Investigating the causal role of MRE11A p.E506* in breast and ovarian cancer

The nuclease MRE11A is often included in genetic test panels for hereditary breast and ovarian cancer (HBOC) due to its BRCA1-related molecular function in the DNA repair pathway. However, whether MRE11A is a true predisposition gene for HBOC is still questionable. We determined to investigate this notion by dissecting the molecular genetics of the c.1516G > T;p.E506* truncating MRE11A variant, that we pinpointed in two unrelated French-Canadian (FC) HBOC patients. We performed a case–control study for the variant in ~ 2500 breast, ovarian, and endometrial cancer patients from the founder FC population of Quebec. Furthermore, we looked for the presence of second somatic alterations in the MRE11A gene in the tumors of the carriers. In summary, these investigations suggested that the identified variant is not associated with an increased risk of developing breast or ovarian cancer. We finally performed a systematic review for all the previously reported MRE11A variants in breast and ovarian cancer. We found that MRE11A germline variants annotated as pathogenic on ClinVar often lacked evidence for such classification, hence misleading the clinical management for affected patients. In summary, our report suggests the lack of clinical utility of MRE11A testing in HBOC, at least in the White/Caucasian populations.

In this study, we identified the truncating c.1516G > T;p.E506* MRE11A variant (rs587781384) in two unrelated French-Canadian women with breast or ovarian cancer following whole-exome sequencing and a broad clinical HBOC panel, respectively. Since, studying founder populations can help in risk estimation and as we have been studying French Canadians (FCs) for some years 9,10 , we thought we could tackle the questionable MRE11A HBOC risk candidacy by investigating the molecular genetics of this single truncating variant, which appears to be overrepresented in the FC population of Quebec compared with other populations studied thus far.

Results
Identifying the truncating c.1516G > T;p.E506* MRE11A variant in two unrelated HBOC patients. A 36 year-old woman was referred to the cancer genetics clinic with a diagnosis of invasive breast ductal carcinoma. Her clinical test for germline pathogenic mutations in BRCA1/2 and PALB2 resulted negative. We then performed whole exome sequencing (WES) in DNA from her peripheral blood mononuclear cells (pbmc) and identified a germline truncating variant c.1516G > T;p.E506* in the MRE11A gene (Methodology, and Table S1). A few months later, a 55 years-old woman diagnosed with ovarian high-grade serous carcinoma tested negative for BRCA1/2 and PALB2 in a broad HBOC clinical panel (Ref. 11 for the panel), however the same truncating variant c.1516G > T;p.E506* in MRE11A was identified (Fig. 1a,b). Both probands were of a French-Canadian ancestry. The c.1516G > T;p.E506* variant was previously reported in three BRCA1/2 negative breast and/or ovarian cancer patients 6,12,13 and twice in ATLD cases on ClinVar. The variant also has a gnomAD frequency of 3.19E-05 and 4E-05 in the UK biobank European-ancestry individuals 14 . Given the variant's low frequency in public databases, we hypothesized that this rare MRE11A variant might be a candidate founder pathogenic variant (PV) for HBOC in FCs.
Further genetic analysis of the c.1516G > T;p.E506* MRE11A variant. During the discovery phase of the project, a segregation analysis was done on the available members of the two families. In family A, the unaffected mother of the proband resulted to be a carrier of the variant. In family B, two healthy relatives of the proband (a maternal aunt and uncle) tested negative (Fig. 1d,e).
To address the possibility of this variant being a founder PV in FCs, we performed a case-control study including 1925 breast, 341 ovarian, and 367 endometrial cancer patients, in addition to 2,287 adult controls and 1932 newborns, all of FC origin ( Table 1). The c.1516G > T;p.E506* variant was not observed in any of the cancer patients, but it was found in 4 control subjects (0.002%; 2 males and 2 females). This suggested that c.1516G > T;p. E506*, which is the most reported likely pathogenic variant in MRE11A on ClinVar, is overrepresented in the FC population, but is unlikely to be associated with a risk of breast or ovarian cancer.
Nonsense-mediated mRNA decay (NMD) of alleles harboring a truncating mutation has been postulated as a molecular mechanism by which tumor suppressor genes get inactivated 15,16 . According to the recentlyestablished four rules of NMD, the c.1516G > T;p.E506* variant is predicted to trigger NMD 15,16 . Hence, we carried out a nonsense-mediated mRNA decay (NMD) assay, where a lymphoblastoid cell line derived from the proband of Family A was treated either with cycloheximide to inhibit mRNA degradation or DMSO as a control, as previously described 17 . This assay confirmed that the c.1516G > T;p.E506* variant can indeed trigger NMD as expected (Fig. 1c).
We next investigated the presence of second pathogenic alterations in the tumors of the two probands. No other somatic mutations in MRE11A was found by Sanger sequencing and none of the tumors showed any signs of loss-of-heterozygosity, an expected genetic event in the course of developing tumors through inactivating  www.nature.com/scientificreports/ tumor suppressor genes. We then performed WES in the breast tumor and mutational signature analysis 18 showed that the breast tumor lacked the characteristic Homologous Recombination Repair Deficiency (HRD) mutational signature, MutSign3, arguing against the involvement of c.1516G > T;p.E506* in breast cancer tumorigenesis (Fig. 1f). DNA quality of the ovarian tumor did not meet requirements for WES. At the tumor level, immunohistochemistry staining demonstrated that the breast tumor sample retained MRE11A protein expression (Fig. 1a). In contrast, despite the lack of additional somatic hits in the MRE11A gene, the ovarian tumor showed loss of MRE11A protein expression (Fig. 1a). However, the mechanism is uncertain given that the wild-type allele is retained at the DNA level. Altogether, these results indicated that the c.1516G > T;p.E506* variant does not increase the risk for breast and ovarian cancer in FCs.

Review of the reported MRE11A variants in breast and/or ovarian cancer.
To further complement our investigation and to generally address the initial question of the debatable pathogenicity and clinical utility of MRE11A testing in HBOC, we performed a systematic review of all previously reported MRE11A germline variants in breast and ovarian cancer patients. A total of 43 distinct MRE11A germline variants have been reported in such patients over the past 17 years (Table S2). Eight were annotated as pathogenic variants in ClinVar. The main criteria for pathogenic annotation in ClinVar included being a truncating allele, undetectable/ absent in control populations, and/or being reported in ATLD or in HBOC patients. Five of those variants had been published or reported in ClinVar as commonly altered among ATLD and cancer cases ( Fig. 2 and Table S2). The c.1516G > T;p.E506* variant was classified as pathogenic in two ATLD cases. However, it had conflicting classifications in hereditary cancer cases. One submission annotated the variant as pathogenic and another as a variant of uncertain significance.
Altogether, these observations demonstrate a clear limitation in the pathogenicity classification of the MRE11A germline variants on ClinVar, particularly as applied to HBOC.

Discussion
Advancement in DNA sequencing technologies has enabled the parallel testing of up to 100 genes within multiple-gene sequencing panels 19 . However, whether all the tested genes are true risk/predisposition genes is questionable. Two main criteria were previously proposed to guide indication for clinical gene screening: clinical validity and utility 20,21 . Both require the candidate to possess genetic variants that are associated with a phenotype (i.e. increased risk of developing cancer) and to predict the clinical outcome and help guide patient management. One of the questionable genes is MRE11A that has been included in HBOC gene panels for over a decade mainly for its BRCA1-closely related DNA repair molecular actions. In this report, using a well-studied founder population, we provide strong support for the notion that protein truncating germline variants in MRE11A are not associated with an increased risk of breast, ovarian, or likely, endometrial cancer. This is in accordance with a recently published retrospective study that analyzed the associated risk with predicted pathogenic variants of 32 cancer predisposition genes, including MRE11A, in 165,000 persons 8 . Seventy two percent (~ 120,000 persons) had cancer, of which ~ 89,000 were women with breast cancer, ~ 13,000 with ovarian cancer, in addition to ~ 5500 women with endometrial cancer. The team identified 140 predicted pathogenic MRE11A variants in total in different cancers (90 in breast cancer, 11 in ovarian cancer, and 5 in endometrial cancer). The authors did not identify a significant association between the MRE11A predicted pathogenic variants and any cancer phenotype, including breast, ovarian, and endometrial cancer 8 , suggesting a lack of MRE11A screening utility in HBOC and likely endometrial cancer patients. In another recent study with a similar objective 22 , Lee et al. investigated the association between the frequently included genes in genetic screening panels, including MRE11A, and familial breast and ovarian cancer. The authors used a point-based curation system that takes into account genetic and experimental evidence to score the gene association with the tested cancer type. The categories included Definitive, Strong, Moderate, Limited, Refuted, Disputed, or No Reported Evidence for association. MRE11A association with familial breast and/or ovarian cancer was disputed, denoting to the conflicting data and views of its association with the cancer. Our findings, taken together with the recent report of LaDuca et al. 8 call for re-evaluating the MRE11A classification in HBOC. Additional investigations targeting rare missense variants in MRE11A, such as those listed in Table S2 and shown in Fig. 2, and their possible association with HBOC are, however, needed to complete the picture for MRE11A and HBOC risk. Additionally, limited by the relatively low number of tested endometrial cancer patients in the current report, we cannot completely exclude a role for MRE11A truncating variants in the development of endometrial cancer. It is also worth noting in this context that most of the studies focused on Caucasian populations and rarely focused on Black populations. Importantly, Hart et al. recently reported threefold and twofold increase in MRE11A variants prevalence in ovarian and breast cancer, respectively, in Black population versus Non-Hispanic Whites 23 .
Our systematic review highlights some of the gaps in our current knowledge of the validity and utility of MRE11A testing. Classifying the MRE11A ATLD and truncated variants as pathogenic in HBOC should be supported, as previously recommended 24 , by further clinical evidence (e.g., population and/or segregation studies) and/or functional studies to assess the possible associated MRE11A function perturbation. Notably, the truncating c.1516G > T;p.E506* variant, investigated here, was not associated with HRD (Fig. 1f), an expected consequence of a disfunctional MRE11A.
MRE11A variants are, however, associated with a clinically important cancer-related phenotype: clonal hematopoiesis. Two independent studies have found that heterozygous rare variants in MRE11A are subject to copyneutral loss of heterozygosity, which is strongly associated with clonal hematopoiesis 25,26 . These clones become much more evident with age, such that clonal hematopoiesis affects 40% of Japanese persons over age 90 years. Therefore, MRE11A variants deserve further attention as they are associated with adverse health outcomes.
In conclusion, we report the detailed investigation of a truncating variant in the candidate breast and ovarian cancer susceptibility gene, MRE11A. Our findings, taken together with other recently published studies, show that truncating variants in MRE11A are not a cause of breast or ovarian cancer. Based on the current evidence, testing for MRE11A variants should not be offered to women with, or at risk for, breast or ovarian cancer.

Patients and methods
Patients and samples. In total, 2633 cancer patients and a control group of 2287 adults and 1932 newborns of French-Canadian ancestry were included in this study, as explained in detail in Table 1  Newborn blood left-over after delivery was collected at the Hôpital Saint-François d' Assise maternity unit between 1996 and 2003. DNA was extracted and a biobank with over 7000 samples was constituted. These samples were made anonymous and unlinked. About 15% of the samples were randomly excluded from the collection in order to preserve even further the anonymity of the samples because any woman having given birth in this hospital during that period has a 15% chance of not being part of the studied samples. The Hôpital Saint-François d' Assise Clinical Research Ethics Committee approved the research project. For the present study a subset of n = 1932 samples from the collection was randomly selected based uniquely on amount of DNA available as it is not possible to identify the subset of samples included in the study, sex information is not available.
Samples from healthy controls (1891 individuals) were obtained through CARTaGENE biobank 27  Index patients and their families. The proband of family A was diagnosed at the age of 36 with invasive ductal carcinoma. This patient is part of a 51-high risk BRCA1/2 negative BC patients study previously carried out by our team 28 . The patient had been screened for germline variants by whole exome sequencing (WES) revealing the presence of a likely pathogenic variant in MRE11A (c.1516G > T;p.E506*) (Fig. 1a,b). Proband of Family B was a 55-year old woman diagnosed with a high-grade serous carcinoma (HGSC) that was referred to the cancer genetics clinic from the gynecology clinic at the Jewish General Hospital (Montreal, Canada) for genetic testing (Fig. 1a,b). The patient had a family history strongly suggestive of hereditary breast and ovarian cancer (HBOC) (mother and aunt diagnosed with breast cancer). A clinical HBOC screening panel identified the same nonsense MRE11A c.1516G > T;p.E506*variant independently found in the proband of family A. Both women were of French-Canadian ancestry. Whole exome sequencing (WES). The proband A was among a 51-high risk BC patient series we previously screened for germline variants in genomic DNA extracted from peripheral blood leukocytes as previously outlined in detail 28 . Breast cancer from the proband studied here was subjected to WES that was performed at the McGill University and Genome Quebec Innovation Centre (MUGQIC). Breast cancer FFPE-derived DNA sample underwent exome capture (Nextera Rapid Capture Exome Kit), followed by 100 bp paired-end sequencing on Illumina HiSeq 2500 (details in Ref 26 ). Bioinformatics analysis of exome sequencing data was performed using our WES pipeline as previously described 9,[32][33][34] . In brief, alignment of sequenced reads to the reference genome (hg19) was performed using BWA 35 (v. 0.5.9). Subsequently, the Genome Analysis Toolkit (GATK) was used to perform local realignment of reads around small insertions and deletions (indels) and to assess capture efficiency and coverage for all samples. The coverage statistics are listed in Table 2. Likely pathogenic variants in 152 known cancer susceptibility genes from Huang et al. 36 and in 468 cancer panel gene list elaborated by the MSK-IMPACT were selected. Candidate somatic mutations were subjected to several filtering steps and eliminated if they fulfilled any one of the following criteria: (1) genomic position of variant covered by < 5-reads,  www.nature.com/scientificreports/ tumours (n = 34) or ascites (n = 2) were genotyped using The Custom TaqMan SNP Genotyping Assay protocol performed as previously described 43 . Using a similar pipeline to our previous work 28 , we genotyped samples from the below 70 years old breast cancer group (n = 512), newborns of French Canadian ancestry (n = 1932) in addition to the healthy control adults (CARTaGENE biobank; n = 1891) for MRE11A c.1516G > T; p.E506* allele using the iPLEX MassARRAY platform (Sequenom) at the McGill University and Genome Quebec Innovation Centre (MUGQIC). A high-resolution melting (HRM) assay was used to test all the remaining patient and control samples included in this study as previously described 9 . For validations, Sanger sequencing was used, and chromatograms were aligned with the NM_005591.4 RefSeq transcript. Probes and primers sequences are available upon request.

Study population (case-control study
Databases and software used in this report. gnomAD. We interrogated the gnomAD database 44 to assess the frequency of the p.E506* MRE11A variant and its prevalence in the reported ethnicities. gno-mAD v2.1.1 was last accessed on 30 April 2020 and the MRE11 p.E506* variant (variant ID:11-94189489-C-A; GRCh37) prevalence was detected as detailed in the text. The PhenoTips software was used to generate pedigrees (https ://pheno tips.com/index .html). The ProteinPaint tool 45 from https ://prote inpai nt.stjud e.org/ was used for elaboration of Fig. 2. Immunohistochemistry. Formalin-fixed, paraffin-embedded (FFPE) samples from the breast and ovarian tumors were studied by IHC. MRE11A (Abcam #ab214, 1:1500) was optimized in a Ventana machine following recommended protocols. MRE11A expression and localization was analyzed by a pathologist (OA) independently for diagnosis and mutational analysis. Aperio ImageScope software was used to obtain the images. NMD assay. A lymphoblastoid cell line was established from a blood sample from the proband of Family A. The cells were cultured using RPMI medium. For assessing whether the mutant allele undergoes NMD, cells were plated in 6 well plates, and either treated with cycloheximide (28 ug/ml) 17 or (DMSO) for 3 h. At the experimental endpoint, RNA was extracted from both the treated or untreated cells using Trizol and RT-PCR was performed to obtain cDNA. cDNA was then subjected to sanger sequencing.
MRE11A germline variants literature review. We did a literature review for the reported MRE11A germline variants as of May 13 2020. We found 21 studies reporting different variants of MRE11A in mainly breast and ovarian cancers over the past 17 years (from 2003 to 2020). One study was excluded as it did not focus on HBOC. All the related info and references are included in Table S2.