonymous variants; others are located in introns or in non-translated regions (rs3743674, rs33963999, rs2276330, rs1801026) but to date no effect on the transcription process has been reported.
An important aspect to consider is that, with the exception of variant -285C > A (or -160C > A) [
21], no other variants have been described for the Mexican population. It would be important to investigate the frequencies of the variants described in this population (Additional file
4).
CDH1 loss of heterozygosity
LOH on specific chromosomal regions is related to the inactivation of tumor suppressor genes. In fact, focal genomic deletions in tumor suppressor genes such as
PTEN, SMAD4, PARK2, RB1, CDKN2A, and
ARID1A have been reported in GC [
22‐
24]. Importantly, allelic loss in the 16q22.1
locus is a genetic mechanism implicated in the inactivation of the
CDH1 gene; up to 91% of the GC cases with LOH in that locus present a reduced expression of E-cadherin [
25]. In our study, the frequency of LOH in 16q22.1 was 13.3%, corresponding to two DGC cases, which is in accordance with ranges reported between 4.5 and 39% [
4,
25,
26]. In both patients, we assume that the LOH found does not correspond to intragenic deletions as it involves only the 3′ of the gene’s
locus. This is corroborated by the fact that some of the variants identified by NGS, for these patients, were observed in heterozygote status (Additional file
2). Additionally, both samples were also analyzed by Multiplex Ligation-dependent Probe Amplification (MLPA) in the tumor DNA (probe SALSA P083-C2 CDH1, MRC Holland) and no major
CDH1 deletions were found.
CDH1 methylation
Methylation of the
CDH1 promoter is the most common epigenetic modification associated with the loss of heterozygosity in DGC cases. It also has been reported that 82% of cases with methylation have reduced expression of E-cadherin [
25]. The frequency of
CDH1 methylation in GC patients has been reported in 25.4 to 76% in Asian and Caucasian populations [
4,
25,
27]. In this study, we observed a frequency of 64.7% (
n = 11 cases) and non-significant differences were observed regarding the gastric cancer histotype. Further,
H. pylori infection has been strongly associated with DNA methylation, specifically with the
CDH1 gene [
28]; in our study, DGC cases positives for H pylori infection, 57.1% of them had
CDH1 promoter methylation and E-cadherin deficiency, and of the MGC cases positives for H pylori, 66.6% had both methylation and E-cadherin deficiency.
Multiple CDH1 alterations
More than one second hit mechanism has been reported in patients with hereditary DGC [
12]. In our study, one DGC patient (men of 57 years old) was carrier of three alterations in the
CDH1 gene: methylation, LOH and the new variant c.-137C > A, although it has no family history of GC. Most probably the finding of concomitant somatic inactivating mechanisms results from intratumor heterogeneity.
Finally, although the small number of samples and the absence of histopathological images constitute limitations to our study, the results it provides, regarding the alterations in the CDH1 gene and its importance for the Mexican population with diffuse and mixed gastric cancer, sheds light on a topic which has not been studied so far.