Molecular and biochemical biomarkers for diagnosis and therapy monitorization of Niemann-Pick type C patients.

BACKGROUND: Niemann-Pick type C (NP-C), one of 50 inherited lysosomal storage disorders, is caused by NPC protein impairment that leads to unesterified cholesterol accumulation in late endosomal/lysosomal compartments. The clinical manifestations of NP-C include hepatosplenomegaly, neurological and psychiatric symptoms. Current diagnosis for NP-C is based on observation of the accumulated cholesterol in fibroblasts of affected individuals, using an invasive and time expensive test, called Filipin staining. Lately, two metabolites that are markedly increased in NP-C patients are arising as biomarkers for this disease screening: 7-ketocholesterol and cholestane-3ß,5α,6ß-triol, both oxidized cholesterol products. OBJECTIVE: In this work, we aimed to evaluate the performance of cholestane-3ß,5α,6ß-triol analysis for the screening and monitoring of NPC patients, correlating it with chitotriosidase levels, Filipin staining and molecular analysis. It was investigated 76 non-treated individuals with NP-C suspicion and also 7 patients with previous NP-C diagnosis under treatment with miglustat, in order to verify the cholestane-3ß,5α,6ß-triol value as a tool for therapy monitoring. RESULTS: Considering molecular assay as golden standard, it was verified that cholestane-3ß,5α,6ß-triol analysis presented 88% of sensitivity, 96.08% of specificity, a positive and negative predictive value calculated in 91.67% and 94.23%, respectively, for the diagnosis of NP-C. Chitotriosidase levels were increased in patients with positive molecular analysis for NP-C. For Filipin staining, it was found 1 false positive, 7 false negative and 24 inconclusive cases, showing that this assay has important limitations for NP-C diagnosis. Besides, we found a significant decrease in cholestane-3ß,5α,6ß-triol concentrations in NP-C patients under therapy with miglustat when compared to non-treated patients. CONCLUSION: Taken together, the present data show that cholestane-3ß,5α,6ß-triol analysis has a high potential to be an important NP-C screening assay, and also can be used for therapy monitorization with miglustat in NP-C patients.

Niemann-Pick disease type C: a case series of Brazilian patients.

UNLABELLED: The aim of the study was to analyze a series of Brazilian patients with Niemann-Pick disease type C (NP-C). METHOD: Correlations between clinical findings, laboratory data, molecular findings and treatment response are presented. RESULT: The sample consisted of 5 patients aged 8 to 26 years. Vertical supranuclear gaze palsy, cerebellar ataxia, dementia, dystonia and dysarthria were present in all cases. Filipin staining showed the "classical" pattern in two patients and a "variant" pattern in three patients. Molecular analysis found mutations in the NPC1 gene in all alleles. Miglustat treatment was administered to 4 patients. CONCLUSION: Although filipin staining should be used to confirm the diagnosis, bone marrow sea-blue histiocytes often help to diagnosis of NP-C. The p.P1007A mutation seems to be correlated with the "variant" pattern in filipin staining. Miglustat treatment response seems to be correlated with the age at disease onset and disability scale score at diagnosis.

Niemann-Pick disease type C: a case series of Brazilian patients / Doenca de Niemann-Pick tipo C: serie de casos de pacientes brasileiros

The aim of the study was to analyze a series of Brazilian patients with Niemann-Pick disease type C (NP-C). Method Correlations between clinical findings, laboratory data, molecular findings and treatment response are presented. Result The sample consisted of 5 patients aged 8 to 26 years. Vertical supranuclear gaze palsy, cerebellar ataxia, dementia, dystonia and dysarthria were present in all cases. Filipin staining showed the “classical” pattern in two patients and a “variant” pattern in three patients. Molecular analysis found mutations in the NPC1 gene in all alleles. Miglustat treatment was administered to 4 patients. Conclusion Although filipin staining should be used to confirm the diagnosis, bone marrow sea-blue histiocytes often help to diagnosis of NP-C. The p.P1007A mutation seems to be correlated with the “variant” pattern in filipin staining. Miglustat treatment response seems to be correlated with the age at disease onset and disability scale score at diagnosis. .

O objetivo desse estudo foi analisar uma série de casos de pacientes brasileiros com doença de Niemann-Pick tipo C (NP-C). Método Correlação entre manifestações clínicas, alterações laboratoriais, estudo molecular e resposta ao tratamento foram realizadas. Resultado A amostra consiste de 5 pacientes com idade entre 8 e 26 anos. Paralisia do olhar vertical supranuclear, ataxia cerebelar, demência, distonia e disartria estavam presentes em todos os casos. Coloração de filipina na cultura de fibroblastos mostrou padrão “clássico” em dois pacientes e padrão “variante” em três casos. O estudo molecular encontrou mutações no gene NPC1 em todos os alelos. O tratamento com miglustate foi realizado em 4 pacientes. Conclusão Embora coloração de filipina seja utilizada para confirmar o diagnóstico, o histiócito azul-marinho no aspirado de medula óssea frequentemente auxilia a confirmar o diagnóstico de NP-C. A mutação p.P1007A está correlacionada com o padrão “ variante” na coloração de filipina. A resposta ao tratamento com miglustate parece estar correlacionada com a idade e escore de desabilidade no momento do diagnóstico. .

Comparison of the measurement of lysosomal hydrolase activity in mycoplasma-contaminated and non-contaminated human fibroblast cultures treated with mycoplasma removal agent.

OBJECTIVES: To investigate the effect of both mycoplasma contamination and of its remover (MRA), through human fibroblasts culture over the activity of some lysosomal hydrolases. DESIGN AND METHODS: Activity was measured in contaminated fibroblasts before and after the addition of MRA. Results were compared with the enzymatic activity in control fibroblasts with and without MRA. RESULTS: Only beta-glucosidase showed no significant alteration in the presence of either mycoplasma or MRA. Total hexosaminidase and beta-galactosidase underwent significant interference in the presence of the mycoplasma and the MRA. The % of hexosaminidase A and arylsulphatase A altered their activity only in the presence of MRA. Beta-glucuronidase changed its activity only in the presence of mycoplasma. CONCLUSIONS: The fibroblast enzymes behaved differently in the presence of MRA and/or mycoplasma, demonstrating the sensitivity of these hydrolases. Our work suggests that mycoplasma and MRA alter the activity of some lysosomal hydrolases.

Effect of amikacin, cephalothin, clindamycin and vancomycin on in vitro fibroblast growth

The effect of four antibiotics (amikacin, clindamycin, cephalothin and vancomycin) was investigated considering that bacterial infection in fibroblasts cultures is a very frequent event. The investigation included the effect of the antibiotics on fibroblast growth and on the activity of the enzyme glucocerebrosidase. The antibiotics were added to the fibroblast cultures and cell growth was evaluated by counting the number of cells and their viability. After cell harvesting, the enzyme activity and content of protein were measured. The results allowed us to conclude that none of the antibiotics affected the cellular number nor the cellular viability. The content of protein decreased when cephalothin and clindamycin were added to the cultures, and glucocerebrosidase was affected in the presence of amikacin. Vancomycin did not interfere with any of the parameters analyzed, so it was chosen to be used in cell cultures to prevent the contamination by gram positive bacteria.