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Vacuolar-type H⁺-ATPase (V-ATPase) is a multimeric complex present in a variety of cellular membranes that acts as an ATP-dependent proton pump and plays a key role in pH homeostasis and intracellular signalling pathways. In humans, 22 autosomal genes encode for a redundant set of subunits allowing the composition of diverse V-ATPase complexes with specific properties and expression. Sixteen subunits have been linked to human disease. Here we describe 26 patients harbouring 20 distinct pathogenic de novo missense ATP6V1A variants, mainly clustering within the ATP synthase α/β family-nucleotide-binding domain. At a mean age of 7 years (extremes: 6 weeks, youngest deceased patient to 22 years, oldest patient) clinical pictures included early lethal encephalopathies with rapidly progressive massive brain atrophy, severe developmental epileptic encephalopathies and static intellectual disability with epilepsy. The first clinical manifestation was early hypotonia, in 70%; 81% developed epilepsy, manifested as developmental epileptic encephalopathies in 58% of the cohort and with infantile spasms in 62%; 63% of developmental epileptic encephalopathies failed to achieve any developmental, communicative or motor skills. Less severe outcomes were observed in 23% of patients who, at a mean age of 10 years and 6 months, exhibited moderate intellectual disability, with independent walking and variable epilepsy. None of the patients developed communicative language. Microcephaly (38%) and amelogenesis imperfecta/enamel dysplasia (42%) were additional clinical features. Brain MRI demonstrated hypomyelination and generalized atrophy in 68%. Atrophy was progressive in all eight individuals undergoing repeated MRIs. Fibroblasts of two patients with developmental epileptic encephalopathies showed decreased LAMP1 expression, Lysotracker staining and increased organelle pH, consistent with lysosomal impairment and loss of V-ATPase function. Fibroblasts of two patients with milder disease, exhibited a different phenotype with increased Lysotracker staining, decreased organelle pH and no significant modification in LAMP1 expression. Quantification of substrates for lysosomal enzymes in cellular extracts from four patients revealed discrete accumulation. Transmission electron microscopy of fibroblasts of four patients with variable severity and of induced pluripotent stem cell-derived neurons from two patients with developmental epileptic encephalopathies showed electron-dense inclusions, lipid droplets, osmiophilic material and lamellated membrane structures resembling phospholipids. Quantitative assessment in induced pluripotent stem cell-derived neurons identified significantly smaller lysosomes. ATP6V1A-related encephalopathy represents a new paradigm among lysosomal disorders. It results from a dysfunctional endo-lysosomal membrane protein causing altered pH homeostasis. Its pathophysiology implies intracellular accumulation of substrates whose composition remains unclear, and a combination of developmental brain abnormalities and neurodegenerative changes established during prenatal and early postanal development, whose severity is variably determined by specific pathogenic variants.

Congenital disorders of glycosylation (CDG) are due to defective glycosylation of glycoconjugates. Conserved oligomeric Golgi (COG)-CDG are genetic diseases due to defects of the COG complex subunits 1-8 causing N-glycan and O-glycan processing abnormalities. In COG-CDG, isoelectric focusing separation of undersialylated glycoforms of serumtransferrin and apolipoprotein C-III (apoC-III) allows to detect N-glycosylation and O-glycosylation defects, respectively. COG5-CDG (COG5 subunit deficiency) is a multisystem disease with dysmorphic features, intellectual disability of variable degree, seizures, acquired microcephaly, sensory defects and autistic behavior. We applied matrix-assisted laser desorption/ionization-MS for a high-throughput screening of differential serum O-glycoform and N-glycoform in five patients with COG5-CDG. When compared with age-matched controls, COG5-CDG showed a significant increase of apoC-III0a (aglycosylated glycoform), whereas apoC-III1 (mono-sialylated glycoform) decreased significantly. Serum N-glycome of COG5-CDG patients was characterized by the relative abundance of undersialylated and undergalactosylated biantennary and triantennary glycans as well as slight increase of high-mannose structures and hybrid glycans. Using advanced and well-established MS-based approaches, the present findings reveal novel aspects on O-glycan and N-glycan profiling in COG5-CDG patients, thus providing an increase of current knowledge on glycosylation defects caused by impairment of COG subunits, in support of clinical diagnosis. Copyright (C) 2017 John Wiley & Sons, Ltd.

Item does not contain fulltext OBJECTIVE: Congenital disorders of glycosylation (CDG) are a group of metabolic diseases due to defects in protein and lipid glycosylation. We searched for the primary defect in 3 children from 2 families with a severe neurological phenotype, including profound developmental delay, intractable epilepsy, progressive microcephaly, severe hypotonia with elevated blood creatine kinase levels, and early fatal outcome. There was clinical evidence of a muscular dystrophy-dystroglycanopathy syndrome, supported by deficient O-mannosylation by muscle immunohistochemistry. METHODS: Biochemical and molecular methods were combined to pinpoint the defect in the glycosylation pathway in the endoplasmic reticulum. RESULTS: Metabolic investigations revealed CDG-I, pointing to a defect in protein N-glycosylation in the endoplasmic reticulum. Analysis of lipid-linked oligosaccharides in fibroblasts showed accumulation of Dol-PP-GlcNAc(2) -Man(5) . DNA analysis revealed mutations in DPM2, 1 of the subunits of the dolichol-phosphate-mannose (DPM) synthase; the patient in the first family is compound heterozygous for 2 mutations (c.68A>G, predicting a missense mutation p.Y23C and c.4-1G>C, a splice mutation), whereas the patients in the second family are homozygous for the same missense mutation (c.68A>G, p.Y23C). INTERPRETATION: We describe a new CDG, due to a deficiency of DPM2. Hence, mutations have now been described in the genes for the 3 subunits of DPM: DPM1, DPM2, and DPM3, whereby DPM2-CDG links the congenital disorders of glycosylation to the congenital muscular dystrophies. ANN NEUROL 2012;72:550-558.

In Italy the debate on the potential of biomass energy resources and the first hypothesis on their use for this purpose are developed in the late Nineties, mainly in response to the guidelines set out in the Kyoto Protocol. In order to estimate the potential contribution of agriculture to energy needs, the paper analyses the structure and organization of the Energy Action Plan of the different Italian regions, with particular attention to forecasts of supply of energy from biomass in the area with a necessary rescheduling of investments between food and no-food crops.

The analysis of the economic instrument "incentive" for the development of bioenergy from forestry sector is the aim of this work. It analyses the structure and operative possibilities within the existing public support system for the development of agricultural activities and production of agro-bio-energies. The study also points out the use of Payments for Environmental Services (PES) for the multifunctionality of agriculture and examines the Rural Development Plan 2000-2006 and the 2007-2013 Rural Development Programmes of some Italian regions.

Investigation for inherited metabolic diseases are rarely performed in epileptic children. Usually these patients are classified on the basis of the seizures phenotype and drug response. Seizures are described in Congenital Disorders of Glycosylation (CDG). In the past years (2005-2011) we analyzed more than three thousand samples from Italian patients by transferrin IEF, positive ones were typed by MALDI-TOF. We identified 21 patients with PMM2-CDG (CDG Ia) and seven with different types of non-PMM2 CDG. Among these, drug resistant epilepsy in the first year of life was the presenting sign in six, commonly infantile spasms, myoclonic or tonic-clonic seizures. Dysmorphisms were a constant finding (7/7), followed by microcephaly (6/7), severe mental retardation (6/7), visual impairment (5/7) and only 1/7 had cerebellar atrophy. Serum transferrin IEF and MALDI-TOF defined a type I underglycoslation profile in these patients but PMM2 was normal. Definitive diagnosis was ALG6- CDG Ic; ALG3-CDG Id and DPM2 in one patient respectively and is pending in four (CDG Ix). We wish to underline that early onset epileptic encephalopathies and drugresistant epilepsy, in the first months of life, should arouse the suspicion of non PMM2-CDG. This could allow the recognition of a number of undiagnosed CDG cases.