Background Loss-of-function germline MEN1 gene mutations account for 75–95% of patients with multiple endocrine neoplasia type 1 (MEN1). It has been postulated that mutations in non-coding regions of MEN1 might occur in some of the remaining patients; however, this hypothesis has not yet been fully investigated. Objective To sequence for the entire MEN1 including promoter, exons and introns in a large MEN1 cohort and determine the mutation profile. Methods and patients A target next-generation sequencing (tNGS) assay comprising 7.2 kb of the full MEN1 was developed to investigate germline mutations in 76 unrelated MEN1 probands (49 familial, 27 sporadic). tNGS results were validated by Sanger sequencing (SS), and multiplex ligation-dependent probe amplification (MLPA) assay was applied when no mutations were identifiable by both tNGS and SS. Results Germline MEN1 variants were verified in coding region and splicing sites of 57/76 patients (74%) by both tNGS and SS (100% reproducibility). Thirty-eight different pathogenic or likely pathogenic variants were identified, including 13 new and six recurrent variants. Three large deletions were detected by MLPA only. No mutation was detected in 16 patients. In untranslated, regulatory or in deep intronic MEN1 regions of the 76 MEN1 cases, no point or short indel pathogenic variants were found in untranslated, although 33 benign/likely benign and three new VUS variants were detected. Conclusions Our study documents that point or short indel mutations in non-coding regions of MEN1 are very rare events. Also, tNGS proved to be a highly effective technology for routine genetic MEN1 testing.

Background/Introduction Myocardial protection is the group of strategies aimed at reducing ischemia-reperfusion lesions and its consequences. There is much discussion regarding different methods and their features, while there is no unanimity about which is most appropriate.

seguimento e tratamento de casos-índices portadores de mutação e de MEN1(-). Descritores: Sequenciamento genético; Diagnóstico genético; Neoplasia endócrina múltipla tipo 1; Sequenciamento de nucleotídeos em larga escala; Doenças raras.

Background: Index cases clinically diagnosed with multiple endocrine neoplasia type 1 (MEN1) are declared as MEN1 phenocopies if no germline MEN1 mutation is identified. In comparison with positive-mutation cases, most phenocopies have been diagnosed in older age, mainly by association of primary hyperparathyroidism (HPT) and pituitary adenoma (PIT), with HPT predominantly diagnosed as uniglandular disease. Besides that, phenocopies rarely develop a third MEN1-related tumor and are associated with lower morbidity and longer survival. However, all these data are mainly derived of genetic studies by Sanger targeted to MEN1 gene from a strict number of MEN1 phenocopies. Objectives: to recognize strong clinical profiles capable of predicting the occurrence or absence of germline MEN1 mutation refining the clinical differentiation of phenocopies and mutation-positive cases before genetic testing disclosure. Casuistic/Methods: 143 MEN1 index cases: 87 MEN1-positive and 56 true MEN1 phenocopies (excluded mutations for MLPA assay and by a mini-painel based on long-range PCR and next generation sequencing of 6 MEN1-related genes (MEN1, p15, p18, p21, p27, AIP) covering full coding and non-coding regions. Results: High detectability rate of MEN1 mutation was associated with the presence of ≥ 4 organs affected for primary tumors (100%), association of HPT/PET (neuroendocrine pancreatic tumor)/PIT (93%), HPT/PET (81%), positive familial history (88% vs. 48%), presence of PET (84%) as malign (80%) as multifocal (95%), two different PETs (100%), multiglandular HPT (81%) and diagnosis of one MEN1-related tumor (93%) or of two/three MEN1 tumors diagnosed before 21y (100%). The combination HPT/PIT has the lowest rate of detection of mutation (33%), it is even lower if PIT was acromegaly (12%) or age at the diagnosis of HPT and PIT was, respectively, > 45y and >30y (8%) and absent if it is added uniglandular HPT (0%) or if there was association HPT/PIT (age-independent) with uniglandular HPT (0%). The prediction for detection of mutation increases if these HPT (> 45y)/PIT (> 30y) cases have multiglandular HPT (20%) and it is 100% with association HPT (< 30y)/PIT (< 21y). A p <0.05 was observed to all data above. Conclusions: By integration of phenotypic clues and full genetic analysis applied to the largest MEN1 phenocopy series, we identified strong clinical predictors capable of anticipate the potential risk rate for mutation detection revealing the estimated chance of an index case harbor a mutation or be classified as phenocopy. By their peculiarities, the management/treatment of phenocopies should potentially be different of that recommended to mutation-positive cases.

Context: Guidelines has systematically suggested genetic testing for a wide spectrum of phenotypes in order to confirm or exclude surely the diagnosis of multiple endocrine neoplasia type 1 (MEN1). However, the probability of find a germline MEN1 mutation has showed extremely variable (5-95%) when different phenotypes are considered. The prompt recognition of potential clinical predictors could anticipate the result of the genetic testing in the different clinical scenarios. Objective: To investigate if specific phenotypic features may predict the presence of germline MEN1 mutations in index-cases with clinical diagnosis of MEN1 syndrome. Methods and Patients: 110 MEN1 index cases were included: amplicons based on long-range PCR covering the full MEN1 open reading frame of 94 MEN1 index cases (49 familial, 45 sporadic) were sequenced by targeted-Next Generation Sequencing (tNGS) MiSeq Illumina platform while Sanger Sequencing (SS) was approached to the other 16 MEN1 index cases (2 familial, 14 sporadic). MLPA assay was supported to 31 MEN1 -negative patients by tNGS/SS whose DNA samples were available. Results: 68 index cases (62%) had germline MEN1 mutations. With the tNGS/SS/MLPA protocol, the mutation detectability rate was higher in familial MEN1 (90%, 46/51 vs. 37%; 22/59; p, < 0.05). In comparison with MEN1 -positive patients, MEN1 -negative cases were older (53±12 vs. 39±13; p, < 0.05) and most of them had no more than two MEN1-related tumors (76%, 32/42 vs. 10%, 7/68; p, < 0.05). Considering the subset of 59 patients with negative familial history, the 22 MEN1 -positive patients revealed clinical predictors for detection of MEN1 mutation when they were compared with 37 truly non -MEN1 mutation carriers. Thus, sporadic MEN1 -positive patients had higher significantly prevalence of the following predictors. 1) ≥ 3 tumors, 86% (19/22) versus 11% (4/37); 2) PETs, 86% (19/22) versus 22% (8/37); 3) which were multifocal, 100% versus 1/8 (9%); 4) with frequent synchronic association of functioning and NF/PETs, 47% (9/19) versus 0% and; 5) high frequency of malignancies, 58% (11/19) versus 5% (2/37). Indeed, there was confirmation of these predictors comparing all 68 MEN1 -positive carriers against 42 MEN1 -negative cases. Conclusions: The strategy adopted to support the diagnosis of MEN1 based on analysis of clinical predictors and genetic testing for tNGS/SS-MLPA underscored that the positive familial history, occurrence of three or more MEN1 tumors, presence of PETs, especially multifocal PETs, and malignancies are strong predictors to detection of MEN1 mutations. ...

Agradeço a Deus por me acompanhar em todo o meu caminho, me amparando em momentos difíceis, até quando até eu duvidada de mim mesmo. Gostaria de agradecer aos meus pais, Marta e Marivaldo, por me ensinarem o verdadeiro significado de caráter, integridade, respeito e, acima de tudo amor, o maior dos ensinamentos. Obrigado pelo apoio incondicional. Agradeço a Bruna, minha noiva, por todo o carinho, compreensão e cumplicidade ao longo de todos estes anos ao meu lado. Agradeço ao meu orientador, Prof. Dr. Delmar, por me receber no serviço e por todos os ensinamentos e conhecimentos a mim transmitidos. Agradeço a todos do LIM 25, em especial Prof. Dr. Alexander, Elisângela, Lucas, Lilian, Betsaida, Eliete, Graça e Geni pela ajuda na execução do projeto, pelas horas de descontração no laboratório e pela amizade verdadeira. Agradeço a todos do LIM 15 e do SELA, em especial a Profa. Dra. Suely Marie e Profa. Dra. Sueli Shinjo pela ajuda e apoio na execução do projeto. Agradeço a todos os residentes e assistentes que colaboraram, de alguma forma, direta ou indiretamente para a execução do projeto. Agradeço a cada professor que já tive e terei, pois sem eles não chegaria aqui. Agradeço a FAPESP pelo apoio financeiro destinado a este trabalho.