In den vergangen Jahren ist durch eine verbesserte Bildgebung die Anzahl der zufällig entdeckten adrenalen Raumforderungen beständig angewachsen. Eine wichtige Differenzialdiagnose sowohl bei adrenalen Inzidentalomen als auch bei der refraktären Hypertonie stellt das Phäochromozytom dar. Aus diesem Hintergrund heraus leitet sich der Wunsch nach Methoden zur sensitiven und spezifischen Bestimmung biochemischer Parameter in der Diagnostik des Phäochromozytoms ab. Derzeit werden hierzu die freien Plasmametanephrine durch High Pressure Liquid Chromatography (HPLC) als Mittel der Wahl angesehen. In der vorliegenden Arbeit wurden bei 113 Patienten mit klinischem Verdacht auf bzw. zum Ausschluss eines Phäochromozytoms die freien Plasmametanephrine mit Hilfe eines Radioimmunoassays (RIA) untersucht. Ziel war zum einen die Evaluierung der freien Metanephrine im Plasma mittels RIA als auch der Vergleich dieser mit anderen biochemischen Parametern und diagnostischen Verfahren hinsichtlich Sensitivität und Spezifität. Der hier verwendete RIA ist relativ zeitnah durchzuführen, kostengünstig und dadurch breit verfügbar. Er zeigt eine hohe diagnostische Sensitivität und Spezifität bei der Bestimmung der freien Metanephrine im Plasma und bietet sich somit für den Einsatz in der Routinediagnostik des Phäochromozytoms an, wodurch verbesserte Aussagen zum Ausschluss bzw. zur Bestätigung der Anwesenheit des Tumors möglich werden.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24314号 / 医博第4908号 / 新制||医||1062(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 小林 恭, 教授 松田 文彦, 教授 柳田 素子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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 / 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

Os feocromocitomas são tumores neuroendócrinos constituídos de células cromafins secretoras de catecolaminas e neuropeptídeos diversos, cuja manifestação clínica mais comum é a hipertensão. Doze a 24% dos tumores aparentemente esporádicos, apresentam mutações germinativas em genes até então associados a síndromes herdadas, como, RET, VHL, SDHB e SDHD. A doença de von Hippel-Lindau é causada por mutações no gene VHL. As proteínas codificadas pelos genes SDHB e SDHD fazem parte do complexo mitocondrial II e da cadeia aeróbica de transporte de elétrons. O objetivo deste projeto de pesquisa foi o rastreamento de mutações nos genes VHL, SDHB e SDHD em pacientes portadores de feocromocitoma ou, também, paraganglioma esporádicos acompanhados no Serviço de Endocrinologia do Hospital das Clínicas da FMUSP. Todos os exons dos três genes estudados foram amplificados por PCR e analisados por dHPLC. Os amplicons que apresentaram cromatogramas suspeitos ao dHPLC foram submetidos ao seqüenciamento automático. Nenhuma mutação foi encontrada no gene VHL, apenas dois polimorfismos previamente descritos no exon 1, c. -77 C>T em dois pacientes e c - 195 G>A em 58,6% do total de alelos dos pacientes estudados. No gene SDHB foram encontrados dois polimorfismos previamente descritos (c. 201-36 G>T e c.487 T>C) em quatro pacientes, uma mutação silenciosa não descrita (c.540 G>A) e uma mutação previamente descrita em portadores de feocromocitoma (c. 293 G>A). Um mesmo paciente apresentou a mutação silenciosa c.540 G>A e o polimorfismo c.201-36 G>T. No gene SDHD foram encontrados dois polimorfismos descritos (c.204 C>T e c.315-32 T>C) em um paciente cada, uma variante alélica descrita na literatura na região 3\' não codificante cuja freqüência nunca foi estudada em outras populações (c.*612 C>T) e duas substituições nunca descritas na região 3\' não codificante (c.*799 T>C e c.*803 A>G). As variantes c.*612 C>T e c.*799 T>C foram detectadas em apenas um paciente cada e não foram encontradas em 200 alelos de controles normais estudados. A variante c.*803 A>G foi encontrada em nove de 76 alelos dos 38 pacientes (11,8%) e em cinco de 200 alelos de 100 controles não afetados (2,5%), sendo, portanto, um polimorfismo significativamente mais freqüente entre os portadores de feocromocitoma ou paraganglioma. Dentre os sete pacientes portadores do polimorfismo c.*803 A>G, três pacientes heterozigotos para este polimorfismo apresentaram um segundo polimorfismo no gene SDHD, sendo que um desses pacientes também apresentava uma mutação no gene SDHB. Dentre os demais quatro pacientes, dois apresentavam o polimorfismo c.*803 A>G em homozigose. Este polimorfismo ocorre no nucleotídeo localizado na posição -1 imediatamente 5\' ao Sítio de Clivagem do pré-mRNA para que ocorra a inserção da cauda Poli(A), fundamental para a estabilidade do mRNA. A substituição da base A pela base G muito provavelmente apresenta uma repercussão funcional, pois a base A na posição -1 é considerada como a mais eficiente na promoção da clivagem do pré-mRNA, enquanto a base G é considerada a menos eficiente (ordem de eficiência de clivagem A > U > C > G). Desta forma, a possibilidade desse polimorfismo conferir susceptibilidade ao desenvolvimento do feocromocitoma e do paraganglioma não está descartada, sendo provável que outros eventos genéticos sejam necessários para promover a tumorigênese. Em conclusão, esse estudo evidenciou uma baixa freqüência de mutações nas regiões codificantes dos genes VHL (mutações não detectadas), SDHB (5,2%) e SDHD (mutações não detectadas) nessa série de pacientes com feocromocitomas e paragangliomas esporádicos, porém, encontrou um polimorfismo na região 3\' não codificante do gene SDHD significativamente mais freqüente nos portadores desses tumores que em indivíduos controles não afetados, e que, por suas características, pode estar relacionado à etiopatogenia do feocromocitoma e do paraganglioma. / Pheochromocytomas are neuroendocrine tumors composed of chromaffin cells that produce and secrete catecholamines as well as a variety of neuropeptides, whose most common clinical manifestation is arterial hypertension. Twelve to 24% of the apparently sporadic pheochromocytomas, present germline mutations in genes previously associated to inherited familiar syndromes, such as, RET, VHL, SDHB e SDHD. The von Hippel-Lindau (VHL) disease occurs upon the VHL gene mutation - a tumor suppressor gene whose product encodes complexes with other proteins leading proteic substracts to the proteolysis. The proteins encoded by the SDHD and SDHB genes are parts of the complex mitochondrial II, as well as the aerobic chain of the electron transport. The aim of the present study was the screening of mutations in the VHL, SDHB and SDHD genes in patients harboring sporadic pheochromocytoma and/or paraganglioma, followed by the Endocrinology Service of Hospital das Clínicas of the University of São Paulo School of Medicine. All the three studied gene exons were amplified by polymerase chain reaction (PCR) and were analyzed by dHPLC, which was the method used for screen mutations. The samples with altered eluting progress were directly sequenced. No mutations were found in the VHL gene, only two polymorphisms previously described in the exon 1, c. -77 C>T in two patients and c - 195 G> in 58.6% out of the total alleles of the studied patients. Two polymorphisms previously described (c. 201-36 G>T and c.487 T>C) in the SDHB gene were found in four patients, as well as silent mutation not yet described (c.540 G>A) and a mutation previously described in patients with pheochromocytoma (c. 293 G>A). A particular patient presented the silent mutation c.540 G>A and the polymorphism c.201-36 G>T. In the SDHD gene two polymorpfisms previously described (c.204 C>T and c.315-32 T>C) were found, one in each patient, as well as an allelic variant previously described in the 3\' non-coding region whose frequency has never been studied in other populations (c.*612 C>T) and two substitutions never described in the 3\' non-coding region (c.*799 T>C and c.*803 A>G). The variants c.*612 C>T and c.*799 T>C were detected in only one patient each and have not been found in 200 alleles of normal control subjects studied. The variant c.*803 A>G was found in nine out of 76 alleles from 38 patients (11.8%) and in five out of 200 alleles from 100 non-affected subjects (2.5%), being, then, a polymorphism significantly more frequent among patients with pheochromocytoma or paraganglioma. Among those seven patients with the polymorphism c.*803 A>G, three patients heterozygotous for the polymorphism presented a second polymorphism in the SDHD gene and one of those patients also presented a mutation in the SDHB gene. Out of the other four patients, two presented the polymorphism c.*803 A>G in heterozygosis. This polymorphism occurs in the nucleotide localized in the position -1 immediately 5\' to the site where the pre-mRNA is cleaved for the insertion of the poly(A) tail, which is essencial to the mRNA stability. The substitution of the A to the G probably presents a functional repercussion, because the A in the position -1 is considered as the most efficient nucleotide in the pre-mRNA cleavage promotion, while the G is considered the least efficient one (scale of cleavage efficiency A > U > C > G). Therefore, the possibility of this polymorphism be associated with susceptibility to the development of pheochromocytoma and paraganglioma is not discarded, being possible that other genetic events are necessary to promote tumorigenesis. In conclusion, this study evidenced a low frequency of mutations in the coding regions of the genes VHL (mutations not detected), SDHB (5,2%) and SDHD (mutations not detected) in this series of patients with sporadic pheochromocytomas and paragangliomas, however, a polymorphism significantly more frequent in patients harboring those tumors was found in the 3\' non-coding region of the SDHD gene and, for its specific characteristics, it can very well be related to the etiopathogenesis of the pheochromocytoma and paraganglioma

Les phéochromocytomes et les paragangliomes (PCC/PGL) sont des tumeurs neuroendocrines rares qui se développent aux dépens de la médullosurrénale et des paraganglions sympathiques et parasympathiques. Près de 40% des patients ont une prédisposition génétique, et, à ce jour 13 gènes de prédisposition ont été identifiés. Les mutations du gène SDHB sont un facteur de risque de malignité et de mauvais pronostic qui conduisent à une perte de fonction de la succinate déshydrogénase (SDH), enzyme du complexe mitochondrial II. Il en résulte une accumulation intracellulaire de succinate responsable du phénomène dit de « pseudohypoxie » qui stimule la production de VEGF et donc l’angiogenèse, ainsi que l’expression du transporteur membranaire du glucose GLUT-1. Le surrisque de malignité liée à SDHB et l’absence de traitement curatif des formes métastatiques de PCC/PGL justifient l’élaboration d’un modèle murin permettant de mettre en place des essais précliniques. L’obtention d’un modèle murin prédisposé au PCC/PGL par mutation du gène Sdhb s’étant révélée infructueuse, notre équipe a finalement généré des cellules chromaffines Sdhb-/- immortalisées (imCC Sdhb-/-) dont l’implantation dans le coussinet adipeux sous-cutané (Fat-pad) de souris nudes NMRI a permis l’établissement du premier modèle de tumeurs porteuses d’une inactivation complète du gène Sdhb. La caractérisation phénotypique du modèle a été réalisée par imagerie multimodalité in vivo, comparativement à un groupe contrôle de souris ayant reçu des imCC non mutées (WT). L’angiogenèse tumorale évaluée par imagerie optique retrouve une expression des intégrines αvβ3 plus marquée dans le groupe Sdhb-/- avec une rétention du traceur prolongée 12 h après injection. L’IRM dynamique de contraste (IRM-DCE), montre un rehaussement tumoral global nettement plus important dans le modèle Sdhb-/-, que le post-traitement par le logiciel PhysioD3D permet d’attribuer à une augmentation du volume capillaire intratumoral. Sur le plan métabolique, la consommation globale de glucose mesurée par TEP au 18FDG est également plus marquée dans les tumeurs Sdhb-/-. Enfin, la spectroscopie par résonance magnétique (1H-MRS) a mis en évidence une accumulation de succinate dans les tumeurs Sdhb-/-, non retrouvée dans les tumeurs WT. Ce résultat a été confirmé par spectrométrie de masse et cette technique innovante de détection du succinate in vivo a été transférée avec succès en clinique pour l’exploration des patients porteurs de PCC/PGL. En conclusion, l’imagerie in vivo a permis de distinguer le phénotype des tumeurs Sdhb-/- de celui des WT, avec une néoangiogenèse, une microcirculation et un métabolisme glucidique augmentés. Ces résultats permettent de proposer des études précliniques de réponse précoce aux traitements. La détection de l’accumulation de succinate par 1H-MRS ouvre la possibilité d’un diagnostic « métabolique » préopératoire pour détecter les patients de mauvais pronostic. / Pheochromocytomas and paragangliomas (PCC/PGL) are rare neuroendocrine tumours that arise from chromaffin cells of the adrenal medulla, sympathetic and parasympathetic paraganglia respectively. Around 15% of PCC are malignant. SDHB mutations are associated with malignancy and poor prognosis. SDH deficiency leads to succinate accumulation that induces a cellular pseudohypoxic phenotype, promoting in particular VEGF and GLUT-1 expression and increasing angiogenesis and glucose metabolism. The high malignancy hazard associated with SDHB and the absence of curative treatment of metastatic forms of the disease make it essential to develop a mouse model for preclinical trials launching. The quest for a predisposed mouse model of Sdhb-deficient tumors being unsuccessful, Sdhb-/- and wild-type (WT) immortalized mouse chromaffin cells previously generated in the laboratory were propagated in the fat pad of NMRI nude mice, thereby providing the first pattern of Sdhb- deficient tumors. These mice were compared to a control group receiving non-mutated imCC (WT) and characterization was performed in vivo by multimodality imaging. Optical imaging assessing the tumor angiogenesis with Angiostamp®, an RGD fluorescent peptide, found an increased expression of integrins αvβ3 in the Sdhb-/- group 12 h after tracer injection. Dynamic contrast enhanced MRI (DCE-MRI) showed an overall tumor enhancement significantly higher in the Sdhb-/- model secondary to an increase of the tumor blood flow (F) and of the intratumoral capillary volume fraction (Vb) (compartmental analysis using PhysioD3D software). Metabolic imaging assessed by 18FDG-PET confirmed the expected high glucose consumption by Sdhb-/- tumors. Finally, magnetic resonance spectroscopy (1H-MRS) detected succinate accumulation in Sdhb-/- tumors and not in WT tumors. This result was confirmed by mass spectrometry and this innovative procedure for in vivo detection of succinate was translated into patients suffering from PCC/PGL. A succinate peak was specifically observed in SDHx-related PCC/PGL patients. In conclusion, these results show strong differences between Sdhb-/- and WT allografts and suggest that preclinical therapeutic studies could be implemented in this unique model of Sdhb-deficient tumour. Our noninvasive, highly sensitive and specific method allowing in vivo detection of succinate, the major biomarker of SDHx-mutated tumors was translated into clinical imaging.

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 / 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

Yung, Him Shun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 133-146). / Abstracts in English and Chinese. / Abstract --- p.i / 論文摘要 --- p.iv / Acknowledgements --- p.vi / Publications based on work in this thesis --- p.vii / Abbreviations --- p.viii / Contents --- p.xi / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Objectives and overview of this study --- p.1 / Chapter 1.2 --- Rat pheochromocytoma (PC12) cells --- p.3 / Chapter 1.3 --- Prostanoids and their receptors --- p.4 / Chapter 1.4 --- Roles of prostanoids --- p.7 / Chapter 1.5 --- Nerve growth factor (NGF) and its receptors --- p.9 / Chapter 1.6 --- Change of gene expressions by NGF in PC12 cells --- p.10 / Chapter 1.7 --- Signaling pathways involved in NGF-induced differentiation of PC12 cells --- p.12 / Chapter 1.8 --- Classification of adenylyl cyclases --- p.14 / Chapter 1.9 --- Methods to study differentiation of PCI 2 cells --- p.15 / Chapter Chapter 2 --- Materials and Methods --- p.19 / Chapter 2.1 --- Materials --- p.19 / Chapter 2.2 --- Cell culture medium and buffers --- p.25 / Chapter 2.3 --- Buffers and solutions for assay of [3H]inositoI phosphates ([3H]IP) production --- p.25 / Chapter 2.4 --- Buffers and solutions for assay of [3H]cAMP production --- p.27 / Chapter 2.5 --- Buffers and solutions for Western blotting --- p.28 / Chapter 2.6 --- Methods --- p.30 / Chapter 2.6.1 --- Maintenance of PC12 cells --- p.30 / Chapter 2.6.2 --- General culture condition of PCI2 cells for NGF treatment --- p.31 / Chapter 2.6.3 --- Determination of phospholipase C activity in PC12 cells --- p.31 / Chapter 2.6.3.1 --- Principle of assay --- p.31 / Chapter 2.6.3.2 --- Column preparation --- p.32 / Chapter 2.6.3.3 --- Measurement of [3H]IP production --- p.33 / Chapter 2.6.3.4 --- Data analysis --- p.34 / Chapter 2.6.4 --- Determination of adenylyl cyclase activity in PC12 cells --- p.35 / Chapter 2.6.4.1 --- Principle of assay --- p.35 / Chapter 2.6.4.2 --- Column preparation --- p.35 / Chapter 2.6.4.3 --- Measurement of [3H]cAMP production --- p.36 / Chapter 2.6.4.4 --- Data analysis --- p.37 / Chapter 2.6.5 --- Determination of neurofilament protein expression in PC12 cells by Western blotting --- p.38 / Chapter 2.6.6 --- Determination of adenylyl cyclase isoform expression in PC12 cells by reverse transcriptase-polymerase chain reaction (RT-PCR) --- p.39 / Chapter 2.6.6.1 --- Isolation of total cellular RNA --- p.39 / Chapter 2.6.6.2 --- Synthesis of first strand cDNA by reverse transcription (RT) --- p.40 / Chapter 2.6.6.3 --- Polymerase Chain Reaction (PCR) --- p.41 / Chapter 2.6.6.4 --- Agarose gel electrophoresis --- p.41 / Chapter 2.6.7 --- Neurite quantification --- p.42 / Chapter 2.6.8 --- Trypan blue exclusion test --- p.42 / Chapter Chapter 3 --- Results --- p.45 / Chapter 3.1 --- Characterization of prostanoid receptor expression in PC12 cells . --- p.45 / Chapter 3.1.1 --- Study of the presence of Gq-coupled prostanoid receptors --- p.45 / Chapter 3.1.2 --- Study of the presence of Gs-co»pled prostanoid receptors --- p.47 / Chapter 3.1.3 --- Study of the presence of Gi-coupled prostanoid receptors --- p.48 / Chapter 3.1.4 --- Further proof of EP3 expression in PC12 cells --- p.50 / Chapter 3.1.5 --- Discussion --- p.51 / Chapter 3.2 --- Time course effect of NGF on PC12 cells --- p.65 / Chapter 3.2.1 --- Effect of NGF on PGE2-mediated inhibition of forskolin-stimulated [3H]cAMP production --- p.65 / Chapter 3.2.2 --- Effect of NGF on basal and forskolin-stimulated [3H]cAMP production --- p.67 / Chapter 3.2.3 --- Acute effect of NGF on [3H]cAMP production --- p.70 / Chapter 3.2.4 --- Effect of NGF withdrawal on basal and forskolin-stimulated [3H]cAMP production --- p.71 / Chapter 3.2.5 --- Effect of NGF on adenylyl cyclase gene expression --- p.72 / Chapter 3.2.6 --- Discussion --- p.74 / Chapter 3.3 --- Quantification of the degree of differentiation of PC12 cells --- p.89 / Chapter 3.3.1 --- Expression of neurofilament protein as a marker of differentiation --- p.89 / Chapter 3.3.2 --- Neurite assays --- p.90 / Chapter 3.3.2.1 --- Manual assessment of PC12 cells --- p.90 / Chapter 3.3.2.2 --- Quantification of images of PC1 2 cells --- p.91 / Chapter 3.3.3 --- Discussion --- p.93 / Chapter 3.4 --- Adenosine A2a receptor activity in PC12 cells --- p.106 / Chapter 3.4.1 --- Effect of NGF on A2Areceptor-mediated [3H]cAMP production --- p.106 / Chapter 3.4.2 --- Synergistic activation of adenylyl cyclase by A2A receptor and forskolin --- p.108 / Chapter 3.4.3 --- Chronic and acute effect of ADA and ZM241385 on [3H]cAMP production --- p.109 / Chapter 3.4.3.1 --- Chronic effect of ADA and ZM241385 --- p.110 / Chapter 3.4.3.2 --- Acute effect of ADA and ZM241385 --- p.111 / Chapter 3.4.4 --- Discussion --- p.112 / Chapter Chapter 4 --- Discussion and future perspectives --- p.121 / Chapter 4.1 --- Discussion --- p.121 / Chapter 4.2 --- Future perspectives --- p.131 / References --- p.133

The human genome is a dynamic structure, recently recognized to present with significant large-scale structural variation. DNA-copy number changes represent one common type of such variation and is found both between individuals and within the somatic cells of the same individual, especially in disease states like cancer.  Apart from DNA-rearrangements, epigenomic changes are increasingly acknowledged as important events in the maintenance of genomic integrity. In this thesis, different array-based methods have been applied for global genomic and epigenomic profiling of both normal and cancer cells. In paper I, a genomic microarray was established and used to determine DNA-copy number variants (CNVs) in a cohort of 76 healthy individuals from three ethnic populations. We identified 315 CNV regions that in total encompassed ~3,5% of the genome. In paper II, the array was utilized to discover CNVs within several differentiated tissues from the same subject. Six variants were identified providing evidence for somatic mosaicism. In paper III and IV we studied pheochromocytomas and paragangliomas, rare endocrine tumours that most often present as benign and sporadic with unclear genetic/epigenetic cause. Genome-wide DNA-copy number analysis of 53 benign and malignant samples in paper III revealed numerous common and novel chromosomal regions of losses and gains. High frequencies of relatively small overlapping regions of deletions were detected on chromosome 1p arm, encompassing several candidate tumour suppressor genes. In paper IV, an epigenomic map for two histone modifications associated with silent (H3K27me3) or active (H3K4me3) gene transcription, was generated for one malignant pheochromocytoma. Integrated analysis of global histone methylation, copy number alterations and gene expression data aided in the identification of candidate tumour genes. In conclusion, the performed studies have contributed to gain knowledge of CNVs in healthy individuals, and identified regions and genes which are likely associated with the development and progression of pheochromocytoma/paraganglioma.

published_or_final_version / abstract / toc / Zoology / Master / Master of Philosophy

Endocrine tumors arise from endocrine glands. Most endocrine tumors are benign but malignant variants exist. Several endocrine neoplasms display loss of parts of chromosome 11 or 18, produce hormones and responds poorly to conventional chemotherapeutics. The multiple endocrine neoplasia syndromes are mainly confined to endocrine tumors. This opens the question if there exists a single or several endocrine tumor genes. The aim of the study was to describe genetic derangements in endocrine tumors. Paper I: Investigation of mutational status of SDHAF2 in parathyroid tumors. SDHAF2 is located in the proximity of 11q13, a region that frequently displays loss in parathyroid tumors. We established that mutations in SDHAF2 are infrequent in parathyroid tumors. Paper II: Study of SDHAF2 gene expression in a cohort of benign pheochromocytomas (PCC) (n=40) and malignant PCC (n=10). We discovered a subset of  benign PCC (28/40) and all malignant PCC (10/10) with significantly lower SDHAF2 expression. Benign PCC with low SDHAF2 expression and malignant tumors consistently expressing low levels of SDHAF2 were methylated in the promoter region. SDHAF2 expression was restored in vitro after treatment with 5- aza-2-deoxycytidine. Paper III: HumanMethylation27 array (Illumina) covering 27578 CpG sites spanning over 14495 genes were analyzed in a discovery cohort of 10 primary small neuroendocrine tumors (SI-NETs) with matched metastases. 2697 genes showed different methylation pattern between the primary tumor and its metastasis. We identified several hypermethylated genes in key regions. Unsupervised clustering of the tumors identified three distinct clusters, one with a highly malignant behavior. Paper IV: Loss of chromosome 18 is the most frequent genetic aberration in SI-NETs. DNA from SI-NETs were subjected to whole exome capture sequencing and high resolution SNP array. Genomic profiling revealed loss of chromosome 18 in 5 out of 7 SI-NETs. No tumor-specific somatic mutation on chromosome 18 was identified which suggests involvement of other mechanisms than point mutations in SI-NET tumorigenesis. Paper V: The cost for diagnostic genetic screening of common susceptibility genes in PCC is expensive and labor intensive. Three PCC from three patients with no known family history were chosen for exome capture sequencing. We identified three variants in known candidate genes. We suggest that exome-capture sequencing is a quick and cost-effective tool.