Critérios de avaliação de prognóstico a longo prazo em doentes com fibrose quística : revisão de literatura e uma visão crítica

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Síndrome de Leigh : descrição de um caso clínico e revisão teórica

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Anemia hemolítica : desafio diagnóstico

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Dificuldades no diagnóstico de fibrose quística : dois casos clínicos

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Fibrose quística : fenótipo e genótipo : descrição de caso clínico

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Défice do transportador de creatina : até onde investigar a causa de um atraso do desenvolvimento?

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Bases moléculaires et cellulaires d’un trouble neurodéveloppemental causé par l’haploinsuffisance de SYNGAP1

La déficience intellectuelle est la cause d’handicap la plus fréquente chez l’enfant. De nombreuses évidences convergent vers l’idée selon laquelle des altérations dans les gènes synaptiques puissent expliquer une fraction significative des affections neurodéveloppementales telles que la déficience intellectuelle ou encore l’autisme. Jusqu’à récemment, la majorité des mutations associées à la déficience intellectuelle a été liée au chromosome X ou à la transmission autosomique récessive. D’un autre côté, plusieurs études récentes suggèrent que des mutations de novo dans des gènes à transmission autosomique dominante, requis dans les processus de la plasticité synaptique peuvent être à la source d’une importante fraction des cas de déficience intellectuelle non syndromique. Par des techniques permettant la capture de l’exome et le séquençage de l’ADN génomique, notre laboratoire a précédemment reporté les premières mutations pathogéniques dans le gène à transmission autosomique dominante SYNGAP1. Ces dernières ont été associées à des troubles comportementaux tels que la déficience intellectuelle, l’inattention, des problèmes d’humeur, d’impulsivité et d’agressions physiques. D’autres patients sont diagnostiqués avec des troubles autistiques et/ou des formes particulières d’épilepsie généralisée. Chez la souris, le knock-out constitutif de Syngap1 (souris Syngap1+/-) résulte en des déficits comme l’hyperactivité locomotrice, une réduction du comportement associée à l’anxiété, une augmentation du réflexe de sursaut, une propension à l’isolation, des problèmes dans le conditionnement à la peur, des troubles dans les mémoires de travail, de référence et social. Ainsi, la souris Syngap1+/- représente un modèle approprié pour l’étude des effets délétères causés par l’haploinsuffisance de SYNGAP1 sur le développement de circuits neuronaux. D’autre part, il est de première importance de statuer si les mutations humaines aboutissent à l’haploinsuffisance de la protéine. SYNGAP1 encode pour une protéine à activité GTPase pour Ras. Son haploinsuffisance entraîne l’augmentation des niveaux d’activité de Ras, de phosphorylation de ERK, cause une morphogenèse anormale des épines dendritiques et un excès dans la concentration des récepteurs AMPA à la membrane postsynaptique des neurones excitateurs. Plusieurs études suggèrent que l’augmentation précoce de l’insertion des récepteurs AMPA au sein des synapses glutamatergiques contribue à certains phénotypes observés chez la souris Syngap1+/-. En revanche, les conséquences de l’haploinsuffisance de SYNGAP1 sur les circuits neuronaux GABAergiques restent inconnues. Les enjeux de mon projet de PhD sont: 1) d’identifier l’impact de mutations humaines dans la fonction de SYNGAP1; 2) de déterminer si SYNGAP1 contribue au développement et à la fonction des circuits GABAergiques; 3) de révéler comment l’haploinsuffisance de Syngap1 restreinte aux circuits GABAergiques affecte le comportement et la cognition. Nous avons publié les premières mutations humaines de type faux-sens dans le gène SYNGAP1 (c.1084T>C [p.W362R]; c.1685C>T [p.P562L]) ainsi que deux nouvelles mutations tronquantes (c.2212_2213del [p.S738X]; c.283dupC [p.H95PfsX5]). Ces dernières sont toutes de novo à l’exception de c.283dupC, héritée d’un père mosaïque pour la même mutation. Dans cette étude, nous avons confirmé que les patients pourvus de mutations dans SYNGAP1 présentent, entre autre, des phénotypes associés à des troubles comportementaux relatifs à la déficience intellectuelle. En culture organotypique, la transfection biolistique de l’ADNc de Syngap1 wild-type dans des cellules pyramidales corticales réduit significativement les niveaux de pERK, en fonction de l’activité neuronale. Au contraire les constructions plasmidiques exprimant les mutations W362R, P562L, ou celle précédemment répertoriée R579X, n’engendre aucun effet significatif sur les niveaux de pERK. Ces résultats suggèrent que ces mutations faux-sens et tronquante résultent en la perte de la fonction de SYNGAP1 ayant fort probablement pour conséquences d’affecter la régulation du développement cérébral. Plusieurs études publiées suggèrent que les déficits cognitifs associés à l’haploinsuffisance de SYNGAP1 peuvent émerger d’altérations dans le développement des neurones excitateurs glutamatergiques. Toutefois, si, et auquel cas, de quelle manière ces mutations affectent le développement des interneurones GABAergiques résultant en un déséquilibre entre l’excitation et l’inhibition et aux déficits cognitifs restent sujet de controverses. Par conséquent, nous avons examiné la contribution de Syngap1 dans le développement des circuits GABAergiques. A cette fin, nous avons généré une souris mutante knockout conditionnelle dans laquelle un allèle de Syngap1 est spécifiquement excisé dans les interneurones GABAergiques issus de l’éminence ganglionnaire médiale (souris Tg(Nkx2.1-Cre);Syngap1flox/+). En culture organotypique, nous avons démontré que la réduction de Syngap1 restreinte aux interneurones inhibiteurs résulte en des altérations au niveau de leur arborisation axonale et dans leur densité synaptique. De plus, réalisés sur des coupes de cerveau de souris Tg(Nkx2.1-Cre);Syngap1flox/+, les enregistrements des courants inhibiteurs postsynaptiques miniatures (mIPSC) ou encore de ceux évoqués au moyen de l’optogénétique (oIPSC) dévoilent une réduction significative de la neurotransmission inhibitrice corticale. Enfin, nous avons comparé les performances de souris jeunes adultes Syngap1+/-, Tg(Nkx2.1-Cre);Syngap1flox/+ à celles de leurs congénères contrôles dans une batterie de tests comportementaux. À l’inverse des souris Syngap1+/-, les souris Tg(Nkx2.1-Cre);Syngap1flox/+ ne présentent pas d’hyperactivité locomotrice, ni de comportement associé à l’anxiété. Cependant, elles démontrent des déficits similaires dans la mémoire de travail et de reconnaissance sociale, suggérant que l’haploinsuffisance de Syngap1 restreinte aux interneurones GABAergiques dérivés de l’éminence ganglionnaire médiale récapitule en partie certains des phénotypes cognitifs observés chez la souris Syngap1+/-. Mes travaux de PhD établissent pour la première fois que les mutations humaines dans le gène SYNGAP1 associés à la déficience intellectuelle causent la perte de fonction de la protéine. Mes études dévoilent, également pour la première fois, l’influence significative de ce gène dans la régulation du développement et de la fonction des interneurones. D’admettre l’atteinte des cellules GABAergiques illustre plus réalistement la complexité de la déficience intellectuelle non syndromique causée par l’haploinsuffisance de SYNGAP1. Ainsi, seule une compréhension raffinée de cette condition neurodéveloppementale pourra mener à une approche thérapeutique adéquate.

Hemorheological alterations in sickle cell anemia and their clinical consequences: the role of genetic modulators

Sickle cell anemia (SCA) is an autosomal recessive chronic hemolytic anemia, caused by homozygosity for the HBB:c.20A>T mutation. The disease presents with high clinical heterogeneity, stroke being the most devastating manifestation. This study aimed to identify genetic modulators of severe hemolysis and stroke risk in children with SCA, as well as understand their consequences at the hemorheological level. Sixty-six children with SCA were categorised according to their degree of cerebral vasculopathy (Stroke/Risk/Control). Relevant data were collected from patients’ medical records. Several polymorphic regions in genes related to vascular cell adhesion and tonus were characterized by molecular methodologies. Data analyses were performed using R software. Several in silico tools (e.g. TFBind, MatInspector) were applied to investigate the main variant consequences. Some genetic variants in vascular adhesion molecule-1 gene promoter and endothelial nitric oxide synthase gene were associated with higher levels of hemolysis and stroke events. They modify important transcription factor binding sites or disturb the corresponding protein structure/function. Our findings emphasize the relevance of the genetic variants in modulating the degree of hemolysis and development of cerebral vasculopathy due to their effect on gene expression, modification of protein biological activities related with erythrocyte/endothelial interactions and consequent hemorheological abnormalities in SCA.

Initial characterization of stiff skin-like syndrome in West Highland white terriers

BACKGROUND Stiff skin syndrome and systemic or localized scleroderma are cutaneous disorders characterized by dermal fibrosis and present clinically with induration of the skin, with or without joint, internal organ or vascular involvement. OBJECTIVES To provide clinical, histological and preliminary genetic analysis of two West Highland white terrier siblings presenting with indurated skin resembling stiff skin syndrome in humans. ANIMALS Two client owned full sibling West Highland white terriers from two different litters. METHODS Clinical examination, histopathological examination and whole genome sequencing analysis of affected and unaffected West Highland white terriers. RESULTS Affected dogs exhibited markedly indurated skin that was attached firmly to the underlying tissue and incomplete closure of the mouth and eyes. No abnormalities were found by neurological or orthopaedic examination, radiographs of the head or whole body computed tomography. Histologically, the dermis and pannicular septa were thickened by a marked increase in coarse collagen fibres and a mild to moderate increase in collagen fibre diameter. The syndrome most likely follows an autosomal recessive mode of inheritance. The sequence analysis did not reveal any obvious causative variant in the investigated candidate genes ADAMTSL2 and FBN1. CONCLUSION AND CLINICAL IMPORTANCE The clinical phenotype and histopathological features of two West Highland white terrier siblings resembled stiff skin syndrome in humans. Unlike in humans, or previously described beagles with stiff skin, there was no restriction of joint mobility. Genetic analysis did not detect a candidate causative variant and warrants further research.

Pulmonary hypoplasia and anasarca syndrome in Cika cattle

BACKGROUND: Hydrops foetalis is defined as excessive fluid accumulation within the foetal extravascular compartments and body cavities. It has been described in human and veterinary medicine, but despite several descriptive studies its aetiology is still not fully clarified. Pulmonary hypoplasia and anasarca (PHA) syndrome is a rare congenital abnormality in cattle that is characterised by hydrops foetalis including extreme subcutaneous oedema (anasarca) and undeveloped or poorly formed lungs (pulmonary hypoplasia). Until now, sporadic cases of PHA were reported in cattle breeds like Australian Dexter, Belted Galloway, Maine-Anjou, and Shorthorn. This report describes the first known cases of PHA syndrome in Slovenian Cika cattle. CASE PRESENTATION: A 13-year-old cow aborted a male calf in the seventh month of pregnancy, while a male calf was delivered by caesarean section on the due date from a 14-year-old cow. The pedigree analysis showed that the calves were sired by the same bull, the dams were paternal half-sisters and the second calf was the product of a dam-son mating. Gross lesions were similar in both cases and characterized by severe anasarca, hydrothorax, hydropericardium, ascites, hypoplastic lungs, absence of lymph nodes, and an enlarged heart. The first calf was also athymic. Histopathology of the second affected calf confirmed severe oedema of the subcutis and interstitium of the organs, and pulmonary hypoplasia. The lymph vessels in the subcutis and other organs were severely dilated. Histopathology of the second calf revealed also lack of bronchus associated lymphoid tissue and adrenal gland hypoplasia. CONCLUSIONS: The findings were consistent with known forms of the bovine PHA syndrome. This is the first report of the PHA syndrome occurring in the local endangered breed of Cika cattle. Observed inbreeding practice supports that this lethal defect most likely follows an autosomal recessive mode of inheritance. In the light of the disease phenotype it is assumed that a mutation causing an impaired development of lymph vessels is responsible for the hydrops foetalis associated malformations in bovine PHA.

Disruption of the BLM gene in ATM-null DT40 cells does not exacerbate either phenotype

Bloom syndrome and ataxia-telangiectasia are autosomal recessive human disorders characterized by immunodeficiency, genome instability and predisposition to develop cancer. Recent data reveal that the products of these two genes, BLM and ATM, interact and function together in recognizing abnormal DNA structures. To investigate the function of these two molecules in DNA damage recognition, we generated double knockouts of ATM(-/-) BLM-/- in the DT40 chicken B-lymphocyte cell line. The double mutant cells were viable and exhibited a variety of characteristics of both ATM(-/-) and BLM-/- cells. There was no evidence for exacerbation of either phenotype; however, the more extreme radiosensitivity seen in ATM(-/-) and the elevated sister chromatid exchange seen in BLM-/- cells were retained in the double mutants. These results suggest that ATM and BLM have largely distinct roles in recognizing different forms of damage in DNA, but are also compatible with partially overlapping functions in recognizing breaks in radiation-damaged DNA.

Inherited copper toxicosis with emphasis on copper toxicosis in Bedlington terriers

Canine copper toxicosis is an important inherited disease in Bedlington terriers, because of its high prevalence rate and similarity to human copper storage disease. It can lead to chronic liver disease and occasional haemolytic anaemia due to impaired copper excretion. The responsible gene for copper toxicosis in Bedlington terriers has been recently identified and was found not to be related to human Wilson's disease gene ATP7B. Although our understanding of copper metabolism in mammals has improved through genetic molecular technology, the diversity of gene mutation related to copper metabolism in animals will help identify the responsible genes for non-Wilsonian copper toxicoses in human. This review paper discusses our knowledge of normal copper metabolism and the pathogenesis, molecular genetics and current research into copper toxicosis in Bedlington terriers, other animals and humans. (C) 2004 Elsevier GmbH. All rights reserved.

HFE genotyping demonstrates a significant incidence of hemochromatosis in up differentiated arthritis

Objective Hereditary hemochromatosis is a common autosomal recessive disorder of iron metabolism. Among Northern Europeans the carrier frequency is estimated to be I in 10, while up to 1 in 200 is affected by the disease. Arthropathy is one early clinical manifestation of this disease, but the articular features are often misdiagnosed. In this study the two frequent mutations of the HLA-linked hemochromatosis gene (HFE) were investigated, in a rheumatology clinic population. Methods Two hundred and six consecutive patients (mean age 57.7 years; 38 male/168 female) attending a rheumatology clinic over a period of 14 months were screened for HFE mutations (C282Y and H63D). All standard diagnostic procedures were used to identify the aetiology: of the arthropathy. Mutations were evaluated by separation on PAGE of digested PCR amplificates of DNA (by SnapI and Bcl-I, for C282Y and H63D, respectively) obtained from PBMCs. Results The C282Y and H63D allele frequencies were 4.5 and 12.8 inpatients with rheumatic diseases. Five patients were homozygote for H63D (2.4%), and one,for C282Y (0.5%). Five patients were compound heterozygous (2.4%). The observed C282Y allele frequency in rheumatic patients with undifferentiated arthritis was 12.9 and exceeded that of healthy subjects (p = 0.01). Conclusions Determination of the HFE genotype is clinically useful in patients with arthritis of unknown origin, to allow early diagnosis of hemochromatosis.

Increased C282Y heterozygosity in gestational diabetes

Background. Hereditary hemochromatosis is an autosomal recessive disorder of iron metabolism that is characterized by excess accumulation of iron in various organs and often leads to diabetes mellitus (DM). To study whether mutations in the hemochromatosis gene (HFE) could be a risk factor for the development of gestational diabetes mellitus (GDM), the prevalence of HFE mutations in patients with GDM was compared to that of healthy pregnant controls. Methods: GDM was diagnosed in 208 of 2,421 pregnant woman screened between the 24th and 28th week of gestation over a period of 18 months. Patients and 170 matched control subjects were screened for the HFE gene mutations C282Y and H63D. Results: In North and Central European GDM patients, the allele frequency of the C282Y mutation (7.7%) was higher than in pregnant controls (2.9%; p = 0.04), while the frequency of the H63D mutation was not different (p = 0.45). Three patients with GDM were homozygous for H63D (3.1%), 1 patient was homozygous for C282Y (1.0%), 2 patients were compound heterozygous (2.0%) and 26 were heterozygous [11 C282Y (11.2%) and 15 H63D (15.3%)]. C282Y and H63D allele frequencies were not different between controls and GDIVI patients of Southern European or non-European origin. Irrespective of the HIFE-mutation status, serum ferritin levels were increased in patients with GDM compared to healthy pregnant controls (p = 0.01), while transferrin saturation was similar in both groups. Conclusions: In North and Central European patients with GDM, the C282Y allele frequency is higherthan in healthy pregnant women, suggesting a genetic susceptibility to the development of GDM. Copyright (c) 2005 S. Karger AG, Basel.

Liposomes as carriers for polymyxins in the treatment of cystic fibrosis lung infections

Cystic fibrosis (CF) is the most common autosomal recessive disorder affecting Caucasian populations. The pathophysiology of this disorder predisposes the lungs of affected patients to chronic infection, typically by Pseudomonas aeruginosa, which is the main cause of morbidity and mortality. Recently, attention has focused on aerosolised polymyxins, which are given prophylactically in an effort to limit infection and subsequent lung damage. This class of antimicrobial compounds is highly active against P. aeruginosa and possess the advantage that resistance rarely develops. However, the rapid lung clearance of antibiotics is a well documented phenomenon and it was postulated that polymyxin treatment could be further improved by liposomal encapsulation. As part of the development of liposomal polymyxin B, analytical methodology (radiolabelling, HPLC and protein assay) applicable to liposomal formulations was established. Liposomes were prepared by the dehydration-rehydration method and encapsulation efficiencies were determined for a number of phospholipid compositions. Vesicles were characterised with respect to size, zeta potential, morphology and release characteristics. The surface hydrophobicity of vesicles was quantified by hydrophobic interaction chromatography and it was found that this method produced comparable results to techniques conventionally used to assess this property. In vivo testing of liposomal polymyxins demonstrated that encapsulation successfully prevented the rapid pulmonary clearance of PXB. Antimicrobial activity of liposomal formulations was quantified and found to be dependent on both the vesicle surface characteristics and their release profile. Investigation of the interaction of PXB with lipopolysaccharide was undertaken and results demonstrated that PXB caused significant structural distortion of the lipid A region. This may be sufficient to abrogate the potentiating action of LPS in the inflammatory cascade.