Made available in DSpace on 2014-08-26T17:31:46Z (GMT). No. of bitstreams: 2 license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) 69585.pdf: 1181357 bytes, checksum: 50462d1ba789c7b199ee8460ca90f3b8 (MD5) / Fundação Oswaldo Cruz. Instituto Fernandes Figueira. Departamento de Ensino. Programa de Pós-Graduação em Saúde da Criança e da Mulher. Rio de Janeiro, RJ, Brasil / A Doença de Gaucher (DG) é uma Doença de Depósito Lisossômico (DDL) e seu tratamento baseia - se na terapia de reposição enzimática. Tal terapia foi um marco na vida de pacientes e especialistas, pois mudou a história da evolução da doença, caracterizando um a nova era na Genética Médica. Este trabalho tem como objeto de pesquisa as perspectivas trazidas por profissionais, com experiência em trata r a Doença de Gaucher no Sistema Único de Saúde no estado do Rio de Janeiro. Uma vez que a DG é a única condição d o grupo das DDL a ser contemplada por uma Política Ministerial, promovendo acesso a drogas de alto custo através de um Protocolo Clínico e Diretrizes Terapêuticas (PCDT). O o bjetivo geral foi a nalisar a prática da aplicação do protocolo oficial de tratame nto da DG e o seu entendimento a partir da ótica dos médicos tratadores, profissionais de saúde e gestores do Centro de Referência , o Instituto Estadual de Hematologia Arthur de Siqueira Cavalcanti (HEMORIO). Os objetivos específicos foram primeiramente id entificar a formaçã o profissional dos envolvidos no programa, analisar a ótica desses profissionais sob re as recomendações do PCDT e como estes situam o Centro de Referência (CR) e m seu atual funcionamento , e d iscutir de maneira crítica a visão dos profiss i onais a respeito dos benefícios e de possíveis falhas do programa. Foram realizadas entrevistas temáticas semiestruturadas e a elas aplicou - se a análise de conteúdo. No que tange ao entendimento sobre o PCDT - DG e o seu viii funcionamento, os resultados apontam a importância da existência de um balizador, um programa robusto governamental, revisado por especialistas bem capacitados no tema. O PCDT - DG foi um avanço na saúde, oficializando e garantindo o acesso à medicação de maneira embasada, controlada por câmar as técnicas estaduais, permitindo a efetuação de pregões públicos, uma maneira transparente de aquisiçã o de drogas de alto custo comparada a medidas judiciais . Os sujeitos da pesquisa são favoráveis ao programa, no entanto possuem uma abordagem crítica ao sistema de saúde no que diz respeit o a entraves na rede de assistência cirúrgica e de reabilitação. Um grande gargalo atualmente no SUS não é exclusivo ao programa da DG: certos questionamentos éticos na fomentação do diagnóstico laboratorial por parte da indústria farmacêutica, apesar de haver relações amigáveis entre esses dois atores no CR. Concluímos que muitos avanços foram conquistados a partir da implementação do protocolo e que talvez este possa servir como modelo para garantir acesso ao tratamento de outras DDL. Algumas incongruências do siste ma são questionáveis e discutida s entre gestores, médicos e usuários, entretanto ainda são muito poucos os estudos publicados no Brasil sobre o tema . / Gaucher disease (GD) is a Lysosomal Storage Disease (LSD) and its treatment is based on enzyme replacement therapy. Such therapy was a milestone in patients `s lives and experts in the field, changing the disease natural history . This work aims at present ing the treatment of GD in the Unified Health System i n the state of Rio de Janeiro, as it is the only LSD to be covered by a Ministerial policy , which promotes access to high cost drugs through a Clinical Gui deline (CG) . The overall objective was to analyze the practical application of the CG protocol in the treatment of GD, and how this guideline was interpreted and used by the medical c haracters , health professionals and ma nagers of the Reference Center and the State Institute of Hematology Arthur de Siquei ra Cavalcanti (HEMORIO ). The specific objectives were to identify the training of those involved in the program, to analyze how professionals viewed the recommendations included in the CP, what they thought about the Reference Center for GD and to critical ly discuss the benefits and possi ble shortcomings of the program . Thematic semi - structured interviews were conducted, and the content analysis was applied. Regarding the understanding of the CP - GD and its op eration, the results point the importance of t he existence of a robust government program, reviewed by well - trained experts in the subject. The CP - GD was a health`s breakthrough , ensuring access to medication, controlled by state technical chambers, a llowing the practice of public auctions, a transpar ent way of purchasing high - cost drugs when compared to individual litigation. The steakeholder`s research were x favo rable to the program, although they criticized the health network constraints for specialized care, such as surgical services and rehabilitat ion. Another major bottleneck in the health system, not exclusive for G D is ethical issues regarding laboratory diagnosis by the pharmaceutical industry. We conclude d that many advances have been achieved from the implementation of the CP, and that hopeful ly this can serve as a model to ensure access t o treatment for other LSD. Managers, physicians and users point out some inconsistencies in the system although there is still limited published data on this subject in Brazil.

The inherited form of primary open angle glaucoma, a disorder characterized by increased intraocular pressure and retina degeneration, is linked to mutations in the olfactomedin (OLF) domain of the myocilin gene. Disease-causing myocilin variants accumulate within trabecular meshwork cells instead of being secreted to the trabecular extracellular matrix thought to regulate aqueous humor flow and control intraocular pressure. Like other diseases of protein misfolding, we hypothesize myocilin toxicity originates from defects in protein biophysical properties. In this thesis, the first preparative recombinant high-yield expression and purification system for the C-terminal OLF domain of myocilin (myoc-OLF) is described. To determine the relative stability of wild-type (WT) and mutant OLF domains, a fluorescence thermal stability assay was adapted to provide the first direct evidence that mutated OLF is folded but less thermally stable than WT. In addition, mutant myocilin can be stabilized by chemical chaperones. Together, this work provides the first quantitative demonstration of compromised stability among identified OLF variants and placing myocilin glaucoma in the context of other complex diseases of protein misfolding. Subsequent investigations into the biophysical properties of WT myoc-OLF provide insight into its structure and function. In particular, myoc-OLF is stable in the presence of glycosaminoglycans (GAGs), as well as over a wide pH range in buffers with functional groups reminiscent of such GAGs. Myoc-OLF contains significant â-sheet and â-turn secondary structure as revealed by circular dichroism analysis. At neutral pH, thermal melts indicate a highly cooperative transition with a melting temperature of ~55°C. A compact core structural domain of OLF was identified by limited proteolysis and consists of approximately residues 238-461, which retains the single disulfide bond and is as stable as the full myoc-OLF construct. This construct also is capable of generating 3D crystals for structure determination. This data, presented in Chapter 3, inform new testable hypotheses for interactions with specific trabecular extracellular matrix components. To gain further insight into the biological function of myoc-OLF, a facile fluorescence chemical stability assay was designed to identify possible ligands and drug candidates. In the assay described in Chapter 4, the target protein is initially destabilized with a chemical denaturant and is tested for re-stabilization upon the addition of small molecules. The assay requires no prior knowledge of the structure and/or function of the target protein, and it is amendable to high-throughput screening. Application of the assay using a library of 1,280 compounds revealed 14 possible ligands and drug candidates for myoc-OLF that may also generate insights into myoc-OLF function. Due to the high â-sheet content of monomeric myoc-OLF and presence of an aggregated species upon myoc-OLF purification, the ability of myoc-OLF to form amyloid fibrils was suspected and verified. The fibril forming region was confirmed to reside in the OLF domain of myocilin. Kinetic analyses of fibril formation reveal a self-propagating process common to amyloid. The presence of an aggregated species was confirmed in cells transfected with WT myocilin, but to a greater extent in cells transfected with P370L mutant myocilin. Both cell lines stained positive for amyloid. Taken together, these results provide further insights into the structure of myocilin and suggest a new hypothesis for glaucoma pathogenesis. Finally, in a related study, small molecule drug candidates were investigated to treat acid â-glucosidase (GCase), the deficient lysosomal enzyme in Gaucher disease, another protein conformational disorder. Three new GCase active-site directed 3,4,5,6-tetrahydroxylazepane inhibitors were synthesized that exhibit half inhibitory concentrations (IC50) in the low millimolar to low micromolar range. Although the compounds thermally stabilize GCase at pH 7.4, only one of the synthesized analogs exhibits chaperoning activity under typical assay conditions. This successful pharmacological chaperone is also one in which GCase is in its proposed active conformation as revealed by X-ray crystallography. Probing the plasticity of the active-site of GCase offers additional insight into possible molecular determinants for an effective small molecule therapy for GD.

Aquest treball és una contribució als estudis de diversitat del genoma humà i pretén estudiar la variació genètica existent a nivell mundial en dos gens, causants de malaltia, el gen CFTR i el gen GBA, en cromosomes d'individus sans. Mutacions en aquests gens produeixen la fibrosi quística i la malaltia de Gaucher respectivament. La fibrosi quística és la malaltia autosòmica recessiva més comuna en poblacions europees. La malaltia de Gaucher és la malaltia lisosòmica d'acumulació lipídica més freqüent. L'estudi analitza la variació genètica en diferents polimorfismes d'ambdós gens; reconstrueix els haplotips i analitza la seva distribució geogràfica; i analitza l'extensió i distribució geogràfica del desequilibri de lligament entre loci. Pel gen GBA, hem ampliat la regió, abastant fins al gen PKLR (que codifica per a la piruvat quinasa). A més a més, pel cas de CFTR, pot ajudar a entendre l'origen de les mutacions més freqüents causants de fibrosi quística. / This work is a contribution to human genome diversity studies and it aims to study the world-wide genetic variation that exists in two disease genes, CFTR and GBA gene, in healthy chromosomes. Mutations in these genes are known to cause cystic fibrosis and Gaucher disease respectively. Cystic fibrosis is the most common severe autosomal recessive disease in patients of European descent. Gaucher disease is the most frequent lysosomal storage disorder. The study analyzes the genetic variation in CFTR and GBA polymorphisms; estimates haplotype frequencies and describes their geographic distribution; and measures linkage disequilibrium between loci. For GBA gene, we have extended the analysis covering PKLR gene (that encodes for a pyruvate kinase). Moreover, for CFTR gene, we have tried to understand the origin of the most common cystic fibrosis causing mutations.

Ceramid, ein zentrales Signalmolekül des Sphingolipidstoffwechsels, ist neben der de novo Synthese über die enzymatische Spaltung von Sphingomyelin und Glucosylceramid zugänglich. Genetische Mutationen, die eine Fehlfaltung der verantwortlichen Enzyme saure Sphingomyelinase (aSMase) und Glucocerebrosidase (GCase) begünstigen, könnten somit zu einer Dysregulation des gesamten Sphingolipidstoffwechsels und den damit verbundenen Signaltransduktionsprozessen führen. Niedermolekulare Inhibitoren können in Zellstudien einen Einblick in diese Prozesse geben und den Defekt eines Enzyms simulieren oder eine etwaige Überaktivität derselben Enzyme verhindern. Für derartige Studien ist die Möglichkeit einer zeitaufgelösten Inhibition von Vorteil. Für diese Methode müssten photolabile Schutzgruppen in eine bereits bekannte Inhibitorstruktur integriert werden. Im Fall der aSMase würden sich hierfür myo-Inosit-bisphosphat-Derivate anbieten, die starke, kompetitive Inhibitoren des Enzyms darstellen. Auf dieser Grundlage werden in der vorliegenden Arbeit die Synthese sowie die in vitro und in cellulo Wirkung des ersten zellpermeablen, photoaktivierbaren Inhibitors für die aSMase präsentiert. Kompetitive Inhibitoren können ebenso als sogenannte pharmakologische Chaperone fungieren, welche Proteine durch Herabsetzung der freien Energie des jeweiligen Faltungszustandes stabilisieren. Dies ist besonders bei von Mutationen betroffenen lysosomalen Enzymen von Interesse, um diese vor einem proteasomalen Abbau zu bewahren und einen geregelten Transport in die Lysosomen zu gewährleisten. So wurden in der vorliegenden Arbeit verschiedene myo-Inositderivate als potenzielle pharmakologische Chaperone für die aSMase und GCase synthetisiert. Um eine Verdrängung der Verbindungen vom aktiven Zentrum des Enzyms durch das natürliche Substrat zu beschleunigen, wurde eine Orthoesterfunktion in die Seitenkette der Inhibitorstruktur integriert, die im sauren Milieu der Lysosomen gespalten werden kann. / Ceramide, a central signaling molecule in sphingolipid metabolism, is in addition to the novo synthesis accessible via the enzymatic cleavage of sphingomyelin and glucosylceramide. Genetic mutations that promote misfolding of the responsible enzymes acid sphingomyelinase (aSMase) and glucocerebrosidase (GCase) could thus lead to a dysregulation of the entire sphingolipid metabolism and the associated signal transduction processes. Small molecule inhibitors can provide insight into these processes in cell studies and simulate the defect of an enzyme or prevent eventual overactivity of the same enzyme. For such studies, the possibility of a time-resolved inhibition would be advantageous. For this method, photolabile protecting groups would have to be integrated into the structure of a known inhibitor. In the case of aSMase, myo-inositol-diphosphate derivatives, which represent strong, competitive inhibitors of the enzyme, would be suitable for this purpose. On this basis, the synthesis as well as the in vitro and in cellulo effects of the first cell-permeable photocaged inhibitor for acid sphingomyelinase are presented in this work. Competitive inhibitors can also act as so-called pharmacological chaperones, which stabilize proteins by reducing the free energy of the respective folding state. This is of particular interest in the case of lysosomal enzymes affected by mutations, in order to protect them from proteasomal degradation and to ensure regulated transport into the lysosomes. In the present work, various myo-inositol derivatives were synthesized as potential pharmacological chaperones for aSMase and GCase. To accelerate displacement of the compounds from the enzyme's active site by the natural substrate, an orthoester function was integrated into the side chain of the inhibitor structure, which can be cleaved in the acidic environment of the lysosome.

Introdução: As Doenças lisossômicas (DLs) são condições genéticas, herdadas na sua maioria de forma autossômica recessiva, caracterizadas usualmente pela deficiência de enzimas lisossômicas específicas, envolvidas na síntese, degradação, armazenamento ou transporte de macromoléculas necessárias para o funcionamento normal do organismo. Nas situações mais típicas, o substrato não degradado acumula-se progressivamente nos lisossomos, com repercussões estruturais e funcionais, levando a sinais e sintomas característicos. Os pacientes apresentam um amplo espectro de manifestações clínicas, que podem incluir disfunção de órgãos, anormalidades esqueléticas, envolvimento neuronal, entre outras. O diagnóstico é usualmente obtido pela identificação da deficiência enzimática específica em leucócitos obtidos do sangue periférico, usualmente realizado em laboratórios de referência. O transporte da amostra pode ser um obstáculo quando o serviço requisitante está situado longe do centro de referência ou em outro país, situação em que a amostra de sangue pode chegar ao laboratório já sem condições de ser analisada. Objetivo: Este estudo teve como objetivo principal, tornar disponível um método mais simples, seguro e acessível que utiliza amostras de leucócitos impregnados em papel filtro (LIPF) como uma nova ferramenta para o diagnóstico bioquímico de pacientes com DLs. Métodos: O estudo envolveu amostras de pacientes com diagnóstico previamente confirmado de DLs (amostra de conveniência, por se tratarem de doenças raras, com incidências individuais ao redor de 1:100.000 recém-nascidos vivos). Foram incluídos no estudo os pacientes com diagnóstico já estabelecido de DLs selecionadas (MPS IVA, Doença de Krabbe, Doença de Gaucher e Doença de Pompe), independente do sexo e/ou idade, atendidos no Serviço de Genética Médica do HCPA, que concordaram em participar do estudo. O grupo de referência negativo foi constituído pelas amostras de 50 indivíduos hígidos, adultos, de ambos os sexos. Resultados: Os resultados obtidos nos ensaios enzimáticos de pacientes com MPS IVA, Doença de Krabbe, Doença de Gaucher e Doença de Pompe, indicaram que as enzimas analisadas em amostras de LIPF permitiram a identificação de todos os pacientes, com sensibilidade de 100%. Os testes de estabilidade realizados nas amostras de LIPF indicaram que as amostras, quando mantidas a 4ºC, se mostram estáveis por pelo menos 30 dias. Conclusões: Nas condições utilizadas, amostras de LIPF se mostraram adequadas para a identificação segura de pacientes com MPS tipo IVA, Doença de Krabbe, Doença de Gaucher e Doença de Pompe. As amostras de leucócitos secos em papel filtro são mais estáveis e seguras para o transporte, indicando que possa ser esta uma importante ferramenta para facilitar a identificação de pacientes com DLDs, especialmente daqueles que vivem em áreas que tem dificuldades para a remessa de amostras líquidas para serviços de referência. / Background: Lysosomal Disorders (LDs) are genetic conditions, mostly inherited in autosomal recessive fashion, usually characterized by a deficiency of specific lysosomal enzymes involved in the synthesis, degradation, storage or transportation of macromolecules necessary for normal functioning of the organism. Typically, the non-degraded substrate is progressively accumulated in lysosomes, with structural and functional repercussions, leading to characteristic signs and symptoms. Affected patients present a wide range of clinical manifestations, which may include organ dysfunction, skeletal anomalies, neuronal involvement, etc. The diagnosis is normally made through identification of the specific enzyme deficiency in white blood cells from a sample of peripheral blood, usually performed in reference laboratories. The transporting of a liquid sample can be a problem when the test orderer is located far from the reference center or in a foreign country, as often the blood sample arrives at the laboratory in poor condition and cannot be properly analyzed. Aim: The main aim of this study was to make available a new technique that is simpler, safer and more accessible, using leukocytes impregnated on filter paper (LIFP) as a new tool for the biochemical diagnosis of patients with LSDs. Methods: This study involved samples of patients with previously confirmed diagnosis of selected LSDs (a convenience sample, as these are rare diseases, with individual incidences around 1:100.000 live newborns). Patients with an established diagnosis of MPS IVA, Krabbe Disease, Gaucher’s disease and Pompe disease regardless of sex and/or age, cared for at the Genetics Service of HCPA and who agreed to participate were included in the study. The negative reference group comprised blood samples from 50 healthy adults of both genders. Results: The results obtained in the enzymatic assays of patients with MPS IVA, Krabbe Disease, Gaucher’s disease, and Pompe disease indicated that the analyzed enzymes in LIFP samples allowed the identification of all patients, with sensitivity of 100%. The stability tests performed in LIFP samples indicated that samples, when maintained at 4ºC, were stable for at least 30 days. Conclusions: In the conditions used, LIFP samples were shown to be adequate for a reliable identification of patients with MPS IVA, Krabbe Disease, Gaucher’s disease, and Pompe disease. Blood samples on filter paper are more stable and reliable for transportation, indicating that this may be an important tool to facilitate the identification of patients with LSDs, particularly those living in areas with difficulties for the shipment of liquid samples to reference cervices.

Metachromatic leukodystrophy (MLD) and Gaucher disease (GD) are caused by a deficiency of arylsulphatase A (ASA) and b-glucocerebrosidase (GBA), respectively. They are lysosomal storage disorders with a heterogeneous clinical spectrum encompassing visceral, skeletal and neurologic involvement resulting in high morbidity and mortality. The overall aim of this study is to elucidate the genetic component/s of high ASA and GBA enzyme activity in normal healthy individuals with the ultimate goal of using this information to produce greater protein activity from a recombinant protein. A wide variation in ASA and GBA enzyme activity levels has been observed in the normal population. The first objective of this project was to identify and characterise single nucleotide polymorphisms (SNPs) in the arylsulphatase A (ARSA) and glucocerebrosidase (GBA) genes that are responsible for determining the levels of expressed enzyme activity in the normal population. The second objective was to assess the contribution of transcriptional regulation and TCP80 mediated translational control to normal enzyme variation. TCP80, a translational control protein that interacts with the GBA coding region, is a splice variant of the interleukin binding factor 3 (ILF3) gene. Ten samples from individuals with high ASA activity and twenty samples from individuals with high GBA activity were screened for polymorphisms via denaturing high pressure liquid chromatography (dHPLC) and sequencing. The frequency of these polymorphisms in the normal population was determined using dot-blot hybridisation. Fifteen ARSA polymorphisms (4 promoter, 5 coding, 5 intronic and 1 poly(A) signal) and two GBA polymorphisms (1 intronic and 1 in 3¢-UTR) were identified. Two low frequency ASA polymorphisms (2723A > G, W193C) were found to be correlated with low activity, while another low frequency ASA polymorphism (1101+123C > T) was found to be correlated with high activity in a population of 113 individuals. Real time PCR was used to measure mRNA levels of GBA, ASA and LF3 along with enzyme activity levels of GBA and ASA in two cell types (leucocytes and skin fibroblasts) from four healthy individuals and seven cell lines (HL60, THP1, Huh7, U118, SW1353, Hep G2, and B-cells). Transcriptional control was evident for all three genes with GBA mRNA levels varying over 30 fold, ASA mRNA levels varying over seven fold and ILF3 levels varying more than 24 fold. The 5¢-flanking region of GBA was investigated for the cis-elements responsible for tissue-specific expression. However, it was not possible to demonstrate that the cis-element region was influencing GBA expression. Translational efficiency was measured using the magnitude of the mRNA:enzyme activity ratio as an indicator. GBA translational inefficiency was most pronounced in B cells which require four times more mRNA molecules than hepatocytes (Hep G2) and over 25 times more mRNA molecules than chondrocytes (SW1353) to produce one unit of GBA enzyme activity. Except in B-cells, GBA translational efficiency appears to increase as ILF3 mRNA levels decrease. The tissue-specific variation observed in the protein levels of the ILF3 splice variants, TCP80 and DRBP76, may play a role. The correlation of several low frequency SNPs with low ASA enzyme activity or high ASA activity indicates a role in determining the distribution of enzyme activity levels in the normal population. However, there do not appear to be any common high activity polymorphisms. Knowledge of the exact mechanisms responsible for the observed transcriptional and translational control of these lysosomal genes will greatly enhance the understanding of genotype-phenotype correlation and the contribution of genetic variants to natural variation.

Introdução: As Doenças lisossômicas (DLs) são condições genéticas, herdadas na sua maioria de forma autossômica recessiva, caracterizadas usualmente pela deficiência de enzimas lisossômicas específicas, envolvidas na síntese, degradação, armazenamento ou transporte de macromoléculas necessárias para o funcionamento normal do organismo. Nas situações mais típicas, o substrato não degradado acumula-se progressivamente nos lisossomos, com repercussões estruturais e funcionais, levando a sinais e sintomas característicos. Os pacientes apresentam um amplo espectro de manifestações clínicas, que podem incluir disfunção de órgãos, anormalidades esqueléticas, envolvimento neuronal, entre outras. O diagnóstico é usualmente obtido pela identificação da deficiência enzimática específica em leucócitos obtidos do sangue periférico, usualmente realizado em laboratórios de referência. O transporte da amostra pode ser um obstáculo quando o serviço requisitante está situado longe do centro de referência ou em outro país, situação em que a amostra de sangue pode chegar ao laboratório já sem condições de ser analisada. Objetivo: Este estudo teve como objetivo principal, tornar disponível um método mais simples, seguro e acessível que utiliza amostras de leucócitos impregnados em papel filtro (LIPF) como uma nova ferramenta para o diagnóstico bioquímico de pacientes com DLs. Métodos: O estudo envolveu amostras de pacientes com diagnóstico previamente confirmado de DLs (amostra de conveniência, por se tratarem de doenças raras, com incidências individuais ao redor de 1:100.000 recém-nascidos vivos). Foram incluídos no estudo os pacientes com diagnóstico já estabelecido de DLs selecionadas (MPS IVA, Doença de Krabbe, Doença de Gaucher e Doença de Pompe), independente do sexo e/ou idade, atendidos no Serviço de Genética Médica do HCPA, que concordaram em participar do estudo. O grupo de referência negativo foi constituído pelas amostras de 50 indivíduos hígidos, adultos, de ambos os sexos. Resultados: Os resultados obtidos nos ensaios enzimáticos de pacientes com MPS IVA, Doença de Krabbe, Doença de Gaucher e Doença de Pompe, indicaram que as enzimas analisadas em amostras de LIPF permitiram a identificação de todos os pacientes, com sensibilidade de 100%. Os testes de estabilidade realizados nas amostras de LIPF indicaram que as amostras, quando mantidas a 4ºC, se mostram estáveis por pelo menos 30 dias. Conclusões: Nas condições utilizadas, amostras de LIPF se mostraram adequadas para a identificação segura de pacientes com MPS tipo IVA, Doença de Krabbe, Doença de Gaucher e Doença de Pompe. As amostras de leucócitos secos em papel filtro são mais estáveis e seguras para o transporte, indicando que possa ser esta uma importante ferramenta para facilitar a identificação de pacientes com DLDs, especialmente daqueles que vivem em áreas que tem dificuldades para a remessa de amostras líquidas para serviços de referência. / Background: Lysosomal Disorders (LDs) are genetic conditions, mostly inherited in autosomal recessive fashion, usually characterized by a deficiency of specific lysosomal enzymes involved in the synthesis, degradation, storage or transportation of macromolecules necessary for normal functioning of the organism. Typically, the non-degraded substrate is progressively accumulated in lysosomes, with structural and functional repercussions, leading to characteristic signs and symptoms. Affected patients present a wide range of clinical manifestations, which may include organ dysfunction, skeletal anomalies, neuronal involvement, etc. The diagnosis is normally made through identification of the specific enzyme deficiency in white blood cells from a sample of peripheral blood, usually performed in reference laboratories. The transporting of a liquid sample can be a problem when the test orderer is located far from the reference center or in a foreign country, as often the blood sample arrives at the laboratory in poor condition and cannot be properly analyzed. Aim: The main aim of this study was to make available a new technique that is simpler, safer and more accessible, using leukocytes impregnated on filter paper (LIFP) as a new tool for the biochemical diagnosis of patients with LSDs. Methods: This study involved samples of patients with previously confirmed diagnosis of selected LSDs (a convenience sample, as these are rare diseases, with individual incidences around 1:100.000 live newborns). Patients with an established diagnosis of MPS IVA, Krabbe Disease, Gaucher’s disease and Pompe disease regardless of sex and/or age, cared for at the Genetics Service of HCPA and who agreed to participate were included in the study. The negative reference group comprised blood samples from 50 healthy adults of both genders. Results: The results obtained in the enzymatic assays of patients with MPS IVA, Krabbe Disease, Gaucher’s disease, and Pompe disease indicated that the analyzed enzymes in LIFP samples allowed the identification of all patients, with sensitivity of 100%. The stability tests performed in LIFP samples indicated that samples, when maintained at 4ºC, were stable for at least 30 days. Conclusions: In the conditions used, LIFP samples were shown to be adequate for a reliable identification of patients with MPS IVA, Krabbe Disease, Gaucher’s disease, and Pompe disease. Blood samples on filter paper are more stable and reliable for transportation, indicating that this may be an important tool to facilitate the identification of patients with LSDs, particularly those living in areas with difficulties for the shipment of liquid samples to reference cervices.

Introdução: As Doenças lisossômicas (DLs) são condições genéticas, herdadas na sua maioria de forma autossômica recessiva, caracterizadas usualmente pela deficiência de enzimas lisossômicas específicas, envolvidas na síntese, degradação, armazenamento ou transporte de macromoléculas necessárias para o funcionamento normal do organismo. Nas situações mais típicas, o substrato não degradado acumula-se progressivamente nos lisossomos, com repercussões estruturais e funcionais, levando a sinais e sintomas característicos. Os pacientes apresentam um amplo espectro de manifestações clínicas, que podem incluir disfunção de órgãos, anormalidades esqueléticas, envolvimento neuronal, entre outras. O diagnóstico é usualmente obtido pela identificação da deficiência enzimática específica em leucócitos obtidos do sangue periférico, usualmente realizado em laboratórios de referência. O transporte da amostra pode ser um obstáculo quando o serviço requisitante está situado longe do centro de referência ou em outro país, situação em que a amostra de sangue pode chegar ao laboratório já sem condições de ser analisada. Objetivo: Este estudo teve como objetivo principal, tornar disponível um método mais simples, seguro e acessível que utiliza amostras de leucócitos impregnados em papel filtro (LIPF) como uma nova ferramenta para o diagnóstico bioquímico de pacientes com DLs. Métodos: O estudo envolveu amostras de pacientes com diagnóstico previamente confirmado de DLs (amostra de conveniência, por se tratarem de doenças raras, com incidências individuais ao redor de 1:100.000 recém-nascidos vivos). Foram incluídos no estudo os pacientes com diagnóstico já estabelecido de DLs selecionadas (MPS IVA, Doença de Krabbe, Doença de Gaucher e Doença de Pompe), independente do sexo e/ou idade, atendidos no Serviço de Genética Médica do HCPA, que concordaram em participar do estudo. O grupo de referência negativo foi constituído pelas amostras de 50 indivíduos hígidos, adultos, de ambos os sexos. Resultados: Os resultados obtidos nos ensaios enzimáticos de pacientes com MPS IVA, Doença de Krabbe, Doença de Gaucher e Doença de Pompe, indicaram que as enzimas analisadas em amostras de LIPF permitiram a identificação de todos os pacientes, com sensibilidade de 100%. Os testes de estabilidade realizados nas amostras de LIPF indicaram que as amostras, quando mantidas a 4ºC, se mostram estáveis por pelo menos 30 dias. Conclusões: Nas condições utilizadas, amostras de LIPF se mostraram adequadas para a identificação segura de pacientes com MPS tipo IVA, Doença de Krabbe, Doença de Gaucher e Doença de Pompe. As amostras de leucócitos secos em papel filtro são mais estáveis e seguras para o transporte, indicando que possa ser esta uma importante ferramenta para facilitar a identificação de pacientes com DLDs, especialmente daqueles que vivem em áreas que tem dificuldades para a remessa de amostras líquidas para serviços de referência. / Background: Lysosomal Disorders (LDs) are genetic conditions, mostly inherited in autosomal recessive fashion, usually characterized by a deficiency of specific lysosomal enzymes involved in the synthesis, degradation, storage or transportation of macromolecules necessary for normal functioning of the organism. Typically, the non-degraded substrate is progressively accumulated in lysosomes, with structural and functional repercussions, leading to characteristic signs and symptoms. Affected patients present a wide range of clinical manifestations, which may include organ dysfunction, skeletal anomalies, neuronal involvement, etc. The diagnosis is normally made through identification of the specific enzyme deficiency in white blood cells from a sample of peripheral blood, usually performed in reference laboratories. The transporting of a liquid sample can be a problem when the test orderer is located far from the reference center or in a foreign country, as often the blood sample arrives at the laboratory in poor condition and cannot be properly analyzed. Aim: The main aim of this study was to make available a new technique that is simpler, safer and more accessible, using leukocytes impregnated on filter paper (LIFP) as a new tool for the biochemical diagnosis of patients with LSDs. Methods: This study involved samples of patients with previously confirmed diagnosis of selected LSDs (a convenience sample, as these are rare diseases, with individual incidences around 1:100.000 live newborns). Patients with an established diagnosis of MPS IVA, Krabbe Disease, Gaucher’s disease and Pompe disease regardless of sex and/or age, cared for at the Genetics Service of HCPA and who agreed to participate were included in the study. The negative reference group comprised blood samples from 50 healthy adults of both genders. Results: The results obtained in the enzymatic assays of patients with MPS IVA, Krabbe Disease, Gaucher’s disease, and Pompe disease indicated that the analyzed enzymes in LIFP samples allowed the identification of all patients, with sensitivity of 100%. The stability tests performed in LIFP samples indicated that samples, when maintained at 4ºC, were stable for at least 30 days. Conclusions: In the conditions used, LIFP samples were shown to be adequate for a reliable identification of patients with MPS IVA, Krabbe Disease, Gaucher’s disease, and Pompe disease. Blood samples on filter paper are more stable and reliable for transportation, indicating that this may be an important tool to facilitate the identification of patients with LSDs, particularly those living in areas with difficulties for the shipment of liquid samples to reference cervices.

Pathogenesis of mucopolysaccharidosis type IIIC (MPS IIIC) and Gaucher disease has not been yet fully clarified, and the causes of phenotypical variability between the patients with the same genotype in Gaucher disease remain obscure. Because the variants in the regulatory regions of genes can cause phenotypical differences mentioned above, I have studied promoter regions of HGSNAT and GBA genes mutated in these lysosomal disorders. I have shown that there is an alternative promoter of GBA (P2). Additional studies were aimed to elucidate possible physiological functions of P2, and its possible role in the pathogenesis of Gaucher disease. I have found that P2 is not tissue specific, and that its variants do not influence the variability of phenotype in Gaucher patients with the same genotype. P2 is used differentially neither during the differentiation of monocytes to macrophages nor in macrophages from controls and Gaucher patients, in whom there is a prominent storage only in cells of macrophage origin. We have thus not found any changes that would suggest a role for P2 in the pathogenesis of Gaucher disease. I have characterized the promoter region of HGSNAT and shown that the binding of Sp1 transcription factor is important for its expression. Sequence variants found in HGSNAT promoter in...

5 Abstract Definitive diagnosis of inherited metabolic disorders commonly depends on the measurement of enzyme activity (which is often complicated) and/or molecular genetic testing. Yet even the standard mutation analysis can bring false negative results in the case of gross chromosomal rearrangements or incorrect regulation of gene expression due to the mutations in regulatory regions. In the present study I focused on characterization of complex mutations affecting the gene encoding ornithin transcarbamylase (OTC) followed by studies of regulatory regions of OTC and GBA (the gene encoding β-glucocerebrosidase). In the first study we identified 14 novel mutations including three large deletions in a cohort of 37 patients with OTC deficiency (OTCD). Subsequently we evaluated clinical significance of all these mutations. We also found a heterozygote carrying a hypomorphic mutation and manifesting OTCD most likely due to unfavorable X-inactivation which was observed independently in three different peripheral tissues. In order to evaluate the clinical significance of a promoter variation c.-366A>G found in a family with mild OTCD we identified three alternative transcription start sites (TSSs) of human OTC and delimited the promoter. We also found a distal enhancer and performed functional analysis of both...