Surviving without BRCA2: MLH1 gets R-looped in to curtail genomic instability.

While breast cancer 2 (BRCA2) loss of heterozygosity (LOH) promotes cancer initiation, it can also induce death in nontransformed cells. In contrast, mismatch repair gene mutL homolog 1 (MLH1) is a tumor-suppressor gene that protects cells from cancer development through repairing mismatched base pairs during DNA mismatch repair (MMR). Sengodan et al., in this issue of the JCI, reveal an interplay between the 2 genes: MLH1 promoted the survival of BRCA2-deficient cells independently of its MMR function. MLH1 protected replication forks from degradation, while also resolving R-loops, thereby reducing genomic instability. Moreover, MLH1 expression was regulated directly by estrogen, shedding light into the hormone-responsive nature of many BRCA2 mutant breast cancers. These results provide important insight into the genetics that drive the initiation of BRCA2-mutated breast cancers.

Clonal origin and genomic diversity in Lynch syndrome-associated endometrial cancer with multiple synchronous tumors: Identification of the pathogenicity of MLH1 p.L582H.

Lynch syndrome-associated endometrial cancer patients often present multiple synchronous tumors and this assessment can affect treatment strategies. We present a case of a 27-year-old woman with tumors in the uterine corpus, cervix, and ovaries who was diagnosed with endometrial cancer and exhibited cervical invasion and ovarian metastasis. Her family history suggested Lynch syndrome, and genetic testing identified a variant of uncertain significance, MLH1 p.L582H. We conducted immunohistochemical staining, microsatellite instability analysis, and Sanger sequencing for Lynch syndrome-associated cancers in three generations of the family and identified consistent MLH1 loss. Whole-exome sequencing for the corpus, cervical, and ovarian tumors of the proband identified a copy-neutral loss of heterozygosity (LOH) occurring at the MLH1 position in all tumors. This indicated that the germline variant and the copy-neutral LOH led to biallelic loss of MLH1 and was the cause of cancer initiation. All tumors shared a portion of somatic mutations with high mutant allele frequencies, suggesting a common clonal origin. There were no mutations shared only between the cervix and ovary samples. The profiles of mutant allele frequencies shared between the corpus and cervix or ovary indicated that two different subclones originating from the corpus independently metastasized to the cervix or ovary. Additionally, all tumors presented unique mutations in endometrial cancer-associated genes such as ARID1A and PIK3CA. In conclusion, we demonstrated clonal origin and genomic diversity in a Lynch syndrome-associated endometrial cancer, suggesting the importance of evaluating multiple sites in Lynch syndrome patients with synchronous tumors.

Epigenetic MLH1 silencing concurs with mismatch repair deficiency in sporadic, naturally occurring colorectal cancer in rhesus macaques.

BACKGROUND: Naturally occurring colorectal cancers (CRC) in rhesus macaques share many features with their human counterparts and are useful models for cancer immunotherapy; but mechanistic data are lacking regarding the comparative molecular pathogenesis of these cancers. METHODS: We conducted state-of-the-art imaging including CT and PET, clinical assessments, and pathological review of 24 rhesus macaques with naturally occurring CRC. Additionally, we molecularly characterized these tumors utilizing immunohistochemistry (IHC), microsatellite instability assays, DNAseq, transcriptomics, and developed a DNA methylation-specific qPCR assay for MLH1, CACNA1G, CDKN2A, CRABP1, and NEUROG1, human markers for CpG island methylator phenotype (CIMP). We furthermore employed Monte-Carlo simulations to in-silico model alterations in DNA topology in transcription-factor binding site-rich promoter regions upon experimentally demonstrated DNA methylation. RESULTS: Similar cancer histology, progression patterns, and co-morbidities could be observed in rhesus as reported for human CRC patients. IHC identified loss of MLH1 and PMS2 in all cases, with functional microsatellite instability. DNA sequencing revealed the close genetic relatedness to human CRCs, including a similar mutational signature, chromosomal instability, and functionally-relevant mutations affecting KRAS (G12D), TP53 (R175H, R273*), APC, AMER1, ALK, and ARID1A. Interestingly, MLH1 mutations were rarely identified on a somatic or germline level. Transcriptomics not only corroborated the similarities of rhesus and human CRCs, but also demonstrated the significant downregulation of MLH1 but not MSH2, MSH6, or PMS2 in rhesus CRCs. Methylation-specific qPCR suggested CIMP-positivity in 9/16 rhesus CRCs, but all 16/16 exhibited significant MLH1 promoter hypermethylation. DNA hypermethylation was modelled to affect DNA topology, particularly propeller twist and roll profiles. Modelling the DNA topology of a transcription factor binding motif (TFAP2A) in the MLH1 promoter that overlapped with a methylation-specific probe, we observed significant differences in DNA topology upon experimentally shown DNA methylation. This suggests a role of transcription factor binding interference in epigenetic silencing of MLH1 in rhesus CRCs. CONCLUSIONS: These data indicate that epigenetic silencing suppresses MLH1 transcription, induces the loss of MLH1 protein, abrogates mismatch repair, and drives genomic instability in naturally occurring CRC in rhesus macaques. We consider this spontaneous, uninduced CRC in immunocompetent, treatment-naïve rhesus macaques to be a uniquely informative model for human CRC.

Mutant huntingtin protein induces MLH1 degradation, DNA hyperexcision, and cGAS-STING-dependent apoptosis.

Huntington's disease (HD) is an inherited neurodegenerative disorder caused by an expanded CAG repeat in the huntingtin (HTT) gene. The repeat-expanded HTT encodes a mutated HTT (mHTT), which is known to induce DNA double-strand breaks (DSBs), activation of the cGAS-STING pathway, and apoptosis in HD. However, the mechanism by which mHTT triggers these events is unknown. Here, we show that HTT interacts with both exonuclease 1 (Exo1) and MutLα (MLH1-PMS2), a negative regulator of Exo1. While the HTT-Exo1 interaction suppresses the Exo1-catalyzed DNA end resection during DSB repair, the HTT-MutLα interaction functions to stabilize MLH1. However, mHTT displays a significantly reduced interaction with Exo1 or MutLα, thereby losing the ability to regulate Exo1. Thus, cells expressing mHTT exhibit rapid MLH1 degradation and hyperactive DNA excision, which causes severe DNA damage and cytosolic DNA accumulation. This activates the cGAS-STING pathway to mediate apoptosis. Therefore, we have identified unique functions for both HTT and mHTT in modulating DNA repair and the cGAS-STING pathway-mediated apoptosis by interacting with MLH1. Our work elucidates the mechanism by which mHTT causes HD.

Association between colorectal cancer, the frequency of Bacteroides fragilis, and the level of mismatch repair genes expression in the biopsy samples of Iranian patients.

BACKGROUND: Deficient DNA mismatch repair (MMR) can cause microsatellite instability (MSI) and is more common in colorectal cancer (CRC) patients. Understanding the carcinogenic mechanism of bacteria and their impact on cancer cells is crucial. Bacteroides fragilis (B. fragilis) has been identified as a potential promoter of tumorigenesis through the alteration of signaling pathways. This study aims to assess the expression levels of msh2, msh6, mlh1, and the relative frequency of B. fragilis in biopsy samples from CRC patients. MATERIALS AND METHODS: Based on the sequence of mlh1, msh2, and msh6 genes, B. fragilis specific 16srRNA and bacterial universal 16srRNA specific primers were selected, and the expression levels of the target genes were analyzed using the Real-Time PCR method. RESULTS: Significant increases in the expression levels of mlh1, msh2, and msh6 genes were observed in the cancer group. Additionally, the expression of these MMR genes showed a significant elevation in samples positive for B. fragilis presence. The relative frequency of B. fragilis in the cancer group demonstrated a significant rise compared to the control group. CONCLUSION: The findings suggest a potential correlation between the abundance of B. fragilis and alterations in the expression of MMR genes. Since these genes can play a role in modifying colon cancer, investigating microbial characteristics and gene expression changes in CRC could offer a viable solution for CRC diagnosis.

[Characteristics and clinical analysis of MLH1 c.463dupC gene mutation in a Lynch syndrome family].

In this study, a case of Lynch syndrome (LS) family line with a novel mutation site in the MLH1 c.463dupC gene was reported and the clinical and pathogenic genetic features of this family were analyzed. A 40-year-old female patient with colon cancer diagnosed at the First Affiliated Hospital of Kunming Medical University on October 2, 2020 was retrospectively included. The clinical data of the family were collected and the family lineage was drawn. The family tumor history met the Amsterdam Criteria Ⅱ and the diagnostic criteria of LS in Chinese, which was a typical LS family lineage. A germline code-shift missense mutation c.463dupC in the MLH1 gene located in exon 6, a possible pathogenic variant, was detected by second-generation sequencing (NGS) in the patient. Subsequently, Sanger sequencing was performed on a total of 20 direct lineage members of the family of the MLH1 gene, 7 cases were found to harbor the mutation and included in the LS high-risk control. Follow-up to October 2023 showed that the patient had endometrial and cervical polyps, one case had colorectal cancer, and two cases had intestinal polyps, all were treated with early intervention and therapy; two cases did not show any clinical symptoms. This study is the first to report a new mutation site for the potentially pathogenic MLH1 c.463dupC, providing a rationale for the pathogenicity of the mutation and standardized health management for familial carriers.

The relationship between DNA mismatch repair gene and other prognostic parameters in pancreatic adenocarcinoma.

The aim of the study was to determine DNA mismatch repair (MMR) proteins by immunohistochemically using MLH1, MSH2, MSH6, and PMS2 antibodies in patients diagnosed as pancreatic ductal adenocarcinoma and to assess its relationship with histopathological and clinical prognostic parameters. Fifty cases with a diagnosis of pancreatic ductal adenocarcinoma who underwent surgical resection, were included in the study. Demographic and histopathological features of the patients were collected from the medical records. The relationships between microsatellite status and prognostic parameters were determined. The mean age of the patients was 66.5 ± 9.5 years (range: 47-87) and male/female ratio was 1.63 (31/19). No errors were detected in DNA MMR proteins in any of the cases, and were classified as microsatellite stable. The mean tumor diameter was 4.01 ± 1.77 cm and 74% of the tumors were localized in the pancreatic head. All of the cases had lymphatic invasion, whereas vascular invasion was detected in only 78% and perineural invasion in 98% of the patients. When the relationship between prognostic parameters and survival was evaluated, statistically significant correlation was observed in patient age and histopathological parameters such as tumor diameter, status of surgical margins, and vascular invasion (p < 0.05). Age, tumor size, presence of tumor at surgical margins, vascular invasion, and adjuvant treatment were correlated with survival. Although microsatellite instability was not detected in our cases, it is important to determine the microsatellite status by immunohistochemistry for predicting the chemotherapy response and determining the immunotherapy option in pancreatic adenocarcinomas.

Mismatch repair protein MLH1 suppresses replicative stress in BRCA2-deficient breast tumors.

Loss of BRCA2 (breast cancer 2) is lethal for normal cells. Yet it remains poorly understood how, in BRCA2 mutation carriers, cells undergoing loss of heterozygosity overcome the lethality and undergo tissue-specific neoplastic transformation. Here, we identified mismatch repair gene mutL homolog 1 (MLH1) as a genetic interactor of BRCA2 whose overexpression supports the viability of Brca2-null cells. Mechanistically, we showed that MLH1 interacts with Flap endonuclease 1 (FEN1) and competes to process the RNA flaps of Okazaki fragments. Together, they restrained the DNA2 nuclease activity on the reversed forks of lagging strands, leading to replication fork (RF) stability in BRCA2-deficient cells. In these cells, MLH1 also attenuated R-loops, allowing the progression of stable RFs, which suppressed genomic instability and supported cell viability. We demonstrated the significance of their genetic interaction by the lethality of Brca2-mutant mice and inhibition of Brca2-deficient tumor growth in mice by Mlh1 loss. Furthermore, we described estrogen as inducing MLH1 expression through estrogen receptor α (ERα), which might explain why the majority of BRCA2 mutation carriers develop ER-positive breast cancer. Taken together, our findings reveal a role of MLH1 in relieving replicative stress and show how it may contribute to the establishment of BRCA2-deficient breast tumors.

Molecular and Clinicopathologic Characterization of Mismatch Repair-Deficient Endometrial Carcinoma Not Related to MLH1 Promoter Hypermethylation.

Universal tumor screening in endometrial carcinoma (EC) is increasingly adopted to identify individuals at risk of Lynch syndrome (LS). These cases involve mismatch repair-deficient (MMRd) EC without MLH1 promoter hypermethylation (PHM). LS is confirmed through the identification of germline MMR pathogenic variants (PV). In cases where these are not detected, emerging evidence highlights the significance of double-somatic MMR gene alterations as a sporadic cause of MMRd, alongside POLE/POLD1 exonuclease domain (EDM) PV leading to secondary MMR PV. Our understanding of the incidence of different MMRd EC origins not related to MLH1-PHM, their associations with clinicopathologic characteristics, and the prognostic implications remains limited. In a combined analysis of the PORTEC-1, -2, and -3 trials (n = 1254), 84 MMRd EC not related to MLH1-PHM were identified that successfully underwent paired tumor-normal tissue next-generation sequencing of the MMR and POLE/POLD1 genes. Among these, 37% were LS associated (LS-MMRd EC), 38% were due to double-somatic hits (DS-MMRd EC), and 25% remained unexplained. LS-MMRd EC exhibited higher rates of MSH6 (52% vs 19%) or PMS2 loss (29% vs 3%) than DS-MMRd EC, and exclusively showed MMR-deficient gland foci. DS-MMRd EC had higher rates of combined MSH2/MSH6 loss (47% vs 16%), loss of >2 MMR proteins (16% vs 3%), and somatic POLE-EDM PV (25% vs 3%) than LS-MMRd EC. Clinicopathologic characteristics, including age at tumor onset and prognosis, did not differ among the various groups. Our study validates the use of paired tumor-normal next-generation sequencing to identify definitive sporadic causes in MMRd EC unrelated to MLH1-PHM. MMR immunohistochemistry and POLE-EDM mutation status can aid in the differentiation between LS-MMRd EC and DS-MMRd EC. These findings emphasize the need for integrating tumor sequencing into LS diagnostics, along with clear interpretation guidelines, to improve clinical management. Although not impacting prognosis, confirmation of DS-MMRd EC may release patients and relatives from burdensome LS surveillance.

Molecular analysis of cancer genomes in children with Lynch syndrome: Exploring causal associations.

Lynch syndrome (LS) predisposes to cancer in adulthood and is caused by heterozygous germline variants in a mismatch repair (MMR) gene. Recent studies show an increased prevalence of LS among children with cancer, suggesting a causal relationship. For LS-spectrum (LSS) cancers, including high-grade gliomas and colorectal cancer, causality has been supported by typical MMR-related tumor characteristics, but for non-LSS cancers, causality is unclear. We characterized 20 malignant tumors of 18 children with LS, including 16 non-LSS tumors. We investigated second hits, tumor mutational load, mutational signatures and MMR protein expression. In all LSS tumors and three non-LSS tumors, we detected MMR deficiency caused by second hit somatic alterations. Furthermore, these MMR-deficient tumors carried driver variants that likely originated as a consequence of MMR deficiency. However, in 13 non-LSS tumors (81%), a second hit and MMR deficiency were absent, thus a causal link between LS and cancer development in these children is lacking. These findings demonstrate that causality of LS in children with cancer, which can be determined by molecular tumor characterization, seems to be restricted to specific tumor types. Large molecular and epidemiological studies are needed to further refine the tumor spectrum in children with LS.

Classification of MLH1 Missense VUS Using Protein Structure-Based Deep Learning-Ramachandran Plot-Molecular Dynamics Simulations Method.

Pathogenic variation in DNA mismatch repair (MMR) gene MLH1 is associated with Lynch syndrome (LS), an autosomal dominant hereditary cancer. Of the 3798 MLH1 germline variants collected in the ClinVar database, 38.7% (1469) were missense variants, of which 81.6% (1199) were classified as Variants of Uncertain Significance (VUS) due to the lack of functional evidence. Further determination of the impact of VUS on MLH1 function is important for the VUS carriers to take preventive action. We recently developed a protein structure-based method named "Deep Learning-Ramachandran Plot-Molecular Dynamics Simulation (DL-RP-MDS)" to evaluate the deleteriousness of MLH1 missense VUS. The method extracts protein structural information by using the Ramachandran plot-molecular dynamics simulation (RP-MDS) method, then combines the variation data with an unsupervised learning model composed of auto-encoder and neural network classifier to identify the variants causing significant change in protein structure. In this report, we applied the method to classify 447 MLH1 missense VUS. We predicted 126/447 (28.2%) MLH1 missense VUS were deleterious. Our study demonstrates that DL-RP-MDS is able to classify the missense VUS based solely on their impact on protein structure.

Rare germline mutation and MSH2-&MSH6 + expression in a double primary carcinoma of colorectal carcinoma and endometrial carcinoma: a case report.

BACKGROUND: Multiple primary malignancies are rare in cancer patients, and risk factors may include genetics, viral infection, smoking, radiation, and other environmental factors. Lynch syndrome (LS) is the most prevalent form of hereditary predisposition to double primary colorectal and endometrial cancer in females. LS, also known as hereditary nonpolyposis colorectal cancer (HNPCC), is a common autosomal dominant condition. Pathogenic germline variants in the DNA mismatch repair (MMR) genes, namely MLH1, MSH2, MSH6, and PMS2, and less frequently, deletions in the 3' end of EPCAM cause LS. It manifested itself as loss of MMR nuclear tumor staining (MMR protein deficient, dMMR). CASE PRESENTATION: This case study describes a double primary carcinoma in a 49-year-old female. In June 2022, the patient was diagnosed with highly to moderately differentiated endometrioid adenocarcinoma. The patient's mother died of esophageal cancer at age 50, and the father died of undefined reasons at age 70. Immunohistochemical stainings found ER (++), PR (++), P53 (+), MSH2 (-), MSH6 (+), MLH1 (+), and PMS2 (+). MMR gene sequencing was performed on endometrial tumor and peripheral blood samples from this patient. The patient carried two pathogenic somatic mutations in the endometrial tumor, MSH6 c.3261dupC (p.Phe1088LeufsTer5) and MSH2 c.445_448dup (p.Val150fs), in addition to a rare germline mutation MSH6 c.133G > C (p.Gly45Arg). Two years ago, the patient was diagnosed with moderately differentiated adenocarcinoma in the left-half colon. Immunohistochemical stainings found MSH2(-), MSH6(+), MLH1(+), and PMS2(+) (data not shown). CONCLUSIONS: In the case of a patient with double primary EC and CRC, a careful evaluation of the IHC and the genetic data was presented. The patient carried rare compound heterozygous variants, a germline missense mutation, and a somatic frameshift mutation of MSH6, combined with a novel somatic null variant of MSH2. Our study broadened the variant spectrum of double primary cancer and provided insight into the molecular basis for abnormal MSH2 protein loss and double primary carcinoma.

Association of a novel frameshift variant and a known deleterious variant in MMR genes with Lynch syndrome in Chinese families.

BACKGROUND: Lynch syndrome (LS) is the most common hereditary colorectal cancer (CRC) syndrome. This condition is characterized by germline variants in DNA mismatch repair (MMR) genes, including MLH1, MSH2, MSH6, and PMS2. In this study, we analyzed the molecular defects and clinical manifestations of two families affected with CRC and proposed appropriate individual preventive strategies for all carriers of the variant. METHODS: We recruited two families diagnosed with CRC and combined their family history and immunohistochemical results to analyze the variants of probands and those of other family members by using whole exome sequencing. Subsequently, gene variants in each family were screened by comparing them with the variants available in the public database. Sanger sequencing was performed to verify the variant sites. An online platform ( https://www.uniprot.org ) was used to analyze the functional domains of mutant proteins. RESULTS: A novel frameshift variant (NM_001281492, c.1129_1130del, p.R377fs) in MSH6 and a known deleterious variant (NM_000249.4:c.1731G > A, p.S577S) in MLH1 were identified in the two families with CRC. Using bioinformatics tools, we noted that the frameshift variant reduced the number of amino acids in the MSH6 protein from 1230 to 383, thereby leading to no MSH6 protein expression. The silent variant caused splicing defects and was strongly associated with LS. 5-Fluorouracil-based adjuvant chemotherapy is not recommended for patients with LS. CONCLUSIONS: The novel frameshift variant (MSH6, c.1129_1130del, p.R377fs) is likely pathogenic to LS, and the variant (MLH1, c.1731G > A, p.S577S) has been further confirmed to be pathogenic to LS. Our findings underscore the significance of genetic testing for LS and recommend that genetic consultation and regular follow-ups be conducted to guide individualized treatment for cancer-afflicted families, especially those with a deficiency in MMR expression.

Rare single-nucleotide variants of MLH1 and MSH2 genes in patients with Lynch syndrome.

BACKGROUND: Approximately 5% of colorectal cancers (CRCs) are hereditary. Lynch syndrome (LS), also known as hereditary nonpolyposis colorectal cancer (HNPCC), is the most common form of recognized hereditary CRC. Although Iran, as a developing country, has a high incidence of CRC, the spectrum of variants has yet to be thoroughly investigated. AIMS: This study aimed to investigate pathogenic and non-pathogenic variants in MLH1 and MSH2 genes in Iranian patients with suspected Lynch syndrome (sLS). METHODS AND RESULTS: In the present study, 25 peripheral blood samples were collected from patients with sLS and high microsatellite instability (MSI-H). After DNA extraction, all samples underwent polymerase chain reaction and Sanger sequencing to identify the variants in the exons of MLH1 and MSH2 genes. The identified variants were interpreted using prediction tools, and were finally reported under ACMG guidelines. In our study population, 13 variants were found in the MLH1 gene and 8 in the MSH2 gene. Interestingly, 7 of the 13 MLH1 variants and 3 of the 8 MSH2 variants were novel, whereas the remaining variants were previously reported or available in databases. In addition, some patients with sLS did not have variants in the exons of the MLH1 and MSH2 genes. The variants detected in the MLH1 and MSH2 genes had specific characteristics regarding the number, area of occurrence, and their relationship with demographic and clinicopathologic features. CONCLUSION: Overall, our results suggest that analysis of MLH1 and MSH2 genes alone is insufficient in the Iranian population, and more comprehensive tests are recommended for detecting LS.

Functional and phenotypic consequences of an unusual inversion in MSH2.

Lynch syndrome is an autosomal dominant disorder that usually results from a pathogenic germline variant in one of four genes (MSH2, MSH6, MLH1, PMS2) involved in DNA mismatch repair. Carriers of such variants are at risk of developing numerous cancers during adulthood. Here we report on a family suspected of having Lynch syndrome due to a history of endometrial adenocarcinoma, ovarian clear cell carcinoma, and adenocarcinoma of the duodenum in whom we identified a germline 29 nucleotide in-frame inversion in exon 3 of MSH2. We further show that this variant is almost completely absent at the protein level, and that the associated cancers have complete loss of MSH2 and MSH6 expression by immunohistochemistry. Functional investigation of this inversion in a laboratory setting revealed a resultant abnormal protein function. Thus, we have identified an unusual, small germline inversion in a mismatch repair gene that does not lead to a premature stop codon yet appears likely to be causal for the observed cancers.

MLH1 Promotor Hypermethylation in Colorectal and Endometrial Carcinomas from Patients with Lynch Syndrome.

Screening for Lynch syndrome (LS) in colorectal cancer (CRC) and endometrial cancer patients generally involves immunohistochemical staining of the mismatch repair (MMR) proteins. In case of MLH1 protein loss, MLH1 promotor hypermethylation (MLH1-PM) testing is performed to indirectly distinguish the constitutional MLH1 variants from somatic epimutations. Recently, multiple studies have reported that MLH1-PM and pathogenic constitutional MMR variants are not mutually exclusive. This study describes 6 new and 86 previously reported MLH1-PM CRCs or endometrial cancers in LS patients. Of these, methylation of the MLH1 gene promotor C region was reported in 30 MLH1, 6 MSH2, 6 MSH6, and 3 PMS2 variant carriers at a median age at diagnosis of 48.5 years [interquartile range (IQR), 39-56.75 years], 39 years (IQR, 29-51 years), 58 years (IQR, 53.5-67 years), and 68 years (IQR, 65.6-68.5 years), respectively. For 31 MLH1-PM CRCs in LS patients from the literature, only the B region of the MLH1 gene promotor was tested, whereas for 13 cases in the literature the tested region was not specified. Collectively, these data indicate that a diagnosis of LS should not be excluded when MLH1-PM is detected. Clinicians should carefully consider whether follow-up genetic MMR gene testing should be offered, with age <60 to 70 years and/or a positive family history among other factors being suggestive for a potential constitutional MMR gene defect.

De novo germline pathogenic variant in Lynch Syndrome: A rare event or the tip of the iceberg?

Lynch Syndrome is an autosomal dominant cancer predisposition syndrome caused by germline pathogenic variants or epimutation in one of the DNA mismatch repair genes. De novo pathogenic variants in mismatch repair genes have been described as a rare event in Lynch Syndrome (1-5%), although the prevalence of de novo pathogenic variants in Lynch Syndrome is probably underestimated. The de novo pathogenic variant was identified in a 26-year-old woman diagnosed with an adenocarcinoma of the caecum with mismatch repair protein deficiency at immunohistochemistry and a synchronous neuroendocrine tumor of the appendix with normal expression of mismatch repair proteins. DNA testing revealed deletion of exon 6 of the MLH1 gene. It appeared to be a de novo event, as the deletion was not detected in the patient's parents. The presence of a mosaicism in the patient was excluded and haplotype analysis demonstrated the paternal origin of the chromosome harboring the deletion. The de novo deletion probably originated either from a very early postzygotic or a single prezygotic mutational event, or from a gonadal mosaicism. In conclusion, the identification of de novo pathogenic variants is crucial to allow proper genetic counseling and appropriate management of the patient's family.

Comparison of immediate germline sequencing and multi-step screening for Lynch syndrome detection in high-risk endometrial and colorectal cancer patients.

OBJECTIVE: Lynch syndrome (LS) is a hereditary cancer predisposition syndrome with a significantly increased risk of colorectal and endometrial cancers. Current standard practice involves universal screening for LS in patients with newly diagnosed colorectal or endometrial cancer using a multi-step screening protocol (MSP). However, MSP may not always accurately identify LS cases. To address this limitation, we compared the diagnostic performance of immediate germline sequencing (IGS) with MSP in a high-risk group. METHODS: A total of 31 Taiwanese women with synchronous or metachronous endometrial and colorectal malignancies underwent MSP which included immunohistochemical staining of DNA mismatch repair (MMR) proteins, MLH1 promoter hypermethylation analysis, and germline sequencing to identify pathogenic variants. All patients who were excluded during MSP received germline sequencing for MMR genes to simulate IGS for the detection of LS. RESULTS: Our findings indicate that IGS surpassed MSP in terms of diagnostic yield (29.0% vs. 19.4%, respectively) and sensitivity (90% vs. 60%, respectively). Specifically, IGS successfully identified nine LS cases, which is 50% more than the number detected through MSP. Additionally, germline methylation analysis revealed one more LS case with constitutional MLH1 promoter hypermethylation, bringing the total LS cases to ten (32.3%). Intriguingly, we observed no significant differences in clinical characteristics or overall survival between patients with and without LS in our cohort. CONCLUSION: Our study suggests that IGS may potentially offer a more effective approach compared to MSP in identifying LS among high-risk patients. This advantage is evident when patients have been pre-selected utilizing specific clinical criteria.