Introdução: A Doença da Urina do Xarope do Bordo (DXB) é um Erro Inato do Metabolsimo (EIM), causada pela deficiência da atividade do complexo enzimático desidrogenase dos L-cetoácidos de cadeia ramificada (CACR). O programa público brasileiro de triagem neonatal não inclui o diagnóstico e o tratamento da DXB. Objetivo: Caracterizar aspectos relacionados ao diagnóstico e ao tratamento de pacientes brasileiros com DXB. Metodologia: Estudo retrospectivo. Os pacientes foram identificados a partir dos registros de laboratório considerado referência nacional para o diagnóstico de DXB, e de contato com demais serviços nacionais de referência em genética médica. O diagnóstico foi realizado entre 1992-2011. Os dados analisados foram obtidos a partir da revisão de prontuários. Resultados: Oitenta e três pacientes, oriundos de 75 famílias, foram incluídos no estudo (mediana de idade= 3 anos; IQ25- 75= 0,57-7). A mediana da idade de início dos sintomas foi de 10 dias (IQ= 5-30), enquanto que a mediana da idade ao diagnóstico foi de 60 dias (IQ= 29-240, p= 0,001). Apenas três (3,6%) pacientes foram diagnosticados antes de desenvolverem manifestações clínicas. A comparação entre os pacientes com (n=12) e sem (n=71) diagnóstico precoce mostrou que o mesmo associa-se com a presença de história familiar positiva e diminuição da prevalência de manifestações clínicas ao diagnóstico, mas não com melhor resultado; além disso, a maioria desses diagnósticos foi realizada entre 2002-2011 (n= 10/12). Considerando a amostra total, 98,8% dos pacientes apresentam atraso de desenvolvimento neuropsicomotor. Conclusão: Os pacientes com DXB são diagnosticados tardiamente no Brasil, de forma que as complicações associadas a esta condição não são prevenidas. Entretanto, existem indícios de que está havendo um melhora gradual desse panorama desde a última década. Os nossos dados indicam que o diagnóstico precoce dessa condição, mesmo que em fase sintomática, associa-se a um melhor prognóstico do paciente. Sugere-se a elaboração de políticas públicas específicas para doenças raras no país. / Introduction: Maple syrup urine disease (MSUD) is an Inborn Errors of Metabolism, is caused by a deficiency in activity of the branched-chain L-keto acid dehydrogenase complex. The Brazilian neonatal screening program does not include the diagnosis and treatment of MSUD. Objective: To draw a profile of aspects related to the diagnosis and treatment of Brazilian patients who received. Methods: In this retrospective study, patients were identified through a search of records from a national reference laboratory for the diagnosis of MSUD and through contact with other medical genetics services across Brazil. A diagnosis of MSUD between 1992 and 2011. Data were collected by means of a chart review. Results: Eighty-three patients from 75 families were enrolled in the study (median age 3 years; interquartile range, 0.57–7). Median age at onset of symptoms was 10 days (IQR 5–30), whereas median age at diagnosis was 60 days (IQR 29–240, p=0.001). Only three (3.6%) patients were diagnosed before the onset of clinical manifestations. A comparison between patients with (n=12) and without (n=71) an early diagnosis show that early diagnosis is associated with the presence of positive familial history and decreased prevalence of clinical manifestations at the time of diagnosis, but not with a better outcome. Overall, 98.8% of patients have some psychomotor or neurodevelopmental delay. Conclusion: In Brazil, patients with MSUD receive a late diagnosis and show neurological compromise and poor survival even with an early diagnosis. We suggest that specific public policies for rare diseases should be developed and implemented in the country.

Introdução: A Doença da Urina do Xarope do Bordo (DXB) é um Erro Inato do Metabolsimo (EIM), causada pela deficiência da atividade do complexo enzimático desidrogenase dos L-cetoácidos de cadeia ramificada (CACR). O programa público brasileiro de triagem neonatal não inclui o diagnóstico e o tratamento da DXB. Objetivo: Caracterizar aspectos relacionados ao diagnóstico e ao tratamento de pacientes brasileiros com DXB. Metodologia: Estudo retrospectivo. Os pacientes foram identificados a partir dos registros de laboratório considerado referência nacional para o diagnóstico de DXB, e de contato com demais serviços nacionais de referência em genética médica. O diagnóstico foi realizado entre 1992-2011. Os dados analisados foram obtidos a partir da revisão de prontuários. Resultados: Oitenta e três pacientes, oriundos de 75 famílias, foram incluídos no estudo (mediana de idade= 3 anos; IQ25- 75= 0,57-7). A mediana da idade de início dos sintomas foi de 10 dias (IQ= 5-30), enquanto que a mediana da idade ao diagnóstico foi de 60 dias (IQ= 29-240, p= 0,001). Apenas três (3,6%) pacientes foram diagnosticados antes de desenvolverem manifestações clínicas. A comparação entre os pacientes com (n=12) e sem (n=71) diagnóstico precoce mostrou que o mesmo associa-se com a presença de história familiar positiva e diminuição da prevalência de manifestações clínicas ao diagnóstico, mas não com melhor resultado; além disso, a maioria desses diagnósticos foi realizada entre 2002-2011 (n= 10/12). Considerando a amostra total, 98,8% dos pacientes apresentam atraso de desenvolvimento neuropsicomotor. Conclusão: Os pacientes com DXB são diagnosticados tardiamente no Brasil, de forma que as complicações associadas a esta condição não são prevenidas. Entretanto, existem indícios de que está havendo um melhora gradual desse panorama desde a última década. Os nossos dados indicam que o diagnóstico precoce dessa condição, mesmo que em fase sintomática, associa-se a um melhor prognóstico do paciente. Sugere-se a elaboração de políticas públicas específicas para doenças raras no país. / Introduction: Maple syrup urine disease (MSUD) is an Inborn Errors of Metabolism, is caused by a deficiency in activity of the branched-chain L-keto acid dehydrogenase complex. The Brazilian neonatal screening program does not include the diagnosis and treatment of MSUD. Objective: To draw a profile of aspects related to the diagnosis and treatment of Brazilian patients who received. Methods: In this retrospective study, patients were identified through a search of records from a national reference laboratory for the diagnosis of MSUD and through contact with other medical genetics services across Brazil. A diagnosis of MSUD between 1992 and 2011. Data were collected by means of a chart review. Results: Eighty-three patients from 75 families were enrolled in the study (median age 3 years; interquartile range, 0.57–7). Median age at onset of symptoms was 10 days (IQR 5–30), whereas median age at diagnosis was 60 days (IQR 29–240, p=0.001). Only three (3.6%) patients were diagnosed before the onset of clinical manifestations. A comparison between patients with (n=12) and without (n=71) an early diagnosis show that early diagnosis is associated with the presence of positive familial history and decreased prevalence of clinical manifestations at the time of diagnosis, but not with a better outcome. Overall, 98.8% of patients have some psychomotor or neurodevelopmental delay. Conclusion: In Brazil, patients with MSUD receive a late diagnosis and show neurological compromise and poor survival even with an early diagnosis. We suggest that specific public policies for rare diseases should be developed and implemented in the country.

Introdução: A Doença da Urina do Xarope do Bordo (DXB) é um Erro Inato do Metabolsimo (EIM), causada pela deficiência da atividade do complexo enzimático desidrogenase dos L-cetoácidos de cadeia ramificada (CACR). O programa público brasileiro de triagem neonatal não inclui o diagnóstico e o tratamento da DXB. Objetivo: Caracterizar aspectos relacionados ao diagnóstico e ao tratamento de pacientes brasileiros com DXB. Metodologia: Estudo retrospectivo. Os pacientes foram identificados a partir dos registros de laboratório considerado referência nacional para o diagnóstico de DXB, e de contato com demais serviços nacionais de referência em genética médica. O diagnóstico foi realizado entre 1992-2011. Os dados analisados foram obtidos a partir da revisão de prontuários. Resultados: Oitenta e três pacientes, oriundos de 75 famílias, foram incluídos no estudo (mediana de idade= 3 anos; IQ25- 75= 0,57-7). A mediana da idade de início dos sintomas foi de 10 dias (IQ= 5-30), enquanto que a mediana da idade ao diagnóstico foi de 60 dias (IQ= 29-240, p= 0,001). Apenas três (3,6%) pacientes foram diagnosticados antes de desenvolverem manifestações clínicas. A comparação entre os pacientes com (n=12) e sem (n=71) diagnóstico precoce mostrou que o mesmo associa-se com a presença de história familiar positiva e diminuição da prevalência de manifestações clínicas ao diagnóstico, mas não com melhor resultado; além disso, a maioria desses diagnósticos foi realizada entre 2002-2011 (n= 10/12). Considerando a amostra total, 98,8% dos pacientes apresentam atraso de desenvolvimento neuropsicomotor. Conclusão: Os pacientes com DXB são diagnosticados tardiamente no Brasil, de forma que as complicações associadas a esta condição não são prevenidas. Entretanto, existem indícios de que está havendo um melhora gradual desse panorama desde a última década. Os nossos dados indicam que o diagnóstico precoce dessa condição, mesmo que em fase sintomática, associa-se a um melhor prognóstico do paciente. Sugere-se a elaboração de políticas públicas específicas para doenças raras no país. / Introduction: Maple syrup urine disease (MSUD) is an Inborn Errors of Metabolism, is caused by a deficiency in activity of the branched-chain L-keto acid dehydrogenase complex. The Brazilian neonatal screening program does not include the diagnosis and treatment of MSUD. Objective: To draw a profile of aspects related to the diagnosis and treatment of Brazilian patients who received. Methods: In this retrospective study, patients were identified through a search of records from a national reference laboratory for the diagnosis of MSUD and through contact with other medical genetics services across Brazil. A diagnosis of MSUD between 1992 and 2011. Data were collected by means of a chart review. Results: Eighty-three patients from 75 families were enrolled in the study (median age 3 years; interquartile range, 0.57–7). Median age at onset of symptoms was 10 days (IQR 5–30), whereas median age at diagnosis was 60 days (IQR 29–240, p=0.001). Only three (3.6%) patients were diagnosed before the onset of clinical manifestations. A comparison between patients with (n=12) and without (n=71) an early diagnosis show that early diagnosis is associated with the presence of positive familial history and decreased prevalence of clinical manifestations at the time of diagnosis, but not with a better outcome. Overall, 98.8% of patients have some psychomotor or neurodevelopmental delay. Conclusion: In Brazil, patients with MSUD receive a late diagnosis and show neurological compromise and poor survival even with an early diagnosis. We suggest that specific public policies for rare diseases should be developed and implemented in the country.

Abstract The mitochondrial oxidative phosphorylation system is composed of five multisubunit enzyme complexes. Complex I is the first and largest of these, containing 46 subunits, seven encoded by mitochondrial DNA (mtDNA) and the rest by nuclear DNA. Isolated complex I deficiency is a major cause of metabolic errors in infancy and childhood, presenting as encephalomyopathies or multisystem disorders. Due to the bigenomic origin of complex I, the genetic causes of these defects can be either mitochondrial or nuclear. The object of the present work was to identify the underlying genetic cause in cases of children with complex I deficiency and to obtain more information on the structurally and functionally important sites of complex I subunits. The complete coding region of mtDNA was analysed by conformation-sensitive gel electrophoresis and subsequent sequencing. In addition, nine nuclear genes encoding conserved subunits of complex I were sequenced. The structural and functional consequences of the new sequence variants were further elucidated using mutagenesis of homologous residue in bacterial NDH-1 or by studying complex I assembly and expression in patient cell lines. Analysis of the mtDNA coding region in 50 children revealed four definitely pathogenic mutations, 3460G>A, 10191T>C, 11778G>A and 14487T>C, in seven patients. In addition, two novel mtDNA base pair substitutions were identified, 3866T>C in a patient with muscle weakness and short stature and 4681T>C in a patient with Leigh syndrome. The latter mutation causes a Leu71Pro amino acid exchange in the ND2 subunit. Cybrid clones harbouring this mutation retained the complex I defect, and reduced amounts of fully assembled complex I were detected in patient cell lines. The 3866T>C mutation leads to a Ile187Thr amino acid substitution in the ND1 subunit, and functional studies of the homologous amino acid substitution in E. coli showed that this had an effect on the assembly or stability of the NDH-1 holoenzyme. Sequencing of the nine nuclear-encoded complex I genes revealed only one novel base pair substitution with pathogenic potential. Further studies are needed, however, to establish the role of the Arg18Cys substitution in the mitochondrial leading peptide of the TYKY subunit. The above findings emphasize the contribution of mtDNA mutations to the aetiology of pediatric patients with complex I deficiency. Furthermore, two LHON primary mutations were identified in the present cohort of patients, although the clinical signs differed considerably from the classical symptoms of LHON. This suggests that the phenotype caused by primary LHON mutations is more variable than has so far been thought.

Orientador: Denise Pontes Cavalcanti / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-22T09:53:23Z (GMT). No. of bitstreams: 1 Moreno_CarolinaAraujo_M.pdf: 4072523 bytes, checksum: 09921eee06d7c1098048c98a3f891b33 (MD5) Previous issue date: 2013 / Resumo: A hidropisia fetal não-imune (HFNI) é causada por um grupo heterogêneo de condições e atualmente corresponde à maior parte dos casos de hidropisia fetal. Em função da ampla diversidade etiopatogênica, a investigação dos casos de HFNI constitui um desafio diagnóstico. Esse estudo teve como objetivo a avaliação prospectiva e sistemática de uma série de casos de HFNI a partir de um protocolo de investigação ampliado, que incluiu a pesquisa de doenças metabólicas. O presente estudo também incluiu a revisão dos casos de HFNI registrados previamente pelo Programa de Genética Perinatal na maternidade da Unicamp. Durante aproximadamente dois anos (2010-2012), foram identificados 53 casos de HFNI. Nesse período, ocorreram 6.129 nascimentos na maternidade local, com registro de HFNI em 37 recém-nascidos, conferindo uma prevalência de 60 por 10.000 nascimentos, valor maior do que o observado no período anterior ao estudo (1987 a 2009). Para o restante da análise, quatro casos foram excluídos devido à impossibilidade de estudá-los adequadamente. A maioria dos hidrópicos nasceu pré-termo (43 - 73,5%). Houve registro de 23 nativivos (47%), 10 óbitos no período neonatal e 26 óbitos durante a gestação (53%), resultado em uma mortalidade geral (pré-natal e neonatal) de 73,4%. A hidropisia foi identificada no pré-natal na maioria dos casos (44 - 89,8%) e, apesar da condição ser comumente associada a mau prognóstico, em três pacientes (6,1%) houve resolução completa e espontânea da hidropisia durante a gestação. Os principais grupos diagnósticos encontrados foram: anomalias cromossômicas (17 casos - 34,7%), quadros sindrômicos (16,4% - oito casos), cardiopatias e infecções congênitas (8,2% - quatro casos cada). Os erros inatos do metabolismo (EIM) corresponderam a 6,1% da amostra (três casos de doenças de depósito lisossômico). Três casos (6,1%) foram classificados como idiopáticos. Comparando os diagnósticos da casuística prospectiva com aqueles observados no período 1987-2009, observou-se que os grupos diagnósticos mais frequentes, ou seja, anomalias cromossômicas, quadros sindrômicos e cardiopatias, foram os mesmos. No entanto, algumas diferenças foram observadas na casuística prospectiva, como a frequência maior de anomalias cromossômicas e de EIM, que podem ser explicadas respectivamente pela inclusão de abortos e pesquisa sistemática de doenças metabólicas. Por outro lado, a menor frequência do grupo de idiopáticos foi decorrente da ampliação do protocolo de investigação e da eliminação de casos inadequadamente estudados. Ressalta-se também que a frequência de EIM registrada no trabalho prospectivo (6%) foi superior à usualmente descrita na literatura e provavelmente aproxima-se da frequência real das doenças metabólicas em HFNI, justificando desse modo a pesquisa das mesmas na investigação de hidrópicos de causa não-imune após exclusão das condições mais comuns, como as anomalias cromossômicas, cardiopatias isoladas, infecções congênitas e síndromes conhecidas. Sendo assim, observou-se que o protocolo de investigação proposto permitiu o diagnóstico clínico-etiológico ou patogênico de mais de 90% dos casos, evidenciando que uma avaliação ampla e sistemática é capaz de identificar a maior parte dos fatores etiopatogênicos envolvidos na HFNI / Abstract: Non-immune hydrops fetalis (NIHF) is caused by a hetereogenous group of conditions, currently accounting for the most cases of hydrops fetalis. Because of the wide etiopathogenic diversity, the investigation of NIHF cases constitutes a real diagnostic challenge. This study aimed to evaluate prospectively and systematically a series of NIHF cases from an expanded research protocol including the investigation of metabolic diseases. The present study also aimed to revise the NIHF cases previously recorded by Perinatal Genetics Program (PGP) in the maternity hospital of Unicamp. During approximately two years (2010-2012), 53 cases were identified. In this period, among 6,129 births that occurred in our hospital, NIHF was identified in 37 newborns, given a birth prevalence of 60 per 10,000, higher than that was observed in the previous period - 23:10,000 (1987-2009). For purpose of all other analysis, four of the 53 cases evaluated had to be excluded due to inability to assess them correctly. Most hydropic individuals were born preterm (43 - 73.5%). Twenty-three patients (47%) were live births, 10 of them died before hospital discharge; and 26 (53%) died in the prenatal period, given an overall mortality of 73.4%. The hydrops were identified in prenatal period in most cases (44 - 89.8%), and despite being commonly associated with poor prognosis, three cases (6.1%) had complete and spontaneous resolution of hydrops during pregnancy. The main diagnostic groups were chromosomal abnormalities (17 - 34.7%), syndromic (8 - 16.4%), isolated heart defects (4 - 8.2%), and congenital infections (4 - 8.2%). Inborn errors of metabolism (IEM) occurred in three cases (6.1%), all represented by lysosomal storage diseases. Three cases (6.1%) were classified as idiopathic. The comparison of these diagnostic groups with those found during the retrospective period (1987-2009) showed that the most frequent groups, i.e. chromosomal abnormalities, syndromic and isolated cardiopathy, were the same. However, some differences were observed in the prospective series. For instance, the higher frequency of chromosomal abnormalities and IEM can be respectively explained because of the inclusion of abortions and due to systematic investigation of metabolic diseases. The lower frequency of idiopathic group, by the other hand, can be regarded as close related, first, to the expanded investigation and, second, to the exclusion of cases poorly studied. It is noteworthy that the recorded IEM frequency in the present prospective series (6%) was higher than the usually reported in the literature and seems to be more realistic, thus, justifying the inclusion of a systematic approach of these conditions in NIHF. This investigation, however, should be initiated after the exclusion of the more common causes, i.e., chromosomal abnormalities, isolated cardiopathy, congenital infections and known syndromes. In conclusion, the proposed investigation protocol allowed the clinical-etiological or pathogenic diagnosis in more than 90% of the cases, suggesting that the most etiopathogenic factors related to NIHF can be identified if a wide and systematic evaluation is performed / Mestrado / Genetica Medica / Mestra em Ciências Médicas

The role of biological markers (biomarkers) in public health, pediatric medicine and clinical toxicology cannot be understated. Clinically validated biomarkers used in newborn screening (NBS) serve to detect individuals at risk for a disease in the population, pre-symptomatically diagnose affected neonates early in life and/or accurately predict disease progression and treatment responses to therapy. However, there is urgent need for the discovery of more specific biomarkers that can improve screening accuracy in a high throughput, cost-effective yet ethical manner. The major objectives of this thesis were to develop innovative nontargeted metabolite profiling methodologies based on multisegment injection-capillary electrophoresis-mass spectrometry (MSI-CE-MS) for early detection of treatable genetic diseases, as well as comprehensive surveillance of drugs of abuse (DoA) in high risk subjects. Chapter II introduces a multiplexed MSI-CE-MS strategy for confirmatory testing of known biomarkers for various inborn errors of metabolism from a dried blood spot (DBS) that was rigorously validated using proficiency test specimens from Centres for Disease Control and Prevention (CDC) and authentic neonatal samples from Newborn Screening Ontario (NSO) with quality assurance. Additionally, MSI-CE-MS together with temporal signal pattern recognition revealed for the first time a novel class of pathognomonic marker elevated in galactosemia, namely N-galactated amino acids. Chapter III describes an untargeted metabolomic study to discover biomarkers of cystic fibrosis (CF) to reduce the high false positive rate and widespread carrier identification by current two-tiered NBS algorithms that rely on genetic testing. A panel of metabolites from retrospective DBS specimens, including several amino acids, ophthalmic acid and an unknown peptide, allowed for differentiation of asymptomatic CF neonates from screen-positive yet unaffected carriers and transient hypertrypsinogenemic cases. Chapter IV develops and validates a high throughput MSI-CE-MS assay for rapid screening for DoA and their metabolites in human urine with improved specificity and broad spectrum coverage as compared to classic targeted immunoassays. This method can also applied to ensure compliance, drug efficacy and patient safety while detecting for potential substitution or adulteration when using high resolution MS/MS. In summary, this thesis contributes an innovative methodology and data workflow for biomarker discovery for improved neonatal screening of rare genetic diseases in the population, which was also applied for more effective drug surveillance strategies in public health given the alarming worldwide opioid crisis. / Thesis / Doctor of Philosophy (PhD)

Introdução: A Doença da Urina do Xarope de Bordo é uma doença hereditária do metabolismo dos aminoácidos de cadeia ramificada, de caráter autossômico recessivo. O diagnóstico precoce é fundamental na prevenção da deterioração neurológica, que se dá pela ausência da implementação do tratamento nutricional adequado. Objetivo: Realizar triagem das mutações em todos os éxons dos três genes envolvidos na Doença da Urina do Xarope de Bordo (BCKDHA, BCKDHB e DBT) através do sequenciamento gênico direto e correlacionar com a heterogeneidade fenotípica. Métodos: Estudo clínico transversal com pacientes chilenos diagnosticados com Doença da Urina do Xarope de Bordo. A genotipagem foi realizada com produtos purificados de reação em cadeia da polimerase (PCR) de DNA.Foi realizada análise in silico das substituições de nucleotídeos através dos softwares MutPred® v1.2, Polyphen-2® - Polymorphism Phenotyping v2 e SIFT®. As características clínicas dos pacientes foram fornecidas pela equipe de nutrição do Instituto de Nutrição e Tecnologia da Universidade de Chile (INTA). Foi realizado um teste exato de Fisher no grupo de pacientes portadores da mutação mais prevalente na amostragem, a I214K com a intenção de avaliar o grau de correlação entre algumas variáveis clínicas e genéticas para verificar a possibilidade de se estabelecer uma relação fenótipo/genótipo. Resultados: Dos 18 pacientes 88% apresentaram mutação no gene BCKDHB, 1 pacientes 5% apresentou mutação no gene BCKDHA e 1 (5%) paciente apresentou mutação no gene DBT. Foram encontradas um total de 8 mutações na amostra e 4 novas mutações (50%). Não se pode afirmar que há correlação de nenhuma das variáveis clínicas com os genótipos encontrados nessa amostragem. Conclusão: Este estudo reportou 4 novas mutações em pacientes portadores de DXB na população chilena, o que pode ajudar em futuros diagnósticos genéticos da doença. Se a DXB fosse diagnosticada de forma mais rápida, na triagem neonatal, talvez fosse possível estabelecer uma relação genótipo-fenótipo de forma mais eficiente / Introduction: Maple Syrup Urine Disease (MSUD) is an autossomal recessive hereditary disease of the branched-chain amino acid metabolism. Early diagnosis is essential in preventing neurological deterioration, which occurs due to inadequate nutritional implametation treatment. Purpose: Screen mutations in all exons from the three genes involved in MSUD (BCKDHA, BCKDHB and DBT) through direct gene sequencing and correlation with phenotypic heterogeneity. Methods: A cross-sectional study with Chilean patients diagnosed with Maple Syrup Urine Disease. Genotyping was performed using purified polymerase chain reaction (PCR) DNA. Nucleotide substitutions In Silico analysis was performed using MutPred® v1.2, Polyphen-2® - Polymorphism Phenotyping v2 and SIFT® softwares. Patients clinical characteristics were provided by the nutrition team from Chile University, Nutriton and Technology Institute (INTA). Results: Of the 18 patients, 88% presented mutation in BCKDHB gene, 1 patient had mutation in BCKDHA gene and 1 patient (5%) presented a mutation in DBT gene. A total of 8 mutations in the sample and 4 new mutations (50%) were found. It can not be affirmed that there is correlation between clinical variables and genotypes in this sample. Conclusion: This study reported 4 new mutations in patients with MSUD in Chilean population, which may help in future genetic diagnosis. If MSUD was diagnosed more rapidly in neonatal screening, it might be possible to establish a genotype-phenotype relationship more efficiently

Orientador: Roger Frigerio Castilho / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-11T10:41:34Z (GMT). No. of bitstreams: 1 Mirandola_SandraRegina_D.pdf: 3705031 bytes, checksum: e1e34dd5e1b3949d9c308b3c20a19787 (MD5) Previous issue date: 2008 / Resumo: A acidemia metilmalônica (MMAemia) é uma desordem metabólica hereditária do metabolismo de aminoácidos com cadeia ramificada e de ácidos graxos com cadeia ímpar, envolvendo um defeito na conversão de metilmalonil-CoA a succinil-CoA. Manifestações sistêmicas e neurológicas nesta doença são relacionadas com o acúmulo de metilmalonato (MMA) em tecidos e fluidos biológicos e com o comprometimento do metabolismo energético. Neste trabalho, verificou-se que o MMA inibiu com grande intensidade a conversão de lactato a piruvato catalisada pela enzima lactato desidrogenase (LDH) em homogenatos de fígado e cérebro de rato. A conversão de piruvato a lactato, catalisada pela LDH, foi menos sensível à inibição por MMA. Estudos de cinética enzimática sobre a inibição da LDH de cérebro, utilizando-se lactato como substrato, indicaram que o MMA inibe esta enzima competitivamente (Ki = 3,02 ± 0,59 mM). Propôs-se que a inibição da conversão lactato/piruvato por MMA contribui para a fisiopatologia da MMAemia, resultando, dentre outras alterações, em acúmulo de lactato e acidemia metabólica. Mostrou-se que, em mitocôndrias isoladas de cérebro e músculo de rato, concentrações milimolares de MMA inibiram o consumo de O2 mantido por succinato, enquanto nenhum efeito inibitório foi observado quando substratos para os complexos I ou IV foram utilizados. Notadamente, o efeito inibitório de MMA, mas não de malonato, no consumo mitocondrial de O2 mantido por succinato foi minimizado quando uma permeabilização não-seletiva das mitocôndrias foi induzida por alameticina. Em adição, o MMA apresentou apenas um pequeno efeito inibitório no consumo de O2 por partículas submitocondriais invertidas na presença de succinato. Não se obteve evidência de produção de malonato nas mitocôndrias tratadas com MMA. Conclui-se que o MMA inibe o consumo mitocondrial de O2 na presença de succinato por interferir na captação deste substrato pela mitocôndria. A inibição do transporte mitocondrial de substratos, induzida pelo MMA, através do carreador de dicarboxilatos, pode ter importantes implicações fisiopatológicas na MMAemia. Comparou-se a suscetibilidade de mitocôndrias isoladas de fígado, rim e coração de rato, assim como de diferentes subregiões cerebrais quanto à transição de permeabilidade mitocondrial (MPT) induzida por 3-nitropropionato (3-NP) e Ca2+. A MPT foi estimada pela queda do potencial elétrico transmembrana e inchamento mitocondrial sensíveis à ciclosporina A. Mitocôndrias de cérebro e coração foram mais suscetíveis à MPT induzida por 3-NP e Ca2+ que organelas isoladas de fígado e rim. A comparação de mitocôndrias de diferentes regiões cerebrais indicou que uma inibição parcial da respiração por 3-NP resultou em MPT mais rapidamente em organelas estriatais que corticais ou cerebelares. Em ratos tratados sistemicamente com 3-NP, verificou-se uma inibição de mesma magnitude da succinato desidrogenase em todos os tecidos estudados. Notadamente, mitocôndrias isoladas de cérebro de ratos tratados sistemicamente com 3-NP apresentaram uma maior suscetibilidade à MPT induzida por Ca2+ quanto comparadas a controles. Propôs-se que a maior suscetibilidade do estriado à neurodegeneração induzida por 3-NP pode ser, pelo menos em parte, explicada por uma maior vulnerabilidade desta região cerebral à MPT, juntamente com a vulnerabilidade desta região ao influxo de Ca2+ citosólico mediado pelo estímulo de receptores de glutamato / Abstract: Methylmalonic acidemia (MMAemia) is an inherited metabolic disorder of branched amino acid and odd-chain fatty acid metabolism, involving a defect in the conversion of methylmalonyl-coenzyme A to succinyl-coenzyme A. Systemic and neurological manifestations in this disease are thought to be associated with the accumulation of methylmalonate (MMA) in tissues and biological fluids with consequent impairment of energy metabolism. In the present work it was observed that MMA potently inhibited lactate dehydrogenase (LDH)-catalyzed conversion of lactate to pyruvate in liver and brain homogenates. LDH was about one order of magnitude less sensitive to inhibition by MMA when catalyzing the conversion of pyruvate to lactate. Kinetic studies on the inhibition of brain LDH indicated that MMA inhibits this enzyme competitively with lactate as a substrate (Ki = 3.02 ± ?0.59 mM). We proposed that inhibition of the lactate/pyruvate conversion by MMA contributes to the MMAemia physiophatology, leading to lactate accumulation and metabolic acidemia. While millimolar concentrations of MMA inhibit succinate-supported O2 consumption by isolated rat brain or muscle mitochondria, there is no effect when either a pool of NADH-linked substrates or N,N,N',N'-tetramethyl-p-phenylendiamine (TMPD)/ascorbate were used as electron donors. Interestingly, the inhibitory effect of MMA, but not of malonate, on succinate-supported brain mitochondrial O2 consumption was minimized when nonselective permeabilization of mitochondrial membranes was induced by alamethicin. In addition, only a slight inhibitory effect of MMA was observed on succinate-supported O2 consumption by inside-out submitochondrial particles. Under our experimental conditions, there was no evidence of malonate production in MMA-treated mitochondria. We conclude that MMA inhibits succinate-supported mitochondrial O2 consumption by interfering with the uptake of this substrate. MMA-induced inhibition of substrate transport by the mitochondrial dicarboxylate carrier may have important physiopatological implications. The susceptibility of isolated mitochondria from liver, kidney and heart and different rat brain regions (striatum, cortex and cerebellum) was compared regarding to mitochondrial permeability transition (MPT) evoked by 3-nitropropionate (3-NP) and Ca2+ ions. In general, isolated brain mitochondria from different regions were more sensitive to 3-NP and Ca2+ toxicity than mitochondria from liver and kidney as estimated by decrease in the transmembrane electrical potential and mitochondrial swelling. The comparision of different brain regions revealed that the inhibition of 50% of the mitochondrial succinate-supported respiration elicited by 3-NP resulted in a Ca2+-induced MPT pore opening, inhibited by cyclosporin A, faster in striatal than in cortical and cerebellar mitochondria. It was verified an inhibition of succinate dehydrogenase activity from the same magnitude in all tissues studied after a 3-NP systemic treatment. Interestingly, isolated forebrain mitochondria obtained from rats systemically treated with 3-NP showed a more pronounced susceptibility to Ca2+-induced MPT pore opening when compared to control rats. We proposed that the increased susceptibility of rat striatum to 3-NP-induced neurodegeneration could be in part explain by a region-specific susceptibility to MPT together with increase vulnerability of this brain region to glutamate receptors-mediated cytosolic Ca2+ influx / Doutorado / Biologia Estrutural, Celular, Molecular e do Desenvolvimento / Doutor em Fisiopatologia Medica

La médecine de précision (MP) est un nouveau paradigme qui révolutionne la pratique médicale actuelle et remodèle complètement la médecine de demain. La MP aspire à placer le patient au centre du parcours de soins en y intégrant les données médicales et biologiques individuelles tout en tenant compte de la grande diversité interindividuelle. La prédiction des états pathologiques chez les patients nécessite une compréhension dynamique et systémique. Les erreurs innées du métabolisme (EIM) sont des troubles génétiques résultant de défauts dans une voie biochimique donnée en raison de la déficience d'une enzyme, de son cofacteur ou d’un transporteur. Les EIM ne sont plus considérées comme des maladies monogéniques mais tendent à être plus complexes et multifactorielles. Le profil métabolomique permet le dépistage d’une pathologie, la recherche de biomarqueurs et l’exploration des voies métaboliques mises en jeu. Dans ce travail de thèse, nous avons utilisé l’approche métabolomique qui est particulièrement pertinente pour les EIM compte tenu de leur physiopathologie de base qui est étroitement liée au métabolisme. Ce travail a permis la mise en place d’une méthodologie métabolomique non ciblée basée sur une stratégie analytique multidimensionnelle comportant la spectrométrie de masse à haute résolution couplée à la chromatographie liquide ultra-haute performance et la mobilité ionique. La mise en place de la méthodologie de prétraitement, d’analyse et d’exploitation des données générées avec des outils de design expérimental et d’analyses multivariées ont été aussi établies. Enfin, cette approche a été appliquée pour l’exploration des EIM avec les mucopolysaccharidoses comme preuve de concept. Les résultats obtenus suggèrent un remodelage majeur du métabolisme des acides aminés dans la mucopolysaccharidose de type I. En résumé, la métabolomique pourrait être un outil complémentaire pertinent en appui à l’approche génomique dans l’exploration des EIM. / The new field of precision medicine is revolutionizing current medical practice and reshaping future medicine. Precision medicine intends to put the patient as the central driver of healthcare by broadening biological knowledge and acknowledging the great diversity of individuals. The prediction of physiological and pathological states in patients requires a dynamic and systemic understanding of these interactions. Inborn errors of metabolism (IEM) are genetic disorders resulting from defects in a given biochemical pathway due to the deficiency of an enzyme, its cofactor or a transporter. IEM are no longer considered to be monogenic diseases, which adds another layer of complexity to their characterization and diagnosis. To meet this need for faster screening, the metabolic profile can be a promising candidate given its ability in disease screening, biomarker discovery and metabolic pathway investigation. In this thesis, we used a metabolomic approach which is particularly relevant for IEM given their basic pathophysiology that is tightly related to metabolism. This thesis allowed the implementation of an untargeted metabolomic methodology based on a multidimensional analytical strategy including high-resolution mass spectrometry coupled with ultra-high-performance liquid chromatography and ion mobility. This work also set a methodology for preprocessing, analysis and interpretation of the generated data using experimental design and multivariate data analysis. Finally, the strategy is applied to the exploration of IEM with mucopolysaccharidoses as a proof of concept. The results suggest a major remodeling of the amino acid metabolisms in mucopolysaccharidosis type I. In summary, metabolomic is a relevant complementary tool to support the genomic approach in the functional investigations and diagnosis of IEM.

Chez l’homme, les erreurs innées du métabolisme des lipides sont dues à des déficits enzymatiques, entraînant une accumulation intracellulaire de substrats lipidiques. Il en résulte un large éventail de symptômes tels que des atteintes viscérales, osseuses et dans certains cas neurologiques. En outre, de nombreux patients atteints de ce type de maladie présentent des anomalies hématologiques et vasculaires attribuées à des anomalies rhéologiques du globule rouge (GR). Ces observations ont conduit à l’hypothèse de l’existence d’un lien entre les propriétés anormales du GR et sa composition lipidique. Or actuellement, le profil lipidique du GR normal reste méconnu. Cependant, le diagnostic précoce de ces troubles est d’une importance capitale pour la prise en charge des patients, notamment dans les cas où un traitement correctif est disponible. La maladie de Gaucher (MG) de type 1, qui est une maladie lysosomale caractérisée par un déficit en β-glucocérébrosidase et pour laquelle un traitement enzymatique substitutif (ERT) est proposé, en est le meilleur exemple. D’où l’intérêt de disposer d’un outil simple et rapide de diagnostic de ce type de maladie.Dans le cas de la MG, le diagnostic repose encore sur la mise en évidence, laborieuse, du déficit enzymatique. Néanmoins, des travaux récents suggèrent que les anomalies rhéologiques du GR pourraient être dues à l’accumulation de quatre sphingolipides, le glucosylcéramide, la glucosylsphingosine, la sphingosine et la sphingosine-1-phosphate, qui seraient de bons candidats biomarqueurs. Or, les méthodes actuelles de dosage de ces sphingolipides nécessitent au moins deux étapes chromatographiques, avec pour chacune une étape longue et fastidieuse de préparation de l’échantillon, ce qui ne facilite guère une approche lipidomique de ce sujet. En outre, seul le glucosylcéramide a été dosé dans le GR tandis que les trois autres sphingolipides n’ont été dosés que dans le plasma. Ces candidats biomarqueurs restent donc à valider.Dans cette thèse, nous avons développé et validé une méthode simple et rapide, par UHPLC-MS/MS, de dosage simultané des 4 sphingolipides impliqués dans la MG. L’application de cette méthode à des GR provenant de patients atteints de la MG, en collaboration avec l’Institut National de Transfusion Sanguine et la société Shire, nous a permis de : 1- valider un biomarqueur parmi les quatre proposés et de montrer que les trois autres n’étaient pas suffisamment spécifiques ; 2- vérifier l’efficacité du traitement ERT actuellement proposé et 3- confirmer l’hypothèse de départ reliant les anomalies rhéologiques du GR à sa composition lipidique.De même, une étude systématique des conditions opératoires nous a permis de généraliser la méthode proposée à l’identification et au dosage de l’ensemble des sphingolipides présents dans un GR ainsi que des phospholipides, constituants majoritaires de sa membrane. Appliquée à la quantification simultanée d’une trentaine de sphingolipides et de phospholipides dans le GR normal et celui de la MG, cette méthode nous a permis de mettre en évidence l’implication d’autres lipides polaires dans la maladie de Gaucher, outre les 4 sphingolipides jusqu’alors proposés. De même, il est prévu de l’adapter à moyen terme pour le profilage total, par classe, de tous les lipides présents dans le GR.Enfin, nous avons évalué d’autres techniques de SM telles que la haute résolution et la mobilité ionique (TWIMS et DIMS) dans le but d’affiner la recherche de nouveaux biomarqueurs, notamment par l’identification des lipides isomères non discriminables par les techniques de MS conventionnelles. Grâce à une collaboration avec le Laboratoire de Chimie Physique (LCP, CNRS UMR 8000) nous avons montré la faisabilité de cette approche en séparant en DIMS deux isomères : la galactosylsphingosine 18:1 et la glucosylsphingosine 18:1 et nous poursuivons actuellement cette étude pour séparer d’autres couples d’isomères. / In humans, hereditary disorders of lipid metabolism are due to enzyme deficiencies, resulting in intracellular accumulation of lipid substrates. This results in a wide range of symptoms such as visceral, bone and in some cases neurological disorders. Furthermore, many patients suffering such diseases have hematologic and vascular symptoms attributed to red blood cell (RBC) rheological abnormalities. These observations led to a hypothesis linking RBC abnormal properties to its lipid composition. However, the lipid profile of normal RBC remains unknown to date. Early diagnosis of these conditions is of importance notably when a therapy is available. This is the case for Gaucher disease (GD) type 1, a lysosomal disorder characterized by β-glucocerebrosidase deficiency, where an enzyme replacement therapy (ERT) is proposed. Hence, the availability of a simple and rapid tool of diagnosis of such a disorder is of great importance, notably for a better patient care and monitoring.To the best of our knowledge, standard diagnosis procedures and monitoring of GD patients are still based on the tedious evaluation of enzyme deficiency. Nevertheless, recent works suggest that these rheological disorders may be due to the accumulation of four sphingolipids, glucosylceramide, glucosylsphingosine, sphingosine and sphingosine-1-phosphate, which could be considered as relevant biomarkers. However, most of current determination methods of these sphingolipids require at least two liquid chromatographic runs, each with a time-consuming sample preparation step that does not facilitate a lipidomic approach. In addition, only glucosylceramide was quantified in RBC while the other three sphingolipids were quantified only in plasma. Thus, these biomarker candidates remain to be validated.In this PhD, we describe a simple and rapid UHPLC-MS/MS method of simultaneous determination of the 4 sphingolipids involved in GD in both plasma and RBC. The application of this method to RBC from GD patients, in collaboration with the Institut National de Transfusion Sanguine and Shire (USA), allowed us: 1- to validate one biomarker among the four proposed candidates and to show that the other three candidates are not specific; 2- to check the efficiency of the proposed ERT and 3- to confirm the initial hypothesis linking the RBC rheological abnormalities to its lipid composition.Also, a systematic study of the operating conditions allowed us to generalize the proposed method to the determination of not only all the sphingolipids present in RBC but also all phospholipids, which are the major constituents of its membrane. The application of the later method to the simultaneous quantification of thirty sphingolipids and phospholipids in normal and GD RBCs, allowed us to validate it and to unravel the involvement of other candidate biomarkers of GD, different from the 4 previous sphingolipids. Providing appropriate modifications, this method is intended to be used for the profiling of all lipid classes in plasma and RBC. This is our main objective in the medium-term.Finally, we evaluated other modern MS techniques such as high resolution (HRMS) and ion mobility (TWIMS and DIMS) in order to refine the investigation of new biomarker candidates, including the separation of lipid isomers that cannot be discriminated by conventional MS techniques. Indeed, in collaboration with the Laboratoire de Chimie Physique (LCP, CNRS UMR 8000), we here show the feasibility of this approach by achieving the separation of two isomers, by the DIMS technique: galactosylsphingosine 18:1 and glucosylsphingosine 18:1, which cannot be separated by conventional methods. We are currently pursuing these investigations in order to separate other isomers.