Characterisation of a Portuguese origin founder missense variant in MSH6

Characterisation of a Portuguese origin founder missense variant in MSH6 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Brief Communication Characterisation of a Portuguese origin founder missense variant in MSH6 William Foulkes, Kenzie Melvill, Leora Witkowski, Céline Domecq, and 12 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9172566/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract In certain populations, founder pathogenic variants are a common cause of Lynch syndrome. Here, we report the identification of a novel founder variant, NM_000179.3 (MSH6):c.2061T > G (p.Cys687Trp). Our study examined 14 probands and 18 additional family members who carry this variant. With one exception, haplotype data are consistent with a single origin for all heterozygotes. We investigated the clinicopathological consequences of this variant and found that, as previously reported for other MSH6 pathogenic variants a) presence of the founder pathogenic variant is not reliably associated with loss of MSH6 expression or microsatellite instability and b) endometrial cancer is the most common associated phenotype. Based on our findings, we recommend reclassifying MSH6 c.2061T > G from missense variant of unknown significance to likely pathogenic according to CanVIG-UK guidelines. Biological sciences/Cancer/Cancer genetics Health sciences/Diseases/Cancer/Gastrointestinal cancer/Colorectal cancer Biological sciences/Cancer/Gynaecological cancer/Endometrial cancer Biological sciences/Genetics/Cancer genetics Biological sciences/Cancer/Cancer screening Founder variant Lynch syndrome variant of uncertain significance immunohistochemistry haplotype endometrial cancer Figures Figure 1 Introduction Lynch syndrome (LS) is an autosomal dominant cancer predisposition syndrome which increases risk for certain cancers, most commonly colorectal (CRC) and endometrial cancer (EC). LS is most often caused by germline pathogenic variants in the DNA mismatch repair (MMR) genes MLH1 , MSH2 , PMS2 , and MSH6 ; MSH6 accounts for about 18% of cases [ 1 , 2 ]. Compared with MLH1 and MSH2 , MSH6 -associated LS is less penetrant and often diagnosed at later ages [ 3 ]. Notably, the risk for EC is significantly greater than the risk for CRC in female MSH6 heterozygotes, which is not the case for other MMR genes [ 3 ]. MMR genes are tumor suppressors. Loss of MMR expression is a hallmark of LS tumors [ 1 ]. Testing for MMR deficiency through immunohistochemical (IHC) staining of MMR proteins or microsatellite instability (MSI) is standard in certain tumor types to identify patients at risk for LS. MMR status can also be used to assess germline variants in MMR genes [ 1 ]; however, the clinical utility of MMR/MSI status in screening for patients at risk for LS and subsequent variant interpretation varies. Malignancies resulting from MLH1 or MSH2 germline variants are more likely to demonstrate MMR deficiency than those resulting from MSH6 or PMS2 germline variants [ 1 , 4 ]. Additionally, nearly half of reported MSH6 variants are missense [ 5 ]. Classification of MSH6 missense variants is further complicated by the often-attenuated LS phenotype, making co-segregation studies less convincing. Founder variants are genetic variants present at increased frequency in certain populations, usually due to genetic drift from a common ancestor. MSH6 pathogenic founder variants (PFVs) have been identified in the Ashkenazi Jewish and French-Canadian populations [ 6 , 7 ]. Portuguese PFVs have been described in MLH1 and in MSH2 [ 8 ]. Historically, characterization of PFVs allowed first-line screening strategies to lower the cost of molecular diagnosis. With the decreasing costs of testing, many centers now favour full gene sequencing via multi-gene panels to avoid multiple lines of screening. Identifying and correctly interpreting germline variants remains important to ensure proper screening for LS families. We report on the missense variant MSH6 c.2061T > G, which we first identified in a Montreal-based woman of Portuguese descent with multiple primary cancers consistent with LS. After contacting researchers and commercial laboratories, we identified 13 additional probands with this variant. Where possible, we collected information on MSI, IHC and loss of heterozygosity as well as genealogy and segregation of the variant. We also performed haplotype analysis to investigate a potential founder origin of the variant. Materials and Methods Patients and Samples This study includes 14 apparently unrelated families (Table S1 ). All are either of Portuguese descent (n = 10) or have origins consistent with Portuguese descent (n = 4; Table 1). Data was collected from centers in Canada, the USA, Portugal, and France. Each family includes at least one individual who tested positive for NM_000179.3( MSH6 ):c.2061T > G (p.Cys687Trp). All participants provided informed consent to their respective centres to participate in research. Ethical approval for this study was granted by the McGill University Health Centre Research Ethics Board (studies MP-37-2019-4865 and MP-37-2023-9071). Molecular Results Germline testing for 14 probands was performed by each centre using in-house or external laboratories. Segregation analysis was performed in 18 additional individuals from five families (Table 1). Two tumours were also analyzed for somatic mutations (Table 1). Analysis of MMR Status All centers used standard procedures to analyze healthy and cancerous tissues. MMR proficiency was analyzed by IHC staining for MLH1, MSH2, MSH6, and PMS2. Biocartis MSI (Mechelen, Belgium) was employed in two cases (Table 1). Haplotype analysis Genotyping of 15 microsatellite markers was performed using 20 DNA samples from ten families (Table 2). These markers covered the region 12.3 Mb upstream and 9.5 Mb downstream of MSH6 . When available, segregation analysis was used to phase alleles and establish haplotypes, as previously described [ 6 ]. Results Characterization of families with MSH6 c.2061T > G Of the 14 families, one fulfilled Amsterdam criteria, four fulfilled Amsterdam II criteria, and six fulfilled Bethesda criteria (Table S1 ). No additional family history data was available for two of the remaining cases. Five of the seven families identified at centres in Portugal originate from the Leiria District of Portugal, situated north of Lisbon. One additional family has origins in a nearby region. The variant was identified in 23 individuals (Table 1). The variant co-segregated with a LS-spectrum cancer in 15 heterozygotes with an average age of 52.2 (range: 34–81) years at first diagnosis. The variant was also identified in an individual with a clinical diagnosis of constitutional mismatch repair deficiency (CMMRD) who carries a second MSH6 VUS in trans with c.2061T > G (Table 1) [ 9 ]. This variant was additionally present in two individuals with a personal history of at least one colorectal polyp (Table 1). Four individuals are, to our knowledge, unaffected to date. Clinicopathological features The most common diagnosis in heterozygous females (9/16) was EC with average age at diagnosis of 56.1 (range: 43–82). The most common diagnosis in heterozygous males was CRC (4/6) with average age at diagnosis of 51.3 (range: 34–69). Of the 15 tumours evaluated by IHC, six (40%) demonstrated loss of MSH6 expression. Loss or retention of MSH6 was not consistent across tumours in individuals with multiple LS-spectrum cancers (Table 1, Fig. 1 ). Two of the tumours that retained MSH6 were microsatellite stable (Table 1). Two tumours with loss of MSH6 had somatic genetic second hits. The proband of Family VIII demonstrated loss of MSH6 expression in non-cancerous tissue, consistent with the clinical diagnosis CMMRD [ 9 ]. Variant Interpretation We employed the Bayesian point system described in Tavtigian et al [ 10 ] to interpret our variant according to both the ClinGen Variant Curation Expert Panel specifications for the American College of Medical Genetics (ACMG) 2015 recommendations and the MMR-specific CanVIG-UK guidelines [ 11 , 12 ]. MSH6 c.2061T > G is classified as a “Variant of Uncertain Significance” per the ACMG criteria but as “Likely Pathogenic” according to the CanVIG-UK guidelines (Table S2). Haplotype analysis A shared haplotype was identified in 15 heterozygous individuals from 9 families (Table 2). This conserved haplotype spans a maximum of 10.4 Mb and a minimum of 4.9 Mb. One additional affected heterozygote (Family V) carried some alleles in common with this haplotype, suggesting a possible distant connection to the other carrier families. The provided sample was confirmed to carry MSH6 c.2061T > G by Sanger sequencing, but no additional family V members were available for genotyping and haplotyping. Discussion This study evaluated the clinical impact of a novel Portuguese missense founder variant causing LS, MSH6 c.2061T > G, in 14 families. We identified a haplotype shared among nine families, suggesting a common ancestor. Based on the origins of several families, this variant likely originated in the Leiria District of Portugal. We first identified this variant in 2008 as a VUS in a proband (Family I) with a convincing personal history of LS-spectrum tumours; however, not all tumours demonstrated loss of MSH6 (Table 1, Fig. 1 ). The variant has since been identified in thirteen additional families, the majority of which (11/14) meet at least one of the clinical criteria for LS (Table S1 ). Despite this, it has been difficult to clarify this variant’s pathogenicity. Like approximately 70% of MMR VUS, the variant results from a missense mutation, which at its foundation makes interpretation more difficult [ 13 ]. Missense variants in the MMR genes are statistically more likely to demonstrate unexpected staining patterns than truncating variants, possibly due to the presence of non-functional protein that cannot be differentiated from functional protein through IHC [ 5 ]. Adding to the challenge, the degree of MSI is lower in MSH6 -associated CRCs and the most common cancer in female heterozygotes (EC) is more likely to have inconsistent MMR/MSI results when compared to the other MMR genes [ 14 , 15 ]. This begs the question of whether MSI/IHC of tumour tissue is a sufficient and effective screening method for the less-penetrant LS genes. This may also explain why MSH6 has the highest proportion of VUS among the MMR genes [ 16 ]. Nevertheless, MSI/IHC evidence is still considered a hallmark feature of tumours in all LS patients, and MSI/IHC results are considered important evidence of pathogenicity in both ACMG and CanVIG-UK criteria [ 11 , 12 ] We present the third known Portuguese founder variant causing LS, and the first in MSH6 . This variant is characterized by classic MSH6 -associated LS, including incomplete penetrance, an often-attenuated phenotype, and higher risk for EC than CRC in females. As observed for other missense variants in MMR genes and studies of MSH6 -associated LS specifically, this variant does not reliably lead to MSI or loss of MSH6 expression by IHC in tumours [ 5 , 14 , 15 ]) (Fig. 1 , Figure S1 , Figure S2). The emphasis of MMR proficiency as evidence against pathogenicity under the ACMG criteria is therefore in direct opposition to substantial prior research. It is plausible that similar missense variants have gone undiagnosed in several families who would benefit from recommended LS screening regiments. In fact, some are calling for the division of LS into gene-specific syndromes to avoid this very issue [ 17 ]. We hope the observations from our study help to facilitate the identification of such families and prompts a broader discussion of the limitations of IHC and MSI as a method for stratifying the risk of LS. This study has limitations, including our inability to include allelic data (PM3) despite the variant being present in an individual with a clinical diagnosis of CMMRD, due to its being in trans with a second MSH6 VUS. There are also currently no functional data available to support pathogenicity of MSH6 c.2061T > G. Nevertheless, the variant reaches a pathogenic score using CanVIG-UK criteria. Declarations Funding This study was supported by funding from the McGill University Health Centre Foundation and its generous donors. Author contributions KM prepared the manuscript, tables, and figures. LW performed variant interpretation and reviewed the manuscript. CD and NH designed and performed haplotype analyses and also contributed and reviewed the manuscript. JM, JLE, KS, SM, MRT, CA, IF, LGR, OCT, KM, and AC provided cases, data and samples for haplotype analysis where possible, and reviewed the manuscript. GC performed molecular characterization for Family I and reviewed the manuscript. VM provided expertise on IHC images and reviewed the manuscript. WDF designed study and oversaw data collection and manuscript preparation. Acknowledgements The authors wish to thank Ana Bonilha for her help with translations and Brianna Lemieux for preliminary data collection. We are also thankful to Myriad Genetics and Ambry Genetics for contributing cases. We would like to acknowledge the contributions of Dr. Aaron Pollet, Ms. Kara Semotiuk, and Dr. Jordan Lerner-Ellis (Mount Sinai Hospital, Toronto) in providing IHC images, molecular data and additional family history information. We thank Dr. Ricardo Fonseca (Instituto Português de Oncologia de Lisboa, Portugal) in providing IHC images. We thank John Lee (Cedars-Sinai Medical Center, Los Angeles) for connecting us with cases and their healthcare providers. Data availability statement Data are available from the principal investigator/corresponding author (WDF) upon reasonable request. References Gallon, R., et al., How Should We Test for Lynch Syndrome? A Review of Current Guidelines and Future Strategies. Cancers (Basel), 2021. 13 (3). Peltomaki, P., Update on Lynch syndrome genomics. Fam Cancer, 2016. 15 (3): p. 385-93. Dominguez-Valentin, M., et al., Cancer risks by gene, age, and gender in 6350 carriers of pathogenic mismatch repair variants: findings from the Prospective Lynch Syndrome Database. Genet Med, 2020. 22 (1): p. 15-25. Elze, L., et al., Microsatellite instability in noncolorectal and nonendometrial malignancies in patients with Lynch syndrome. J Natl Cancer Inst, 2023. 115 (7): p. 853-860. Chen, W., et al., Unexpected expression of mismatch repair protein is more commonly seen with pathogenic missense than with other mutations in Lynch syndrome. Hum Pathol, 2020. 103 : p. 34-41. Castellsague, E., et al., Characterization of a novel founder MSH6 mutation causing Lynch syndrome in the French Canadian population. Clin Genet, 2015. 87 (6): p. 536-42. Raskin, L., et al., Characterization of two Ashkenazi Jewish founder mutations in MSH6 gene causing Lynch syndrome. Clin Genet, 2011. 79 (6): p. 512-22. Pinto, C., et al., Co-occurrence of nonsense mutations in MSH6 and MSH2 in Lynch syndrome families evidencing that not all truncating mutations are equal. J Hum Genet, 2016. 61 (2): p. 151-6. Pinheiro, M., et al., A novel exonic rearrangement affecting MLH1 and the contiguous LRRFIP2 is a founder mutation in Portuguese Lynch syndrome families. Genet Med, 2011. 13 (10): p. 895-902. Pinheiro, M., et al., The nonsense mutation MSH2 c.2152C>T shows a founder effect in Portuguese Lynch syndrome families. Genes Chromosomes Cancer, 2019. 58 (9): p. 657-664. Pinheiro, M., et al., The MSH2 c.388_389del mutation shows a founder effect in Portuguese Lynch syndrome families. Clin Genet, 2013. 84 (3): p. 244-50. Guerrini-Rousseau, L., et al., Constitutional mismatch repair deficiency-associated brain tumors: report from the European C4CMMRD consortium. Neurooncol Adv, 2019. 1 (1): p. vdz033. Tavtigian, S.V., et al., Fitting a naturally scaled point system to the ACMG/AMP variant classification guidelines. Hum Mutat, 2020. 41 (10): p. 1734-1737. Richards, S., et al., Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med, 2015. 17 (5): p. 405-24. CanVIG-UK. Mismatch Repair Genes: CanVIG-UK Gene-Specific Guidance . Available from: https://www.cangene-canvaruk.org/gene-specific-recommendations. Thompson, B.A., et al., Application of a 5-tiered scheme for standardized classification of 2,360 unique mismatch repair gene variants in the InSiGHT locus-specific database. Nat Genet, 2014. 46 (2): p. 107-115. Wang, C., et al., Evaluation of microsatellite instability patterns in mismatch repair deficiency: a retrospective analysis of 285 endometrial cancers. Front Immunol, 2025. 16 : p. 1628979. Helderman, N.C., et al., Lower Degree of Microsatellite Instability in Colorectal Carcinomas From MSH6-Associated Lynch Syndrome Patients. Mod Pathol, 2025. 38 (7): p. 100757. Frederiksen, J.H., et al., Classification of MSH6 Variants of Uncertain Significance Using Functional Assays. Int J Mol Sci, 2021. 22 (16). Moller, P., et al., Dominantly inherited micro-satellite instable cancer - the four Lynch syndromes - an EHTG, PLSD position statement. Hered Cancer Clin Pract, 2023. 21 (1): p. 19. Tables Table 1 and 2 are available in the Supplementary Files section. Additional Declarations There is no duality of interest Supplementary Files Table1FINALNEWVM.xlsx Table 1 Table2FINALNEW.xlsx Table 2 SupplementalMaterialsFINALagerangesV2.pdf Supplemental Material Cite Share Download PDF Status: Under Review Version 1 posted Review # 2 received at journal 05 May, 2026 Review # 1 received at journal 28 Apr, 2026 Reviewer # 3 agreed at journal 24 Apr, 2026 Reviewer # 2 agreed at journal 21 Apr, 2026 Reviewer # 1 agreed at journal 20 Apr, 2026 Reviewers invited by journal 14 Apr, 2026 First submitted to journal 06 Apr, 2026 Submission checks completed at journal 24 Mar, 2026 Unknown event 23 Mar, 2026 Editor assigned by journal 19 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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19:30:57","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9172566/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9172566/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107399111,"identity":"02f69545-5251-4604-be3b-54599acb2233","added_by":"auto","created_at":"2026-04-21 07:13:40","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":257366,"visible":true,"origin":"","legend":"\u003cp\u003eImmunohistochemistry of MMR proteins in cancers from proband of Family I\u003c/p\u003e\n\u003cp\u003eFemale proband was diagnosed with MSH6-deficient endometrioid-type endometrial adenocarcinoma with squamous differentiation in her 40s and MMR-proficient colorectal cancer in her 50s. A) MLH1 retention in both the endometrial adenocarcinoma with squamous differentiation (left) and the non-neoplastic endometrial glands (right); B) absence MSH6 in endometrial carcinoma, arrow indicates tumor with squamous differentiation; C) retention of MSH6 in colorectal tumor; D) retention of PMS2 in endometrial tumor; E) retention of MSH2 in endometrial tumor; F) retention of MSH2 in colorectal tumor; Magnification 200x. IHC performed using antibodies G168-728 (MLH1; Cell Marque), G219-1129 (MSH2; Cell Marque), 44 (MSH6; Cell Marque), and MRQ-28 (PMS2; Cell Marque). This individual is known to carry \u003cem\u003eMSH6 \u003c/em\u003ec.2061T\u0026gt;G in the germline.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9172566/v1/0cb04e2a97fc5d24bbba0f6e.jpg"},{"id":107489321,"identity":"3ad15254-d6e4-4265-9902-801f8655480b","added_by":"auto","created_at":"2026-04-22 02:47:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":451437,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9172566/v1/b578a3df-86dc-4dbf-b2de-352a651c5308.pdf"},{"id":107487745,"identity":"b21d0eba-07a7-4546-bee5-3a04e8d04028","added_by":"auto","created_at":"2026-04-22 02:42:39","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":16905,"visible":true,"origin":"","legend":"Table 1","description":"","filename":"Table1FINALNEWVM.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9172566/v1/b20962cb895a7d409d095c9e.xlsx"},{"id":107399114,"identity":"e89584f7-77ee-4db5-8500-220da8eec43f","added_by":"auto","created_at":"2026-04-21 07:13:40","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":12995,"visible":true,"origin":"","legend":"Table 2","description":"","filename":"Table2FINALNEW.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9172566/v1/37531febbc411963b5fb35b8.xlsx"},{"id":107399113,"identity":"3873c45c-a55b-4612-8bd4-3df01d83b550","added_by":"auto","created_at":"2026-04-21 07:13:40","extension":"pdf","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":2471373,"visible":true,"origin":"","legend":"Supplemental Material","description":"","filename":"SupplementalMaterialsFINALagerangesV2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9172566/v1/213ccebf6ba616ebee01d057.pdf"}],"financialInterests":"There is no duality of interest","formattedTitle":"Characterisation of a Portuguese origin founder missense variant in MSH6","fulltext":[{"header":"Introduction","content":"\u003cp\u003eLynch syndrome (LS) is an autosomal dominant cancer predisposition syndrome which increases risk for certain cancers, most commonly colorectal (CRC) and endometrial cancer (EC). LS is most often caused by germline pathogenic variants in the DNA mismatch repair (MMR) genes \u003cem\u003eMLH1\u003c/em\u003e, \u003cem\u003eMSH2\u003c/em\u003e, \u003cem\u003ePMS2\u003c/em\u003e, and \u003cem\u003eMSH6\u003c/em\u003e; \u003cem\u003eMSH6\u003c/em\u003e accounts for about 18% of cases [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Compared with \u003cem\u003eMLH1\u003c/em\u003e and \u003cem\u003eMSH2\u003c/em\u003e, \u003cem\u003eMSH6\u003c/em\u003e-associated LS is less penetrant and often diagnosed at later ages [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Notably, the risk for EC is significantly greater than the risk for CRC in female \u003cem\u003eMSH6\u003c/em\u003e heterozygotes, which is not the case for other MMR genes [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMMR genes are tumor suppressors. Loss of MMR expression is a hallmark of LS tumors [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Testing for MMR deficiency through immunohistochemical (IHC) staining of MMR proteins or microsatellite instability (MSI) is standard in certain tumor types to identify patients at risk for LS. MMR status can also be used to assess germline variants in MMR genes [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]; however, the clinical utility of MMR/MSI status in screening for patients at risk for LS and subsequent variant interpretation varies. Malignancies resulting from \u003cem\u003eMLH1\u003c/em\u003e or \u003cem\u003eMSH2\u003c/em\u003e germline variants are more likely to demonstrate MMR deficiency than those resulting from \u003cem\u003eMSH6\u003c/em\u003e or \u003cem\u003ePMS2\u003c/em\u003e germline variants [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Additionally, nearly half of reported \u003cem\u003eMSH6\u003c/em\u003e variants are missense [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Classification of \u003cem\u003eMSH6\u003c/em\u003e missense variants is further complicated by the often-attenuated LS phenotype, making co-segregation studies less convincing.\u003c/p\u003e \u003cp\u003eFounder variants are genetic variants present at increased frequency in certain populations, usually due to genetic drift from a common ancestor. \u003cem\u003eMSH6\u003c/em\u003e pathogenic founder variants (PFVs) have been identified in the Ashkenazi Jewish and French-Canadian populations [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Portuguese PFVs have been described in \u003cem\u003eMLH1\u003c/em\u003e and in \u003cem\u003eMSH2\u003c/em\u003e [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHistorically, characterization of PFVs allowed first-line screening strategies to lower the cost of molecular diagnosis. With the decreasing costs of testing, many centers now favour full gene sequencing via multi-gene panels to avoid multiple lines of screening. Identifying and correctly interpreting germline variants remains important to ensure proper screening for LS families.\u003c/p\u003e \u003cp\u003eWe report on the missense variant \u003cem\u003eMSH6\u003c/em\u003e c.2061T\u0026thinsp;\u0026gt;\u0026thinsp;G, which we first identified in a Montreal-based woman of Portuguese descent with multiple primary cancers consistent with LS. After contacting researchers and commercial laboratories, we identified 13 additional probands with this variant. Where possible, we collected information on MSI, IHC and loss of heterozygosity as well as genealogy and segregation of the variant. We also performed haplotype analysis to investigate a potential founder origin of the variant.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePatients and Samples\u003c/h2\u003e \u003cp\u003eThis study includes 14 apparently unrelated families (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). All are either of Portuguese descent (n\u0026thinsp;=\u0026thinsp;10) or have origins consistent with Portuguese descent (n\u0026thinsp;=\u0026thinsp;4; Table\u0026nbsp;1). Data was collected from centers in Canada, the USA, Portugal, and France. Each family includes at least one individual who tested positive for NM_000179.3(\u003cem\u003eMSH6\u003c/em\u003e):c.2061T\u0026thinsp;\u0026gt;\u0026thinsp;G (p.Cys687Trp). All participants provided informed consent to their respective centres to participate in research. Ethical approval for this study was granted by the McGill University Health Centre Research Ethics Board (studies MP-37-2019-4865 and MP-37-2023-9071).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMolecular Results\u003c/h3\u003e\n\u003cp\u003eGermline testing for 14 probands was performed by each centre using in-house or external laboratories. Segregation analysis was performed in 18 additional individuals from five families (Table\u0026nbsp;1). Two tumours were also analyzed for somatic mutations (Table\u0026nbsp;1).\u003c/p\u003e\n\u003ch3\u003eAnalysis of MMR Status\u003c/h3\u003e\n\u003cp\u003eAll centers used standard procedures to analyze healthy and cancerous tissues. MMR proficiency was analyzed by IHC staining for MLH1, MSH2, MSH6, and PMS2. Biocartis MSI (Mechelen, Belgium) was employed in two cases (Table\u0026nbsp;1).\u003c/p\u003e\n\u003ch3\u003eHaplotype analysis\u003c/h3\u003e\n\u003cp\u003eGenotyping of 15 microsatellite markers was performed using 20 DNA samples from ten families (Table\u0026nbsp;2). These markers covered the region 12.3 Mb upstream and 9.5 Mb downstream of \u003cem\u003eMSH6\u003c/em\u003e. When available, segregation analysis was used to phase alleles and establish haplotypes, as previously described [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003eCharacterization of families with MSH6\u003c/span\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ec.2061T\u0026thinsp;\u0026gt;\u0026thinsp;G\u003c/span\u003e\u003c/p\u003e \u003cp\u003eOf the 14 families, one fulfilled Amsterdam criteria, four fulfilled Amsterdam II criteria, and six fulfilled Bethesda criteria (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). No additional family history data was available for two of the remaining cases. Five of the seven families identified at centres in Portugal originate from the Leiria District of Portugal, situated north of Lisbon. One additional family has origins in a nearby region.\u003c/p\u003e \u003cp\u003eThe variant was identified in 23 individuals (Table\u0026nbsp;1). The variant co-segregated with a LS-spectrum cancer in 15 heterozygotes with an average age of 52.2 (range: 34\u0026ndash;81) years at first diagnosis. The variant was also identified in an individual with a clinical diagnosis of constitutional mismatch repair deficiency (CMMRD) who carries a second \u003cem\u003eMSH6\u003c/em\u003e VUS \u003cem\u003ein trans\u003c/em\u003e with c.2061T\u0026thinsp;\u0026gt;\u0026thinsp;G (Table\u0026nbsp;1) [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. This variant was additionally present in two individuals with a personal history of at least one colorectal polyp (Table\u0026nbsp;1). Four individuals are, to our knowledge, unaffected to date.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eClinicopathological features\u003c/h2\u003e \u003cp\u003eThe most common diagnosis in heterozygous females (9/16) was EC with average age at diagnosis of 56.1 (range: 43\u0026ndash;82). The most common diagnosis in heterozygous males was CRC (4/6) with average age at diagnosis of 51.3 (range: 34\u0026ndash;69). Of the 15 tumours evaluated by IHC, six (40%) demonstrated loss of MSH6 expression. Loss or retention of MSH6 was not consistent across tumours in individuals with multiple LS-spectrum cancers (Table\u0026nbsp;1, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Two of the tumours that retained MSH6 were microsatellite stable (Table\u0026nbsp;1). Two tumours with loss of MSH6 had somatic genetic second hits. The proband of Family VIII demonstrated loss of MSH6 expression in non-cancerous tissue, consistent with the clinical diagnosis CMMRD [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eVariant Interpretation\u003c/h3\u003e\n\u003cp\u003eWe employed the Bayesian point system described in Tavtigian et al [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] to interpret our variant according to both the ClinGen Variant Curation Expert Panel specifications for the American College of Medical Genetics (ACMG) 2015 recommendations and the MMR-specific CanVIG-UK guidelines [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. \u003cem\u003eMSH6\u003c/em\u003e c.2061T\u0026thinsp;\u0026gt;\u0026thinsp;G is classified as a \u0026ldquo;Variant of Uncertain Significance\u0026rdquo; per the ACMG criteria but as \u0026ldquo;Likely Pathogenic\u0026rdquo; according to the CanVIG-UK guidelines (Table S2).\u003c/p\u003e\n\u003ch3\u003eHaplotype analysis\u003c/h3\u003e\n\u003cp\u003eA shared haplotype was identified in 15 heterozygous individuals from 9 families (Table\u0026nbsp;2). This conserved haplotype spans a maximum of 10.4 Mb and a minimum of 4.9 Mb. One additional affected heterozygote (Family V) carried some alleles in common with this haplotype, suggesting a possible distant connection to the other carrier families. The provided sample was confirmed to carry \u003cem\u003eMSH6\u003c/em\u003e c.2061T\u0026thinsp;\u0026gt;\u0026thinsp;G by Sanger sequencing, but no additional family V members were available for genotyping and haplotyping.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study evaluated the clinical impact of a novel Portuguese missense founder variant causing LS, \u003cem\u003eMSH6\u003c/em\u003e c.2061T\u0026thinsp;\u0026gt;\u0026thinsp;G, in 14 families. We identified a haplotype shared among nine families, suggesting a common ancestor. Based on the origins of several families, this variant likely originated in the Leiria District of Portugal.\u003c/p\u003e \u003cp\u003eWe first identified this variant in 2008 as a VUS in a proband (Family I) with a convincing personal history of LS-spectrum tumours; however, not all tumours demonstrated loss of MSH6 (Table\u0026nbsp;1, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The variant has since been identified in thirteen additional families, the majority of which (11/14) meet at least one of the clinical criteria for LS (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Despite this, it has been difficult to clarify this variant\u0026rsquo;s pathogenicity. Like approximately 70% of MMR VUS, the variant results from a missense mutation, which at its foundation makes interpretation more difficult [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Missense variants in the MMR genes are statistically more likely to demonstrate unexpected staining patterns than truncating variants, possibly due to the presence of non-functional protein that cannot be differentiated from functional protein through IHC [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAdding to the challenge, the degree of MSI is lower in \u003cem\u003eMSH6\u003c/em\u003e-associated CRCs and the most common cancer in female heterozygotes (EC) is more likely to have inconsistent MMR/MSI results when compared to the other MMR genes [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. This begs the question of whether MSI/IHC of tumour tissue is a sufficient and effective screening method for the less-penetrant LS genes. This may also explain why \u003cem\u003eMSH6\u003c/em\u003e has the highest proportion of VUS among the MMR genes [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Nevertheless, MSI/IHC evidence is still considered a hallmark feature of tumours in all LS patients, and MSI/IHC results are considered important evidence of pathogenicity in both ACMG and CanVIG-UK criteria [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eWe present the third known Portuguese founder variant causing LS, and the first in \u003cem\u003eMSH6\u003c/em\u003e. This variant is characterized by classic \u003cem\u003eMSH6\u003c/em\u003e-associated LS, including incomplete penetrance, an often-attenuated phenotype, and higher risk for EC than CRC in females. As observed for other missense variants in MMR genes and studies of \u003cem\u003eMSH6\u003c/em\u003e-associated LS specifically, this variant does not reliably lead to MSI or loss of MSH6 expression by IHC in tumours [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, Figure S2). The emphasis of MMR proficiency as evidence against pathogenicity under the ACMG criteria is therefore in direct opposition to substantial prior research. It is plausible that similar missense variants have gone undiagnosed in several families who would benefit from recommended LS screening regiments. In fact, some are calling for the division of LS into gene-specific syndromes to avoid this very issue [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. We hope the observations from our study help to facilitate the identification of such families and prompts a broader discussion of the limitations of IHC and MSI as a method for stratifying the risk of LS.\u003c/p\u003e \u003cp\u003eThis study has limitations, including our inability to include allelic data (PM3) despite the variant being present in an individual with a clinical diagnosis of CMMRD, due to its being in \u003cem\u003etrans\u003c/em\u003e with a second \u003cem\u003eMSH6\u003c/em\u003e VUS. There are also currently no functional data available to support pathogenicity of \u003cem\u003eMSH6\u003c/em\u003e c.2061T\u0026thinsp;\u0026gt;\u0026thinsp;G. Nevertheless, the variant reaches a pathogenic score using CanVIG-UK criteria.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis study was supported by funding from the McGill University Health Centre Foundation and its generous donors.\u003c/p\u003e\u003ch2\u003eAuthor contributions\u003c/h2\u003e \u003cp\u003eKM prepared the manuscript, tables, and figures. LW performed variant interpretation and reviewed the manuscript. CD and NH designed and performed haplotype analyses and also contributed and reviewed the manuscript. JM, JLE, KS, SM, MRT, CA, IF, LGR, OCT, KM, and AC provided cases, data and samples for haplotype analysis where possible, and reviewed the manuscript. GC performed molecular characterization for Family I and reviewed the manuscript. VM provided expertise on IHC images and reviewed the manuscript. WDF designed study and oversaw data collection and manuscript preparation.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThe authors wish to thank Ana Bonilha for her help with translations and Brianna Lemieux for preliminary data collection. We are also thankful to Myriad Genetics and Ambry Genetics for contributing cases. We would like to acknowledge the contributions of Dr. Aaron Pollet, Ms. Kara Semotiuk, and Dr. Jordan Lerner-Ellis (Mount Sinai Hospital, Toronto) in providing IHC images, molecular data and additional family history information. We thank Dr. Ricardo Fonseca (Instituto Portugu\u0026ecirc;s de Oncologia de Lisboa, Portugal) in providing IHC images. We thank John Lee (Cedars-Sinai Medical Center, Los Angeles) for connecting us with cases and their healthcare providers.\u003c/p\u003e\u003ch2\u003eData availability statement\u003c/h2\u003e \u003cp\u003eData are available from the principal investigator/corresponding author (WDF) upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eGallon, R., et al., \u003cem\u003eHow Should We Test for Lynch Syndrome? A Review of Current Guidelines and Future Strategies.\u003c/em\u003e Cancers (Basel), 2021. \u003cstrong\u003e13\u003c/strong\u003e(3).\u003c/li\u003e\n \u003cli\u003ePeltomaki, P., \u003cem\u003eUpdate on Lynch syndrome genomics.\u003c/em\u003e Fam Cancer, 2016. \u003cstrong\u003e15\u003c/strong\u003e(3): p. 385-93.\u003c/li\u003e\n \u003cli\u003eDominguez-Valentin, M., et al., \u003cem\u003eCancer risks by gene, age, and gender in 6350 carriers of pathogenic mismatch repair variants: findings from the Prospective Lynch Syndrome Database.\u003c/em\u003e Genet Med, 2020. \u003cstrong\u003e22\u003c/strong\u003e(1): p. 15-25.\u003c/li\u003e\n \u003cli\u003eElze, L., et al., \u003cem\u003eMicrosatellite instability in noncolorectal and nonendometrial malignancies in patients with Lynch syndrome.\u003c/em\u003e J Natl Cancer Inst, 2023. \u003cstrong\u003e115\u003c/strong\u003e(7): p. 853-860.\u003c/li\u003e\n \u003cli\u003eChen, W., et al., \u003cem\u003eUnexpected expression of mismatch repair protein is more commonly seen with pathogenic missense than with other mutations in Lynch syndrome.\u003c/em\u003e Hum Pathol, 2020. \u003cstrong\u003e103\u003c/strong\u003e: p. 34-41.\u003c/li\u003e\n \u003cli\u003eCastellsague, E., et al., \u003cem\u003eCharacterization of a novel founder MSH6 mutation causing Lynch syndrome in the French Canadian population.\u003c/em\u003e Clin Genet, 2015. \u003cstrong\u003e87\u003c/strong\u003e(6): p. 536-42.\u003c/li\u003e\n \u003cli\u003eRaskin, L., et al., \u003cem\u003eCharacterization of two Ashkenazi Jewish founder mutations in MSH6 gene causing Lynch syndrome.\u003c/em\u003e Clin Genet, 2011. \u003cstrong\u003e79\u003c/strong\u003e(6): p. 512-22.\u003c/li\u003e\n \u003cli\u003ePinto, C., et al., \u003cem\u003eCo-occurrence of nonsense mutations in MSH6 and MSH2 in Lynch syndrome families evidencing that not all truncating mutations are equal.\u003c/em\u003e J Hum Genet, 2016. \u003cstrong\u003e61\u003c/strong\u003e(2): p. 151-6.\u003c/li\u003e\n \u003cli\u003ePinheiro, M., et al., \u003cem\u003eA novel exonic rearrangement affecting MLH1 and the contiguous LRRFIP2 is a founder mutation in Portuguese Lynch syndrome families.\u003c/em\u003e Genet Med, 2011. \u003cstrong\u003e13\u003c/strong\u003e(10): p. 895-902.\u003c/li\u003e\n \u003cli\u003ePinheiro, M., et al., \u003cem\u003eThe nonsense mutation MSH2 c.2152C\u0026gt;T shows a founder effect in Portuguese Lynch syndrome families.\u003c/em\u003e Genes Chromosomes Cancer, 2019. \u003cstrong\u003e58\u003c/strong\u003e(9): p. 657-664.\u003c/li\u003e\n \u003cli\u003ePinheiro, M., et al., \u003cem\u003eThe MSH2 c.388_389del mutation shows a founder effect in Portuguese Lynch syndrome families.\u003c/em\u003e Clin Genet, 2013. \u003cstrong\u003e84\u003c/strong\u003e(3): p. 244-50.\u003c/li\u003e\n \u003cli\u003eGuerrini-Rousseau, L., et al., \u003cem\u003eConstitutional mismatch repair deficiency-associated brain tumors: report from the European C4CMMRD consortium.\u003c/em\u003e Neurooncol Adv, 2019. \u003cstrong\u003e1\u003c/strong\u003e(1): p. vdz033.\u003c/li\u003e\n \u003cli\u003eTavtigian, S.V., et al., \u003cem\u003eFitting a naturally scaled point system to the ACMG/AMP variant classification guidelines.\u003c/em\u003e Hum Mutat, 2020. \u003cstrong\u003e41\u003c/strong\u003e(10): p. 1734-1737.\u003c/li\u003e\n \u003cli\u003eRichards, S., et al., \u003cem\u003eStandards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology.\u003c/em\u003e Genet Med, 2015. \u003cstrong\u003e17\u003c/strong\u003e(5): p. 405-24.\u003c/li\u003e\n \u003cli\u003eCanVIG-UK. \u003cem\u003eMismatch Repair Genes: CanVIG-UK Gene-Specific Guidance\u003c/em\u003e. Available from: https://www.cangene-canvaruk.org/gene-specific-recommendations.\u003c/li\u003e\n \u003cli\u003eThompson, B.A., et al., \u003cem\u003eApplication of a 5-tiered scheme for standardized classification of 2,360 unique mismatch repair gene variants in the InSiGHT locus-specific database.\u003c/em\u003e Nat Genet, 2014. \u003cstrong\u003e46\u003c/strong\u003e(2): p. 107-115.\u003c/li\u003e\n \u003cli\u003eWang, C., et al., \u003cem\u003eEvaluation of microsatellite instability patterns in mismatch repair deficiency: a retrospective analysis of 285 endometrial cancers.\u003c/em\u003e Front Immunol, 2025. \u003cstrong\u003e16\u003c/strong\u003e: p. 1628979.\u003c/li\u003e\n \u003cli\u003eHelderman, N.C., et al., \u003cem\u003eLower Degree of Microsatellite Instability in Colorectal Carcinomas From MSH6-Associated Lynch Syndrome Patients.\u003c/em\u003e Mod Pathol, 2025. \u003cstrong\u003e38\u003c/strong\u003e(7): p. 100757.\u003c/li\u003e\n \u003cli\u003eFrederiksen, J.H., et al., \u003cem\u003eClassification of MSH6 Variants of Uncertain Significance Using Functional Assays.\u003c/em\u003e Int J Mol Sci, 2021. \u003cstrong\u003e22\u003c/strong\u003e(16).\u003c/li\u003e\n \u003cli\u003eMoller, P., et al., \u003cem\u003eDominantly inherited micro-satellite instable cancer - the four Lynch syndromes - an EHTG, PLSD position statement.\u003c/em\u003e Hered Cancer Clin Pract, 2023. \u003cstrong\u003e21\u003c/strong\u003e(1): p. 19.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 and 2 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"european-journal-of-human-genetics","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"ejhg","sideBox":"Learn more about [European Journal of Human Genetics](http://www.nature.com/ejhg/)","snPcode":"41431","submissionUrl":"https://mts-ejhg.nature.com/cgi-bin/main.plex","title":"European Journal of Human Genetics","twitterHandle":"@ejhg_journal","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Founder variant, Lynch syndrome, variant of uncertain significance, immunohistochemistry, haplotype, endometrial cancer","lastPublishedDoi":"10.21203/rs.3.rs-9172566/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9172566/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn certain populations, founder pathogenic variants are a common cause of Lynch syndrome. Here, we report the identification of a novel founder variant, NM_000179.3 (MSH6):c.2061T\u0026thinsp;\u0026gt;\u0026thinsp;G (p.Cys687Trp). Our study examined 14 probands and 18 additional family members who carry this variant. With one exception, haplotype data are consistent with a single origin for all heterozygotes. We investigated the clinicopathological consequences of this variant and found that, as previously reported for other \u003cem\u003eMSH6\u003c/em\u003e pathogenic variants a) presence of the founder pathogenic variant is not reliably associated with loss of MSH6 expression or microsatellite instability and b) endometrial cancer is the most common associated phenotype. Based on our findings, we recommend reclassifying \u003cem\u003eMSH6\u003c/em\u003e c.2061T\u0026thinsp;\u0026gt;\u0026thinsp;G from missense variant of unknown significance to likely pathogenic according to CanVIG-UK guidelines.\u003c/p\u003e","manuscriptTitle":"Characterisation of a Portuguese origin founder missense variant in MSH6","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-21 07:13:36","doi":"10.21203/rs.3.rs-9172566/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-05-05T12:48:47+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-04-28T09:27:35+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-04-24T07:13:30+00:00","index":3,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-04-21T10:44:57+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-04-20T05:57:19+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2026-04-14T11:02:37+00:00","index":"","fulltext":""},{"type":"submitted","content":"European Journal of Human Genetics","date":"2026-04-06T19:08:58+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-24T10:12:28+00:00","index":"","fulltext":""},{"type":"checksFailed","content":"","date":"2026-03-23T14:44:40+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-19T19:26:33+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"european-journal-of-human-genetics","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"ejhg","sideBox":"Learn more about [European Journal of Human Genetics](http://www.nature.com/ejhg/)","snPcode":"41431","submissionUrl":"https://mts-ejhg.nature.com/cgi-bin/main.plex","title":"European Journal of Human Genetics","twitterHandle":"@ejhg_journal","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"1164505d-f249-4adf-b787-fc7ed611d20d","owner":[],"postedDate":"April 21st, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-05-05T12:48:47+00:00","index":2,"fulltext":"This content is not available."}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":65806898,"name":"Biological sciences/Cancer/Cancer genetics"},{"id":65806899,"name":"Health sciences/Diseases/Cancer/Gastrointestinal cancer/Colorectal cancer"},{"id":65806900,"name":"Biological sciences/Cancer/Gynaecological cancer/Endometrial cancer"},{"id":65806901,"name":"Biological sciences/Genetics/Cancer genetics"},{"id":65806902,"name":"Biological sciences/Cancer/Cancer screening"}],"tags":[],"updatedAt":"2026-04-21T07:13:36+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-21 07:13:36","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9172566","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9172566","identity":"rs-9172566","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}