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Master's Dissertation
DOI
https://doi.org/10.11606/D.5.2020.tde-29012020-122521
Document
Author
Full name
Betsaida Urtremari
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2019
Supervisor
Committee
Lourenço Junior, Delmar Muniz (President)
Marco, Luiz Armando Cunha de
Achatz, Maria Isabel Alves de Souza Waddington
Camacho, Cléber Pinto
Title in Portuguese
Padronização e aplicação de painel baseado em sequenciamento paralelo em larga escala direcionado ao diagnóstico genético de neoplasia endócrina múltipla tipo 1
Keywords in Portuguese
Biologia molecular
Doenças genéticas
Endocrinologia
Genética
Neoplasia endócrina múltipla tipo 1
Sequenciamento paralelo em larga escala
Abstract in Portuguese
A neoplasia endócrina múltipla tipo 1 (MEN1) é uma doença genética, de herança autossômica dominante, caracterizada pelo desenvolvimento de tumores endócrinos que acometem, principalmente, hipófise, paratireoide e pâncreas/duodeno endócrinos. Entretanto, tumores acometendo outras glândulas endócrinas bem como vários outros tecidos não endócrinos têm sido reportados incluindo tumores nas adrenais, em pulmão, timo, entre outros. MEN1 é causada, principalmente, por mutação germinativa no gene MEN1 (11q13). Subsequentemente, outros genes (CDKN2B/p15, CDKN2C/p18 CDKN1A/p21, CDKN1B/p27 e AIP) foram relacionados à síndrome MEN1 por fenótipos condizentes com MEN1. Consensos internacionais tem sistematicamente sugerido a análise do gene MEN1 e, mais recentemente, dos genes relacionados a MEN1, em um número crescente de fenótipos que não encontram os critérios clínicos de diagnóstico de MEN1, mas que são suspeitos para esta síndrome. Alguns exemplos destas condições são: hiperparatireoidismo primário (HPT) por adenoma diagnosticado em idade jovem ( < 30 anos) ou por hiperplasia independente de idade, HPT recorrente, gastrinomas, tumores endócrinos multifocais do pâncreas, apresentações atípicas de MEN1, entre outras. Entretanto, não se tem observado sistematicamente, na prática clínica, a realização de análise genética destes diversos grupos de pacientes com graus variáveis de risco potencial de MEN1. De fato, análise de mutação germinativa tem sido dirigida, principalmente, para pacientes com diagnóstico clínico de MEN1 familial clássica. Isto se deve, em parte, pelo desconhecimento de clínicos sobre a indicação de análise genética destes subgrupos de pacientes, pelas limitações (necessidade de sequenciamento de múltiplos genes e ausência de "hot spots" mutacionais) e elevada custo-efetividade do Sequenciamento Sanger (SS) para suportar o diagnóstico genético de MEN1 para todas estas condições na prática clínica. Assim, neste estudo, nós pretendemos incorporar à prática médica um painel genético baseado em sequenciamento paralelo em larga escala que vem sendo validado em nosso Serviço. A padronização de análise por NGS direcionada ao gene MEN1 resultou em elevada acurácia na detecção de mutações e em melhor custo-efetividade com capacidade potencial para substituir o SS. Os objetivos do presente estudo são: 1) padronizar técnica de sequenciamento gênico capaz de proporcionar a análise genética completa e integrada de todos os genes relacionados a MEN1 conhecidos (genes MEN1, CDKN2B/p15, CDKN2C/p18 CDKN1A/p21, CDKN1B/p27Kip1 e AIP ) permitindo tanto a avaliação de casos com MEN1 familial como de todos os casos com MEN1 ou condições clínicas associadas a MEN1 (MEN1 esporádica, casos atípicos ou suspeitos de MEN1 e indivíduos com tumores MEN1-relacionados aparentemente esporádicos); 2) propiciar, pela avaliação genética completa, o aprimoramento do aconselhamento genético destas diferentes populações. Foram analisados, inicialmente, pelo menos 40 pacientes com fenótipo de MEN1 esporádica ou com suspeita clínica de MEN1; 20 pacientes com tumores neuroendócrinos pancreáticos aparentemente esporádicos e/ou multifocais, 20 pacientes com HPT primário por idade jovem ou por hiperplasia ou por recorrência.. Em etapa inicial do presente projeto foi necessária a otimização da amplificação de "long range PCR" para todos genes MEN1-relacionados com nova enzima em decorrência de descontinuação do uso de enzima anterior que já havia sido otimizada previamente em nosso Serviço. Também, um painel por sequenciamento paralelo em larga escala foi conduzido para análise de 25 pacientes com MEN1 esporádica e variantes patogênicas/potencialmente patogênicas foram identificadas no gene MEN1 em 2 pacientes. Posteriormente mais 39 pacientes foram submetidos a sequenciamento e nenhuma alteração patogênica foi encontrada
Title in English
Standardization and application of a panel based on large scale parallel sequencing directed to the genetic diagnosis of multiple endocrine neoplasia type 1
Keywords in English
Endocrinology
Genetic diseases
Genetics
Large-scale parallel sequencing
Molecular biology
Multiple endocrine neoplasia type 1
Abstract in English
Multiple endocrine neoplasia type 1 (MEN1) is a genetic disease of autosomal dominant inheritance, characterized by the development of endocrine tumors that mainly affect pituitary, parathyroid and endocrine pancreas/duodenum. However, tumors affecting other endocrine glands as well as several other non-endocrine tissues have been reported, including tumors in the adrenal, lung, thymus, among others. MEN1 is mainly caused by germline mutation in the MEN1 gene (11q13). Subsequently, other genes (CDKN2B/p15, CDKN2C/p18, CDKN1A /p21, CDKN1B/p27 and AIP) were related to MEN1 syndrome by MEN1-like phenotypes. International consensus has systematically suggested the analysis of the MEN1 gene and, more recently, of the MEN1-related genes to a growing number of phenotypes that do not meet the clinical criteria for diagnosis of MEN1, but which are suspect for this syndrome. Some examples of these conditions are: primary hyperparathyroidism (HPT) for adenoma diagnosed at young age ( < 30 years) or by age-independent parathyroid hyperplasia, recurrent HPT, gastrinomas, multifocal pancreatic endocrine tumors, atypical MEN1 presentations, among others. However, it has not been systematically observed, in clinical practice, the genetic analysis of these different groups of patients with varying degrees of potential risk of MEN1. In fact, germline mutation analysis has been directed, mainly, to patients with clinical diagnosis of classic familial MEN1. This is due in part to the lack of clinical knowledge about the indication of genetic analysis of these subgroups of patients, limitations (need for sequencing of multiple genes and absence of mutational hot spots) and high cost-effectiveness of Sanger Sequencing (SS) to support the genetic diagnosis of MEN1 for all these conditions in clinical practice. Thus, in this study, we intend to incorporate at the medical practice a genetic panel based large scale parallel sequencing that has been validated in our Service. The standardization large scale parallel sequencing analysis directed to the MEN1 gene resulted in high accuracy in the detection of mutations and in better cost-effectiveness with potential capacity to replace the SS. The objectives of the present study are: 1) standardize gene sequencing technique capable of providing complete and integrated genetic analysis of all known MEN1-related genes (MEN1, CDKN2B / p15, CDKN2C / p18, CDKN1A / p21, CDKN1B / p27Kip1 and AIP genes) allowing for the evaluation of both familial MEN1 cases as of all MEN1 cases or MEN1-associated clinical conditions (sporadic MEN1, atypical or suspected MEN1 cases, and individuals with apparently sporadic MEN1-related tumors); 2) provide, through the complete genetic evaluation, the improvement of genetic counseling of these different populations. The achievement of these objectives has the potential to improve and expand the genetic diagnosis of MEN1 in our country, to define better the subgroups of patients that benefit from the genetic panel as well as to improve and modify the clinical management, treatment and genetic counseling of these different subgroups in potential risk for MEN1. At least 40 patients with sporadic MEN1 phenotype or with clinical suspicion of MEN1 will be initially analyzed; 20 patients with apparently sporadic and/or multifocal pancreatic neuroendocrine tumors, 20 young patients with primary HPT or with parathyroid hyperplasia or recurrent HPT In the initial stage of the present project, the optimization of the long range PCR amplification for all MEN1-related genes with a new enzyme was necessary due to the discontinuation of production for the enzyme that had been previously optimized in our Service. Also, a panel by NGS was conducted for analysis of 25 patients with sporadic MEN1 and pathogenic/potentially pathogenic variants were identified in the MEN1 gene in 2 patients. Subsequently 39 more patients underwent sequencing and no pathogenic changes were found
 
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Publishing Date
2020-01-29
 
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