- Email: [email protected]
Hereditary diffuse gastric cancer and E-cadherin: Description of the first germline mutation in an Italian family
EJSO 33 (2007) 448e451
www.ejso.com
Hereditary diffuse gastric cancer and E-cadherin: Description of the first germline mutation in an Italian family F. Roviello a, G. Corso a, C. Pedrazzani a,*, D. Marrelli a, G. De Falco b,c, A. Berardi a, L. Garosi a, G. Suriano d, C. Vindigni b, A. De Stefano a, L. Leoncini b, R. Seruca d, E. Pinto a a
Department of Human Pathology and Oncology, Unit of Surgical Oncology, University of Siena, Viale Bracci 2, 53100 Siena, Italy b Department of Human Pathology and Oncology, Unit of Pathology, University of Siena, Italy c College of Science and Technology Sbarro Institute for Cancer Research and Molecular Medicine, Temple University, Philadelphia, PA, USA d Institute of Molecular Pathology and Immunology of the University of Porto, University of Porto, Portugal Accepted 16 October 2006 Available online 28 November 2006
Abstract Aims: Germline mutation of the E-cadherin gene (CDH1) accounts for the Hereditary Diffuse Gastric Cancer (HDGC) syndrome. Fourteen pedigrees with Diffuse Gastric Cancer that fulfilled the International Gastric Cancer Linkage Consortium (IGCLC) criteria were selected and screened for CDH1 germline mutations. Methods: The entire coding region of the CDH1 gene and all introneexon boundaries were analyzed by direct sequencing in the 14 families fulfilling the IGCLC criteria. E-cadherin immunohistochemical expression was evaluated on tumour as well as normal formalin-fixed paraffin embedded tissues. Results: A novel germline missense mutation was found. It was a single C / T substitution in exon 8, resulting in a transition of CCG / CTG (C1118T; Pro373Leu) demonstrated in the proband and her brother. At immunohistochemical analysis, the staining intensity was reduced and considered weakly positive (15%). Conclusions: The first CDH1 germline mutation of an Italian family is herein reported. The present missense mutation has never been described so far. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: E-cadherin; CDH1; Hereditary; Diffuse gastric cancer
Introduction Hereditary Diffuse Gastric Cancer (HDGC) is an autosomal dominant familial syndrome associated with the diffuse histotype and caused by germline mutations in the Ecadherin gene (CDH1).1 CDH1 gene maps to chromosome 16q22.1 and comprises 16 exons encoding for E-cadherin protein.2 E-cadherin is a transmembrane glycoprotein responsible for cell-to-cell adhesion and its deregulation is correlated with infiltrative and metastatic ability of the tumour.3,4 Originally, CDH1 germline mutation was described in three Maori families by Guilford and colleagues in 19985; subsequently, additional germline mutations have been identified in families from several * Corresponding author. Tel.: þ39 577 585156; fax: þ39 577 585157. E-mail address: [email protected] (C. Pedrazzani). 0748-7983/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.ejso.2006.10.028
countries.6 No patients with CDH1 germline mutations have been described in Italy so far.7 This study reports the first novel CDH1 germline missense mutation identified in Italian families. Methods Familial history was retrospectively investigated according to the International Gastric Cancer Linkage Consortium (IGCLC) criteria in order to search for families with potential Hereditary Diffuse Gastric Cancer (HDGC) in 238 patients affected by primary gastric cancer and operated on at the Division of General Surgery and Surgical Oncology, University of Siena, Italy, between January 1988 and December 2004. The adopted criteria were the following: two or more documented cases of diffuse gastric cancer
F. Roviello et al. / EJSO 33 (2007) 448e451
Sequencing analysis
in first or second degree relatives, with at least one diagnosed before the age of 50 or, three or more documented cases of diffuse gastric cancer in first or second degree relatives, independent of age of onset.6 In cases suspicious for HDGC the histopathological report was obtained for all the relatives affected by gastric cancer in order to confirm the diffuse type.
2 ml aliquot of purified PCR product was cycle sequenced using Big Dye Terminator Mix (Applied Biosystems, Foster City, CA) in a total volume of 20 ml. Reaction products were purified with ethanol/sodium acetate and washed with 70% ethanol; and finally 5 ml of product was resuspended in 15 ml of double distilled water before loading on ABI capillary sequencers. Sequence reads were base-called using Chromas program. Comparison between the germline sequence present in gene bank and sample sequence was made using the BLAST2 seq program (www.ncbi.nlm.nih.gov/blast/bl2seq/ wblast2.cgi)
DNA extraction Genomic DNA was purified from peripheral white blood cells or frozen normal gastric mucosa. Puregene DNA Purification Kit (Gentra Systems) was used and the manufacturer’s manual was followed for genomic DNA isolation as previously described in detail.8
Immunohistochemical analysis Formalin-fixed paraffin embedded tissues were used for the evaluation of E-cadherin immunohistochemical (IHC) expression adopting a mouse monoclonal antibody against E-cadherin (HECD-1, diluted 1:200; Zymed, San Francisco, CA, USA), following the modification of the avidinebiotin complex and diaminobenzidine. Briefly, the intensity of the yellowish brown staining was considered as positive (more than 90% of the tumour cells exhibited an intense staining), weakly positive (10e90%) and negative (<10%).9 E-cadherin IHC expression was evaluated on tumoural as well as normal tissue.
Polymerase chain reaction (PCR) amplification Each exon of the CDH1 gene including introneexon boundaries was amplified with forward and reverse primers. Primers and conditions were as previously reported.1 Briefly, PCR reactions were carried out in a volume of 50 ml containing 100 ng genomic DNA template, 20 mM of each PCR primer, 10 mM dNTPs, Phusion HF or GC Buffer 5 and Taq Phusion High-Fidelity DNA Polymerase (Finnzymes, Finland) 2 U/ml. The reaction was performed in programmable thermocyclers according to protocol standard. A 5 ml aliquot of each PCR reaction was run on a 2% agarose gel to confirm the size, quantity, and purity of each PCR product. The remaining 45 ml of PCR product was purified using Jet Quick Gel Extraction Spin Kit (Celbio, Italy) and eluted in a volume of 30 ml of TE (TriseEDTA pH 8.0) according to the manufacturer’s instructions.
Results Fourteen families fulfilled the IGCLC clinical criteria. Eight families (57.1%) had two first-degree members with histologically confirmed diffuse gastric cancer of which at least one diagnosed before the age of 50 and 6 (42.9%)
I-1
I-2
(+)
II-1
II-2
II-3
449
(+)
II-5
II-4
(?)
II-6
II-7
(-)
(?)
= Male = Female
II-3 III-1
III-2
(-)
(-)
= Gastric Cancer = Colon Cancer
IV-1
IV-2
= Ductal Breast Cancer = Dead = Proband Figure 1. Pedigree of family with the novel missense germline CDH1 mutation. II-1 (75 years old); II-2 (70 years old); II-3 (72 years old); II-5 (73 years old); II-7 (79 years old); III-3 (65 years old); (þ) CDH1 mutation carrier; () no CDH1 mutation carrier; (?) biological material not obtained.
450
F. Roviello et al. / EJSO 33 (2007) 448e451
Figure 2. Chromatogram of sequence analysis of the exon 8 of the patient with the novel missense germline CDH1 mutation. The sequence variation is C/T, a substitution of C for T (missense mutation).
had three first-degree relatives diagnosed with diffuse gastric cancer independent of the age of onset. The 14 HDGC patients were screened for CDH1 germline mutations and a novel missense mutation was identified in one family. It was a single C / T substitution in exon 8, resulting in a transition of CCG / CTG (Pro373Leu) observed in the proband and her brother (Figs. 1 and 2). A silent exonic sequence variant previously reported10 was detected in an other patient: CAC / CAT (His632His) in exon 12. IHC of the patient with CDH1 mutation showed a moderate/scarce positivity in membranous staining; the staining intensity was reduced and considered weakly positive; staining intensity of tumour was about 15% of normal mucosa (Fig. 3).
taking into consideration the unknown pathogenic role of this missense mutation and in consideration of his advanced age (Fig. 1). Strict follow-up with yearly chromoendoscopy was preferred.
Discussion In 1998 Guilford and colleagues described the first germline mutation of E-cadherin gene in three Maori families with predisposition to autosomal dominant diffuse gastric cancer.5 According to the most recent review, 58 CDH1 germline mutations have been described, of which 19 (33%) are of the missense type.11 The CDH1 germline mutation herein reported is a missense mutation never described so far; this results as a single C / T substitution in exon 8, causing a transition of CCG / CTG (C1118T; Pro373Leu). The mutation was identified in the proband and her brother. The C1118T change observed in this family represents a novel missense change of unknown pathogenic relevance. Germline missense mutations represent a clinical burden in genetic counselling since their pathogenic role must be demonstrated. In particular, several concerns continue on the management of asymptomatic carriers. Current surveillance techniques are unsatisfactory and prophylactic total gastrectomy remains the treatment of choice for carriers of pathogenic CDH1 germline mutations.1 No prophylactic gastrectomy was proposed to the brother of our patient
Figure 3. E-cadherin immunohistochemistry (200 magnification) of the patient with the novel missense germline CDH1 mutation. (A) Normal mucosa. (B) Tumour tissue.
F. Roviello et al. / EJSO 33 (2007) 448e451
Suriano and colleagues realized a method for demonstrating the pathogenic role of missense mutations via in silico analysis and in vitro functional assays.12 To date, 13 out of the 19 described missense mutations have been studied with the above mentioned technique. The majority of them (11) showed an impairment of cell-to-cell adhesion and a loss of invasion ability supporting their pathogenic nature.12 Immunohistochemical analysis of our patient showed a significant ipoexpression (15%) of E-cadherin protein in tumour tissue compared to normal mucosa. Somatic inactivation of E-cadherin is due to the well-known phenomenon of ‘second inactivating hit’. The most frequent epigenetic mechanism is the hypermethylation of the promoter region of the CDH1 gene; other possible but less frequent mechanisms are somatic mutations and loss of heterozygosity.13,14 Overall, this study identifies a novel missense mutation of the CDH1 gene in the first family diagnosed with HDGC in Italy. The pathogenic role of the herein described mutation must be demonstrated.
2.
3. 4. 5. 6.
7.
8.
9.
10.
Acknowledgements
11.
This work has been supported by grant PAR 2004, University of Siena, and by grant MIUR 2005 to Unit of Surgical Oncology, University of Siena, Italy. Conflict of interest: None declared.
13.
References
14.
1. Brooks-Wilson AR, Kaurah P, Suriano G, et al. Germline E-cadherin mutations in hereditary diffuse gastric cancer: assessment of 42 new
12.
451
families and review of genetic screening criteria. J Med Genet 2004; 41:508–17. Berx G, Cleton-Jansen AM, Nollet F, et al. E-cadherin is a tumour/ invasion suppressor gene mutated in human lobular breast cancers. EMBO J 1995;14:6107–15. Takeichi M. Cadherin cell adhesion receptors as a morphogenetic regulator. Science 1991;251:1451–5. Takeichi M. Cadherins in cancer: implications for invasion and metastasis. Curr Opin Cell Biol 1993;5:806–11. Guilford P, Hopkins J, Harraway J, et al. E-cadherin germline mutations in familial gastric cancer. Nature 1998;392:402–5. Caldas C, Carneiro F, Lynch HT, et al. Familial gastric cancer: overview and guidelines for management. J Med Genet 1999;36: 873–80. Graziano F, Ruzzo AM, Bearzi I, Testa E, Lai V, Magnani M. Screening E-cadherin germline mutations in Italian patients with familial diffuse gastric cancer: an analysis in the District of Urbino, Region Marche, Central Italy. Tumori 2003;89:255–8. Suriano G, Yew S, Ferreira P, et al. Characterization of a recurrent germ line mutation of the E-cadherin gene: implications for genetic testing and clinical management. Clin Cancer Res 2005;11: 5401–9. Liu YC, Shen CY, Wu HS, et al. Mechanisms inactivating the gene for E-cadherin in sporadic gastric carcinomas. World J Gastroenterol 2006;12:2168–73. Berx G, Becker KF, Hofler H, van Roy F. Mutations of the human E-cadherin (CDH1) gene. Hum Mutat 1998;12:226–37. Suriano G, Ferreira P, Mateus AR, Correia J, Henriques L, Seruca R. Genetics of hereditary diffuse gastric cancer: progress and future challenges. Future Oncol 2006;2:363–70. Suriano G, Mulholland D, de Wever O, et al. The intracellular E-cadherin germline mutation V832 M lacks the ability to mediate cellecell adhesion and to suppress invasion. Oncogene 2003;22:5716–9. Oliveira C, de Bruin J, Nabais S, et al. Intragenic deletion of CDH1 as the inactivating mechanism of the wild-type allele in an HDGC tumour. Oncogene 2004;23:2236–40. Rodriguez-Sanjuan JC, Fontalba A, Mayorga M, et al. A novel mutation in the E-cadherin gene in the first family with hereditary diffuse gastric cancer reported in Spain. Eur J Surg Oncol 2006 Jul 24 [Epub ahead of print].
www.ejso.com
Hereditary diffuse gastric cancer and E-cadherin: Description of the first germline mutation in an Italian family F. Roviello a, G. Corso a, C. Pedrazzani a,*, D. Marrelli a, G. De Falco b,c, A. Berardi a, L. Garosi a, G. Suriano d, C. Vindigni b, A. De Stefano a, L. Leoncini b, R. Seruca d, E. Pinto a a
Department of Human Pathology and Oncology, Unit of Surgical Oncology, University of Siena, Viale Bracci 2, 53100 Siena, Italy b Department of Human Pathology and Oncology, Unit of Pathology, University of Siena, Italy c College of Science and Technology Sbarro Institute for Cancer Research and Molecular Medicine, Temple University, Philadelphia, PA, USA d Institute of Molecular Pathology and Immunology of the University of Porto, University of Porto, Portugal Accepted 16 October 2006 Available online 28 November 2006
Abstract Aims: Germline mutation of the E-cadherin gene (CDH1) accounts for the Hereditary Diffuse Gastric Cancer (HDGC) syndrome. Fourteen pedigrees with Diffuse Gastric Cancer that fulfilled the International Gastric Cancer Linkage Consortium (IGCLC) criteria were selected and screened for CDH1 germline mutations. Methods: The entire coding region of the CDH1 gene and all introneexon boundaries were analyzed by direct sequencing in the 14 families fulfilling the IGCLC criteria. E-cadherin immunohistochemical expression was evaluated on tumour as well as normal formalin-fixed paraffin embedded tissues. Results: A novel germline missense mutation was found. It was a single C / T substitution in exon 8, resulting in a transition of CCG / CTG (C1118T; Pro373Leu) demonstrated in the proband and her brother. At immunohistochemical analysis, the staining intensity was reduced and considered weakly positive (15%). Conclusions: The first CDH1 germline mutation of an Italian family is herein reported. The present missense mutation has never been described so far. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: E-cadherin; CDH1; Hereditary; Diffuse gastric cancer
Introduction Hereditary Diffuse Gastric Cancer (HDGC) is an autosomal dominant familial syndrome associated with the diffuse histotype and caused by germline mutations in the Ecadherin gene (CDH1).1 CDH1 gene maps to chromosome 16q22.1 and comprises 16 exons encoding for E-cadherin protein.2 E-cadherin is a transmembrane glycoprotein responsible for cell-to-cell adhesion and its deregulation is correlated with infiltrative and metastatic ability of the tumour.3,4 Originally, CDH1 germline mutation was described in three Maori families by Guilford and colleagues in 19985; subsequently, additional germline mutations have been identified in families from several * Corresponding author. Tel.: þ39 577 585156; fax: þ39 577 585157. E-mail address: [email protected] (C. Pedrazzani). 0748-7983/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.ejso.2006.10.028
countries.6 No patients with CDH1 germline mutations have been described in Italy so far.7 This study reports the first novel CDH1 germline missense mutation identified in Italian families. Methods Familial history was retrospectively investigated according to the International Gastric Cancer Linkage Consortium (IGCLC) criteria in order to search for families with potential Hereditary Diffuse Gastric Cancer (HDGC) in 238 patients affected by primary gastric cancer and operated on at the Division of General Surgery and Surgical Oncology, University of Siena, Italy, between January 1988 and December 2004. The adopted criteria were the following: two or more documented cases of diffuse gastric cancer
F. Roviello et al. / EJSO 33 (2007) 448e451
Sequencing analysis
in first or second degree relatives, with at least one diagnosed before the age of 50 or, three or more documented cases of diffuse gastric cancer in first or second degree relatives, independent of age of onset.6 In cases suspicious for HDGC the histopathological report was obtained for all the relatives affected by gastric cancer in order to confirm the diffuse type.
2 ml aliquot of purified PCR product was cycle sequenced using Big Dye Terminator Mix (Applied Biosystems, Foster City, CA) in a total volume of 20 ml. Reaction products were purified with ethanol/sodium acetate and washed with 70% ethanol; and finally 5 ml of product was resuspended in 15 ml of double distilled water before loading on ABI capillary sequencers. Sequence reads were base-called using Chromas program. Comparison between the germline sequence present in gene bank and sample sequence was made using the BLAST2 seq program (www.ncbi.nlm.nih.gov/blast/bl2seq/ wblast2.cgi)
DNA extraction Genomic DNA was purified from peripheral white blood cells or frozen normal gastric mucosa. Puregene DNA Purification Kit (Gentra Systems) was used and the manufacturer’s manual was followed for genomic DNA isolation as previously described in detail.8
Immunohistochemical analysis Formalin-fixed paraffin embedded tissues were used for the evaluation of E-cadherin immunohistochemical (IHC) expression adopting a mouse monoclonal antibody against E-cadherin (HECD-1, diluted 1:200; Zymed, San Francisco, CA, USA), following the modification of the avidinebiotin complex and diaminobenzidine. Briefly, the intensity of the yellowish brown staining was considered as positive (more than 90% of the tumour cells exhibited an intense staining), weakly positive (10e90%) and negative (<10%).9 E-cadherin IHC expression was evaluated on tumoural as well as normal tissue.
Polymerase chain reaction (PCR) amplification Each exon of the CDH1 gene including introneexon boundaries was amplified with forward and reverse primers. Primers and conditions were as previously reported.1 Briefly, PCR reactions were carried out in a volume of 50 ml containing 100 ng genomic DNA template, 20 mM of each PCR primer, 10 mM dNTPs, Phusion HF or GC Buffer 5 and Taq Phusion High-Fidelity DNA Polymerase (Finnzymes, Finland) 2 U/ml. The reaction was performed in programmable thermocyclers according to protocol standard. A 5 ml aliquot of each PCR reaction was run on a 2% agarose gel to confirm the size, quantity, and purity of each PCR product. The remaining 45 ml of PCR product was purified using Jet Quick Gel Extraction Spin Kit (Celbio, Italy) and eluted in a volume of 30 ml of TE (TriseEDTA pH 8.0) according to the manufacturer’s instructions.
Results Fourteen families fulfilled the IGCLC clinical criteria. Eight families (57.1%) had two first-degree members with histologically confirmed diffuse gastric cancer of which at least one diagnosed before the age of 50 and 6 (42.9%)
I-1
I-2
(+)
II-1
II-2
II-3
449
(+)
II-5
II-4
(?)
II-6
II-7
(-)
(?)
= Male = Female
II-3 III-1
III-2
(-)
(-)
= Gastric Cancer = Colon Cancer
IV-1
IV-2
= Ductal Breast Cancer = Dead = Proband Figure 1. Pedigree of family with the novel missense germline CDH1 mutation. II-1 (75 years old); II-2 (70 years old); II-3 (72 years old); II-5 (73 years old); II-7 (79 years old); III-3 (65 years old); (þ) CDH1 mutation carrier; () no CDH1 mutation carrier; (?) biological material not obtained.
450
F. Roviello et al. / EJSO 33 (2007) 448e451
Figure 2. Chromatogram of sequence analysis of the exon 8 of the patient with the novel missense germline CDH1 mutation. The sequence variation is C/T, a substitution of C for T (missense mutation).
had three first-degree relatives diagnosed with diffuse gastric cancer independent of the age of onset. The 14 HDGC patients were screened for CDH1 germline mutations and a novel missense mutation was identified in one family. It was a single C / T substitution in exon 8, resulting in a transition of CCG / CTG (Pro373Leu) observed in the proband and her brother (Figs. 1 and 2). A silent exonic sequence variant previously reported10 was detected in an other patient: CAC / CAT (His632His) in exon 12. IHC of the patient with CDH1 mutation showed a moderate/scarce positivity in membranous staining; the staining intensity was reduced and considered weakly positive; staining intensity of tumour was about 15% of normal mucosa (Fig. 3).
taking into consideration the unknown pathogenic role of this missense mutation and in consideration of his advanced age (Fig. 1). Strict follow-up with yearly chromoendoscopy was preferred.
Discussion In 1998 Guilford and colleagues described the first germline mutation of E-cadherin gene in three Maori families with predisposition to autosomal dominant diffuse gastric cancer.5 According to the most recent review, 58 CDH1 germline mutations have been described, of which 19 (33%) are of the missense type.11 The CDH1 germline mutation herein reported is a missense mutation never described so far; this results as a single C / T substitution in exon 8, causing a transition of CCG / CTG (C1118T; Pro373Leu). The mutation was identified in the proband and her brother. The C1118T change observed in this family represents a novel missense change of unknown pathogenic relevance. Germline missense mutations represent a clinical burden in genetic counselling since their pathogenic role must be demonstrated. In particular, several concerns continue on the management of asymptomatic carriers. Current surveillance techniques are unsatisfactory and prophylactic total gastrectomy remains the treatment of choice for carriers of pathogenic CDH1 germline mutations.1 No prophylactic gastrectomy was proposed to the brother of our patient
Figure 3. E-cadherin immunohistochemistry (200 magnification) of the patient with the novel missense germline CDH1 mutation. (A) Normal mucosa. (B) Tumour tissue.
F. Roviello et al. / EJSO 33 (2007) 448e451
Suriano and colleagues realized a method for demonstrating the pathogenic role of missense mutations via in silico analysis and in vitro functional assays.12 To date, 13 out of the 19 described missense mutations have been studied with the above mentioned technique. The majority of them (11) showed an impairment of cell-to-cell adhesion and a loss of invasion ability supporting their pathogenic nature.12 Immunohistochemical analysis of our patient showed a significant ipoexpression (15%) of E-cadherin protein in tumour tissue compared to normal mucosa. Somatic inactivation of E-cadherin is due to the well-known phenomenon of ‘second inactivating hit’. The most frequent epigenetic mechanism is the hypermethylation of the promoter region of the CDH1 gene; other possible but less frequent mechanisms are somatic mutations and loss of heterozygosity.13,14 Overall, this study identifies a novel missense mutation of the CDH1 gene in the first family diagnosed with HDGC in Italy. The pathogenic role of the herein described mutation must be demonstrated.
2.
3. 4. 5. 6.
7.
8.
9.
10.
Acknowledgements
11.
This work has been supported by grant PAR 2004, University of Siena, and by grant MIUR 2005 to Unit of Surgical Oncology, University of Siena, Italy. Conflict of interest: None declared.
13.
References
14.
1. Brooks-Wilson AR, Kaurah P, Suriano G, et al. Germline E-cadherin mutations in hereditary diffuse gastric cancer: assessment of 42 new
12.
451
families and review of genetic screening criteria. J Med Genet 2004; 41:508–17. Berx G, Cleton-Jansen AM, Nollet F, et al. E-cadherin is a tumour/ invasion suppressor gene mutated in human lobular breast cancers. EMBO J 1995;14:6107–15. Takeichi M. Cadherin cell adhesion receptors as a morphogenetic regulator. Science 1991;251:1451–5. Takeichi M. Cadherins in cancer: implications for invasion and metastasis. Curr Opin Cell Biol 1993;5:806–11. Guilford P, Hopkins J, Harraway J, et al. E-cadherin germline mutations in familial gastric cancer. Nature 1998;392:402–5. Caldas C, Carneiro F, Lynch HT, et al. Familial gastric cancer: overview and guidelines for management. J Med Genet 1999;36: 873–80. Graziano F, Ruzzo AM, Bearzi I, Testa E, Lai V, Magnani M. Screening E-cadherin germline mutations in Italian patients with familial diffuse gastric cancer: an analysis in the District of Urbino, Region Marche, Central Italy. Tumori 2003;89:255–8. Suriano G, Yew S, Ferreira P, et al. Characterization of a recurrent germ line mutation of the E-cadherin gene: implications for genetic testing and clinical management. Clin Cancer Res 2005;11: 5401–9. Liu YC, Shen CY, Wu HS, et al. Mechanisms inactivating the gene for E-cadherin in sporadic gastric carcinomas. World J Gastroenterol 2006;12:2168–73. Berx G, Becker KF, Hofler H, van Roy F. Mutations of the human E-cadherin (CDH1) gene. Hum Mutat 1998;12:226–37. Suriano G, Ferreira P, Mateus AR, Correia J, Henriques L, Seruca R. Genetics of hereditary diffuse gastric cancer: progress and future challenges. Future Oncol 2006;2:363–70. Suriano G, Mulholland D, de Wever O, et al. The intracellular E-cadherin germline mutation V832 M lacks the ability to mediate cellecell adhesion and to suppress invasion. Oncogene 2003;22:5716–9. Oliveira C, de Bruin J, Nabais S, et al. Intragenic deletion of CDH1 as the inactivating mechanism of the wild-type allele in an HDGC tumour. Oncogene 2004;23:2236–40. Rodriguez-Sanjuan JC, Fontalba A, Mayorga M, et al. A novel mutation in the E-cadherin gene in the first family with hereditary diffuse gastric cancer reported in Spain. Eur J Surg Oncol 2006 Jul 24 [Epub ahead of print].