Pharmacokinetics and Changes in Lipid Mediator Profiling after Consumption of Specialized Pro-Resolving Lipid-Mediator-Enriched Marine Oil in Healthy Subjects.

Omega-3 polyunsaturated fatty acids (PUFAs) play a vital role in human health, well-being, and the management of inflammatory diseases. Insufficient intake of omega-3 is linked to disease development. Specialized pro-resolving mediators (SPMs) are derived from omega-3 PUFAs and expedite the resolution of inflammation. They fall into categories known as resolvins, maresins, protectins, and lipoxins. The actions of SPMs in the resolution of inflammation involve restricting neutrophil infiltration, facilitating the removal of apoptotic cells and cellular debris, promoting efferocytosis and phagocytosis, counteracting the production of pro-inflammatory molecules like chemokines and cytokines, and encouraging a pro-resolving macrophage phenotype. This is an experimental pilot study in which ten healthy subjects were enrolled and received a single dose of 6 g of an oral SPM-enriched marine oil emulsion. Peripheral blood was collected at baseline, 3, 6, 9, 12, and 24 h post-administration. Temporal increases in plasma and serum SPM levels were found by using LC-MS/MS lipid profiling. Additionally, we characterized the temporal increases in omega-3 levels and established fundamental pharmacokinetics in both aforementioned matrices. These findings provide substantial evidence of the time-dependent elevation of SPMs, reinforcing the notion that oral supplementation with SPM-enriched products represents a valuable source of essential bioactive SPMs.

Serum lipid mediator profiles in COVID-19 patients and lung disease severity: a pilot study.

Coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 infection is highly heterogeneous, ranging from asymptomatic to severe and fatal cases. COVID-19 has been characterized by an increase of serum pro-inflammatory cytokine levels which seems to be associated with fatal cases. By contrast, the role of pro-resolving lipid mediators (SPMs), involved in the attenuation of inflammatory responses, has been scarcely investigated, so further studies are needed to understand SPMs metabolism in COVID-19 and other infectious diseases. Our aim was to analyse the lipid mediator metabolome, quantifying pro- and anti-inflammatory serum bioactive lipids by LC-MS/MS in 7 non-infected subjects and 24 COVID-19 patients divided into mild, moderate, and severe groups according to the pulmonary involvement, to better understand the disease outcome and the severity of the pulmonary manifestations. Statistical analysis was performed with the R programming language (R Foundation for Statistical Computing, Vienna, Austria). All COVID-19 patients had increased levels of Prostaglandin E2. Severe patients showed a significant increase versus controls, mild- and moderate-affected patients, expressed as median (interquartile range), in resolvin E1 [112.6 (502.7) vs 0.0 (0.0) pg/ml in the other groups], as well as in maresin 2 [14.5 (7.0) vs 8.1 (4.2), 5.5 (4.3), and 3.0 (4.0) pg/ml, respectively]. Moreover, 14-hydroxy docosahexaenoic acid (14-HDHA) levels were also increased in severe vs control and mild-affected patients [24.7 (38.2) vs 2.4 (2.2) and 3.7 (6.4) ng/mL, respectively]. Resolvin D5 was also significantly elevated in both moderate [15.0 (22.4) pg/ml] and severe patients [24.0 (24.1) pg/ml] versus controls [0.0 (0.0) pg/ml]. These results were confirmed by sparse partial least squares discriminant analysis which highlighted the contribution of these mediators to the separation between each of the groups. In conclusion, the potent inflammatory response to SARS-CoV-2 infection involves not only pro- but also anti-inflammatory lipid mediators that can be quantified in easily accessible serum samples, suggesting the need to perform future research on their generation pathways that will help us to discover new therapeutic targets.

Pediatric Gaucher disease with intermediate type 2-3 phenotype associated with parkinsonian features and levodopa responsiveness.

INTRODUCTION: Gaucher disease (GD) is an autosomal recessive lysosomal storage disorder caused by a deficiency of acid ß-glucosidase encoded by the GBA gene. In patients with GD, childhood onset parkinsonian features have been rarely described. METHODS: Twin siblings with GD are described, including clinical follow-up and treatment response. Bone marrow, enzyme activity studies and genotyping were performed. RESULTS: By age 9 months, symptoms at onset were thrombocytopenia and splenomegaly. By age 2, hypokinesia, bradykinesia and oculomotor apraxia were observed. By age 5 a complete rigid hypokinetic syndrome was stablished in both patients, including bradykinesia, tremor and rigidity. Treatment with imiglucerase, miglustat, ambroxol and levodopa were performed. Levodopa showed a good response with improvement in motor and non-motor skills. Foamy cells were found in the bone marrow study. Glucocerebrosidase activity was 28% and 26%. Sanger sequencing analysis identified a missense mutation and a complex allele (NP_000148: p.[(Asp448His)]; [(Leu422Profs*4)]) in compound heterozygosity in GBA gene. CONCLUSIONS: Two siblings with neuronopathic GD with an intermediate form between type 2 and 3, with a systemic and neurological phenotype are described. The complex neurological picture included a hypokinetic-rigid and tremor syndrome that improved with levodopa treatment. These conditions together have not been previously described in pediatric GD. We suggest that in children with parkinsonian features, lysosomal storage disorders must be considered, and a levodopa trial must be performed. Moreover, this report give support to the finding that GBA and parkinsonian features share biological pathways and highlight the importance of lysosomal mechanisms in parkinsonism pathogenesis, what might have therapeutic implications.

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.

Omega-3 Polyunsaturated Fatty Acids and Their Bioactive Metabolites in Gastrointestinal Malignancies Related to Unresolved Inflammation. A Review.

Chronic inflammation takes part in the pathogenesis of some malignancies of the gastrointestinal tract including colorectal (CRC), gastric, and esophageal cancers. The use of ω3 polyunsaturated fatty acid (ω3-PUFA) supplements for chemoprevention or adjuvant therapy of gastrointestinal cancers is being investigated in recent years. Most evidence has been reported in CRC, although their protective role has also been reported for Helicobacter pylori-induced gastric cancer or Barrett's esophagus-derived adenocarcinoma. Studies based on ω3-PUFA supplementation in animal models of familial adenomatous polyposis (FAP) and CRC revealed positive effects on cancer prevention, reducing the number and size of tumors, down-regulating arachidonic acid-derived eicosanoids, upregulating anti-oxidant enzymes, and reducing lipid peroxidation, whereas contradictory results have been found in induced colitis and colitis-associated cancer. Beneficial effects have also been found in FAP and ulcerative colitis patients. Of special interest is their positive effect as adjuvants on radio- and chemo-sensitivity, specificity, and prevention of treatment complications. Some controversial results obtained in CRC might be justified by different dietary sources, extraction and preparation procedures of ω3-PUFAs, difficulties on filling out food questionnaires, daily dose and type of PUFAs, adenoma subtype, location of CRC, sex differences, and genetic factors. Studies using animal models of inflammatory bowel disease have confirmed that exogenous administration of active metabolites derived from PUFAs called pro-resolving mediators like lipoxin A4, arachidonic acid-derived, resolvins derived from eicosapentaenoic (EPA), docosahexaenoic (DHA), and docosapentaenoic (DPA) acids as well as maresin 1 and protectins DHA- and DPA-derived improve disease and inflammatory outcomes without causing immunosuppression or other side effects.

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.

Evaluation of two approaches to lysosomal acid lipase deficiency patient identification: An observational retrospective study.

BACKGROUND AND AIMS: Lysosomal acid lipase deficiency (LALD) leads to the accumulation of cholesteryl esters and/or triglycerides (TG) in lysosomes due to the lack of the enzyme codified by the LIPA gene. The most common symptoms are dyslipidaemia and hypertransaminasemia, together with manifestations common to other lysosomal storage disorders (LSDs), including visceromegalies and elevated plasma biomarkers. Alteration of the lipid-liver profile (LLP) has been widely applied as a criterion for LALD screening, but the usefulness of biomarkers has not yet been explored. Our purpose was to explore the utility of plasma chitotriosidase activity (ChT) and CCL18/PARC concentration in addition to LLP to identify LALD patients in an observational retrospective study of two different sample collections. METHODS: Biological samples refining: Collection 1 (primary hypercholesterolemia suspected) included unrelated individuals with hyperlipidaemia and without LDLR, APOB and PCSK9 gene mutations (Set 1), and Collection 2 (LSD suspected) included individuals without definitive LSD diagnosis (Set 2). We assessed plasma LLP (total cholesterol and its fractions, TG concentration and transaminases activities), as well as plasma ChT and CCL18/PARC. All subjects with anomalous LLP and/or biomarker levels were LIPA sequenced. RESULTS: Twenty-four subjects showed altered LLP and/or biomarkers. We identified two LALD patients (one homozygous and one compound heterozygous) and one carrier of a novel LIPA variant. CONCLUSIONS: The measurement of plasma ChT and CCL18/PARC combined with LLP will be a useful approach to identifying LALD patients in retrospective LALD patient studies.

Evaluation of Chitotriosidase and CC-Chemokine Ligand 18 as Biomarkers of Microglia Activation in Amyotrophic Lateral Sclerosis.

BACKGROUND: The development of biomarkers for use in diagnosing, monitoring disease progression and analyzing therapeutic trials response in amyotrophic lateral sclerosis (ALS) is essential. OBJECTIVE: The aim of this study was to identify inflammatory factors in plasma or cerebrospinal fluid (CSF) from patients with ALS with particular attention to specific markers of microglia activation as chitotriosidase (ChT) and chemokine (C-C motif) ligand 18 (CCL18) to determine its potential as ALS biomarkers. METHODS: We studied CSF and plasma samples from 32 patients and 42 healthy controls. We assayed the ChT activity by a spectrofluorometric method and protein levels of other inflammatory -biomarkers (tumor necrosis factor [TNF]-alpha, interleukin [IL]-6 and CCL18) by enzyme-linked immunosorbent assay. CHIT1 gene polymorphism in exon 10 (c.1049_1072dup24) encoding inactive ChT enzyme was genotyped in all subjects. RESULTS: ChT activity and TNF-alpha protein levels were significantly higher in CSF of ALS patients, but we found no correlation with the severity and progression of the disease. Nevertheless, we did not found any differences in CCL18 or IL-6 protein levels between both groups in CSF or plasma. In our sample, only 3% of subjects were homozygous carriers for the CHIT1 exon 10 duplication associated with defective enzyme. CONCLUSIONS: High ChT activity in CSF of patients with ALS may reflect microglia activation and could be a potential biomarker of the disease. We did not find any significant difference regarding CCL-18, another specific marker of microglia activation that is related with M2-like microglia phenotype. Deepening the understanding of the activation state of microglia (M1 and M2) may contribute to the knowledge about the specific role of neuroinflammation in ALS and future therapeutic strategies.

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.

Twelve years of experience with miglustat in the treatment of type 1 Gaucher disease: The Spanish ZAGAL project.

We report data from a prospective, observational study (ZAGAL) evaluating miglustat 100mg three times daily orally. in treatment-naïve patients and patients with type 1 Gaucher Disease (GD1) switched from previous enzyme replacement therapy (ERT). Clinical evolution, changes in organ size, blood counts, disease biomarkers, bone marrow infiltration (S-MRI), bone mineral density by broadband ultrasound densitometry (BMD), safety and tolerability annual reports were analysed. Between May 2004 and April 2016, 63 patients received miglustat therapy; 20 (32%) untreated and 43 (68%) switched. At the time of this report 39 patients (14 [36%] treatment-naïve; 25 [64%] switch) remain on miglustat. With over 12-year follow-up, hematologic counts, liver and spleen volumes remained stable. In total, 80% of patients achieved current GD1 therapeutic goals. Plasma chitotriosidase activity and CCL-18/PARC concentration showed a trend towards a slight increase. Reductions on S-MRI (p=0.042) with an increase in BMD (p<0.01) were registered. Gastrointestinal disturbances were reported in 25/63 (40%), causing miglustat suspension in 11/63 (17.5%) cases. Thirty-eight patients (60%) experienced a fine hand tremor and two a reversible peripheral neuropathy. Overall, miglustat was effective as a long-term therapy in mild to moderate naïve and ERT stabilized patients. No unexpected safety signals were identified during 12-years follow-up.

Diagnosis features of pediatric Gaucher disease patients in the era of enzymatic therapy, a national-base study from the Spanish Registry of Gaucher Disease.

BACKGROUND: The enzymatic replacement therapy (ERT) availability for Gaucher disease (GD) has changed the landscape of the disease, several countries have screening programs. These actions have promoted the early diagnosis and avoided many complications in pediatric patients. In Spain ERT has been available since 1993 and 386 patients have been included in the Spanish Registry of Gaucher Disease (SpRGD). The aim of this study is to analyze the impact of ERT on the characteristics at time of diagnosis and initial complications in pediatric Gaucher disease patients. AIM: To analyze the impact of ERT on the characteristics at time of diagnosis and initial complications in pediatric Gaucher disease patients. METHODS: A review of data in SpRGD from patients' diagnosed before 18 years old was performed. The cohort was split according the year of diagnosis (≤1994, cohort A; ≥1995, cohort B). RESULTS: A total of 98 pediatric patients were included, GD1: 80, GD3: 18; mean age: 7.2 (0.17-16.5) years, 58 (59.2%) males and 40 (40.8%) females. Forty-five were diagnosed ≤ 1994 and 53 ≥ 1995. Genotype: N370S/N370S: 2 (2.0%), N370S/L444P: 27 (27.5%), N370S/other: 47 (48%), L444P/L444P: 7 (7.1%), L444P/D409H: 2 (2.0%), L444P/other: 3 (6.2%), other/other: 10 (10.2%). The mean age at diagnosis was earlier in patients diagnosed after 1995 (p < 0.001) and different between the subtypes, GD1: 8.2 (0.2-16.5) years and GD3: 2.8 (0.17-10.2) years (p < 0.001). There were more severe patients in the group diagnosed before 1994 (p = 0.045) carrying L444P (2), D409H (2), G377S (1), G195W (1) or the recombinant mutation. The patients' diagnosed ≤1994 showed worse cytopenias, higher chance of bone vascular complications at diagnosis and previous spleen removal. The patients started ERT at a median time after diagnosis of 5.2 years [cohort A] and 1.6 years [cohort B] (p < 0.001). CONCLUSIONS: The early diagnosis of Gaucher disease in the era of ERT availability has permitted to reduce the incidence of severe and irreversible initial complication in pediatric patients, and this has permitted better development of these patients. This is the largest pediatric cohort from a national registry.

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.

Assessment of plasma chitotriosidase activity, CCL18/PARC concentration and NP-C suspicion index in the diagnosis of Niemann-Pick disease type C: a prospective observational study.

BACKGROUND: Niemann-Pick disease type C (NP-C) is a rare, autosomal recessive neurodegenerative disease caused by mutations in either the NPC1 or NPC2 genes. The diagnosis of NP-C remains challenging due to the non-specific, heterogeneous nature of signs/symptoms. This study assessed the utility of plasma chitotriosidase (ChT) and Chemokine (C-C motif) ligand 18 (CCL18)/pulmonary and activation-regulated chemokine (PARC) in conjunction with the NP-C suspicion index (NP-C SI) for guiding confirmatory laboratory testing in patients with suspected NP-C. METHODS: In a prospective observational cohort study, incorporating a retrospective determination of NP-C SI scores, two different diagnostic approaches were applied in two separate groups of unrelated patients from 51 Spanish medical centers (n = 118 in both groups). From Jan 2010 to Apr 2012 (Period 1), patients with ≥2 clinical signs/symptoms of NP-C were considered 'suspected NP-C' cases, and NPC1/NPC2 sequencing, plasma chitotriosidase (ChT), CCL18/PARC and sphingomyelinase levels were assessed. Based on findings in Period 1, plasma ChT and CCL18/PARC, and NP-C SI prediction scores were determined in a second group of patients between May 2012 and Apr 2014 (Period 2), and NPC1 and NPC2 were sequenced only in those with elevated ChT and/or elevated CCL18/PARC and/or NP-C SI ≥70. Filipin staining and 7-ketocholesterol (7-KC) measurements were performed in all patients with NP-C gene mutations, where possible. RESULTS: In total across Periods 1 and 2, 10/236 (4%) patients had a confirmed diagnosis o NP-C based on gene sequencing (5/118 [4.2%] in each Period): all of these patients had two causal NPC1 mutations. Single mutant NPC1 alleles were detected in 8/236 (3%) patients, overall. Positive filipin staining results comprised three classical and five variant biochemical phenotypes. No NPC2 mutations were detected. All patients with NPC1 mutations had high ChT activity, high CCL18/PARC concentrations and/or NP-C SI scores ≥70. Plasma 7-KC was higher than control cut-off values in all patients with two NPC1 mutations, and in the majority of patients with single mutations. Family studies identified three further NP-C patients. CONCLUSION: This approach may be very useful for laboratories that do not have mass spectrometry facilities and therefore, they cannot use other NP-C biomarkers for diagnosis.

Lyso-glycosphingolipid abnormalities in different murine models of lysosomal storage disorders.

In lysosomal glycosphingolipid storage disorders, marked elevations in corresponding glycosphingoid bases (lyso-glycosphingolipids) have been reported, such as galactosylsphingosine in Krabbe disease, glucosylsphingosine in Gaucher disease and globotriaosylsphingosine in Fabry disease. Using LC­MS/MS, we comparatively investigated the occurrence of abnormal lyso-glycosphingolipids in tissues and plasma of mice with deficiencies in lysosomal α-galactosidase A, glucocerebrosidase and galactocerebrosidase. The nature and specificity of lyso-glycosphingolipid abnormalities are reported and compared to that in correspondingly more abundant N-acylated glycosphingolipids. Specific elevations in tissue and plasma globotriaosylsphingosine were detected in α-galactosidase A-deficient mice; glucosylsphingosine in glucocerebrosidase-deficient mice and galactosylsphingosine in galactocerebrosidase-deficient animals. A similar investigation was conducted for two mouse models of Niemann Pick type C (Npc1nih and Npc1nmf164), revealing significant tissue elevation of several neutral glycosphingolipids and concomitant increased plasma glucosylsphingosine. This latter finding was recapitulated by analysis of plasma of NPC patients. The value of plasma glucosylsphingosine in biochemical confirmation of the diagnosis of NPC is discussed.