Effect of 3'UTR RET variants on RET mRNA secondary structure and disease presentation in medullary thyroid carcinoma

The RET S836S variant has been associated with early onset and increased risk for metastatic disease in medullary thyroid carcinoma (MTC). However, the mechanism by which this variant modulates MTC pathogenesis is still open to discuss. Of interest, strong linkage disequilibrium (LD) between RET S836S and 3'UTR variants has been reported in Hirschsprung's disease patients. Objective To evaluate the frequency of the RET 3’UTR variants (rs76759170 and rs3026785) in MTC patients and to determine whether these variants are in LD with S836S polymorphism. Methods Our sample comprised 152 patients with sporadic MTC. The RET S836S and 3’UTR (rs76759170 and rs3026785) variants were genotyped using Custom TaqMan Genotyping Assays. Haplotypes were inferred using the phase 2.1 program. RET mRNA structure was assessed by Vienna Package. Results The mean age of MTC diagnosis was 48.5±15.5 years and 57.9%were women. The minor allele frequencies of RET polymorphisms were as follows: S836S, 5.6%; rs76759170, 5.6%; rs3026785, 6.2%. We observed a strong LD among S836S and 3’UTR variants (|D’| = -1, r2 = 1 and |D’| = -1, r2 = 0,967). Patients harboring the S836S/3’UTR variants presented a higher percentage of lymph node and distant metastasis (P = 0.013 and P<0.001, respectively). Accordingly, RNA folding analyses demonstrated different RNA secondarystructure predictions for WT(TCCGT), S836S(TTCGT) or 3’UTR(GTCAC) haplotypes. The S836S/3’UTR haplotype presented a greater number of double helices sections and lower levels of minimal free energy when compared to the wild-type haplotype, suggesting that these variants provides the most thermodynamically stable mRNA structure, which may have functional consequences on the rate of mRNA degradation. Conclusion The RET S836S polymorphism is in LD with 3’UTR variants. In silico analysis indicate that the 3’UTR variants may affect the secondary structure of RET mRNA, suggesting that these variants might play a role in posttranscriptional control of the RET transcripts

A systematic CRISPR screen defines mutational mechanisms underpinning signatures caused by replication errors and endogenous DNA damage.

Mutational signatures are imprints of pathophysiological processes arising through tumorigenesis. We generated isogenic CRISPR-Cas9 knockouts (Δ) of 43 genes in human induced pluripotent stem cells, cultured them in the absence of added DNA damage, and performed whole-genome sequencing of 173 subclones. ΔOGG1, ΔUNG, ΔEXO1, ΔRNF168, ΔMLH1, ΔMSH2, ΔMSH6, ΔPMS1, and ΔPMS2 produced marked mutational signatures indicative of being critical mitigators of endogenous DNA modifications. Detailed analyses revealed mutational mechanistic insights, including how 8-oxo-dG elimination is sequence-context-specific while uracil clearance is sequence-context-independent. Mismatch repair (MMR) deficiency signatures are engendered by oxidative damage (C>A transversions), differential misincorporation by replicative polymerases (T>C and C>T transitions), and we propose a 'reverse template slippage' model for T>A transversions. ΔMLH1, ΔMSH6, and ΔMSH2 signatures were similar to each other but distinct from ΔPMS2. Finally, we developed a classifier, MMRDetect, where application to 7,695 WGS cancers showed enhanced detection of MMR-deficient tumors, with implications for responsiveness to immunotherapies

Process evaluation of the Bridging the Age Gap in Breast Cancer decision support intervention cluster randomized trial [abstract only]

Aims/Objectives: Shared decision making on the choice of treatment for older women with breast cancer involves many factors. Comprehensive geriatric assessment (CGA) is recognised to have a role in older patients with cancer, but how this should be utilised is still debatable. A pilot study involving older women newly diagnosed with early operable primary breast cancer was conducted aiming to explore the potential value of CGA. Methods: Decision of primary treatment followed consultation with the clinical team and was not guided by any aspect of this study. CGA, using a validated cancer-specific tool from our collaborator, A Hurria, was conducted within 6 weeks after diagnosis, regardless of date of surgery/first treatment. A total of 178 female patients aged ≥70 years with a new diagnosis of early (stage 1 or 2; cT0-2, N0-1, M0) operable primary breast cancer proven histologically, were thus far recruited from three UK centres. Results: Among these 178 patients, 149 underwent primary surgery and 29 received non-surgical treatment (primary endocrine therapy (N=28) or radiotherapy (N=1)). CGA determined that increasing age (p=0.006), reduced independence with activities of daily living (ADLs) (p=0.001) and independent activities of daily living (IADLS) (p=0.001), increased number and severity of comorbidity (p=0.043), reduced Karnofsky performance status when rated both by the patient (p=0.001) and physician (p=0.003), were significantly related to non-surgical treatment within 6 weeks after diagnosis. Other CGA parameters measured which were not significant include number of daily medications, level of social support, level of social activity, cognition, number of falls, 'Timed up and go' score. Conclusions: The pilot study has confirmed that CGA may have value in assessing this cohort of patients. Generally, it appears that patients receiving non-surgical treatment are more frail than their counterparts undergoing surgery. The study is ongoing and has expanded to include an international centre

Modelo de regulação dos nucleotídeos extracelulares no líquido superficial das vias respiratórias

Exportado OPUSMade available in DSpace on 2019-08-14T12:00:38Z (GMT). No. of bitstreams: 1 disserta__o__tauanne_amarante.pdf: 2121982 bytes, checksum: 95bb930aefb9c0e7989d691937dfb048 (MD5) Previous issue date: 9Os nucleotídeos extracelulares desempenham um papel fundamental na regulação do transporte mucociliar e, dessa forma, são essenciais na defesa das vias respiratórias contra patógenos inalados. Os nucleotídeos adenosina trifostafato (ATP), adenosina difosfato (ADP) e adenosina monofosfato (AMP) são secretados pelo epitélio respiratório para o líquido superficial das vias respiratórias, onde são metabolizados por ecto-enzimas e transformados nos nucleosídeos adenosina (ADO) e inosina (INO) que, por sua vez, são absorvidos pelas células do epitélio respiratório. São as concentrações de ATP e ADO extracelulares que, ao ativar os receptores P2Y2 e A2B na membrana celular, regulam a taxa de secreção de muco, a frequência de batimento ciliar e a hidratação e viscosidade do muco. Esta rede bioquímica de regulação das concentrações de ATP e ADO extracelulares foi recentemente descrita quantitativamente por um modelo computacional baseado em dados experimentais coletados em culturas de células do epitélio respiratório humano. Embora este modelo tenha reproduzido dados experimentais in vitro, ele não podia ser usado para investigar a regulação de nucleotídeos extracelulares in vivo, já que se baseou em culturas de célula contendo líquido a uma altura de ~1500 mm, enquanto in vivo a altura do líquido superficial é da ordem de 30 mm. Com o intuito de adaptar o modelo computacional para descrever o comportamento do sistema in vivo, selecionamos dados experimentais da literatura em que a rede de regulação foi investigada para alturas do líquido superficial variando de 14 mm a 2730 mm. A nova versão do modelo, proposta neste trabalho, consiste na solução da equação de reação-difusão em coordenadas cilíndricas, com condições de contorno que expressam as reações catalisadas pelas ectonucleotidases na superfície do epitélio bronquial. O conjunto de equações diferencias acopladas foi resolvido numericamente usando o método de volumes finitos. A método foi validado comparando a solução numérica com a solução analítica de dois casos particulares de condição de contorno: barreiras refletoras e fluxo constante em z=0. Utilizando o modelo, investigamos o comportamento dos nucleotídeos após choque hipotônico, obtendo uma boa concordância com os dados experimentais. Este modelo pode ser importante para o desenvolvimento de novas terapias para tratamento de patologias respiratórias, especialmente da Fibrose Cística. Os indivíduos portadores dessa doença genética apresentam quadro de infecção bacteriana crônica devido a uma falha no transporte mucociliar, que inclui a desidratação do muco e a secreção de muco em excessoExtracellular nucleotides play a key role in the regulation of mucociliary clearance and thus are essential in protecting the airways against inhaled pathogens. Adenosine triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP) nucleotides are secreted by the respiratory epithelium to the airway surface liquid, where they are metabolized by ecto-enzymes and converted into the nucleosides adenosine (ADO) and inosine (INO) which in turn are absorbed by the cells of the respiratory epithelium. The concentrations of extracellular ATP and ADO, by activating the membrane receptors P2Y2 and A2B, regulate the mucus secretion rate, the ciliary beating frequency, and mucus hydration and mucus viscosity. The biochemical network that regulates the concentrations of extracellular ATP and ADO was recently described quantitatively by a computer model based on experimental data collected in cultures of human respiratory epithelial cells. Although this model reproduced the experimental data, it could not be used to investigate the regulation of extracellular nucleotides in vivo, because it was based on cell cultures containing _uid at a height of _ 1500_m, while in vivo the height of the airway surface liquid is approximately 30_m. Aiming to adapt the computer model to describe the behavior of the system in vivo, we selected from the literature experimental data where the regulatory network was investigated for liquid surface heights ranging from 14_m to 2730_m. The new version of the model, proposed in this work, consists in the solution of the reactiondi _usion equation in cylindrical coordinates, with boundary conditions that express the reactions catalyzed by ectonucleotidases on the surface of the bronchial epithelium. The set of coupled di_erential equations was solved numerically using the _nite volume method. The numerical solution was validated by comparing it with the analytical solution of two particular cases of boundary conditions: re_ective barriers and constant _ux at z = 0. Using the model, we investigate the behavior of the nucleotides after hypotonic challenge, the results are in good agreement with the experimental data. This model can be important for the development of new therapies for respiratory pathologies, especially cystic _brosis. Individuals with this genetic disease have chronic bacterial infection in the lungs due to a failure in mucociliary clearance, that includes mucus dehydration and excessive mucus secretion

Análise da estabilidade termodinâmica e de parâmetros estruturais de DNA e RNA por modelos mesoscópicos

Exportado OPUSMade available in DSpace on 2019-08-14T03:27:18Z (GMT). No. of bitstreams: 1 tesecorrigida_posdefesa_com_artigos.pdf: 2535851 bytes, checksum: 427cea796d53008b9de2914c3e6fca71 (MD5) Previous issue date: 6Modelos mesoscopicos como o proposto por Peyrard e Bishop apresentam uma relevancia significativa no estudo da estabilidade termica de DNA e RNA. A sua simplicidade computacional permite a analise desses oligonucleotideos por periodos mais longos e de importancia fisiologica e experimental inacessiveis por tecnicas mais sofisticadas. Recentemente houve bastante progresso na parametrizacao dos pares de bases canonicos CG e AT em DNA e CG e AU em RNA para este modelo. No entanto, por serem mais estaveis do que pares nao-canonicos, essas bases sao relativamente simplesde modelar. Nosso estudo viabilizou a aplicacao do modelo PB alem das bases canonicas, bem como a investigacao de parametros estruturais. O primeiro sistema que foi estudado e o de guanina-uracila (GU) em RNA. GU desempenha um papel biologico importante, atuando como um local de reconhecimento para biomoleculas, alem de ser o par nao complementar mais comum em RNA. Devido a nao isostericidadedo par GU, sua estabilidade e influenciada pelo contexto da sequencia. GU apresenta diversas possibilidades conformacionais, inclusive assumindo um numero diferente de ligacoes hidrogenio dependendo das bases vizinhas. Para considerar todas as possibilidades de contexto de GU em RNA e necessario lidar com uma quantidademuito grande de parametros. Esse problema e contornado ao realizarmos a otimizacao de parametros correlacionando com dados de temperatura de desnaturacao obtidos da literatura. Desenvolvemos uma estrategia de reducao do espaco de busca de parametros que possibilitou a determinacao de grupos de configuracao de GU ordenados por intensidade de ligacao de hidrogenio. Em comparacao com dados experimentais da literatura obtivemos uma grande concordancia nas ligacoes de hidrogenio, em especial com as tecnicas de NMR. Assim, pudemos nao apenas concluir a parametrizacao deGU em RNA mas tambem demonstrar que a tecnica permite prever ligacoes de hidrogenio de pares de base nao-canonicos. No segundo tema nos abordamos a limitacao da Hamiltoniana 2D Peyrard-Bishop que nao inclui qualquer parametro estrutural. Mostramos que e possivel partir de uma formulacao Hamiltoniana helicoidal e obter uma Hamiltoniana modificada em 2D que inclui a informacao sobre o passo da helice de DNA, tecnicamente chamado de rise. Nesse estudo tambem usamos o metodo de otimizacao correlacionando com dados de desnaturacao disponiveis na literatura para obter valores de rise. Para realizar a comparacao dos nossos resultados com os obtidospor medidas experimentais, desenvolvemos uma ferramenta de pesquisa que acessa a base de dados no Nucleic Acids Database (NDB) e seleciona as sequencias de interesse para obter valores de rise oriundos de raios-x e NMR. A concordancia dos nossos resultadosde rise foi em geral semelhante com os do NDB com excecao de AT seguido de TA em DNA. Alem disto, pudemos estudar a variacao do rise em funcao de concentracao salina. Os nossos resultados evidenciam a possibilidade de realizar estudos estruturais em oligonucleotideos usando temperaturas de desnaturacao.Mesoscopic models, like the one proposed by Peyrard and Bishop, are important for the study of the thermal stability of DNA and RNA. Its computational simplicity allows to analyse these oligonucleotides over longer time scales of physiological and experimental importance which are not accessible by more sophisticated techniques. Recently, there has been progress in the parametrization of canonical base pairs for this model. However, as they are more stable than non-canonical pairs, these bases are relatively easy to model. Our study aimed at applying the PB model beyond the canonical bases, as well as to investigate structural parameters. The first system we analysed was guanine-uracil (GU) in RNA. GU has an important biological role, acting as a recognition site for biomolecules and is also the most common non-complementarybase pair in RNA. Due to the non-isostericity of GU, its stability is influenced by the sequence context. GU has several conformational possibilities and may even have different hydrogen bonds depending on neighbouring bases. To consider all context possibilities for GU in RNA it becomes necessary to deal with a very large number of parameters. This problem is dealt with by optimizing the parameters correlatingwith melting temperature data from the literature. We developed a strategy to reduce the parameter search space which allowed us to determine GU configuration groups sorted by hydrogen bond intensity. When comparing with experimental results from the literature we obtain a good agreement for the hydrogen bonds, especially from NMR. Therefore, we not only were able to conclude the parametrization of GU inRNA but also to demonstrate that the technique allows to predict hydrogen bonds for non-canonical base pairs. In the second subject we approached a limitation of the 2D Peyrard-Bishop Hamiltonian which is the lack of structural parameters. We showed that it is possible to start from a helicoidal Hamiltonian and obtain a modified 2D Hamiltonian which includes information about the helix step, technically known asrise. In this study we also use the optimization method where we correlate melting temperature data from the literature to obtain the values for rise in DNA. To perform the comparison of our results with those obtained from experimental measurements we developed a query tool which accesses the Nucleic Acids Database (NDB) and selectssequences of interest to obtain values of rise coming from X-ray and NMR. In general our results agreed well with those from the NDB except for AT followed by TA in DNA. Furthermore, we were able to study the variation of rise with salt concentration. Our results show the possibility of performing structural studies in oligonucleotides using melting temperatures

Salt Dependent Mesoscopic Model for RNA with Multiple Strand Concentrations

Mesoscopic models can be used for the description of the thermodynamic properties of RNA duplexes. With the use of experimental melting temperatures, its parametrization can provide important insights into its hydrogen bonds and stacking interactions as has been done for high sodium concentrations. However, the RNA parametrization for lower salt concentrations is still missing due to the limited amount of published melting temperature data. While the Peyrard-Bishop (PB) parametrization was found to be largely independent of strand concentrations, it requires that all temperatures are provided at the same strand concentrations. Here we adapted the PB model to handle multiple strand concentrations and in this way we were able to make use of an experimental set of temperatures to model the hydrogen bond and stacking interactions at low and intermediate sodium concentrations. For the parametrizations we make a distinction between terminal and internal base pairs, and the resulting potentials were qualitatively similar as we obtained previously for DNA. The main difference from DNA parameters, was the Morse potentials at low sodium concentrations for terminal r(AU) which is stronger than d(AT), suggesting higher hydrogen bond strength

Substantial somatic genomic variation and selection for BCOR mutations in human induced pluripotent stem cells

Funder: Dr Josef Steiner Cancer Research Award 2019, Medical Research Council (MRC) Grant-in-Aid to the MRC Cancer unit, CRUK Pioneer AwardAbstract: We explored human induced pluripotent stem cells (hiPSCs) derived from different tissues to gain insights into genomic integrity at single-nucleotide resolution. We used genome sequencing data from two large hiPSC repositories involving 696 hiPSCs and daughter subclones. We find ultraviolet light (UV)-related damage in ~72% of skin fibroblast-derived hiPSCs (F-hiPSCs), occasionally resulting in substantial mutagenesis (up to 15 mutations per megabase). We demonstrate remarkable genomic heterogeneity between independent F-hiPSC clones derived during the same round of reprogramming due to oligoclonal fibroblast populations. In contrast, blood-derived hiPSCs (B-hiPSCs) had fewer mutations and no UV damage but a high prevalence of acquired BCOR mutations (26.9% of lines). We reveal strong selection pressure for BCOR mutations in F-hiPSCs and B-hiPSCs and provide evidence that they arise in vitro. Directed differentiation of hiPSCs and RNA sequencing showed that BCOR mutations have functional consequences. Our work strongly suggests that detailed nucleotide-resolution characterization is essential before using hiPSCs

A practical framework and online tool for mutational signature analyses show intertissue variation and driver dependencies

Mutational signatures are patterns of mutations that arise during tumorigenesis. We present an enhanced, practical framework for mutational signature analyses. Applying these methods to 3,107 whole-genome-sequenced (WGS) primary cancers of 21 organs reveals known signatures and nine previously undescribed rearrangement signatures. We highlight interorgan variability of signatures and present a way of visualizing that diversity, reinforcing our findings in an independent analysis of 3,096 WGS metastatic cancers. Signatures with a high level of genomic instability are dependent on TP53 dysregulation. We illustrate how uncertainty in mutational signature identification and assignment to samples affects tumor classification, reinforcing that using multiple orthogonal mutational signature data is not only beneficial, but is also essential for accurate tumor stratification. Finally, we present a reference web-based tool for cancer and experimentally generated mutational signatures, called Signal (https://signal.mutationalsignatures.com), that also supports performing mutational signature analyses