BackgroundMucopolysaccharidoses (MPS) are lysosomal storage diseases in which mutations of genes encoding for lysosomal enzymes cause defects in the degradation of glycosaminoglycans (GAGs). The accumulation of GAGs in lysosomes results in cellular dysfunction and clinical abnormalities. The early initiation of enzyme replacement therapy (ERT) can slow or prevent the development of severe clinical manifestations. MPS I and II newborn screening has been available in Taiwan since August 2015. Infants who failed the recheck at recall were referred to MacKay Memorial Hospital for a detailed confirmatory diagnosis.MethodsFrom August 2015 to November 2017, 294,196 and 153,032 infants were screened using tandem mass spectrometry for MPS I and MPS II, respectively. Of these infants, 84 suspected cases (eight for MPS I; 76 for MPS II) were referred for confirmation. Urinary first-line biochemistry examinations were performed first, including urinary GAG quantification, two-dimensional electrophoresis, and tandem mass spectrometry assay for predominant disaccharides derived from GAGs. If the results were positive, a confirmative diagnosis was made according to the results of leukocyte enzymatic assay and molecular DNA analysis. Leukocyte pellets were isolated from EDTA blood and used for fluorescent α-iduronidase (IDUA) or iduronate-2-sulfatase (IDS) enzymatic assay. DNA sequencing analysis was also performed.ResultsNormal IDS and IDUA enzyme activities were found in most of the referred cases except for four who were strongly suspected of having MPS I and three who were strongly suspected of having MPS II. Of these infants, three with novel mutations of the IDS gene (c.817C > T, c.1025A > G, and c.311A > T) and four with two missense mutations of the IDUA gene (C.300-3C > G, c.1874A > C; c.1037 T > G, c.1091C > T) showed significant deficiencies in IDS and IDUA enzyme activities (< 5% of mean normal activity), respectively. Urinary dermatan sulfate and heparan sulfate quantitative analyses by tandem mass spectrometry also demonstrated significant elevations. The prevalence rates of MPS I and MPS II in Taiwan were 1.35 and 1.96 per 100,000 live births, respectively.ConclusionsThe early initiation of ERT for MPS can result in better clinical outcomes. An early confirmatory diagnosis increases the probability of receiving appropriate medical care such as ERT quickly enough to avoid irreversible manifestations. All high risk infants identified in this study so far remain asymptomatic and are presumed to be affected with the attenuated disease variants.

Dysfunction of mitochondria causes defects in oxidative phosphorylation system (OXPHOS) and increased production of reactive oxygen species (ROS) triggering the activation of the cell death pathway that underlies the pathogenesis of aging and various diseases. The process of autophagy to degrade damaged cytoplasmic components as well as dysfunctional mitochondria is essential for ensuring cell survival. We analyzed the role of autophagy inpatient-specific induced pluripotent stem (iPS) cells generated from fibroblasts of patients with mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) with well-characterized mitochondrial DNA mutations and distinct OXPHOS defects. MELAS iPS cells recapitulated the pathogenesis of MELAS syndrome, and showed an increase of autophagy in comparison with its isogenic normal counterpart, whereas mitophagy is very scarce at the basal condition. Our results indicated that the existence of pathogenic mtDNA alone in mitochondrial disease was not sufficient to elicit the degradation of dysfunctional mitochondria. Nonetheless, oxidative insults induced bulk macroautophagy with the accumulation of autophagosomes and autolysosomes upon marked elevation of ROS, overload of intracellular calcium, and robust depolarization of mitochondrial membrane potential, while mitochondria respiratory function was impaired and widespread mitophagy compromised cell viability. Collectively, our studies provide insights into the dysfunction of autophagy and activation of mitophagy contributing to the pathological mechanism of mitochondrial disease.

Impairment of the ubiquitin-proteasome-system (UPS) and autophagy causing cytoplasmic aggregation of ubiquitin andp62 have been implicated in the pathogenesis of most neurodegenerative disorders, yet, they have not been fully elucidated in leukodystrophies. The relationship among impairment of UPS, autophagy, and globoid cell leukodystrophy (GLD), one of the most common demyelinating leukodystrophies, is clarified in this study. We examined the ubiquitin and autophagy markers in the brains of twitcher mice, a murine model of infantile GLD, and in human oligodendrocytes incubated with psychosine. Immunohistochemical examinations showed spatiotemporal accumulation of ubiquitin- and p62-aggregates mainly in the white matter of brain and spinal cord at disease progression. Western blot analysis demonstrated a significant accumulation of ubiquitin, p62, and LC3-II in insoluble fraction in parallel with progressive demyelination and neuroinflammation in twitcher brains. In vitro study validated a dose- and time-dependent cytotoxicity of psychosine upon autophagy and UPS machinery. Inhibition of autophagy and UPS exacerbated the accumulation of insoluble ubiquitin, p62, and LC3-II proteins mediated by psychosine cytotoxicity as well as increased cytoplasmic deposition of ubiquitin- and p62-aggregates, and accumulation of autophagosomes and autolysosomes. Further, the subsequent accumulation of reactive oxygen species and reduction of mitochondrial respiration led to cell death. Our studies validate the impairment of proteasome and autophagy underlying the pathogenesis of GLD. These findings provide a novel insight into pathogenesis of GLD and suggest a specific pathomechanism as an ideal target for therapeutic approaches.

Background/Aims: Indoxyl sulfate (IS) is a protein-bound uremic toxin that accumulates in patients with chronic kidney disease (CKD). We explored the effect of IS on human early endothelial progenitor cells (EPCs) and analyzed the correlation between serum IS levels and parameters of vascular function, including endothelial function in a CKD-based cohort. Methods: A cross-sectional study with 128 stable CKD patients was conducted. Flow-mediated dilation (FMD), pulse wave velocity (PWV), ankle brachial index, serum IS and other biochemical parameters were measured and analyzed. In parallel, the activity of early EPCs was also evaluated after exposure to IS. Results: In human EPCs, a concentration-dependent inhibitory effect of IS on chemotactic motility and colony formation was observed. Additionally, serum IS levels were significantly correlated with CKD stages. The total IS (T-IS) and free IS (F-IS) were strongly associated with age, hypertension, cardiovascular disease, blood pressure, PWV, blood urea nitrogen, creatine and phosphate but negatively correlated with FMD, the estimated glomerular filtration rate (eGFR), hemoglobin, hematocrit, and calcium. A multivariate linear regression analysis also showed that FMD was significantly associated with IS after adjusting for other confounding factors. Conclusions: In humans, IS impairs early EPCs and was strongly correlated with vascular dysfunction. Thus, we speculate that this adverse effect of IS may partly result from the inhibition of early EPCs.

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BackgroundThe aim of this study was to use the liquid chromatography/tandem mass spectrometry (LC‐MS/MS) method to quantitate levels of three urinary glycosaminoglycans (GAGs; dermatan sulfate [DS], heparan sulfate [HS], and keratan sulfate [KS]) to help make a correct diagnosis of mucopolysaccharidosis (MPS).MethodsWe analyzed the relationships between phenotypes and levels of urinary GAGs of 79 patients with different types of MPS.ResultsThe patients with mental retardation (n = 21) had significantly higher levels of HS than those without mental retardation (n = 58; 328.8 vs. 3.2 μg/ml, p < 0.001). The DS levels in the patients with hernia, hepatosplenomegaly, claw hands, coarse face, valvular heart disease, and joint stiffness were higher than those without. Twenty patients received enzyme replacement therapy (ERT) for 1–12.3 years. After ERT, the KS level decreased by 90% in the patients with MPS IVA compared to a 31% decrease in the change of dimethylmethylene blue (DMB) ratio. The DS level decreased by 79% after ERT in the patients with MPS VI compared to a 66% decrease in the change of DMB ratio.ConclusionsThe measurement of GAG fractionation biomarkers using the LC‐MS/MS method is a more sensitive and reliable tool than the DMB ratio for MPS high‐risk screening, diagnosis, subclass identification, and monitoring the efficacy of ERT.

Mucopolysaccharidosis (MPS) is caused by the deficiency of a specific hydrolytic enzyme that catalyzes the step-wise degradation of glycosaminoglycans (GAGs). In this study, we propose an empirical method to calculate levels of GAG-derived disaccharides based on the quantity (peak areas) of chondroitin sulfate (CS) with the aim of making a diagnosis of MPS more accurate and reducing the occurrence of false positive and false negative results. In this study, levels of urinary GAG-derived disaccharides were measured in 67 patients with different types of MPS and 165 controls without MPS using a tandem mass spectrometry assay. Two different methods of reporting GAG-derived disaccharides were assessed; normalization to urinary CS (in μg/mL), and normalization to μg/mg creatinine. CS-normalization yielded more consistent values than creatinine-normalization. In particular, levels of urinary dermatan sulfate (DS), heparan sulfate (HS), and keratan sulfate (KS) significantly varied because of changes in urine creatinine levels, which were proportional to age but inversely proportional to DS, HS, and KS measurements. Using CS-normalization revealed the actual status of DS, HS, and KS without the influence of factors such as age, urine creatinine, and other physiological conditions. It could discriminate between the patients with MPS and controls without MPS, and also to evaluate changes in GAG levels pre- and post-enzyme replacement therapy.

Background/Aims: Advanced glycation end products (AGEs) are pro-inflammatory and pro-oxidative compounds that play a critical role in endothelial dysfunction and atherosclerosis. Protein-bound uremic toxins, indoxyl sulfate (IS) and p-cresyl sulfate (PCS), inhibit endothelial function. We explored the association of IS and PCS with AGEs in a hemodialysis (HD) cohort. Methods: This study was a cross-sectional study that recruited 129 stable patients on maintenance HD in a single medical center from July 1 to July 15, 2011. Serum levels of total and free IS, PCS and AGEs were measured concurrently. General laboratory results and patient background were also investigated. Results: Serum levels of AGEs were associated with total IS (r = 2.7, p < 0.01) but not total PCS (r = 0.01, NS), free IS (r = 0.11, NS) or free PCS (r = 0.04, NS) using Pearson's analysis. Multiple linear regression analysis showed that total IS was significantly related to AGEs (β = 0.296, p < 0.01), free IS (β = 0.502, p < 0.01) and creatinine (β = 0.294, p < 0.01). Serum AGEs levels were also independently correlated with diabetes status (β = 0.250, p = 0.01) and total IS (β = 0.341, p < 0.01) concentrations after adjusting for other confounding variables. Moreover, patients with diabetes had higher serum AGEs levels than patients without diabetes (p < 0.01). Conclusions: These findings suggest that serum levels of total IS were associated with AGEs levels, which may participate in the process of atherosclerosis.