32 research outputs found
Clinical efficacy of Enzyme Replacement Therapy in paediatric Hunter patients, an independent study of 3.5 years
BACKGROUND: Hunter Syndrome is an X-linked lysosomal storage disorder due to the deficit of iduronate 2-sulfatase, an enzyme catalysing the degradation of the glycosaminoglycans (GAG) dermatan- and heparan-sulfate. Treatment of the disease is mainly performed by Enzyme Replacement Therapy (ERT) with idursulfase, in use since 2006. Clinical efficacy of ERT has been monitored mainly by the Hunter Outcome Survey (HOS) while very few independent studies have been so far conducted. The present study is a 3.5-years independent follow-up of 27 Hunter patients, starting ERT between 1.6 and 27 years of age, with the primary aim to evaluate efficacy of the therapy started at an early age (<12 years). METHODS: In this study, we evaluated: urinary GAG content, hepato/splenomegaly, heart valvulopathies, otorinolaryngological symptoms, joint range of motion, growth, distance covered in the 6-minute walk test, neurological involvement. For data analysis, the 27 patients were divided into three groups according to the age at start of ERT: â€5 years, >5 andââ€â12 years andâ>â12 years. Patients were analysed both as 3 separate groups and also as one group; in addition, the 20 patients who started ERT up to 12 years of age were analysed as one group. Finally, patients presenting a âsevereâ phenotype were compared with âattenuatedâ ones. RESULTS: Data analysis revealed a statistically significant reduction of the urinary GAG in patients â€5 years andââ€â12 years and of the hepatomegaly in the group aged >5 andââ€â12 years. Although other clinical signs improved in some of the patients monitored, statistical analysis of their variation did not reveal any significant changes following enzyme administration. The evaluation of ERT efficacy in relation to the severity of the disease evidenced slightly higher improvements as for hepatomegaly, splenomegaly, otological disorders and adenotonsillar hypertrophy in severe vs attenuated patients. CONCLUSIONS: Although the present protocol of idursulfase administration may result efficacious in delaying the MPS II somatic disease progression at some extent, in this study we observed that several signs and symptoms did not improve during the therapy. Therefore, a strict monitoring of the efficacy obtained in the patients under ERT is becoming mandatory for clinical, ethical and economic reasons. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13023-014-0129-1) contains supplementary material, which is available to authorized users
Analisi molecolare dei geni implicati nelle mucopolisaccaridosi: valutazione della casistica di uno studio multicentrico.
Introduzione
Nell'ambito di un Progetto PRIN, sono state rivalutate le diagnosi molecolari dei pazienti arruolati nello studio, per un totale di 60 soggetti, affetti da diverse forme di MPS.
Metodi
I dati genetici dei singoli pazienti, resi disponibili dalle diverse UnitĂ Cliniche, sono stati ri-analizzati. Le varianti sono state ri-annottate secondo l'annotazione HGVS corrente.
Risultati
La diagnosi molecolare Ăš risultata disponibile per 50 dei 60 pazienti. Per i 10 pazienti non caratterizzati era disponibile l'analisi enzimatica, sulla quale si era basata la formulazione della diagnosi di malattia.
Sono state identificate 86 varianti causanti patologia (55 diverse). Il 70% erano missenso, 11.6% non senso e una variante senso; il 9.3% erano ampie delezioni/riarrangiamenti, il 3.5% piccole delezioni/inserzioni, il 4.6% varianti di splicing. Nove varianti non erano riportate in letteratura.
Una spiccata disomogeneitĂ nell'annotazione delle varianti Ăš risultata evidente; ciĂČ Ăš imputabile al fatto che l'analisi genetica di molti pazienti risale a periodi storici diversi, in cui erano in vigore regole di annotazione differenti. Tale aspetto potrebbe causare difficoltĂ nell'interpretazione delle varianti, con inevitabili ricadute sulla consulenza genetica.
Discussione
Lo studio evidenzia la necessitĂ di completare la diagnosi molecolare nei pazienti MPS giĂ diagnosticati in passato con procedure di tipo biochimico. Emerge poi la necessitĂ di aggiornare periodicamente l'annotazione delle varianti e di depositarle presso database dedicati (LOVD , ClinVAr, ecc.) in modo da renderle disponibili alla comunitĂ scientifica. E' necessario, inoltre, stabilire un percorso diagnostico-molecolare condiviso, che preveda anche la ricerca delle varianti nei genitori per facilitare la diagnosi e consentire di identificare anche le varianti de novo, che richiedono un diverso percorso di consulenza
A novel CRISPR/Cas9-based iduronate-2-sulfatase (IDS) knockout human neuronal cell line reveals earliest pathological changes
Abstract Multiple complex intracellular cascades contributing to Hunter syndrome (mucopolysaccharidosis type II) pathogenesis have been recognized and documented in the past years. However, the hierarchy of early cellular abnormalities leading to irreversible neuronal damage is far from being completely understood. To tackle this issue, we have generated two novel iduronate-2-sulfatase (IDS) loss of function human neuronal cell lines by means of genome editing. We show that both neuronal cell lines exhibit no enzymatic activity and increased GAG storage despite a completely different genotype. At a cellular level, they display reduced differentiation, significantly decreased LAMP1 and RAB7 protein levels, impaired lysosomal acidification and increased lipid storage. Moreover, one of the two clones is characterized by a marked decrease of the autophagic marker p62, while none of the two mutants exhibit marked oxidative stress and mitochondrial morphological changes. Based on our preliminary findings, we hypothesize that neuronal differentiation might be significantly affected by IDS functional impairment
A1-42 fragment induces an up-regulation of NCX3 activity that prevents caspase-12 activation.
The molecular mechanisms responsible for A1-42-peptide induced intracellular Ca2+ homeostasis dysregulation still remain unclear. We report data obtained in mouse hippocampal neurons and NGF-differentiated PC-12 cells suggesting that the extracellular-dependent early increase(30minutes) in intracellular calcium concentration ([Ca2+]i), following A1-42 exposure, caused the activation of calpain that in turn elicited a cleavage of the Na+/Ca2+ exchanger(NCX3). This cleavage generated a hyperfunctional form of the antiporter and increased NCX currents(INCX) in the reverse mode of operation. Interestingly, this NCX3 calpain-dependent cleavage was essential for the A1-42-dependent INCX increase. Indeed, the calpain inhibitor calpeptin and the removal of the calpain-cleavage recognition sequence, via site-directed mutagenesis, abolished this effect. Moreover, the enhanced NCX3-activity was paralleled by an increased Ca2+ content in the ER-stores. Remarkably, the silencing or knocking-out of the ncx3 gene prevented the enhancement of both INCX and Ca2+ content in the ER-stores, suggesting that NCX3 was involved in the increase of ER Ca2+-content stimulated by A1-42. By contrast, in the late phase (72hours), when the NCX3 proteolytic cleavage abruptly ceased, the occurrence of a parallel reduction in ER Ca2+ content triggered ER-stress, as revealed by caspase-12 activation. Concomitantly, the late increase in [Ca2+]i coincided with neuronal death. Interestingly, NCX3 silencing caused an earlier activation of A1-42-induced caspase-12. Indeed, in NCX3 silenced neurons, A1-42 exposure hastened caspase-dependent apoptosis, thus reinforcing neuronal cell death. These results suggest that A1-42, through Ca2+-dependent calpain activation, generates a hyperfunctional form of NCX3 that by increasing Ca2+ content into ER delays caspase-12 activation, and thus neuronal death
. Ab1-42 fragment induces an up-regulation of NCX3 activity that prevents caspase-12 activation.
The molecular mechanisms responsible for A1-42-peptide induced intracellular Ca2+ homeostasis dysregulation still remain unclear. We report data obtained in mouse hippocampal neurons and NGF-differentiated PC-12 cells suggesting that the extracellular-dependent early increase(30minutes) in intracellular calcium concentration ([Ca2+]i), following A1-42 exposure, caused the activation of calpain that in turn elicited a cleavage of the Na+/Ca2+ exchanger(NCX3). This cleavage generated a hyperfunctional form of the antiporter and increased NCX currents(INCX) in the reverse mode of operation. Interestingly, this NCX3 calpain-dependent cleavage was essential for the A1-42-dependent INCX increase. Indeed, the calpain inhibitor calpeptin and the removal of the calpain-cleavage recognition sequence, via site-directed mutagenesis, abolished this effect. Moreover, the enhanced NCX3-activity was paralleled by an increased Ca2+ content in the ER-stores. Remarkably, the silencing or knocking-out of the ncx3 gene prevented the enhancement of both INCX and Ca2+ content in the ER-stores, suggesting that NCX3 was involved in the increase of ER Ca2+-content stimulated by A1-42. By contrast, in the late phase (72hours), when the NCX3 proteolytic cleavage abruptly ceased, the occurrence of a parallel reduction in ER Ca2+ content triggered ER-stress, as revealed by caspase-12 activation. Concomitantly, the late increase in [Ca2+]i coincided with neuronal death. Interestingly, NCX3 silencing caused an earlier activation of A1-42-induced caspase-12. Indeed, in NCX3 silenced neurons, A1-42 exposure hastened caspase-dependent apoptosis, thus reinforcing neuronal cell death. These results suggest that A1-42, through Ca2+-dependent calpain activation, generates a hyperfunctional form of NCX3 that by increasing Ca2+ content into ER delays caspase-12 activation, and thus neuronal death
AÎČ1-42 fragment induces an up-regulation of NCX3 activity that prevents caspase-12 activation
The molecular mechanisms responsible for A1-42-peptide induced intracellular Ca2+ homeostasis dysregulation still remain unclear. We report data obtained in mouse hippocampal neurons and NGF-differentiated PC-12 cells suggesting that the extracellular-dependent early increase(30minutes) in intracellular calcium concentration ([Ca2+]i), following A1-42 exposure, caused the activation of calpain that in turn elicited a cleavage of the Na+/Ca2+ exchanger(NCX3). This cleavage generated a hyperfunctional form of the antiporter and increased NCX currents(INCX) in the reverse mode of operation. Interestingly, this NCX3 calpain-dependent cleavage was essential for the A1-42-dependent INCX increase. Indeed, the calpain inhibitor calpeptin and the removal of the calpain-cleavage recognition sequence, via site-directed mutagenesis, abolished this effect. Moreover, the enhanced NCX3-activity was paralleled by an increased Ca2+ content in the ER-stores. Remarkably, the silencing or knocking-out of the ncx3 gene prevented the enhancement of both INCX and Ca2+ content in the ER-stores, suggesting that NCX3 was involved in the increase of ER Ca2+-content stimulated by A1-42. By contrast, in the late phase (72hours), when the NCX3 proteolytic cleavage abruptly ceased, the occurrence of a parallel reduction in ER Ca2+ content triggered ER-stress, as revealed by caspase-12 activation. Concomitantly, the late increase in [Ca2+]i coincided with neuronal death. Interestingly, NCX3 silencing caused an earlier activation of A1-42-induced caspase-12. Indeed, in NCX3 silenced neurons, A1-42 exposure hastened caspase-dependent apoptosis, thus reinforcing neuronal cell death. These results suggest that A1-42, through Ca2+-dependent calpain activation, generates a hyperfunctional form of NCX3 that by increasing Ca2+ content into ER delays caspase-12 activation, and thus neuronal death