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Congenital disorders of glycosylation (CDG) are genetic diseases characterized by deficient synthesis (CDG type I) and/or abnormal processing (CDG type II) of glycan moieties linked to protein and lipids. The impact of the molecular defects on protein glycosylation and in turn on the clinical phenotypes of patients with CDG is not yet understood. ALG12-CDG is due to deficiency of ALG12 α1,6-mannosyltransferase that adds the eighth mannose residue on the dolichol-PP-oligosaccharide precursor in the endoplasmic reticulum. ALG12-CDG is a severe multisystem disease associated with low to deficient serum immunoglobulins and recurrent infections. We thoroughly investigated the glycophenotype in a patient with novel ALG12 variants and immunodeficiency. We analyzed serum native transferrin, as first line test for CDG and we profiled serum IgG and total serum N-glycans by a combination of consolidated (N-glycan analysis by MALDI MS) and innovative mass spectrometry-based protocols, such as GlycoWorks RapiFluor N-glycan analysis coupled with LC-ESI MS. Intact serum transferrin showed, as expected for a CDG type I defect, underoccupancy of N-glycosylation sites. Surprisingly, total serum proteins and IgG N-glycans showed some specific changes, consisting in accumulating amounts of definite high-mannose and hybrid structures. As a whole, ALG12-CDG behaves as a dual CDG (CDG-I and II defects) and it is associated with distinct, abnormal glycosylation of total serum and IgG N-glycans. Glycan profiling of target glycoproteins may endorse the molecular defect unraveling the complex clinical phenotype of CDG patients.

Congenital disorders of glycosylation (CDG) are inherited metabolic diseases affecting the glycan biosynthesis of glycoconjugates [1]. They represent an expanding group of multisystemic diseases with variable phenotypes and prevalent neurological involvement. Among the genetic disorders associated to the protein N-glycosylation pathway, CDG type I (CDG-I) occur in the cytosol or in the endoplasmic reticulum (ER) affecting dolichol-linked oligosaccharide synthesis, whereas CDG type II (CDG-II) involve the N-linked oligosaccharide processing in the Golgi. ALG12-CDG is caused by deficiency of ALG12 ?1,6-mannosyltransferase that adds the eighth mannose residue on the dolichol-PP-oligosaccharide precursor in the ER, thus ensuring its correct shape and branching before the oligosaccharyl-transferase (OST) complex action. We investigated the glyco-phenotype of a novel ALG12-CDG patient with unreported variants, sharing most of the clinical signs of ALG12 deficiency, including recurrent infections and hypogammaglobulinemia. By a combination of consolidated and innovative mass spectrometry-based protocols [2-4], we analyzed serum native transferrin and profiled serum IgG and total serum N-glycans. MALDI MS analysis of intact transferrin showed underoccupancy of N-glycosylation sites, a typical feature of CDG-I. Moreover, N-glycome analysis either on total serum N-glycan pool and on IgG released N-glycans, revealed the abnormal occurrence of high-mannose and hybrid N-glycan species. These accumulating glycoforms, further analyzed by UHPLC-ESI MS, corresponded to unbranched structures with ?1,2-terminal mannose residues, previously identified in serum of patients with MAN1B1-CDG (CDG-II defect) [5]. Glycosylation analyses on the observed ALG12-CDG patient revealed a combination of CDG-I and CDG-II defects, these last associated with abnormal IgG N-glycan profile, consistent with the immunophenotype. As a whole, glycan characterization of target glycoproteins may endorse the molecular defect unraveling the complex clinical phenotype of CDG patients.

Congenital disorders of glycosylation (CDG) are inherited metabolic diseases affecting the glycan biosynthesis of glycoconjugates. They represent an expanding group of multisystemic diseases with variable phenotypes and prevalent neurological involvement. More than 120 genetic disorders have been associated to defective glycosylation, mainly in the protein N-glycosylation pathway. Among these, CDG type I (CDG-I) occur in the cytosol or in the endoplasmic reticulum (ER) affecting dolichol-linked oligosaccharide synthesis, whereas CDG type II (CDG-II) involve the N-linked oligosaccharide processing in the Golgi. ALG12-CDG (MIM: 607143) is caused by mutations of the human orthologue of the yeast asparagine-linked glycosylation (ALG)12 gene, encoding the mannosyltransferase which adds in the ER an