Identification of CDH1 germline missense mutations associated with functional inactivation of the E-cadherin protein in young gastric cancer probands

E-cadherin is involved in the formation of cell-junctions and the maintenance of epithelial integrity. Direct evidence of E-cadherin mutations triggering tumorigenesis has come from the finding of inactivating germline mutations of the gene (CDH1) in hereditary diffuse gastric cancer (HDGC). We screened a series of 66 young gastric cancer probands for germline CDH1 mutations, and two novel missense alterations together with an intronic variant were identified. We then analysed the functional significance of the two exonic missense variants found here as well as a third germline missense variant that we previously identified in a HGDC family. cDNAs encoding either the wild-type protein or mutant forms of E-cadherin were stably transfected into CHO (Chinese hamster ovary) E-cadherin-negative cells. Transfected cell-lines were characterized in terms of aggregation, motility and invasion. We show that a proportion of apparently sporadic early-onset diffuse gastric carcinomas are associated with germline alterations of the E-cadherin gene. We also demonstrate that a proportion of missense variants are associated with significant functional consequences, suggesting that our cell model can be used as an adjunct in deciding on the potential pathogenic role of identified E-cadherin germline alterations.     

Germline E-cadherin gene (CDH1) mutations predispose to familial gastric cancer and colorectal cancer

Inherited mutations in the E-cadherin gene ( CDH1 ) were described recently in three Maori kindreds with familial gastric cancer. Familial gastric cancer is genetically heterogeneous and it is not clear what proportion of gastric cancer susceptibility in non-Maori populations is due to germline CDH1 mutations. Therefore, we screened eight familial gastric cancer kindreds of British and Irish origin for germline CDH1 mutations, by SSCP analysis of all 16 exons and flanking sequences. Each family contained: (i) two cases of gastric cancer in first degree relatives with one affected before age 50 years; or (ii) three or more cases of gastric cancer. Novel germline CDH1 mutations (a nonsense and a splice site) were detected in two families (25%). Both mutations were predicted to truncate the E-cadherin protein in the signal peptide domain. In one family there was evidence of non-penetrance and susceptibility to both gastric and colorectal cancer; thus, in addition to six cases of gastric cancer, a CDH1 mutation carrier developed colorectal cancer at age 30 years. We have confirmed that germline mutations in the CDH1 gene cause familial gastric cancer in non-Maori populations. However, only a minority of familial gastric cancers can be accounted for by CDH1 mutations. Loss of E-cadherin function has been implicated in the pathogenesis of sporadic colorectal and other cancers, and our findings provide evidence that germline CDH1 mutations predispose to early onset colorectal cancer. Thus, CDH1 should be investigated as a cause of inherited susceptibility to both gastric and colorectal cancers.     

Identification of seven novel germline mutations in the human E-cadherin (CDH1) gene

Hereditary diffuse gastric cancer (HDGC) is a cancer predisposition syndrome caused by germline mutation of the gene encoding the tumour-suppressor E-cadherin (CDH1). We describe the search for CDH1 mutations in 36 new diffuse gastric cancer families. All 16 CDH1 exons, neighbouring intronic sequence and an essential promoter region were screened by DNA sequencing. We detected nine different mutations, seven of which were novel. Of the seven novel mutations, five were identified in families who met the IGCLC clinical criteria for HDGC. Two mutations resulted in a premature stop codon and truncation of the protein. Three mutations affected splice sites; two of the splice-site mutations were shown by RT-PCR to disturb normal CDH1 splicing, while the third splice-site mutation was present in two unrelated HDGC families. The remaining two mutations resulted in amino acid substitutions and impaired the ability of E-cadherin protein to form cellular aggregates and suppress invasion in vitro. Together with the occurrence of extra-gastric tumours such as lobular breast and colorectal cancer, these findings further extend the types of CDH1 mutations and the spectrum of tumours associated with HDGC.     

Screening E-cadherin in gastric cancer families reveals germline mutations only in hereditary diffuse gastric cancer kindred

The International Gastric Cancer Linkage Consortium (IGCLC) predicted that up to 25% of families fulfilling the criteria for hereditary diffuse gastric cancer (HDGC) would harbor CDH1 germline mutations. This was based on observations from the low number of diffuse gastric cancer families described at the time, and its validation would require analysis of larger numbers. Here we report the results of germline CDH1 mutation screening in 39 kindred with familial aggregation of gastric cancer, a subset of which fulfills the criteria defined by the IGCLC for HDGC. CDH1 germline mutations were detected in four of 11 (36.4%) HDGC families. No mutations were identified in 63.6% of HDGC families or in kindred with familial aggregation of gastric cancer not fulfilling criteria for HDGC. These results add support to the evidence that only HDGC families harbor germline mutations in CDH1 and that genes other than CDH1 remain to be identified.     

ATM germline mutations in Spanish early-onset breast cancer patients negative for BRCA1/BRCA2 mutations

Heterozygous carriers of ATM (ataxia telangiectasia mutated gene) mutations have increased risk of breast cancer (BC). We have estimated the prevalence of mutations in the ATM gene among Spanish patients with early-onset BC. Forty-three patients diagnosed with BC before the age of 46 years, and negative for BRCA1 and BRCA2 mutations, were analysed for the presence of ATM mutations. A total of 34 ATM sequence variants were detected: 1 deleterious mutation, 10 unclassified variants and 23 polymorphisms. One patient (2.3%) carried the ATM deleterious mutation (3802delG that causes ataxia telangiectasia in the homozygous state) and 13 patients carried the 10 ATM unclassified variants. The truncating mutation 3802delG and eight of the rare variants were not detected in a control group of 150 individuals. Different bioinformatic sequence analysis tools were used to evaluate the effects of the unclassified ATM changes on RNA splicing and function protein. This in silico analysis predicted that the missense variants 7653 T>C and 8156 G>A could alter the splicing by disrupting an exonic splicing enhancer motif and the 3763 T>G, 6314 G>C, and 8156 G>A variants would affect the ATM protein function. These are the initial results concerning the prevalence of germline mutations in the ATM gene among BC cases in a Spanish population, and they suggest that ATM mutations can confer increased susceptibility to early-onset BC.     

De novo CDH1 mutation in a family presenting with early-onset diffuse gastric cancer

In this report, we describe the first concluded case of a de novo germline mutation in CDH1 in a hereditary diffuse gastric cancer (HDGC) kindred. The incident case was a woman with a personal history of Hodgkin's lymphoma and diffuse gastric cancer, who was then confirmed to have a CDH1 mutation (c.1792 C>T (R598X)). The patient's mother was found to have the same CDH1 germline mutation; however, neither maternal grandparent was found to carry the mutation, thus leading to a conclusion that the proband's mother's mutation is of de novo origin. This case highlights the importance of recognition of the HDGC syndrome and of testing for CDH1 germline mutations in young individuals with diffuse gastric cancer without a family history of the disease.     

A novel CDH1 germline missense mutation in a sporadic gastric cancer patient in north-east of Italy

It is well documented that germline mutations in the E-cadherin (CDH1) gene are linked to hereditary diffuse gastric cancer (HDGC). Despite the known molecular genetic causes, most gastric cancers are sporadic and poorly investigated for susceptibility genes. We report the finding of a novel germline missense mutation in exon 6, c. 820 G > A (p.G274S) in one sporadic gastric cancer patient. This new variant does not affect cryptic splicing of CDH1 as demonstrated by molecular assay. Immunohistochemical analysis shows a mixed pattern of E-cadherin staining (membranous and cytoplasmic) in the intestinal component, while in the diffuse counterpart, the membranous staining was prevalent and a reduced membranous expression of ß-catenin was observed. In vitro assays suggest that the mutant G274S does not affect the E-cadherin protein function, its expression pattern or subcellular localization. This new variant is present in EC2 extracellular domain of the protein (p.G120S in mature protein). The molecular modelling shows that this point mutation is not dramatic for local structure. However, p.S120 is located on the surface of the protein close to the functional calcium sites and in the region of interaction with EC1 domain of another E-cadherin molecule involved in the formation of the intercellular junction. Moreover, p.S120 residue could be involved in posttranslational modifications, such as phosphorylation or glycosylation, with possible effects on stability and integrity of adhesive properties of E-cadherin. In conclusion, the pathogenicity of this mutation is unlikely; probably we found a new germline CDH1 missense mutation with potential impact, however, of uncertain significance.     

Similar contributions of BRCA1 and BRCA2 germline mutations to early-onset breast cancer in Germany

This study was undertaken to investigate the prevalence of BRCA1 and BRCA2 germline mutations in 91 German patients unselected for family history, who were diagnosed with breast cancer before the age of 41 years. Clinical information and blood samples were obtained from all patients. A comprehensive BRCA1 and BRCA2 mutational analysis was performed using the protein truncation assay and single-strand conformational polymorphism analysis followed by DNA sequencing of variant signals detected by these assays. Five different deleterious germline mutations including four frameshift mutations and one missense mutation were identified, three in BRCA1 (3.3%) and two mutations (2.2%) in BRCA2. Both BRCA2 mutations are novel and might be specific for the German population. An additional BRCA1 missense mutation previously described and classified as an unknown variant was found. This mutation was also detected in two breast cancer patients of family P 328 and not in 140 healthy controls suggesting that it is disease associated. In addition, one common polymorphism and five novel intronic sequence variants with unknown significance were found. Our findings show that mutations in BRCA1 and BRCA2 may contribute similarly to early-onset breast cancer in Germany. Given current constraints on health-care resources, these results support the notion that BRCA1 and BRCA2 mutation screening may have the strongest impact on health-care when targeted to high-risk populations.     

A germline E-cadherin mutation in a family with gastric and colon cancer

Inactivating mutations have been found in the cell-cell adhesion molecule E-cadherin (CDH1), which acts as a tumor suppressor gene in different kinds of cancers, e.g. primarily diffuse gastric cancer and lobular breast cancer. In this study, we screened for germline alterations in familial gastric and colon cancer cases. In total, 20 gastric and 18 colon cancer patients with both familial gastric and colon cancer were tested for germline E-cadherin alterations by using PCR/SSCP, specific restriction digestion test and sequencing. No pathogenic mutations were identified in the gastric cancer patients. In two colon cancer patients, a missense mutation in exon 12, codon 592 (Ala592Thr) was found. This alteration segregated with diffuse gastric cancer and colon cancer in one of the families. The prevalence of this alteration in the general population and colon cancer cases was almost the same. However, the fact that this alteration (Ala592Thr) segregated with colon cancer and diffuse gastric cancer in one big family, suggests that this E-cadherin missense alteration, beside predisposing to diffuse gastric cancer, also may play a role in colorectal carcinogenesis.     

Contribution of BRCA1 and BRCA2 germline mutations to the incidence of early-onset breast cancer in Cyprus

In Cyprus, the prevalence of breast cancer associated with BRCA1 and BRCA2 mutations in young women is unknown. In this study, we present the results of mutational analysis of the BRCA1 and BRCA2 genes in 26 Cypriot women diagnosed with breast cancer by the age of 40. The entire coding regions, including splice sites, of the BRCA1 and BRCA2 genes were sequenced using cycle sequencing. We identified four pathogenic mutations: two in BRCA1 [c.1840A>T (K614X), c.5310delG (5429delG)] and two in BRCA2 [c.3531-3534delCAGC (3758del4), c.8755delG (8984delG)] in six of 26 unrelated patients. The BRCA2 mutation c.3531-3534delCAGC (3758del4) is novel and the BRCA1 mutation c.1840A>T (K614X) is reported for the first time in Cypriot patients. The BRCA2 Cypriot founder mutation c.8755delG (8984delG) was detected in three unrelated patients. Additionally, we identified one novel BRCA1 missense mutation, two novel polymorphisms and three novel intronic variants of which BRCA1 c.4185+3A>G (IVS12+3A>G) may be pathogenic. Of the six BRCA1/2 mutation carriers, only four had a family history. These results show that the prevalence of BRCA1 and BRCA2 mutations in Cypriot women diagnosed with early-onset breast cancer is high. We conclude that Cypriot women with early-onset breast cancer should be offered BRCA1/2 testing irrespective of their family history.     

早发性乳腺癌/卵巢癌BRCA1基因突变分析

目的探讨中国北方地区汉族人群早发性乳腺癌和卵巢癌与BRCA1基因的关系.方法应用聚合酶链反应-单链构象多态性分析和DNA直接测序法检测BRCA1基因外显子2和外显子11部分序列.结果对12例早发性乳腺癌/卵巢癌患者PCR-SSCP的检测中,在外显子2发现1例突变,经DNA测序证实突变为第166碱基插入碱基"C".结论 BRCA1基因突变可能与北方地区汉族人群早发性乳腺癌有关.     

CDH1 mutations in gastric cancers are not associated with family history

CDH1 mutation is the most frequent genetic alteration in hereditary diffuse gastric cancer (GC) and early onset diffuse GC patients. However, the incidence of CDH1 mutations in sporadic GC with or without family history has not been studied. This retrospective study includes a total of 993 Korean patients with primary advanced GC who underwent surgery and received palliative chemotherapy. Targeted deep sequencing was performed in all cases and family history of GC was searched with survival analysis. We found CDH1 alterations in 146 of 993 patients (14.7 %) and 8 were germline (0.8 %). Out of 146 patients with CDH1 mutations, 25 (17.1 %) had a family history of GC in one of their first relatives, and 12 patients (8.2 %) were diagnosed with familial GC (FGC). All cases with FGC were diffuse type by Lauren classification, and only one harbored a previously reported germline mutation of CDH1 (c.2638 G > A) and the remaining 11 harbored known somatic CDH1 mutations. Among all patients with CDH1 mutation, there was no significant survival difference between patients with family history or FGC. In the 847 patients without CDH1 mutation, 189 (22.3 %) had a family history of GC and 92 patients (10.9 %) were FGC. CDH1 mutations were more frequent in patients with early onset (<45 years) GC (45.5 %) compared with patients with late onset GC (10.9 %) (p = 0.001), but were not significantly associated with the family history of GC (p > 0.05). CDH1 mutations are mostly somatic and typically are not associated with family history.     

Germline mutations of E-cadherin gene in Korean familial gastric cancer patients

Gastric cancer is the most common cancer in Korea. Germline mutations of the E-cadherin gene have recently been identified in familial gastric cancer patients. We screened five Korean familial gastric cancer patients to investigate germline mutations of the E-cadherin gene. These patients fulfilled the following criteria: presence of at least two gastric cancer patients within first-degree relatives and one patient diagnosed before the age of 50 years. Abnormal band patterns were found in exons 6 and 10 in two familial gastric cancer patients by polymerase chain reaction-single strand conformation polymorphism analysis (probands from the SNU-G2 and SNU-G1001 families, respectively). DNA sequencing analysis of the E-cadherin gene of these two patients revealed missense mutations in each exon. The SNU-G2 proband harbored a missense mutation from aspartic acid (GAT) to glycine (GGT) at codon 244 in exon 6 of the E-cadherin gene, and the SNU-G1001 proband had a missense mutation from valine (GTG) to alanine (GCG) at codon 487 in exon 10. The SNU-G2 proband was diagnosed with gastric cancer at the age of 38; three brothers and two sisters had died of gastric cancer under the age of 50, and their mother had died of gastric cancer at the age of 63. The SNU-G1001 proband was diagnosed with gastric cancer at the age of 42 and one brother had died of gastric cancer at the age of 49. In summary, we found germline mutations of the E-cadherin gene in two of five Korean familial gastric cancer patients screened. Received: January 4, 1999 / Accepted: February 4, 1999     

MLH1 germline epimutations in selected patients with early-onset non-polyposis colorectal cancer

Recently, the germline epigenetic inactivation of MLH1 has been reported in a number of patients with early-onset colorectal cancer among other characteristics. The aim of the present study is to evaluate the presence of MLH1 germline epimutations in selected colorectal cancer patients suspected of hereditary non-polyposis colorectal cancer (HNPCC) in order to determine in which patients the MLH1 epigenetic test should be performed. From a total of 109 microsatellite instability (MSI)-positive HNPCC-suspected patients, 11 showed a lack of MLH1 expression in tumor tissue and no germline mutations in the mismatch repair (MMR) genes. In nine of these cases and in three additional patients with multiple tumors, the study of the germline MLH1 promoter hypermethylation was performed by means of methylation-specific PCR and combined bisulfite-restriction analysis techniques. One of the selected patients resulted positive for the MLH1 epimutation, which was confirmed in the DNA extracted from buccal lavage. The patient with the epimutation had developed an epidermoid lip carcinoma and an early-onset colorectal tumor with MSI, no MLH1 expression, and loss of heterozygosity of the gene. Parents and siblings did not carry the epigenetic alteration, suggesting a de novo mechanism. Although germline MLH1 epimutations seem to be mostly uncommon, when the cases are well selected, the probability of finding them increases. Thus, taking into account ours and previous reports, we propose that screening for MLH1 epimutations in blood DNA could be performed in early-onset colorectal cancer patients with MSI, lack of MLH1 expression in the tumor, and no germline mutations in the MMR genes.     

BRCA1 germline mutations in Indian familial breast cancer

Germline mutation analysis of BRCA1 gene has demonstrated significant allelic heterogeneity. These differences represent historical influences of migration, population structure and geographic or cultural isolation. To date, there have been no reports of Indian families with mutations in BRCA1. We have screened for mutations in selected coding exons of BRCA1 and their flanking intron regions in three breast or breast and ovarian cancer families with family history of three or more cases of breast cancer under age 45 and/or ovarian cancer at any age. We have also analyzed 10 female patients with sporadic breast cancer regardless of age and family history, as well as 50 unrelated normal individuals as controls. Thus a total of 90 samples were analyzed for BRCA1 mutations using polymerase chain reaction-mediated site directed mutagenesis (PSM) and single stranded conformation polymorphism (SSCP) analysis for various selected exons followed by sequencing of variant bands. Eight point mutations were identified. Two deleterious pathogenic, protein truncating non-sense mutations were detected in exon 11 (E1250X) and exon 20 (E1754X) and six novel and unique amino acid substitutions (F1734S, D1739Y, V1741G, Q1747H, P1749A, R1753K). One complex missense mutation of exon 20 [V1741G; P1749A] was seen in two out of three families and another complex combination of missense and non-sense mutations of the same exon [V1741G; E1754X] was observed in only one family. These complex mutations exist only in breast cancer families but not in control populations of women. Three splice site variants (IVS20+3A>C, IVS20+4A>T, IVS20+5A>T) and two intronic variants (IVS20+21_22insG, IVS20+21T>G) were also detected. In the group of 10 sporadic female patients no mutations were found.