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ALG3-CDG is one of the very rare types of congenital disorder of glycosylation (CDG) caused by variants in the ER-mannosyltransferase ALG3. Here, we summarize the clinical, biochemical, and genetic data of four new ALG3-CDG patients, who were identified by a type I pattern of serum transferrin and the accumulation of Man 5 GlcNAc 2 -PP-dolichol in LLO analysis. Additional clinical symptoms observed in our patients comprise sensorineural hearing loss, right-descending aorta, obstructive cardiomyopathy, macroglossia, and muscular hypertonia. We add four new biochemically confirmed variants to the list of ALG3-CDG inducing variants: c.350G>C (p.R117P), c.1263G>A (p.W421*), c.1037A>G (p.N346S), and the intron variant c.296+4A>G. Furthermore, in Patient 1 an additional open-reading frame of 141 bp (AAGRP) in the coding region of ALG3 was identified. Additionally, we show that control cells synthesize, to a minor degree, a hybrid protein composed of the polypeptide AAGRP and ALG3 (AAGRP-ALG3), while in Patient 1 expression of this hybrid protein is significantly increased due to the homozygous variant c.160_196del (g.165C>T). By reviewing the literature and combining our findings with previously published data, we further expand the knowledge of this rare glycosylation defect., (© 2019 Wiley Periodicals, Inc.)

Background: Congenital disorders of glycosylation (CDG) includes ALG8 deficiency, a protein N-glycosylation defect with a broad clinical spectrum. If most of the 15 previously reported patients present an early-onset multisystem severe disease and early death, three patients including the cas princeps, present long-term survival and less severe symptoms., Methods: In order to further characterize ALG8-CDG, two new ALG8 patients are described and mRNA analyses of the ALG8-CDG cas princeps were effected., Results: One new patient exhibited a hepato-intestinal and neurological phenotype with two novel variants (c.91A > C p.Thr31Pro; c.139dup p.Thr47Asnfs*12). The other new patient, homozygous for a known variant (c.845C > T p.Ala282Val), presented a neurological phenotype with epilepsy, intellectual disability and retinis pigmentosa. The cas princeps ALG8-CDG patient was reported to have two heterozygous frameshift variants predicted to be without activity. We now described a novel ALG8 transcript variant in this patient and the 3D model of the putative encoded protein reveals no major difference with that of the normal ALG8 protein., Conclusion: The description of the two new ALG8 patients affirms that ALG8-CDG is a severe disease. In the cas princeps, as the originally described frameshift variants are degraded, the novel variant is promoted and could explain a milder phenotype.

An efficient and convergent method for the synthesis of mannose-1-phosphate prodrugs is described as a potential therapy for congenital disorders of glycosylation-Ia (CDG-Ia). The key feature of the proposed approach is the silver assisted nucleophilic substitution of 2,3,4,6-tetra-O-protected-alpha-d-mannopyranosyl bromides with various silver phosphate salts to afford mono, di, and tri-mannopyranosyl phosphates. A preliminary biological evaluation of the synthesized phosphate prodrugs has been carried out.

Congenital disorders of glycosylation (CDG) type I (CDG I) are rare autosomal recessive diseases caused by deficiencies in the assembly of the dolichol-linked oligosaccharide (DLO) that is required for N-glycoprotein biosynthesis. CDG Ie is due to a defect in the synthesis of dolichyl-phosphoryl-mannose (Dol-P-Man), which is needed for DLO biosynthesis as well as for other glycosylation pathways. Human Dol-P-Man synthase is a heterotrimeric complex composed of DPM1p, DPM2p, and DPM3p, with DPM1p being the catalytic subunit. Here, we report two new CDG Ie patients who present milder symptoms than the five other CDG Ie patients described to date. The clinical pictures of the patients MS and his sister MT are dominated by major ataxia, with no notable hepatic involvement. MS cells accumulate the immature DLO species Dol-PP-GlcNAc2Man5 and possess only residual Dol-P-Man synthase activity. One homozygous intronic mutation, g.IVS4-5T>A, was found in the DPM1 gene, leading to exon skipping and transcription of a shortened transcript. Moreover, DPM1 expression was reduced by more than 90% in MS cells, in a nonsense-mediated mRNA decay (NMD)-independent manner. Full analysis of the DPM2 and DPM3 genes revealed a decrease in DPM2 expression and normal expression of DPM3. This description emphasizes the large spectrum of symptoms characterizing CDG I patients.

Mammalian protein N-glycosylation requires the transfer of an oligosaccharide containing 2 residues of N-acetylglucosamine, 9 residues of mannose and 3 residues of glucose (Glc3Man9 GlcNAc2) from Glc3Man9GlcNAc2-diphospho (PP)-dolichol (DLO) onto proteins in the endoplasmic reticulum (ER). Under some pathophysiological conditions, DLO biosynthesis is perturbed, and truncated DLO is hydrolyzed to yield oligosaccharyl phosphates (OSP) via unidentified mechanisms. DLO diphosphatase activity (DLODP) was described in vitro, but its characterization is hampered by a lack of convenient non-radioactive substrates. Our objective was to develop a fluorescence-based assay for DLO hydrolysis. Using a vancomycin-based solid-phase extraction procedure coupled with thin layer chromatography (TLC) and mass spectrometry, we demonstrate that mouse liver membrane extracts hydrolyze fluorescent bacterial lipid II (LII: GlcNAc-MurNAc(dansyl-pentapeptide)-PP-undecaprenol) to yield GlcNAc-MurNAc(dansyl-pentapeptide)-P (GM5P). GM5P production by solubilized liver microsomal proteins shows similar biochemical characteristics to those reported for human hepatocellular carcinoma HepG2 cell DLODP activity. To conclude, we show, for the first time, hydrolysis of lipid II by a eukaryotic enzyme. As LII and DLO are hydrolyzed by the same, or closely related, enzymes, fluorescent lipid II analogs are convenient non-radioactive substrates for investigating DLODP and DLODP-like activities. [ABSTRACT FROM AUTHOR]