Browsing by Author "Rocha, H."
Now showing 1 - 8 of 8
Results Per Page
Sort Options
- Characterization of mitochondrial proteome in a severe case of ETF-QO deficiencyPublication . Rocha, H.; Ferreira, R.; Carvalho, J.; Vitorino, R.; Santa, C.; Lopes, L.; Gregersen, N.; Vilarinho, L.; Amado, F.Multiple acyl-CoA dehydrogenase deficiency (MADD) is a mitochondrial fatty acid oxidation disorder caused by mutations that affect electron transfer flavoprotein (ETF) or ETF:ubiquinone oxidoreductase (ETF-QO) or even due to unidentified disturbances of riboflavin metabolism. Besides all the available data on the molecular basis of FAO disorders, including MADD, the pathophysiological mechanisms underlying clinical phenotype development, namely at the mitochondrial level, are poorly understood. In order to contribute to the elucidation of these mechanisms, we isolated mitochondria from cultured fibroblasts, from a patient with a severe MADD presentation due to ETF-QO deficiency, characterize its mitochondrial proteome and compare it with normal controls. The used approach (2-DE-MS/MS) allowed the positive identification of 287 proteins in both patient and controls, presenting 35 of the significant differences in their relative abundance. Among the differentially expressed are proteins associated to binding/folding functions, mitochondrial antioxidant enzymes as well as proteins associated to apoptotic events. The overexpression of chaperones like Hsp60 or mitochondrial Grp75, antioxidant enzymes and apoptotic proteins reflects the mitochondrial response to a complete absence of ETF-QO. Our study provides a global perspective of the mitochondrial proteome plasticity in a severe case of MADD and highlights the main molecular pathways involved in its pathogenesis.
- Diagnosis of a patient with a kinetic variant of medium and short-chain 3-hydroxyacyl-CoA dehydrogenase deficiency by newborn screeningPublication . Vilarinho, L.; Sales Marques, J.; Rocha, H.; Ramos, A.; Lopes, L.; Narayan, S.B.; Bennett, M.J.Medium and short-chain 3-hydroxyacyl-CoA dehydrogenase deficiency is a rare cause of impaired mitochondrial fatty acid oxidation. We present a case report of a patient with hyperinsulinism and homozygosity for a novel mutation causing a kinetic variant of the enzyme. The diagnosis was initially inferred by abnormal newborn screening acylcarnitine analysis with elevated C4-hydroxyacylcarnitine.
- Enhanced interpretation of newborn screening results without analyte cutoff valuesPublication . Marquardt, G.; Currier, R.; McHugh, D.M.; Gavrilov, D.; Magera, M.J.; Matern, D.; Oglesbee, D.; Raymond, K.; Rinaldo, P.; Smith, E.H.; Tortorelli, S.; Turgeon, C.T.; Lorey, F.; Wilcken, B.; Wiley, V.; Greed, L.C.; Lewis, B.; Boemer, F.; Schoos, R.; Marie, S.; Vincent, M.F.; Sica, Y.C.; Domingos, M.T.; Al-Thihli, K.; Sinclair, G.; Al-Dirbashi, O.Y.; Chakraborty, P.; Dymerski, M.; Porter, C.; Manning, A.; Seashore, M.R.; Quesada, J.; Reuben, A.; Chrastina, P.; Hornik, P.; Atef Mandour, I.; Atty Sharaf, S.A.; Bodamer, O.; Dy, B.; Torres, J.; Zori, R.; Cheillan, D.; Vianey-Saban, C.; Ludvigson, D.; Stembridge, A.; Bonham, J.; Downing, M.; Dotsikas, Y.; Loukas, Y.L.; Papakonstantinou, V.; Zacharioudakis, G.S.; Baráth, Á.; Karg, E.; Franzson, L.; Jonsson, J.J.; Breen, N.N.; Lesko, B.G.; Berberich, S.L.; Turner, K.; Ruoppolo, M.; Scolamiero, E.; Antonozzi, I.; Carducci, C.; Caruso, U.; Cassanello, M.; la Marca, G.; Pasquini, E.; Di Gangi, I.M.; Giordano, G.; Camilot, M.; Teofoli, F.; Manos, S.M.; Peterson, C.K.; Mayfield Gibson, S.K.; Sevier, D.W.; Lee, S.Y.; Park, H.D.; Khneisser, I.; Browning, P.; Gulamali-Majid, F.; Watson, M.S.; Eaton, R.B.; Sahai, I.; Ruiz, C.; Torres, R.; Seeterlin, S.M.A.; Stanley, E.L.; Hietala, A.; McCann, M.; Campbell, C.; Hopkins, P.V.; de Sain-Van der Velden, M.G.; Elvers, B.; Morrissey, M.A.; Sunny, S.; Knoll, D.; Webster, D.; Frazier, D.M.; McClure, J.D.; Sesser, D.E.; Willis, S.A.; Rocha, H.; Vilarinho, L.; John, C.; Lim, J.; Caldwell, S.G.; Tomashitis, K.; Castiñeiras Ramos, D.E.; Cocho de Juan, J.A.; Rueda Fernández, I.; Yahyaoui Macías, R.; Egea-Mellado, J.M.; González-Gallego, I.; Delgado Pecellin, C.; García-Valdecasas Bermejo, M.S.; Chien, Y.H.; Hwu, W.L.; Childs, T.; McKeever, C.D.; Tanyalcin, T.; Abdulrahman, M.; Queijo, C.; Lemes, A.; Davis, T.; Hoffman, W.; Baker, M.; Hoffman, G.L.PURPOSE: To improve quality of newborn screening by tandem mass spectrometry with a novel approach made possible by the collaboration of 154 laboratories in 49 countries. METHODS: A database of 767,464 results from 12,721 cases affected with 60 conditions was used to build multivariate pattern recognition software that generates tools integrating multiple clinically significant results into a single score. This score is determined by the overlap between normal and disease ranges, penetration within the disease range, differences between conditions, and weighted correction factors. RESULTS: Ninety tools target either a single condition or the differential diagnosis between multiple conditions. Scores are expressed as the percentile rank among all cases with the same condition and are compared to interpretation guidelines. Retrospective evaluation of past cases suggests that these tools could have avoided at least half of 279 false-positive outcomes caused by carrier status for fatty-acid oxidation disorders and could have prevented 88% of known false-negative events. CONCLUSION: Application of this computational approach to raw data is independent from single analyte cutoff values. In Minnesota, the tools have been a major contributing factor to the sustained achievement of a false-positive rate below 0.1% and a positive predictive value above 60%.
- Integrative approaches for the diagnosis of inborn errors of metabolismPublication . Rocha, H.Inborn errors of metabolism (IEM) are a group of inherited genetic disorders affecting Human metabolism. Several examples will be presented illustrating the integration of the diagnostic approaches for different disease groups.
- Mitochondria proteome profiling: a comparative analysis between gel- and gel-free approachesPublication . Ferreira, R.; Rocha, H.; Almeida, V.; Padrão, A.L.; Santa, C.; Vilarinho, L.; Amado, F.; Vitorino, R.Mitochondrial proteomics emerged aiming to disclose the dynamics of mitochondria under various pathophysiological conditions. In the present study we investigated the relative merits of gel-based (2DE and SDS-LC) and gel-free (2D-LC) protein separation approaches and protein identification algorithms (Mascot and Paragon) in the proteome profiling of mitochondria isolated from cultured fibroblasts, a sample traditionally used for diagnosis purposes. Combining data retrieved from 2DE, 2D-LC and SDS-LC and search methods, a total of 696 non-redundant proteins were identified. An overlap of only 19% between the proteins identified by the three different methods was observed when Mascot and Paragon were used. Regarding protein ID, a consistency in the number of identified proteins per sample was noticed for 2DE approach. Independent of the methodological approach chosen, it was noticed that the predominance in mitochondria of hydrophilic proteins with 20-50 kDa and pI 5-6 and 8-9; however, 2D-LC and SDS-LC allowed the enrichment of proteins with a mass below 30 kDa and of basic proteins with pI values above 8. In conclusion, data from the present study highlight the power of integrating different separation technologies and protein identification algorithms.
- Molecular picture of cobalamin C/D defects before and after newborn screening eraPublication . Nogueira, C.; Marcão, A.; Rocha, H.; Sousa, C.; Fonseca, H.; Valongo, C.; Vilarinho, L.Objective: Birth prevalence of Cobalamin (Cbl) C or D defects in Portugal is an estimated 1:85,000, one of the highest worldwide. We compared the genotype/phenotype of patients identified with CblC or CblD before and after the implementation of expanded newborn screening. Methods: Twenty-five Portuguese CblC/D patients, 14 symptomatic and 11 identified through screening, were diagnosed using gas chromatography or tandem mass spectrometry. Molecular characterization was performed through the study of MMACHC and MMADHC genes. Results: The most common MMACHC mutation, c.271dupA, was present in 100% of MMACHC alleles of all CblC screened patients, in contrast with the 61% identified before expanded newborn screening. All studied cases (except one, who presented a CblD deficiency) presented a CblC defect. More CblC late-onset patients were diagnosed before the introduction of newborn screening than in the post newborn screening era, probably because some early onset patients died without a definitive diagnosis. Conclusion: The molecular data found in this cohort contribute to the improvement of screening and diagnosis of Cbl defects and would enable a confirmatory diagnosis of these patients, reducing the need for complex, costly, laborious, and time-consuming biochemical/enzymatic tests.
- Mutations at the flavin binding site of ETF:QO yield a MADD-like severe phenotype in DrosophilaPublication . Alves, E.; Henriques, B.J.; Rogrigues, J.V.; Prudêncio, P.; Rocha, H.; Vilarinho, L.; Martinho, R.G.; Gomes, C.M.Following a screening on EMS-induced Drosophila mutants defective for formation and morphogenesis of epithelial cells, we have identified three lethal mutants defective for the production of embryonic cuticle. The mutants are allelic to the CG12140 gene, the fly homologue of electron transfer flavoprotein:ubiquinone oxidoreductase (ETF:QO). In humans, inherited defects in this inner membrane protein account for multiple acyl-CoA dehydrogenase deficiency (MADD), a metabolic disease of β-oxidation, with a broad range of clinical phenotypes, varying from embryonic lethal to mild forms. The three mutant alleles carried distinct missense mutations in ETF:QO (G65E, A68V and S104F) and maternal mutant embryos for ETF:QO showed lethal morphogenetic defects and a significant induction of apoptosis following germ-band elongation. This phenotype is accompanied by an embryonic accumulation of short- and medium-chain acylcarnitines (C4, C8 and C12) as well as long-chain acylcarnitines (C14 and C16:1), whose elevation is also found in severe MADD forms in humans under intense metabolic decompensation. In agreement the ETF:QO activity in the mutant embryos is markedly decreased in relation to wild type activity. Amino acid sequence analysis and structural mapping into a molecular model of ETF:QO show that all mutations map at FAD interacting residues, two of which at the nucleotide-binding Rossmann fold. This structural domain is composed by a β-strand connected by a short loop to an α-helix, and its perturbation results in impaired cofactor association via structural destabilisation and consequently enzymatic inactivation. This work thus pinpoints the molecular origins of a severe MADD-like phenotype in the fruit fly and establishes the proof of concept concerning the suitability of this organism as a potential model organism for MADD.
- Retrospective study of the medium-chain acyl-CoA dehydrogenase deficiency in PortugalPublication . Ventura, F.V.; Leandro, P.; Luz, A.; Rivera, I.A.; Silva, M.F.; Ramos, R.; Rocha, H.; Lopes, A.; Fonseca, H.; Gaspar, A.; Diogo, L.; Martins, E.; Leão-Teles, E.; Vilarinho, L.; Tavares de Almeida, I.Medium-chain acyl-CoA dehydrogenase deficiency (MCADD) is the commonest genetic defect of mitochondrial fatty acid β-oxidation. About 60% of MCADD patients are homozygous for the c.985A>G (p.Lys329Glu) mutation in the ACADM gene (G985 allele). Herein, we present the first report on the molecular and biochemical spectrum of Portuguese MCADD population. From the 109 patients studied, 83 were diagnosed after inclusion of MCADD in the national newborn screening, 8 following the onset of symptoms and 18 through segregation studies. Gypsy ancestry was identified in 85/109 patients. The G985 allele was found in homozygosity in 102/109 patients, in compound heterozygosity in 6/109 and was absent in one patient. Segregation studies in the Gypsy families showed that 93/123 relatives were carriers of the G985 allele, suggesting its high prevalence in this ethnic group. Additionally, three new substitutions-c.218A>G (p.Tyr73Cys), c.503A>T (p.Asp168Val) and c.1205G>T (p.Gly402Val)-were identified. Despite the particularity of the MCADD population investigated, the G985 allele was found in linkage disequilibrium with H1(112) haplotype. Furthermore, two novel haplotypes, H5(212) and H6(122) were revealed.