Browsing by Author "Martins, Esmeralda"
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- Assessing Lysosomal Disorders in the NGS Era: Identification of Novel Rare VariantsPublication . Encarnação, Marisa; Coutinho, Maria Francisca; Silva, Lisbeth; Ribeiro, Diogo; Ouesleti, Souad; Campos, Teresa; Santos, Helena; Martins, Esmeralda; Cardoso, Maria Teresa; Vilarinho, Laura; Alves, SandraLysosomal storage diseases (LSDs) are a heterogeneous group of genetic disorders with variable degrees of severity and a broad phenotypic spectrum, which may overlap with a number of other conditions. While individually rare, as a group LSDs affect a significant number of patients, placing an important burden on affected individuals and their families but also on national health care systems worldwide. Here, we present our results on the use of an in-house customized next-generation sequencing (NGS) panel of genes related to lysosome function as a first-line molecular test for the diagnosis of LSDs. Ultimately, our goal is to provide a fast and effective tool to screen for virtually all LSDs in a single run, thus contributing to decrease the diagnostic odyssey, accelerating the time to diagnosis. Our study enrolled a group of 23 patients with variable degrees of clinical and/or biochemical suspicion of LSD. Briefly, NGS analysis data workflow, followed by segregation analysis allowed the characterization of approximately 41% of the analyzed patients and the identification of 10 different pathogenic variants, underlying nine LSDs. Importantly, four of those variants were novel, and, when applicable, their effect over protein structure was evaluated through in silico analysis. One of the novel pathogenic variants was identified in the GM2A gene, which is associated with an ultra-rare (or misdiagnosed) LSD, the AB variant of GM2 Gangliosidosis. Overall, this case series highlights not only the major advantages of NGS-based diagnostic approaches but also, to some extent, its limitations ultimately promoting a reflection on the role of targeted panels as a primary tool for the prompt characterization of LSD patients.
- Caso electroencefalográfico: epilepsia mioclónica precocePublication . Monteiro, Tânia; Martins, Esmeralda; Chorão, RuiABSTRACT Introduction: The suppression-burst (SB) electroencephalographic pattern is rather common during the neonatal period and suggests severe encephalopathy. When significant hypoxic-ischemic insult is excluded, brain malformations and metabolic disorders have to be ruled out. Two distinctive epileptic syndromes are described: early epileptic encephalopathy with SB (Ohtahara syndrome) and early myoclonic epilepsy (EME). The later is frequently associated with neurometabolic disorders, one of the most common being nonketotic hyperglycinemia (NKH). Case report: A baby girl presented with multiple erratic clonic and myoclonic seizures from the second day of life, refractory to antiepileptic drugs. She was hypotonic, lethargic and had episodes of apnea. The electroencephalogram (EEG) showed multiple bursts of multifocal epileptiform activity with long periods of almost flat tracing; this pattern persisted beyond the neonatal period, it was present at the last EEG performed at age four months. Barbiturate-induced coma with mechanical ventilation was induced. She died at the age of five months. The second but not the first sample of cerebrospinal fluid (CSF) and blood revealed an increased CSF/serum glycine ratio (0,11 – normal<0,03). Post-morten liver tissue biopsy found a defi cit at the glycine cleavage system (GCS) (6,6 mkat/ kg - normal 45,0-195,0) and molecular studies detected a mutation in the gene GLDC molecular testing. This result allowed better parent’s genetic counseling. Conclusions: Early myoclonic epilepsy presents with multifocal seizures and SB on pattern on the EEG in the neonatal period, metabolic causes must be investigated, namely the neonatal form of NHK. CSF and plasma aminoacids, including glycine levels, should be measured, simultaneously and sometimes repeatedly. Enzymatic and molecular analysis may confirm this diagnosis and are useful for parent’s genetic counseling.
- Congenital disorders of glycosylationPublication . Mendes, Ana Raquel; Quelhas, D; Correia, Joana; Paiva Coelho, Margarida; Bandeira, Anabela; Martins, EsmeraldaCongenital disorders of glycosylation are a highly variable, rapidly expanding family of genetic diseases that result from defects in the synthesis of glycans. The vast majority of these monogenic diseases are inherited in an autosomal recessive way, but some types follow an autosomal dominant or X-linked inheritance. The present work aimed to review the state of the art of congenital disorders of glycosylation, including available therapeutic options, and present a simplified diagnostic approach to this group of diseases. Congenital disorders of glycosylation can be classified into four categories: N-linked glycosylation defects, O-linked glycosylation defects, combined glycosylation defects, and glycosphingolipid and glycosylphosphatidylinositol anchor synthesis defects. The phenotype may range from mild to severe, depending on disease severity. Clinical features include dysmorphic features, neurologic, dermatologic, cardiac, endocrine, immunologic, hematologic, gastrointestinal and liver involvement, and skeletal muscle abnormalities. As there is no universal or pathognomonic sign or symptom and no sensitive diagnostic test, it is of foremost importance to keep a high index of suspicion of these diseases. When a congenital disorder of glycosylation is suspected, the first step in screening is to perform serum transferrin isoelectric focusing. Molecular genetic testing is the most specific diagnostic test. Treatment is usually symptomatic, with specific treatment only available for some of these disorders. Since congenital defects of glycosylation may affect any organ at any age and have variable clinical presentation, they should be considered in the differential diagnosis of any patient with multiorgan involvement.
- Continuous use of glycomacropeptide in the nutritional management of patients with phenylketonuria: a clinical perspectivePublication . Pena, Maria João; Pinto, Alex; de Almeida, Manuela Ferreira; de Sousa Barbosa, Catarina; Ramos, Paula Cristina; Rocha, Sara; Guimas, Arlindo; Ribeiro, Rosa; Martins, Esmeralda; Bandeira, Anabela; Dias, Cláudia Camila; MacDonald, Anita; Borges, Nuno; Rocha, Júlio CésarBackground: In phenylketonuria (PKU), modified casein glycomacropeptide supplements (CGMP-AA) are used as an alternative to the traditional phenylalanine (Phe)-free L-amino acid supplements (L-AA). However, studies focusing on the long-term nutritional status of CGMP-AA are lacking. This retrospective study evaluated the long-term impact of CGMP-AA over a mean of 29 months in 11 patients with a mean age at CGMP-AA onset of 28 years (range 15-43) [8 females; 2 hyperphenylalaninaemia (HPA), 3 mild PKU, 3 classical PKU and 3 late-diagnosed]. Outcome measures included metabolic control, anthropometry, body composition and biochemical parameters. Results: CGMP-AA, providing 66% of protein equivalent intake from protein substitute, was associated with no significant change in blood Phe with CGMP-AA compared with baseline (562 ± 289 µmol/L vs 628 ± 317 µmol/L; p = 0.065). In contrast, blood tyrosine significantly increased on CGMP-AA (52.0 ± 19.2 μmol/L vs 61.4 ± 23.8 μmol/L; p = 0.027). Conclusions: Biochemical nutritional markers remained unchanged which is an encouraging finding in adults with PKU, many of whom are unable to maintain full adherence with nutritionally fortified protein substitutes. Longitudinal, prospective studies with larger sample sizes are necessary to fully understand the metabolic impact of using CGMP-AA in PKU.
- Doenças hereditárias do metabolismo: importância de um diagnóstico precoce para a criança e para a famíliaPublication . Martins, EsmeraldaRESUMO: O sucesso do rastreio neonatal da fenilcetonúria nas décadas de 70/80, veio permitir a partir da década de 90, com o recurso à espectrometria de massa em tandem (MS/MS), a pesquisa de um grande número de doenças hereditárias do metabolismo (DHM). A possibilidade de obter um diagnóstico pré-sintomático para estas doenças, que dispõem de tratamento, é potencialmente gratificante, havendo uma tendência para alargar cada vez mais o painel das doenças rastreáveis. Assim em Portugal, o Programa Nacional de Diagnóstico Precoce (PNDP) passou a incluir 24 patologias a partir de 2004. Presentemente, o benefício do rastreio é inequívoco para um grupo restrito de doenças, em que se inclui a fenilcetonúria e o défice da desidrogenase dos ácidos gordos de cadeia média (MCAD), e não consensual para as restantes, com diferentes painéis de rastreio nos vários países. Neste trabalho, proposemo-nos avaliar o contributo de um diagnóstico pré-sintomático na saúde das crianças rastreadas e a importância deste diagnóstico na vida das respectivas famílias. Este estudo incluiu 253 doentes com DHM, sendo alvo de especial relevância 93 que são afectados pelas patologias recentemente incluídas no painel de rastreio metabólico neonatal alargado em Portugal. Estes 93 doentes pertencem a dois grupos, os diagnosticados na fase sintomática com base na apresentação clínica (n=45) e os rastreados pelo PNDP, na sua maioria em fase pré-sintomática (n=48). Foram comparados em paralelo os doentes com a mesma DHM em termos de morbilidade e mortalidade. A fenilcetonúria é a única DHM em que já existem registos consistentes do seguimento a longo prazo e cujo sucesso serviu de ponto de partida para o rastreio das restantes doenças. Na avaliação do benefício para o doente foram especialmente consideradas quatro patologias: leucinose, argininemia, acidúria 3-hidroxi- 3-metilglutárica (3HMG) e défice de MCAD, por serem as mais relevantes na nossa experiência. Concluímos que o diagnóstico precoce contribuiu para a redução da morbilidade e da mortalidade aguda e crónica associadas a estas DHM sendo o ganho para a criança mais efectivo na leucinose, argininemia e défice de MCAD. No impacto para a família, destaca-se a importância do diagnóstico dos demais membros afectados, muitas das vezes ainda assintomáticos ou com sintomas minor, mas também nalgumas situações com quadros incapacitantes e arrastados ao longo dos anos sem diagnóstico etiológico. Nestes casos, o aconselhamento genético e o diagnóstico prénatal são uma mais-valia inquestionável. O rastreio neonatal permite o diagnóstico de todas as variantes da patologia, desde os casos mais graves aos mais ligeiros, assim como um real conhecimento epidemiológico das DHM. Estes dados suportam a decisão de incluir no painel português, doenças muito raras noutros países europeus e que, pelo contrário, são particularmente frequentes entre nós, nomeadamente a argininemia e a 3HMG. Uma reflexão sobre o trabalho desenvolvido, permite-nos concluir que a comunicação do resultado anómalo à família por parte de um clínico experiente no diagnóstico e tratamento destas patologias, o acompanhamento multidisciplinar, o aconselhamento e estudo familiar quer a nível bioquímico quer genético e a possibilidade de diagnóstico pré-natal, fazem desta intervenção integrada na área da pediatria, uma mais-valia para os doentes do foro metabólico. ABSTRACT: After the success of neonatal screening for phenylketonuria in the 70s/80s and, with the application in the 90's, of the tandem mass spectrometry (MS/MS), it became possible to search for a large number of inherited metabolic diseases (IMD). The possibility of having a early diagnosis for these treatable disorders is potentially rewarding, which lead to a tendency to extend the panel of screening disorders. Hence, since 2004, in Portugal, the Programa Nacional de Diagnóstico Precoce (National Neonatal Screening Program) includes 24 IMD. Currently, the benefit is unequivocal for a limited group of diseases – including phenylketonuria and medium chain acyl-CoA dehydrogenase deficiency (MCAD), but the remaining disorders are still subject of discussion, which explains the existence of heterogeneous screening panels in different countries. In this study, we proposed ourselves to assess the contribution of the neonatal screening for the health of children with pre-symptomatic diagnosis and to evaluate the impact of this diagnosis in the life of their families. This study included 253 metabolic patients, with particular attention to the 93 patients affected with the diseases recently included in the Portuguese expanded neonatal metabolic screening panel. These 93 patients were divided into two groups: those who have been diagnosed in a symptomatic stage (n=45) and those that have been detected by the neonatal screening program, most of them in pre-symptomatic stage (n=48). Within the same IMD, patients of the two groups have been compared for morbidity and mortality. Phenylketonuria is the only IMD with solid records of long-term follow-up and whose successful treatment was used as a starting point for the screening of the remaining diseases. Concerning the evaluation of the benefit for the patient, four diseases were specifically assessed: MSUD, hyperargininemia, 3-hydroxy-3-methylglutaric aciduria (3HMG) and MCAD deficiency - because they are the most assumed relevant IMD according to our experience. We have verified that the early diagnosis contributed to a reduction of the mortality and morbidity associated with acute and chronic presentations of these IMD. The gain has been most significant for children with MSUD, hyperargininemia and MCAD deficiency. Regarding the impact in the family, we highlight the importance of diagnosing affected relatives, often still asymptomatic or presenting minor symptoms, but also, in a few cases, with long time disabling symptoms without adequated diagnosis. In these cases, genetic counseling and prenatal diagnosis are unquestionably valuable. Expanded neonatal screening allows for the diagnosis in most of the diseases in its whole spectrum - from severe to mild – and also for the knowledge of the real epidemiology of the IMD. These data support the decision of inclusion, in the Portuguese panel, of very rare diseases in other European countries, which are particularly frequent among us - such as hyperargininemia and 3HMG. Some reflection on the work already developed allowed us to conclude that the communication of abnormal results to the family by an expert clinician in the diagnosis and treatment of these disorders, the multidisciplinary follow up, the counseling and familial study (both biochemical and genetic) and the possibility of prenatal diagnosis, make this integrated intervention in the area of Pediatrics an advantage for metabolic patients.
- Fatty Liver Caused by Glycogen Storage Disease Type IX: A Small Series of Cases in ChildrenPublication . Leuzinger Dias, C.; Maio, I.; Brandão, J.; Tomás, E.; Martins, Esmeralda; Santos Silva, E.Background: The prevalence of non-alcoholic fatty liver disease (NAFLD) affecting children and adolescents has increased dramatically in recent years. This increase is most probably related to the obesity pandemic and the high consumption of fructose. However, hepatic steatosis has some rare causes (e.g., some metabolic diseases) of which clinicians should be aware, particularly (but not only) when patients are non-obese or non-overweight. Differential diagnosis is notably important when pathologies have a specific treatment, such as for glycogenosis type IX (GSD-IX). Aims: To contribute to the knowledge on the differential diagnosis of NAFLD in paediatric age and to the clinical, biochemical, molecular, and histological characterisations of GSD-IX, a rare metabolic disorder. Methods: We performed a retrospective study of a small series of cases (n = 3) of GSD-IX diagnosed in the past 6 years, who were currently being followed up in the Units of Gastroenterology or Metabolic Diseases of the Paediatric Division of our hospital and whose clinical presentation was NAFLD in paediatric age. Results: Three male patients were diagnosed with NAFLD before 2 years of age, 2 with confirmed diagnosis before the age of 3 years (alanine aminotransferase [ALT], liver ultrasound, and molecular analysis) and 1 whose diagnosis was confirmed at 11 years (ALT, liver ultrasound, liver histology, and molecular analysis). None of the patients were obese or overweight, and the daily fructose consumption was unknown. The outcome was favourable in all 3 patients, with follow-up periods ranging from 2 to 6 years. Conclusion: The decision on how far the search for secondary causes of NAFLD should go can be difficult, and GSD-IX must be on the list of possible causes.
- Genes, Children and PediatriciansPublication . Coelho, Margarida Paiva; Soares, Ana Rita; Magalhães, Catarina; Martins, Esmeralda
- Inherited metabolic disorders: A century of evolutionPublication . Martins, Esmeralda
- Leigh Syndrome Spectrum: A Portuguese Population Cohort in an Evolutionary Genetic EraPublication . Baldo, Manuela Schubert; Nogueira, Célia; Pereira, Cristina; Janeiro, Patrícia; Ferreira, Sara; Lourenço, Charles M.; Bandeira, Anabela; Martins, Esmeralda; Magalhães, Marina; Rodrigues, Esmeralda; Santos, Helena; Ferreira, Ana Cristina; Vilarinho, LauraMitochondrial diseases are the most common inherited inborn error of metabolism resulting in deficient ATP generation, due to failure in homeostasis and proper bioenergetics. The most frequent mitochondrial disease manifestation in children is Leigh syndrome (LS), encompassing clinical, neuroradiological, biochemical, and molecular features. It typically affects infants but occurs anytime in life. Considering recent updates, LS clinical presentation has been stretched, and is now named LS spectrum (LSS), including classical LS and Leigh-like presentations. Apart from clinical diagnosis challenges, the molecular characterization also progressed from Sanger techniques to NGS (next-generation sequencing), encompassing analysis of nuclear (nDNA) and mitochondrial DNA (mtDNA). This upgrade resumed steps and favored diagnosis. Hereby, our paper presents molecular and clinical data on a Portuguese cohort of 40 positive cases of LSS. A total of 28 patients presented mutation in mtDNA and 12 in nDNA, with novel mutations identified in a heterogeneous group of genes. The present results contribute to the better knowledge of the molecular basis of LS and expand the clinical spectrum associated with this syndrome.
- Lipid Antigen Presentation by CD1b and CD1d in Lysosomal Storage Disease PatientsPublication . Pereira, C.; Pérez-Cabezas, B.; Ribeiro, H.; Maia, M.; Cardoso, M.; Dias, A.; Azevedo, O.; Ferreira, M.; Garcia, P.; Rodrigues, E.; Castro-Chaves, P.; Martins, Esmeralda; Aguiar, P.; Pineda, M.; Amraoui, Y.; Fecarotta, S.; Leão-Teles, E.; Deng, S.; Savage, P.; Macedo, M.The lysosome has a key role in the presentation of lipid antigens by CD1 molecules. While defects in lipid antigen presentation and in invariant Natural Killer T (iNKT) cell response were detected in several mouse models of lysosomal storage diseases (LSD), the impact of lysosomal engorgement in human lipid antigen presentation is poorly characterized. Here, we analyzed the capacity of monocyte-derived dendritic cells (Mo-DCs) from Fabry, Gaucher, Niemann Pick type C and Mucopolysaccharidosis type VI disease patients to present exogenous antigens to lipid-specific T cells. The CD1b- and CD1d-restricted presentation of lipid antigens by Mo-DCs revealed an ability of LSD patients to induce CD1-restricted T cell responses within the control range. Similarly, freshly isolated monocytes from Fabry and Gaucher disease patients had a normal ability to present α-Galactosylceramide (α-GalCer) antigen by CD1d. Gaucher disease patients' monocytes had an increased capacity to present α-Gal-(1-2)-αGalCer, an antigen that needs internalization and processing to become antigenic. In summary, our results show that Fabry, Gaucher, Niemann Pick type C, and Mucopolysaccharidosis type VI disease patients do not present a decreased capacity to present CD1d-restricted lipid antigens. These observations are in contrast to what was observed in mouse models of LSD. The percentage of total iNKT cells in the peripheral blood of these patients is also similar to control individuals. In addition, we show that the presentation of exogenous lipids that directly bind CD1b, the human CD1 isoform with an intracellular trafficking to the lysosome, is normal in these patients.