Original Article Middle East Journal of Cancer; October July 20152020 6(3):11(4): 493-501 New Variants in the CDH1 Gene in Iranian Families with Hereditary Diffuse Gastric Cancer Majid Kheirollahi*, Maryam Saneipour**, Mohammad Amin Tabatabaiefar*, Mehrdad Zeinalian*, Mohammad Minakari***, Abbas Moridnia**♦ *Pediatric Inherited Diseases Research Center, Research Institute for Primordial Prevention of Non-communicable Disease and Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran **Department of Genetics and Molecular Biology, School of Medicine, Dezful University of Medical Sciences, Dezful, Iran ***Department of Internal Medicine, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran Please cite this article as: Kheirollahi M, Saneipour M, Tabatabaiefar MA, Zeinalian M, Minakari M, Moridnia A. New variants in the CDH1 gene in Iranian families with hereditary diffuse gastric cancer. Middle East J Cancer. 2020;11(4): 493-501. doi: 10. 30476/mejc.2020.81478.1016. ♦Corresponding Author: Abbas Moridnia, PhD Department of Genetics and Molecular Biology, School of Medicine, Dezful University of Medical Sciences, Dezful, Iran Tel: +986142423791 Email: moridnia.a@dums.ac.ir Abstract Background: Hereditary diffuse gastric cancer (HDGC) is a hereditable form of diffuse gastric cancer with very aggressive tumors, poor prognosis, and delayed clinical signs. Method: We assessed 17 probands identified with HDGC upon gastrectomy according to the histopathological criteria confirmed by a pathologist and familial history. We extracted DNA from peripheral blood and formalin fixed paraffin-embedded tissues. DNA sequencing was done following PCR amplification of 16 exons and exon/intron boundaries of the CDH1 gene and exon 2 of CTNNA1 gene. The Multiplex Ligation-dependent Probe Amplification technique was performed on patients with no pathogenic variants in sequencing. Results: Totally, 17 probands comprising seven males and 10 females were assessed. In three patients, we recognized the tumors in the early TNM stage (I, II), while in 14 cases, tumors were observed in the late stages (III, IV). Overall, DNA sequencing of the CDH1 gene identified 16 variants (seven exonic including five new variants and nine intronic containing six new variants). Moreover, Multiplex Ligation-dependent Probe Amplification detected one deletion in exon 1 of two patients. Conclusion: Our results showed that E-cadherin deficiency in HDGC was related to CDH1 gene point mutations and large deletion with high heterogeneity, which should be considered in the diagnosis and treatment of HDGC patients. Keywords: CDH1 gene, Diffuse gastric cancer, Iranian families, Hereditary, Mutation Received: April 23, 2019; Accepted: January 13, 2020 Majid Kheirollahi et al. Introduction Gastric cancer (GC) is the fourth common cancer with 952,000 new cases and 723,000 deaths during 2012 and the second cause of mortality among all cancers.1,2 In addition, GC is estimated to be the eleventh cause of all deaths and account for 1.8% of all deaths by 2030.2 GC is the most common cause of cancer-related mortality in Iran, the second prevalent cancer in males (14%), and the fourth in females (7%).2, 3 Most of GC cases are sporadic, and familial aggregation could be observed in approximately 10% of the cases.4, 5 Hereditary pattern is observed in a few cases (13%). 6 According to Lauren histological classification, GC is divided into intestinal and diffuse type of adenocarcinoma.7 Hereditary diffuse gastric cancer (HDGC) is an autosomal dominant inherited form of DGC, a highly invasive tumor with a poor prognosis, high penetrance, and infiltrating pattern. This causes gastric wall thickening (Linitis plastica) without forming a definite mass. Signet ring cell carcinoma (SRCC) or isolated cell type carcinoma are common histopathologic features of diffuse GC (DGC).8 Mutations in the CDH1 gene is the most prevalent cause of HDGC and sporadic DGC.9 CTNNA1 (encoding the alpha-E-catenin) is the only identified gene involved in HDGC other than CDH1 germline mutations. This gene has been reported in a large HDGC pedigree as a 2 bp germline deletion in exon 2. 10 Germline mutations in CTNNA1, BRCA2, STK11, SDHB, PRSS1, ATM, MSR1, and PALB2 genes were reported in HDGC patients.11 Furthermore, several genes including LMTK3, RHOA, PIK3CA, MED1, ARID1A, and MCTP22 were detected in the HDGC patients with somatic mutations.10 More than 80% of the HDGC carriers for CDH1 mutations, whether male or female, might be inflicted with GC until the age of 80. Moreover, there is a 60% risk of lobular breast cancer until Figure. 1 (a): Sequence electropherogram of exon 3 of the CDH1 gene. Arrow indicates the location of the base substitution at c.181G>A ( p.61T>A). (b): Sequence electropherogram of exon 15 of the CDH1 gene. Arrow indicates the location of the base substitution at c.2331C>G ( p.777D>E). 1 (c): Sequence electropherogram of exon 7 of the CDH1 gene. Arrow indicates the location of the base deletion at c.889delA. (d): Sequence electropherogram of exon 9 of the CDH1 gene. Arrow indicates the location of the base deletion at c.1177delA. 494 Middle East J Cancer 2020; 11(4): 493-501 CDH1 Gene and Hereditary Diffuse Gastric Cancer Table 1. Epidemiological and clinicopathological features of HDGC patients Sample ID A B 4 14 15 18 20 22 29 37 40 41 42 43 44 45 48 Tissue type Blood Blood FFPE FFPE FFPE FFPE FFPE FFPE FFPE FFPE FFPE FFPE FFPE Blood Blood FFPE FFPE Gender Female Female Male Male Male Female Male Male Female Female Male Female Female Male Female Female Female Age of diagnosis 31 34 49 36 61 37 78 31 70 30 51 57 27 41 29 37 72 the age of 80 in women with CDH1 germline mutation.12 CDH1 gene is located on 16q22.1, including 16 exons encodingE-cadherin.13, 14 E-cadherin is Stage IV IV IIIA IIIB IV IIIA IIIC IIIC IIIC IIIB IV IIIC IIIA IIIB II I II Histopathological type Signet ring cell carcinoma Signet ring cell carcinoma Signet ring cell carcinoma Signet ring cell carcinoma Signet ring cell carcinoma Signet ring cell carcinoma Signet ring cell carcinoma Signet ring cell carcinoma Poorly differentiated adenocarcinoma Poorly differentiated adenocarcinoma Signet ring cell carcinom Poorly differentiated adenocarcinoma Poorly differentiated adenocarcinoma Signet ring cell carcinoma Signet ring cell carcinoma Signet ring cell carcinoma Signet ring cell carcinoma a transmembrane protein that plays a pivotal role in cell adhesion and tumor suppression.15 The CDH1 promoter hypermethylation is the most common epigenetic inactivation mechanism of Figure. 2 (a): Sequence electropherogram of the exon 3 of the CDH1 gene. Arrow indicates the location of the base substitution at c.348G>A (p.116L>L). (b): Sequence electropherogram of exon 13 of the CDH1 gene. Arrow indicates the location of the base substitution at c.2076T>C ( p.692A>A). (c): Sequence electropherogram of exon 14 of the CDH1 gene. Arrow indicates the location of the base substitution at c.2292C>T (p.764D>D). (d): Sequence electropherogram of exon 13 and the intron boundary of the CDH1 gene. Arrow indicates the location of the base deletion at c.1937-58delA. Middle East J Cancer 2020; 11(4): 493-501 495 Majid Kheirollahi et al. Table 2. Exonic and intronic variants in CDH1 gene among HDGC patients Sample ID 14, 15, 20, 48. 20 A, B B 14, 29, B, 15, 40, 41, 42, 46 B Type of Exon or exon/ tissue intron boundary FFPE 3 FFPE 3 Blood 7 Blood 9 FFPE/ Blood 13 Substitution G>A A>G T>C Deletion Heterozygosity Heterozygote* Heterozygote* A Homozygote* A Homozygote* - Heterozygote/ Homozygote Blood 14 C> - Heterozygote 29 15 FFPE FFPE 15 4 C>G G>C - Heterozygote* Heterozygote 15, 22, A, B, 40, 41, 43, 46 22 FFPE/ Blood 13 A Homozygote* FFPE 13 G> - Heterozygote* 22 FFPE 13 G>A - Heterozygote* 14, 15, 20, 22, 44, FFPE 15 C>G - Heterozygote* 14, 15, 20, 22, FFPE/ Blood 44, 48 14, 15, 20, 22, 44, 48 FFPE/ Blood 15 T>G - Heterozygote* 15 C>G - Heterozygote 40 FFPE 15 T>G - Heterozygote* 43, 46 FFPE/ Blood 15 G>A - Heterozygote - Amino acid Chromosome location L116L T61A Exonic Exonic. A692A NM_004360.3:c.348G>A NM_004360.3:c.181A>G NM_004360.4:c.889delA NM_004360.4:c.1177delA NM_004360.:c.2076T>C rs:1801552 D764D NM_004360:c.2292C>T rs: 61747636 D777E NM_004360:c.2331C>G NM_004360:c.531+10G>C rs: 33963999 NM_004360.3:c.193758delA NM_004360.4: c.2164+48G>A NM_004360.4: c.2164+51G>A NM_004360.4:c.229648C>G NM_004360.4:c.229644T>G NM_004360:c.229622C>G rs:763184960 NM_004360.3 :c.2439+105T>G NM_004360.4: c.2439+52G>A rs:33965115 *New mutation, FFPE: Formalin fixed-paraffin embedded the gene as the second hit in HDGC; however, other secondary substitution or insertion/deletion mutations have been reported with less frequency.14 To date, the majority of the germline mutations have been identified as single nucleotide substitutions.16 On average, 5% of the familial DGC cases are due to the large deletions up to several exons of the CDH1 gene.17, 18 Large deletions in CDH1 gene have been described as a reason for cancer susceptibility in Japanese,19 Canadian, and European familial GC patients.20 In this study, we reported several new CDH1 variants and a large deletion in Iranian patients with HDGC. province, central Iran, and Alaa cancer control center, a charity-based foundation for cancer patients in Isfahan. Four samples belonged to blood’s probands and 13 samples of probands were formalin fixed-paraffin embedded (FFPE) tumor tissues. We extracted the DNA of blood samples by Prime Prep Genomic DNA Isolation Kit (GeNet Bio, Korea). FFPE tumor samples were cut to 5-l0µm thickness sections for DNA extraction using SDS-proteinase K digestion, phenol chloroform, and ethanol precipitation. All patients participating in the study or their families completed informed consent forms. The Review Board of Isfahan University of Medical Sciences approved this study with code number 394479. Materials and Methods Patients and sampling We identified 17 probands with HDGC using histological features and clinical criteria based on IGCLC 21. A pathologist confirmed DGC. We selected the samples from GC patients who, from January 2011 to April 2016, referred to AlZahra Hospital, a referral hospital in Isfahan 496 DNA sequencing We assessed the obtained DNA to sequence CDH1 and CTNNA1 genes in all the samples. Polymerase chain reaction (PCR) amplified the DNA. Specific primers amplified all coding exons and exon/intron boundary regions of the CDH1 gene (NM_004360.4) and exon 2 of CTNNA1 Middle East J Cancer 2020; 11(4): 493-501 CDH1 Gene and Hereditary Diffuse Gastric Cancer Table 3. Effect of non-synonymous variants on protein function of CDH1 gene Amino acid subsitution PolyPhen 2 T61A Benign: 0.005 D777E. SIFT Tolerated: 0.78 Probably damaging: Tolerated: 1 0.985 I-Mutant v2.0 Decrease stability DDG: -0.99 Decrease stability DDG: -0.48 Mutation taster PROVEAN Polymorphism. Neutral Disease causing. Neutral Mutation assessor PhD-SNP ConSurf lowNeutral FI score: 1.085 lowNeutral FI score: 1.245. 1 7 Ranges of score variation are depicted below the program names. Categorical predictions based on the following cut-off values: Polyphen-2: Benign= 0-0.2, Possibly damaging=0.2-0.85, Probably damaging=0.85-1; SIFT: Damaging if <=0.05, I-Mutant: Free Energy Change Value (DDG)<0= Decrease stability, DDG>0=Increase stability; Mutation Taster: disease causing= probably deleterious, disease causing automatic = known to be deleterious, polymorphism= probably harmless, polymorphism automatic= known to be harmless, PROVEAN: Deleterious if <= -2.5, Mutation assessor: Functional impact of a variant (Func. Ipmact)= predicted functional (high, medium), predicted non-functional (low, neutral).;PhD-SNP: Neutral: Neutral polymorphism, Disease: Disease-related polymorphism; ConSurf: The conservation scale: Variable=1-3, Average=46, Conserved=7-9 gene (NM_001903.4). We sequenced the PCR product of each reaction through the use of ABI 3130XL capillary sequencing platform (Applied Biosystems/Life Technologies, Carlsbad, CA, USA). The obtained sequences were analyzed using Chromas software, version 2.31. recognized the tumors at an early TNM stage (I, II), and in 14 cases (82%), the tumors were detected at late stages (III, IV). SRCC and “poorly differentiated adenocarcinoma” were reported as the histopathological type of the tumor in 13 (76.5%) and 4 (23.5%) cases, respectively (Table 1). In silico investigation of pathogenicity We assessed the effect of non-synonymous variants on protein function using bioinformatics software tools, including Polyphen2, SIFT, IMutant, Mutation taster, Mutation assessor, PROVEAN, ConSurf, and PhD-SNP. Human Splice Finder (HSF) version 3 evaluated the effect of intronic variants on the splicing site. Sequence analysis DNA sequencing of the amplified PCR products exhibited eleven new variants (5 exonic and 6 intronic) in the CDH1 gene of the samples (Table 2). Two exonic variants were nonsynonymous (T61A and D777E), three were synonymous (L116L, A692A and D764D), and two were one-nucleotide deletion (c.889delA and c.1177delA). No single nucleotide changes occurred in exon 2 of CTNNA1. Using DNA sequencing, we further found one base pair deletion in CDH1 gene at c.1937-58delA in eight samples. Interestingly, seven of these samples also carried exonic variants, two of which were predicted to be pathogenic. This could favor the benign effect of the intronic variant. Multiplex ligation-dependent probe amplification (MLPA) We examined samples with no identified pathogenic point mutations to identify large deletions/duplications in CDH1 and CTNNA1 genes using SALSA P083-C2 CDH1 MLPA kit (MRC-Holland, Amsterdam, Netherlands). The reactions were performed according to the manufacturer’s instruction. Probe ratio (PR) described deletions or duplications. A PR of less than 0.7 presented a gene dosage reduction, and a PR of more than 1.3 indicated an increase in gene dosage. Results Clinicopathologic characteristics Altogether, the mean age of the cases at diagnosis was 45.4 years (49.6 in men and 42.4 in women) and seven of the 17 (41%) patients were older than 45. In three patients (18%), we Middle East J Cancer 2020; 11(4): 493-501 Amino acid substitutions We detected two non-synonymous variants, including a single base pair substitution, A to G transition resulting in a single amino acid substitution at codon 61 as p.61T>A (Figure 1a), and a single base pair substitution C to G transversion leading to an amino acid substitution at codon 777 as p.777D>E (Figure 1b) (Table. 2). Moreover, we detected two deletions with one-nucleotide at c.889delA (Figure 1c) and c.1177delA (Figure 1d). The discovered synonymous variants were: a single base pair 497 Majid Kheirollahi et al. Figure 3. Coffalyser electropherogram of the CDH1 gene. Above electrophoretogram relates to normal controls and the bottom belongs to the patients. Arrow indicates deletion in exon 1. 498 Middle East J Cancer 2020; 11(4): 493-501 CDH1 Gene and Hereditary Diffuse Gastric Cancer Table 4. Intronic variants effect on splicing site Sample ID NM_004360.4:c.1937-58delA NM_004360.4:c.2164+51G>A NM_004360.4:c.2296-48C>G NM_004360.4:c.2296-44T>G- c.2296-48C>G NM_004360.4:c.2296-22C>G NM_004360.4:c.2439+52G>A NM_004360.3:c.2439+105G>T Splice site type Acceptor Donor Acceptor Donor Acceptor Acceptor Donor Consensus value (0-100) 70.07 72.25 69.78 73.78 82.33 66.32 66.12 Consensus value: splice site if >= 65 substitution, G to A nucleotide transition resulting in synonymous substitution at codon 116 (p.116L>L) (Figure 2a), a single base pair substitution, T to C nucleotide transition leading to a synonymous substitution at codon 692 (p.692A>A) (Figure 2b), and finally, a single base pair substitution, C to T transition leading to a synonymous substitution at codon 764 (p.764D>D) (Figure 2c) (Table. 2). Effect of non-synonymous variants on protein function Bioinformatics analysis by PolyPhen2, IMutant, Mutation taster, Mutation assessor, and ConSurf software tools suggested the p.777D>E substitution can have pathogenicity effect on the protein function (Table 3). Intronic substitutions and splicing site-effects Moreover, we identified certain likely benign variants in intronic sites including a single base pair deletion at c.1937-58delA (Figure 2d) and several single base pair substitutions containing the following: c.531+10G>C, (c.2164+48G>A, c.2164+51G>Ac.2296-48C>G, c.2296-44T>G c.2296-22C>G, c.2439+52G>A and finally (.2439+105T>G. The evaluation of intronic variants showed the potential effect of seven different variants on the splicing site (Table 4). MLPA results Using the MLPA technique, we found one large deletion in exon 1 (CDH1 probe 12651L19938) in two patients (Figure 3). This change was confirmed by quantitative PCR by primers of exon 1 CDH1 gene in patients. From the same Middle East J Cancer 2020; 11(4): 493-501 patient, we obtained the normal sample as the negative control for normalization. To analyze the results, we utilized the ABI Step One Plus (Applied Biosystems, Foster City, CA, USA) instrument and the ΔΔCt method. Discussion Most GC cases are sporadic and in approximately 10% of cases, a familial aggregation is observed. Therefore, hereditary GC encompasses just a minority of cases.4-6 Almost 15 to 50% of families presenting with HDGC criteria (according to International Gastric Cancer Linkage Consortium) presented germline mutations in CDH1 gene.22 Until 2010, the rate of CDH1 gene mutations in DGC was reported 25 to 50%;16 however, using updated criteria, the rate of mutation was reduced down to 10-18% in countries with low GC incidence.22, 23 The HDGC incidence rate is unknown in Iran. In the present study, we identified somatic and germline variants in Iranian patients with HDGC in 15 of 17 (88.2%) cases. Regarding certain variants and single nucleotide polymorphisms in CDH1 gene, there is a correspondence between our findings and other studies. For instance, substitutions at position c.2076T>C (A692A), 24 c.2292C>T (D764D), c.531+10G>C,25 and c.2439+52G>A were previously described.26 Based on table 2, other exonic and intronic variants marked with an asterisk were novel in our study. Corso et al. detected mutations in two Italian patients with early onset DGC. In their study, one of the mutations was missense p.Arg224Cys and the other was a substitution -63C>A. Also, they found five out of 21 tumors in early TNM 499 Majid Kheirollahi et al. stage (I, II), and 15 out of 21 in the late stages (III, IV).27 Until now, only one study has evaluated CDH1 gene mutations in a single family with HDGC in Iran, reporting a truncating mutation.28 We found certain substitutions, small and large deletions in our samples located on exons 1, 3, 7, 9, 13, 14, and 15 of CDH1 gene. Bioinformatics analysis suggested that the p.777D>E substitution in exon 15 showed that this mutation would negatively affect the protein function. Exon 1 of CDH1 gene encodes the signal peptide domain and exon 3 of this gene encodes the propeptide of E-cadherin protein. The signal peptide domain is necessary for the import of the protein into the endoplasmic reticulum. Exons 7, 9, and 13 of CDH1 gene encode the extracellular domain of E-cadherin, essential for cell-cell adhesion;24 also, changes in this domain can destroy the cell adhesion. Exons 14 and 15 of CDH1 encode the cytoplasmic domain of E-cadherin. Cytoplasmic domain binds to β-catenin and plays a fundamental role in tumor silencing.28 Approximately 5% of CDH1 mutations in HDGC patients were large deletions.17, 18 In the present study, we detected one large deletion in exon 1 in two cases by the MLPA technique. In 2009, Oliveira et al. identified some large deletions in exons 1, 2, 14, 15, and 16 of CDH1 gene in HDGC families.18 In 2014, a large genomic deletion (c.1566-?_1711+?del) in exon 11 of the CDH1 gene in a patient with no familial history of gastric cancer was reported;29 moreover, Molinaro et al. reported a deletion with 1642 bp length, with breakpoints in introns 6 and 8 of CDH1 gene (c.833-476_1138-463del).30 We found several intronic variants in CDH1 gene, including c.1937-58delA, c.2164+51G>A, c.2296-48C>G, c.2296-44T>G, c.2296-22C>G, c.2439+52G>A, and c.2439+105G>T. Bioinformatics analysis by HSF showed that they might generate new potential splice sites leading to incorrect splicing. Further functional data is required to reach a conclusion on the effects of these variants. In conclusion, our results showed that Ecadherin deficiency in HDGC is associated with CDH1 gene point mutations and large deletions with high heterogeneity. This should be considered 500 in the diagnosis and treatment of HDGC patients. These results highlight the important roles of CDH1 gene, development of molecular and genetic testing by PCR sequencing, MLPA, and high throughput techniques such as whole exome sequencing and whole genome sequencing for an early diagnosis and prevention of the potentially lethal effects of HDGC in high risk individuals. Acknowledgements We sincerely thank the patients and the professional staff of the pathology lab in Al-Zahra Hospital (Isfahan, Iran) and Alaa Cancer Control Center (Isfahan, Iran) for their cooperation in this study. Conflict of Interest None declared. References 1. 2. 3. 4. 5. 6. 7. Bray F, Ferlay J, Soerjomataram I, Seige IRL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2018;68(6):394-424. Mehrabani D, Hosseini SV, Rezaianzadeh A, Amini M, Mehrabani G, Tarrahi MJ. 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