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Copyright © 2019 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited, Regenerative medicine using stem cell technology is an emerging field that is currently tested for inborn and acquired liver diseases. Objective. This phase I/II prospective, open label, multicenter, randomized trial aimed primarily at evaluating the safety of Heterologous Human Adult Liver–derived Progenitor Cells (HepaStem) in pediatric patients with urea cycle disorders (UCDs) or Crigler-Najjar (CN) syndrome 6 months posttransplantation. The secondary objective included the assessment of safety up to 12 months postinfusion and of preliminary efficacy. Methods. Fourteen patients with UCDs and 6 with CN syndrome were divided into 3 cohorts by body weight and intraportally infused with 3 doses of HepaStem. Clinical status, portal vein hemodynamics, morphology of the liver, de novo detection of circulating anti– human leukocyte antigen antibodies, and clinically significant adverse events (AEs) and serious adverse events to infusion were evaluated by using an intent-to-treat analysis. Results. The overall safety of HepaStem was confirmed. For the entire study period, patient-month incidence rate was 1.76 for the AEs and 0.21 for the serious adverse events, of which 38% occurred within 1 month postinfusion. There was a trend of higher events in UCD as compared with CN patients. Segmental left portal vein thrombosis occurred in 1 patient and intraluminal local transient thrombus in a second patient. The other AEs were in line with expectations for catheter placement, cell infusion, concomitant medications, age, and underlying diseases. Conclusions. This study led to European clinical trial authorization for a phase II study in a homogeneous patient cohort, with repeated infusions and intermediate doses.

The increased survival of urea cycle disorders (UCDs) patients has led the attention to clinical manifestations that characterize the long-term disease course. Acute and chronic liver disease have been anecdotally reported since the very first description of UCDs. However, a detailed analysis of long-term liver involvement in large patient cohorts is still needed. Chronic liver damage in UCDs has probably a multifactorial origin, but the specific underlying mechanisms of liver disease have not yet been well elucidated. In this study, we report on chronic liver involvement and on associated metabolic abnormalities in a large cohort of 102 UCD patients, followed by two reference centers in Italy. Chronic liver involvement was observed in over 60% of UCDs patients, and comparison between individual diseases showed a significant higher frequency in argininosuccinate lyase deficiency (ASLD) and in hyperornithinemia-hyperammonemia-homocitrullinemia (HHH) syndrome with elevation of transaminases and of gamma-GT in ASLD, and of alpha-fetoprotein in HHH syndrome. Also, consistent with a chronic hepatic dysfunction, ultrasound examination revealed more pronounced abnormalities in ASLD and in HHH syndrome, when compared to other UCDs. Our study highlights in a large UCDs patients' cohort that chronic liver disease is a common finding in UCDs, often with a distinct phenotype between different diseases. Furthers studies are needed to elucidate the specific involvement of different metabolic pathways in the pathogenesis of liver dysfunction in UCDs.

Cobalamin C (cblC) defect is the most common inherited disorder of cobalamin metabolism. Developmental delay, behavioral problems, and maculopathy are common, but they have not been systematically investigated. The aim of this study was to define early neurodevelopment in cblC patients and the possible contribution of different factors, such as mode of diagnosis, age at diagnosis, presence of brain lesions and epilepsy. Children up to the age of 4 years with a visual acuity ≥1/10 were evaluated using the Griffiths' Mental Development Scales. Eighteen children were enrolled (age range 12-48 months). Four were diagnosed by newborn screening (NBS); in the others mean age at diagnosis was 3.5 months (range 0.3-18 months). Eight had seizures: three in the first year, and five after the second year of life. Fourteen had brain lesions on magnetic resonance imaging (MRI). Neurovisual assessment evidenced low visual acuity (

A 5-day old newborn boy is admitted at the Emergency Department of our Hospital with cardiorespiratory failure and coma. High levels of plasma ammonium are found and a metabolic congenital defect is suspected. Medical treatment and diagnostic workup are started. The non-response to medical treatment requires prompt initiation of dialysis.

Transient infantile hypertriglyceridemia (HTGT1; OMIM #614480) is a rare autosomal recessive disorder, which manifests in early infancy with transient hypertriglyceridemia, hepatomegaly, elevated liver enzymes, persistent fatty liver and hepatic fibrosis. This rare clinical entity is caused by inactivating mutations in the GPD1 gene, which encodes the cytosolic isoform of glycerol-3-phosphate dehydrogenase. Here we report on four patients from three unrelated families of diverse ethnic origins, who presented with hepatomegaly, liver steatosis, hypertriglyceridemia, with or without fasting ketotic hypoglycemia. Whole exome sequencing revealed the affected individuals to harbor deleterious biallelic mutations in the GPD1 gene, including the previously undescribed c.806G > A (p.Arg269Gln) and c.640T > C (p.Cys214Arg) mutations. The clinical features in three of our patients showed several differences compared to the original reports. One subject presented with recurrent episodes of fasting hypoglycemia along with hepatomegaly, hypetriglyceridemia, and elevated liver enzymes; the second showed a severe liver disease, with intrahepatic cholestasis associated with kidney involvement; finally, the third presented persistent hypertriglyceridemia at the age of 30 years. These findings expand the current knowledge of this rare disorder, both with regard to the phenotype and molecular basis. The enlarged phenotypic spectrum of glycerol-3-phosphate dehydrogenase 1 deficiency can mimic other inborn errors of metabolism with liver involvement and should alert clinicians to recognize this entity by considering GPD1 mutations in appropriate clinical settings.

Peroxisomal disorders (PDs) present with wide phenotypic variability. An appropriate diagnosis requires a complete analysis of peroxisomal metabolites. We developed a multiplex LC-MS/MS method, using atmospheric pressure chemical ionization allowing the simultaneous determination in plasma of very-long-chain fatty acids, phytanic, pristanic, docosahexaenoic acids and di- and tri-hydroxycolestanoic bile acids. Two hundred microliters of plasma extracted with acetonitrile and 200μl extracted with hexane after an acid hydrolysis were combined, evaporated, dissolved in 10μl of methanol and analyzed. The acquisition was in negative-ion mode using multiple reaction monitoring. The method was validated analytically and clinically. Linearity was 0.1-200μmol/l for docosanoic, cis-13-docosenoic, tetracosanoic, cis-15-tetracosenoic and phytanic acids; 0.01-10μmol/l for hexacosanoic acid; 0.02-20μmol/l for di-hydroxycolestanoic, tri-hydroxycolestanoic and pristanic acids; 0.3-300μmol/l for docosahexaenoic acid. Intra-day and inter-day CVs were below 3.88 and 3.98 respectively for all compounds. Samples from patients with known peroxisomal disorders were compared with controls and the method allowed to confirm the diagnosis in all subjects with a 100% sensitivity. The advantage of this multiplex method is to allow in a single chromatographic run the simultaneous determination of a large number of peroxisome biomarkers with a simple preparative phase without derivatization.

NDUFB11, a component of mitochondrial complex I, is a relatively small integral membrane protein, belonging to the "supernumerary" group of subunits, but proved to be absolutely essential for the assembly of an active complex I. Mutations in the X-linked nuclear-encoded NDUFB11 gene have recently been discovered in association with two distinct phenotypes, i.e. microphthalmia with linear skin defects and histiocytoid cardiomyopathy. We report on a male with complex I deficiency, caused by a de novo mutation in NDUFB11 and displaying early-onset sideroblastic anemia as the unique feature. This is the third report that describes a mutation in NDUFB11, but all are associated with a different phenotype. Our results further expand the molecular spectrum and associated clinical phenotype of NDUFB11 defects.

Item does not contain fulltext AIM: Epilepsy is commonly observed in congenital disorders of glycosylation (CDG), but no distinctive electroclinical pattern has been recognized. We aimed at identifying a characteristic clinical presentation that might help targeted diagnostic work-up. METHOD: Based on the initial observation of an index case with CDG and migrating partial seizures, we evaluated 16 additional children with CDG and analysed their clinical course, biochemical, genetic, electrographic, and imaging findings. RESULTS: Four of 17 consecutively observed children with CDG (three females, one male) were first referred between the first and fourth month of life, after early onset of migrating partial seizures. All four patients manifested developmental delay, microcephaly, and multi-organ involvement. Magnetic resonance imaging disclosed cerebral and cerebellar atrophy. Isoelectrofocusing of transferrin, enzymatic studies, and lipid-linked oligosaccharide analysis indicated CDG-I. Genetic testing demonstrated either homozygous or compound heterozygous variants involving the ALG3 gene in patients 1 and 3, the RFT1 gene in patient 2, and the ALG1 gene in patient 4. At last follow-up, patients 1 and 2 were 5 and 3(1/2) years old. Patients 3 and 4 had died due to respiratory failure during pneumonia and refractory status epilepticus respectively. INTERPRETATION: Children with migrating partial seizures and concomitant multisystem involvement should be investigated for CDG.

Barth syndrome (BTHS) is an X-linked inborn error of metabolism which affects males. The main manifestations are cardiomyopathy, myopathy, hypotonia, growth delay, intermittent neutropenia and 3-methylglutaconic aciduria. Diagnosis is confirmed by mutational analysis of the TAZ gene and biochemical dosage of the monolysocardiolipin/tetralinoleoyl cardiolipin (MLCL:L4-CL) ratio. We report a 6-year-old boy who presented with severe hypoglycemia, lactic acidosis and severe dilated cardiomyopathy soon after birth. The MLCL:L4-CL ratio confirmed BTHS (3.90 on patient's fibroblast, normal: 0-0.3). Subsequent sequencing of the TAZ gene revealed only the new synonymous variant NM_000116.3 (TAZ):c.348C>T p.(Gly116Gly), which did not appear to affect the protein sequence. In silico prediction analysis suggested the new c.348C>T nucleotide change could alter the TAZ mRNA splicing processing. We analyzed TAZ mRNAs in the patient's fibroblasts and found an abnormal skipping of 24 bases (NM_000116.3:c.346_371), with the consequent ablation of 8 amino acid residues in the tafazzin protein (NP_000107.1:p.Lys117_Gly124del). Molecular analysis of at risk female family members identified the patient's sister and mother as heterozygous carriers. Apparently harmless synonymous variants in the TAZ gene can damage gene expression. Such findings widen our knowledge of molecular heterogeneity in BTHS.

This study focused on the molecular characterization of patients with leukoencephalopathy associated with a specific biochemical defect of mitochondrial respiratory chain complex III, and explores the impact of a distinct magnetic resonance imaging pattern of leukoencephalopathy to detect biallelic mutations in LYRM7 in patients with biochemically unclassified leukoencephalopathy. ‘Targeted resequencing’ of a custom panel including genes coding for mitochondrial proteins was performed in patients with complex III deficiency without a molecular genetic diagnosis. Based on brain magnetic resonance imaging findings in these patients, we selected additional patients from a database of unclassified leukoencephalopathies who were scanned for mutations in LYRM7 by Sanger sequencing. Targeted sequencing revealed homozygous mutations in LYRM7 , encoding mitochondrial LYR motif-containing protein 7, in four patients from three unrelated families who had a leukoencephalopathy and complex III deficiency. Two subjects harboured previously unreported variants predicted to be damaging, while two siblings carried an already reported pathogenic homozygous missense change. Sanger sequencing performed in the second cohort of patients revealed LYRM7 mutations in three additional patients, who were selected on the basis of the magnetic resonance imaging pattern. All patients had a consistent magnetic resonance imaging pattern of progressive signal abnormalities with multifocal small cavitations in the periventricular and deep cerebral white matter. Early motor development was delayed in half of the patients. All patients but one presented with subacute neurological deterioration in infancy or childhood, preceded by a febrile infection, and most patients had repeated episodes of subacute encephalopathy with motor regression, irritability and stupor or coma resulting in major handicap or death. LYRM7 protein was strongly reduced in available samples from patients; decreased complex III holocomplex was observed in fibroblasts from a patient carrying a splice site variant; functional studies in yeast confirmed the pathogenicity of two novel mutations. Mutations in LYRM7 were previously found in a single patient with a severe form of infantile onset encephalopathy. We provide new molecular, clinical, and neuroimaging data allowing us to characterize more accurately the molecular spectrum of LYRM7 mutations highlighting that a distinct and recognizable magnetic resonance imaging pattern is related to mutations in this gene. Inter- and intrafamilial variability exists and we observed one patient who was asymptomatic by the age of 6 years.

Therapeutic drug monitoring (TDM) should be adopted in all neonatal intensive care units (NICUs), where the most preterm and fragile babies are hospitalized and treated with many drugs, considering that organs and metabolic pathways undergo deep and progressive maturation processes after birth. Different developmental changes are involved in interindividual variability in response to drugs. A crucial point of TDM is the choice of the bioanalytical method and of the sample to use. TDM in neonates is primarily used for antibiotics, antifungals, and antiepileptic drugs in clinical practice. TDM appears to be particularly promising in specific populations: neonates who undergo therapeutic hypothermia or extracorporeal life support, preterm infants, infants who need a tailored dose of anticancer drugs. This review provides an overview of the latest advances in this field, showing options for a personalized therapy in newborns and infants.

Methylmalonic Acidurias (MMAs) are a group of inborn errors of metabolism (IEMs), specifically of propionate catabolism characterized by gastrointestinal and neurometabolic manifestations resulting from a deficiency in the function of methylmalonyl-CoA mutase, methylmalonyl-CoA epimerase, and cobalamin metabolism. In Expanded Newborn Screening (NBS), increased levels of propionylcarnitine (C3) and/or of its ratios by MS/MS analysis of dried blood spots (DBS) samples are suggestive for either Propionic Acidemia or MMAs. C3 elevation is not considered a specific marker for these disorders, resulting in high false-positive rates. The use of analyte ratios improves specificity, but it still cannot resolve the diagnostic issue. Second-tier testing are strongly recommended as confirmation of primary NBS results and for a differential diagnosis. LC-MS/MS analysis allows the quantification of more specific markers of the disorder. Here, we report the case of a newborn with a suspected MMA at Expanded NBS and at second-tier test. Given the urgent situation, in-depth diagnostic investigations were performed. Further investigations surprisingly revealed a Vitamin B12 deficiency due to a maternal malnutrition during pregnancy. This case emphasized that metabolic alterations at NBS may not only be influenced by genome and related to IEMs, but also to external factors and to maternal conditions.

Cristina Dallabona, Truus E. M. Abbink, Rosalba Carrozzo, Alessandra Torraco, Andrea Legati, Carola G. M. van Berkel, Marcello Niceta, Tiziana Langella, Daniela Verrigni, Teresa Rizza, Daria Diodato, Fiorella Piemonte, Eleonora Lamantea, Mingyan Fang, Jianguo Zhang, Diego Martinelli, Elsa Bevivino, Carlo Dionisi-Vici, Adeline Vanderver, Sunny G. Philip, Manju A. Kurian, Ishwar C. Verma, Sunita Bijarnia-Mahay, Sandra Jacinto, Fatima Furtado, Patrizia Accorsi, Anna Ardissone, Isabella Moroni, Ileana Ferrero, Marco Tartaglia, Paola Goffrini, Daniele Ghezzi, Marjo S. van der Knaap and Enrico Bertini. LYRM7 mutations cause a multifocal cavitating leukoencephalopathy with distinct MRI appearance. Brain 2016; 139: 782-794. doi:10.1093/brain/awv392. The authors apologize for mixing the mutations of Patients 5 and 7 on pages 788 and 789 in Figure 2 and its legend, in the main text and the online Supplementary Tables. This has now been corrected online. On page 788, the text should read: Sanger sequencing of the entire coding sequence of LYRM7 and intron-exon boundaries was performed in three additional patients. Patient 5 was found homozygous for the c.37delA (p.Thr13Hisfs∗17) mutation, which is predicted to dramatically affect recognition of the splice donor motif of exon 4 and likely impair proper RNA splicing (Fig. 2M and N), while Patients 6 and 7 were found to be homozygous for the c.214C4T (p.Q72∗) (Fig. 2L), and c.243-244 + 2delGAGT (p.?) (Fig. 2H) mutations, respectively. The Supplementary Tables have been corrected online. On page 789, Figure 2 and legend should be as follows.

Due to an unfortunate error during the typesetting process, the collaborators were presented incorrectly.

Objectives To evaluate the role of next generation sequencing in genetic diagnosis of pediatric patients with persistent hypoglycemia. Study design Sixty-four patients investigated through an extensive workup were divided in 3 diagnostic classes based on the likelihood of a genetic diagnosis: (1) single candidate gene (9/64); (2) multiple candidate genes (43/64); and (3) no candidate gene (12/64). Subsequently, patients were tested through a custom gene panel of 65 targeted genes, which included 5 disease categories: (1) hyperinsulinemic hypoglycemia, (2) fatty acid-oxidation defects and ketogenesis defects, (3) ketolysis defects, (4) glycogen storage diseases and other disorders of carbohydrate metabolism, and (5) mitochondrial disorders. Molecular data were compared with clinical and biochemical data. Results A proven diagnosis was obtained in 78% of patients with suspicion for a single candidate gene, in 49% with multiple candidate genes, and in 33% with no candidate gene. The diagnostic yield was 48% for hyperinsulinemic hypoglycemia, 66% per fatty acid-oxidation and ketogenesis defects, 59% for glycogen storage diseases and other carbohydrate disorders, and 67% for mitochondrial disorders. Conclusions This approach provided a diagnosis in ~50% of patients in whom clinical and laboratory evaluation did not allow identification of a single candidate gene and a diagnosis was established in 33% of patients belonging to the no candidate gene class. Next generation sequencing technique is cost-effective compared with Sanger sequencing of multiple genes and represents a powerful tool for the diagnosis of inborn errors of metabolism presenting with persistent hypoglycemia.

AIFM1 is a gene located on the X chromosome, coding for AIF (Apoptosis-Inducing Factor), a mitochondrial flavoprotein involved in caspase-independent cell death. AIFM1 mutations have been associated with different clinical phenotypes: a severe infantile encephalopathy with combined oxidative phosphorylation deficiency and the Cowchock syndrome, an X-linked Charcot-Marie-Tooth disease (CMTX4) with axonal sensorimotor neuropathy, deafness and cognitive impairment. In two male cousins with early-onset mitochondrial encephalopathy and cytochrome c oxidase (COX) deficiency, we identified a novel AIFM1 mutation. Muscle biopsies and electromyography in both patients showed signs of severe denervation. Our patients manifested a phenotype that included signs of both cortical and motor neuron involvement. These observations emphasize the role of AIF in the development and function of neurons.

Methylmalonic acidemia with homocystinuria, cobalamin deficiency type C (cblC) (MMACHC) is the most common inborn error of cobalamin metabolism. Despite a multidrug treatment, the long-term follow-up of early-onset patients is often unsatisfactory, with progression of neurological and ocular impairment. Here, the in-vivo proteome of control and MMACHC lymphocytes (obtained from patients under standard treatment with OHCbl, betaine, folate and L-carnitine) was quantitatively examined by two dimensional differential in-gel electrophoresis (2D-DIGE) and mass spectrometry. Twenty three proteins were found up-regulated and 38 proteins were down-regulated. Consistent with in vivo studies showing disturbance of glutathione metabolism, a deregulation in proteins involved in cellular detoxification, especially in glutathione metabolism was found. In addition, relevant changes were observed in the expression levels of proteins involved in intracellular trafficking and protein folding, energy metabolism, cytoskeleton organization and assembly. This study demonstrates relevant changes in the proteome profile of circulating lymphocytes isolated from treated cblC patients. Some results confirm previous observations in vivo on fibroblast, thus concluding that some dysregulation is ubiquitous. On the other hand, new findings could be tissue-specific. These observations expand our current understanding of the cblC disease and may ignite new research and therapeutic strategies to treat this disorder.

Vici syndrome is a human inherited multi-system disorder caused by recessive mutations in EPG5, encoding the EPG5 protein that mediates the fusion of autophagosomes with lysosomes. Immunodeficiency characterized by lack of memory B cells and increased susceptibility to infection is an integral part of the condition, but the role of EPG5 in the immune system remains unknown. Here we show that EPG5 is indispensable for the transport of the TLR9 ligand CpG to the late endosomal-lysosomal compartment, and for TLR9-initiated signaling, a step essential for the survival of human memory B cells and their ultimate differentiation into plasma cells. Moreover, the predicted structure of EPG5 includes a membrane remodeling domain and a karyopherin-like domain, thus explaining its function as a carrier between separate vesicular compartments. Our findings indicate that EPG5, by controlling nucleic acids intracellular trafficking, links macroautophagy/autophagy to innate and adaptive immunity.

Hereditary tyrosinemia type 1 (HT1) is characterized by severe progressive liver disease and renal tubular dysfunction. Kidney involvement is characterized by hypophosphatemic rickets and Fanconi syndrome. Different animal models were useful to investigate the pathophysiology of the disease and the effects of NTBC therapy on liver and kidney function. NTBC has revolutionized the prognosis of HT1 and its acute and chronic effects on renal tubular function have been proved, with normalization of tubular function within a few weeks, particularly hypophosphatemia and proteinuria. NTBC therapy is highly effective in improving renal function both at short and long-term. However, its efficacy critically depends on the age at start of treatment with normal outcome in patients diagnosed at birth by newborn screening.

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.

Disclaimer: This diagnostic guideline is intended as an educational resource and represents the opinions of the authors, and is not representative of recommendations or policy of the American College of Medical Genetics and Genomics (ACMG). The information should be considered a consensus based on expert opinion, as more comprehensive levels of evidence were not available in the literature in all cases. Background: Acid sphingomyelinase deficiency (ASMD) is a rare, progressive, and often fatal lysosomal storage disease. The underlying metabolic defect is deficiency of the enzyme acid sphingomyelinase that results in progressive accumulation of sphingomyelin in target tissues. ASMD manifests as a spectrum of severity ranging from rapidly progressive severe neurovisceral disease that is uniformly fatal to more slowly progressive chronic neurovisceral and chronic visceral forms. Disease management is aimed at symptom control and regular assessments for multisystem involvement. Purpose and methods: An international panel of experts in the clinical and laboratory evaluation, diagnosis, treatment/management, and genetic aspects of ASMD convened to review the evidence base and share personal experience in order to develop a guideline for diagnosis of the various ASMD phenotypes. Conclusions: Although care of ASMD patients is typically provided by metabolic disease specialists, the guideline is directed at a wide range of providers because it is important for primary care providers (e.g., pediatricians and internists) and specialists (e.g., pulmonologists, hepatologists, and hematologists) to be able to identify ASMD. Genet Med advance online publication 13 April 2017

PMC Free: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5203859/ Background: Remethylation defects are rare inherited disorders in which impaired remethylation of homocysteine to methionine leads to accumulation of homocysteine and perturbation of numerous methylation reactions. Objective: To summarise clinical and biochemical characteristics of these severe disorders and to provide guidelines on diagnosis and management. Data sources: Review, evaluation and discussion of the medical literature (Medline, Cochrane databases) by a panel of experts on these rare diseases following the GRADE approach. Key recommendations: We strongly recommend measuring plasma total homocysteine in any patient presenting with the combination of neurological and/or visual and/or haematological symptoms, subacute spinal cord degeneration, atypical haemolytic uraemic syndrome or unexplained vascular thrombosis. We strongly recommend to initiate treatment with parenteral hydroxocobalamin without delay in any suspected remethylation disorder; it significantly improves survival and incidence of severe complications. We strongly recommend betaine treatment in individuals with MTHFR deficiency; it improves the outcome and prevents disease when given early. info:eu-repo/semantics/publishedVersion

Purpose of review:Niemann-Pick disease type C (NP-C) is a neurovisceral disorder that may be more prevalent than earlier estimates. Diagnosis of NP-C is often delayed; a key aim for clinical practice is to reduce this delay. Recently, substantial progress has been made in the field of NP-C screening and diagnosis, justifying an update to the existing recommendations for clinical practice.Recent findings:New biomarker profiling and genetic analysis technologies are included as first-line diagnostic tests for NP-C. Most diagnoses can now be confirmed by combination of biomarker and genetic analyses. Filipin staining may facilitate diagnosis in uncertain cases. Recommendations are provided for psychiatrists, neuro-ophthalmologists, and radiologists, and on screening within specific at-risk patient cohorts. The NP-C diagnostic algorithm has been updated and simplified.Summary:This publication provides expert recommendations for clinicians who may see patients presenting with the signs and symptoms of NP-C, including general practitioners, pediatricians, neurologists, and psychiatrists.

Background:Lysosphingolipids (LysoSLs) are derivatives of sphingolipids which have lost the amide-linked acyl chain. More recently, LysoSLs have been identified as storage compounds in several sphingolipidoses, including Gaucher, Fabry and Niemann-Pick diseases. To date, different methods have been developed to measure each individual lysosphingolipid in plasma. This report describes a rapid liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) assay for simultaneous quantification of several LysoSLs in plasma.Methods:We analyzed the following compounds: hexosylsphingosine (HexSph), globotriaosylsphingosine (LysoGb3), lysosphingomyelin (LysoSM) and lysosphingomyelin-509 (LysoSM-509). The sample preparation requires only 100 μL of plasma and consists of an extraction with a mixture of MeOH/acetone/HResults:The method validation showed high sensitivity, an excellent accuracy and precision. Reference ranges were determined in healthy adult and pediatric population. The results demonstrate that the LC-MS/MS method can quantify different LysoSLs and can be used to identify patients with Fabry (LysoGb3), Gaucher and Krabbe (HexSph) diseases, prosaposine deficiency (LysoGb3 and HexSph), and Niemann-Pick disease types A/B and C (LysoSM and LysoSM-509).Conclusions:This LC-MS/MS method allows a rapid and simultaneous quantification of LysoSLs and is useful as a biochemical diagnostic tool for sphingolipidoses.

Hereditary tyrosinemia type 1 (HT1) is characterized by severe progressive liver disease and renal tubular dysfunction. Kidney involvement is characterized by hypophosphatemic rickets and Fanconi syndrome. Different animal models were useful to investigate the pathophysiology of the disease and the effects of NTBC therapy on liver and kidney function. NTBC has revolutionized the prognosis of HT1 and its acute and chronic effects on renal tubular function have been proved, with normalization of tubular function within a few weeks, particularly hypophosphatemia and proteinuria. NTBC therapy is highly effective in improving renal function both at short and long-term. However, its efficacy critically depends on the age at start of treatment with normal outcome in patients diagnosed at birth by newborn screening.

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

Non-alcoholic fatty liver disease (NAFLD) is a spectrum of metabolic abnormalities ranging from simple triglyceride accumulation in the hepatocytes to hepatic steatosis with inflammation, ballooning and fibrosis. It has been demonstrated that the pathogenesis of NAFLD involves increased oxidative stress, with consumption of the major cellular antioxidant, glutathione (GSH). Liver has a fundamental role in sulfur compound metabolism, although the data reported on plasma thiols status in NAFLD are conflicting. We recruited 63 NAFLD patients, and we analyzed all plasma thiols, such as homocysteine (Hcy), cysteine (Cys), cysteinylglycine (CysGly) and GSH, by high-performance liquid chromatography (HPLC) with fluorescence detection. Hcy, Cys and CysGly plasma levels increased in NAFLD patients (p &lt, 0.0001), whereas GSH levels were decreased in NAFLD patients when compared to controls (p &lt, 0.0001). On the contrary, patients with steatohepatitis exhibited lower levels of Hcy and Cys than subjects without. Furthermore, a positive correlation was found between Hcy and Cys and the presence of fibrosis in children with NAFLD. Taken together, these data demonstrated a defective hepatic sulfur metabolism in children with NAFLD, and that high levels of Hcy and Cys probably correlates with a pattern of more severe histological liver damage, due to mechanisms that require further studies.

Background Hereditary tyrosinemia type 1 (HT1) is characterized by severe progressive liver disease and renal tubular dysfunction. NTBC therapy has revolutionized the management of HT1 but its effect on renal tubular function has so far been poorly investigated. The aim of this study was to describe the early effect of NTBC on renal tubular disease in patients with HT1. Methods Five HT1 patients (age between 5 and 53 months) with different types of presentation were evaluated before and during the first 2 weeks of therapy with NTBC in a retrospective case analysis for phosphate metabolism and renal tubular function. Results Before starting NTBC therapy, all children manifested signs of renal dysfunction which included hypophosphatemia, acidosis, reduced phosphate reabsorption, aminoaciduria, glycosuria (Fanconi syndrome), and variable degree of proteinuria. Some patients also presented increased urinary calcium/creatinine ratio and raised fractional excretion of sodium. Starting of NTBC therapy resulted in the rapid normalization of plasma phosphate within one week from its initiation in majority of patients and in all patients during the second week of therapy. TmP/GFR normalized in 48 h, while the other markers of renal dysfunction showed an improving trend over 2 weeks. Conclusions NTBC is an efficient treatment for renal tubular dysfunction in HT1, allowing the return to normal function within a few weeks. Its early effect on renal tubular cells appeared to be very rapid, particularly in normalizing plasma phosphate and TmP/GFR. In our series of patients, the TmP/GFR resulted as the most reliable index of tubular function.

TMEM70 deficiency is the most common nuclear-encoded defect affecting the ATP synthase. In this multicentre retrospective study we characterise the natural history of the disease, treatment and outcome in 48 patients with mutations in TMEM70. Eleven centers from eight European countries, Turkey and Israel participated.All 27 Roma and eight non-Roma patients were homozygous for the common mutation c.317-2A G. Five patients were compound heterozygotes for the common mutation and mutations c.470 T A, c.628A C, c.118_119insGT or c.251delC. Six Arab Muslims and two Turkish patients were homozygous for mutations c.238C T, c.316 + 1G T, c.336 T A, c.578_579delCA, c.535C T, c.359delC. Age of onset was neonatal in 41 patients, infantile in six cases and two years in one child. The most frequent symptoms at onset were poor feeding, hypotonia, lethargy, respiratory and heart failure, accompanied by lactic acidosis, 3-methylglutaconic aciduria and hyperammonaemia. Symptoms further included: developmental delay (98%), hypotonia (95%), faltering growth (94%), short stature (89%), non-progressive cardiomyopathy (89%), microcephaly (71%), facial dysmorphism (66%), hypospadias (50% of the males), persistent pulmonary hypertension of the newborn (22%) and Wolff-Parkinson-White syndrome (13%). One or more acute metabolic crises occurred in 24 surviving children, frequently followed by developmental regression. Hyperammonaemic episodes responded well to infusion with glucose and lipid emulsion, and ammonia scavengers or haemodiafiltration. Ten-year survival was 63%, importantly for prognostication, no child died after the age of five years.TMEM70 deficiency is a panethnic, multisystemic disease with variable outcome depending mainly on adequate management of hyperammonaemic crises in the neonatal period and early childhood.

Dihydrolipoamide dehydrogenase (DLD, E3) is a flavoprotein common to pyruvate, α-ketoglutarate and branched-chain α-keto acid dehydrogenases. We found two novel DLD mutations (p.I40Lfs*4; p.G461E) in a 19 year-old patient with lactic acidosis and a complex amino- and organic aciduria consistent with DLD deficiency, manifesting progressive exertional fatigue. Muscle biopsy showed mitochondrial proliferation and lack of DLD cross-reacting material. Riboflavin supplementation determined the complete resolution of exercise intolerance with the partial restoration of the DLD protein and disappearance of mitochondrial proliferation in the muscle. Morphological and functional studies support the riboflavin chaperon-like role in stabilizing DLD protein with rescue of its expression in the muscle.

Hereditary fructose intolerance (HFI) symptoms develop at first introduction of fruit during weaning. We report on an infant with suspected HFI who presented with repeated episodes of vomiting and hypotension after ingestion of fruit-containing meals. The first episode occurred at age 4 months. Despite negative genetic testing for HFI, strict avoidance of fruit ingestion resulted in lack of recurrence of symptoms. Oral-fructose-tolerance testing conducted with an apple mousse did not determine hypoglycemia or fructosuria but caused severe hypotension. Allergy evaluations were negative, and the history was diagnostic for fruit-induced food protein–induced enterocolitis syndrome. Because this non-immunoglobulin E–mediated gastrointestinal food hypersensitivity manifests as profuse, repetitive vomiting, often with diarrhea, leading to acute dehydration and lethargy, it may be misinterpreted as HFI. We advise pediatricians to consider food protein–induced enterocolitis syndrome in the differential diagnosis when there is a suspicion of HFI.

Purpose of review To assess all the possible differential diagnosis of food protein-induced enterocolitis syndrome (FPIES), both in acute and chronic presentation, reviewing the data reported in published studies. Recent findings There is an increase of reported cases of FPIES in recent years. As the disease presents with nonspecific symptoms, it can be misunderstood in many ways. The differential diagnosis includes, in acute presentations, the following: sepsis, other infectious diseases, acute gastrointestinal episodes, surgical emergencies, food allergies. In its chronic forms, FPIES may mimic malabsorption syndromes, metabolic disorders, primary immunodeficiencies, neurological conditions, coagulation defects, and other types of non-IgE-mediated food allergy. Summary A thorough clinical evaluation, including symptoms, signs, and laboratory findings, is necessary to lead the clinicians toward the diagnosis of FPIES. The major reason for delayed diagnosis appears to be the lack of knowledge of the disease.

MEDNIK (mental retardation, enteropathy, deafness, neuropathy, ichthyosis, and keratodermia) syndrome has been recently described as a new disorder of copper metabolism. This multisystem disease combines clinical and biochemical signs of both Menkes and Wilson's diseases, in which liver copper overload is treatable using zinc acetate therapy. MEDNIK syndrome is caused by mutation of the AP1S1 gene, which codes for the σ1A subunit of adaptor protein complex 1, and directs intracellular trafficking of copper pumps ATP7A and ATP7B. Adaptor protein complexes regulate clathrin-coated vesicle assembly, protein cargo sorting, and vesicular trafficking between organelles in eukaryotic cells. A growing number of diseases have been associated with mutations in genes coding for adaptor protein complexes subunits and we propose for them the term adaptinopathies, as a new organic category of disorders of intracellular trafficking, which offers the opportunity to dissect the mechanisms involved in the crosstalk between the Golgi apparatus and the other organelles.

Methylmalonic aciduria (MMA) is one of the most frequent organic acidurias, a class of diseases caused by enzymatic defects mainly involved in the catabolism of branched-chain amino acids. Recently, mild MMA and C4-dicarboxylyl-carnitine (C4DC-C) accumulation have been reported in patients carrying mutation in genes encoding the α-subunit (SUCLG1) and the β-subunit (SUCLA2) of the ADP-forming succinyl-CoA synthetase (SCS). We developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify in dried blood spot the two isobaric compounds of C4DC-C, succinyl-carnitine and methylmalonyl-carnitine, to allow the differential diagnosis between classical MMA and SCS-related defects. This method, with an easy liquid-phase extraction and derivatization procedure, has been validated to demonstrate the specificity, linearity, recovery, lowest limit of quantification (LLOQ), accuracy and precision for quantitative determination of blood succinyl-carnitine and methylmalonyl-carnitine. The assay was linear over a concentration range of 0.025-10 μmol/L and achieved the LLOQ of 0.025 μmol/L for both metabolites. The average slope, intercept, and coefficient of linear regression (r(2)) were respectively: 0.3389 (95% confidence interval 0.2888-0.3889), 0.0113 (95% confidence interval -0.0157 to 0.0384), 0.9995 (95% confidence interval 0.9990-1.0000) for succinyl-carnitine and 0.5699 (95% confidence interval 0.5263-0.6134), 0.0319 (95% confidence interval -0.0038 to 0.0677), 0.9997 (95% confidence interval 0.9995-1.0000) for methylmalonyl-carnitine. Within-day and between-day coefficients of variation (CV) were 1.94% and 3.19% for succinyl-carnitine and 3.21%, and 2.56 for methylmalonyl-carnitine. This method is accurate and provides a new tool to differentiate patients with classical methylmalonic acidemia from those with SCS-related defects.

Objective: Congenital hyperinsulinism (CHI) requires rapid diagnosis and treatment to avoid irreversible neurological sequelae due to hypoglycaemia. Aetiological diagnosis is instrumental in directing the appropriate therapy. Current diagnostic algorithms provide a complete set of diagnostic tools including (i) biochemical assays, (ii) genetic facility and (iii) state-of-the-art imaging. They consider the response to a therapeutic diazoxide trial an early, crucial step before proceeding (or not) to specific genetic testing and eventually imaging, aimed at distinguishing diffuse vs focal CHI. However, interpretation of the diazoxide test is not trivial and can vary between research groups, which may lead to inappropriate decisions. Objective of this report is proposing a new algorithm in which early genetic screening, rather than diazoxide trial, dictates subsequent clinical decisions. Patients, Methods and Results: Two CHI patients weaned from parenteral glucose infusion and glucagon after starting diazoxide. No hypoglycaemia was registered during a 72-h continuous glucose monitoring (CGMS), or hypoglycaemic episodes were present for no longer than 3% of 72-h. Normoglycaemia was obtained by low–medium dose diazoxide combined with frequent carbohydrate feeds for several years. We identified monoallelic, paternally inherited mutations in KATP channel genes, and 18F-DOPA PET-CT revealed a focal lesion that was surgically resected, resulting in complete remission of hypoglycaemia. Conclusions: Although rare, some patients with focal lesions may be responsive to diazoxide. As a consequence, we propose an algorithm that is not based on a ‘formal’ diazoxide response but on genetic testing, in which patients carrying paternally inherited ABCC8 or KCNJ11 mutations should always be subjected to 18F-DOPA PET-CT.

Background Mitochondrial disease is a family of genetic disorders characterized by defects in the generation and regulation of energy. Epilepsy is a common symptom of mitochondrial disease, and in the vast majority of cases, refractory to commonly used antiepileptic drugs. Ferroptosis is a recently-described form of iron- and lipid-dependent regulated cell death associated with glutathione depletion and production of lipid peroxides by lipoxygenase enzymes. Activation of the ferroptosis pathway has been implicated in a growing number of disorders, including epilepsy. Given that ferroptosis is regulated by balancing the activities of glutathione peroxidase-4 (GPX4) and 15-lipoxygenase (15-LO), targeting these enzymes may provide a rational therapeutic strategy to modulate seizure. The clinical-stage therapeutic vatiquinone (EPI-743, α-tocotrienol quinone) was reported to reduce seizure frequency and associated morbidity in children with the mitochondrial disorder pontocerebellar hypoplasia type 6. We sought to elucidate the molecular mechanism of EPI-743 and explore the potential of targeting 15-LO to treat additional mitochondrial disease-associated epilepsies. Methods Primary fibroblasts and B-lymphocytes derived from patients with mitochondrial disease-associated epilepsy were cultured under standardized conditions. Ferroptosis was induced by treatment with the irreversible GPX4 inhibitor RSL3 or a combination of pharmacological glutathione depletion and excess iron. EPI-743 was co-administered and endpoints, including cell viability and 15-LO-dependent lipid oxidation, were measured. Results EPI-743 potently prevented ferroptosis in patient cells representing five distinct pediatric disease syndromes with associated epilepsy. Cytoprotection was preceded by a dose-dependent decrease in general lipid oxidation and the specific 15-LO product 15-hydroxyeicosatetraenoic acid (15-HETE). Conclusions These findings support the continued clinical evaluation of EPI-743 as a therapeutic agent for PCH6 and other mitochondrial diseases with associated epilepsy.

Background Glycogen storage disease type III (GSDIII) is caused by mutations of AGL gene with debranching enzyme deficiency. Patients with GSDIII manifest fasting hypoglycemia, hepatomegaly, hepatopathy, myopathy, and cardiomyopathy. We report on an 18‐year‐old boy with a profound growth retardation (

Cystinuria is an autosomal recessive disease that causes l-cystine precipitation in urine and nephrolithiasis. Disease severity is highly variable; it is known, however, that cystinuria has a more severe course in males. The aim of this study was to compare l-cystine metastability in first-morning urine collected from 24 normal female and 24 normal male subjects. Samples were buffered at pH 5 and loaded with l-cystine (0.4 and 4 mM final concentration) to calculate the amount remaining in solution after overnight incubation at 4 °C; results were expressed as Z scores reflecting the l-cystine solubility in each sample. In addition, metabolomic analyses were performed to identify candidate compounds that influence l-cystine solubility. l-cystine solubility Z score was +0.44 ± 1.1 and −0.44 ± 0.70 in female and male samples, respectively (p

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.

Cobalamin C (cblC) defect is the most common inborn error of vitamin B12 metabolism. Clinical features vary as does the severity of the disease. In most cases, the clinical symptoms of cblC defect tend to appear during infancy or early childhood as a multisystem disease with severe neurologic, ocular, hematologic, renal, and gastrointestinal signs. The neurologic findings are common and include hypotonia, developmental delay, microcephaly, seizures hydrocephalus, and brain MRI abnormalities. We report a case of a young boy with cblC defect, who did not undergo newborn screening, presenting at the age of 2 years with isolated pulmonary hypertension as the leading symptom. This novel way of presentation of cblC defect enlarges the spectrum of inherited diseases that must be considered in the differential diagnosis of pulmonary hypertension.

Congenital hyperinsulinism (CHI) is a genetic disorder characterized by profound hypoglycemia related to an inappropriate insulin secretion. It is a heterogeneous disease classified into two major subgroups: "channelopathies" due to defects in ATP-sensitive potassium channel, encoded by ABCC8 and KCNJ11 genes, and "metabolopathies" caused by mutation of several genes (GLUD1, GCK, HADH, SLC16A1, HNF4A and HNF1A) and involved in different metabolic pathways. To elucidate the genetic etiology of CHI in the Italian population, we conducted an extensive sequencing analysis of the CHI-related genes in a large cohort of 36 patients: Twenty-nine suffering from classic hyperinsulinism (HI) and seven from hyperinsulinism-hyperammonemia (HI/HA). Seventeen mutations have been found in fifteen HI patients and five mutations in five HI/HA patients. Our data confirm the major role of ATP-sensitive potassium channel in the pathogenesis of Italian cases (~70%) while the remaining percentage should be attributed to other. A better knowledge of molecular basis of CHI would lead to improve strategies for genetic screening and prenatal diagnosis. Moreover, genetic analysis might also help to distinguish the two histopathological forms of CHI, which would lead to a clear improvement in the treatment and in genetic counseling.

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.

Methylmalonic aciduria with homocystinuria, cblC defect, is the most frequent disorder of vitamin B12 metabolism. CblC patients are commonly treated with a multidrug therapy to reduce metabolite accumulation and to increase deficient substrates. However the long-term outcome is often unsatisfactory especially in patients with early onset, with frequent progression of neurological and ocular impairment. Recent studies, have shown perturbation of cellular redox status in cblC. To evaluate the potential contribution of oxidative stress into the patophysiology of cblC defect, we have analyzed the in vivo glutathione metabolism in a large series of cblC deficient individuals.Levels of different forms of glutathione were measured in lymphocytes obtained from 18 cblC patients and compared with age-matched controls. Furthermore, we also analyzed plasma cysteine and total homocysteine.We found an imbalance of glutathione metabolism in cblC patients with a significant decrease of total and reduced glutathione, along with a significant increase of different oxidized glutathione forms.These findings show a relevant in vivo disturbance of glutathione metabolism underlining the contribution of glutathione pool depletion to the redox imbalance in treated cblC patients. Our study may be helpful in addressing future research to better understanding the pathogenetic mechanism of the disease and in developing new therapeutic approaches, including the use of novel vitamin B₁₂ derivatives.

Congenital hyperinsulinism (CHI) occurs as a consequence of unregulated insulin secretion from the pancreatic beta-cells. Severe recessive mutations and milder dominant mutations have been described in the ABCC8 and KCNJ11 genes encoding SUR1 and Kir6.2 subunits of the beta-cell ATP-sensitive K(+) channel. Here we report two patients with CHI unresponsive to medical therapy with diazoxide. Sequencing analysis identified a compound heterozygous mutation in ABCC8 in both patients. The first one is a carrier for the known mild dominant mutation p.Glu1506Lys jointly with the novel mutation p.Glu1323Lys. The second carries the p.Glu1323Lys mutation and a second novel mutation, p.Met1394Arg. Functional studies of both novel alleles showed reduced or null cell surface expression, typical of recessive mutations. Compound heterozygous mutations in congenital hyperinsulinism result in complex interactions. Studying these mechanisms can improve the knowledge of this disease and modify its therapy.