Glycoproteomics and Functional Characterization of Novel Variants in Siblings with ALG1-CDG.

Congenital disorders of glycosylation (CDGs) are a growing group of inborn errors caused by gene defects in glycan biosynthesis pathways. Genetic testing of two patients from the same family harboring ALG1-related CDG (ALG1-CDG) revealed heterozygous variations in the c.1129 A>C and c.1263+3 A>T. Although most CDGs with abnormal N-glycosylation can be detected by transferrin screening, Western blotting of serum transferrin from the two siblings did not reveal significant glycosylation deficiency. In this study, the biochemical phenotype and pathological molecular basis of the CDGs in the siblings were further explored. Analysis of the patients' ALG1 cDNA indicated that the associated heterozygous variants were c.1129 A>C (p.Met377Leu) and c.1188 T>A (p.Cys396X). Single-molecule real-time (SMRT) sequencing indicated that the variant frequencies of M377L and C396X were 50% and 36%, respectively. In addition, glycoproteomics analysis suggested that the glycoforms of transferrin were altered in the patients and that glycosylation site occupancy was decreased. It was also confirmed that N-glycosylation was altered in the patients' serum transferrin. Conserved amino acid variants of yeast Alg1 were recombinantly overexpressed, purified and subjected to an in vitro quantitative activity assay. The results showed that these variants caused a decrease in enzymatic activity, suggesting pathogenicity.