Co‐occurrence of germline pathogenic variants for different hereditary cancer syndromes in patients with Lynch syndrome

Background: Lynch syndrome (LS) is a hereditary condition characterized by a high risk of colorectal cancer, endometrial cancer, and other neoplasia associated with germline alterations in DNA mismatch repair genes. The classical genetic diagnostic strategy for LS consists of the Sanger sequencing of genes associated with the suspected syndrome. Next‐generation sequencing (NGS) enables the simultaneous sequencing of a large number of hereditary cancer genes. Here, we aimed to study whether other germline pathogenic variants of hereditary cancer genes are present in patients with LS. Methods: A cohort of 84 probands with a previous genetic diagnosis of LS by Sanger sequencing was reanalyzed using NGS via a commercial panel of 94 hereditary cancer genes by hybrid capture. The American College of Medical Genetics and Genomics criteria were used to classify the clinical significance of the variants. The findings of NGS were confirmed by Sanger sequencing. When possible, genetic analyses of the new findings in the proband's relatives were also performed by Sanger sequencing. Results: We identified five families (6%), out of 84, with at least two germline pathogenic variants conferring to high or moderate risk in different dominant cancer‐predisposing genes: [MLH1‐BRCA2‐NBN], [MLH1‐BRCA1], [MSH2‐ATM], [MSH6‐NF1], and [MLH1‐FANCA]. Interestingly, only one out of these five families exhibited a clinical phenotype associated with the new pathogenic variants. The family with three pathogenic variants of the [MLH1‐BRCA2‐NBN] genes showed a high aggregation of tumors associated with LS and breast and ovarian cancer syndrome. Conclusions: Our results showed that the co‐occurrence of more than one pathogenic variant in cancer‐predisposing genes was remarkable among cases of LS. In most cases, no clinicial manifestations were associated with the secondary pathogenic variants. Further studies are needed to confirm these findings and elucidate their clinical impact. Reanalysis of LS families should be considered only in families with mixed clinical phenotypes.RFA is a recipient of a Fellowship from the Consellería Educación of the Valencian Community. The studies of ED and ACA are funded by the Acción Juvenil from Consellería Educación of the Valencian Community and the Spanish Ministry of Economy and Competitiveness, respectively. VDO is a recipient of a Fellowship from the Spanish Association Against Cancer (AECC). This work was supported by grants from the Foundation for the Promotion of Health and Biomedical Research in the Valencian Region, FISABIO (Grants: UGP‐14‐192 and UGP‐16‐146), and the Carlos III Health Institute (Grant AES: PI17/01082)

MLH1-methylated endometrial cancer under 60 years of age as the “sentinel” cancer in female carriers of high-risk constitutional MLH1 epimutation

Objective. Universal screening of endometrial carcinoma (EC) for mismatch repair deficiency (MMRd) and Lynch syndrome uses presence of MLH1 methylation to omit common sporadic cases from follow-up germline testing. However, this overlooks rare cases with high-risk constitutional MLH1 methylation (epimutation), a poorly-recognized mechanism that predisposes to Lynch-type cancers with MLH1 methylation. We aimed to de-termine the role and frequency of constitutional MLH1 methylation among EC cases with MMRd, MLH1- methylated tumors.Methods. We screened blood for constitutional MLH1 methylation using pyrosequencing and real-time methylation-specific PCR in patients with MMRd, MLH1-methylated EC ascertained from (i) cancer clinics (n = 4, <60 years), and (ii) two population-based cohorts; Columbus-area (n = 68, all ages) and Ohio Colo-rectal Cancer Prevention Initiative (OCCPI) (n = 24, <60 years).Results. Constitutional MLH1 methylation was identified in three out of four patients diagnosed between 36 and 59 years from cancer clinics. Two had mono-/hemiallelic epimutation (similar to 50% alleles methylated). One with multiple primaries had low-level mosaicism in normal tissues and somatic second-hits affecting the unmethylated allele in all tumors, demonstrating causation. In the population-based cohorts, all 68 cases from the Columbus-area cohort were negative and low-level mosaic constitutional MLH1 methylation was identified in one patient aged 36 years out of 24 from the OCCPI cohort, representing one of six (similar to 17%) patients <50 years and one of 45 patients (similar to 2%) <60 years in the combined cohorts. EC was the first/dual-first cancer in three pa-tients with underlying constitutional MLH1 methylation.Conclusions. A correct diagnosis at first presentation of cancer is important as it will significantly alter clinical management. Screening for constitutional MLH1 methylation is warranted in patients with early-onset EC or syn-chronous/metachronous tumors (any age) displaying MLH1 methylation.(c) 2023 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/)

Genetic and clinical characterization of a novel FH founder mutation in families with hereditary leiomyomatosis and renal cell cancer syndrome

Hereditary leiomyomatosis and renal cell cancer syndrome is a rare autosomal dominant hereditary syndrome. Previously, we published the largest cohort of FH mutation carriers in Spain and observed a highly recurrent missense heterozygous variant, FH(NM_000143.4):c.1118A > G p.(Asn373Ser), in 104 individuals from 31 apparently unrelated families. Here, we aimed to establish its founder effect and characterize the associated clinical phenotype. Results Haplotype analysis confirmed that families shared a common haplotype (32/38 markers) spanning 0.61-0.82 Mb, indicating this recurrent variant was inherited from a founder ancestor. Cutaneous and uterine leiomyomatosis were diagnosed in 64.6% (64/99) and 98% (50/51) of patients, respectively, and renal cell cancer was present in 10.4% (10/96). The pathogenic FH_c.1118A > G variant is a Spanish founder mutation that originated 12-26 generations ago. We estimate that the variant may have appeared between 1370 and 1720. Individuals carrying this founder mutation had similar frequency of renal cell cancer and a higher frequency of renal cysts and leiomyomas than those in other cohorts of this syndrome. Conclusions In the Spanish province of Alicante there is a high prevalence of HLRCC because of the founder mutation FH c.1118A > G; p.(Asn373Ser). The characterization of founder mutations provides accurate and specific information regarding their penetrance and expressivity. In individuals with suspected HLRCC from the province of Alicante, genetic testing by direct analysis of the founder FH c.1118A > G; p.(Asn373Ser) mutation may be a faster and more efficient diagnostic tool compared with complete gene sequencing

Primary constitutional MLH1 epimutations: a focal epigenetic event

BACKGROUND: Constitutional MLH1 epimutations are characterised by monoallelic methylation of the MLH1 promoter throughout normal tissues, accompanied by allele-specific silencing. The mechanism underlying primary MLH1 epimutations is currently unknown. The aim of this study was to perform an in-depth characterisation of constitutional MLH1 epimutations targeting the aberrantly methylated region around MLH1 and other genomic loci. METHODS: Twelve MLH1 epimutation carriers, 61 Lynch syndrome patients, and 41 healthy controls, were analysed by Infinium 450 K array. Targeted molecular techniques were used to characterise the MLH1 epimutation carriers and their inheritance pattern. RESULTS: No nucleotide or structural variants were identified in-cis on the epimutated allele in 10 carriers, in which inter- generational methylation erasure was demonstrated in two, suggesting primary type of epimutation. CNVs outside the MLH1 locus were found in two cases. EPM2AIP1-MLH1 CpG island was identified as the sole differentially methylated region in MLH1 epimutation carriers compared to controls. CONCLUSION: Primary constitutional MLH1 epimutations arise as a focal epigenetic event at the EPM2AIP1-MLH1 CpG island in the absence of cis-acting genetic variants. Further molecular characterisation is needed to elucidate the mechanistic basis of MLH1 epimutations and their heritability/reversibility

Highly sensitive MLH1 methylation analysis in blood identifies a cancer patient with low-level mosaic MLH1 epimutation

Constitutional MLH1 methylation (epimutation) is a rare cause of Lynch syndrome. Low-level methylation (<= 10%) has occasionally been described. This study aimed to identify low-level constitutional MLH1 epimutations and determine its causal role in patients with MLH1-hypermethylated colorectal cancer. Eighteen patients with MLH1-hypermethylated colorectal tumors in whom MLH1 methylation was previously undetected in blood by methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) were screened for MLH1 methylation using highly sensitive MS-melting curve analysis (MS-MCA). Constitutional methylation was characterized by different approaches. MS-MCA identified one patient (5.6%) with low-level MLH1 methylation ( 1%) in blood and other normal tissues, which was confirmed by clonal bisulfite sequencing in blood. The patient had developed three clonally related gastrointestinal MLH1-methylated tumor lesions at 22, 24, and 25 years of age. The methylated region in normal tissues overlapped with that reported for other carriers of constitutional MLH1 epimutations. Low-level MLH1 methylation and reduced allelic expression were linked to the same genetic haplotype, whereas the opposite allele was lost in patient's tumors. Mutation screening of MLH1 and other hereditary cancer genes was negative. Herein, a highly sensitive MS-MCA-based approach has demonstrated its utility for the identification of low-level constitutional MLH1 epigenetic mosaicism. The eventual identification and characterization of additional cases will be critical to ascertain the cancer risks associated with constitutional MLH1 epigenetic mosaicism

Comprehensive Constitutional Genetic and Epigenetic Characterization of Lynch-Like Individuals

The causal mechanism for cancer predisposition in Lynch-like syndrome (LLS) remains unknown. Our aim was to elucidate the constitutional basis of mismatch repair (MMR) deficiency in LLS patients throughout a comprehensive (epi)genetic analysis. One hundred and fifteen LLS patients harboring MMR-deficient tumors and no germline MMR mutations were included. Mutational analysis of 26 colorectal cancer (CRC)-associated genes was performed. Pathogenicity of MMR variants was assessed by splicing and multifactorial likelihood analyses. Genome-wide methylome analysis was performed by the Infinium Human Methylation 450K Bead Chip. The multigene panel analysis revealed the presence of two MMR gene truncating mutations not previously found. Of a total of 15 additional MMR variants identified, five -present in 6 unrelated individuals- were reclassified as pathogenic. In addition, 13 predicted deleterious variants in other CRC-predisposing genes were found in 12 probands. Methylome analysis detected one constitutionalMLH1epimutation, but no additional differentially methylated regions were identified in LLS compared to LS patients or cancer-free individuals. In conclusion, the use of an ad-hoc designed gene panel combined with pathogenicity assessment of variants allowed the identification of deleterious MMR mutations as well as new LLS candidate causal genes. Constitutional epimutations in non-LS-associated genes are not responsible for LLS

Identificación y caracterización de alteraciones epigenéticas responsables de síndrome de Lynch y Lynch-like

[spa] La deficiencia reparadora es una característica de los tumores de individuos con síndrome de Lynch, los cuales son portadores de mutaciones o epimutaciones en línea germinal de los genes reparadores de desapareamientos del DNA. Sin embargo, más del 50% de pacientes con tumores con deficiencia reparadora no pueden ser diagnosticados debido a la ausencia de mutaciones germinales que expliquen su predisposición, siendo considerados pacientes “Lynch-like”. Nuestra primera hipótesis es que, en estos pacientes, la presencia de epimutaciones germinales podría ser responsable en algunos casos de la predisposición a desarrollar tumores. Por su parte, la epimutación primaria en MLH1 aparece de novo en el individuo y no sigue un patrón de herencia mendeliano. Sus portadores están considerados individuos con síndrome de Lynch, sin embargo, el riesgo de los familiares de primer grado es desconocido. Estos pacientes, además, presentan mucha variabilidad en el porcentaje de metilación presente en el promotor de MLH1, lo que se conoce como mosaicismo epigenético, y que en ocasiones dificulta la también la identificación de los pacientes portadores. También hipotetizamos que una mejor identificación y la caracterización exhaustiva de las epimutaciones constitucionales en MLH1 permitirá mejorar el manejo clínico de estos pacientes y su consejo genético. Así pues, los objetivos de este trabajo han sido buscar la presencia de alteraciones epigenéticas constitucionales responsables de síndrome de Lynch en individuos Lynch-like e identificar y caracterizar molecularmente epimutaciones constitucionales en MLH1. Para ello, hemos realizado un análisis exhaustivo de 121 individuos y 12 pacientes con epimutación en MLH1, tanto a nivel genético como epigenético y hemos estudiado mediante técnicas de alta sensibilidad la metilación constitucional en 22 pacientes con tumores MLH1- metilados menores de 50 años. En la serie Lynch-like, hemos identificado un portador de epimutación en MLH1, 3 individuos con mutaciones no identificadas en los genes reparadores y 19 variantes de significado desconocido en genes reparadores de las cuales reclasificamos 8, diagnosticando a 10 familias de nuestra serie y 5 adicionales. El estudio somático dde 5 tumores encontró dobles mutaciones somáticas en 2 de ellos. También identificamos variantes patogénicas y predichas patogénicas por su análisis in-silico en otros genes de predisposición a cáncer colorrectal: MUTYH, BUB1, SETD2, FAN1, EXO1, MSH3, APC y POLD1Las alteraciones constitucionales epigenéticas fuera de los genes reparadores no parecen tener ningún papel en la patología Lynch-like. La caracterización genética de los portadores de epimutación en MLH1 permitió su clasificación como epimutación primaria, demostrando la aparición de novo de la epimutación en 3 casos y el borrado intergeracional en un nuevo caso. El estudio del metiloma de estos pacientes puso de manifiesto que las mutaciones primarias en MLH1 son un evento epigenético focal de extensión constante (Chr3: 37,033,791-37,035,400; 1,6Kb), específico de este tipo de pacientes y mantenido tanto en los tejidos normales del individuo como sus tumores colorrectales. Estos resultados apoyan que exista un mecanismo causal subyacente vinculado a las reprogramaciones epigenéticas de los primeros estadios de la vida humana. La puesta a punto de una técnica de alta sensibilidad permitió la identificación de un nuevo caso de epimutación en MLH1 con un 1% de metilación germinal, demostrando que las epimutaciones pueden tener una prevalencia más elevada de la reportada hasta el momento, debido al infradiagnóstico de pacientes con mosaicismo germinal, lo que nos ha llevado a proponer una modificación del algoritmo diagnóstico para epimutaciones en síndrome de Lynch. El presente estudio ofrece una perspectiva amplia sobre la diversidad de mecanismos causales que pueden dar lugar a la deficiencia reparadora en tumores y destaca la relevancia de estudiar de manera exhaustiva todos los pacientes con el fin de poder identificar todos los individuos con una predisposición genética o epigenética al cáncer.[eng] Lynch-like syndrome individuals develop MMR deficient tumors but no germline mutations or epimutations at MMR genes have been identified. Our first hypothesis is that their cancer predisposition may be caused in some of them by unidentified epimutations in cancer predisposition genes. Primary MLH1 epimutation carriers are considered as Lynch syndrome patients. However, it is not possible to estimate the risk for their first-degree relatives due to the complex patterns of inheritance. A better understanding of MLH1 epimutations will benefit the clinical management of these families. We aimed to search for the presence of constitutional epimutations responsible of Lynch-like predisposition and to deeply characterize MLH1 epimutations identified. 121 Lynch-like patients and 12 MLH1 epimutation carriers were submitted to a exhaustive genetic and epigenetic characterization. In addition, 22 patients with MLH1-methylated tumors were analyzed by high-sensitive methylation detection techniques. In three patients MMR pathogenic variants not previously identified were found. Nineteen MMR VUS were identified and we performed the reclassification of 8 of them, giving diagnostic for a total of 15 families. Double somatic mutations were found in 2 of 5 analyzed tumors. A new case of MLH1 epimutation was identified. Pathogenic or predicted pathogenic variants were discovered in new-proposed cancer predisposition genes in a subset of Lynch-like cases. However, epimutations outside MMR genes do not seem to play a role. MLH1 epimutation was found as a focal epigenetic event, with constant length throughout tissues and specific of MLH1 epimutation carriers, suggesting a common causal mechanism. High sensitivity methylation techniques allow the identification of a new case of MLH1 epimutation with epigenetic mosaicism (≈1% methylation). The current study provides a global perspective on the heterogeneity of causal mechanisms leading to MMR deficiency and highlights the relevance of comprehensive characterization to diagnose individuals with genetic or epigenetic predisposition to Lynch syndrome

Identificación y caracterización de alteraciones epigenéticas responsables de síndrome de Lynch y Lynch-like

La deficiencia reparadora es una característica de los tumores de individuos con síndrome de Lynch, los cuales son portadores de mutaciones o epimutaciones en línea germinal de los genes reparadores de desapareamientos del DNA. Sin embargo, más del 50% de pacientes con tumores con deficiencia reparadora no pueden ser diagnosticados debido a la ausencia de mutaciones germinales que expliquen su predisposición, siendo considerados pacientes “Lynch-like”. Nuestra primera hipótesis es que, en estos pacientes, la presencia de epimutaciones germinales podría ser responsable en algunos casos de la predisposición a desarrollar tumores. Por su parte, la epimutación primaria en MLH1 aparece de novo en el individuo y no sigue un patrón de herencia mendeliano. Sus portadores están considerados individuos con síndrome de Lynch, sin embargo, el riesgo de los familiares de primer grado es desconocido. Estos pacientes, además, presentan mucha variabilidad en el porcentaje de metilación presente en el promotor de MLH1, lo que se conoce como mosaicismo epigenético, y que en ocasiones dificulta la también la identificación de los pacientes portadores. También hipotetizamos que una mejor identificación y la caracterización exhaustiva de las epimutaciones constitucionales en MLH1 permitirá mejorar el manejo clínico de estos pacientes y su consejo genético. Así pues, los objetivos de este trabajo han sido buscar la presencia de alteraciones epigenéticas constitucionales responsables de síndrome de Lynch en individuos Lynch-like e identificar y caracterizar molecularmente epimutaciones constitucionales en MLH1. Para ello, hemos realizado un análisis exhaustivo de 121 individuos y 12 pacientes con epimutación en MLH1, tanto a nivel genético como epigenético y hemos estudiado mediante técnicas de alta sensibilidad la metilación constitucional en 22 pacientes con tumores MLH1- metilados menores de 50 años. En la serie Lynch-like, hemos identificado un portador de epimutación en MLH1, 3 individuos con mutaciones no identificadas en los genes reparadores y 19 variantes de significado desconocido en genes reparadores de las cuales reclasificamos 8, diagnosticando a 10 familias de nuestra serie y 5 adicionales. El estudio somático dde 5 tumores encontró dobles mutaciones somáticas en 2 de ellos. También identificamos variantes patogénicas y predichas patogénicas por su análisis in-silico en otros genes de predisposición a cáncer colorrectal: MUTYH, BUB1, SETD2, FAN1, EXO1, MSH3, APC y POLD1Las alteraciones constitucionales epigenéticas fuera de los genes reparadores no parecen tener ningún papel en la patología Lynch-like. La caracterización genética de los portadores de epimutación en MLH1 permitió su clasificación como epimutación primaria, demostrando la aparición de novo de la epimutación en 3 casos y el borrado intergeracional en un nuevo caso. El estudio del metiloma de estos pacientes puso de manifiesto que las mutaciones primarias en MLH1 son un evento epigenético focal de extensión constante (Chr3: 37,033,791-37,035,400; 1,6Kb), específico de este tipo de pacientes y mantenido tanto en los tejidos normales del individuo como sus tumores colorrectales. Estos resultados apoyan que exista un mecanismo causal subyacente vinculado a las reprogramaciones epigenéticas de los primeros estadios de la vida humana. La puesta a punto de una técnica de alta sensibilidad permitió la identificación de un nuevo caso de epimutación en MLH1 con un 1% de metilación germinal, demostrando que las epimutaciones pueden tener una prevalencia más elevada de la reportada hasta el momento, debido al infradiagnóstico de pacientes con mosaicismo germinal, lo que nos ha llevado a proponer una modificación del algoritmo diagnóstico para epimutaciones en síndrome de Lynch. El presente estudio ofrece una perspectiva amplia sobre la diversidad de mecanismos causales que pueden dar lugar a la deficiencia reparadora en tumores y destaca la relevancia de estudiar de manera exhaustiva todos los pacientes con el fin de poder identificar todos los individuos con una predisposición genética o epigenética al cáncer.Lynch-like syndrome individuals develop MMR deficient tumors but no germline mutations or epimutations at MMR genes have been identified. Our first hypothesis is that their cancer predisposition may be caused in some of them by unidentified epimutations in cancer predisposition genes. Primary MLH1 epimutation carriers are considered as Lynch syndrome patients. However, it is not possible to estimate the risk for their first-degree relatives due to the complex patterns of inheritance. A better understanding of MLH1 epimutations will benefit the clinical management of these families. We aimed to search for the presence of constitutional epimutations responsible of Lynch-like predisposition and to deeply characterize MLH1 epimutations identified. 121 Lynch-like patients and 12 MLH1 epimutation carriers were submitted to a exhaustive genetic and epigenetic characterization. In addition, 22 patients with MLH1-methylated tumors were analyzed by high-sensitive methylation detection techniques. In three patients MMR pathogenic variants not previously identified were found. Nineteen MMR VUS were identified and we performed the reclassification of 8 of them, giving diagnostic for a total of 15 families. Double somatic mutations were found in 2 of 5 analyzed tumors. A new case of MLH1 epimutation was identified. Pathogenic or predicted pathogenic variants were discovered in new-proposed cancer predisposition genes in a subset of Lynch-like cases. However, epimutations outside MMR genes do not seem to play a role. MLH1 epimutation was found as a focal epigenetic event, with constant length throughout tissues and specific of MLH1 epimutation carriers, suggesting a common causal mechanism. High sensitivity methylation techniques allow the identification of a new case of MLH1 epimutation with epigenetic mosaicism (≈1% methylation). The current study provides a global perspective on the heterogeneity of causal mechanisms leading to MMR deficiency and highlights the relevance of comprehensive characterization to diagnose individuals with genetic or epigenetic predisposition to Lynch syndrome

Identificación y caracterización de alteraciones epigenéticas responsables de síndrome de Lynch y Lynch-like

La deficiencia reparadora es una característica de los tumores de individuos con síndrome de Lynch, los cuales son portadores de mutaciones o epimutaciones en línea germinal de los genes reparadores de desapareamientos del DNA. Sin embargo, más del 50% de pacientes con tumores con deficiencia reparadora no pueden ser diagnosticados debido a la ausencia de mutaciones germinales que expliquen su predisposición, siendo considerados pacientes “Lynch-like”. Nuestra primera hipótesis es que, en estos pacientes, la presencia de epimutaciones germinales podría ser responsable en algunos casos de la predisposición a desarrollar tumores. Por su parte, la epimutación primaria en MLH1 aparece de novo en el individuo y no sigue un patrón de herencia mendeliano. Sus portadores están considerados individuos con síndrome de Lynch, sin embargo, el riesgo de los familiares de primer grado es desconocido. Estos pacientes, además, presentan mucha variabilidad en el porcentaje de metilación presente en el promotor de MLH1, lo que se conoce como mosaicismo epigenético, y que en ocasiones dificulta la también la identificación de los pacientes portadores. También hipotetizamos que una mejor identificación y la caracterización exhaustiva de las epimutaciones constitucionales en MLH1 permitirá mejorar el manejo clínico de estos pacientes y su consejo genético. Así pues, los objetivos de este trabajo han sido buscar la presencia de alteraciones epigenéticas constitucionales responsables de síndrome de Lynch en individuos Lynch-like e identificar y caracterizar molecularmente epimutaciones constitucionales en MLH1. Para ello, hemos realizado un análisis exhaustivo de 121 individuos y 12 pacientes con epimutación en MLH1, tanto a nivel genético como epigenético y hemos estudiado mediante técnicas de alta sensibilidad la metilación constitucional en 22 pacientes con tumores MLH1- metilados menores de 50 años. En la serie Lynch-like, hemos identificado un portador de epimutación en MLH1, 3 individuos con mutaciones no identificadas en los genes reparadores y 19 variantes de significado desconocido en genes reparadores de las cuales reclasificamos 8, diagnosticando a 10 familias de nuestra serie y 5 adicionales. El estudio somático dde 5 tumores encontró dobles mutaciones somáticas en 2 de ellos. También identificamos variantes patogénicas y predichas patogénicas por su análisis in-silico en otros genes de predisposición a cáncer colorrectal: MUTYH, BUB1, SETD2, FAN1, EXO1, MSH3, APC y POLD1Las alteraciones constitucionales epigenéticas fuera de los genes reparadores no parecen tener ningún papel en la patología Lynch-like. La caracterización genética de los portadores de epimutación en MLH1 permitió su clasificación como epimutación primaria, demostrando la aparición de novo de la epimutación en 3 casos y el borrado intergeracional en un nuevo caso. El estudio del metiloma de estos pacientes puso de manifiesto que las mutaciones primarias en MLH1 son un evento epigenético focal de extensión constante (Chr3: 37,033,791-37,035,400; 1,6Kb), específico de este tipo de pacientes y mantenido tanto en los tejidos normales del individuo como sus tumores colorrectales. Estos resultados apoyan que exista un mecanismo causal subyacente vinculado a las reprogramaciones epigenéticas de los primeros estadios de la vida humana. La puesta a punto de una técnica de alta sensibilidad permitió la identificación de un nuevo caso de epimutación en MLH1 con un 1% de metilación germinal, demostrando que las epimutaciones pueden tener una prevalencia más elevada de la reportada hasta el momento, debido al infradiagnóstico de pacientes con mosaicismo germinal, lo que nos ha llevado a proponer una modificación del algoritmo diagnóstico para epimutaciones en síndrome de Lynch. El presente estudio ofrece una perspectiva amplia sobre la diversidad de mecanismos causales que pueden dar lugar a la deficiencia reparadora en tumores y destaca la relevancia de estudiar de manera exhaustiva todos los pacientes con el fin de poder identificar todos los individuos con una predisposición genética o epigenética al cáncer.Lynch-like syndrome individuals develop MMR deficient tumors but no germline mutations or epimutations at MMR genes have been identified. Our first hypothesis is that their cancer predisposition may be caused in some of them by unidentified epimutations in cancer predisposition genes. Primary MLH1 epimutation carriers are considered as Lynch syndrome patients. However, it is not possible to estimate the risk for their first-degree relatives due to the complex patterns of inheritance. A better understanding of MLH1 epimutations will benefit the clinical management of these families. We aimed to search for the presence of constitutional epimutations responsible of Lynch-like predisposition and to deeply characterize MLH1 epimutations identified. 121 Lynch-like patients and 12 MLH1 epimutation carriers were submitted to a exhaustive genetic and epigenetic characterization. In addition, 22 patients with MLH1-methylated tumors were analyzed by high-sensitive methylation detection techniques. In three patients MMR pathogenic variants not previously identified were found. Nineteen MMR VUS were identified and we performed the reclassification of 8 of them, giving diagnostic for a total of 15 families. Double somatic mutations were found in 2 of 5 analyzed tumors. A new case of MLH1 epimutation was identified. Pathogenic or predicted pathogenic variants were discovered in new-proposed cancer predisposition genes in a subset of Lynch-like cases. However, epimutations outside MMR genes do not seem to play a role. MLH1 epimutation was found as a focal epigenetic event, with constant length throughout tissues and specific of MLH1 epimutation carriers, suggesting a common causal mechanism. High sensitivity methylation techniques allow the identification of a new case of MLH1 epimutation with epigenetic mosaicism (≈1% methylation). The current study provides a global perspective on the heterogeneity of causal mechanisms leading to MMR deficiency and highlights the relevance of comprehensive characterization to diagnose individuals with genetic or epigenetic predisposition to Lynch syndrome

Comprehensive Constitutional Genetic and Epigenetic Characterization of Lynch-Like Individuals

Síndrome de Lynch; Panell de gens del càncer; EpimutacióSíndrome de Lynch; Panel de genes del cáncer; EpimutaciónLynch syndrome; Cancer genes panel; EpimutationThe causal mechanism for cancer predisposition in Lynch-like syndrome (LLS) remains unknown. Our aim was to elucidate the constitutional basis of mismatch repair (MMR) deficiency in LLS patients throughout a comprehensive (epi)genetic analysis. One hundred and fifteen LLS patients harboring MMR-deficient tumors and no germline MMR mutations were included. Mutational analysis of 26 colorectal cancer (CRC)-associated genes was performed. Pathogenicity of MMR variants was assessed by splicing and multifactorial likelihood analyses. Genome-wide methylome analysis was performed by the Infinium Human Methylation 450K Bead Chip. The multigene panel analysis revealed the presence of two MMR gene truncating mutations not previously found. Of a total of 15 additional MMR variants identified, five -present in 6 unrelated individuals- were reclassified as pathogenic. In addition, 13 predicted deleterious variants in other CRC-predisposing genes were found in 12 probands. Methylome analysis detected one constitutional MLH1 epimutation, but no additional differentially methylated regions were identified in LLS compared to LS patients or cancer-free individuals. In conclusion, the use of an ad-hoc designed gene panel combined with pathogenicity assessment of variants allowed the identification of deleterious MMR mutations as well as new LLS candidate causal genes. Constitutional epimutations in non-LS-associated genes are not responsible for LLS.This work was funded by the Spanish Ministry of Economy and Competitiveness and cofunded by FEDER funds -a way to build Europe- (grants SAF2012-33636, SAF2015-68016-R and SAF2016-80888-R), CIBERONC, RTICC Network (RD12/0036/0031 and RD12/0036/0008), the Spanish Association Against Cancer (AECC) (080253), the Government of Catalonia (grant 2014SGR338, 2017SGR1282 and PERIS SLT002/16/0037), Fundación Mutua Madrileña (grant AP114252013). We thank CERCA Programme for institutional support. ED was supported by a grant from the Spanish Ministry of Economy and Competitiveness. The AECC fellowship to MG-A. AF was supported by a grant from the Catalonian Health Department (SLT002/16/00409). FM was supported by CIBERONC. The Mexican National Council for Science and Technology (CONACyT) fellowship to GV