ORIGINAL RESEARCH published: 07 June 2021 doi: 10.3389/fonc.2021.648649 WRN Germline Mutation Is the Likely Inherited Etiology of Various Cancer Types in One Iranian Family Mahnaz Norouzi 1, Mohammad Shafiei 1*†,ZeinabAbdollahi2,PanizMiar2, Hamid Galehdari 1, Mohammad Hasan Emami 3, Mehrdad Zeinalian 2 and Mohammad Amin Tabatabaiefar 2,4*† Edited by: 1 Department of Biology, Faculty of Sciences, Shahid Chamran University of Ahvaz, Ahvaz, Iran, 2 Department of Genetics Israel Gomy, and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran, 3 Department of Dana–Farber Cancer Institute, Gastroenterology, Poursina Hakim Digestive Disease Research Center, Isfahan University of Medical Sciences, Isfahan, Iran, United States 4 Pediatric Inherited Diseases Research Center, Research Institute for Primordial Prevention of Noncommunicable Disease, Reviewed by: Isfahan University of Medical Sciences, Isfahan, Iran Mev Dominguez-Valentin, Oslo University Hospital, Norway Background: Familial cancers comprise a considerable distribution of colorectal cancers Reza Ghasemi, Washington University School of (CRCs), of which only about 5% occurs through well-established hereditary syndromes. It Medicine in St. Louis, has been demonstrated that deleterious variants at the newly identified cancer- United States predisposing genes could describe the etiology of undefined familial cancers. *Correspondence: Mohammad Amin Tabatabaiefar Methods: The present study aimed to identify the genetic etiology in a 32-year-old man [email protected]; with early onset familial CRC employing several molecular diagnostic techniques. DNA [email protected] Mohammad Shafiei was extracted from tumoral and normal formalin-fixed-paraffin-embedded (FFPE) shafi[email protected] blocks, and microsatellite instability (MSI) was evaluated. Immunohistochemistry † staining of MMR proteins was performed on tumoral FFPE blocks. Next-generation These authors have contributed fi equally to this work and share sequencing (NGS), multiplex ligation-dependent ampli cation (MLPA) assay, and senior authorship Sanger sequencing were applied on the genomic DNA extracted from peripheral blood. Data analysis was performed using bioinformatics tools. Genetic variants Specialty section: This article was submitted to interpretation was based on ACMG. Cancer Genetics, Results: MSI analysis indicated MSI-H phenotype, and IHC staining proved no a section of the journal Frontiers in Oncology expressions of MSH2 and MSH6 proteins. MLPA and NGS data showed no Received: 01 January 2021 pathogenic variants in MMR genes. Further analysis of NGS data revealed a candidate Accepted: 05 May 2021 WRN frameshift variant (p.R389Efs*3), which was validated with Sanger sequencing. The Published: 07 June 2021 variant was interpreted as pathogenic since it met the criteria based on the ACMG Citation: guideline including very strong (PVS1), strong (PS3), and moderate (PM2). Norouzi M, Shafiei M, Abdollahi Z, Miar P, Galehdari H, Emami MH, Conclusion: WRN is a DNA helicase participating in DNA repair pathways to sustain Zeinalian M and Tabatabaiefar MA fi (2021) WRN Germline Mutation Is the genomic stability. WRN de cient function may contribute to CRC development that is valuable Likely Inherited Etiology of Various for further investigation as a candidate gene in hereditary cancer syndrome diagnosis. Cancer Types in One Iranian Family. Front. Oncol. 11:648649. Keywords: colorectal cancer, microsatellite instability, hereditary non-polyposis colorectal cancer, next-generation doi: 10.3389/fonc.2021.648649 sequencing, WRN Frontiers in Oncology | www.frontiersin.org 1 June 2021 | Volume 11 | Article 648649 Norouzi et al. WRN Cause of Hereditary Cancer INTRODUCTION colorectal tumors, which typically arise in patients with late-onset cancer (12, 13). Unexplained dMMR CRC, termed “Lynch-like Hereditary cancer syndromes account for a small percentage of all syndrome”, is another MSI colorectal tumor for which neither the cancers diagnosed each year, although they are common in some deleterious germline variant of MMR genes nor the cancer forms, such as colorectal and breast cancers (1, 2). Colorectal hypermethylation of the MLH1 gene promoter can be identified cancer (CRC) is considered one of the most frequent malignancies (12, 15, 23).Somaticbi-allelicmutationsintheMMRgenesmay worldwide, and a family history of the condition is a major risk explain a large number of these Lynch-like dMMR tumors (nearly factor (1, 3). Notably, the relative risk of developing CRC increases 70%). However, the etiology of a large number of these unclassified in individuals with a family history of CRC. Alongside screening MSI/dMMR tumors is unclear (12, 23, 24). Moreover, dMMR CRCs and surveillance, another advantage of determining the molecular encompass around 60% of HNPCC cases. About 40% of HNPCC basis of familial CRC is its therapeutic relevance (4, 5). Meanwhile, patients do not represent MMR defects, and they are accordingly chromosomal instability arises in a remarkable portion of CRC introduced as familial colorectal cancer type X (FCCTX) with tumors. Microsatellite instability (MSI), which is caused by the undefined etiology. FCCTX is a group of heterogeneous diseases aberrant function of the DNA mismatch repair (MMR) system, characterized by microsatellite stable (MSS) and late-onset clinically accounts for nearly 15% of all CRC patients. In fact, insertion or different from LS (21, 25, 26). Despite the recent advances in gene deletion of a repetitive unit in a short tandemly repeated sequence identification technologies, the genetic etiology of many hereditary (STR), designated as MSI, is a robust diagnostic marker for cancers remains undefined, especially FCCTX groups (27, 28). identifying Lynch syndrome (LS) suspicious individuals (6–8). By the poor clinical diagnostic approaches, it is difficult to LS is an autosomal dominant hereditary cancer syndrome differentiate Lynch CRC tumors from non-hereditary ones due to caused by germline defects in the DNA mismatch repair genes, the high frequency of MSI occurrence in sporadic tumors. This including MLH1, MSH2, MSH6, PMS2, and EPCAM (9, 10). situation becomes more complicated when there is a positive family Most of the pathogenic variants of MMR genes include small history of developing CRC or different related cancer types (19). In insertions/deletions or large genetic rearrangements, missense, the present study, a CRC-affected individual from a fulfilled and nonsense variants (11–13). The majority of these gene Amsterdam II criteria pedigree, showing Lynch-associated MSI defects were found in MLH1 and MSH2 (60 to 80%) genes. and IHC phenotypes, underwent whole-exome sequencing (WES). Other detrimental variants in MSH6 or PMS2 and, rarely, to The analysis of WES data revealed no pathogenic variants in any of EpCAM genes have been identified associated with Lynch the MMR genes. Further evaluation unveiled a frameshift variant phenotype. Nevertheless, recent population studies have found in WRN (RECQL2; HGNC ID: 12791) gene as a potential candidate a higher frequency for germline pathogenic variants in MSH6 gene deserving subsequent considerations. and PMS2 genes (14–16). It was demonstrated that these genes WRN encodes a DNA helicase enzyme from the highly encompass lower penetrance pathogenic variants, and MSH6 conserved RECQ gene family and has a pivotal role in DNA deleterious variant carriers represent late-onset cancer compared repair and maintenance of genomic stability (29, 30). The WRN to MLH1 or MSH2 carriers (13). Moreover, an increased risk of has both helicase and exonuclease activity and has been extracolonic tumors, particularly endometrial cancer, has been considered a genomic caretaker (31). Homozygous truncating shown in MSH2 pathogenic variant carriers (13). Endometrial variants in the WRN gene cause Werner syndrome (WS; OMIM and ovarian cancers are the most prevalent Lynch-associated 277700) manifests, which are characterized by early onset age- tumors. Other associated cancers include pancreatic, biliary tract, associated diseases such as cancer (32, 33). In addition to the ureter, renal pelvis, gastric, small intestine, brain, sebaceous elevated risk of developing cancer in WS patients, several studies adenomas, sebaceous carcinomas, and keratoacanthomas (9). have indicated a tumor suppressor-like function for WRN, while LS is also known as hereditary non-polyposis colorectal cancer making it a prominent cancer-predisposing gene (34, 35). More (HNPCC), with an approximate prevalence of one in 600 to one in importantly, various evaluations have suggested a crucial role for 3,000 individuals (17, 18). HNPCC is a heterogeneous group of WRN associated with different types of familial cancers, disorders with overlapping features that are clinically evaluated using specifically breast and ovarian cancers (36–38). However, the Amsterdam (I or II) or Bethesda guidelines (19–22). Clinical association of germline WRN gene variants with familial CRC guidelines are based on family and personal history of cancer, and has not been established well. Herein, we aimed to introduce a their use as diagnostic tools for LS is restricted due to their low truncating variant in WRN (NM_000553:exon9:p.R389Efs*3) as sensitivity. Patients with HNPPCC are at increased risk for a pathogenic candidate based on ACMG standard guideline (39) metachronous or synchronous colon and extracolonic Lynch- in a patient affected by early onset familial CRC.