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HUMAN MUTATION Mutation in Brief #944 (2007) Online

MUTATION IN BRIEF

Identification of Seven Novel Germline Mutations in the Human E-Cadherin (CDH1) Gene H. More1, B. Humar1, W. Weber2, R. Ward3, A. Christian4, C. Lintott4, F. Graziano5, A-M. Ruzzo6, E. Acosta7, B. Boman8, M. Harlan8, P. Ferreira9, R. Seruca9,10, G. Suriano9,10, and P. Guilford1* 1

Cancer Genetics Laboratory, University of Otago, Dunedin, Aotearoa, New Zealand; 2Human Genetics, Center of Biomedicine DKBW, Basel University Hospital, Basel, Switzerland; 3Department of Medical Oncology, St. Vincent’s Hospital, Darlinghurst, Sydney, Australia; 4Central and Southern Regional Genetic Services, Christchurch Women's Hospital and Wellington Hospital, Aotearoa, New Zealand; 5Medical Oncology Unit, Hospital of Urbino, Urbino, Italy; 6Institute of Biochemistry “G Fornaini”, University of Urbino, Urbino, Italy; 7 Surgical Oncology UPAEP School of Medicine, Azcarate Puebla, Puebla, Mexico; 8Division of Genetics and Preventive Medicine, Jefferson Medical College, Philadelphia, Pennsylvania; 9Institute of Molecular Pathology and Immunology of the University of Porto, IPATIMU, Porto, Portugal; 10Faculty of Medicine, University of Porto, Porto, Portugal *Correspondence to: Parry Guilford, Cancer Genetics Laboratory, University of Otago, Dunedin, Aotearoa, New Zealand; E-mail: [email protected] Grant sponsor: Health Research Council of New Zealand; Grant number: 03/265 Communicated by Albert de la Chapelle

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 splicesite 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 extragastric 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. © 2007 Wiley-Liss, Inc. KEY WORDS: CDH1; E-cadherin; mutation; hereditary diffuse gastric cancer

INTRODUCTION

Gastric cancer is one of the leading causes of cancer-related mortality worldwide (WHO-IRC, 2003). The most widely used classification system (Lauren 1965) distinguishes two histological types of gastric carcinoma, the Received 21 May 2006; accepted revised manuscript 11 September 2006.

© 2007 WILEY-LISS, INC. DOI: 10.1002/humu.9473

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intestinal and the diffuse form. While the incidence of intestinal gastric cancer has gradually declined in the last decades, the incidence of diffuse gastric cancer (DGC) has remained steady (WHO-IRC, 2003), consistent with a larger contribution of inherited genetic risk to this form of the disease. About 10% of gastric cancers display some form of familial clustering and the majority of families with autosomal dominant clustering are associated with DGC (Caldas et al., 1999). The only known cancer syndrome dominated by gastric cancer is Hereditary Diffuse Gastric Cancer (HDGC). HDGC is defined by the presence of germline mutations in the gene (CDH1; MIM# 192090) encoding the cell-tocell adhesion protein E-cadherin (Guilford et al, 1998; Blair et al., 2006). E-cadherin deficiency provides an obvious explanation for the diffuse, scattered growth of HDGC tumours, as the protein is the central component of epithelial cell-to-cell adhesion junctions and as such is required for the integrity of epithelial layers (D’Souza-Schorey, 2005). In CDH1 germline mutation carriers, the lifetime penetrance is estimated to be around 70% (Pharoah et al., 2001). It is not clear why CDH1 mutations preferentially affect gastric epithelia, however, tumours do occur outside the stomach in HDGC families. The spectrum and frequency of extra-gastric disease is uncertain, as the number of identified HDGC families is too small to establish significant associations, however lobular breast cancer does appear to occur at an increased rate (Blair et al., 2006). In this report, we present the results on CDH1 mutation screening in 36 families with a high DGC incidence or a very early age of DGC onset. METHODS Sample Collection and DNA Extraction

DNA from affected and non-affected members of 36 families was sent to our laboratory for CDH1 mutation detection. DNA samples, tumour tissue, family history and pathological reports were obtained with informed consent. DNA from paraffin-embedded tumour tissue was extracted after overnight digestion with proteinase K in a Tween-KCl buffer and purification in 10% Chelex-100 resin (Biorad, Hercules, CA; http://www.bio-rad.com). CDH1 Mutation Detection

Germline DNA was screened for CDH1 mutations by PCR amplification and sequencing of the 16 CDH1 exons as previously described (Humar et al., 2002a; Guilford et al., 1999). PCR amplification of the CDH1 promoter region (from –353 to +45 of the transcription start site) was performed as described in Humar et al. (2002b). Sequencing was performed on a LiCor 4000 DNA Sequencer (LiCor, Lincoln, NE; http://www.licor.com). For splice site and missense mutations, CDH1 sequence changes found in affected patients were examined for sequence conservation amongst higher eukaryotes (Padgett et al., 1986; Ng and Henikoff, 2003). CDH1 mutations were confirmed by independent PCR amplification, reverse sequencing and, where possible, by restriction enzyme digest. RT-PCR was used to investigate putative splice site mutations. cDNA was generated from blood RNA using SUPERSCRIPT II reverse transcriptase according to the manufacturer’s protocol (Invitrogen, Carlsbad, CA; http://www.invitrogen.com). To study the mutation 49-2C, a cDNA fragment from exon 1 to exon 3 was amplified with the following primers, Ex1F 5’- ATG GGC CCT TGG AGC and Ex3R 5’- AAT AGG CTG TCC TTT GTC G. PCR conditions were 40 cycles at 55°C annealing using FastStart Taq (Roche Diagnostics, Mannheim, Germany; http://www.roche.com) and 2mM Mg++. For the mutation 2440-6C>G, a cDNA fragment from exon 15 to 16 was amplified with primers Ex15F 5’- TGA CTC GTA ACG ACG TTG CAC CAA and Hec211 5’- GTC TGA GCT CCC TGA ACT CC at 2mM Mg++ and 60°C annealing for 35 cycles. The effect of missense mutations on E-cadherin function was predicted using the SIFT (Sorting Intolerant From Tolerant) database (Ng and Henikoff, 2003). The functional significance of the E-cadherin missense changes was assessed with respect to aggregation and invasion suppression capacity as described in Suriano et al. (2003b). Briefly, mutant CHO cells were stably transfected with cDNAs encoding mutant or wild type E-cadherin and examined using soft-agar aggregation and Matrigel invasion assays. Western blots were used to measure Ecadherin protein levels in the transfected cell lines. Nucleotide and codon numbering refers to the coding DNA sequence starting at the translation initiation site.

CDH1 Germline Mutations 3

RESULTS AND DISCUSSION

We analysed the 16 exons of the CDH1 gene including the exon intron boundaries and an essential region of the CDH1 promoter in 36 DGC families. Following the IGCLC clinical criteria for HDGC (Caldas et al., 1999), the 36 families divided into 24 that met the criteria and 12 that did not. CDH1 mutations were found in seven families that met the IGCLC criteria, and in three that did not. The pedigrees are described in Figure 1 and an overview of the detected mutations is summarised in Table 1. Seven of the detected mutations are novel, with one of them identified in two unrelated DGC families, while two mutations have previously been described in two unrelated families (c.1901C>T, Suriano et al., 2003a and c.2095C>T, Guilford et al., 1998). Table 1. CDH1 Germline Mutations Family

Ethnicity

IGCLC criteria1

Modified criteria2

Other cancers3

Region

Nucleotide change4 (c.)

1

Caucasian

yes

1a

Br, Cr S, Pr, Bl, Pa, T

intron 1

2

Caucasian

no

no

3

Caucasian

yes

1a

4

Hispanic

yes

1a

5 6

Caucasian Caucasian

no no

no 3

7

Caucasian

yes

1a

8

Maori

yes

9

Chinese

10

Caucasian

Mutation type

49-2A>C

Predicted protein change (p.) ND

exon 3

353C>G

Thr118Arg

missense

exon 6

715G>A

Gly239Arg

missense

exon 8

1107delC

Asn369LysfsX392

stop

exon 8 exon 8

1137G>A 1137G>A

ND ND

splice site splice site

exon 10

1391_1392delTC

Leu472HisfsX481

stop

1

exon 12

1901C>T5

Ala634Val

missense

yes

1

exon 13

2095C>T6

Gln699X

stop

yes

1a

intron 15

2440-6C>G

ND

splice site

Cr

Br, Cr, SG, BiL Br

L

splice site

1

Criterion 1: two or more documented cases of DGC in first or second degree relatives with at least one diagnosed before the age of 50 years. Criterion 2: three or more documented cases of DGC in first or second degree relatives independent of age of onset. 2Brooks-Wilson et al., 2004. 3In most cases samples were not available for mutation testing. Br: breast, Cr: colorectal, S: skin, Pr: prostate, Bl: bladder, Pa: pancreas, T: throat, SG: salivary gland, L: lung, BiL: Bilateral lung cancer. 4The numbering is based on the cDNA sequence and places +1 at the translation start site for both nucleotides and codons. GenBank accession number AC009082 version AC009082.7. 5Vécsey-Semjén et al., 2002; Suriano et al., 2003a. 6Guilford et al., 1998. Family 1

The proband is a Caucasian female with DGC diagnosis at 42 years and a family history of gastric and extragastric cancers. The mutation c.49-2A>C occurs in the CDH1 intron 1 splice acceptor site. The wild type A at c.49-2 is 100% conserved amongst higher eukaryotes (Padgett et al., 1986). The mutation was found in two other family members with DGC, the proband’s father who was diagnosed at 68 years and an aunt diagnosed at 54 years. The mutation was also present in a paraffin-embedded sample of a second aunt with a colorectal cancer diagnosis at 55 years. We did not find the mutation in an asymptomatic sister. Blood RNA from the aunt with DGC was analysed for splicing defects. RT-PCR using exon spanning primers demonstrated abnormal CDH1 transcripts compared to mRNA from two healthy, unrelated controls (Fig. 2a). In addition to the DGC and colorectal colon cancer there is a broad spectrum of extra-gastric cancers in this family, some of which could be due to the CDH1 mutation and include the following sites: breast (age at diagnosis 50y), skin (41y), skin (age unknown), prostate (age unknown), bladder (67y), and pancreas (60y). Two unrelated HDGC families with mutations at the same nucleotide position have been reported (c.49-2A>G, Moran et al., 2005; Richards et al., 1999). In one of the families the occurrence of lung and rectal cancer was reported (Richards et al., 1999), but the detailed history of the other family was not described (Moran et al., 2005). A somatic 49-2A>C mutation has been shown in a breast tumour of both ductal and lobular histology (Salahshor et al., 2001).

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Family 2

The proband is a Caucasian male who was diagnosed with DGC at 55 years. The patient’s father died of gastric cancer at 64 years. The missense mutation occurs at c.353C>G in exon 3 coding for the precursor region of the Ecadherin protein and causes an amino acid substitution p.Thr118>Arg. This region is not highly conserved across species (Berx et al., 1995).The SIFT database (Ng and Henikoff, 2003) predicted no deleterious effect for this mutation. However, CHO cells transfected with the mutant CDH1 cDNA did not form cellular aggregates, whereas compaction was observed for cells transfected with wild type CDH1 after 24 hours incubation (Fig. 2b). Compared to the wild type transfectants, CHO cells transfected with the mutant cDNA also displayed 10 times more cells able to invade matrigel (Fig. 2b). The mutation therefore impairs E-cadherin ability to mediate cell-to-cell adhesion and to suppress invasion in vitro. The first 129 amino acids of E-cadherin are normally cleaved during intracellular proteolytic processing to generate the mature protein. The p.Thr118Arg change in the precursor segment could affect this process (Ozawa and Kemler, 1990), producing a form of E-cadherin with less active adhesive function. Interestingly, three out of four siblings are mutation carriers, aged over 50y and still asymptomatic, suggesting the penetrance of this mutation could be lower than that of truncating mutations. Although DGC risk assessment for this family is difficult, the information from the functional studies satisfied requirements for pre-symptomatic mutation testing and clinical management in this family. Family 3

The proband, a Caucasian female, presented with DGC at 30 years. Her mother died of DGC at the age of 29 years. The missense mutation c.715G>A in exon 6 causes an amino acid substitution p.Gly239Arg in the extracellular domain of the protein. Gly239 is 100% conserved amongst higher eukaryotes (Padgett et al., 1986) and the SIFT database (Ng and Henikoff, 2003) predicted zero tolerance for this change. Accordingly, no cell-tocell aggregates were observed in CHO cells transfected with mutant cDNA in contrast to the wild type controls (Fig. 2). Cells expressing mutant E-cadherin had on average 13 times more matrigel-invading cells compared to the wild type CDH1 controls (Fig. 2b). The mutation thus confers deficient E-cadherin function with respect to intercellular adhesion and suppression of invasion in vitro. Though both affected family members were diagnosed with DGC at an early age, the proband's grandmother is a confirmed mutation carrier and still disease-free at the age of 79y. Family 4

The index patient of family 4 is a Hispanic female with DGC diagnosed at 48 years and a sister who died of DGC at 27 years and a mother with colorectal cancer at 64 years. The mutation is a one base pair deletion c.1107delC in exon 8 coding for the extracellular domain of E-cadherin. The deletion causes a frameshift resulting in a stop at p.392 in exon 9 and leads to truncation of the extracellular E-cadherin domain. The family meets the IGCLC criteria, as is usually observed for truncating mutations. The colorectal cancer however, did not occur in the family branch with DGC (Fig. 1). Families 5 and 6

The index patients of families 5 and 6 both carried the same CDH1 mutation (c.1137G>A). The change affects the last nucleotide of exon 8. G at this position is conserved in 78% of higher eukaryotes compared to only 4% for the A (Padgett et al., 1986). The A allele was not detected by BsaJI digest in a survey of 140 unrelated individuals. The mutation is expected to alter normal splicing, however, no RNA was available to confirm a splicing defect. The patient from family 5 is a Caucasian male with a DGC diagnosis and a DGC family history. Further clinical data and additional samples were not available for further investigation of the mutation. In family 6, the mutation was detected in a Caucasian female who was diagnosed with DGC at 29 years and relapsed four years later. Her 28 year-old sister is an asymptomatic mutation carrier. The mutation was not detected in her healthy mother but the mutation was observed in a tumour of the proband’s father who presented with a salivary gland tumour in his tongue at age 36. Only the 1137A allele was detected in a paraffin-embedded sample of this tumour, suggesting that the wild type CDH1 allele has been deleted in this tumour. While no other DGC cases have been recorded, other tumours that might be due to CDH1 mutation in this family include a breast and a colorectal cancer, though both were diagnosed at an older age. The lung cancers documented in two cousins of the proband are likely to be the result of a genetic predisposition, as the cancers were bilateral in both cases (Fig. 1). The risk of developing DGC for carriers of this mutation is unclear. However, the presence of the mutation in two cancer-prone families

CDH1 Germline Mutations 5

and its absence in unrelated healthy controls, together with the observed loss of heterozygosity, provides evidence for the aetiologic nature of the c.1137G>A change. Accordingly, genetic testing led to a total prophylactic gastrectomy in the asymptomatic mutation carrier of family 6 (Fig. 1). Family 7

The proband is a Caucasian male who presented with DGC at 34 years. His father died of DGC aged 61 years, and one great aunt died at 78 years diagnosed with breast cancer of unknown histology. The mutation is a two base pair deletion c.1391_1392delTC in exon 10 within the extracellular domain of the E-cadherin protein. The resulting frameshift generates a stop at p.481 causing truncation of the extracellular domain. The family meets the IGCLC criteria, with the breast cancer possibly related to the CDH1 mutation. Family 8

The proband is a Maori female who presented with DGC at 35 years. Her mother was also reported to have died of DGC at a young age. The exon 12 missense mutation c.1901C>T (p.Ala634Val) detected in this IGCLC family has previously been described in an early onset DGC patient (Suriano et al., 2003a) and in a colon cancer cell line (Vécsey-Semjén et al., 2002). The mutation has been shown to result in deficient E-cadherin function in vitro (Suriano et al., 2003b). Family 9

The proband from this IGCLC family is a Chinese male with DGC diagnosed at 39 years. His mother died of DGC at 55 years, while his brother presented with DGC at 24 years. The splice site mutation c.2095C>T has been previously described in a family of Maori origin (Guilford et al., 1998). Family 10

The proband from family 10 is a Caucasian male who presented with DGC at 36 years and a family history of DGC and lung cancer fulfilling the IGCLC criteria (Fig. 1). The mutation alters the splice acceptor site within intron 15 (c.2440-6C>G) and causes abnormal RNA transcripts compared to RNA from a healthy unrelated control individual (Fig. 2a). The mutation was not present in the healthy mother of the proband and was presumably inherited from the father, who had lung cancer and whose own father had DGC.

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Figure 1. Pedigree of families screened for CDH1 mutations. Arrow points to the proband.

CDH1 Germline Mutations 7

Figure 2. Functional analyses of splice site and missense mutations. A: RT-PCR analysis of splice site mutations indicates abnormal splicing. N = negative control, C = wild type control. B: The functional consequences of CDH1 missense mutations as assessed by aggregate formation and matrigel invasion. Western Blot analysis demonstrates similar Ecadherin expression levels in the transfected cell lines.

Germline mutations in genes other than CDH1 such as the mismatch repair genes, APC and p53 have been detected in cancer-prone families with the occurrence of gastric tumours (Shinmura et al., 1999). However, their relative contribution to inherited gastric cancer is minor and presently leaves CDH1 the only gene that is systematically screened in families with a high incidence of gastric cancer. In this report, we have investigated a series of 36 DGC families for CDH1 germline mutations. Two of the detected mutations (families 8 and 9) have been observed before (Vécsey-Semjén et al., 2002; Guilford et al., 1998), while one novel mutation (the splice site c.1137G>A) was present in two unrelated families. Together, one third of the detected mutations were present in more than one family. However, previously published reports (reviewed in Blair et al., 2006) support that the majority of CDH1 mutations have arisen independently in the absence of significant founder effects or mutational hotspots. In the described HDGC families, extra-gastric tumours were recorded at two different sites (tongue and colon) in individuals with demonstrated CDH1 mutations. The wide spectrum of sites, the low frequency of tumour incidence and the absence of genetic material for the majority of extra-gastric cancers does not allow for assessment of any significant association. However, the cancer spectrum in family 6 suggests that an apparent predisposition to cancer at various sites, combined with one or more cases of early onset DGC, may be an occasional clinical presentation associated with HDGC. Excluding the families with undocumented history, the seven novel germline mutations were detected in five families that met the IGCLC criteria and in two that did not. Together, CDH1 mutations were present in 29% (7 out of 24) of families meeting the IGCLC criteria. Strict application of the IGCLC guidelines for the selection of DGC families for CDH1 screening would have resulted in two out of seven CDH1 mutations remaining undetected. Brooks-Wilson et al. have proposed a more detailed set of study criteria (Brooks-Wilson et al., 2004). Family 6, which represents a single case of DGC below the age of 45, would fall under criteria no 3, while family 2 (2 cases of DGC >50y) would again fail to meet these revised criteria. It is therefore clear that comprehensive screening of families with DGC for CDH1 mutations requires that borderline families be included in mutation searching. REFERENCES Berx G, Staes K, Van Hengel J, Molemans F, Bussemakers M, Van Bokhoven A, Van Roy F. 1995. Cloning and Characterization of the Human Invasion Suppressor Gene E-Cadherin (CDH1). Genomics 26:281-289.

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