E-Cadherin (CDH1) and p53 rather than SMAD4 and Caspase-10 germline mutations contribute to genetic predisposition in Portuguese gastric cancer patients

Abstract

Approximately 30% of all hereditary diffuse gastric cancer (HDGC) families carry CDH1 germline mutations. The other two thirds remain genetically unexplained and are probably caused by alterations in other genes. Using polymerase chain reaction (PCR)/single-strand conformation polymorphism (SSCP)/sequencing, we screened 32 Portuguese families with a history of gastric cancer and 23 patients with early onset gastric cancer for CDH1 germline mutations. In probands negative for CDH1 mutations, we screened genes involved in hereditary cancer syndromes in which gastric cancer may be one of the component tumours, namely p53 (Li-Fraumeni Syndrome) and hMLH1 and hMSH2 (HNPCC). We also screened in these patients for mutations in Caspase-10, a gene inactivated in sporadic gastric cancer, and SMAD4, a gene whose inactivation in mice is associated with signet-ring cell carcinoma of the stomach. One of the families fulfilling the HDGC criteria harboured a CDH1 germline mutation, and one of the families with incomplete criteria harboured a p53 germline mutation. No mutations were identified in hMLH1 and hMSH2, and only sequence variants were found in SMAD4 and Caspase-10. The present work reports for the first time CDH1 germline mutations in Portuguese gastric cancer families, and highlights the need for p53 mutation screening in families lacking CDH1 germline mutations, in a country with one of the highest incidences of gastric cancer in the world. No evidence was found for a role of germline mutations in SMAD4 and Caspase-10 in families lacking CDH1 mutations.

Introduction

The incidence of gastric cancer has been decreasing in older patients, but in younger patients and cases with familial clustering the level remains stable [1]. Aggregation of gastric cancer within families is observed in approximately 10% of the cases [2], [3], but only 1–3% of gastric carcinomas arise as a result of inherited gastric cancer predisposition syndromes [4].

In 1999, clinical criteria for the hereditary diffuse gastric cancer (HDGC) syndrome were defined by the International Gastric Cancer Linkage Consortium (IGCLC) [5]. However, gastric cancer may also be seen as part of the tumour spectrum in other inherited cancer predisposition syndromes, such as: hereditary non-polyposis colorectal cancer syndrome (HNPCC), Li-Fraumeni syndrome (LFS), Familial Adenomatous Polyposis (FAP), Cowden syndrome and Peutz-Jeghers syndrome (PJS) [6], [7], [8], [9], [10].

The presence of germline CDH1 mutations in affected family members was shown to be, the genetic defect responsible for a proportion of families with HDGC as first described by Guilford and collaborators in 1998 [11]. Approximately one third of families with an aggregation of gastric cancer, fulfilling the IGCLC criteria for HDGC studied so far, show germline CDH1 mutations in affected individuals (reviewed in [12]). Most of these families carry truncating mutations, whereas a small percentage carry missense mutations [12].

Approximately two thirds of HDGC families remain genetically unexplained. In kindred negative for CDH1 germline mutations, other genes are probably involved. In some inherited predisposition syndromes characterised by a higher incidence of gastric cancer, germline mutations of different tumour-related genes have been demonstrated to segregate with the disease. HNPCC occurs due to inactivating alterations of mismatch repair genes (MMR) leading to instability at short tandem repeat sequences – microsatellites – microsatellite instability (MSI), a typical molecular manifestation of MMR deficiency in the tumour tissue of HNPCC patients [13]. In approximately 70% of Li-Fraumeni kindred, which occasionally present with gastric cancer cases, germline mutations in p53 are found [14]. SMAD4 has been found to be inactivated in a percentage of PJS, and knockout studies revealed the presence of foci of signet-ring carcinoma cells in the stomach of SMAD4 heterozygous mice [15]. Each of the aforementioned genes, as well as genes found to be inactivated in sporadic gastric cancer (like Caspase-10 [16]), remain good candidates for familial gastric cancer.

We have selected a series of Portuguese families with a positive history of gastric cancer (n=32), and a series of Portuguese early onset gastric cancer patients (n=23) and screened all probands for CDH1 germline mutations. In probands negative for CDH1, we have screened the entire coding sequence and splice-sites of several candidate genes, namely SMAD4, Caspase-10 and the mutational hotspots corresponding to exons 5–8 of p53. Whenever tumour tissue was available, we performed MSI analysis, and in MSI-positive cases hMLH1 and hMSH2 coding sequences were studied to exclude HNPCC.