A importância da fenotipagem aprofundada no diagnóstico de síndromes genéticas

Tese (Doutorado) — Universidade de Brasília, Faculdade de Ciências da Saúde, Programa de Pós-Graduação em Ciências Da Saúde, 2022.A descoberta do genoma humano e os avanços tecnológicos no sequenciamento genômico foram o marco inicial para que se estabelecesse a prática de “deep phenotyping Na prática clínica “deep phenotyping” é avaliado a partir de dados descritos em prontuário, exames complementares, avaliações morfocelulares e quaisquer outras análises que possam auxiliar na definição de uma hipótese diagnóstica. Utilizando essa metodologia de “deep phenotyping” foram abordados dois pacientes que não tinham diagnóstico definido. Neste trabalho foi relatado o caso clínico de uma paciente do sexo feminino com hiperpigmentação disseminada na pele, baixa estatura, dismorfia facial, ulcerações em boca e ânus, voz rouca e atraso no desenvolvimento neuropsicomotor, que teve como diagnóstico a deficiência congênita da vitamina B12. O sequenciamento do exoma revelou uma variante c.515_516del (p.Thr172fs*10) em homozigose no gene LMBRD1. A biópsia de pele mostrou inúmeros melanossomos em vacúolos autofágicos em derme papilar. A hipótese que explica a hiperpigmentação cutânea é a incontinência pigmentar da melanina resultado do aumento do estresse oxidativo em virtude da hiperprodução da homocisteína e o desbalanço da glutationa. A deficiência da vitamina B12 deve ser considerada para aqueles pacientes que apresentam hiperpigmentação cutânea com ou sem baixa estatura e atraso no desenvolvimento neuropsicomotor. O tratamento se dá pela suplementação da vitamina B12. Este trabalho também descreveu um paciente portador de uma variante patogênica no ENPP5 com comprometimento esquelético e anomalias dentárias. O paciente do sexo masculino apresenta alterações morfológicas e cognitivas, crises convulsivas, macrocefalia, crescimento anormal do complexo maxilomandibular, hiperplasia gengival e hipercementose em todos os dentes. Nos exames radiográficos foi evidenciado hiperostose da calota craniana, osteopenia em membros superiores e inferiores e em corpos vertebrais, cifoescoliose, alargamento da região metafisária distal e afilamento da cortical óssea dos ossos longos, crescimento aumentado da pré-maxila e hipercementose nos molares inferiores. O sequenciamento do exoma concluiu tratar-se de uma variante patogênica missense (c.173G>T; p.Gly58Val) em homozigose no gene ENPP5. Esse paciente é o primeiro relato na literatura que descreve essa variante como causa de uma displasia esquelética. Pouco se sabe sobre a função do ENPP5 e sua influência no processo de biomineralização e nas anomalias dentárias, necessitando de estudos futuros para melhor compreender a patogênese desta nova displasia esquelética. Portanto, este trabalho ressalta a importância da fenotipagem aprofundada em dois pacientes. Com esta estratégia foi possível orientar a análise molecular e concluir o diagnóstico deles e, assim, permitir o tratamento e a conduta terapêutica específica para esses dois pacientes.The discovery of the human genome and technological advances in genomic sequencing were the starting point for establishing the practice of “deep phenotyping”. In clinical practice deep phenotyping is based on data described in medical records, complementary exams, morphocellular assessments and any other analysis that may help to define a diagnostic hypothesis.Using this strategy we studied two patients who had no defined diagnosis. In this work we report the clinical case of a female patient with widespread hyperpigmentation in the skin, short stature, facial dysmorphia, ulcerations in the mouth and anus, hoarse voice and delay in neuropsychomotor development, whose diagnosis was congenital vitamin B12 deficiency. Exome sequencing revealed a homozygous c.515_516del variant (p.Thr172fs*10) in the LMBRD1 gene. Skin biopsy showed numerous melanosomes in autophagic vacuoles in papillary dermis. The hypothesis that explains cutaneous hyperpigmentation is the pigmentary incontinence of melanin resulting from the increase in oxidative stress due to the hyperproduction of homocysteine and the imbalance of glutathione. Vitamin B12 deficiency should be considered for those patients who present with cutaneous hyperpigmentation with or without short stature and delayed neuropsychomotor development. Treatment is with vitamin B12 supplementation. This work also described a patient carrying a pathogenic variant in ENPP5 with skeletal involvement and dental anomalies. The male patient presents morphological and cognitive alterations, seizures, macrocephaly, abnormal growth of the maxillomandibular complex, gingival hyperplasia and hypercementosis in all teeth. The radiographic exams showed hyperostosis of the skullcap, osteopenia in the upper and lower limbs and vertebral bodies, kyphoscoliosis, enlargement of the distal metaphyseal region and thinning of the cortical bone of the long bones, increased growth of the premaxilla and hypercementosis in the lower molars. The exome sequencing revealed a missense pathogenic variant (c.173G>T; p.Gly58Val) in homozygosis in the ENPP5 gene. This patient is the first report in the literature that describes this variant as the cause of skeletal dysplasia. Little is known about the role of ENPP5 and its influence on the biomineralization process and on dental anomalies, requiring further studies to better understand the pathogenesis of this new skeletal dysplasia. Therefore, this work highlights the importance of in-depth phenotyping in two patients. With this strategy, it was possible to guide the molecular analysis and conclude their diagnosis and, thus, allow the treatment and specific therapeutic conduct for these two patients

Avaliação morfológica de baço, linfonodo e fígado de macacos-prego cebus apella tratados com nanopartículas magnéticas recobertas com DMSA

Dissertação (mestrado)—Universidade de Brasília, Faculdade de Medicina, Programa de Pós-Graduação em Patologia Molecular, 2008.Os fluidos magnéticos (FMs) são suspensões coloidais formadas por nanopartículas magnéticas (NPMs), constituídas por ferritas, tais como a magnetita (Fe3O4) e a maghemita ( Fe2O4), dispersas em meio líquido. Nos últimos anos, o uso de FM tem sido apontado como uma ferramenta promissora para diversas aplicações biomédicas. No entanto, é necessário avaliar os efeitos causados pelo uso dos FM no organismo para que estes possam se tornar uma ferramenta amplamente utilizada. Diante disso, o presente trabalho tem por finalidade avaliar as alterações morfológicas de baço, linfonodo e fígado após a administração endovenosa de fluido agnético (FM-DMSA) composto por nanopartículas magnéticas a base de maguemita recobertas por ácido meso-2,3-dimercaptosuccínico (DMSA) em macacos-prego (Cebus apella). Para isso, amostras de baço, linfonodo e fígado de animais controle (AC), tratado 12 horas (AE12h) e tratado 90 dias (AE90d) foram observadas através de mediante Microscopia Óptica (MO) e Microscopia Eletrônica de Transmissão (MET), a fim de avaliar as possíveis alterações morfológicas e ultraestruturais, bem como citolocalizar as NPMs nos tecidos selecionados. O baço e linfonodo não apresentaram alterações morfológicas, nem mesmo infiltrado inflamatório nas amostras de AE12h e AE90d. Porém, amostras de fígado para o AE90d mostraram alterações da estrutura tecidual, com aumento dos espaços de Disse. Nas análises realizadas, o FM-DMSA se mostrou presente internalizado em vesículas no citoplasma, com aspecto granular típico. Foram realizados testes morfométricos das mitocôndrias hepáticas para todos os animais, sendo que em AE90d a área ocupada por essas organelas assumiu valor aproximadamente triplicado comparado com as amostras do animal controle, o que sugere alteração metabólica nas mesmas. O FM-DMSA não mostrou ser uma substância completamente inócua em todos os órgãos analisados, uma vez que alterações hepáticas no AE90d foram imputadas à presença do FM no tecido. Por outro lado, análises realizadas em baço e linfonodo não apontaram qualquer traço de toxicidade da amostra. Assim, não foi constatada formação de processos patológicos severos em nenhum dos órgãos analisados, o que atesta em favor da biocompatibilidade da amostra e possibilidade de sua utilização com fins diagnósticos e terapêuticos em humanos, uma vez que esses dados são inéditos no que se refere à a sua utilização em primatas. Novos testes de toxicidade e biodistribuição para o FM-DMSA devem ser realizados a fim de confirmar o potencial uso desta ferramenta para aplicações biomédicas. _____________________________________________________________________________ ABSTRACTMagnetic fluids (MF) are colloidal suspensions formed by magnetic iron oxide nanoparticles (MNPs) such as magnetite (Fe3O4) and maghemite (γ Fe2O4), dispersed in a liquid medium. In recent years, the use of magnetic fluid has been suggested as a promising tool for various biomedical applications; however, it is necessary to evaluate the effects caused by the use of MF in the organism, so that this tool can be widely used. Therefore, the present study aims to assess the morphological changes of spleen, liver and lymph nodes after treatment with magnetitebased magnetic fluid stabilized with DMSA (2,3-dimercaptosuccinic acid) in capuchin monkeys (Cebus apella). Samples of spleen, liver and lymph node from control animal (CA), 12 hours treatment animal (EA12h) and 90 days treatment animal (EA90d) were observed by Light and Transmission Electron Microscopy (TEM), to assess the possible morphological and ultrastructural alterations, and cytolocalization of MNPs in the selected tissues. Samples of spleen and lymph node showed no morphological changes and did not display inflammatory infiltrate for EA12d and EA90d. Nevertheless, for EA90d liver samples, morphological changes in the tissue structure and Disse space were shown. In all analyses performed, the DMSA-MF was internalized in cytoplasmic vesicles. However, for EA90d liver samples, secondary lysossomes were also observed. Morphometrical tests of hepatic mitochondria were performed and analyzed for all animals, being that for EA90d these are organelles tripled its size, compared with CA. This suggests a metabolic alteration in the liver mitochondria. DMSA-MF was not proven to be completely harmless, due to the fact that hepatic alterations were attributed to the MF presence in the tissue. On the other hand, spleen and lymph nodes did not show any kind of toxic effects to their cells and any pathological process was observed, demonstrating the biocompatibles of MF. This study demonstrated the possibility of utilization of FM in diagnostic and therapies but new tests of toxicity and body distribution need to be conducted in order to evaluate the potential use of DMSA-FM as a tool for biomedical applications

Translocation and insecticidal activity of Bacillus thuringiensis living inside of plants

The major biological pesticide for the control of insect infestations of crops, Bacillus thuringiensis was found to be present naturally within cotton plants from fields that had never been treated with commercial formulations of this bacterium. The ability of B. thuringiensis to colonize plants as an endophyte was further established by the introduction of a strain marked by production of green fluorescent protein (GFP). After inoculation of this preparation close to the roots of cotton and cabbage seedlings, GFP-marked bacteria could be re-isolated from all parts of the plant, having entered the roots and migrated through the xylem. Leaves taken from the treated plants were able to cause toxicity when fed to the Lepidoptera Spodoptera frugiperda (cotton) and Plutella xylostella (cabbage). These results open up new horizons for understanding the natural ecology and evolution of B. thuringiensis and use of B. thuringiensis in insect control

Electron micrograph of capuchin monkey lymph node tissue.

<p>(A) Control animal. Lymphocyte showing normal ultrastructure. (B) EA12h with DMSA-MNPs internalized in lymphocyte cytoplasm. (C) EA90d sample, showing few electron dense material internalized in macrophage. Scale bars: (A & C): 2 µm; (B): 1 µm.</p

Morphological Analysis of Reticuloendothelial System in Capuchin Monkeys (<i>Sapajus</i> spp.) after Meso-2,3-Dimercaptosuccinic Acid (DMSA) Coated Magnetic Nanoparticles Administration

<div><p>Magnetic nanoparticles can be used for numerous <i>in vitro</i> and <i>in vivo</i> applications. However, since uptake by the reticuloendothelial system represents an obstacle for the achievement of nanoparticle diagnostic and therapeutic goals, the aim of the present study was to evaluate the uptake of dimercaptosuccinic acid coated magnetic nanoparticles by reticuloendothelial system phagocytic cells present in lymph nodes, spleen, and liver tissue and how the presence of these particles could have an impact on the morphology of these organs in capuchin monkeys (<i>Sapajus</i> spp.). Animals were intravenously injected with dimercaptosuccinic acid coated magnetic nanoparticles and euthanized 12 hours and 90 days post-injection. Organs were processed by transmission electron microscopy and histological techniques. Samples of spleen and lymph nodes showed no morphological changes. Nevertheless, liver samples collected 90 days post-administration showed slight morphological alteration in space of Disse. Moreover, morphometrical analysis of hepatic mitochondria was performed, suggesting a clear positive correlation between mitochondrial area and dimercaptosuccinic acid coated magnetic nanoparticles administration time. The present results are directly relevant to current safety considerations in clinical diagnostic and therapeutic uses of magnetic nanoparticles.</p></div

Mitochondrial morphometry of liver samples from control and treated animals.

<p>Electron micrographs show hepatocyte mitochondria area (µm²) in control animal (CA) (n=1) and experimental animals euthanized 12 hours after magnetic nanoparticle administration (EA12h) (n =1) and 90 days after injection (EA90d) (n =1). Note the swollen appearance of mitochondria in animal EA90d when compared to the other two groups. Graph shows mitochondrial area (mean ± SD) for the different treatments. (*) indicates differences among treatments, P < 0.0001. Scale bars: 1 µm.</p

Electron micrograph of capuchin monkey liver tissue.

<p>(A and B) Control animal samples with intact hepatocytes. In (B) note the normal appearance of hepatocyte, sinusoids containing red blood cells and normal space of Disse (arrow). (C and D) Liver from EA12h showing the presence of MNPs inside hepatocytes (black arrows), either enveloped by a membrane (inset in C) or free in the cell cytoplasm. In (D) also observe a more dilated space of Disse (white arrowhead). (E and F) Liver from EA90d showing MNPs inside hepatocytes and lysosomes (E). In (F) note the slightly dilated space of Disse (white arrowhead). MNP agglomerates were also observed in the cytoplasm of endothelial cells (black arrows). Scale bars: (A, C, D & F): 2 µm; (B & E): 5 µm. Insets: (C): 0.2 µm and (E): 0.5 µm. N = 1 for control and for each experimental condition.</p