|Ahead of print publication
Germline likely pathogenic variants in ataxia-telangiectasia-mutated gene in an Iranian family with hereditary diffuse gastric cancer without CDH1 mutation
Majid Kheirollahi1, Maryam Saneipour2, Abbas Moridnia2
1 Pediatric Inherited Diseases Research Center, Research Institute for Primordial Prevention of Non-Communicable Disease, Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
2 Department of Genetics and Molecular Biology, School of Medicine, Dezful University of Medical Sciences, Dezful, Iran
|Date of Submission||18-May-2019|
|Date of Decision||08-Jan-2020|
|Date of Acceptance||12-Apr-2020|
|Date of Web Publication||27-Apr-2021|
Department of Genetics and Molecular Biology, School of Medicine, Dezful University of Medical Sciences, Dezful
Source of Support: None, Conflict of Interest: None
Background: Gastric cancer (GC) is the fourth common cancer in the world and the second cause of cancer-related mortality. Germline mutations in the E-cadherin gene (CDH1) are the most common cause of hereditary diffuse GC (HDGC) and explain 25%–30% of cases. In HDGC families without the pathogenic CDH1 variant, there is poor management and therapeutic strategies, and detect other genetic defects in HDGC, except CDH1 gene will be useful for further clarification of the disease mechanisms and risk-reducing strategies. Here, we reported an Iranian pedigree with familial HDGC to assess the fundamental genetic causes by whole-exome sequencing (WES).
Materials and Methods: WES performed in an Iranian with a history of familial GC in whom no pathogenic variants or indels has been found in CDH1 and CTNNA1 genes with Sanger sequencing and multiplex ligation-dependent probe amplification methods.
Results: Prioritizing genes associate with HDGC recognized several variants include c.2572T>C, and c.3161C>G in ataxia-telangiectasia mutated (ATM), c.1114A>C in BRCA2, and finally c.1173A>G in PIK3CA. Protein function prediction software tools reveal that c.3161C>G in ATM is likely pathogen.
Conclusion: The results of this study suggested a role for the known cancer predisposition gene ATM in families with HDGC with no pathogenic variant in CDH1. Our results suggested that mutations in ATM and other genes, particularly the mutations found in this study, should be considered even in one case of positive familial status of HDGC disease. The presence of these mutations in patients with familial history raises important issues regarding genetic counseling.
Keywords: Ataxia-telangiectasia-mutated gene, diffuse gastric cancer, whole-exome sequencing
|How to cite this URL:|
Kheirollahi M, Saneipour M, Moridnia A. Germline likely pathogenic variants in ataxia-telangiectasia-mutated gene in an Iranian family with hereditary diffuse gastric cancer without CDH1 mutation. J Can Res Ther [Epub ahead of print] [cited 2021 Jun 22]. Available from: https://www.cancerjournal.net/preprintarticle.asp?id=314862
| > Introduction|| |
Gastric cancer (GC) is one of the most common cancers in the world and the second cause of mortality among all cancers. The GC is a common cause of cancer-related deaths in Iran and its prevalence rate is high, particularly in the North and Northwest., The sporadic form of GC is a dominant type, and the hereditary form of GC accounts for up to 10% of cases (HGC). The GCs are categorized into two groups, consisting of intestinal and diffuse type, according to the Lauren histological classification. Diffuse GC (DGC) accounts for approximately 30% of all GCs and has a poor prognosis, especially in young patients. Hereditary DGC (HDGC) is an autosomal dominant hereditary form of DGC that defined on the basis of family history of DGC, poor prognosis, high penetrance, highly aggressive tumors, delayed clinical signs, and associated with lobular breast cancer (LBC)., The CDH1 mutations are the most common cause of HDGC disease. This gene located on 16q22.1 position and contains 16 exons that encode for E-cadherin protein, which has three domains including extracellular, transmembrane, and cytoplasmic that shows a substantial role in cell–cell adhesion and tumor suppression.
Other than the CDH1, germline mutations of CTNNA1 (α-E-catenin) and BRCA2, STK11, SDHB, PRSS1, ataxia-telangiectasia mutated (ATM), MSR1, and PALB2 genes have been reported in HDGC patients. Furthermore, several somatic mutations, including LMTK3, RHOA, PIK3CA, MED1, ARID1A, and MCTP22 genes, were detected in the HDGC patients. However, given the rarity of these variants, the associated risk of DGC is hard to quantify, and these variants are not used in routine clinical testing to aid the management of these families.
For families with HDGC and known pathogenic CDH1 mutations, guidelines exist for risk assessment, disease management, surveillance (including regular endoscopies), and risk-reducing therapies (including prophylactic gastrectomy). However, for families with no pathogenic variant in CDH1, the risk assessment is uncertain, therefore making decisions and assessing the efficacy of risk-reducing strategies is challenging. Here, we aimed to identify genetic variants for predisposition to HDGC in an Iranian with a positive family history of DGC without pathogenic CDH1 variants and recognized a rare, likely pathogenic variant in ATM gene.
| > Materials And Methods|| |
Statement of ethics
The present study was approved by the Local Ethics Committee of Isfahan University of Medical Sciences (IRAN) and was in accordance with the ethical standards as laid down in the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards. Informed consent form was completed for each participant in the study.
According to the International GC Linkage Consortium, a family pedigree for HDGC with an autosomal dominant pattern of inheritance from Isfahan province (central of Iran) was selected [Figure 1]. The proband is a patient with HDGC diagnosed at 57 years and has a sister with LBC under 50 years old. Informed consent forms were completed by patients participating in the study or their families.
|Figure 1: Familial pedigree of hereditary diffuse gastric cancer in the studied family. Age of affected family members is indicated by an Asterisk|
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High-quality genomic DNA from peripheral blood samples was extracted of two affected sisters in whom no pathogenic variants or indels have been found in CDH1 and CTNNA1 genes with Sanger sequencing and multiplex ligation-dependent probe amplification methods using PrimePrep Genomic DNA Isolation Kit (Genet Bio, Korea). The whole-exome sequencing (WES) was performed using a custom-designed Nimbelgen chip capturing (Agilent Technologies, Inc., Santa Clara, CA, USA), followed by a paired-end, high-throughput sequencing using Illumina HiSeq 4000 with coverage ×100 mean depth (Macrogen Company, South Korea). All exons and 10 bp flanking were detected and analyzed. The detected variations included point mutations, deletion, and duplication (<250 bp). Briefly, the variant analysis has been done through mapping the text files of sequences read to the reference genome (UCSC hg19) using the Burrows–Wheeler Alignment software with default parameters. Following alignment, Genome Analysis Toolkit software library was used to identify the single-nucleotide polymorphism and insertion/deletion of WES data. Then, applying public databases, including 1000 Genome Project, HapMap samples, and dbSNP, identified variants with frequency >1% and synonymous substitutions filtered out. Variants with a minor allele frequency <0.1% were filtered to downstream analysis. The resulting list of variants were annotated by Annovar software. Annotated variations were filtered according to their frequency, chromosomal location, functional consequences, inheritance pattern, and clinical history.
Cosegregation study through Sanger sequencing
Sanger sequencing was used to confirm the accuracy of detecting variants in the proband and her sister's affected. Segregation analysis of the variants in the proband's parents was performed by PCR and Sanger sequencing.
| > Results|| |
Whole-exome sequencing results
The total number of variants of the proband in the final VCF file was 101,252. An in-house step-wise filtering process was performed to find the genetic cause of DGC disease, according to the genes reported in the DGC, pathogenicity, variant's effect, nonsynonymous, upstream/downstream, and exonic/intronic variants. Prioritizing genes that association with DGC identified several gene mutations include one T/C heterozygote substitution (c.2572T>C, p. Phe858 Leu) in exon 17, and C/G heterozygote substitution (c.3161C>G, p. Pro1054Arg) in exon 22 of ATM gene, A/C heterozygote substitution (c.1114A>C, p. Asn372His) in exon 10 of BRCA2 gene, and one A/G heterozygote substitution (c.1173A>G, p. Ile391Met) in exon 7 of PIK3CA gene [Table 1].
|Table 1: Variants in the PIK3CA, BRCA2, and ATM genes in hereditary diffuse gastric cancer family|
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The heterozygous C to G transversion for the c.3161C>G (p. Pro1054Arg (in exon 22 of ATM was predicted likely pathogenic by PolyPhen-2, SIFT, I-Mutant, Mutation Taster, PROVEAN, Mutation Assessor, PhD-SNP, and ConSurf software. Sanger sequencing results confirmed the detected variant in both affected family members [Table 2] and [Figure 2].
|Figure 2: The Sanger sequencing electropherograms. (a) Heterozygous c.3161C>G mutation in forward strand. (b) Heterozygous c.3161C>G mutation in reverse strand|
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| > Discussion|| |
Families with a deleterious genetic mutation are advised to have genetic counseling, clinical monitoring, and recommendations for prophylactic surgery. In spite of known mutations in the CDH1 gene as the most frequent etiology of HDGC, remarkably in up to 70% of HDGC families, the genetic etiology remains unknown. In this condition, the next-generation sequencing (NGS) method permits to find novel genes and variants involved in genetic diseases such as cancer., WES is a new emerging method of NGS for the identification of causative genes in Mendelian disorders, even in the lack of enough information about the inheritance pattern or exact clinical diagnosis. According to the catalog of somatic mutations in cancer, the top genes often mutated in GC that are identified by NGS technique include TP53, APC, CDH1, TRRAP, PIK3CA, MLL3, RNF213, KMT2D, MLL, CTNNB1, CREBBP, AKAP9, CACNA1D, MYH9, ZNF521, SETBP1, KRAS, CDH11, and ATM. The several of cancer-related genes that frequently mutated in GC identified by the WES technique. In this regard, the frequent mutations were reported by Wang et al., including TP53, PTEN, ARID1A, APC, CTNNB1, and CDH1, and by Zang et al., including TP53, PI3KCA, CTNNB1, ARID1A, KMT2C, and FAT4.
Since the predictive testing in asymptomatic at risk individual of the pedigree is provided the inherited pathogenic mutations, in this study, WES was performed in the proband and then cosegregation of the detected variants in the pedigree followed by a stepwise in-house filtering process, and identified a likely pathogenic c.3161C>G heterozygote variant that produces p. Pro1054Arg in exon 22 of the ATM gene in an Iranian family with DGC disease. Furthermore, several variants detected in other genes include BRCA2 and PIK3CA. The previous studies demonstrated the effect of these genes in cancer, for example, PIK3CA mutations associated with bone metastasis recurrence and reduced responsiveness to EGF-targeted therapies and also BRCA2 mutations reported as candidate genes in HDGC families.
| > Conclusion|| |
Our study expands the spectrum of ATM likely pathogenic variants in DGC patients with a positive family history and confirms the utility of targeted NGS sequencing in genetic diagnosis. However, confirmation of the pathogenicity status of missense variants that mentioned above, will be recommended in future studies by several analyses such as determining variant frequencies in a healthy control population, co-segregation of the variants in the pedigree, recurrence of the variants in independent families, in silico predictions, and in vitro functional studies.
We sincerely thank the patients and the professional staff of the Alaa Cancer Control Center, a charity-based foundation for cancer patients in Isfahan, Iran.
Financial support and sponsorship
This study was conducted with the support of Isfahan University of Medical Sciences (IRAN) with grant number 394479.
Conflicts of interest
There are no conflicts of interest.
| > References|| |
Mehrabani D, Hosseini SV, Rezaianzadeh A, Amini M, Mehrabani G, Tarrahi MJ. Prevalence of stomach cancer in Shiraz, Southern Iran. J Res Med Sci 2013;18:335-7.
Almasi Z, Rafiemanesh H, Salehiniya H. Epidemiology characteristics and trends of incidence and morphology of stomach cancer in Iran. Asian Pac J Cancer Prev 2015;16:2757-61.
Henson DE, Dittus C, Younes M, Nguyen H, Albores-Saavedra J. Differential trends in the intestinal and diffuse types of gastric carcinoma in the United States, 1973-2000: Increase in the signet ring cell type. Arch Pathol Lab Med 2004;128:765-70.
Donner I, Kiviluoto T, Ristimäki A, Aaltonen LA, Vahteristo P. Exome sequencing reveals three novel candidate predisposition genes for diffuse gastric cancer. Fam Cancer 2015;14:241-6.
Oliveira C, Sousa S, Pinheiro H, Karam R, Bordeira-Carriço R, Senz J, et al
. Quantification of epigenetic and genetic 2nd
hits in CDH1 during hereditary diffuse gastric cancer syndrome progression. Gastroenterology 2009;136:2137-48.
van der Post RS, Vogelaar IP, Carneiro F, Guilford P, Huntsman D, Hoogerbrugge N, et al
. Hereditary diffuse gastric cancer: Updated clinical guidelines with an emphasis on germline CDH1 mutation carriers. J Med Genet 2015;52:361-74.
Feroce I, Serrano D, Biffi R, Andreoni B, Galimberti V, Sonzogni A, et al
. Hereditary diffuse gastric cancer in two families: A case report. Oncol Lett 2017;14:1671-4.
Worthley DL, Phillips KD, Wayte N, Schrader KA, Healey S, Kaurah P, et al
. Gastric adenocarcinoma and proximal polyposis of the stomach (GAPPS): A new autosomal dominant syndrome. Gut 2012;61:774-9.
Pinheiro H, Oliveira C, Seruca R, Carneiro F. Hereditary diffuse gastric cancer-pathophysiology and clinical management. Best Pract Res Clin Gastroenterol 2014;28:1055-68.
Majewski IJ, Kluijt I, Cats A, Scerri TS, Jong D, Kluin RJ, et al
. An α-E-catenin (CTNNA1) mutation in hereditary diffuse gastric cancer. J Pathol 2013;229:621-9.
Hansford S, Kaurah P, Li-Chang H, Woo M, Senz J, Pinheiro H, et al
. Hereditary diffuse gastric cancer syndrome: CDH1 mutations and beyond. JAMA Oncol 2015;1:23-32.
Li H, Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 2009;25:1754-60.
Vogelaar IP, van der Post RS, Bisseling TM, van Krieken JHJ, Ligtenberg MJ, Hoogerbrugge N. Familial gastric cancer: Detection of a hereditary cause helps to understand its etiology. Hered Cancer Clin Pract 2012;10:18.
Gaston D, Hansford S, Oliveira C, Nightingale M, Pinheiro H, Macgillivray C, et al
. Germline mutations in MAP3K6 are associated with familial gastric cancer. PLoS Genet 2014;10:e1004669.
Schrauwen I, Helfmann S, Inagaki A, Predoehl F, Tabatabaiefar MA, Picher MM, et al
. A mutation in CABP2, expressed in cochlear hair cells, causes autosomal-recessive hearing impairment. Am J Hum Genet 2012;91:636-45.
Sawyer S, Hartley T, Dyment D, Beaulieu C, Schwartzentruber J, Smith A, et al
. Utility of whole-exome sequencing for those near the end of the diagnostic odyssey: Time to address gaps in care. Clin Genet 2016;89:275-84.
Lianos GD, Mangano A, Cho WC, Roukos DH. From standard to new genome-based therapy of gastric cancer. Expert Rev Gastroenterol Hepatol 2015;9:1023-6.
Wang K, Kan J, Yuen ST, Shi ST, Chu KM, Law S, et al
. Exome sequencing identifies frequent mutation of ARID1A in molecular subtypes of gastric cancer. Nat Genet 2011;43:1219-23.
Zang ZJ, Cutcutache I, Poon SL, Zhang SL, McPherson JR, Tao J, et al
. Exome sequencing of gastric adenocarcinoma identifies recurrent somatic mutations in cell adhesion and chromatin remodeling genes. Nat Genet 2012;44:570-4.
Kuboki Y, Yamashita S, Niwa T, Ushijima T, Nagatsuma A, Kuwata T, et al
. Comprehensive analyses using next-generation sequencing and immunohistochemistry enable precise treatment in advanced gastric cancer. Ann Oncol 2016;27:127-33.
Markman B, Atzori F, Pérez-García J, Tabernero J, Baselga J. Status of PI3K inhibition and biomarker development in cancer therapeutics. Ann Oncol 2010;21:683-91.
[Figure 1], [Figure 2]
[Table 1], [Table 2]