Identificación de variante genética causal para síndromes de cáncer colorrectal hereditario; secuenciación masiva en paralelo y aplicación de herramientas bioinformáticas
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Date
2023
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Facultad de Ciencias Agrarias-Universidad Nacional de Rosario
Abstract
El cáncer colorrectal (CCR) tiene una elevada incidencia y mortalidad a nivel mundial
y en Argentina es la segunda causa de muerte por cáncer (10,6%). Los Síndromes de CCR
hereditario se dividen en Cáncer Colorrectal Hereditario No Polipósico (CCHNP) y síndromes
de Poliposis, siendo el síndrome de Lynch (SL) y la Poliposis Adenomatosa Familiar (PAF) las
formas de CCR hereditario más comunes. La utilización de las técnicas de secuenciación de
nueva generación (NGS) para guiar la prevención, el diagnóstico y el tratamiento de
enfermedades basadas en los genes individuales de una persona o una familia, su medio
ambiente y su estilo de vida, se conoce con el nombre de medicina de precisión. A pesar de este
nuevo paradigma de la Medicina de Precisión son pocos los reportes y la aplicación de estas
técnicas en América Latina. Particularmente, el grupo de investigación REM-ProCanHe del
cual formo parte, lleva adelante desde 1996 el desarrollo de investigación clínica para la
identificación temprana de CCR en pos de alcanzar la medicina de precisión. La vinculación
internacional de este grupo con especialistas en la temática de la universidad de Helsinki
(Finlandia) ha permitido realizar técnicas de secuenciación masiva en paralelo en muestras
argentinas. El objetivo general del presente trabajo fue aplicar herramientas bioinformáticas
para realizar un análisis preciso y rápido con el objetivo de identificar a nivel germinal la
variante causal asociada con aumento de susceptibilidad a desarrollar CCR hereditario,
partiendo de resultados genómicos derivados de secuenciación de nueva generación. Se
estudiaron 21 casos de pacientes clínicamente diagnosticados con síndrome de Poliposis
provenientes del Registro de Poliposis Adenomatosa Familiar del Hospital de
Gastroenterología Dr. Carlos Bonorino Udaondo. Se analizaron de manera secuencial por i)
secuenciación con el método de Sanger del exón 15 del gen APC (directamente relacionado con
PAF); ii) luego con la técnica de MLPA para evaluar presencia de grandes rearreglos; iii)
finalmente, para aquellas muestras aun negativas sin alteración genética-causal identificada
(mediante métodos i y ii) se realizó la secuenciación del exoma completo. Como resultado, a
través de la técnica de secuenciación por Sanger, en 6 casos identificamos la variante genético causal en el gen APC, siendo todas variantes novel, las cuales podrían ser verificadas a nivel
funcional por otras técnicas o incorporar mayor información genética ya sea de muestras de la
familia en estudio u otras muestras independientes que presenten la misma variante. Además,
para otros 4 casos y mediante la secuenciación del exoma completo pudimos identificar la
variante genético-causal candidata para predisposición en genes con asociación establecida para
la patología. Específicamente, 2 casos presentaron alteración en genes relacionados con la
patología, pero por el fenotipo clínico no pudieron confirmarse como genético-causal. Los otros
2 casos presentaron variantes en dos genes que se relacionan con el funcionamiento
homeostático del intestino por lo cual necesitamos mayor evidencia sobre los mismos para
poder adjudicarlos como genético-causal. Po otra parte, no encontramos genes que se relacionan
directamente con el fenotipo en 7 casos los cuales todavía siguen en estudio. A partir de los
datos obtenidos en este trabajo, se evidenciaron los parámetros, los pasos a seguir para llevar el
resultado obtenido desde el secuenciador hasta el archivo VCF, como así también el diseño de
un pipeline para la priorización de variante. Se espera que las herramientas y procedimientos
obtenidos en el presente trabajo contribuyan de manera significativa en la medicina de precisión
posibilitando el desarrollo de nuevas estrategias para el estudio del Síndrome de CCR
hereditario.
Colorectal cancer (CRC) has a high incidence and mortality worldwide and, in Argentina, it is the second cause of death from cancer (10.6%). Hereditary CRC Syndromes are divided into Hereditary Non-Polyposis Colorectal Cancer (HNPCC) and Polyposis syndromes, with Lynch syndrome (LS) and Familial Adenomatous Polyposis (FAP) being the most common forms of hereditary CRC. The use of NGS techniques to guide the prevention, diagnosis, and treatment of disease based on a person's or family's individual genes, environment, and lifestyle is known as precision medicine. Despite this new paradigm of precision medicine, there are few reports and applications of these techniques in Latin America. In particular, the REM-ProCanHe research group, of which I am a member, has been carrying out precision medicine for the early detection of CRC since 1996. The international linkage of this group with specialists in the subject from the University of Helsinki (Finland) has made it possible to carry out massive parallel sequencing techniques in Argentine samples. Therefore, the general objective of this work was to apply bioinformatic tools to perform a precise and rapid analysis to identify at the germinal level the causal variant associated with increased susceptibility to develop hereditary CRC, based on genomic results derived from new sequencing techniques. 21 samples were taken from patients diagnosed with polyposis syndromes from the Family Adenomatous Polyposis Registry of the Dr Carlos Bonorino Udaondo Hospital of Gastroenterology, first performing the sequencing by the Sanger method of exon 15 of the APC gene (directly related to PAF), then by the MLPA technique and finally those negative samples for both methods we performed whole exome sequencing. We were able to find, through the Sanger sequencing technique, 6 samples with a genetic-causal variant in the APC gene with a “novel” variant, which could be verified at a functional level by other techniques or incorporate more genetic information either from samples of the family under study or other independent samples that present the same variant. Moreover, by whole exome sequencing technique: a) 4 samples with a genetic-causal variant in genes related to the pathology were found; b) 2 samples in genes related to the pathology that cannot be identified as genetic-causal because of the clinical phenotype; c) 2 samples present variants in two genes that are related to the homeostatic functioning of the intestine, for which we need more evidence about them to be able to adjudicate them as genetic-causal. However, we did not find genes that are directly related to the phenotype in 7 samples which are still under study. From the data obtained in this work, the parameters were evidenced, and the steps to follow to take the result obtained from the sequencer to the VCF file, as well as the design of a pipeline for variant prioritization. Therefore, based on the tools obtained in this work, will allow us to apply for precision medicine in our research group, enabling new strategies to study and analyze cases of hereditary CRC syndrome.
Colorectal cancer (CRC) has a high incidence and mortality worldwide and, in Argentina, it is the second cause of death from cancer (10.6%). Hereditary CRC Syndromes are divided into Hereditary Non-Polyposis Colorectal Cancer (HNPCC) and Polyposis syndromes, with Lynch syndrome (LS) and Familial Adenomatous Polyposis (FAP) being the most common forms of hereditary CRC. The use of NGS techniques to guide the prevention, diagnosis, and treatment of disease based on a person's or family's individual genes, environment, and lifestyle is known as precision medicine. Despite this new paradigm of precision medicine, there are few reports and applications of these techniques in Latin America. In particular, the REM-ProCanHe research group, of which I am a member, has been carrying out precision medicine for the early detection of CRC since 1996. The international linkage of this group with specialists in the subject from the University of Helsinki (Finland) has made it possible to carry out massive parallel sequencing techniques in Argentine samples. Therefore, the general objective of this work was to apply bioinformatic tools to perform a precise and rapid analysis to identify at the germinal level the causal variant associated with increased susceptibility to develop hereditary CRC, based on genomic results derived from new sequencing techniques. 21 samples were taken from patients diagnosed with polyposis syndromes from the Family Adenomatous Polyposis Registry of the Dr Carlos Bonorino Udaondo Hospital of Gastroenterology, first performing the sequencing by the Sanger method of exon 15 of the APC gene (directly related to PAF), then by the MLPA technique and finally those negative samples for both methods we performed whole exome sequencing. We were able to find, through the Sanger sequencing technique, 6 samples with a genetic-causal variant in the APC gene with a “novel” variant, which could be verified at a functional level by other techniques or incorporate more genetic information either from samples of the family under study or other independent samples that present the same variant. Moreover, by whole exome sequencing technique: a) 4 samples with a genetic-causal variant in genes related to the pathology were found; b) 2 samples in genes related to the pathology that cannot be identified as genetic-causal because of the clinical phenotype; c) 2 samples present variants in two genes that are related to the homeostatic functioning of the intestine, for which we need more evidence about them to be able to adjudicate them as genetic-causal. However, we did not find genes that are directly related to the phenotype in 7 samples which are still under study. From the data obtained in this work, the parameters were evidenced, and the steps to follow to take the result obtained from the sequencer to the VCF file, as well as the design of a pipeline for variant prioritization. Therefore, based on the tools obtained in this work, will allow us to apply for precision medicine in our research group, enabling new strategies to study and analyze cases of hereditary CRC syndrome.
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Keywords
Bioinformática, Medicina de precisión, Secuenciación, Variación genética, Neoplasmas, Síndromes de colon rectal