Coexisting germline variants of MLH1 and MSH6 in a patient with Lynch syndrome, endometrial cancer, and ovarian cancer

Coexisting germline variants of MLH1 and MSH6 in a patient with Lynch syndrome, endometrial cancer, and ovarian cancer | 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 Research Article Coexisting germline variants of MLH1 and MSH6 in a patient with Lynch syndrome, endometrial cancer, and ovarian cancer Sho Umegaki, Masanobu Takahashi, Junko Hasegawa-Minato, Maako Kawamura, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3926364/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Lynch syndrome is an autosomal dominant disorder caused by a heterozygous pathogenic germline variant in mismatch repair (MMR) genes, including MLH1 , MSH2 , MSH6 , PMS2 , and EPCAM . Lynch syndrome often causes a familial cluster of patients with malignant tumors. We describe a 37-year-old woman who presented with endometrioid carcinoma in the ovary and endometrium associated with Lynch syndrome. She carried two germline pathogenic variants, a recurrently reported MLH1 c.2250C > G (p. Tyr750*) and previously unreported MSH6 c.2385del (p. Ile795Metfs*15). The tumor cells showed microsatellite instability. Immunohistochemistry showed decreased MLH1 expression, loss of PMS2 expression, retained MSH2 expression, and loss of MSH6 expression, indicating that both variants impair protein stability, causing MMR deficiency. Whether these variants were inherited from her parents or occurred de novo is unknown. The tumor cells had somatic variants BRCA1 c.1016del and BRCA2 c.36dupT, which might be due to secondary mutation by MMR deficiency. The use of pembrolizumab, an immune checkpoint inhibitor, resulted in a durable partial response of metastatic lung tumors. This case highlights the rare possibility of multiple germline variants in MMR genes in individuals with Lynch syndrome. Lynch syndrome MLH1 MSH6 coexisting germline variants Figures Figure 1 Figure 2 Introduction Lynch syndrome is a cancer predisposition syndrome due to a heterozygous germline pathogenic variant of the mismatch repair (MMR) genes, which develops various types of malignant tumors, including colorectal and endometrial cancer [ 1 , 2 ]. Because a single pathogenic variant of a single MMR gene causes this disease and is inherited in an autosomal dominant manner, multiple germline variants of MMR genes are rarely detected in individuals with Lynch syndrome. We report a case of a patient with Lynch syndrome who presented with ovarian and endometrial cancers and carried two independent germline variants of MLH1 and MSH6 . Case presentation A 37-year-old woman presented with lower abdominal pain and visited a local hospital. Computed tomography (CT) scan showed a right ovarian tumor and thickened endometrium (Fig. 1 a-c). Subsequently, she underwent right ovarian tumor resection with intraperitoneal collapse, followed by resection of the uterus, left ovary, and omentum at our hospital. Peritoneal dissemination and right ovarian and endometrial tumors were pathologically diagnosed as endometrioid carcinoma. Ovarian and endometrial cancers were considered as primary lesions although we could not exclude the possibility that either ovary or endometrial tumor was a metastatic lesion. After surgery, the patient received systemic chemotherapy with carboplatin and paclitaxel. Somatic variants of BRCA1 c.1016del and BRCA2 c.36dupT were detected in the endometrial tumor tissue using myChoice testing (Myriad Genetics, Salt Lake City, UT, USA), but germline variants of BRCA1/2 were not observed using BRACAnalysis (Myriad Genetics). Microsatellite instability (MSI) testing showed MSI-high, and immunostaining of the tumor tissues showed decreased MLH1 expression, loss of PMS2 expression, retained MSH2 expression, and loss of MSH6 expression (Fig. 2 a). Her mother had gastric cancer in her thirties and died of ovarian cancer at the age of 54 years. In addition, her maternal grandfather had a history of gastric and colorectal cancers at an unknown age, and her maternal grandmother had colorectal cancer at an unknown age (Fig. 2 b). The MMR gene screening test with next-generation sequencing and multiplex ligation-dependent probe amplification analysis (FALCO Biosystems Ltd., Kyoto, Japan) showed two germline variants of MLH1 c.2250 C > G (p. Tyr750*) and MSH6 c.2385del (p. Ile795Metfs*15). Consequently, she was diagnosed with Lynch syndrome. She had a complete response to chemotherapy and continued to be treated with bevacizumab plus olaparib, a PARP inhibitor, as maintenance therapy for metastatic ovarian cancer. However, she complained of headache and vertigo 8 months after the maintenance therapy started. CT scan showed a massive brain tumor in her right parietal lobe (Fig. 1 d). We performed brain tumor resection considering the possibility of its association with Lynch syndrome. Thus, the histological type was also endometrioid carcinoma diagnosed as brain metastasis. Immunostaining of this tissue also revealed decreased MLH1, negative PMS2, positive MSH2, and negative MSH6 expressions, which was the same pattern as that of the primary tumor tissue. She received whole-brain radiation therapy and continued treatment with olaparib, but she developed multiple lung metastases after 2 months (Fig. 1 e, f). Soon after tumor relapse, she started receiving pembrolizumab and lenvatinib for metastatic endometrial cancer, which led to a significant shrinkage of multiple lung metastases, the best response with partial response without any other new lesions for 1 year and 10 months. We intend to perform a genetic analysis for her sister and her sister’s daughter in the future. Discussion Lynch syndrome is caused by a germline heterozygous pathogenic variant of a single MMR gene [ 1 , 2 ]. More than one pathological germline variant of the MMR gene is not usually detected in individuals with Lynch syndrome. However, our patient had two germline variants of MLH1 c.2250C > G and MSH6 c.2385del. These variants were considered pathogenic based on the immunostaining of MMR proteins in tumor tissues with decreased MLH1 expression and loss of MSH6 expression (Fig. 2 a), and the MSI-high. These variants of MLH1 and MSH6 could be transmitted as follows: (1) each variant was inherited from one parent each; (2) one variant was inherited from one parent and the other occurred de novo in the patient; and (3) both variants were inherited from one parent. Considering that the occurrence of de novo germline mutations in MMR genes is rare [ 3 ] and that there are multiple family histories of gastrointestinal and gynecological cancers in the maternal line (Fig. 2 b), we considered that at least one pathogenic variant of MLH1 or MSH6 was inherited from her mother. However, we could not perform genetic testing for her mother because she had already died. Her father never had cancer until the age of 68 years, and his parents, brother, and sister had no malignant tumors, indicating that the variants were not inherited from the paternal line. Thus, one or two of the variants were likely to have been inherited from her mother, although we cannot confirm how two germline variants of MMR genes occurred. Kast et al. reported a patient with endometrial cancer at the age of 46 years who carried both germline variants in BRCA1 (p.Arg71Serfs*21) and MSH6 (p. Ile172fs*10), which are both considered frameshift mutations and pathogenic [ 4 ]. Pinto et al. reported a case series of the co-occurrence of MSH6 (p.Gln344*) and MSH2 (p. Arg929*) nonsense variants in eight probands with Lynch syndrome [ 5 ]. They proposed that this MSH2 C-terminal nonsense variant expected to encode a protein lacking six amino acid residues is a variant of unknown significance because five tumors of the eight probands retained MSH2 expression [ 5 ]. Ferrer-Avargues et al. recently showed that 5 of 84 families (6%) with Lynch syndrome had multiple germline variants of MMR genes and other different cancer-disposing genes, such as MLH1-BRCA2-NBN , MLH1-BRCA1 , MSH2-ATM , MSH6-NF1 , and MLH1-FANCA [ 6 ]. Each variant of MMR genes was considered pathogenic and had high penetrance, whereas the penetrance of cancer-disposing genes other than BRCA1 was considered moderate [ 6 ]. The detection of multiple germline pathogenic variants of different MMR genes in a single individual, similar to our case, is quite rare. Individuals with multiple germline pathogenic variants are more frequently diagnosed because of the increasingly widespread use of genetic testing, including multiple germline gene panels, tumor-only panels, tumor and germline panels, and whole genome sequencing. The MLH1 protein consists of 756 amino acids and binds to PMS2 in the C-terminal region to form a MutLα heterodimer, which exhibits DNA cleavage activity for DNA MMR [ 2 ]. MLH1 c.2250C > G (p. Tyr750*) is a C-terminal nonsense variant predicted to defect the last seven amino acid residues of the C-terminal domain and is a variant of unknown significance based on the InSiGHT database with recurrently reported data [ 7 ]. For example, MLH1 c.2250C > G was detected in an individual with suspected Lynch syndrome who fulfilled the Amsterdam criteria [ 8 ]; however, it was not determined to be pathogenic because no tumor was available to assess MSI status. This germline variant was also detected in a Chinese family that did not fulfill the Amsterdam criteria but included three individuals with onset of colorectal cancer in their 40s [ 9 ]. In contrast, Kosinski et al. showed in their in vitro analysis that MLH1 c.2250C > G retained MLH1 protein expression but reduced dimerization with PMS2, leading to impaired MMR activity [ 10 ]. Another study also supported this variant’s inability to stabilize the PMS2 protein [ 11 ]. In our case, immunohistochemical analysis showed decreased MLH1 expression but not complete loss of expression, indicating that this variant, at least in part, impairs MLH1 protein stability in tumor cells. A previous study reported that 32% (14/44) of MLH1 variant carriers showed a weakly positive pattern of MLH1 protein expression in tumor tissues [ 12 ]. Consistently, our previous functional analysis demonstrated that 42 of 100 MLH1 variants partially impair protein stability (25–75% relative expression compared with wild type) and 24 of 42 variants impair MMR activity ( G may reduce the dimerization ability of PMS2 in tumor cells, causing MMR deficiency. The MSH6 protein consists of 1360 amino acids and forms a MutSα heterodimer with MSH2 to recognize DNA mismatch sites [ 2 ]. MSH6 c.2385del (p. Ile795Metfs*15) is a frameshift variant in the MutS core domain region and considered a pathological variant, although this variant has not been reported in the InSiGHT database or elsewhere. The onset of endometrial and ovarian cancers in our patient conforms to the consensus that the incidence of endometrial cancer is higher in women with the germline variant of MSH6 [ 14 , 15 ]. MMR deficiency results in the accumulation of somatic mutations, including point mutations and insertion/deletion mutations, at repetitive nucleotide sequences termed microsatellite regions, leading to the onset of Lynch syndrome-associated tumors [ 1 , 16 ]. We also detected somatic homologous recombination gene variants, BRCA1 c.1016del and BRCA2 c.36dupT, in tumor tissues. We speculate that these base insertion/deletion mutations, located in repetitive sequences, are secondary mutations caused by MMR deficiency. The detection of BRCA1 and BRCA2 variants concurs with the finding that approximately 50% of MSI-high colorectal cancers have BRCA2 somatic mutations, and approximately one-fifth have BRCA1 somatic mutations [ 17 ]. Recent advances in genetic testing by next-generation sequencing and molecularly targeted drugs have enabled personalized treatment of tumors with disrupted DNA repair mechanisms, such as MMR [ 18 ] and homologous recombination repair [ 19 ]. MMR-deficient tumors accumulate genetic mutations and subsequent generation of highly immunogenic tumor antigens, which explains the benefit of immune checkpoint inhibitors to some patients with MMR-deficient tumors [ 18 ]. Consistently, our patient showed a favorable response to pembrolizumab. Although the tumor carried pathogenic somatic BRCA1 and BRCA2 variants, she did not benefit from olaparib but had a transient response to platinum-based chemotherapy. Sokol et al. analyzed > 200,000 patients with cancer and reported that homologous recombination deficiency and MSI-high phenotype were generally mutually exclusive across cancer types but may rarely co-occur, particularly in prostate cancer [ 20 ]. These variants were generally monoallelic and not associated with homologous recombination deficiency, indicating that most BRCA1/2 variants coexisting with MSI are likely bystander events that may not lead to sensitivity to PARP inhibitors [ 20 ]. Conclusion We reported a case of a patient with Lynch syndrome who carried coexisting germline variants of MMR genes, a recurrently reported MLH1 c.2250 C > G (p. Tyr750*) and a previously unreported MSH6 c.2385del (p. Ile795Metfs*15), as well as somatic pathogenic variants of BRCA1 and BRCA2 in the tumor tissues. The patient benefited from an immune checkpoint inhibitor but not a PARP inhibitor. Clinicians should pay attention to the rare possibility of multiple germline variants of MMR genes for the genetic analysis of patients and their family members. Declarations Acknowledgment We thank the patient and her families and all the clinicians who allowed us to publish this case report. We thank Enago (www.enago.jp) for the English language review. This study was not supported by funding. We report conflicts of interest as follows: M. Takahashi has received research funding from Ono Pharmaceutical, Chugai Pharmaceutical, and MSD, and lecture fees from Daiichi Sankyo, Ono Pharmaceutical, Bristol Meyers Squibb, and Taiho Pharmaceutical Company. C. Ishioka has received research funding from Chugai Pharmaceutical, Taiho Pharmaceutical, Asahi‐Kasei Pharma, Ono Pharmaceutical, Daiichi Sankyo, Takeda Pharmaceutical, and Hitachi Ltd, and lecture fees from Chugai Pharmaceutical and Daiichi Sankyo. Authors’ contributions SU collected the data, contributed to the conception of the work, and draft the manuscript. MT saw the patient and got consent form the patient for the genetic analysis and this work, collected the data, contributed to the conception of the work, and draft and revised the manuscript. JHM, HT, MS were involved in surgery and drug therapy, and revised the manuscript. MK and YA performed genetic counselling and revised the manuscript. ST, KT, KO, HI, KS1 interpreted data and revised the manuscript. FF performed IHC and interpreted data. CI interpreted data, supervised the work, and revised the manuscript. All authors reviewed the manuscript. Ethics approval and consent to participate This work was approved by the IRB of the Ethics Committee Tohoku University Hospital. The patient has consented to the publication of this work. Competing interests This study was not supported by funding. We report conflicts of interest as follows: M. Takahashi has received research funding from Ono Pharmaceutical, Chugai Pharmaceutical, and MSD, and lecture fees from Daiichi Sankyo, Ono Pharmaceutical, Bristol Meyers Squibb, and Taiho Pharmaceutical Company. C. Ishioka has received research funding from Chugai Pharmaceutical, Taiho Pharmaceutical, Asahi‐Kasei Pharma, Ono Pharmaceutical, Daiichi Sankyo, Takeda Pharmaceutical, and Hitachi Ltd, and lecture fees from Chugai Pharmaceutical and Daiichi Sankyo. References Lynch HT, Snyder CL, Shaw TG, Heinen CD, Hitchins MP (2015) Milestones of Lynch syndrome: 1895–2015. Nat Rev Cancer 15:181–194. https://doi.org/10.1038/nrc3878 Tamura K, Kaneda M, Futagawa M et al (2019) Genetic and genomic basis of the mismatch repair system involved in Lynch syndrome. Int J Clin Oncol 24:999–1011. https://doi.org/10.1007/s10147-019-01494-y Win AK, Jenkins MA, Buchanan DD et al (2011) Determining the frequency of de novo germline mutations in DNA mismatch repair genes. J Med Genet 48:530–534. https://doi.org/10.1136/jmedgenet-2011-100082 Kast K, Neuhann TM, Görgens H et al (2012) Germline truncating-mutations in BRCA1 and MSH6 in a patient with early onset endometrial cancer. BMC Cancer 12:1–5. https://doi.org/10.1186/1471-2407-12-531 Pinto C, Pinheiro M, Peixoto A et al (2016) Co-occurrence of nonsense mutations in MSH6 and MSH2 in Lynch syndrome families evidencing that not all truncating mutations are equal. J Hum Genet 61:151–156. https://doi.org/10.1038/jhg.2015.124 Ferrer-Avargues R, Castillejo MI, Dámaso E et al (2021) Co-occurrence of germline pathogenic variants for different hereditary cancer syndromes in patients with Lynch syndrome. Cancer Commun 41:218–228. https://doi.org/10.1002/cac2.12134 Thompson BA, Spurdle AB, Plazzer JP et al (2014) 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 46:107–115. https://doi.org/10.1038/ng.2854 Mueller J, Gazzoli I, Bandipalliam P et al (2009) Comprehensive molecular analysis of mismatch repair gene defects in suspected Lynch syndrome (hereditary nonpolyposis colorectal cancer) cases. Cancer Res 69:7053–7061. https://doi.org/10.1158/0008-5472.CAN-09-0358 Yuan Y, Huang YQ, Cai SR et al (2004) Genetic characterization of Chinese hereditary non-polyposis colorectal cancer by DHPLC and multiplex PCR. Jpn J Clin Oncol 34:660–666. https://doi.org/10.1093/jjco/hyh121 Kosinski J, Hinrichsen I, Bujnicki JM et al (2010) Identification of Lynch syndrome mutations in the MLH1-PMS2 interface that disturb dimerization and mismatch repair. Hum Mutat 31:975–982. https://doi.org/10.1002/humu.21301 Mohd AB, Palama B, Nelson SE et al (2006) Truncation of the C-terminus of human MLH1 blocks intracellular stabilization of PMS2 and disrupts DNA mismatch repair. DNA Repair (Amst) 5:347–361. https://doi.org/10.1016/j.dnarep.2005.11.001 Mangold E, Pagenstecher C, Friedl W et al (2005) Tumours from MSH2 mutation carriers show loss of MSH2 expression but many tumours from MLH1 mutation carriers exhibit weak positive MLH1 staining. J Pathol 207:385–395. https://doi.org/10.1002/path.1858 Takahashi M, Shimodaira H, Andreutti-Zaugg C et al (2007) Functional analysis of human MLH1 variants using yeast and in vitro mismatch repair assays. Cancer Res 67:4595–4604. https://doi.org/10.1158/0008-5472.CAN-06-3509 Baglietto L, Lindor NM, Dowty JG et al (2010) Risks of Lynch syndrome cancers for MSH6 mutation carriers. J Natl Cancer Inst 102:193–201. https://doi.org/10.1093/jnci/djp473 Goodfellow PJ, Buttin BM, Herzog TJ et al (2003) Prevalence of defective DNA mismatch repair and MSH6 mutation in an unselected series of endometrial cancers. Proc Natl Acad Sci U S A 100:5908–5913. https://doi.org/10.1073/pnas.1030231100 Abildgaard AB, Nielsen SV, Bernstein I et al (2023) Lynch syndrome, molecular mechanisms and variant classification. Br J Cancer 128:726–734. https://doi.org/10.1038/s41416-022-02059-z Deihimi S, Lev A, Slifker M et al (2017) BRCA2, EGFR, and NTRK mutations in mismatch repair-deficient colorectal cancers with MSH2 or MLH1 mutations. Oncotarget 8:39945–39962. https://doi.org/10.18632/oncotarget.18098 Le DT, Durham JN, Smith KN et al (2017) Mismatch repair deficiency predicts response of solid tumors to PD-1 blockade. Science 357:409–413. https://doi.org/10.1126/science.aan6733 Lord CJ, Ashworth A (2017) PARP inhibitors: Synthetic lethality in the clinic. Science 355:1152–1158. https://doi.org/10.1126/science.aam7344 Sokol ES, Jin DX, Fine A et al (2022) PARP Inhibitor Insensitivity to BRCA1/2 Monoallelic Mutations in Microsatellite Instability-High Cancers. JCO Precis Oncol 6:e2100531. https://doi.org/10.1200/po.21.00531 Additional Declarations Competing interest reported. M. Takahashi has received research funding from Ono Pharmaceutical, Chugai Pharmaceutical, and MSD, and lecture fees from Daiichi Sankyo, Ono Pharmaceutical, Bristol Meyers Squibb, and Taiho Pharmaceutical Company. C. Ishioka has received research funding from Chugai Pharmaceutical, Taiho Pharmaceutical, Asahi‐Kasei Pharma, Ono Pharmaceutical, Daiichi Sankyo, Takeda Pharmaceutical, and Hitachi Ltd, and lecture fees from Chugai Pharmaceutical and Daiichi Sankyo. Cite Share Download PDF Status: Posted Version 1 posted 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3926364","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":271177710,"identity":"ca4881e4-eafc-47d5-925f-ed0ea3b5cab0","order_by":0,"name":"Sho Umegaki","email":"","orcid":"","institution":"Tohoku University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Sho","middleName":"","lastName":"Umegaki","suffix":""},{"id":271177711,"identity":"6ae0f594-4d09-42c2-830b-b980505d7329","order_by":1,"name":"Masanobu 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05:29:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3926364/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3926364/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":50815220,"identity":"89dbf565-cdbc-4ce0-9ab0-4ba0840536c3","added_by":"auto","created_at":"2024-02-07 19:41:20","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":324918,"visible":true,"origin":"","legend":"\u003cp\u003eComputed tomography (CT) findings\u003c/p\u003e\n\u003cp\u003eCT scan shows a massive right ovarian tumor (a), thickened endometrium in axial view (b) and coronal view (c), brain metastasis (d), and multiple lung metastases (e, f).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-3926364/v1/f8ef1c3d64c81d6326630164.png"},{"id":50815222,"identity":"c28d54db-12a2-48a9-a0ac-b6797a4a2934","added_by":"auto","created_at":"2024-02-07 19:41:20","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":560139,"visible":true,"origin":"","legend":"\u003cp\u003eImmunohistochemistry of mismatch repair proteins in the endometrial cancer tissues and the family tree of the proband.\u003c/p\u003e\n\u003cp\u003e(a) Immunohistochemistry of mismatch repair proteins of the endometrial cancer shows decreased MLH1, negative PMS2, positive MSH2, and negative MSH6 expressions.\u003c/p\u003e\n\u003cp\u003e(b) The family history included multiple cancers within maternal relatives, including gastric and ovarian cancers from the patient’s mother, gastric and colorectal cancers from her maternal grandfather, and ovarian cancer from her maternal grandmother. Abbreviations: AO, age of onset.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-3926364/v1/b1e3c8b7d69b4c199a386bd4.png"},{"id":55595227,"identity":"df599013-8f64-4afc-8936-6c60dfbfb276","added_by":"auto","created_at":"2024-04-30 10:25:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1191749,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3926364/v1/21a4e0a0-be84-46ac-a23b-c2f70800da1d.pdf"}],"financialInterests":"Competing interest reported. M. Takahashi has received research funding from Ono Pharmaceutical, Chugai Pharmaceutical, and MSD, and lecture fees from Daiichi Sankyo, Ono Pharmaceutical, Bristol Meyers Squibb, and Taiho Pharmaceutical Company. C. Ishioka has received research funding from Chugai Pharmaceutical, Taiho Pharmaceutical, Asahi‐Kasei Pharma, Ono Pharmaceutical, Daiichi Sankyo, Takeda Pharmaceutical, and Hitachi Ltd, and lecture fees from Chugai Pharmaceutical and Daiichi Sankyo.","formattedTitle":"Coexisting germline variants of MLH1 and MSH6 in a patient with Lynch syndrome, endometrial cancer, and ovarian cancer","fulltext":[{"header":"Introduction","content":"\u003cp\u003eLynch syndrome is a cancer predisposition syndrome due to a heterozygous germline pathogenic variant of the mismatch repair (MMR) genes, which develops various types of malignant tumors, including colorectal and endometrial cancer [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Because a single pathogenic variant of a single MMR gene causes this disease and is inherited in an autosomal dominant manner, multiple germline variants of MMR genes are rarely detected in individuals with Lynch syndrome. We report a case of a patient with Lynch syndrome who presented with ovarian and endometrial cancers and carried two independent germline variants of \u003cem\u003eMLH1\u003c/em\u003e and \u003cem\u003eMSH6\u003c/em\u003e.\u003c/p\u003e"},{"header":"Case presentation","content":"\u003cp\u003eA 37-year-old woman presented with lower abdominal pain and visited a local hospital. Computed tomography (CT) scan showed a right ovarian tumor and thickened endometrium (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea-c). Subsequently, she underwent right ovarian tumor resection with intraperitoneal collapse, followed by resection of the uterus, left ovary, and omentum at our hospital. Peritoneal dissemination and right ovarian and endometrial tumors were pathologically diagnosed as endometrioid carcinoma. Ovarian and endometrial cancers were considered as primary lesions although we could not exclude the possibility that either ovary or endometrial tumor was a metastatic lesion. After surgery, the patient received systemic chemotherapy with carboplatin and paclitaxel. Somatic variants of \u003cem\u003eBRCA1\u003c/em\u003e c.1016del and \u003cem\u003eBRCA2\u003c/em\u003e c.36dupT were detected in the endometrial tumor tissue using myChoice testing (Myriad Genetics, Salt Lake City, UT, USA), but germline variants of \u003cem\u003eBRCA1/2\u003c/em\u003e were not observed using BRACAnalysis (Myriad Genetics). Microsatellite instability (MSI) testing showed MSI-high, and immunostaining of the tumor tissues showed decreased MLH1 expression, loss of PMS2 expression, retained MSH2 expression, and loss of MSH6 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHer mother had gastric cancer in her thirties and died of ovarian cancer at the age of 54 years. In addition, her maternal grandfather had a history of gastric and colorectal cancers at an unknown age, and her maternal grandmother had colorectal cancer at an unknown age (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). The MMR gene screening test with next-generation sequencing and multiplex ligation-dependent probe amplification analysis (FALCO Biosystems Ltd., Kyoto, Japan) showed two germline variants of \u003cem\u003eMLH1\u003c/em\u003e c.2250 C\u0026thinsp;\u0026gt;\u0026thinsp;G (p. Tyr750*) and \u003cem\u003eMSH6\u003c/em\u003e c.2385del (p. Ile795Metfs*15). Consequently, she was diagnosed with Lynch syndrome. She had a complete response to chemotherapy and continued to be treated with bevacizumab plus olaparib, a PARP inhibitor, as maintenance therapy for metastatic ovarian cancer. However, she complained of headache and vertigo 8 months after the maintenance therapy started. CT scan showed a massive brain tumor in her right parietal lobe (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). We performed brain tumor resection considering the possibility of its association with Lynch syndrome. Thus, the histological type was also endometrioid carcinoma diagnosed as brain metastasis. Immunostaining of this tissue also revealed decreased MLH1, negative PMS2, positive MSH2, and negative MSH6 expressions, which was the same pattern as that of the primary tumor tissue. She received whole-brain radiation therapy and continued treatment with olaparib, but she developed multiple lung metastases after 2 months (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee, f). Soon after tumor relapse, she started receiving pembrolizumab and lenvatinib for metastatic endometrial cancer, which led to a significant shrinkage of multiple lung metastases, the best response with partial response without any other new lesions for 1 year and 10 months.\u003c/p\u003e \u003cp\u003eWe intend to perform a genetic analysis for her sister and her sister\u0026rsquo;s daughter in the future.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eLynch syndrome is caused by a germline heterozygous pathogenic variant of a single MMR gene [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. More than one pathological germline variant of the MMR gene is not usually detected in individuals with Lynch syndrome. However, our patient had two germline variants of \u003cem\u003eMLH1\u003c/em\u003e c.2250C\u0026thinsp;\u0026gt;\u0026thinsp;G and \u003cem\u003eMSH6\u003c/em\u003e c.2385del. These variants were considered pathogenic based on the immunostaining of MMR proteins in tumor tissues with decreased MLH1 expression and loss of MSH6 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea), and the MSI-high. These variants of \u003cem\u003eMLH1\u003c/em\u003e and \u003cem\u003eMSH6\u003c/em\u003e could be transmitted as follows: (1) each variant was inherited from one parent each; (2) one variant was inherited from one parent and the other occurred de novo in the patient; and (3) both variants were inherited from one parent. Considering that the occurrence of de novo germline mutations in MMR genes is rare [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] and that there are multiple family histories of gastrointestinal and gynecological cancers in the maternal line (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb), we considered that at least one pathogenic variant of \u003cem\u003eMLH1\u003c/em\u003e or \u003cem\u003eMSH6\u003c/em\u003e was inherited from her mother. However, we could not perform genetic testing for her mother because she had already died. Her father never had cancer until the age of 68 years, and his parents, brother, and sister had no malignant tumors, indicating that the variants were not inherited from the paternal line. Thus, one or two of the variants were likely to have been inherited from her mother, although we cannot confirm how two germline variants of MMR genes occurred.\u003c/p\u003e \u003cp\u003eKast et al. reported a patient with endometrial cancer at the age of 46 years who carried both germline variants in \u003cem\u003eBRCA1\u003c/em\u003e (p.Arg71Serfs*21) and \u003cem\u003eMSH6\u003c/em\u003e (p. Ile172fs*10), which are both considered frameshift mutations and pathogenic [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Pinto et al. reported a case series of the co-occurrence of \u003cem\u003eMSH6\u003c/em\u003e (p.Gln344*) and \u003cem\u003eMSH2\u003c/em\u003e (p. Arg929*) nonsense variants in eight probands with Lynch syndrome [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. They proposed that this \u003cem\u003eMSH2\u003c/em\u003e C-terminal nonsense variant expected to encode a protein lacking six amino acid residues is a variant of unknown significance because five tumors of the eight probands retained MSH2 expression [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Ferrer-Avargues et al. recently showed that 5 of 84 families (6%) with Lynch syndrome had multiple germline variants of MMR genes and other different cancer-disposing genes, such as \u003cem\u003eMLH1-BRCA2-NBN\u003c/em\u003e, \u003cem\u003eMLH1-BRCA1\u003c/em\u003e, \u003cem\u003eMSH2-ATM\u003c/em\u003e, \u003cem\u003eMSH6-NF1\u003c/em\u003e, and \u003cem\u003eMLH1-FANCA\u003c/em\u003e [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Each variant of MMR genes was considered pathogenic and had high penetrance, whereas the penetrance of cancer-disposing genes other than \u003cem\u003eBRCA1\u003c/em\u003e was considered moderate [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The detection of multiple germline pathogenic variants of different MMR genes in a single individual, similar to our case, is quite rare. Individuals with multiple germline pathogenic variants are more frequently diagnosed because of the increasingly widespread use of genetic testing, including multiple germline gene panels, tumor-only panels, tumor and germline panels, and whole genome sequencing.\u003c/p\u003e \u003cp\u003eThe MLH1 protein consists of 756 amino acids and binds to PMS2 in the C-terminal region to form a MutLα heterodimer, which exhibits DNA cleavage activity for DNA MMR [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. \u003cem\u003eMLH1\u003c/em\u003e c.2250C\u0026thinsp;\u0026gt;\u0026thinsp;G (p. Tyr750*) is a C-terminal nonsense variant predicted to defect the last seven amino acid residues of the C-terminal domain and is a variant of unknown significance based on the InSiGHT database with recurrently reported data [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. For example, \u003cem\u003eMLH1\u003c/em\u003e c.2250C\u0026thinsp;\u0026gt;\u0026thinsp;G was detected in an individual with suspected Lynch syndrome who fulfilled the Amsterdam criteria [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]; however, it was not determined to be pathogenic because no tumor was available to assess MSI status. This germline variant was also detected in a Chinese family that did not fulfill the Amsterdam criteria but included three individuals with onset of colorectal cancer in their 40s [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In contrast, Kosinski et al. showed in their \u003cem\u003ein vitro\u003c/em\u003e analysis that \u003cem\u003eMLH1\u003c/em\u003e c.2250C\u0026thinsp;\u0026gt;\u0026thinsp;G retained MLH1 protein expression but reduced dimerization with PMS2, leading to impaired MMR activity [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Another study also supported this variant\u0026rsquo;s inability to stabilize the PMS2 protein [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In our case, immunohistochemical analysis showed decreased MLH1 expression but not complete loss of expression, indicating that this variant, at least in part, impairs MLH1 protein stability in tumor cells. A previous study reported that 32% (14/44) of \u003cem\u003eMLH1\u003c/em\u003e variant carriers showed a weakly positive pattern of MLH1 protein expression in tumor tissues [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Consistently, our previous functional analysis demonstrated that 42 of 100 \u003cem\u003eMLH1\u003c/em\u003e variants partially impair protein stability (25\u0026ndash;75% relative expression compared with wild type) and 24 of 42 variants impair MMR activity (\u0026lt;\u0026thinsp;60% of in vitro MMR activity) [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The presumed truncated MLH1 protein (p. Tyr750*) coded by c.2250C\u0026thinsp;\u0026gt;\u0026thinsp;G may reduce the dimerization ability of PMS2 in tumor cells, causing MMR deficiency.\u003c/p\u003e \u003cp\u003eThe MSH6 protein consists of 1360 amino acids and forms a MutSα heterodimer with MSH2 to recognize DNA mismatch sites [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. \u003cem\u003eMSH6\u003c/em\u003e c.2385del (p. Ile795Metfs*15) is a frameshift variant in the MutS core domain region and considered a pathological variant, although this variant has not been reported in the InSiGHT database or elsewhere. The onset of endometrial and ovarian cancers in our patient conforms to the consensus that the incidence of endometrial cancer is higher in women with the germline variant of \u003cem\u003eMSH6\u003c/em\u003e [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMMR deficiency results in the accumulation of somatic mutations, including point mutations and insertion/deletion mutations, at repetitive nucleotide sequences termed microsatellite regions, leading to the onset of Lynch syndrome-associated tumors [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. We also detected somatic homologous recombination gene variants, \u003cem\u003eBRCA1\u003c/em\u003e c.1016del and \u003cem\u003eBRCA2\u003c/em\u003e c.36dupT, in tumor tissues. We speculate that these base insertion/deletion mutations, located in repetitive sequences, are secondary mutations caused by MMR deficiency. The detection of \u003cem\u003eBRCA1\u003c/em\u003e and \u003cem\u003eBRCA2\u003c/em\u003e variants concurs with the finding that approximately 50% of MSI-high colorectal cancers have \u003cem\u003eBRCA2\u003c/em\u003e somatic mutations, and approximately one-fifth have \u003cem\u003eBRCA1\u003c/em\u003e somatic mutations [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRecent advances in genetic testing by next-generation sequencing and molecularly targeted drugs have enabled personalized treatment of tumors with disrupted DNA repair mechanisms, such as MMR [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] and homologous recombination repair [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. MMR-deficient tumors accumulate genetic mutations and subsequent generation of highly immunogenic tumor antigens, which explains the benefit of immune checkpoint inhibitors to some patients with MMR-deficient tumors [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Consistently, our patient showed a favorable response to pembrolizumab. Although the tumor carried pathogenic somatic \u003cem\u003eBRCA1\u003c/em\u003e and \u003cem\u003eBRCA2\u003c/em\u003e variants, she did not benefit from olaparib but had a transient response to platinum-based chemotherapy. Sokol et al. analyzed\u0026thinsp;\u0026gt;\u0026thinsp;200,000 patients with cancer and reported that homologous recombination deficiency and MSI-high phenotype were generally mutually exclusive across cancer types but may rarely co-occur, particularly in prostate cancer [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. These variants were generally monoallelic and not associated with homologous recombination deficiency, indicating that most \u003cem\u003eBRCA1/2\u003c/em\u003e variants coexisting with MSI are likely bystander events that may not lead to sensitivity to PARP inhibitors [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eWe reported a case of a patient with Lynch syndrome who carried coexisting germline variants of MMR genes, a recurrently reported \u003cem\u003eMLH1\u003c/em\u003e c.2250 C\u0026thinsp;\u0026gt;\u0026thinsp;G (p. Tyr750*) and a previously unreported \u003cem\u003eMSH6\u003c/em\u003e c.2385del (p. Ile795Metfs*15), as well as somatic pathogenic variants of \u003cem\u003eBRCA1\u003c/em\u003e and \u003cem\u003eBRCA2\u003c/em\u003e in the tumor tissues. The patient benefited from an immune checkpoint inhibitor but not a PARP inhibitor. Clinicians should pay attention to the rare possibility of multiple germline variants of MMR genes for the genetic analysis of patients and their family members.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the patient and her families and all the clinicians who allowed us to publish this case report. We thank Enago (www.enago.jp) for the English language review.\u003c/p\u003e\n\u003cp\u003eThis study was not supported by funding. We report conflicts of interest as follows: M. Takahashi has received research funding from Ono Pharmaceutical, Chugai Pharmaceutical, and MSD, and lecture fees from Daiichi Sankyo, Ono Pharmaceutical, Bristol Meyers Squibb, and Taiho Pharmaceutical Company. C. Ishioka has received research funding from Chugai Pharmaceutical, Taiho Pharmaceutical, Asahi‐Kasei Pharma, Ono Pharmaceutical, Daiichi Sankyo, Takeda Pharmaceutical, and Hitachi Ltd, and lecture fees from Chugai Pharmaceutical and Daiichi Sankyo.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSU collected the data, contributed to the conception of the work, and draft the manuscript. MT saw the patient and got consent form the patient for the genetic analysis and this work, collected the data, contributed to the conception of the work, and draft and revised the manuscript. JHM, HT, MS were involved in surgery and drug therapy, and revised the manuscript. MK and YA performed genetic counselling and revised the manuscript. ST, KT, KO, HI, KS1 interpreted data and revised the manuscript. FF performed IHC and interpreted data. CI interpreted data, supervised the work, and revised the manuscript. All authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis work was approved by the IRB of the Ethics Committee Tohoku University Hospital. The patient has consented to the publication of this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis study was not supported by funding. We report conflicts of interest as follows: M. Takahashi has received research funding from Ono Pharmaceutical, Chugai Pharmaceutical, and MSD, and lecture fees from Daiichi Sankyo, Ono Pharmaceutical, Bristol Meyers Squibb, and Taiho Pharmaceutical Company. C. Ishioka has received research funding from Chugai Pharmaceutical, Taiho Pharmaceutical, Asahi‐Kasei Pharma, Ono Pharmaceutical, Daiichi Sankyo, Takeda Pharmaceutical, and Hitachi Ltd, and lecture fees from Chugai Pharmaceutical and Daiichi Sankyo.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLynch HT, Snyder CL, Shaw TG, Heinen CD, Hitchins MP (2015) Milestones of Lynch syndrome: 1895\u0026ndash;2015. Nat Rev Cancer 15:181\u0026ndash;194. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/nrc3878\u003c/span\u003e\u003cspan address=\"10.1038/nrc3878\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTamura K, Kaneda M, Futagawa M et al (2019) Genetic and genomic basis of the mismatch repair system involved in Lynch syndrome. Int J Clin Oncol 24:999\u0026ndash;1011. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s10147-019-01494-y\u003c/span\u003e\u003cspan address=\"10.1007/s10147-019-01494-y\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWin AK, Jenkins MA, Buchanan DD et al (2011) Determining the frequency of de novo germline mutations in DNA mismatch repair genes. J Med Genet 48:530\u0026ndash;534. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1136/jmedgenet-2011-100082\u003c/span\u003e\u003cspan address=\"10.1136/jmedgenet-2011-100082\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKast K, Neuhann TM, G\u0026ouml;rgens H et al (2012) Germline truncating-mutations in BRCA1 and MSH6 in a patient with early onset endometrial cancer. BMC Cancer 12:1\u0026ndash;5. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/1471-2407-12-531\u003c/span\u003e\u003cspan address=\"10.1186/1471-2407-12-531\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePinto C, Pinheiro M, Peixoto A et al (2016) Co-occurrence of nonsense mutations in MSH6 and MSH2 in Lynch syndrome families evidencing that not all truncating mutations are equal. J Hum Genet 61:151\u0026ndash;156. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/jhg.2015.124\u003c/span\u003e\u003cspan address=\"10.1038/jhg.2015.124\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFerrer-Avargues R, Castillejo MI, D\u0026aacute;maso E et al (2021) Co-occurrence of germline pathogenic variants for different hereditary cancer syndromes in patients with Lynch syndrome. Cancer Commun 41:218\u0026ndash;228. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/cac2.12134\u003c/span\u003e\u003cspan address=\"10.1002/cac2.12134\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThompson BA, Spurdle AB, Plazzer JP et al (2014) 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 46:107\u0026ndash;115. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/ng.2854\u003c/span\u003e\u003cspan address=\"10.1038/ng.2854\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMueller J, Gazzoli I, Bandipalliam P et al (2009) Comprehensive molecular analysis of mismatch repair gene defects in suspected Lynch syndrome (hereditary nonpolyposis colorectal cancer) cases. Cancer Res 69:7053\u0026ndash;7061. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1158/0008-5472.CAN-09-0358\u003c/span\u003e\u003cspan address=\"10.1158/0008-5472.CAN-09-0358\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYuan Y, Huang YQ, Cai SR et al (2004) Genetic characterization of Chinese hereditary non-polyposis colorectal cancer by DHPLC and multiplex PCR. Jpn J Clin Oncol 34:660\u0026ndash;666. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/jjco/hyh121\u003c/span\u003e\u003cspan address=\"10.1093/jjco/hyh121\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKosinski J, Hinrichsen I, Bujnicki JM et al (2010) Identification of Lynch syndrome mutations in the MLH1-PMS2 interface that disturb dimerization and mismatch repair. Hum Mutat 31:975\u0026ndash;982. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/humu.21301\u003c/span\u003e\u003cspan address=\"10.1002/humu.21301\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMohd AB, Palama B, Nelson SE et al (2006) Truncation of the C-terminus of human MLH1 blocks intracellular stabilization of PMS2 and disrupts DNA mismatch repair. DNA Repair (Amst) 5:347\u0026ndash;361. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.dnarep.2005.11.001\u003c/span\u003e\u003cspan address=\"10.1016/j.dnarep.2005.11.001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMangold E, Pagenstecher C, Friedl W et al (2005) Tumours from MSH2 mutation carriers show loss of MSH2 expression but many tumours from MLH1 mutation carriers exhibit weak positive MLH1 staining. J Pathol 207:385\u0026ndash;395. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/path.1858\u003c/span\u003e\u003cspan address=\"10.1002/path.1858\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTakahashi M, Shimodaira H, Andreutti-Zaugg C et al (2007) Functional analysis of human MLH1 variants using yeast and in vitro mismatch repair assays. Cancer Res 67:4595\u0026ndash;4604. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1158/0008-5472.CAN-06-3509\u003c/span\u003e\u003cspan address=\"10.1158/0008-5472.CAN-06-3509\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBaglietto L, Lindor NM, Dowty JG et al (2010) Risks of Lynch syndrome cancers for MSH6 mutation carriers. J Natl Cancer Inst 102:193\u0026ndash;201. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/jnci/djp473\u003c/span\u003e\u003cspan address=\"10.1093/jnci/djp473\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGoodfellow PJ, Buttin BM, Herzog TJ et al (2003) Prevalence of defective DNA mismatch repair and MSH6 mutation in an unselected series of endometrial cancers. Proc Natl Acad Sci U S A 100:5908\u0026ndash;5913. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1073/pnas.1030231100\u003c/span\u003e\u003cspan address=\"10.1073/pnas.1030231100\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAbildgaard AB, Nielsen SV, Bernstein I et al (2023) Lynch syndrome, molecular mechanisms and variant classification. Br J Cancer 128:726\u0026ndash;734. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41416-022-02059-z\u003c/span\u003e\u003cspan address=\"10.1038/s41416-022-02059-z\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDeihimi S, Lev A, Slifker M et al (2017) BRCA2, EGFR, and NTRK mutations in mismatch repair-deficient colorectal cancers with MSH2 or MLH1 mutations. Oncotarget 8:39945\u0026ndash;39962. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.18632/oncotarget.18098\u003c/span\u003e\u003cspan address=\"10.18632/oncotarget.18098\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLe DT, Durham JN, Smith KN et al (2017) Mismatch repair deficiency predicts response of solid tumors to PD-1 blockade. Science 357:409\u0026ndash;413. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1126/science.aan6733\u003c/span\u003e\u003cspan address=\"10.1126/science.aan6733\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLord CJ, Ashworth A (2017) PARP inhibitors: Synthetic lethality in the clinic. Science 355:1152\u0026ndash;1158. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1126/science.aam7344\u003c/span\u003e\u003cspan address=\"10.1126/science.aam7344\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSokol ES, Jin DX, Fine A et al (2022) PARP Inhibitor Insensitivity to BRCA1/2 Monoallelic Mutations in Microsatellite Instability-High Cancers. JCO Precis Oncol 6:e2100531. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1200/po.21.00531\u003c/span\u003e\u003cspan address=\"10.1200/po.21.00531\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Lynch syndrome, MLH1, MSH6, coexisting germline variants","lastPublishedDoi":"10.21203/rs.3.rs-3926364/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3926364/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eLynch syndrome is an autosomal dominant disorder caused by a heterozygous pathogenic germline variant in mismatch repair (MMR) genes, including \u003cem\u003eMLH1\u003c/em\u003e, \u003cem\u003eMSH2\u003c/em\u003e, \u003cem\u003eMSH6\u003c/em\u003e, \u003cem\u003ePMS2\u003c/em\u003e, and \u003cem\u003eEPCAM\u003c/em\u003e. Lynch syndrome often causes a familial cluster of patients with malignant tumors. We describe a 37-year-old woman who presented with endometrioid carcinoma in the ovary and endometrium associated with Lynch syndrome. She carried two germline pathogenic variants, a recurrently reported \u003cem\u003eMLH1\u003c/em\u003e c.2250C\u0026thinsp;\u0026gt;\u0026thinsp;G (p. Tyr750*) and previously unreported \u003cem\u003eMSH6\u003c/em\u003e c.2385del (p. Ile795Metfs*15). The tumor cells showed microsatellite instability. Immunohistochemistry showed decreased MLH1 expression, loss of PMS2 expression, retained MSH2 expression, and loss of MSH6 expression, indicating that both variants impair protein stability, causing MMR deficiency. Whether these variants were inherited from her parents or occurred de novo is unknown. The tumor cells had somatic variants \u003cem\u003eBRCA1\u003c/em\u003e c.1016del and \u003cem\u003eBRCA2\u003c/em\u003e c.36dupT, which might be due to secondary mutation by MMR deficiency. The use of pembrolizumab, an immune checkpoint inhibitor, resulted in a durable partial response of metastatic lung tumors. This case highlights the rare possibility of multiple germline variants in MMR genes in individuals with Lynch syndrome.\u003c/p\u003e","manuscriptTitle":"Coexisting germline variants of MLH1 and MSH6 in a patient with Lynch syndrome, endometrial cancer, and ovarian cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-07 19:41:16","doi":"10.21203/rs.3.rs-3926364/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c3caa2e2-5200-48e7-bcb3-dfdb547cd43c","owner":[],"postedDate":"February 7th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-04-30T10:17:12+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-07 19:41:16","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3926364","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3926364","identity":"rs-3926364","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}