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Abstract(s)
A doença de Niemann-Pick tipo C (NPC) é uma patologia neurodegenerativa progressiva e transmissão autossómica recessiva. É classificada como uma doença rara apresentando uma prevalência estimada de 1 por cada 100000 nados vivos.
A doença de NPC é caracterizada por uma acumulação de colesterol e vários esfingolípidos no cérebro e nos órgãos viscerais, devido à perda funcional das proteínas NPC1 e/ou NPC2 envolvidas no tráfego de colesterol. Esta acumulação de colesterol constitui o hallmark patognomónico da doença.
A heterogeneidade clínica, associada a sinais e sintomas não específicos e que se sobrepõem com outras patologias, dificultam o diagnóstico, podendo levar a um longo atraso entre a apresentação dos sintomas iniciais e a confirmação laboratorial.
Até muito recentemente, o diagnóstico bioquímico desta patologia era efetuado com recurso à coloração de filipina. Apesar de durante muito tempo ter sido considerado gold standard para o diagnóstico de NPC, este ensaio apresenta limitações: i) implica uma colheita invasiva, pois pressupõe a realização de uma biópsia de pele; ii) morosidade, implica o tempo de cultura da biópsia; iii) tecnicamente exigente; iv) é um método qualitativo e pouco específico. O diagnóstico é posteriormente confirmado pela identificação de variantes patogénicas nos genes NPC1 e NPC2.
Recentemente, e baseado na acumulação de lípidos que ocorre nesta patologia, nomeadamente esteróis, esfingolípidos e lisoesfingolípidos, têm sido desenvolvidas novas abordagens de diagnóstico. Este trabalho teve como objetivo primário a implementação de um teste rápido, quantitativo, sensível e robusto que permitisse o estudo dos biomarcadores Lisoesfingomielina (LysoSM) e do seu análogo carboxilado, Lisoesfingomielina-509 (LysoSM-509), recorrendo ao método de cromatografia líquida acoplada à espectrometria de massa em tandem, LC-MS/MS.
O estudo destes biomarcadores permitiu implementar um método complementar de diagnóstico para os doentes de NPC, além de permitir discriminá-los dos doentes com deficiência em Esfingomielinase Ácida (ASMD). Este método passou a ser utilizado como método de rastreio de primeira linha para estas duas patologias, ultrapassando essencialmente as limitações dos tradicionais testes de diagnóstico, reduzindo o tempo de intervenção do operador, além do custo e tempo de resposta.
Niemann-Pick type C disease (NPC) isa a progressive autosomal recessive neurodegenerative disease. It is classified as a rare disease presenting an estimated prevalence of 1 case per 100000 live births. NPC disease is characterized by an accumulation of cholesterol and several sphingolipids in the brain and visceral organs due to a functional loss of the proteins involved in the cholesterol traffic, NPC1 and/or NPC2 proteins.This cholesterol accumulation constitutes the pathognomonic hall mark. Clinical heterogeneity, associated with nonspecific signs and symptoms that overlap with other pathologies, complicates the diagnostic process, leading to a long delay between the initial symptom presentation and the diagnosis. Until recently, the biochemical diagnosis of this pathology was made using filipin staining. This test was considered gold standard for the diagnosis of NPC for a long time, in spite of his limitations, since it is an invasive method and presupposes the accomplishment of a skin biopsy, in addition to being a tecchnically demanding and time consuming test. The biochemical diagnosis is afterwards confirmed by the identification of pathogenic variants by sequencing the NPC1 and NPC2 genes. Based on the accumulation of lipids that occurs in this pathology, specifically sterols, sphinggolipids and lysosphingolipids, alternative approaches of diagnosis have been developed. This work had as its primary approach the implementation of a rapid, sensitive and robust test that allows the detection of Lysosphingomyelin (LysoSM)and its carboxylated analogue Lysosphingomyelin-509 (LysoSM-509) biomarkers by the analytical technique of liquid chromatography coupled to tandem mass spectrometry, LC-MS/MS. That biomarkers study allowed the discrimination between NPC patients and patients with Acid Sphingomyelinase deficiency (ASMD), and clearly distinguish them from control individuals. This procedure is now used as a first-line creening method for these two pathologies, essentially overcoming the limitations of traditional diagnostic tests, reducing operator intervention time, besides its costs and response time.
Niemann-Pick type C disease (NPC) isa a progressive autosomal recessive neurodegenerative disease. It is classified as a rare disease presenting an estimated prevalence of 1 case per 100000 live births. NPC disease is characterized by an accumulation of cholesterol and several sphingolipids in the brain and visceral organs due to a functional loss of the proteins involved in the cholesterol traffic, NPC1 and/or NPC2 proteins.This cholesterol accumulation constitutes the pathognomonic hall mark. Clinical heterogeneity, associated with nonspecific signs and symptoms that overlap with other pathologies, complicates the diagnostic process, leading to a long delay between the initial symptom presentation and the diagnosis. Until recently, the biochemical diagnosis of this pathology was made using filipin staining. This test was considered gold standard for the diagnosis of NPC for a long time, in spite of his limitations, since it is an invasive method and presupposes the accomplishment of a skin biopsy, in addition to being a tecchnically demanding and time consuming test. The biochemical diagnosis is afterwards confirmed by the identification of pathogenic variants by sequencing the NPC1 and NPC2 genes. Based on the accumulation of lipids that occurs in this pathology, specifically sterols, sphinggolipids and lysosphingolipids, alternative approaches of diagnosis have been developed. This work had as its primary approach the implementation of a rapid, sensitive and robust test that allows the detection of Lysosphingomyelin (LysoSM)and its carboxylated analogue Lysosphingomyelin-509 (LysoSM-509) biomarkers by the analytical technique of liquid chromatography coupled to tandem mass spectrometry, LC-MS/MS. That biomarkers study allowed the discrimination between NPC patients and patients with Acid Sphingomyelinase deficiency (ASMD), and clearly distinguish them from control individuals. This procedure is now used as a first-line creening method for these two pathologies, essentially overcoming the limitations of traditional diagnostic tests, reducing operator intervention time, besides its costs and response time.
Description
Keywords
Niemann-Pick tipo C Spectrometria de massa Lisoesfingomielina Deficiência em esfingomielase ácida 509