Alterações de dosagem no genoma de doentes com atraso mental

Mestrado em BiotecnologiaDevelopment delay/Intellectual disability (DD/ID) is a serious and life-long condition which represents a challenge for families and public health services. It is characterized by suboptimal functioning of the central nervous system resulting in limitations both in intellectual functioning and in adaptive behavior, and it is observed in approximately 2-3% of the population worldwide. Establishing the disease etiology is important for clinical management, genetic counseling and coping strategies of the families. Array-based comparative genomic hybridization technique (aCGH), also called molecular karyotyping (MC), allows us to directly measure genomic copy number variations between the patient and a control DNA. The whole genome of an individual is represented in a high-resolution “virtual karyotype”, allowing the detection of submicroscopic alterations, undetectable by standard or highresolution karyotyping techniques. In Portugal, the guidelines for testing patients with DD/ID indicate the Gbanding karyotyping in first place and, whenever the result is normal, testing for the most common single gene disorders (Fragile X, for instance), for subtelomeric rearrangements and with specific FISH probes. However, the latter technologies are not suitable for whole genome scans in routine diagnosis, both because of the lower resolution levels (the case of conventional karyotyping), the extensive time consumption and high costs (the FISH case). The introduction of aCGH should contribute to the etiological classification of a large proportion of the DD/ID patients as well as to conclude about the utility of using these technologies for diagnosis of idiopathic DD/ID in the clinical context.O atraso mental (AM) é uma doença que apresenta desafios para a vida tanto nas famílias como na sociedade. É caracterizada por um funcionamento subóptimo do sistema nervoso central que manifesta limitações quer ao nível da capacidade intelectual quer do comportamento adaptativo e tem uma incidência de aproximadamente 2-3% de nados vivos em todo o mundo. A determinação da causa da doença é importante para o correcto encaminhamento clínico, aconselhamento genético e estratégias de coping a desenvolver pela família. A técnica de hibridação genómica comparativa (aCGH), também designada de cariótipo molecular (CM), permite medir variações no número de cópias entre o genoma do doente e de um controlo. A totalidade do genoma de um indivíduo é representado num cariótipo virtual de alta resolução, permitindo assim a detecção de alterações submicroscópicas indetectáveis por métodos de cariotipaem standard ou de alta resolução. Em Portugal, as recomendações para os testes genéticos a realizar num doente com AM indicam o cariótipo tradicional de bandas G como primeira abordagem e, sempre que o resultado seja negativo, a pesquisa relativa às patologias unigénicas mais comuns (Síndrome de X frágil, por exemplo), rearranjos subteloméricos e sondas de FISH específicas. No entanto, estas tecnologias não são aplicáveis para análise de todo o genoma no contexto de diagnóstico de rotina, tanto pela sua baixa resolução (caso do cariítpo convencional) como pela elevada laboriosidade e/ou custos monetários (caso do FISH). A introdução de aCGH pretende contribuir para a classificação etiológica de grande parte dos doentes com AM, assim como, concluir acerca da utilidade desta tecnologia para o diagnóstico de AM idiopático no contexto clínico

MYOD1 involvement in myopathy

[Excerpt] Introduction Myogenic Differentiation 1 (MYOD1) encodes a transcription factor that plays an important role in myogenic determination into mature skeletal muscle [1]. The first loss-of-function mutation of MYOD1 in humans was described in three siblings with perinatal lethal fetal akinesia [2].[...]We thank the individual and family. Funding was provided by The Fonds de recherche du Québec - Santé (FRQS) and Canadian Institutes of Health Research (CIHR) to P.M.C., Fundação para a Ciência e Tecnologia (FCT) with the fellowship SFRH/BD/84650/2010 to F.L. and Groupe Pasteur Mutualité Foundation (GPM Foundation) to M.M.info:eu-repo/semantics/publishedVersio

Refining the phenotype associated with biallelic DNAJC21 mutations

Accepted manuscriptInherited bone marrow failure syndromes (IBMFS) are caused by mutations in genes involved in genomic stability. Although they may be recognized by the association of typical clinical features, variable penetrance and expressivity are common, and clinical diagnosis is often challenging. DNAJC21, which is involved in ribosome biogenesis, was recently linked to bone marrow failure. However, the specific phenotype and natural history remain to be defined. We correlate molecular data, phenotype, and clinical history of 5 unreported affected children and all individuals reported in the literature. All patients present features consistent with IBMFS: bone marrow failure, growth retardation, failure to thrive, developmental delay, recurrent infections, and skin, teeth or hair abnormalities. Additional features present in some individuals include retinal abnormalities, pancreatic insufficiency, liver cirrhosis, skeletal abnormalities, congenital hip dysplasia, joint hypermobility, and cryptorchidism. We suggest that DNAJC21-related diseases constitute a distinct IBMFS, with features overlapping Shwachman-Diamond syndrome and Dyskeratosis congenita, and additional characteristics that are specific to DNAJC21 mutations. The full phenotypic spectrum, natural history, and optimal management will require more reports. Considering the aplastic anemia, the possible increased risk for leukemia, and the multisystemic features, we provide a checklist for clinical evaluation at diagnosis and regular follow-up.FCT—Fundação para a Ciência e a Tecnologia (SFRH/BD/84650/2010)info:eu-repo/semantics/publishedVersio

Chitayat-Hall and Schaaf-Yang syndromes: a common aetiology: expanding the phenotype of MAGEL2-related disorders

Chitayat-Hall syndrome, initially described in 1990, is a rare condition characterised by distal arthrogryposis, intellectual disability, dysmorphic features and hypopituitarism, in particular growth hormone deficiency. The genetic aetiology has not been identified.Background Chitayat-Hall syndrome, initially described in 1990, is a rare condition characterised by distal arthrogryposis, intellectual disability, dysmorphic features and hypopituitarism, in particular growth hormone deficiency. The genetic aetiology has not been identified. Methods and results We identified three unrelated families with a total of six affected patients with the clinical manifestations of Chitayat-Hall syndrome. Through whole exome or whole genome sequencing, pathogenic variants in the MAGEL2 gene were identified in all affected patients. All disease-causing sequence variants detected are predicted to result in a truncated protein, including one complex variant that comprised a deletion and inversion. Conclusions Chitayat-Hall syndrome is caused by pathogenic variants in MAGEL2 and shares a common aetiology with the recently described Schaaf-Yang syndrome. The phenotype of MAGEL2-related disorders is expanded to include growth hormone deficiency as an important and treatable complicationhe McLaughlin Centre, University of Toronto, Toronto, Canada, and Fondation Jeanne et Jean- Louis Lévesque (JLM). The Centre for Genetic Medicine, The Hospital for Sick Children, Toronto, Canada. FDL has a fellowship funded by FCT - Fundação para a Ciência e a Tecnologia (SFRH/BD/84650/2010)info:eu-repo/semantics/publishedVersio

Deciphering the genetic basis of intellectual disability through unbiased genomic approaches

Tese de Doutoramento em Ciências da SaúdeNeurodevelopmental disorders arise in childhood and are life-long condition that represents many challenges to the patients, their families and society, through public services. Among those, intellectual disability affects 1% of the population in developed countries, encompassing the most common group of neurodevelopmental disorders. Intellectual disability is characterized by cognitive impairment and limitation in functioning capacity and can be cause by exogenous factors (such as maternal alcohol abuse during pregnancy, infections and malnutrition) but it is well established that genetic factors play important roles in its pathophysiology. In ID, as in many others disorders, detecting the underlying genetic cause is a complex and time consuming process yet of great value to the patients and families because it allows the possibility of genetic counseling. In the last years, advances in two major types of technologies allowed great advances in the discovery of new genomic anomalies causing intellectual disability: array comparative genomic hybridization and massive parallel sequencing. Array comparative genomic hybridization allowed a high resolution genome wide investigation of copy number variations leading to the discovery of many novel microdeletion/microduplication syndromes. Massive parallel sequencing evolved in a way that the sequencing of all the genome (or, at a lower cost, all the exons) is now possible to perform in any genetic laboratory in a time and cost-efficient manner, allowing the discovery of many novel variants in previously known and newly discovered intellectual disability genes. These two approaches have provided significantly new insights into the biological pathways associated with intellectual disability and tremendously improved the diagnostic process. In this study we applied these two technologies to the study of the genetic basis of neurodevelopmental disorders. We studied a big group of patients with idiopathic intellectual disability by aCGH (which included two cohorts with different selection criteria - a research and a clinical cohort), a group of patients with a Rett syndrome-like clinical presentation by exome sequencing and re-analyzed exome data from a pediatric heterogeneous cohort. Array comparative genomic hybridization allowed the detection of previously known microdeletion and microduplication syndromes in patients with until then unexplained intellectual disability, with yields of 13% in the research cohort and 18% in the clinical cohort. Importantly it also allowed the discovery of 12 new loci likely to cause neurodevelopmental disease as well as the gathering of additional patients with overlapping genomic imbalances and phenotypic features, allowing the definition of new (rare) syndromes. Massive parallel sequencing – more specifically whole exome sequencing - was applied to a group of patients sharing similar clinical presentation (Rett syndrome-like) and proved to be very effective, leading to the identification of five new genes possibly involved in intellectual disability (HTT, SMARCA1, GABBR2, RHOBTB2 and EIF4G1). It is currently an accepted fact however, that the data generated by exome sequencing at a certain point in time, may not retrieve the genetic cause of the disease. This limitation is often related with the lack of information regarding the genes detected. Given the always increasing knowledge on genes and pathways involved in neurodevelopmental disorders, the need for reevaluation of older and previously unsolved cases emerges. This strategy was also applied in this work and proven to be extremely useful in the clinical context, adding new patients to help establish the relevance of candidate disease genes and raising new candidate genes (DNAJC21, MYOD1 and PAX7). In summary, this work helped to clarify the genetic basis of disease in several patients until then unsolved, as well as to bring forward new candidate loci and genes for intellectual disability and other neurodevelopmental disorders.This work was supported by Foundation for Science and Technology (FCT) through a PhD studentship (SFRH/BD/90167/2012) and by the Seventh Framework Programme (FP7/2007- 2013) under grant agreement no. 262055. This work was also supported by the FEDER through the Operational Programme Competitiveness Factors - COMPETE and the national funds through the FCT - Foundation for Science and Technology within the projects (POCI-01-0145-FEDER-007038), and by the project NORTE01- 0145-FEDER-000013, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF).As perturbações do neurodesenvolvimento surgem na infância e constituem doenças crónicas, criando inúmeras limitações para os doentes, famílias e sociedade – sob a forma de serviços públicos. Entre estas, o défice intelectual (previamente designado atraso mental) afecta 1% da população dos países desenvolvidos, sendo o tipo de doença do neurodesenvolvimento mais comum. O défice intelectual é caracterizado por uma limitação cognitiva e funcional e pode ser causado por factores exógenos (como o consumo materno de álcool durante a gravidez, infeções e malnutrição), sendo no entanto fatores genéticos reconhecidos como muito importantes para a patofisiologia do défice cognitivo. Neste grupo de doenças, assim como em muitas outras, a deteção da causa genética é um processo complexo e demorado mas de grande valor para os doentes e famílias, uma vez que abre portas á possibilidade de aconselhamento genético. Nos últimos anos, avanços em duas grandes tecnologias de diagnóstico genético permitiram a descoberta de novas anomalias genéticas associadas e défice intelectual: array de hibridação genómica comparativa e sequenciação paralela massiva. Os arrays de hibridação genómica comparativa permitiram a análise de alta resolução de todo o genoma na busca de alterações do número de cópias, o que resultou na descoberta de várias novas síndromes associadas microdeleções/microduplicações. A sequenciação paralela massiva desenvolveu-se de uma forma em que a sequenciação de todo o genoma (ou, de forma mais económica, de todos os exões) é agora possível realizar em qualquer laboratório de genética em tempo útil e com um bom custo/benefício, permitindo a descoberta de variantes tanto em genes já conhecidos como em novos genes causadores de défice intelectual. Estas duas abordagens contribuíram significativamente para novas descobertas em vias moleculares associadas com défice intelectual e para o melhoramento do seu diagnóstico. Neste estudo aplicamos estas duas tecnologias ao estudo da base genética de doenças do neurodesenvolvimento. Estudamos por aCGH um grande grupo de doentes com défice intelectual idiopático (o que incluiu dois coortes com diferentes critérios de seleção – um coorte de investigação e um coorte clinico), estudamos por sequenciação de exoma um grupo de doentes com sintomatologia semelhante à síndrome de Rett, e reanalisamos dados de exoma de um grupo heterogéneo de doentes pediátricos. O array de hibridação genómica comparativa permitiu a deteção de microdeleções e microduplicações já conhecidas em doentes até à data com défice intelectual idiopático, com uma taxa de sucesso de 13% no coorte de investigação e de 18% no coorte clinico. Foi também possível a deteção de novos 12 novos loci passíveis de causar doença do neurodesenvolvimento assim como a recolha de doentes adicionais com desequilíbrios genómico e fenótipo sobreponíveis, contribuindo para a definição de novas síndromes raras. A sequenciação paralela massiva – em particular a sequenciação do exoma - foi aplicada a um grupo de doentes com apresentação clinica semelhante (síndrome de Rett-like), revelando-se bem-sucedida na identificação de cinco novos genes possivelmente envolvidos no défice intelectual (HTT, SMARCA1, GABBR2, RHOBTB2 e EIF4G1). Atualmente é facto aceite que os dados gerados por sequenciação do exoma numa determinada altura poderão não levar á descoberta da causa genética da doença. Esta limitação está muitas vezes relacionada com a escassez de informação relativamente aos genes encontrados. Tendo em conta o sempre crescente conhecimento relativo a genes e vias moleculares envolvidas em doenças do neurodesenvolvimento, surge a necessidade de reavaliação de casos antigos não solucionados. Esta estratégia foi também aplicada neste trabalho e provando ser de grande utilidade no contexto clinico, levando à deteção de mais doentes que contribuem para a determinação dos genes candidatos relevantes assim como para a deteção de novos genes candidatos (DNAJC21 , MYOD1 and PAX7 ). Em resumo, este trabalho contribuiu para a clarificação da causa genética de doença em vários doentes até à data não resolvidos, e propõe novos loci candidatos e genes que contribuem para o défice intelectual e outras doenças do neurodesenvolvimento

Síntese de fosfonatos e avaliação da sua atividade anti-inflamatória e antioxidante

Os fosfonatos são uma classe importante de compostos químicos, utilizados na terapia de patologias ósseas, tais como a doença de Paget, hipercalcémia e osteólise associada a tumores, devido à sua capacidade para inibir a perda óssea. Estes compostos têm também demonstrado atividade anticancerígena in vitro com inibição da proliferação celular de várias linhas tumorais [1,2]. Neste trabalho procedeu-se à síntese de ácidos fosfónicos e ésteres monofosfónicos, sintetizados a partir de aldeídos, bem como à avaliação da atividade antioxidante e da atividade anti-inflamatória dos compostos obtidos. A atividade antioxidante foi avaliada por três métodos diferentes, nomeadamente o método do radical DPPH, o sistema β-caroteno/ácido linoleico e o poder redutor total, com vista a inferir sobre o mecanismo de ação. Os estudos de atividade anti-inflamatória in vitro foram também avaliados por diferentes mecanismos de ação, com determinação da capacidade de inibição da desnaturação da albumina e de inibição da atividade de lipoxigenases. A toxidade dos fosfonatos em estudo foi avaliada utilizando o teste de letalidade em Artemia salina. Os compostos em estudo apresentaram atividade antioxidante, tendo-se observado que o composto (E)-3-(2-nitrofenil)-1-hidroxipropen-2-ilfosfonato de dimetilo foi o que apresentou melhor resultados na capacidade para sequestrar radicais, de poder redutor total, bem como na capacidade de proteção do substrato lipídico. Alguns dos compostos apresentaram também elevada capacidade anti-inflamatória, designadamente os ésteres 1-(4-bromofenil)-1-hidroximetilfosfonato de dimetilo e 1-(4-formilfenil)-1-hidroximetilfosfonato de metilo, os quais apresentaram elevado potencial para inibir a atividade da lipoxigenase

Unravelling the Potential Cytotoxic Effects of Metal Oxide Nanoparticles and Metal(Loid) Mixtures on A549 Human Cell Line

Humans are typically exposed to environmental contaminants' mixtures that result in different toxicity than exposure to the individual counterparts. Yet, the toxicology of chemical mixtures has been overlooked. This work aims at assessing and comparing viability and cell cycle of A549 cells after exposure to single and binary mixtures of: titanium dioxide nanoparticles (TiO2NP) 0.75-75 mg/L; cerium oxide nanoparticles (CeO2NP) 0.0.75-10 μg/L; arsenic (As) 0.75-2.5 mg/L; and mercury (Hg) 5-100 mg/L. Viability was assessed through water-soluble tetrazolium (WST-1) and thiazolyl blue tetrazolium bromide (MTT) (24 h exposure) and clonogenic (seven-day exposure) assays. Cell cycle alterations were explored by flow cytometry. Viability was affected in a dose- and time-dependent manner. Prolonged exposure caused inhibition of cell proliferation even at low concentrations. Cell-cycle progression was affected by TiO2NP 75 mg/L, and As 0.75 and 2.5 μg/L, increasing the cell proportion at G0/G1 phase. Combined exposure of TiO2NP or CeO2NP mitigated As adverse effects, increasing the cell surviving factor, but cell cycle alterations were still observed. Only CeO2NP co-exposure reduced Hg toxicity, translated in a decrease of cells in Sub-G1. Toxicity was diminished for both NPs co-exposure compared to its toxicity alone, but a marked toxicity for the highest concentrations was observed for longer exposures. These findings prove that joint toxicity of contaminants must not be disregarded.Project NanoLegaTox (PTDC/SAU-PUB/29651/2017) cofinanced by COMPETE 2020, Portugal 2020 and European Union, through FEDER.info:eu-repo/semantics/publishedVersio

Cytotoxic effects of single and binary mixtures of metal oxide nanoparticles and metal(loid) on A549 human cell line.

Background, Motivation and Objective: The need to assess the toxicity resulting from exposure to mixtures of chemicals has been recognized by the WHO and EU, as humans are simultaneously exposed to an array of natural and anthropogenic contaminants. Of particular interest are the potential combined effects resulting from interaction of nanoparticles (NPs) and metals. While the first are the current driving force for emerging contaminants, the latter, as legacy contaminants, remain a concern. Metals show strong affinity to NPs, which can change the uptake and toxicity to the organism of each individual contaminant. Studying the effects on the respiratory tract is of upmost relevance because of its constant contact with xenobiotics, resulting in adverse effects on the lung. Considering the above, the objective of this work was to assess and compare viability, cell cycle, and uptake of A549 cells after exposure to single and binary mixtures of titanium dioxide nanoparticles (TiO2NP), cerium oxide nanoparticles (CeO2NP), arsenic (As) and mercury (Hg). These chemicals were chosen because: 1) TiO2NP are among the most abundantly used NPs; 2) CeO2NP have been used in nanomedicine for its high biocompatibility and cytoprotective effect; and 3) As and Hg due to their non‐biodegradable, persistent, and extremely toxic character. This work intends to support adequate human risk assessment resulting from co-exposure to multiple contaminants.NanoLegaTox (PTDC/SAU-PUB/29651/2017) cofinanced by COMPETE2020, Portugal2020 and European Union, through FEDER. A.T.Reis, F.Brandão and M.J.Bessa financed by FCT Grants SFRH/BPD/122112/2016, SFRH/BD/101060/2014 and SFRH/BD/12046/2016. A.C.Estrada and C.B.Lopes funded by national funds (OE), in the scope of the framework contract foreseen in the numbers 4, 5 and 6 of article 23, Decree-Law 57/2016, August 29, changed by Law 57/2017, July 19.info:eu-repo/semantics/publishedVersio