Twice weekly dosing with Sebelipase alfa (Kanuma®) rescues severely ill infants with Wolman disease.

BACKGROUND: Sebelipase alfa (Kanuma®) is approved for patients with Wolman disease (WD) at a dosage of 3-5 mg/kg once weekly. Survival rates in the second of two clinical trials was greater, despite recruiting more severely ill patients, probably related to higher initial and maximal doses. We aimed to evaluate the effective pharmacokinetics and pharmacodynamics of Sebelipase alfa when administered to patients with severe WD at 5 mg/kg twice weekly, an intensive regimen which was not assessed in the trials. METHODS: We recruited 3 patients receiving Sebelipase alfa 5 mg/kg twice weekly. We measured LAL activity in leukocytes and plasma oxysterol concentration in two patients and LAL activity in fibroblasts in one patient. Clinical follow up was also assessed. RESULTS: Analyses of LAL activity and oxysterols demonstrate that there is short-lived enzyme activity post-dosing which is associated with the release of stored lipids. Clinical data demonstrate that 5 mg/kg twice weekly dosing is well tolerated and effective. CONCLUSION: 5 mg/kg twice weekly dosing with Sebelipase alfa rescues severely ill infants with WD by increasing substrate clearance. There is biologically relevant lipid accumulation in the 'trough' periods before the next dosing, even with this intensive regimen.

Serum Phospholipid Profile Changes in Gaucher Disease and Parkinson's Disease.

Alterations in the levels of serum sphingolipids and phospholipids have been reported in Gaucher disease and in Parkinson's disease, suggesting a potential role of these lipids as biomarkers. This project's objective is to detect novel associations and novel candidate biomarkers in the largest Spanish Gaucher and Parkinson diseases of the Iberian Peninsula. For that, 278 participants were included: 100 sporadic Parkinson's patients, 70 Gaucher patients, 15 GBA1-mutation-carrier Parkinson's patients and 93 controls. A serum lipidomics array including 10 phospholipid groups, 368 species, was performed using high-performance liquid chromatography-mass spectrometry. Lipid levels were compared between groups via multiple-regression analyses controlling for clinical and demographic parameters. Additionally, lipid levels were compared within the Gaucher and Parkinson's groups controlling for medication and/or disease severity. Results were controlled for robustness by filtering of non-detectable lipid values. There was an increase in the levels of phosphatidylcholine, with a simultaneous decrease in lyso-phosphatidylcholine, in the Gaucher, Parkinson's and GBA1-mutation-carrier Parkinson's patients vs. controls. Phosphatidylethanolamine, lyso- and plasmalogen-phosphatidylethanolamine were also increased in Gaucher and Parkinson's. Gaucher patients also showed an increase in lyso-phosphatidylserine and phosphatidylglycerol. While in the Gaucher and Parkinson's groups, velaglucerase alpha and dopamine agonists, respectively, showed positive associations with the lipid changes, miglustat treatment in Gaucher patients normalized the altered phosphatidylcholine/lyso-phosphatidylcholine ratio. In conclusion, Gaucher and Parkinson's patients showed changes in various serum phospholipid levels when compared with healthy controls, further supporting the role of such lipids in disease development and, possibly, as putative biomarkers. This hypothesis was reinforced by the normalizing effect of miglustat, and by controlling for data robustness, even though the limited number of participants, especially in the sub-distribution by treatment groups in GD requires validation in a larger number of patients.

Allelic and phenotypic characterization of CYP2D6 and its encoded P450 cytochrome enzyme in a serie of Spanish type 1 Gaucher disease patients / Caracterización alélica y fenotípica del gen y la enzima Cyp2d6 en una cohorte de pacientes españoles con enfermedad de Gaucher tipo 1

BACKGROUND: Cytochrome p450 is the main drug metabolic pathway. CYP2D6 is a highly polymorphic gene that encodes a cytochrome p450 enzyme with three activity levels: null, reduced and normal. Apart from another type of mutations CYP2D6 can suffer duplications and deletions of the entire gene. This is the pathway to metabolize one of the Gaucher disease treatments, whose dose administration is regulated according to the metabolizer phenotype, this being one of the administration limitations. OBJECTIVES: The aim of this paper is to evaluate the allelic frequencies and the metabolizer status of Gaucher type 1 patients in the Spanish population and compare it with the general Spanish population and other Gaucher disease groups. METHODS: In this study, 109 type 1 Gaucher disease patients were analyzed with the xTAG(R)CYP2D6 kit to identify the CYP2D6 gene alleles. RESULTS: We observed that eighty-seven patients could be classified as extensive, 14 as intermediate, 6 as poor and 2 as ultra-rapid metabolizers. The allelic duplication frequency is 5.5% and deletion is 4.5%. The most common allele is wild-type and the second is the null *4 allele. Intermediate phenotype frequency is higher than expected (p < 0.05). CONCLUSIONS: Our Spanish GD series shows an unexpected distribution of some alleles and phenotypic metabolizer status, in contrast to that previously reported in the Spanish population

INTRODUCCIÓN: La superfamilia citocromo P450 es la principal vía de metabolización de fármacos. Uno de los genes que la componen, CYP2D6, es altamente polimórfico y puede producir enzimas con 3 niveles de actividad: nula, reducida o normal. Además de presentar variantes puntuales, este gen puede sufrir duplicidad o deleción. CYP2D6 es la principal vía de metabolización del último tratamiento aprobado para la enfermedad de Gaucher, cuya administración y dosificación depende del estado metabolizador de CYP2D6. OBJETIVOS: El objetivo de este trabajo es evaluar la frecuencia alélica y la distribución de fenotipos metabolizadores en una serie de pacientes españoles con enfermedad de Gaucher, y compararla con los datos publicados para población española general y con otros grupos de pacientes de Gaucher. MÉTODOS: Se han genotipificado 109 pacientes con enfermedad de Gaucher tipo 1 mediante el sistema xTAG(R) CYP2D6. RESULTADOS: Nuestra población se distribuye en 87 pacientes con un fenotipo metabolizador normal, 14 intermedios, 6 lentos y 2 ultrarrápidos. La frecuencia de la duplicación y deleción del gen es del 5,5 y 4,5%, respectivamente. El alelo más común es la forma nativa de la proteína y el segundo el alelo *4 que codifica para una proteína inactiva. La frecuencia de fenotipos intermedios es superior a la esperada en población general (p < 0,05), principalmente a causa de un incremento en la frecuencia de los alelos que codifican enzimas con actividad reducida (p < 0,05). CONCLUSIONES: El grupo español de pacientes con enfermedad de Gaucher muestra una distribución alélica y fenotípica diferente a la esperada para la población española

Serum protein profile analysis in lysosomal storage disorders patients.

INTRODUCTION: Serum protein electrophoresis (SPE) is a well-established technique to identify alterations in plasma protein profiles, caused by diseases as multiple myeloma (MM). In addition, it could be a cost-effective technique to discover new plasma biomarkers. Relation between MM and lysosomal storage diseases (LSDs) as Gaucher disease has been set out but, it has not been evaluated on other LSDs nor the utility of the SPE as first step on LSDs biomarkers discovery projects. MATERIALS AND METHODS: Stored plasma samples at diagnosis from several LSDs patients underwent analysis. Quality control was checked prior to the SPE was analyzed by capillary electrophoresis. The analysis for monoclonal spikes and the differences between each fraction on patients' samples vs the control data previously published, were evaluated. Furthermore, immunoprotein quantification and free light chains ratio were done by nephelometry and turbidimetry. RESULTS: Seventy-five samples of LSD patients at diagnosis, were assessed. The frequency of the MGUS on LSDs patients was not higher than in general population whereas one lysosomal acid lipase deficiency infant showed increased IgA and kappa deviation. Regarding to the usefulness of SPE in biomarkers discovery, statistically significant differences were observed on SPE fractions between LSDs and healthy population. DISCUSSION: The evaluation of SPE fractions can be a useful tool to understand pathophysiologic aspects in LDSs and, to simplify new marker discovery projects. In some of them, the MGUS appearance is a risk factor for the MM development despite its frequency is not increased on the studied LSDs at diagnosis.

Allelic and phenotypic characterization of CYP2D6 and its encoded P450 cytochrome enzyme in a serie of Spanish type 1 Gaucher disease patients.

BACKGROUND: Cytochrome p450 is the main drug metabolic pathway. CYP2D6 is a highly polymorphic gene that encodes a cytochrome p450 enzyme with three activity levels: null, reduced and normal. Apart from another type of mutations CYP2D6 can suffer duplications and deletions of the entire gene. This is the pathway to metabolize one of the Gaucher disease treatments, whose dose administration is regulated according to the metabolizer phenotype, this being one of the administration limitations. OBJECTIVES: The aim of this paper is to evaluate the allelic frequencies and the metabolizer status of Gaucher type 1 patients in the Spanish population and compare it with the general Spanish population and other Gaucher disease groups. METHODS: In this study, 109 type 1 Gaucher disease patients were analyzed with the xTAG®CYP2D6 kit to identify the CYP2D6 gene alleles. RESULTS: We observed that eighty-seven patients could be classified as extensive, 14 as intermediate, 6 as poor and 2 as ultra-rapid metabolizers. The allelic duplication frequency is 5.5% and deletion is 4.5%. The most common allele is wild-type and the second is the null *4 allele. Intermediate phenotype frequency is higher than expected (p<0.05). CONCLUSIONS: Our Spanish GD series shows an unexpected distribution of some alleles and phenotypic metabolizer status, in contrast to that previously reported in the Spanish population.

LC-MS/MS analysis of plasma glucosylsphingosine as a biomarker for diagnosis and follow-up monitoring in Gaucher disease in the Spanish population.

Background Gaucher disease (GD), caused by a deficiency in acid ß-glucosidase, leads to the accumulation of glucosylsphingosine (GluSph), which has been used as a powerful biomarker for the diagnosis and follow-up of GD. Our aim was to perform the first retrospective study of GluSph in Spanish patients, analyzing its relationship with classical biomarkers and other parameters of disease and its utility regarding treatment monitoring. Methods Classical biomarkers were evaluated retrospectively by standard methods in a total of 145 subjects, including 47 GD patients, carriers, healthy controls and patients suffering from other lysosomal lipidoses. GluSph was also measured using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method developed as part of the present study. Results The optimized method presented intra- and inter-assay variations of 3.1 and 11.5%, respectively, overall recovery higher than 96% and linearity up to plasma concentrations of 1000 ng/mL with 100% specificity and sensitivity. Only GD patients displayed GluSph levels above 5.4 ng/mL at diagnosis and this was significantly correlated with the classical biomarkers chitotriosidase (r = 0.560) and the chemokine CCL18/PARC (CCL18/PARC) (ρ = 0.515), as well as with the Spanish magnetic resonance imaging index (S-MRI, r = 0.364), whereas chitotriosidase correlated with liver volume (r = 0.372) and CCL18/PARC increased in patients with bone manifestations (p = 0.005). GluSph levels decreased with treatment in naïve patients. Conclusions Plasma GluSph is the most disease-specific biomarker for GD with demonstrated diagnostic value and responsiveness to therapy. GluSph in the present series of patients failed to demonstrate better correlations with clinical characteristics at onset than classical biomarkers.

New variants in Spanish Niemann-Pick type c disease patients.

Niemann-Pick type C (NPC) disease is a rare inherited disease, with progressive neurodegeneration as the main symptom. It is a lysosomal storage disorder caused by mutations in NPC1 or NPC2 genes, leading to a lysosomal cholesterol trafficking impairment. Disease indicators are the clinical suspicion and biomarker levels. However, a genetic study is mandatory for the diagnosis, which is complicated due to the different variants with unknown significance. The aim of this work was to identify the variants responsible for NPC in our pediatric population. Twenty-two samples from non-related infants believed to have NPC disease were analyzed during the last 3 years. Surrogate biomarkers of the disease were evaluated whenever possible. Sanger sequencing for both genes is reported for all samples. Complementary genetic studies were performed when necessary. NPC disease was confirmed in 31.8% of subjects due to homozygous or compound heterozygous genetic variants in NPC1. The following four novel variants were identified: a gross deletion variant composed of the gene promoter and the first exon, NM_000271.3:c.385delT, NM_000271.3:c.1553+1342_1655-291del, and NM_000271.3:c.1757delA. None had functional activity and all resulted in important structural changes in the protein.

Muscle-tendon weakness contributes to chronic fatigue syndrome in Gaucher's disease.

BACKGROUND: Chronic fatigue (CFg) is a prevalent symptom in Gaucher disease (GD) at diagnosis (79%) and remains in a quarter of patients after years of therapy. Bone abnormalities are present in over 70% and peripheral neuropathy in about 11% of the patients, which contributes to the disabling and debilitating complications. Our hypothesis is that other factors such as muscle-tendinous weakness could have influence in the development of CFg. METHODS: We have evaluated the fiber structure and elasticity of muscle-tendinous unit by strain-elastography (S-ELA) and analyzed their influence in the CFg. S-ELA study was performed in Achilles tendon in 25 type 1 and two type 3 GD patients, all of them with fatigue and were on enzymatic replacement therapy for mean 13 years; simultaneously, bone marrow burden by MRI and calcaneus ultrasound densitometry were evaluated. Blood cell counts, plasma biomarkers, GBA1 genotyping, and SF36 quality of life scale (QoL) were also performed. STATISTICAL ANALYSIS: descriptive and comparative test. RESULTS: All patients showed a normal Achilles tendinous structure. Abnormal stiff grade 2-3 was found in 17/27 (62.9%); in 11/27 (40.7%) of patients, the alteration was bilateral. There were no correlations between the S-ELA results to other variables; nevertheless, a significant correlation between the degree of tendon hardness and the low score on the QoL scales (p = 0.0035) was found. The S-ELA is a sensitive painless, fast, and low cost method to detect muscle-tendinous subclinical dysfunction that could contribute to CFg in GD. The identification of subclinical tendon alteration would be a sign of alarm, focused on the risk of development of bone complications. CONCLUSION: Intratendinous alteration in strain-elastography is an independent variable in GD patients with persistent fatigue.

Web-Based Bioinformatics Predictors: Recommendations to Assess Lysosomal Cholesterol Trafficking Diseases-Related Genes.

INTRODUCTION: The growing number of genetic variants of unknown significance (VUS) and availability of several in silico prediction tools make the evaluation of potentially deleterious gene variants challenging. MATERIALS AND METHODS: We evaluated several programs and software to determine the one that can predict the impact of genetic variants found in lysosomal storage disorders (LSDs) caused by defects in cholesterol trafficking best. We evaluated the sensitivity, specificity, accuracy, precision, and Matthew's correlation coefficient of the most common software. RESULTS: Our findings showed that for exonic variants, only MutPred1 reached 100% accuracy and generated the best predictions (sensitivity and accuracy = 1.00), whereas intronic variants, SROOGLE or Human Splicing Finder (HSF) generated the best predictions (sensitivity = 1.00, and accuracy = 1.00). DISCUSSION: Next-generation sequencing substantially increased the number of detected genetic variants, most of which were considered to be VUS, creating a need for accurate pathogenicity prediction. The focus of the present study is the importance of accurately predicting LSDs, with majority of previously unreported specific mutations. CONCLUSION: We found that the best prediction tool for the NPC1, NPC2, and LIPA variants was MutPred1 for exonic regions and HSF and SROOGLE for intronic regions and splice sites.

Gaucher disease and chronic myeloid leukemia: first reported patient receiving enzyme replacement and tyrosine kinase inhibitor therapies simultaneously.

Report a female diagnosed as type 1 Gaucher disease after a femoral pathologic fracture when she was 55 years old. Enzyme replacement therapy was started, and she achieved therapeutic goals. In 2015, a Ph' CML with numerous pseudo-Gaucher cells in bone marrow appears. BCR/ABL was not present at GD diagnosis.

The erythrocyte osmotic resistance test as screening tool for cholesterol-related lysosomal storage diseases.

BACKGROUND: Erythrocyte volume regulation and membrane elasticity are essential for adaptation to osmotic and mechanical stress, and life span. Here, we evaluated whether defective cholesterol trafficking caused by the rare lysosomal storages diseases (LSDs), Niemann-Pick type C (NPC) and Lysosomal acid lipase (LAL) deficiency (LALD) impairs these properties. Moreover, we tested whether measurements of cholesterol membrane content and osmotic resistance serve as a screening test for these LSDs. METHODS: Patients were genotyped for mutations in NPC1, NPC2, or LIPA genes. We measured LSD plasma biomarkers and LAL activity. Red blood cells (RBC) membrane cholesterol content was evaluated in 73 subjects. Osmotic resistance tests (ORT) were conducted in 121 blood samples from LSD suspected patients and controls. RESULTS: We did not find statistically significant differences between RBC cholesterol content between subjects and controls. However, the ORT, particularly at 0.49% (w/v) hypotonic sodium chloride solution, revealed a significant higher osmotic resistance in LSDs patients than in controls. We established a cut-off value of ≤51% of haemolysis with sensibility and specificity values of 80% and 70%, respectively. CONCLUSIONS: NPC and LALD do not alter cholesterol content in the RBC membrane but increase osmotic resistance. Therefore, ORT serves as screening test for the studied LSDs.

Inhibition of Intermediate-Conductance Calcium-Activated K Channel (KCa3.1) and Fibroblast Mitogenesis by α-Linolenic Acid and Alterations of Channel Expression in the Lysosomal Storage Disorders, Fabry Disease, and Niemann Pick C.

The calcium/calmodulin-gated KCa3.1 channel regulates normal and abnormal mitogenesis by controlling K+-efflux, cell volume, and membrane hyperpolarization-driven calcium-entry. Recent studies suggest modulation of KCa3.1 by omega-3 fatty acids as negative modulators and impaired KCa3.1 functions in the inherited lysosomal storage disorder (LSD), Fabry disease (FD). In the first part of present study, we characterize KCa3.1 in murine and human fibroblasts and test the impact of omega-3 fatty acids on fibroblast proliferation. In the second, we study whether KCa3.1 is altered in the LSDs, FD, and Niemann-Pick disease type C (NPC). Our patch-clamp and mRNA-expression studies on murine and human fibroblasts show functional expression of KCa3.1. KCa currents display the typical pharmacological fingerprint of KCa3.1: Ca2+-activation, potentiation by the positive-gating modulators, SKA-31 and SKA-121, and inhibition by TRAM-34, Senicapoc (ICA-17043), and the negative-gating modulator, 13b. Considering modulation by omega-3 fatty acids we found that α-linolenic acid (α-LA) and docosahexanenoic acid (DHA) inhibit KCa3.1 currents and strongly reduce fibroblast growth. The α-LA-rich linseed oil and γ-LA-rich borage oil at 0.5% produce channel inhibition while α-LA/γ-LA-low oils has no anti-proliferative effect. Concerning KCa3.1 in LSD, mRNA expression studies, and patch-clamp on primary fibroblasts from FD and NPC patients reveal lower KCa3.1-gene expression and membrane expression than in control fibroblasts. In conclusion, the omega-3 fatty acid, α-LA, and α-LA/γ-LA-rich plant oils, inhibit fibroblast KCa3.1 channels and mitogenesis. Reduced fibroblast KCa3.1 functions are a feature and possible biomarker of cell dysfunction in FD and NPC and supports the concept that biased lipid metabolism is capable of negatively modulating KCa3.1 expression.