Academic literature on the topic 'Multiple endocrine neoplasia type 2 (MEN2)'

INTRODUÇÃO: A imensa maioria dos casos com Neoplasia Endócrina Múltipla Tipo 2 (NEM2) é causada por uma única mutação germinativa no proto-oncogene RET. Entretanto, há alguns poucos casos descritos na literatura (~16) que apresentam duplas mutações/polimorfismos no gene RET, geralmente associados a fenótipos atípicos. OBJETIVOS: Os objetivos deste projeto são: a) caracterizar os aspectos clínicos de pacientes advindos de cinco famílias não relacionadas com diagnóstico de NEM2A, nas quais se documentou a presença de uma nova dupla mutação germinativa RET nos códons 634 e 791 e b) realizar rastreamento gênico familiar dos casos sob-risco com a finalidade de identificarmos possíveis casos com esta nova mutação. PACIENTES: Cinco casos-índice foram recentemente descobertos albergando a dupla mutação germinativa RET C634Y/Y791F. Nestas famílias há relato de 208 parentes, potencialmente, sob-risco (~50%) de serem portadores desta mutação. Dentre estes 208 indivíduos, 81 (38,9%) aceitaram participar do rastreamento gênico. MÉTODOS: O estudo foi realizado na Disciplina de Endocrinologia, Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo. O DNA do sangue periférico dos pacientes e de seus parentes sob- risco foi obtido após a obtenção do consentimento livre e esclarecido. Após PCR, foi realizado o sequenciamento gênico direto (ABI 3130x/l Sequencer, Applied Biosystems), abrangendo todos os 20 éxons do RET. Foram investigados potenciais polimorfismos e mutações no RET. Os tumores NEM2-relacionados (carcinoma medular de tireoide, CMT; feocromocitoma, FEO; hiperparatireoidismo primário, HPT) foram estudados quanto a vários parâmetros clínicos, como: sinais/sintomas; dimensão, penetrância e agressividade dos tumores; porcentagem de remissão bioquímica do CMT, e recidiva ou persistência dos tumores. RESULTADOS: Dentre os 81 indivíduos que participaram do rastreamento gênico, documentamos 28 casos (34,5%) como portadores da dupla mutação RET C634Y/Y791F. Além disso, as mutações foram testadas in vitro e um gene de efeito fundador foi descartado. Observou-se que: a) os cinco casos-índice (100%) com mutação germinativa RET C634Y/Y791F apresentaram FEO com características tais como, tumores medindo acima de 5,0 cm, e quatro dentre os cinco casos (80%) tiveram o diagnóstico com idades inferiores a 35 anos. A frequência de FEO nos parentes adultos (a partir da terceira década) foi de 73% e eram bilaterais em 80%; b) as características do CMT nos afetados albergando a mutação RET 634/791 eram similares às que ocorrem em pacientes com a mutação 634 isolada. Adicionalmente, estudo in vitro desta dupla mutação revelou que o grau de fosforilação das células com a dupla mutação celular 634/791 era significativamente maior (p=0,04), quando comparada com a capacidade fosforilativa das mutações 634 e 791 avaliadas individualmente. CONCLUSÃO: Diante dos presentes achados, conclui-se que os pacientes com esta nova dupla mutação tenderam a apresentar FEO de maior agressividade (precocidade, tumores volumosos e bilaterais), enquanto que o CMT, nestes casos, apresentava características usuais aos portadores de mutação RET no códon 634. A nossa hipótese é que os achados relativos ao FEO, nos presentes pacientes, foram influenciados pela presença da segunda mutação RET Y791F, a qual teria atuado como fator modulador do fenótipo. Esta hipótese foi suportada pelos estudos in vitro. Além disso, a ausência de um efeito fundador levou-nos a prever que esta dupla mutação RET pode não ser rara, na área geográfica estudada. Ainda que estes achados devam ser validados, os presentes dados podem ser úteis no diagnóstico genético e no manejo clínico e terapêutico dos pacientes com NEM2A<br>INTRODUCTION: The vast majority of cases with multiple endocrine neoplasia type 2A (MEN2A) is caused by a single germline mutation in the RET proto-oncogene. However, some instances (~16) of double germline RET mutations have been reported, most frequently associated with atypical phenotypes. OBJECTIVES: The main goals of this project were: a) to characterize the phenotype of patients from five unrelated MEN2A families harboring a new RET double germline in codons 634 and 791; b) perform the genetic screening in at-risk family members attempting to search for new similarly affected cases. PATIENTS: Five affected index-cases were recently found with this new double RET mutation C634Y/Y791F. In these five families, 208 first-degree relatives were reported and they were at-risk (~50%) to develop this mutation. Eighty-one individuals out of the 208 at-risk relatives (38.9%) were available and signed the informed consent. METHODS: The present investigation was performed at the Department of Endocrinology, School of Medicine, University of São Paulo. DNA was obtained from the peripheral blood of patients and at-risk relatives, after obtaining the informed consent of all individuals. After PCR, the direct genetic sequence was performed (ABI 3130x/l Sequencer, Applied Biosystems), involving all 20 exons of the RET proto-oncogene. Potential mutations and polymorphisms were investigated. In addition, the mutations were tested in vitro and a potential founder effect was evaluated. The MEN2A- related tumors (medullary thyroid carcinoma, MTC; pheochromocytoma, PHEO; and primary hyperparathyroidism, HPT) were approached using several clinical parameters, as: signs and symptoms; age at the diagnosis; tumor size and aggressiveness; biochemical remission of MTC; tumor relapse or persistence; RESULTS: Within the 81 genetically screened individuals, 28 cases (34.5%) were documented harboring the RET C634Y/Y791F germline mutation. It was observed that: a) the five affected index-cases (100%) were presented with PHEOs with features such as tumors measuring more than 5.0cm, and four out of the five cases (80%) had this diagnosis under the age of 35 years old. The frequency of PHEO in the adult affected relatives was also high (73%), from the third decade on, and were bilateral in 80%; b) the features and outcome of MTC in the affected C634Y/Y791F cases were similar to those MEN2A cases harboring the RET 634 mutation only. In addition, in vitro studies verified that cells with the 634/791 mutation had a significantly higher capacity of phosphorylation than cells harboring each individual mutation (p=0.04). Also, a founding gene was ruled out in these families. CONCLUSION: Our present data indicated that the earlier and more aggressive PHEOs seen in the present cases were most probably due to a phenotype modulation of the second RET 791 mutation. This hypothesis was supported by data from in vitro studies. Also, the absence of a founding couple led us to predict that this double RET mutation may not be rare, in the studied geographic area. Although these findings need to be validated, the present data may be useful in the genetic diagnosis as well as in the clinical and therapeutic management of MEN2A

Introdução: A neoplasia endócrina múltipla tipo 2 (MEN2) é uma doença hereditária autossômica dominante causada por mutação germinativa RET, que cursa com carcinoma medular de tireóide (CMT), feocromocitoma (Feo) e hiperparatireoidismo. O CMT é uma neoplasia maligna, que se desenvolve já na 1ª década de vida, pouco responsiva a quimioterapia/radioterapia. Assim, tireoidectomia profilática é indicada antes dos 5 ou dos 10 anos, dependendo do códon mutado, para assegurar a cura. O CMT é um tumor de crescimento lento e os pacientes convivem com o diagnóstico de câncer por décadas. Além disto, podem desenvolver Feo, predispondoos ao risco de óbito por infarto agudo do miocárdio ou acidente vascular em idade jovem. Somam-se situações de stress como risco de transmissão aos descendentes, expectativa de resultados de exames periódicos e risco de múltiplas cirurgias. Há poucos trabalhos enfocando os aspectos psíquicos em MEN2. O doente oncológico pode desenvolver sintomas psicológicos de: ansiedade, depressão, angústia, medo de recorrência da doença, perturbações psicossomáticas, stress decorrente das cirurgias e auto-conceito negativo. Objetivos: avaliar a sintomatologia ansiosa e depressiva, a qualidade de vida, o ajustamento psicológico, a presença de culpa auto-referida pela transmissão da doença aos filhos, o conhecimento da doença e a adesão ao tratamento. Casuística: Avaliação de 43 pacientes pertencentes a 12 famílias com diagnóstico clínico e gênico de MEN2. Metodologia: Avaliação psicológica por Entrevista semidirigida, Escala Hospitalar de Ansiedade e Depressão (HAD), European Organization for Research and Treatment of Cancer Quality of Life, Escala de Ajustamento Mental para Câncer e Estrutura Fatorial. A análise dos dados foi realizada de modo quantitativo e qualitativo. Resultados: Todos os 43 pacientes com MEN2 apresentavam CMT (100%) e 19 deles tinham diagnóstico prévio ou atual de Feo (44%; 19/43). Dos 43 pacientes, 16 (37%) tratados por CMT eram considerados curados (calcitonina < 2 pg/mL) enquanto que os outros 27 (63%) apresentavam persistência ou recorrência do CMT (25) ou aguardavam tratamento cirúrgico (2). Ao todo, 41 pacientes tinham história de 59 procedimentos cirúrgicos relacionados à MEN2 (1,4 cirurgia/paciente; 1- 6 cirurgias). Também, 31 pacientes (72%) responderam que tiveram medo de morrer como reação à informação do diagnóstico de MEN2. A maioria concordou em realizar o teste genético de seus filhos (21/22; 95%). Dos 16 pacientes com MEN2 que tiveram filhos com resultados positivos para o gene RET 12 (75%) caracterizaram a informação como \"muito difícil\" e \"medo de perda\". Um terço dos pacientes (5/16; 31%) referiam sentimento de culpa pela transmissão genética aos seus filhos. A aderência ao uso contínuo de medicações prescritas foi elevada (37; 86%). Em relação à informação sobre a doença, 32 pacientes (77%) responderam que se sentiam bem informados. O valor médio do estado global de saúde foi 68,1 ± 22,3 (variação de escore: 0-100), considerando tanto os aspectos físicos como emocionais. As frequências de sintomatologia ansiosa e depressiva encontradas pela escala HAD foram, respectivamente, 42% (18/43) e 26% (11/43). Uma correlação positiva estatisticamente significativa foi observada entre depressão e ansiedade. Associação positiva foi observada de ansiedade e depressão com as sub-escalas: desânimo-fraqueza, preocupação ansiosa, evitamento cognitivo, fadiga, dor, insônia, náusea e vômito, dispnéia, perda de apetite e diarréia. Paralelamente, houve uma associação inversa com as sub-escalas de estado global de saúde, desempenho de atividades, funcionamento cognitivo e funcionamento emocional. Os tipos de enfrentamento mais usados pelos pacientes diante do diagnóstico de MEN2 foram o espirito de luta (3,3 ± 0,77; variação de score: 1-4), o evitamento cognitivo (2,56 ± 1,17) e o fatalismo (3,1 ± 0,90). O espírito de luta apresentou correlação direta estatisticamente significativa com fatalismo (r = 0,369; p = 0,015). Conclusões: Pela elevada prevalência, sintomatologia ansiosa e depressiva devem ser ativamente investigadas em pacientes com MEN2. O impacto de ser portador de uma doença genética predispondo a câncer gerou: medo de morte e de perda dos filhos, evitamento cognitivo, fatalismo e forte espirito de luta. Este projeto poderá propiciar o início do desenvolvimento de um ambulatório multidisciplinar envolvendo tanto médicos como psicólogos com manejo em aconselhamento genético<br>Introduction: Multiple endocrine neoplasia type 2 (MEN2) is autosomal-dominant hereditary cancer syndrome caused by germline RET mutation with high susceptibility to develop tumors as medullary thyroid carcinoma (MTC), pheochromocytoma (Pheo), and hyperparathyroidism. The CMT is a malignancy that develops already in the 1st decade of life, poorly responsive to chemotherapy / radiotherapy. Thus, prophylactic thyroidectomy is indicated before 5 or 10 years, depending on the mutated codon to ensure healing. The CMT is a slow-growing tumor and the patients live with the diagnosis of cancer for decades. In addition, they can develop Pheo, predisposing them to risk of death from myocardial infarction or stroke at a young age. Add to stress conditions such as risk of transmission to offspring, expectative for results of periodic examinations and risk of multiple surgeries. It is known that cancer patients can develop psychological symptoms of anxiety, depression, distress, fear of recurrence, stress for surgery and negative self-concept. However, studies focusing on the psychological aspects in MEN2 are strict and mainly related with the time of RET genetic testing and genetic counselling. Objectives: To assess anxious and depressive symptoms, quality of life, psychological adjustment, presence of guilt by self-reported disease transmission to children, knowledge of disease and treatment adherence. Methods: Evaluation of 43 patients from 12 families with clinical and genetic diagnosis of MEN2. Methodology: Psychological assessment by semi-directed interview, Scale Hospital Anxiety and Depression (HAD), European Organization for Research and Treatment of Cancer Quality of Life Scale, Mental Adjustment to Cancer and Factor Structure. Data analysis was performed quantitatively and qualitatively. Results: All 43 patients had CMT MEN2 (100%) and 19 had previous or current diagnosis of pheochromocytoma (44%, 19/43). Of the 43 patients, 16 (37%) treated by CMT were considered cured (calcitonin < 2 pg / ml) while the other 27 (63%) had persistent or recurrent MTC (25) or were awaiting surgery (2). Altogether, 41 patients had a history of 59 surgical procedures related to MEN2 (1.4 Surgery / Patient, 1-6 surgeries). Altogether, 31 patients (72%) responded that they were afraid to die as a reaction to the diagnosis of MEN2 information. Most agreed to conduct genetic testing of their children (21/22, 95%). Of the 16 patients with MEN2 who had children with positive results for the RET gene 12 (75%) characterized the information as \"very difficult\" and \"fear of loss\". One third of patients (5/16, 31%) reported feelings of guilt by genetic transmission to their children. Adherence to continuous use of prescribed medications was high (37, 86%). The frequencies of anxious and depressive symptoms found by the HAD scale were, respectively, 42% (18/43) and 26% (11/43). A statistically significant positive correlation was observed between depression and anxiety. There was a positive association of anxiety/depression with the sub-scales: weakness-dejection, anxious preoccupation, cognitive avoidance, fatigue, pain, insomnia, nausea and vomiting, dyspnea, loss of appetite and diarrhea. Inverse association occurred with the subscales of general health, performance activities, cognitive functioning and operation emocional. The more types of coping used by patients before the diagnosis of MEN2 were fighting spirit (3.3 ± 0.77; change of score: 1-4), cognitive avoidance (2.56 ± 1.17) and fatalism (3.1 ± 0.90). The mean global health status was 68.1 ± 22.3 (range of scores: 0-100), considering both the physical and emotional aspects. Conclusions: Due to its high prevalence, anxious and depressive symptoms should be actively investigated in patients with MEN2. The impact of being a carrier of a genetic disorder predisposing to cancer generated: fear of death and loss of children, cognitive avoidance, fatalism, strong fighting spirit. This project will promote the development of a multidisciplinary clinic involving both physicians and psychologists with management in genetic counseling

INTRODUÇÃO: Na Neoplasia Endócrina Múltipla tipo 2 (NEM2), o desenvolvimento do Carcinoma Medular de Tireoide (CMT), Feocromocitoma (FEO) e Hiperparatireoidismo primário (HPT) está associado à mutações germinativas ativadoras no proto-oncogene RET. Casos de CMT esporádico podem apresentar mutações somáticas no RET (~40%). A variabilidade fenotípica observada em casos de CMT e FEO familiais associados à NEM2 indica o envolvimento de eventos genéticos adicionais que seriam responsáveis pelas diferenças clínicas observadas nos indivíduos afetados (idade de desenvolvimento, progressão e agressividade do tumor). Outras alterações genéticas no RET como duplas mutações, SNPs e haplótipos específicos podem influenciar na susceptibilidade, agressividade e modulação do fenótipo NEM2. Entretanto, os estudos de outros genes envolvidos no processo da tumorigênese NEM2 ainda estão em andamento. Recentemente foi mostrado que RET ativado controla a expressão de proteínas inibidoras do ciclo celular (p18 e p27). Mutações germinativas no gene p27 foram recentemente associadas à susceptibilidade de tumores neuroendócrinos e estão associadas à síndrome NEM4 (Neoplasia endócrina múltipla tipo 4). Mutações somáticas, inativadoras de p27, são raramente encontradas em vários tipos de tumores. Entretanto, diversos estudos documentaram que a redução na expressão e a sublocalização citoplamática de p27 são controladas por alterações pós-transducionais e/ou epigenéticas. OBJETIVOS: o estudo teve como objetivos avaliar a participação de genes, recentemente associados ao RET ativado, em tumores de pacientes com NEM2 e também verificar se polimorfismos no gene p27 estariam atuando como moduladores de fenótipo em uma grande família com NEM2. CASUÍTICA: foram analisadas 66 amostras tumorais advindas de 36 pacientes com diagnóstico clínico e genético de NEM2 e 28 indivíduos pertencentes a uma grande família com NEM2A-CMTF e mutação C620R no gene RET. MÉTODOS: As análises somáticas do p27 e também de p15, p18 e RET foram realizadas por PCR e sequenciamento direto de DNA e análise de microssatélites para p27 foi realizada por PCR e eletroforese capilar. Análises de expressão e localização da proteína p27 celular foram realizadas por Western blot e imunohistoquímica. A análise da modulação de fenótipo na família com NEM2A foi realizada por meio da amplificação do éxon 1 do gene p27 na amostra de sangue total. RESULTADOS: Não foram encontradas mutações somáticas no gene p27 e também nos genes p15 e p18. Entretanto, verificamos baixa expressão proteica de p27 em tumores CMT e FEO, a qual se encontrava relacionada com o tipo e agressividade do códon mutado no RET, principalmente em tumores que apresentavam mutação RET no códon 634 (controle x 634 p=0,05; controle x 634/791 p= 0,032; 620 x 634 p=0,045; 620 x 634/791 p= 0,002; 620 x 634 + 634/791 p=0,036). Notou-se também correlação positiva entre os níveis de expressão de p27 na localização nuclear, analisada por imunohistoquímica, e o genótipo TT do SNP p27 p.V109G (p=0,03). CONCLUSÕES: Alterações moleculares somáticas no gene p27 nos tumores NEM2 não são frequentes. Entretanto, a redução na expressão e a localização citoplasmática de p27 provavelmente estão associadas a alterações somáticas em outros genes que controlam os processos de fosforilação da proteína p27 (eventos pós-transducionais)<br>INTRODUCTION: In Multiple Endocrine Neoplasia type 2 (MEN2) the development of medullary thyroid carcinoma (MTC), pheochromocytoma (PHEO) and primary hyperparathyroidism (HPT) are associated with activating germline mutations in RET proto-oncogene. Cases of sporadic MTC may have somatic RET mutations (~ 40%). The phenotypic variability observed in cases with familial MTC/MEN2 and PHEO/MEN2 indicates the probable involvement of additional genetic events that could be responsible for the clinical differences observed in the affected individuals (age development, progression and aggressiveness of the tumor). Other genetic alterations such as RET double mutations, SNPs and specific haplotypes may influence susceptibility, aggressiveness and MEN2 phenotype modulation. However, studies of other genes involved in the tumorigenesis of MEN2 are still in progress. Recently, it was shown that the activated RET controls the expression of cell cycle inhibitory proteins (p18 and p27). Germline mutations in the p27 gene have recently been associated with the susceptibility to neuroendocrine tumors and are associated with the MEN4 syndrome (Multiple endocrine neoplasia type 4). Somatic inactivating mutations p27 are rarely found in many types of tumors. However, several studies have documented that reduced expression and subcellular location of p27 is controlled by post-transductional changes and/or epigenetic factors. OBJECTIVES: This study aimed to evaluate the role of genes recently associated with RET activated in tumors from MEN2 patients and also check whether polymorphisms in the p27 gene would be acting as modulators of phenotype in a large MEN2 family. PATIENTS: We analyzed 66 tumor samples from 36 patients with clinical and genetic diagnosis of MEN2 and from 28 individuals belonging to a large family with FMTC/MEN2A and RET C620R mutation. METHODS: The analyses of somatic p27, p15, p18 and RET were performed by PCR and direct sequencing of DNA and microsatellite analysis was performed for p27 by PCR and capillary electrophoresis. Expression analysis and subcellular localization of p27 protein were performed by Western blot and immunohistochemistry. The analysis of phenotype modulation in MEN2A families was performed by the amplification of exon 1 of the p27 gene in a whole blood sample. RESULTS: There were no somatic mutations in the p27 gene and also in the p15 and p18 genes. However, we verified a low p27 protein expression in MTC/MEN2 and PHEO/MEN2 that showed a definite correlation with the type and aggressiveness of the mutated RET codon, mainly in those tumors from cases with germline RET codon 634 mutations (control vs 634, p=0,05; control vs 634/791, p= 0,032; 620 vs 634, p=0,045; 620 vs 634/791, p= 0,002; 620 vs 634 + 634/791, p=0,036). It was also verified a positive correlation between the immunohistochemistry expression of nuclear p27 subcellular location and the p27 p.V109G TT genotype (p=0,03). CONCLUSIONS: The reduction in the expression of p27 and its subcellular localization are likely to be associated with somatic changes in other genes that control the processes of phosphorylation of p27 protein through post-transductional events

A neoplasia endócrina múltipla tipo 2 (NEM-2) é uma síndrome tumoral herdada por mutações germinativas no proto-oncogene RET (RET) e transmitida por herança autossômica dominante. Atualmente, a indicação de tireoidectomia total preventiva é recomendada a indivíduos portadores de mutações no RET. Analisamos a aplicação do método Eletroforese em Gel Sensível à Conformação (CSGE) no rastreamento de mutações hot-spots do RET. Sete famílias com NEM-2 foram rastreadas pelo CSGE, seqüenciamento gênico e análise do Polimorfismo Conformacional de Cadeia Simples (SSCP). Usando o CSGE e SSCP, identificamos cinco das seis (83,3%) mutações verificadas pelo seqüenciamento: Cys620Arg, Cys634Arg, Cys634Tyr, Val648Ile e Met918Thr. Foram analisados 128 amplicons englobando mutações hot-spots do RET e 116 dentre 128 (90.6%) concordaram com o seqüenciamento genético. Os polimorfismos 691 e 769 também foram documentados pelo CSGE e SSCP. Os dados obtidos por CSGE e SSCP foram totalmente (100%) concordantes. O CSGE revelou ser metodologia sensível, rápida, fácil de ser executada e de baixo custo na detecção de mutações nos códons 620, 634, 648, e 918, as quais constituem grande maioria (~95%) dos pacientes com NEM-2. Quanto à mutação Val804Met (prevalência na população inferior a 3%), o método necessita ser otimizado. Concluímos que o CSGE é uma metodologia efetiva para o rastreamento de mutações que mais freqüentemente ocorrem no RET como causadora de NEM-2.<br>Multiple endocrine neoplasia type 2 (MEN2) is an autosomal dominant inherited tumor syndrome caused by activating germline mutations in RET proto-oncogene (RET). Presently, the prophylactic total thyroidectomy is recommended to all RET mutations carriers. Here we tested the Conformation Sensitive Gel Electrophoresis (CSGE) as a screening method for the RET hot-spot mutations. Seven MEN2 families were studied by CSGE, as well as by Single Strand Conformational Polymorphism (SSCP) and direct sequencing analysis. Using CSGE and SSCP, we were able to detect five out of the six (83.3%) RET mutations verified by direct sequencing analysis: Cys620Arg, Cys634Arg, Cys634Tyr, Val648Ile and Met918Thr. RET polymorphisms 691 and 769 were verified by CSGE and SSCP. In our sample, data obtained using CSGE were fully concordant (100%) with SSCP findings. Thus, CSGE showed to be a sensitive, fast, low-cost, and ease procedure to detect RET mutations in codons 620, 634, 648, and 918 which are reported as the most prevalent RET variants (~95%) in large MEN2 series. As to the Val804Met mutation (prevalence in the population lower than 3%), this method still needs to be optimized. We concluded that CSGE is an effective screening method for the most frequent RET hot-spot disease-causing mutations.

Les cellules souches pluripotentes induites (CSPi) permettent la modélisation de processus avec, en oncologie, un intérêt potentiel pour la modélisation de syndromes de prédisposition au cancer liés à des mutations germinales d’oncogènes. Nous avons généré des lignées de CSPi à partir de patients atteints de néoplasies endocriniennes multiples de type 2 (NEM2), porteurs de mutations germinales du gène RET : RETC620R, RETC634Y et RETM918T. Nous avons généré une CSPi RETY634C, contrôle isogénique, par correction de la mutation RETC634Y via CRSPR/Cas9. Ces CSPi présentent tous les critères de pluripotence avec un caryotype normal et expriment Ret. L’étude histologique approfondie des tératomes a mis en évidence le développement de cellules C en leur sein et également de cellules neuroendocrines exprimant la Chromogranine A mais sans aspect d’hyperplasie des cellules C ou de carcinome médullaire de la thyroïde ni de tumeur neuroendocrine réminiscente du phénotype des NEM2. L’analyse comparative de l’expression des gènes de ces CSPi a mis en évidence, dès le stade de pluripotence, une activation du réseau transcriptionnel du gène EGR1 qui pourrait constituer un des mécanismes moléculaires responsables de la mise en place du phénotype des NEM2. La différenciation en cellules souches de la crête neurale (CSCN), cellules d’origine cibles des tumeurs développées dans le cadre des NEM2, en particulier le phéochromocytome, était efficace et reproductible pour toutes nos lignées. Nous avons mis en évidence l’activation d’un programme commun invasif au niveau des CSCN avec mutation RETC634Y et RETM918T ainsi qu’une forte dérégulation du réseau des intégrines entraînant une forte dérégulation de l’adhésion cellulaire. Ceci était confirmé par une augmentation des capacités de migration CSCN avec mutation de RET par rapport aux CSCN témoins. Ainsi, la génération de CSPi avec mutation de RET a permis d’identifier des voies de signalisation potentiellement impliquées dans la physiopathologie des NEM2 et constitue une première étape vers la modélisation des NEM2 in vitro<br>Induced pluripotent stem cell (iPSC) offer major perspectives in disease modelling and, in the oncology field, can be used for modelling cancer predisposition syndromes. We generated IPSC lines from somatic cells of patients with multiple endocrine neoplasia type 2 (MEN2) who harboured germline mutations in the RET gene: RETC620R, RETC634Y et RETM918T. We have also generated an isogenic RETY634C iPSC control line by genome engineering using CRSPR/Cas9-mediated method to "correct” C634Y mutation. All iPSC lines exhibited all markers of pluripotency with a normal karyotype and expressed Ret. A thorough histological study of teratomas from these iPSC highlighted the development of C cells and Chromogranin A-expressing neuroendocrine cells within them but without C-cell hyperplasia, medullary thyroid carcinoma or neuroendocrine tumours reminiscent of MEN2 phenotype. Comparative gene expression analysis revealed an activation of the EGR1 transcriptional network, at the pluripotent stem cell stage which could be one of the molecular effector of the phenotype. Neural crest stem cell (NCSC), the cell of origin of some of the tumoral features of MEN2, could be differentiated in vitro from all our RET-mutated iPSC lines effectively. Gene expression analysis revealed an activation of cell invasion program in RETC634Y and RETM918T–mutated NCSC and a deregulation of integrin network causing a strong deregulation of cell adhesion which was confirmed with increased migration capabilities in vitro. Thus, the generation of the first RET-mutated iPSCs allowed the identification of signalling pathways potentially implicated in the pathophysiology of MEN2 and constitute a first step in modelling these tumours in vitro

碩士<br>國立臺灣大學<br>分子醫學研究所<br>94<br>Multiple endocrine neoplasia type 2 (MEN 2) is caused by autosomal dominant gain-of-function mutations in the RET proto-oncogene which has a high penetrance for medullary thyroid carcinoma (MTC). So far, the associations between specific RET mutations (genotype) and the aggressiveness of MTC and variations of other endocrine neoplasia (phenotype) are well expounded. Hot spot mutations in six exons (10, 11, 13, 14, 15, and 16) located in either cysteine-rich or tyrosine kinase domains of RET cause one of three distinctive clinical subtypes: MEN 2A, MEN 2B, and familial MTC. In this study, we propose to elucidate the germ-line RET proto-oncogene mutations and subtypes in Taiwanese subjects with MTC, and evaluate the value of appropriate medical interventions and genetic counseling in afflicted patients and asymptomatic gene carriers. DNA was extracted from the peripheral blood leukocytes of 61 members of 20 unrelated families as having individuals affected by MTC. The suspected carriers and apparently sporadic MTC cases were tested for MEN 2-associated germ-line mutations by polymerase chain reaction (PCR)-based sequencing of the RET gene exons, including 8, 10, 11, 13, 14, 15, and 16. Ten family members in the three MEN 2A kindreds had mutations in codon 634 of exon 11, 1 C > R and 2 C > F. Two family members of one unclassified kindred had the C620F mutation of exon 10. An index case had the C634W germ-line mutation but her family refused the test, could not be defined as hereditary or de novo MEN 2A. Additionally we found 3 de novo cases, including 2 MEN 2B and 1 MEN 2A, with the M918T and C634R mutations, respectively. These mutations were not detected in either of their parents or siblings. The other 12 MTC cases were defined as sporadic MTC whose DNA did not carry any non-synonymous mutations in the 7 exons. We found that all MTC patient with family history or with the other phenotypes of MEN 2 had RET germ-line mutations. For patients with isolated MTC without family history and other endocrine syndromes, we did not find any non-synonymous RET germ-line mutations among the 7 exons. Our findings suggest that all patients with MTC should be screened for the RET proto-oncogene by molecular analysis in order to detect occult or de novo MEN 2 or familial MTC. To date, germ-line genetic testing has become the basis for therapeutic decisions in MEN 2 affected patients and can facilitate the early presymptomatic detections of gene carriers. MEN 2 gives a unique model for early prevention and cure of cancer and for risk stratification of carriers by genetic diagnosis. This provides important insight into the prominent benefits of genetic counseling in cancer therapy.

The dominantly inherited cancer syndrome multiple endocrineneoplasia type 2A (MEN 2A) is characterized by medullary thyroid carcinoma (MTC), pheochromocytoma and parathyroid hyperplasia. The related syndrome MEN 2B comprises MTC, pheochromocytoma and mucosalneuromas. Genes responsible for these cancers map to the pericentromericregion of chromosome 10, as do loci responsible for dominantly inherited MTC without additional clinical features, and MEN 2A associated with askin disorder, cutaneous lichen amyloidosis. The relationship between the genes responsible for these diseases is not known. Nothing is known of the biochemical basis for MEN 2; a positional cloning strategy to identify MEN2 gene(s) is therefore warranted. This thesis makes contributions to several steps of a strategy to identify MEN2. The first objective was to clone DNA markers more closely linked toMEN2A than the known flanking markers, FNRB and RBP3. Repeat element-mediated polymerase chain reaction (REM-PCR), a method of cloning human DNA fragments from hybrid DNA sources, was developed for this purpose. Primers directed to the 3' ends of human Alu and Ll elements provided the basis for amplification of human-specific DNA fragments; DNA from a somatic cell hybrid highly enriched for the MEN2Aregion was used as template. This technique yielded thirty-three REM-PCR clones, two of which mapped to intervals near MEN2A. One of these, pC11/A1S-6-c23, defines a polymorphic locus, D10S94. Somatic cell hybrid mapping of pC11/A1S-6-c23, combined with the results of linkage studies conducted by Drs. Nancy Simpson and Paul Goodfellow, served to localize D10S94 to 10811.2 between the centromere and RBP3. D10S94 is very tightly linked to MEN2A and was not idemonstrated to recombine with the disease locus in these studies. It was used as a starting point for large scale cloning and mapping efforts directed to the identification of genes. Long range restriction mapping by pulsed field gel electrophoresis revealed a dense cluster of CpG islands at D10S94. Genes potentially associated with these CpG islands represent candidate genes for MEN2. To determine whether relatively large sequence alterations, which could serve to direct gene cloning efforts to a small region, were associated with D10S94, a survey of the 180-kb CpG island-rich region at this locus was undertaken in the genomes of MEN 2A and MEN 2B patients. Long range restriction mapping by field inversion gel electrophoresis demonstrated that no alterations of 3.3 kb or more were present at DlOS94 in these patients. Yeast artificial chromosomes (YACs) identified in collaboration with Drs. Ken Kidd and Jay Lichter, expanded DlOS94 and, with genomic long range restriction mapping, established physical linkage to six other DNA markers near MEN2. Recent published reports identify very closely linked flanking markers and refine the minimal region to which MEN2A is localized. The genomic long range restriction map and YAC contig span the entire refined MEN2A candidate region. This map and contig can now serve as a guide and a cloning source, respectively, for experiments designed to identify genes within this region, and an intensive search for potentially disease-causing mutations to identify genes important in the etiology of MEN 2.