ALBERT

Description: Background. Enzymatic replacement therapy (ERT) had modified the natural history of Gaucher disease (GD). Historically the GD patients had growth retardation; frequently spleen removal, and multiple bone complications. It is known that more than 70% of GD patients diagnosed in adulthood have bone complications. Nowadays we have learnt through clinical trials that patients treated regularly with ERT during childhood achieve a normal percentile of growing and avoid complications. In this report we are summarizing the Spanish experience in the last two decades treating children with GD in every day clinical practice. Patients & Methods: From 376 patients included in the Spanish Registry of Gaucher Disease working since 1993, a total of 79 patients were diagnosed under18 years old, 26 of them were classified as GD type 2; 13 developed a GD3 during follow-up and 40 were classified as GD1. For the analysis we have only included GD1 and GD3 patients, 53 patients. Statistical analysis: Descriptive and frequency analysis were performed in a SSPS data base. Results: Baseline characteristics: Gender: 18 females and 35 males. Mean age at diagnosis 5.8 y.o. (range 0.5-17), 78.4% (62) of cases were the initial case for familiar studies, mean age at start ERT 8.5 years (0.5-18), Mutational analysis: L444P/L444P was the most frequent genotype in GD3 and N370S heterozygosis in GD1, there was only one case with N370S homozygosis, Three patients were splenectomised before diagnosis and 60% underwent a bone marrow aspiration or biopsy and 3 liver biopsy during the initial workout. The paediatric Gaucher Score Index (PGSI) (Kallish S, Kaplan P 2012) shows: mild-disease: 62.5%, moderate: 31.25% and severe: 6.25%, missing data 5 patients. Mean chitotriosidase: 12,535 nmol.ml/h (871-49,031), hemoglobine: 11.4g/dL (6.9-17), 43% of patients presented anemia at diagnosis. Mean platelet count: 121 x109 (12,363), 53% of patients presents with 〈 100 x109 platelets. Bone assessment: 49% (26) of patients shows bone manifestations: Erlenmeyer flask deformity, 24.5%; Bone crisis, 13.2%; avascular necrosis, 5.6%; bone infiltration, 7.5%, Bone infarction: 3.7%; isolated bone pain, 13.2%. The mean follow-up was 13 y (2-21), 45 received ERT following diagnosis, mean years on therapy 13.2 (2-21). In first line: Alglucerase, 8.9% (4); Imiglucerase, 75.5% (34); Velaglucerase, 6.7% (3) and Taliglucerase 8.9% (4). Actual therapies: Imiglucerase: 47.1% (25); Velaglucerase: 17% (9); Taliglucerase: 3.7% (2); Miglustat: 7.5% (4), Eliglustat: 1.8% (1), no ERT/ loss of contact: 7.5%. Mean time on ERT: 13,2 years, twenty-one patients had received ERT during 15 or more years (15-21); 12 for 〉 10 years (12-14) and 11〈 10 years (2-9). Actual status: mean haemoglobin levels: 13, 3g/dL, mean platelets count: 162 x109, Chitotriosidase: 497 mg.prot.h, there are no patients with hepatomegaly and only 3 patients have splenomegaly. Events related to therapy: infusion reactions G3/4: 4, all patients had IgG specific antibodies, and all reactions occurs between 1 and 7 months after start therapy; abandonment 2; changes of ERT, 7 (excluding alglucerase to imiglucerase); related to disease: growth retardation 9 (bellow 25 percentile for age/high); bone crisis 3; joint replacement 1, osteopenia 2 cases (after 2 years on ERT), normalization of coagulation factors (VW,FV,FIX,FXI), others: early diabetes 1, early menarche 1, thyroiditis 1, sacroileitis 1, pregnancies 3, lost follow-up 4. Conclusions: The incidence of GD related complications, especially bone complications, in paediatric patients on ERT is lower than in adults, probably related to early ERT onset. Endocrine complications are rare, difficult to correlate to ERT. Growth retardation was resolved with ERT in the majority of cases. The early onset of therapy in symptomatic child is highly recommended. Table 1. Immune reactions and endocrine complications Patient Age at Dx Gender Genotype Time on ERT Type of reaction 1 4 F N370S/84GG 6 m (Imiglucerase) Infusion reaction 2 3 F N370S/IVS4-2ª〉G;c.(-203)A〉G 7 m (Imiglucerase) Infusion reaction 3 3 F N370S/L444P 1 m (Velaglucerase*) Infusion reaction 4 9 F N370S/ G4384delC 3 m(Imiglucerase) Infusion reaction 5 4 M N370S/L444P 72 m (Imiglucerase) Diabetes 6 0,5 F N370S/L444P 12 m (Imiglucerase) Thyroiditis 7 4 F N370S/R47X 48 m (Imiglucerase) Early manarche Disclosures No relevant conflicts of interest to declare.

Description: Background: Gaucher disease (GD), caused by a deficiency of the glucocerebrosidase (GC) enzyme, is characterized by the accumulation of glucosylceramide (GlcCer) within lysosomes, resulting in cellular dysfunction and damage. GlcCer is catalyzed by the glucosylceramide synthase (GCS), also known as UDP-glucose ceramide glucosyltransferase, (UGCG), EC 2.4.1.80, first enzyme in glycosphingolipids biosynthesis. Mutations in the glucocerebrosidase (GBA) gene are required to cause GD, but other factors play an important role. Aims: GCS gene is a logical candidate to hypothesize that its variability could be involved in clinical severity. Patients and methods: We have analyzed GCS variants (g.(−295)C〉T, g.(−222)ins10, g.148A〉G, g.166A〉T, g.25510C〉G, g.29312A〉G and g.34991A〉G) in a group of 80 [N370S]+[L444P] heterozygous and 23 N370S homozygous patients. Results: were related with severity score index (SSI). g.(−295)C〉T and g.166A〉T SNPs were in complete linkage disequilibrium. In the N370S homozygous, carriers of g.(−222)ins10 have higher SSI than non carriers (6.6 vs 2.4, pG had higher SSI than the G-allele carriers (6.7 vs 1.5, p

Description: Gaucher disease (GD) is caused by a deficiency of the lysosomal enzyme acid β-glucosidase (GC) leading to accumulation of glucosylceramide within the macrophages of the reticuloendothelial systems. Plasma of GD patients contains low levels of total cholesterol, LDL-cholesterol and HDL-cholesterol. Enzyme replacement therapy (ERT) with imiglucerase and Substrate Reduction Therapy (SRT) with miglustat restores the haemoglobin levels, platelet counts and organomegalies. Previously we have demonstrated that ERT had significant effects on the concentration and metabolism of plasma lipoproteins. However, the effects of SRT on plasma lipid and other atherogenic profiles of GD patients have not been explored. We report the results of long term (36 months) SRT on plasma lipoprotein concentrations in 26 (11 men and 15 women) GD patients. Mean age of patients was 51 (SD ±20.7; range 22–74) years. Ten patients were therapy-naïve to SRT and 16 switched from previous enzyme replacement therapy (ERT). All patients received 100 mg of Miglustat t.i.d with recommendations of a low hydrocarbonate diet in the first weeks. Total cholesterol (TC), triglycerides (TG), low density lipoprotein cholesterol (LDL-c), high density lipoprotein cholesterol (HDL-C), Apolipoproteins (Apo AI, ApoB and Lp(a)), CRP, MPI1b, CCL18 concentrations and chitotriosidase activity were measured before (baseline) and after 12, 24 and 36 months of follow-up. Treatment resulted in a significant increases in HDL-c concentration in naïve patients during the first 24 months (p

Description: Background and objective Miglustat (Zavesca®), an inhibitor of the enzyme glucosylceramide synthase, is an orally active molecule that has been shown to be a safe and effective therapy for type I GD. Here we report on our experience of miglustat monotherapy for the treatment of type 1 Gaucher disease (GD) patients in Spain. Design and Methods: We designed a prospective, open-label study of 21 patients with mild-to- moderate type 1 GD, to investigate the efficacy and tolerability of miglustat over a 12-month period in therapy-naïve patients and in patients who have previously received enzyme replacement therapy (ERT). Clinical assessments, including haematological parameters and organomegaly, were carried out prior to and after 6 and 12 months of miglustat therapy. In addition, the results were compared with data analysed retrospectively from 40 patients with type 1 GD who had been treated for 6 months with ERT. The data following 6 months of miglustat therapy are presented here. Results At present, 16 patients have completed 6 months of miglustat treatment. All patients with anaemia had improved haemoglobin concentrations, and in the treatment-naive group a mean increase of 1 g/dl was observed. Platelet counts improved in patients with thrombocytopenia and were maintained in patients with counts within normal limits at baseline. Chitotriosidase activity was maintained in switched patients and decreased in naïve patients. Miglustat was well tolerated, and the efficacy after 6 months therapy was comparable to that observed in the clinical trials and in patients treated with ERT for 6 months. Interpretation and conclusions In our experience, properly selected patients with mild to moderate type I GD had a satisfactory clinical and biochemical response to 6 months of miglustat therapy. The therapy was well tolerated, and clinical benefits were comparable to those obtained with ERT.

Description: Abstract 3270 Introduction: Many of the mutations of the lysosomal acid b-glucosidase (b-glucocerebrosidase) associated with Gaucher disease (GD) translate into enzymes that retain partial catalytic activity in vitro but exhibit impaired cellular trafficking as a consequence of aberrant folding. Current investigational therapeutic strategies for include the development of ligands of the enzyme capable of promoting those conformational changes that are required for efficient folding, restoring trafficking. Although somewhat counter intuitive, competitive inhibitors of this b-glucocerebrosidase, at subinhibitory concentrations, can increase steady-state lysosomal levels of active enzyme through this rescuing mechanism, acting as “pharmacological chaperones”. At the massive lysosomal substrate concentration, the inhibitor would be replaced from the active site of the enzyme and the metabolic activity recovered. However, most of the pharmacological chaperones under study are iminosugars that behave as broad spectrum inhibitors, inhibiting simultaneously several glucosidases, which represents a serious inconvenient for clinical applications. An additional problem is that iminosugars and their derivatives are not active as pharmacological chaperones for glucocerebrosidase mutations located outside the domain containing the active site and are associated with neurological involvement, as the L444P mutation. Aim: The aim of this work is to present molecules with a high binding specificity towards b-glucocerebrosidase, with a high ratio of chaperone versus inhibitor activity and capable of producing an increased in the levels of mutant enzymes associated with Gaucher disease, including mutations located outside the catalytic domain. Methods: Different bicyclic derivatives of L-idonojirimycin were designed and chemically synthesized from D-glucose after in silico structural analysis and identification of the most favorable molecular features to interact with the active site of glucocerebrosidase. The chaperone potential of these compounds was evaluated at different concentrations in vitro using a cell model of GDcarrying the more frequent mutations in Gaucher disease, namely N370S and L444P. (P201230804). Results: The obtained results showed an increase in b-glucocerebrosidase activity at various chaperone concentrations, ranging from 1.96 to 4.98 folds for the L444P mutant and from 2.01 to 3.06 folds for the N370S mutation. Comments: The bicyclic derivatives of L-idonojirimycin could be considered as a therapeutic alternative for GD, mainly in patients with mutations located outside the active site of the enzyme and associated with neurologic involvement. Disclosures: Giraldo: Actelion: Membership on an entity's Board of Directors or advisory committees; Genzyme: Research Funding; Shire: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Description: Substrate reduction therapy (SRT) has showed as an useful therapy in type 1 GD patients with mild or moderate disease. The main limitation to use miglustat has been the gastrointestinal adverse events. Since 2004 we have conducted the ZAGAL (Zavesca en Gaucher Leve) study for monitoring the real-life use of miglustat in Spanish adult patients with mild-to moderate disease. The study included GD1 patient’s naïve to therapy as well as patients who have previously been treated with ERT. For follow-up, the therapeutic goals for GD therapy by Pastores GM et al were applied. To date a total of 351 GD1 patients have been diagnosed in Spain (FEETEG unpublished. 2013); from 2004 until 2013, 53 of them have been exposed of miglustat (15.1%). In 16 patients (30.2%) miglustat was the first line of therapy and the remaining 38 switched from ERT. Currently 37 patients (mean age 43.6 y, range 22-83) 50.9% females, are on miglustat therapy (20 at least for 5 years and 13 during more than 8 years). Related to efficacy, the therapy permit to achieve a stable hemoglobin concentration level and spleen reduced volume, but we recorded a decrease of absolute platelet count in 15 patients (28.3%) (mean: 35x109/L, range: 10-86x109/L). Related to biomarkers changes an increase in CT activity was observed in 56.6% of patients (mean: 2,448 nmol/mL.h, range 135-13,687) and 43.4 % for CCL18/PARC (mean: 250 ng/mL, range: 19-1016). Seventeen patients (32.1%) had transitory gastrointestinal disturbances. Sixteen patients (30.2%) discontinued therapy: one of them for pregnancy, two by bone crisis, two by weight loss, one for bad compliance and ten by gastrointestinal discomfort or intolerance (18.8%). 60% of patients has a fine tremor in first months on therapy. 4 died by non-related causes (2 cancer, 1 hearth attack, 1 liver failure), Conclusion The long term follow-up of GD patients treated with Miglustat shows that more than 69% had achieved and maintains the therapeutic goals. A high individual variability had been observed related to miglustat gastrointestinal intolerance apparently no related with GBA genotype, gender and age, but possibly associated with disacaridases inhibition and food habits. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction: Chitotriosidase protein (CT) is the most important biochemical marker described for Gaucher disease (GD). CT activity is increased several hundred-fold in plasma of GD patients and shows a strong positive correlation with the severity of the disease. However, a recessively inherited enzyme deficiency, with an incidence of about 6% in the Caucasian population, means that not all patients with GD can be monitored by measuring CT activity. Materials and Methods: This study was performed with plasma samples from 22 type 1 GD patients and 23 healthy subjects. All patients are from Spanish GD Registry. Applying two dimensional gel electrophoresis (2-DE) we compared the plasma proteome profile of GD patients with that of healthy subjects. Differential proteins were excised and prepared for MALDI-TOF analysis. Results were verify with 2-DE western blot. Results: In this study we have described five CT isoforms in a 2-DE gel from GD plasma samples. We show and compare the 2-DE images of each CT spot in plasma protein samples from healthy controls, GD patients with wild-type CT protein, and GD patients either homozygous or heterozygous for the 24 bp duplication. We propose a connection between the quantitative image analysis of each CT spot and the enzymatic activity of CT in each patient. Lastly, our results verifies that post-translational glycosylation explains at least some of differences between CT isoforms. Conclusion: The possibilities of proteomic technology in the investigation of biological biomarkers of GD and the nature of the CT protein are demonstrated, as well as in detecting new biochemical markers useful for the diagnosis of the disease and for monitoring patient response to therapy.

Description: High density microarrays (HDM) are powerful tools for simultaneously profiling the expression levels of thousands of genes. The application of this technology to study of neoplastic hematological disorders.has identified new sub groups of disease not related previously and new prognosis markers. However there is a limited experience in the gene expression studies using low density microarrays (LDM) in neoplastic hematological disorders. A gene expression analysis system based on a LDM containing 538 oligonucleotides has been developed. Whole technical process was optimized to improve the analysis of differential expression. We have analyzed mRNA from cell line cultures (Jurkat, U937), whole blood samples from healthy subjects and different hematological malignancies (HM) using this chip. A hierarchical clustering procedure applying Welch t-statistics with Bonferroni correction was used to analysis gene expression data The LDM generated a linear response of 2 magnitude orders and a CV values less than 20% for hybridization and label replicates. This procedure detects 0,2 fmols of mRNA. We have found genes with statistically significant differences between Jurkatt and U937 cells cultures, and blood samples from 15 healthy donors, 59 lymphocyte leukemia and 13 myeloid leukemia and myelodisplasia syndrome patients. A classification system based on gene expression data was constructed with an accuracy of 97%.to predict healthy or lymphocyte leukemia status. To identify different subsets of patients in the B-CLL group, whole blood samples from 12 B-CLL patients were collected and defined as stables, according to clinical and analytical criteria at the time of diagnosis, “stable” (n=6) if disease stability was maintained for more than five years after the diagnosis and “progressive” (n=6) if the disease progressed less than one year after the diagnosis. Applying Welch statistical test without correction and a p

Description: Gaucher’s disease (GD) is an autosomal recessive lysosomal storage disorder, characterized by accumulation of glycosphingolipid in so-called Gaucher cells. The clinical manifestations of Gaucher disease are highly variable, and although certain genotypes are often associated with mild or severe symtomps, a defined correlation between genotype and phenotype does not exist. Identification of serum biochemical markers characteristic of disease may be useful in the diagnosis and monitoring of GD, nevertheless about 6% of the population does not express the chitotriosidase (CT) gene. In this study, we analyzed using currently available enzyme analysis the relationship among two known surrogate markers: CT, which utility in initiation and optimization of costly therapeutic interventions has been highly demonstrated, and a newly-described chemokine, pulmonary and activation-regulated chemokine (CCL18/PARC) as associated to Gaucher disease. Patients and methods: We have analysed 21 control samples, 149 samples of GD patients on enzyme replacement therapy (ERT), and 52 samples of GD patients on substrate reduction therapy (SRT), 4 samples of GD on combined therapy (ERT+SRT) and 7 samples of GD patients without therapy. The samples were stored at −80°C in the biobank of Biochemistry and Molecular and Cellular Biology Department of Zaragoza University. The CT activity and CCL18/PARC quantification were performed simultaneously in the samples obtained at baseline and yearly during 7 years under ERT. Results: our results concluded that both markers levels similarly fell with time and their variations correlated strongly each other, mainly in patients under ERT. The poor correlation of both variables in the case of SRT might be due to small number of samples. These findings demonstrated that CCL18 is a good biomarker of monitoring GD, comparable to CT and very useful in patients without expression of CT gene. Conclusion: The availability of sensitive plasma surrogate markers may be of great value for monitoring the efficacy of treatment, especially in cases of deficiencies of some marker.