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Krabbe disease (KD) is a rare lysosomal storage disorder caused by biallelic pathogenic variants in GALC. Most patients manifest the severe classic early-infantile form, while a small percentage of cases have later-onset types. We present two siblings with atypical clinical and neuroimaging phenotypes, compared to the classification of KD, who were found to carry biallelic loss-of-function GALC variants, including a recurrent 30 kb deletion and a previously unreported deep intronic variant that was identified by mRNA sequencing. This family represents a unique description in the KD literature and contributes to expanding the clinical and molecular spectra of this rare disorder.

Niemann-Pick disease (NPD) type A and type B are part of the spectrum disease of the acid sphingomyelinase deficiency (ASMD). Plasma lipid abnormalities are frequently associated with both NPD-A and NPD-B, and include decreased high-density lipoprotein cholesterol (HDL-C), increased low-density lipoprotein cholesterol (LDL-C), and hypertriglyceridemia. The atherogenic lipid profile has been associated to early atherosclerotic vascular disease and coronary artery disease in NPD-B patients. Thus, early treatment of dyslipidemia in these patients is advisable. We present here a pediatric case of NPD-B with an atherogenic lipid profile not responding to lifestyle changes, low fat diet, and daily supplementation with plant sterols. We reviewed the existing literature about the treatment strategies for dyslipidemia in ASMD patients, with a special focus on the pediatric age. Finally, we speculated on the mechanisms underlying dyslipidemia in this disorder. The clinical experiences in lipid-lowering strategies in NPD-B patients are limited, in particular in the pediatric age. Olipudase alfa appears as the most promising candidate for improving lipid profile. Since olipudase alfa is not yet approved and, due to its costs, it will probably not be available for all patients worldwide, further research is needed to broaden our knowledge on this clinical need and to evaluate the efficacy and the long-term effects of lipid-lowering agents in ASMD patients. A deep understanding of the pathophysiology of dyslipidemia in ASMD may promote the identification of new targets and support the identification of new therapeutic strategies.

Acid sphingomyelinase deficiency (ASMD) is an ultra-rare disease, and several gaps of knowledge on various issues remain, particularly at a regional/national level. Expert opinions collected through well-defined consensus methodologies are increasingly used to make available reliable information in the context of rare/ultra-rare diseases. With the aim to provide indications on infantile neurovisceral ASMD (also formerly known as Niemann–Pick disease type A), chronic neurovisceral ASMD (formerly known as Niemann–Pick disease type A/B) and chronic visceral ASMD (formerly known as Niemann–Pick disease type B) in Italy, we conducted a Delphi consensus of experts focused on five main areas: (i) patients and disease characteristics; (ii) unmet needs and quality of life; (iii) diagnostic issues; (iv) treatment-related aspects; and (v) patient journey. Pre-specified, objective criteria were used to outline the multidisciplinary panel, based on 19 Italian experts in ASMD in paediatric and adult patients from different Italian Regions, including both clinicians (n = 16) and ASMD patients’ advocacy or payors with expertise in rare diseases (n = 3). During two Delphi rounds, a high ratio of agreement was found on several topics related to ASMD characteristics, diagnosis, management and disease burden. Our findings may provide valuable indications for management of ASMD at a public health level in Italy.

Niemann-Pick C disease (NPC) is a lysosomal disease caused by mutations in NPC1 or NPC2 genes responsible for intracellular accumulation of free cholesterol and glycosphingolipids in a variety of tissues. We collected plasma samples from 15 NPC1 patients and 15 age-matched controls to analyze the impairment of lipid metabolism. Comprehensive-targeted quantitative lipidomic analysis was per-formed by Ion Mobility Mass Spectrometry, while oxysterols and lyso-sphingolipids, the classical NPC biomarkers, were analyzed by LC-MS/MS. Lipidomic analysis allowed the quantitation of ~1100 lipid species, belonging to 13 different classes. Statistical analysis of collected data showed a significant differentiation between NPC patients and controls. Lipid profiling showed an elevation of arachidonic acid and total diacylglycerols. Conversely, sphingomyelins, phosphatidylethano-lamines, phosphatidylcholines, cholesterylesters, and lactosylceramides were decreased. Indeed, the lipid imbalance was consistent with the increased concentrations of oxysterols and lyso-sphingolipids. Our study revealed a novel disease biosignature suggesting new potential diagnostic biomarkers. The alteration in key lipids molecules involved in inflammatory pathways and in oxidative stress regulation, provides new insights in the complex pathophysiology of the disease, still largely un-known.

Background Oligosaccharidoses are storage disorders due to enzymatic defects involved in the breakdown of the oligosaccharidic component of glycosylated proteins. The defect cause the accumulation of oligosaccharides (OS) and, depending on the lacking enzyme, results in characteristic profiles which are helpful for the diagnosis. We developed a new tandem mass spectrometry method for the screening of urinary OS which was applied to identify a large panel of storage disorders. Methods The method was set-up in urine and dried urine spots (DUS). Samples were analysed, without derivatization and using maltoheptaose as internal standard, by UHPLC-MS/MS with MRM acquisition of target OS transitions, including Glc4, the biomarker of Pompe disease. The chromatographic run was Results The method allowed to confirm the diagnosis of oligosaccharidoses (sialidosis, α-/β-mannosidosis, fucosidosis, aspartylglucosaminuria) and of GM1 and GM2 (Sandhoff type) gangliosidosis, by detecting specific OS profiles. In other storage disorders (mucolipidosis II and III, mucopolysaccharidosis type IVB) the analyisis revealed abnormal OS excretion with non-specific profiles. Besides Pompe disease, the tetrasaccharide Glc4 was increased also in disorders of autophagy (Vici syndrome, Yunis-Varon syndrome, and Danon disease) presenting cardiomuscular involvement with glycogen storage. Overall, results showed a clear separation between patients and controls, both in urine and in DUS. Conclusion This new UHPLC/MS-MS method, which is suitable for rapid and easy screening of OS in urine and DUS, expands the detection of storage disorders from oligosaccharidoses to other diseases, including the novel category of inherited disorders of autophagy.

Pompe disease (PD) is caused by deficiency of the enzyme acid α-glucosidase resulting in glycogen accumulation in lysosomes. Clinical symptoms include skeletal myopathy, respiratory failure, and cardiac hypertrophy. We studied plasma proteomic and lipidomic profiles in 12 PD patients compared to age-matched controls. The proteomic profiles were analyzed by nLC-MS/MS SWATH method. Wide-targeted lipidomic analysis was performed by LC-IMS/MS, allowing to quantify >1100 lipid species, spanning 13 classes. Significant differences were found for 16 proteins, with four showing the most relevant changes (GPLD1, PON1, LDHB, PKM). Lipidomic analysis showed elevated levels of three phosphatidylcholines and of the free fatty acid 22:4, and reduced levels of six lysophosphatidylcholines. Up-regulated glycolytic enzymes (LDHB and PKM) are involved in autophagy and glycogen metabolism, while down-regulated PON1 and GPLD1 combined with lipidomic data indicate an abnormal phospholipid metabolism. Reduced GPLD1 and dysregulation of lipids with acyl-chains characteristic of GPI-anchor structure suggest the potential involvement of GPI-anchor system in PD. Results of proteomic analysis displayed the involvement of multiple cellular functions affecting inflammatory, immune and antioxidant responses, autophagy, Ca2+ -homeostasis, and cell adhesion. The combined multi-omic approach revealed new biosignatures in PD, providing novel insights in disease pathophysiology with potential future clinical application.

Background Infantile cholestasis (IC) is defined as an impairment of bile production or flow occurring in the first months of life. The diagnostic approach in IC is challenging since the differential diagnosis is broad. Methods We retrospectively evaluated 91 cholestatic infants referred to our department from 2014 to 2019. Patients with cholestasis underwent a complete IC diagnostic work-up including quantification of plasma oxysterols 7-ketocholesterol (7-KC) and cholestan-3β,5α,6β-triol (C-Triol). Results Oxysterols concentrations were mildly elevated in IC compared to control population. 7-KC and C-Triol plasma levels presented a linear relationship between them and with Spleen-Z score. Patients with NP-C showed the highest concentrations of both oxysterols compared with other etiologies of IC. Excluding NP-C patients, oxysterols concentrations were similar among all other etiological groups with no correlations found between them and the levels of cholesterol and bilirubin. ROC analysis identified AUCs of 1.0 for both oxysterols in predicting NP-C. Conclusion Infants with IC should undergo a stepwise evaluation in which detailed clinical and deep analytical assessments are the main crossroads. Plasma oxysterols, a simple, reliable, and convenient diagnostic test should be included in the first steps of the diagnostic process in IC.

Niemann-Pick disease type C (NPC) is a rare, genetic, progressive neurodegenerative disorder with high unmet medical need. We investigated the safety and efficacy of arimoclomol, which amplifies the heat shock response to target NPC protein misfolding and improve lysosomal function, in patients with NPC. In a 12-month, prospective, randomised, double-blind, placebo-controlled, phase 2/3 trial (ClinicalTrials.gov identifier: NCT02612129), patients (2-18 years) were randomised 2:1 to arimoclomol:placebo, stratified by miglustat use. Routine clinical care was maintained. Arimoclomol was administered orally three times daily. The primary endpoint was change in 5-domain NPC Clinical Severity Scale (NPCCSS) score from baseline to 12 months. Fifty patients enrolled; 42 completed. At month 12, the mean progression from baseline in the 5-domain NPCCSS was 0.76 with arimoclomol vs 2.15 with placebo. A statistically significant treatment difference in favour of arimoclomol of -1.40 (95% confidence interval: -2.76, -0.03; P=.046) was observed, corresponding to a 65% reduction in annual disease progression. In the prespecified subgroup of patients receiving miglustat as routine care, arimoclomol resulted in stabilisation of disease severity over 12 months with a treatment difference of -2.06 in favour of arimoclomol (P=.006). Adverse events occurred in 30/34 patients (88.2%) receiving arimoclomol and 12/16 (75.0%) receiving placebo. Fewer patients had serious adverse events with arimoclomol (5/34, 14.7%) vs placebo (5/16, 31.3%). Treatment-related serious adverse events (n=2) included urticaria and angioedema. Arimoclomol provided a significant and clinically meaningful treatment effect in NPC and was well tolerated.

Background Classic infantile onset of Pompe disease (c-IOPD) leads to hypotonia and hypertrophic cardiomyopathy within the first days to weeks of life and, without treatment, patients die of cardiorespiratory failure in their first 1–2 years of life. Enzymatic replacement therapy (ERT) with alglucosidase alfa is the only available treatment, but adverse immune reactions can reduce ERT’s effectiveness and safety. It is therefore very important to identify strategies to prevent and manage these complications. Several articles have been written on this disease over the last 10 years, but no univocal indications have been established. Methods Our study presents a review of the current literature on management of immune responses to ERT in c-IOPD as considered by an Italian study group of pediatric metabolists and immunologists in light of our shared patient experience. Results We summarize the protocols for the management of adverse reactions to ERT, analyzing their advantages and disadvantages, and provide expert recommendations for their optimal management, to the best of current knowledge. However, further studies are needed to improve actual management protocols, which still have several limitations.

Mucopolysaccharidosis type I (MPS-I) is a severe genetic disease caused by a deficiency of the alpha-L-iduronidase (IDUA) enzyme. Ex vivo hematopoietic stem cell (HSC) gene therapy is a promising therapeutic approach for MPS-I, as demonstrated by preclinical studies performed in naive MPS-I mice. However, after enzyme replacement therapy (ERT), several MPS-I patients develop anti-IDUA immunity that may jeopardize ex vivo gene therapy efficacy. Here we treat MPS-I mice with an artificial immunization protocol to mimic the ERT effect in patients, and we demonstrate that IDUA-corrected HSC engraftment is impaired in pre-immunized animals by IDUA-specific CD8+ T cells spared by pre-transplant irradiation. Conversely, humoral anti-IDUA immunity does not impact on IDUA-corrected HSC engraftment. The inclusion of lympho-depleting agents in pre-transplant conditioning of pre-immunized hosts allowes rescue of IDUA-corrected HSC engraftment, which is proportional to CD8+ T cell eradication. Overall, these data demonstrate the relevance of pre-existing anti-transgene T cell immunity on ex vivo HSC gene therapy, and they suggest the application of tailored immune-depleting treatments, as well as a deeper immunological characterization of patients, to safeguard the therapeutic effects of ex vivo HSC gene therapy in immunocompetent hosts., MPS-I patients receiving enzyme replacement therapy develop an anti-IDUA immune response that may affect ex vivo gene therapy. Squeri et al. show, in the MPS-I murine model, that pre-existing IDUA-specific CD8+ T cell response impairs LV-transduced HSC engraftment and that the efficacy of ex vivo gene therapy is rescued by lympho-depleting drugs.

Background Mitochondrial respiratory chain consists of five complexes encoded by nuclear and mitochondrial genomes. Mitochondrial aminoacyl-tRNA synthetases are key enzymes in the synthesis of such complexes. Bi-allelic variants of VARS2, a nuclear gene encoding for valyl-tRNA (Val-tRNA) synthetase, are associated to several forms of mitochondrial encephalopathies or cardiomyoencephalopathies. Among these, the rare homozygous c.1100C > T (p.Thr367Ile) mutation variably presents with progressive developmental delay, axial hypotonia, limbs spasticity, drug-resistant epilepsy leading, in some cases, to premature death. Yet only six cases, of which three are siblings, harbouring this homozygous mutation have been described worldwide. Case presentation Hereby, we report two additional cases of two non-related young girls from Sardinia, born from non-consanguineous and healthy parents, carrying the aforesaid homozygous VARS2 variant. At onset both the patients presented with worsening psychomotor delay, muscle hypotonia and brisk tendon reflexes. Standard genetic tests were normal, as well as metabolic investigations. Brain MRI showed unspecific progressive abnormalities, such as corpus callosum hypoplasia (patient A) and cerebellar atrophy (patient A and B). Diagnosis was reached by adopting massive parallel next generation sequencing. Notably clinical phenotype of the first patient appears to be milder compared to previous known cases. The second patient eventually developed refractory epilepsy and currently presents with severe global impairment. Because no specific treatment is available as yet, both patients are treated with supporting antioxidant compounds along with symptomatic therapies. Conclusions Given the paucity of clinical data about this very rare mitochondrial encephalopathy, our report might contribute to broaden the phenotypic spectrum of the disorder. Moreover, noteworthy, three out of five pedigrees so far described belong to the Northern Sardinia ethnicity.

PURPOSE: We studied microRNAs as potential biomarkers for Pompe disease. METHODS: We analyzed microRNA expression by small RNA-seq in tissues from the disease murine model at two different ages (3 and 9 months), and in plasma from Pompe patients. RESULTS: In the mouse model we found 211 microRNAs that were differentially expressed in gastrocnemii and 66 in heart, with a different pattern of expression at different ages. In a preliminary analysis in plasma from six patients 55 microRNAs were differentially expressed. Sixteen of these microRNAs were common to those dysregulated in mouse tissues. These microRNAs are known to modulate the expression of genes involved in relevant pathways for Pompe disease pathophysiology (autophagy, muscle regeneration, muscle atrophy). One of these microRNAs, miR-133a, was selected for further quantitative real-time polymerase chain reaction analysis in plasma samples from 52 patients, obtained from seven Italian and Dutch biobanks. miR-133a levels were significantly higher in Pompe disease patients than in controls and correlated with phenotype severity, with higher levels in infantile compared with late-onset patients. In three infantile patients miR-133a decreased after start of enzyme replacement therapy and evidence of clinical improvement. CONCLUSION: Circulating microRNAs may represent additional biomarkers of Pompe disease severity and of response to therapy.

An amendment to this paper has been published and can be accessed via the original article.

Background Glycogen storage disease type II (GSDII) or Pompe disease is a rare autosomal recessive metabolic disorder that leads to intracellular glycogen storage in many tissues, mainly in skeletal muscle, heart and liver. Facial muscle weakness and altered craniofacial growth are very common in Pompe disease children. In this paper we describe the orofacial features in two children affected by GSDII and illustrate a multidisciplinary approach that involved enzyme replace therapy, non-invasive ventilation (NIV) and pediatric dentistry with 5-year follow-up. Results Two Infantile Pompe Disease children were examined by a pediatric dentist at the age of 4 and 5 years old respectively. The orofacial examination showed typical facies with similar features: hypotonia of facial and tongue muscles, lip incompetence, narrow palate with reduction in transversal dimension of the upper dental arch, macroglossia, low position of the tip of the tongue, concave profile, Class III malocclusion with hypoplasia of maxillary-malar area and mandibular prognathism. Myofunctional therapy and orthodontic treatment consisted in oral muscle exercises associated to intraoral and extraoral orthodontic devices. NIV facial mask was substituted with a nasal pillow mask in order to avoid external pressure on the mid-face which negatively influences craniofacial growth. Conclusions This paper evidences that the pediatric dentist plays an important role in craniofacial growth control, oral function rehabilitation and, therefore, in the improvement of the quality of life of Pompe children and their families. Therefore an early pediatric dental evalutation should be included in the multidisciplinary management of children suffering from Infantile Pompe Disease.

BackgroundNiemann–Pick disease type C (NPC) is a rare, progressive, neurodegenerative disease associated with neurovisceral manifestations resulting from lysosomal dysfunction and aberrant lipid accumulation. A multicentre, prospective observational study (ClinicalTrials.gov ID:NCT02435030) of individuals with genetically confirmed NPC1 or NPC2 receiving routine clinical care was conducted, to prospectively characterize and measure NPC disease progression and to investigate potential NPC-related biomarkers versus healthy individuals. Progression was measured using the abbreviated 5-domain NPC Clinical Severity Scale (NPCCSS), 17-domain NPCCSS and NPC clinical database (NPC-cdb) score. Cholesterol esterification and heat shock protein 70 (HSP70) levels were assessed from peripheral blood mononuclear cells (PBMCs), cholestane-3β,5α-,6β-triol (cholestane-triol) from serum, and unesterified cholesterol from both PBMCs and skin biopsy samples. The inter- and intra-rater reliability of the 5-domain NPCCSS was assessed by 13 expert clinicians’ rating of four participants via video recordings, repeated after ≥3 weeks. Intraclass correlation coefficients (ICCs) were calculated.ResultsOf the 36 individuals with NPC (2–18 years) enrolled, 31 (86.1%) completed the 6–14-month observation period; 30/36 (83.3%) were receiving miglustat as part of routine clinical care. A mean (±SD) increase in 5-domain NPCCSS scores of 1.4 (±2.9) was observed, corresponding to an annualized progression rate of 1.5. On the 17-domain NPCCSS, a mean (±SD) progression of 2.7 (±4.0) was reported. Compared with healthy individuals, the NPC population had significantly lower levels of cholesterol esterification (ppp=0.0006). Cholestane-triol levels were significantly higher in individuals with NPC versus healthy individuals (p=0.008) and correlated with the 5-domain NPCCSS (Spearman’s correlation coefficient=0.265, p=0.0411). The 5-domain NPCCSS showed high ICC agreement in inter-rater reliability (ICC=0.995) and intra-rater reliability (ICC=0.937). ConclusionsProgression rates observed were consistent with other reports on disease progression in NPC. The 5-domain NPCCSS reliability study supports its use as an abbreviated alternative to the 17-domain NPCCSS that focuses on the most relevant domains of the disease. The data support the use of cholestane-triol as a disease monitoring biomarker and the novel methods of measuring unesterified cholesterol could be applicable to support NPC diagnosis. Levels of HSP70 in individuals with NPC were significantly decreased compared with healthy individuals.

Background Niemann–Pick disease type C (NPC) is a rare, progressive, neurodegenerative disease associated with neurovisceral manifestations resulting from lysosomal dysfunction and aberrant lipid accumulation. A multicentre, prospective observational study (Clinical Trials.gov ID: NCT02435030) of individuals with genetically confirmed NPC1 or NPC2 receiving routine clinical care was conducted, to prospectively characterize and measure NPC disease progression and to investigate potential NPC-related biomarkers versus healthy individuals. Progression was measured using the abbreviated 5-domain NPC Clinical Severity Scale (NPCCSS), 17-domain NPCCSS and NPC clinical database (NPC-cdb) score. Cholesterol esterification and heat shock protein 70 (HSP70) levels were assessed from peripheral blood mononuclear cells (PBMCs), cholestane-3β,5α-,6β-triol (cholestane-triol) from serum, and unesterified cholesterol from both PBMCs and skin biopsy samples. The inter- and intra-rater reliability of the 5-domain NPCCSS was assessed by 13 expert clinicians’ rating of four participants via video recordings, repeated after ≥ 3 weeks. Intraclass correlation coefficients (ICCs) were calculated. Results Of the 36 individuals with NPC (2–18 years) enrolled, 31 (86.1%) completed the 6–14-month observation period; 30/36 (83.3%) were receiving miglustat as part of routine clinical care. A mean (± SD) increase in 5-domain NPCCSS scores of 1.4 (± 2.9) was observed, corresponding to an annualized progression rate of 1.5. On the 17-domain NPCCSS, a mean (± SD) progression of 2.7 (± 4.0) was reported. Compared with healthy individuals, the NPC population had significantly lower levels of cholesterol esterification (p p p = 0.0006). Cholestane-triol levels were significantly higher in individuals with NPC versus healthy individuals (p = 0.008) and correlated with the 5-domain NPCCSS (Spearman’s correlation coefficient = 0.265, p = 0.0411). The 5-domain NPCCSS showed high ICC agreement in inter-rater reliability (ICC = 0.995) and intra-rater reliability (ICC = 0.937). Conclusions Progression rates observed were consistent with other reports on disease progression in NPC. The 5-domain NPCCSS reliability study supports its use as an abbreviated alternative to the 17-domain NPCCSS that focuses on the most relevant domains of the disease. The data support the use of cholestane-triol as a disease monitoring biomarker and the novel methods of measuring unesterified cholesterol could be applicable to support NPC diagnosis. Levels of HSP70 in individuals with NPC were significantly decreased compared with healthy individuals. Trial registration CT-ORZY-NPC-001: ClincalTrials.gov NCT02435030, Registered 6 May 2015, https://clinicaltrials.gov/ct2/show/NCT02435030; EudraCT 2014–005,194-37, Registered 28 April 2015, https://www.clinicaltrialsregister.eu/ctr-search/trial/2014-005194-37/DE. OR-REL-NPC-01: Unregistered.

Niemann-Pick type C (NPC) disease is an autosomal recessive lysosomal storage disorder caused by mutations in NPC1 or NPC2 genes. In 2009, the molecular characterization of 44 NPC Italian patients has been published. Here, we present an update of the genetic findings in 105 Italian NPC patients belonging to 83 unrelated families (77 NPC1 and 6 NPC2). NPC1 and NPC2 genes were studied following an algorithm recently published. Eighty-four different NPC1 and five NPC2 alleles were identified. Only two NPC1 alleles remained non detected. Sixty-two percent of NPC1 alleles were due to missense variants. The most frequent NPC1 mutation was the p.F284Lfs*26 (5.8% of the alleles). All NPC2 mutations were found in the homozygous state, and all but one was severe. Among newly diagnosed patients, 18 novel NPC1 mutations were identified. The pathogenic nature of 7/9 missense alleles and 3/4 intronic variants was confirmed by filipin staining and NPC1 protein analysis or mRNA expression in patient’s fibroblasts. Taken together, our previous published data and new results provide an overall picture of the molecular characteristics of NPC patients diagnosed so far in Italy.

Background Although representing two distinct disease entities, Niemann-Pick disease type C (NP-C) disease and acid sphingomyelinase deficiency (ASMD) share several phenotypic features. The lack of biomarkers was responsible in the past of diagnostic delay. Recently, plasma oxysterols, cholestan-3β,5α,6β-triol (Triol) and 7-ketocholesterol (7-KC) and lysosphingolipids, Lyso-sphingomyelin (Lyso-SM) and Lysosphingomyelin-509 (Lyso-SM-509), have been proposed as diagnostic biomarkers. We aimed to assess the diagnostic power of the two biomarkers categories and to evaluate possible correlations with patients' age and clinical phenotypes. Patients and methods We analyzed plasma oxysterols and lysosphingolipids in patients affected by NP-C and ASMD, and compared with healthy controls. Results Oxysterols were always increased in both NP-C and ASMD. In NP-C, Lyso-SM and Lyso-SM-509 were increased in 70%, and 100% of patients, respectively. Biomarkers negatively correlated with patients' age, with highest levels in early-infantile, intermediate in the late-infantile and lowest in the juvenile phenotype. In ASMD, lysosphingolipids were both increased, with a greater order of magnitude than in NP-C, with highest levels in chronic-neurovisceral vs visceral phenotype. Conclusions Lysosphingolipids are useful biomarkers for a rapid and precise diagnosis, allowing clear distinction between NP-C and ASMD. They are more reliable biomarkers than oxysterols and correlate with patients' age and clinical phenotype.

BACKGROUND: Enzyme replacement therapy (ERT) has deeply modified the clinical history of Infantile Onset Pompe Disease (IOPD). However, its long-term effectiveness is still not completely defined. Available data shows a close relationship between clinical outcome and patients' cross-reactive immunological status (CRIM), being CRIM-negative status a negative prognostic factor. At the same time limited data are available on the long-term treatment in CRIM-positive infants. METHODS: A retrospective multicentre observational study was designed to analyse the long-term effectiveness of ERT in IOPD. Thirteen Italian centres spread throughout the country were involved and a cohort of 28 patients (15 females, 13 males, born in the period: February 2002-January 2013) was enrolled. IOPD diagnosis was based on clinical symptoms, enzymatic and molecular analysis. All patients received ERT within the first year of life. Clinical, laboratory, and functional data (motor, cardiac and respiratory) were collected and followed for a median period of 71 months (5 years 11 months). RESULTS: Median age at onset, diagnosis and start of ERT were 2, 3 and 4 months, respectively. CRIM status was available for 24/28 patients: 17/24 (71%) were CRIM-positive. Nineteen patients (67%) survived > 2 years: 4 were CRIM-negative, 14 CRIM-positive and one unknown. Six patients (5 CRIM-positive and one unknown) never needed ventilation support (21,4%) and seven (6 CRIM-positive and one unknown: 25%) developed independent ambulation although one subsequently lost this function. Brain imaging study was performed in 6 patients and showed peri-ventricular white matter abnormalities in all of them. Clinical follow-up confirmed the better prognosis for CRIM-positive patients, though a slow, progressive worsening of motor and/or respiratory functions was detected in 8 patients. CONCLUSIONS: These data are the result of the longest independent retrospective study on ERT in IOPD reported so far outside clinical trials. The data obtained confirmed the better outcome of the CRIM-positive patients but at the same time, showed the inability of the current therapeutic approach to reverse or stabilize the disease progression. The results also evidenced the involvement of central nervous system in Pompe disease. To better understand the disease clinical history and to improve treatment efficacy larger multicentre studies are needed as well as the development of new therapeutic approaches.

Introduction: The aim of this study was to evaluate the muscle MRI pattern of 9 patients (median age: 6.5±2.74 years) affected by classic infantile-onset Pompe disease (IOPD) who were treated with enzyme replacement therapy (ERT). Methods: We performed and qualitatively scored T1-weighted (T1-w) sequences of the facial, shoulder girdle, paravertebral, and lower limb muscles and short-tau inversion recovery (STIR) sequences of the lower limbs using the Mercuri and Morrow scales, respectively. Results: On T1-w images, mild (grade 1) or moderate (grade 2) involvement was found in the tongue in 6 of 6 patients and in the adductor magnus muscle in 6 of 9. STIR hyperintensity was detected in all areas examined, categorized as limited to mild in 5 of 8 patients. Discussion: On T1-w sequences, mild/moderate adipose substitution in the adductor magnus and tongue muscles was documented. STIR edema-like alterations of thigh and calf muscles are novel findings. Correlations with biopsy findings and clinical parameters are needed to fully understand these findings. This article is protected by copyright. All rights reserved.

Propionic acidemia (PA) (MIM# 606054) is a rare inborn error of metabolism due to deficient activity of propionyl-CoA carboxylase. Patients present with life-threatening episodes of acute metabolic decompensation and disease course is characterized by multiorgan involvement with frequent neurologic dysfunction that includes developmental delay, movement disorders/dystonia, seizures, stroke-like episodes, psychiatric symptoms, and basal ganglia lesions. Standard treatment relies on the dietary restriction of propionate precursors combined with carnitine supplementation, which buffers mitochondrial propionate accumulation. To reduce propionate production by intestinal flora,1,2 chronic cyclic metronidazole (MTZ) administration is advised.3

GM1 ganglioside, a monosialic glycosphingolipid and a crucial component of plasma membranes, accumulates in lysosomal storage disorders, primarily in GM1 gangliosidosis. The development of biomarkers for simplifying diagnosis, monitoring disease progression and evaluating drug therapies is an important objective in research into neurodegenerative lysosomal disorders. With this in mind, we established fluorescent imaging and flow-cytometric methods to track changes in GM1 ganglioside levels in patients with GM1 gangliosidosis and in control cells. We also evaluated GM1 ganglioside content in patients’ cells treated with the commercially available Miglustat, a substrate inhibitor potentially suitable for the treatment of late-onset GM1 gangliosidosis. The flow-cytometric method proved to be sensitive, unbiased, and rapid in determining variations in GM1 ganglioside content in human lymphocytes derived from small amounts of fresh blood. We detected a strong correlation between GM1 ganglioside content and the clinical severity of GM1 gangliosidosis. We confirm the ability of Miglustat to act as a substrate reduction agent in the patients’ treated cells. As well as being suitable for diagnosing and managing patients with GM1 gangliosidosis this method could be useful in the diagnosis and management of other lysosomal diseases, such as galactosialidosis, Type C Niemann-Pick, and any other disease with pathologic variations of GM1 ganglioside.

The aim of this review is to summarize the evidence on efficacy, effectiveness and safety of intravenous enzyme replacement therapy (ERT) available for mucopolysaccharidoses (MPSs) I, II, IVA, VI and VII, gained in phase III clinical trials and in observational post-approval studies. Post-marketing data are sometimes conflicting or controversial, possibly depending on disease severity, differently involved organs, age at starting treatment, and development of anti-drug antibodies (ADAs). There is general agreement that ERT is effective in reducing urinary glycosaminoglycans and liver and spleen volume, while heart and joints outcomes are variable in different studies. Effectiveness on cardiac valves, trachea and bronchi, hearing and eyes is definitely poor, probably due to limited penetration in the specific tissues. ERT does not cross the blood–brain barrier, with the consequence that the central nervous system is not cured by intravenously injected ERT. All patients develop ADAs but their role in ERT tolerance and effectiveness has not been well defined yet. Lack of reliable biomarkers contributes to the uncertainties about effectiveness. The data obtained from affected siblings strongly indicates the need of neonatal screening for treatable MPSs. Currently, other treatments are under evaluation and will surely help improve the prognosis of MPS patients.

Background Mucopolysaccharidosis-IVA (Morquio A disease) is a lysosomal disorder in which the abnormal accumulation of keratan sulfate and chondroitin-6-sulfate is consequent to mutations in the galactosamine-6-sulfatase (GALNS) gene. Since standard DNA sequencing analysis fails to detect about 16% of GALNS mutant alleles, gross DNA rearrangement screening and uniparental disomy evaluation are required to complete the molecular diagnosis. Despite this, the second pathogenic GALNS allele generally remains unidentified in ~ 5% of Morquio-A disease patients. Methods In an attempt to bridge the residual gap between clinical and molecular diagnosis, we performed an mRNA-based evaluation of three Morquio-A disease patients in whom the second mutant GALNS allele had not been identified. We also performed sequence analysis of the entire GALNS gene in two patients. Results Different aberrant GALNS mRNA transcripts were characterized in each patient. Analysis of these transcripts then allowed the identification, in one patient, of a disease-causing deep intronic GALNS mutation. The aberrant mRNA products identified in the other two individuals resulted in partial exon loss. Despite sequencing the entire GALNS gene region in these patients, the identity of a single underlying pathological lesion could not be unequivocally determined. We postulate that a combination of multiple variants, acting in cis, may synergise in terms of their impact on the splicing machinery. Conclusions We have identified GALNS variants located within deep intronic regions that have the potential to impact splicing. These findings have prompted us to incorporate mRNA analysis into our diagnostic flow procedure for the molecular analysis of Morquio A disease. Electronic supplementary material The online version of this article (10.1186/s12881-018-0694-6) contains supplementary material, which is available to authorized users.

Background: Gaucher disease (GD) presents with a range of signs and symptoms. Physicians can fail to recognise the early stages of GD owing to a lack of disease awareness, which can lead to significant diagnostic delays and sometimes irreversible but avoidable morbidities. Aim: The Gaucher Earlier Diagnosis Consensus (GED-C) initiative aimed to identify signs and co-variables considered most indicative of early type 1 and type 3 GD, to help non-specialists identify ‘at-risk’ patients who may benefit from diagnostic testing. Methods: An anonymous, three-round Delphi consensus process was deployed among a global panel of 22 specialists in GD (median experience 17.5 years, collectively managing almost 3000 patients). The rounds entailed data gathering, then importance ranking and establishment of consensus, using 5-point Likert scales and scoring thresholds defined a priori. Results: For type 1 disease, seven major signs (splenomegaly, thrombocytopenia, bone-related manifestations, anaemia, hyperferritinaemia, hepatomegaly and gammopathy) and two major co-variables (family history of GD and Ashkenazi-Jewish ancestry) were identified. For type 3 disease, nine major signs (splenomegaly, oculomotor disturbances, thrombocytopenia, epilepsy, anaemia, hepatomegaly, bone pain, motor disturbances and kyphosis) and one major co-variable (family history of GD) were identified. Lack of disease awareness, overlooking mild early signs and failure to consider GD as a diagnostic differential were considered major barriers to early diagnosis. Conclusion: The signs and co-variables identified in the GED-C initiative as potentially indicative of early GD will help to guide non-specialists and raise their index of suspicion in identifying patients potentially suitable for diagnostic testing for GD. Fil: Mehta, Atul. University of California at Los Angeles; Estados Unidos Fil: Kuter, David J.. Harvard Medical School; Estados Unidos Fil: Salek, Sam S.. University Of Hertfordshire; Reino Unido Fil: Belmatoug, Nadia. University Hospital Paris Nord Val de Seine.Referral Center for Lysosomal Diseases; Francia Fil: Bembi, Bruno. Academic Medical Centre Hospital of Udine; Italia Fil: Bright, Jeremy. Oxford PharmaGenesis Ltd. Research Evaluation Unit; Reino Unido Fil: vom Dahl, Stephan. Heinrich-Heine University; Alemania Fil: Deodato, Federica. Bambino Gesù Children’s Hospital; Italia Fil: Di Rocco, Maja. Istituto Giannina Gaslini; Italia Fil: Göker Alpan, Ozlem. No especifíca; Fil: Hughes, Derralynn A.. University of California at Los Angeles; Estados Unidos Fil: Lukina, Elena A.. National Research Center for Hematology; Rusia Fil: Machaczka, Maciej. University Of Rzeszów; Polonia. Karolinska University Hospital Huddinge; Suecia Fil: Mengel, Eugen. Johannes Gutenberg Universitat Mainz; Alemania Fil: Nagral, Aabha. Apollo Hospital; India Fil: Nakamura, Kimitoshi. Kumamoto University; Japón Fil: Narita, Aya. Tottori University. Faculty Of Medicine; Japón Fil: Oliveri, María Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Inmunología, Genética y Metabolismo. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Inmunología, Genética y Metabolismo; Argentina Fil: Pastores, Gregory. The Mater Misericordiae University Hospital; Irlanda Fil: Pérez-López, Jordi. Hospital Vall d’Hebron; España Fil: Ramaswami, Uma. Universidade Federal do Rio Grande do Sul; Brasil Fil: Schwartz, Ida V.. Universidade Federal do Rio Grande do Sul; Brasil Fil: Szer, Jeff. The Royal Melbourne Hospital; Australia Fil: Weinreb, Neal J.. University of Miami; Estados Unidos Fil: Zimran, Ari. Shaare Zedek Medical Center; Israel. Hadassah Medical School; Israel

Mucopolysaccharidosis I-Hurler (MPS I-H) is the most severe form of a metabolic genetic disease caused by mutations of IDUA gene encoding the lysosomal α-L-iduronidase enzyme. MPS I-H is a rare, life-threatening disease, evolving in multisystem morbidity including progressive neurological disease, upper airway obstruction, skeletal deformity and cardiomyopathy. Allogeneic hematopoietic stem cell transplantation (HSCT) is currently the gold standard for the treatment of MPS I-H in patients diagnosed and treated before 2–2.5 years of age, having a high rate of success. Beyond the child’s age, other factors influence the probability of treatment success, including the selection of patients, of graft source and the donor type employed. Enzyme replacement therapy (ERT) with human recombinant laronidase has also been demonstrated to be effective in ameliorating the clinical conditions of pre-transplant MPS I-H patients and in improving HSCT outcome, by peri-transplant co-administration. Nevertheless the long-term clinical outcome even after successful HSCT varies considerably, with a persisting residual disease burden. Other strategies must then be considered to improve the outcome of these patients: one is to pursue early pre-symptomatic diagnosis through newborn screening and another one is the identification of novel treatments. In this perspective, even though newborn screening can be envisaged as a future attractive perspective, presently the best path to be pursued embraces an improved awareness of signs and symptoms of the disorder by primary care providers and pediatricians, in order for the patients’ timely referral to a qualified reference center. Furthermore, sensitive new biochemical markers must be identified to better define the clinical severity of the disease at birth, to support clinical judgement during the follow-up and to compare the effects of the different therapies. A prolonged neuropsychological follow-up of post-transplant cognitive development of children and residual disease burden is needed. In this perspective, the reference center must guarantee a multidisciplinary follow-up with an expert team. Diagnostic and interventional protocols of reference centers should be standardized whenever possible to allow comparison of clinical data and evaluation of results. This review will focus on all these critical issues related to the management of MPS I-H.

Juvenile and adult GM1-gangliosidosis are invariably characterized by progressive neurological deterioration. To date only symptomatic therapies are available. We report for the first time the positive results of Miglustat (OGT 918, N-butyl-deoxynojirimycin) treatment on three Italian GM1-gangliosidosis patients. The first two patients had a juvenile form (enzyme activity ≤5%, GLB1 genotype p.R201H/c.1068 + 1G > T; p.R201H/p.I51N), while the third patient had an adult form (enzyme activity about 7%, p.T329A/p.R442Q). Treatment with Miglustat at the dose of 600 mg/day was started at the age of 10, 17 and 28 years; age at last evaluation was 21, 20 and 38 respectively. Response to treatment was evaluated using neurological examinations in all three patients every 4–6 months, the assessment of Movement Disorder-Childhood Rating Scale (MD-CRS) in the second patient, and the 6-Minute Walking Test (6-MWT) in the third patient. The baseline neurological status was severely impaired, with loss of autonomous ambulation and speech in the first two patients, and gait and language difficulties in the third patient. All three patients showed gradual improvement while being treated; both juvenile patients regained the ability to walk without assistance for few meters, and increased alertness and vocalization. The MD-CRS class score in the second patient decreased from 4 to 2. The third patient improved in movement and speech control, the distance covered during the 6-MWT increased from 338 to 475 m. These results suggest that Miglustat may help slow down or reverse the disease progression in juvenile/adult GM1-gangliosidosis.

Two oxysterols, cholestan-3β,5α,6β-triol (C-triol) and 7-ketocholesterol (7-KC), have been recently proposed as diagnostic markers of Niemann-Pick type C (NP-C) disease, representing a potential alternative diagnostic tool to the more invasive and time consuming filipin test in cultured fibroblasts. Usually, the oxysterols are detected and quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) method using atmospheric pressure chemical ionization (APCI) or electro-spray-ionization (ESI) sources, after a variety of derivatization procedures to enhance sensitivity. We developed a sensitive LC-MS/MS method to quantify the oxysterols in plasma as dimethylaminobutyrate ester, suitable for ESI analysis. This method, with an easy liquid-phase extraction and a short derivatization procedure, has been validated to demonstrate specificity, linearity, recovery, lowest limit of quantification, accuracy and precision. The assay was linear over a concentration range of 0.5-200ng/mL for C-triol and 1.0-200ng/mL for 7-KC. Intra-day and inter-day coefficients of variation (CV%) were

Gaucher disease (GD) is caused by an enzyme deficiency that leads to the accumulation of glycolipids in various organs. Although the signs and symptoms of GD emerge in childhood in the majority of patients, the disease often remains unrecognized for many years with delay of benefits of therapy or development of irreversible complications. Based on published data and data from the International Collaborative Gaucher Group Registry, an algorithm has been drafted for early diagnosis of GD in pediatric patients. It will help hematologists in promoting a timely diagnosis and early access to therapy for pediatric patients with GD.

Niemann–Pick disease type C (NPC) is a lysosomal storage disease characterized by progressive neurological degeneration. Miglustat is the first approved specific therapy and its efficacy in stabilizing or slowing disease progression has been demonstrated in previous studies. We evaluated data from 10 NPC patients treated with Miglustat in a single study centre. All disease manifestations were assessed and patients were stratified according to age at onset of neurological symptoms. Neurological data were recorded by using a modified version of the NP–C disability scale; a “composite score” and a “mean annual change” were calculated to evaluate disease progression. We observed a mean annual change of the composite score of 0.04 in our cohort, indicating slower progression of neurological symptoms if compared with the natural history of the disease. The evidence of slower disease evolution in patients treated with Miglustat suits with previous data and here it is also emphasized by the comparison between disease progression in two early-infantile onset patients receiving different Miglustat dosages. Evaluation of the mean annual change for individual subgroups of patients evidenced minor values in juvenile patients, highlighting better response in such class of patients. Among individual neurological parameters, swallowing showed the minor mean annual change (0.02), indicating better response to therapy. We underline the importance of using a standardized disability scale to quantify and compare neurological features and their evolution over time.

Infantile Pompe disease, resulting from deficiency of lysosomal acid α-glucosidase, requires enzyme replacement therapy with recombinant human acid α-glucosidase. Most patients develop antirecombinant human acid α-glucosidase antibodies, leading to reduced response to enzyme therapy in a subgroup of them. Aiming to improve treatment response, several immune tolerance induction strategies have been explored. We describe a patient with life-threatening infusion-associated reactions presenting anti-recombinant human acid α-glucosidase antibodies. He was successfully treated with an immune tolerance induction protocol, consisting of plasma exchange combined with a single dose of rituximab. Immediate reduction of antibody titer was obtained and enzyme therapy was resumed without infusion-associated reactions. Twenty-two months later, immunoglobulin G titer remained below 1:100. In conclusion, we applied a short-course immune tolerance induction strategy in a patient with severe infusion-associated reactions and anti-recombinant human acid α-glucosidase antibodies, leading to early and persisting reduction of antibody titer, in the absence of significant adverse events.

Extra/intradural strip electrode implantation on motor cortex may be possible minimally invasive neurosurgical method for therapeutic neuromodulation in Parkinson's disease (PD). The aim of this review is to assess motor cortex stimulation (MCS) efficacy and safety in advanced PD. Sixteen published articles were included with a total of 130 PD patients treated. In almost all results are from prospective observational open labeled study, only in two studies blinded assessment was carried out. Negative results are reported in three studies. Significant improvement in motor symptoms with remarkable effect on axial symptoms, L-dopa-induced dyskinesia and quality of life are outlined in thirteen studies. Surgical technique involved implant of four-contact strip electrode over M1 in epidural space with exception of few cases in which implant was carried out in subdural space. Surgical procedure was performed contralateral to most affected side with exception of five patients in which it was carried at dominant hemisphere; in four patients electrode implant was bilateral but stimulation was carried out simultaneously on both sides only in two cases. Complications and adverse events occurred very rarely for extradural MCS whereas with higher rate for subdural MCS. Based on review of current literature extra/intradural MCS represents an alternative to deep brain stimulation (DBS) to surgically treat PD patients who are not candidate for DBS. MCS is a minimally invasive neuromodulation procedure with low morbidity-mortality that can relieve all three major symptoms of PD on both sides simultaneously and bilaterally; it has significant effectiveness on axial symptoms, gait disturbances and therapy complications.