Caracterização clínica, molecular e de vias celulares de pacientes com neurofibromatose e esclerose tuberosa no Brasil

Introdução. As neurofibromatoses tipo 1 (NF1) e tipo 2 (NF2) e a esclerose tuberosa (ET) são as genodermatoses mais comuns, que possuem potencial de desenvolvimento de tumores benignos e malignos. Elas são síndromes hereditárias com padrão de herança autossômico dominante e expressividade altamente variável, causadas, respectivamente, por mutações nos genes supressores tumorais NF1, NF2 e TSC1 ou TSC2, todos com grande número de mutações diferentes descritas em bases de dados, envolvendo rearranjos e mutações de ponto. Objetivos. O objetivo deste trabalho foi caracterizar, do ponto de vista clínico e molecular, os pacientes e familiares com diagnóstico de NF1 e NF2 avaliados no Hospital de Clínicas de Porto Alegre e pacientes com diagnóstico de ET avaliados em centros da Rede Nacional de Câncer Familial, além de avaliar a expressão gênica global e os processos celulares em células com mutações em TSC1 e TSC2. Resultados. Foram recrutados 93 pacientes com NF1 e 7 com NF2, no sul do Brasil, e 53 pacientes com ET oriundos de 4 regiões do Brasil. Na análise molecular, a frequência de grandes rearranjos e mutações de ponto foi de 4,3 e 79,0% em NF1; 14,3 e 29,0% em NF2; e 9,0 e 81,0% em TSC1 e TSC2, sendo 33 variantes novas em NF1 e 20 em TSC1 e TSC2. Correlações genótipo-fenótipo específicas para a população brasileira não foram estabelecidas, mas algumas correlações previamente descritas também foram identificadas em nosso estudo. Hotspots para mutações não foram encontrados. Por fim, análise de processos celulares e expressão gênica com células mutadas e normais (com e sem rapamicina) mostrou que não há morte celular e mudança em fases de ciclo celular; entretanto, em pacientes com esclerose tuberosa, a indução de autofagia é muito maior do que em células controles após o tratamento com rapamicina (p=0,039). Discussão e conclusões. Este é o primeiro estudo do Brasil a realizar a caracterização molecular completa de pacientes com neurofibromatoses e esclerose tuberosa. A caracterização das variantes nesses pacientes é muito importante para confirmar o diagnóstico quando a clínica é duvidosa, para o acompanhamento da doença e a possível prevenção das características multissistêmicas. Além disso, foi demonstrado que a autofagia pode ser o mecanismo inicial que leva a formação de tumores em células com mutações em TSC1 ou TSC2. O processo de autofagia precisa ser estudado com mais detalhe a fim de esclarecer o papel desse mecanismo na esclerose tuberosa, e, possivelemente, levar ao uso de terapia de alvo molecular direcionada à via de autofagia, em conjunto com inibidores de mTOR ou sozinha.Introduction. Neurofibromatosis type 1 (NF1) and type 2 (NF2) and tuberous sclerosis (TSC) are the most common genodermatoses, with the potential to develop benign and malignant tumors. These genodermatoses are hereditary syndromes with an autosomal dominant inheritance pattern and highly variable expressivity. They are caused, respectively, by mutations in the tumor suppressor genes NF1, NF2 and TSC1 or TSC2, all with a large number of different mutations described, including rearrangements and point mutations. Objectives. The aim of this study was to perform clinical and molecular characterization of NF1 and NF2 patients and their relatives, evaluated at Hospital de Clínicas of Porto Alegre and patients diagnosed with TSC evaluated in the Familial Cancer National Network. In addition, we aimed to evaluate the global gene expression and cellular processes in cells with mutations in TSC1 and TSC2. Results. Ninety-three NF1 patients and 7 NF2 patients were recruited from south Brazil; 53 TSC patients were recruited from 4 Brazilian regions. In molecular analysis, the frequency of large rearrangements and point mutations was 4.3 and 79.0% in NF1; 14.3 and 29.0% in NF2; and 9.0 and 81.0% in TSC1 and TSC2, with 33 new variants in NF1 and 20 in TSC1 and TSC2. Specific genotype-phenotype correlations in Brazilian population were not established, but some previously described correlations were also identified in our study. Hotspots for mutations were not found. Finally, analysis of cellular processes and gene expression in normal and mutated cells (with and without rapamycin) showed that there is no cell death and change in cell cycle phases; however, in patients with tuberous sclerosis, the induction of autophagy is much greater than in control cells after treatment with rapamycin (p=0.039). Discussion and conclusion. This is the first Brazilian study to perform the complete molecular characterization of patients with neurofibromatoses and tuberous sclerosis. Variant characterization in these patients is very important to confirm diagnosis when the clinic is doubtful, to follow disease progress and possibly prevent the occurrence of multisystemic characteristics. In addition, we showed that autophagy may be the initial mechanism leading to tumor formation in cells with TSC1 or TSC2 mutations. Autophagy needs to be studied in more detail to clarify the role of this mechanism in tuberous sclerosis, and possibly lead to the use of autophagy-targeted molecular therapy, alone or in conjunction with mTOR inhibitors

TSC1 and TSC2 gene mutations and their implications for tratment in tuberous sclerosis complex : a review

Tuberous sclerosis complex is an autosomal dominant disorder characterized by skin manifestations and formation of multiple tumors in different organs, mainly in the central nervous system. Tuberous sclerosis is caused by the mutation of one of two tumor suppressor genes, TSC1 or TSC2. Currently, the development of novel techniques and great advances in high-throughput genetic analysis made mutation screening of the TSC1 and TSC2 genes more widely available. Extensive studies of the TSC1 and TSC2 genes in patients with TSC worldwide have revealed a wide spectrum of mutations. Consequently, the discovery of the underlying genetic defects in TSC has furthered our understanding of this complex genetic disorder, and genotype-phenotype correlations are becoming possible, although there are still only a few clearly established correlations. This review focuses on the main symptoms and genetic alterations described in TSC patients from 13 countries in three continents, as well as on genotype-phenotype correlations established to date. The determination of genotype-phenotype correlations may contribute to the establishment of successful personalized treatment for TSC

The Role of Co-Deleted Genes in Neurofibromatosis Type 1 Microdeletions: an Evolutive Approach

Neurofibromatosis type 1 (NF1) is a cancer predisposition syndrome that results from dominant loss-of-function mutations mainly in the NF1 gene. Large rearrangements are present in 5–10% of affected patients, generally encompass NF1 neighboring genes, and are correlated with a more severe NF1 phenotype. Evident genotype–phenotype correlations and the importance of the co-deleted genes are difficult to establish. In our study we employed an evolutionary approach to provide further insights into the understanding of the fundamental function of genes that are co-deleted in subjects with NF1 microdeletions. Our goal was to access the ortholog and paralog relationship of these genes in primates and verify if purifying or positive selection are acting on these genes. Fourteen genes were analyzed in twelve mammalian species. Of these, four and ten genes showed positive selection and purifying selection, respectively. The protein, RNF135, showed three sites under positive selection at the RING finger domain, which may have been selected to increase efficiency in ubiquitination routes in primates. The phylogenetic analysis suggests distinct evolutionary constraint between the analyzed genes. With these analyses, we hope to help clarify the correlation of the co-deletion of these genes and the more severe phenotype of NF1

The paradox of autophagy in tuberous sclerosis complex

Tuberous sclerosis complex (TSC) is an autosomal dominant genetic disorder caused by germline mutations in TSC1 or TSC2 genes, which leads to the hyperactivation of the mTORC1 pathway, an important negative regulator of autophagy. This leads to the development of hamartomas in multiple organs. The variability in symptoms presents a challenge for the development of completely effective treatments for TSC. One option is the treatment with mTORC1 inhibitors, which are targeted to block cell growth and restore autophagy. However, the therapeutic effect of rapamycin seems to be more efficient in the early stages of hamartoma development, an effect that seems to be associated with the paradoxical role of autophagy in tumor establishment. Under normal conditions, autophagy is directly inhibited by mTORC1. In situations of bioenergetics stress, mTORC1 releases the Ulk1 complex and initiates the autophagy process. In this way, autophagy promotes the survival of established tumors by supplying metabolic precursors during nutrient deprivation; paradoxically, excessive autophagy has been associated with cell death in some situations. In spite of its paradoxical role, autophagy is an alternative therapeutic strategy that could be explored in TSC. This review compiles the findings related to autophagy and the new therapeutic strategies targeting this pathway in TSC