Combined mass spectrometry methods for serum N-glycoprotein profiling enhance the awareness of the molecular patho-mechanism in ALG12-CDG

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 <-mannosyl residue to the core Man(<1-6) of the dolichol-PP-oligosaccharide precursor, thus ensuring its correct shape and branching before the oligosyltransferase (OST) complex action. Nine patients with ALG12-CDG have been reported so far. We investigated by MALDI MS and UHPLC-ESI MS the glyco-phenotype of a novel patient with unreported variants, sharing most of the clinical signs of patients with ALG12 deficiency, including severe recurrent infections with hypogammaglobulinemia and B cell dysfunction. MALDI MS analysis of native transferrin showed underoccupancy of N-glycosylation sites, a typical feature of CDG-I. N-glycome analysis by MALDI MS, 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). 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. Glycan characterization of target glycoproteins may endorse the molecular defect unraveling the complex clinical phenotype of CDG patients.