Identificación y caracterización de nuevos genes de predisposición al cáncer colorrectal familiar

[spa] INTRODUCCIÓN El cáncer colorrectal (CCR) representa una de las neoplasias más frecuentes a nivel mundial. Los casos de CCR se pueden clasificar en formas esporádicas, hereditarias y familiares en base a la agregación familiar. Las formas hereditarias están causadas por mutaciones germinales de alta penetrancia en genes conocidos que se transmiten en la familia, provocando una fuerte agregación y aparición temprana de la enfermedad. El CCR familiar se caracteriza por la presencia de cierta agregación y causas genéticas de predisposición desconocidas, siendo en algunos casos la agregación familiar tan fuerte como en las formas hereditarias. Estos casos son sugestivos de estar causados por variantes de alta penetrancia en genes aún por identificar. Para detectar la causa germinal de predisposición en las familias con agregación para CCR existen distintas estrategias, siendo la secuenciación de nueva generación una manera de analizar grandes cantidades de datos de forma rápida y económica. La secuenciación del exoma, consistente en secuenciar toda la región codificante del genoma, es una aproximación útil para detectar variantes de alta penetrancia causantes de enfermedades con herencia mendeliana. OBJETIVOS 1) Identificación de nuevos genes candidatos implicados en cáncer colorrectal familiar mediante secuenciación completa del exoma. 2) Implementación de una metodología automática de filtrado y priorización de variantes genéticas a partir de los datos de secuenciación del exoma. 3) Búsqueda de nuevas vías de señalización relacionadas con la aparición de cáncer colorrectal familiar. 4) Cribado de los genes POLE y POLD1 en pacientes con múltiples pólipos o CCR de aparición temprana. ARTÍCULOS (METODOLOGÍA Y RESULTADOS) La metodología y resultados de esta tesis se recogen en tres artículos, siendo los dos primeros estudios de secuenciación del exoma en familias con fuerte agregación para CCR y sin alteraciones germinales en los genes hereditarios APC, MUTYH o los genes de Lynch (Whole-exome sequencing identifies rare pathogenic variants in new predisposition genes for familial colorectal cancer. Genetics in Medicine 2015;17:131-42; The Fanconi anemia DNA damage repair pathway in the spotlight for germline predisposition to colorectal cancer). European Journal of Human Genetics 2016 May 11 doi: 10.1038/ejhg.2016.44). Por otro lado, el tercer estudio se basa en la búsqueda de mutaciones en el dominio exonucleasa de los nuevos genes hereditarios POLE y POLD1 en 155 casos con múltiples pólipos o CCR de aparición temprana mediante secuenciación convencional Sanger (POLE and POLD1 screening in 155 patients with multiple polyps and early-onset colorectal cancer. Scientific reports, en revisión). DISCUSIÓN Los dos primeros estudios, al tener una metodología similar debido a que las técnicas realizadas son las mismas con pequeñas modificaciones en el análisis de datos, se comentan juntos. Se argumenta la adecuación de la tecnología utilizada, la selección de pacientes, la implementación de la metodología de anotación y filtrado de variantes, los genes candidatos de predisposición identificados mediante estos estudios y por último qué estudios adicionales se podrían realizar para reforzar la evidencia de estos descubrimientos. La discusión del tercer artículo incluye la tecnología utilizada, las características de los pacientes secuenciados y las variantes detectadas en nuestro estudio (incluyendo una variante de cambio aminoacídico para la cual se realizan múltiples experimentos para comprobar su patogenicidad).[eng] Colorectal cancer (CRC) is one of the most common tumours and an important cause of mortality in the developed world. It is caused by environmental and genetic factors, with 35% of the variation in CRC susceptibility probably explained by inherited causes. The best known examples of inherited CRC predisposition are Mendelian forms such as Lynch syndrome and familial adenomatous polyposis. They account for ~5% of all cases, and are due to germline mutations in APC, MUTYH and the mismatch repair (MMR) genes, which confer a high risk of developing this disease. However, there is still a considerable number of cases with strong familial CRC aggregation and early disease onset with an unknown inherited genetic basis. Next generation sequencing is a good strategy to identify the germline predisposition cause in these cases, being exome sequencing a cost-efective approximation, useful for detecting high penetrance variants. This thesis is focused on the identification of new CRC predisposition genes through whole-exome sequencing in families with a strong aggregation for CRC and no alterations in the genes responsible for the classical hereditary forms. This strategy led to the publication of two papers: * Whole-exome sequencing identifies rare pathogenic variants in new predisposition genes for familial colorectal cáncer. Genetics in Medicine 2015;17:131-42 * The Fanconi anemia DNA damage repair pathway in the spotlight for germline predisposition to colorectal cancer. European Journal of Human Genetics 2016 May 11 doi: 10.1038/ejhg.2016.44 On the other hand, another study was conducted with the aim to find pathogenic variants in the exonuclease domain of the recently discovered hereditary genes POLE and POLD1, in a cohort of 155 Spanish patients, using conventional Sanger sequencing. A paper including the results of this study has been submitted * POLE and POLD1 screening in 155 patients with multiple polyps and early-onset colorectal cáncer. Scientific Reports (under revision

BRIP1, a Gene Potentially Implicated in Familial Colorectal Cancer Type X

Familial colorectal cancer Type X (FCCTX) comprises a heterogeneous group of families with an increased risk of developing colorectal cancer and other related tumors, but with mismatch repair–proficient, microsatellite-stable (MSS) tumors. Unfortunately, the genetic basis underlying their cancer predisposition remains unknown. Although pathogenic germline variants in BRIP1 increase the risk of developing hereditary ovarian cancer, the involvement of BRIP1 in hereditary colorectal cancer is still not well known. In order to identify new BRIP1 variants associated with inherited colorectal cancer, affected and nonaffected individuals from 18 FCCTX or high-risk MSS colorectal cancer families were evaluated by whole-exome sequencing, and another 62 colorectal cancer patients from FCCTX or high-risk MSS colorectal cancer families were screened by a next-generation sequencing (NGS) multigene panel. The families were recruited at the Genetic Counseling Unit of Hospital Clínico San Carlos of Madrid. A total of three different BRIP1 mutations in three unrelated families were identified. Among them, there were two frameshift variants [c.1702_1703del, p.(Asn568TrpfsTer9) and c.903del, p.(Leu301PhefsTer2)] that result in the truncation of the protein and are thus classified as pathogenic (class 5). The remaining was a missense variant [c.2220G>T, p.(Gln740His)] considered a variant of uncertain significance (class 3). The segregation and lossof-heterozygosity studies provide evidence linking the two BRIP1 frameshift variants to colorectal cancer risk, with suggestive but not definitive evidence that the third variant may be benign. The results here presented suggest that germline BRIP1 pathogenic variants could be associated with hereditary colorectal cancer predisposition

Germline Mutations in FAF1 Are Associated With Hereditary Colorectal Cancer

Background & aims: A significant proportion of colorectal cancer (CRC) cases have familial aggregation but little is known about the genetic factors that contribute to these cases. We performed an exhaustive functional characterization of genetic variants associated with familial CRC. Methods: We performed whole-exome sequencing analyses of 75 patients from 40 families with a history of CRC (including early-onset cases) of an unknown germline basis (discovery cohort). We also sequenced specific genes in DNA from an external replication cohort of 473 families, including 488 patients with colorectal tumors that had normal expression of mismatch repair proteins (validation cohort). We disrupted the Fas-associated factor 1 gene (FAF1) in DLD-1 CRC cells using CRISPR/Cas9 gene editing; some cells were transfected with plasmids that express FAF1 missense variants. Cells were analyzed by immunoblots, quantitative real-time polymerase chain reaction, and functional assays monitoring apoptosis, proliferation, and assays for Wnt signaling or nuclear factor (NF)-kappa-B activity. Results: We identified predicted pathogenic variant in the FAF1 gene (c.1111G>A; p.Asp371Asn) in the discovery cohort; it was present in 4 patients of the same family. We identified a second variant in FAF1 in the validation cohort (c.254G>C; p.Arg85Pro). Both variants encoded unstable FAF1 proteins. Expression of these variants in CRC cells caused them to become resistant to apoptosis, accumulate beta-catenin in the cytoplasm, and translocate NF-kappa-B to the nucleus. Conclusions: In whole-exome sequencing analyses of patients from families with a history of CRC, we identified variants in FAF1 that associate with development of CRC. These variants encode unstable forms of FAF1 that increase resistance of CRC cells to apoptosis and increase activity of beta-catenin and NF-kappa-B

Integrated Analysis of Germline and Tumor DNA Identifies New Candidate Genes Involved in Familial Colorectal Cancer

Colorectal cancer (CRC) shows aggregation in some families but no alterations in the known hereditary CRC genes. We aimed to identify new candidate genes which are potentially involved in germline predisposition to familial CRC. An integrated analysis of germline and tumor whole-exome sequencing data was performed in 18 unrelated CRC families. Deleterious single nucleotide variants (SNV), short insertions and deletions (indels), copy number variants (CNVs) and loss of heterozygosity (LOH) were assessed as candidates for first germline or second somatic hits. Candidate tumor suppressor genes were selected when alterations were detected in both germline and somatic DNA, fulfilling Knudson's two-hit hypothesis. Somatic mutational profiling and signature analysis were also performed. A series of germline-somatic variant pairs were detected. In all cases, the first hit was presented as a rare SNV/indel, whereas the second hit was either a different SNV (3 genes) or LOH affecting the same gene (141 genes). BRCA2, BLM, ERCC2, RECQL, REV3L and RIF1 were among the most promising candidate genes for germline CRC predisposition. The identification of new candidate genes involved in familial CRC could be achieved by our integrated analysis. Further functional studies and replication in additional cohorts are required to confirm the selected candidates

New genes emerging for colorectal cancer predisposition.

Colorectal cancer (CRC) is one of the most frequent neoplasms and an important cause of mortality in the developed world. This cancer is caused by both genetic and environmental factors although 35% of the variation in CRC susceptibility involves inherited genetic differences. Mendelian syndromes account for about 5% of the total burden of CRC, with Lynch syndrome and familial adenomatous polyposis the most common forms. Excluding hereditary forms, there is an important fraction of CRC cases that present familial aggregation for the disease with an unknown germline genetic cause. CRC can be also considered as a complex disease taking into account the common disease-commom variant hypothesis with a polygenic model of inheritance where the genetic components of common complex diseases correspond mostly to variants of low/moderate effect. So far, 30 common, low-penetrance susceptibility variants have been identified for CRC. Recently, new sequencing technologies including exome- and whole-genome sequencing have permitted to add a new approach to facilitate the identification of new genes responsible for human disease predisposition. By using whole-genome sequencing, germline mutations in the POLE and POLD1 genes have been found to be responsible for a new form of CRC genetic predisposition called polymerase proofreading-associated polyposis

The MLH1 c.1852_1853delinsGC (p.K618A) variant in colorectal cancer: genetic association study in 18,723 individuals.

Colorectal cancer is one of the most frequent neoplasms and an important cause of mortality in the developed world. Mendelian syndromes account for about 5% of the total burden of CRC, being Lynch syndrome and familial adenomatous polyposis the most common forms. Lynch syndrome tumors develop mainly as a consequence of defective DNA mismatch repair associated with germline mutations in MLH1, MSH2, MSH6 and PMS2. A significant proportion of variants identified by screening these genes correspond to missense or noncoding changes without a clear pathogenic consequence, and they are designated as "variants of uncertain significance", being the c.1852_1853delinsGC (p.K618A) variant in the MLH1 gene a clear example. The implication of this variant as a low-penetrance risk variant for CRC was assessed in the present study by performing a case-control study within a large cohort from the COGENT consortium-COST Action BM1206 including 18,723 individuals (8,055 colorectal cancer cases and 10,668 controls) and a case-only genotype-phenotype correlation with several clinical and pathological characteristics restricted to the Epicolon cohort. Our results showed no involvement of this variant as a low-penetrance variant for colorectal cancer genetic susceptibility and no association with any clinical and pathological characteristics including family history for this neoplasm or Lynch syndrome

The MLH1 c.1852_1853delinsGC (p.K618A) Variant in Colorectal Cancer:Genetic Association Study in 18,723 Individuals

Colorectal cancer is one of the most frequent neoplasms and an important cause of mortality in the developed world. Mendelian syndromes account for about 5% of the total burden of CRC, being Lynch syndrome and familial adenomatous polyposis the most common forms. Lynch syndrome tumors develop mainly as a consequence of defective DNA mismatch repair associated with germline mutations in MLH1, MSH2, MSH6 and PMS2. A significant proportion of variants identified by screening these genes correspond to missense or noncoding changes without a clear pathogenic consequence, and they are designated as "variants of uncertain significance", being the c.1852_1853delinsGC (p.K618A) variant in the MLH1 gene a clear example. The implication of this variant as a low-penetrance risk variant for CRC was assessed in the present study by performing a case-control study within a large cohort from the COGENT consortium-COST Action BM1206 including 18,723 individuals (8,055 colorectal cancer cases and 10,668 controls) and a case-only genotype-phenotype correlation with several clinical and pathological characteristics restricted to the Epicolon cohort. Our results showed no involvement of this variant as a low-penetrance variant for colorectal cancer genetic susceptibility and no association with any clinical and pathological characteristics including family history for this neoplasm or Lynch syndrome

Whole-exome sequencing identifies rare pathogenic variants in new predisposition genes for familial colorectal cancer

Purpose. Colorectal cancer is an important cause of mortality in the developed world. Hereditary forms are due to germ-line mutations in APC, MUTYH, and the mismatch repair genes, but many cases present familial aggregation but an unknown inherited cause. The hypothesis of rare high-penetrance mutations in new genes is a likely explanation for the underlying predisposition in some of these familial cases. Methods. Exome sequencing was performed in 43 patients with colorectal cancer from 29 families with strong disease aggregation without mutations in known hereditary colorectal cancer genes. Data analysis selected only very rare variants (0–0.1%), producing a putative loss of function and located in genes with a role compatible with cancer. Variants in genes previously involved in hereditary colorectal cancer or nearby previous colorectal cancer genome-wide association study hits were also chosen. Results. Twenty-eight final candidate variants were selected and validated by Sanger sequencing. Correct family segregation and somatic studies were used to categorize the most interesting variants in CDKN1B, XRCC4, EPHX1, NFKBIZ, SMARCA4, and BARD1. Conclusion. We identified new potential colorectal cancer predisposition variants in genes that have a role in cancer predisposition and are involved in DNA repair and the cell cycle, which supports their putative involvement in germ-line predisposition to this neoplasm.We are sincerely grateful to the Centre Nacional d'Anàlisi Genòmica and the Biobank of Hospital Clínic–IDIBAPS, Barcelona, for technical help, and the International Cancer Genome Consortium for access to exome data set. The work was carried out (in part) at the Esther Koplowitz Centre, Barcelona. CEJ and JM are supported by a contract from CIBERehd. MVC is supported by Ministerio de Educación, Cultura y Deporte (FPU12/05138). PG and SCB are supported by a contract from the Fondo de Investigación Sanitaria (JR13/00013 and CP 03-0070, respectively). CIBERehd and CIBERER are funded by the Instituto de Salud Carlos III. This work was supported by grants from the Fondo de Investigación Sanitaria/FEDER (10/00641, 11/00219, 11/00681, RD12/0036/006, 13/02588), the Ministerio de Economía y Competitividad (SAF2010-19273), Fundación Científica de la Asociación Española contra el Cáncer (GCB13131592CAST), COST Action BM1206 (SCB and CRP), Beca Grupo de Trabajo “Oncología” AEG (Asociación Española de Gastroenterología), and Agència de Gestió d'Ajuts Universitaris i de Recerca (Generalitat de Catalunya, 2014SGR255)

TOPIC HIGHLIGHT New genes emerging for colorectal cancer predisposition

Abstract Colorectal cancer (CRC) is one of the most frequent neoplasms and an important cause of mortality in the developed world. This cancer is caused by both genetic and environmental factors although 35% of the variation in CRC susceptibility involves inherited genetic differences. Mendelian syndromes account for about 5% of the total burden of CRC, with Lynch syndrome and familial adenomatous polyposis the most common forms. Excluding hereditary forms, there is an important fraction of CRC cases that present familial aggregation for the disease with an unknown germline genetic cause. CRC can be also considered as a complex disease taking into account the common diseasecommom variant hypothesis with a polygenic model of inheritance where the genetic components of common complex diseases correspond mostly to variants of low/ moderate effect. So far, 30 common, low-penetrance susceptibility variants have been identified for CRC. Recently, new sequencing technologies including exomeand whole-genome sequencing have permitted to add New genes emerging for colorectal cancer predisposition World J Gastroenterol 2014 February 28; 20(8): 1961-1971 ISSN 1007-9327 (print) ISSN 2219-2840 © 2014 Baishideng Publishing Group Co., Limited. All rights reserved. Esteban-Jurado C et al . New genes for colorectal cancer predisposition a new approach to facilitate the identification of new genes responsible for human disease predisposition. By using whole-genome sequencing, germline mutations in the POLE and POLD1 genes have been found to be responsible for a new form of CRC genetic predisposition called polymerase proofreading-associated polyposis