Gaucher disease, the most common lysosomal storage disorder, is caused by β-glucocerebrosidase deficiency. Bone complications are the major cause of morbidity in patients with type 1 Gaucher disease (GD1). Genetic components strongly influence bone remodelling. In addition, chronic inflammation produced by Gaucher cells induces the production of several cytokines, which leads to direct changes in the bone remodelling process and can also affect the process indirectly through other immune cells. In this study, we analysed the association between bone mineral density (BMD), bone marrow burden score, and relevant genetic polymorphisms related to bone metabolism, as well as profiles of proinflammatory cytokines in a GD1 cohort. This study included 83 patients distributed according to bone status. BMD was measured with DXA and broadband ultrasound attenuation; bone marrow involvement was evaluated using MRI. We also analysed 26 SNPs located in 14 genes related to bone metabolism. To assess proinflammatory status, we analysed IL-4, IL-6, IL-7, IL-10, IL-13, MIP-1α, MIP-1β, and TNFα in plasma samples from 71 control participants and GD1 patients. SNP genotype proportions and BMD differed significantly between ESRI c.453-397T>C and VDR c.1024+283G>A variants. We also observed significant associations between GD1 genotypes and bone affectation. When patients were stratified by spleen status, we observed significant correlations between non-/splenectomized groups and Spanish MRI (S-MRI) score. Across genotype proportions of non-/splenectomized patients and S-MRI, we observed significant differences in ESRI c.453-397T>C, VDR c.-83-25988G>A, and TNFRSF11B c.9C>G polymorphisms. We observed different significant proinflammatory profiles between control participants, treatment-naïve patients, and patients on enzyme replacement therapy (ERT); between non-/splenectomized patients (between untreated and ERT-treated patients) and among those with differing GBA genotypes. The data suggest that patients with GD1 have increased susceptibility to developing bone disease owing to the coexistence of genetic variants, and that genetic background in GD1 is fundamental to regulate the impact of proinflammatory status on the development of bone disease.

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. 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. 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. 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.

Background: Gaucher disease (GD), caused by a deficiency in acid β-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.

Accumulation of galactosphingolipids is a general characteristic of Fabry disease, a lysosomal storage disorder caused by the deficient activity of α-galactosidase A encoded by the GLA gene. Although many polymorphic GLA haplotypes have been described, it is still unclear whether some of these variants are causative of disease symptoms. We report the study of an inheritance of a complex intronic haplotype (CIH) (c.-10C > T, c.369 + 990C > A, c.370-81_370-77delCAGCC, c.640-16A > G, c.1000-22C > T) within the GLA gene associated with Fabry-like symptoms and galactosphingolipid accumulation. We analysed α-Gal A activity in plasma, leukocytes and skin fibroblasts in patients, and measured accumulation of galactosphingolipids by enzymatic methods and immunofluorescence techniques. Additionally, we evaluated GLA expression using quantitative PCR, EMSA, and cDNA cloning. CIH carriers had an altered GLA expression pattern, although most of the carriers had high residual enzyme activity in plasma, leukocytes and in skin fibroblasts. Nonetheless, CIH carriers had significant galactosphingolipid accumulation in fibroblasts in comparison with controls, and also glycolipid deposits in renal tubules and glomeruli. EMSA assays indicated that the c.-10C > T variant in the promoter affected a nuclear protein binding site. Thus, inheritance of the CIH caused an mRNA deregulation altering the GLA expression pattern, producing a tissue glycolipid storage.

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.