N-Glycan Abnormalities in Children with Galactosemia

Abstract

Galactose intoxication and over-restriction in galactosemia may affect glycosylation pathways and cause multisystem effects. In this study, we describe an applied hydrophilic interaction chromatography ultra-performance liquid chromatography high-throughput method to analyze whole serum and extracted IgG N-glycans with measurement of agalactosylated (G0), monogalactosylated (G1), and digalactosylated (G2) structures as a quantitative measure of galactose incorporation. This was applied to nine children with severe galactosemia (genotype Q188R/Q188R) and one child with a milder variant (genotype S135L/S135L). The profiles were also compared with those obtained from three age-matched children with PMM2-CDG (congenital disorder of glycosylation type Ia) and nine pediatric control samples. We have observed that severe N-glycan assembly defects correct in the neonate following dietary restriction of galactose. However, treated adult galactosemia patients continue to exhibit ongoing N-glycan processing defects. We have now applied informative galactose incorporation ratios as a method of studying the presence of N-glycan processing defects in children with galactosemia. We identified N-glycan processing defects present in galactosemia children from an early age. For G0/G1, G0/G2, and (G0/G1)/G2 ratios, the difference noted between galactosemia patients and controls was found to be statistically significant (p = 0.002, 0.01, and 0.006, respectively).

Classical galactosaemia: novel insights in IgG N-glycosylation and N-glycan biosynthesis

“…It is not clear if ongoing glycosylation abnormalities further modify the observed outcomes. 21,28 In our previous studies of circulating IgG N-glycans, we demonstrated correction of the gross assembly defects in young galactosaemia patients following treatment with galactose restriction. 28 We also demonstrated the presence of IgG N-glycan processing defects in adults and children with galactosaemia maintained on a galactose restricted diet using IgG galactose incorporation ratios.…”

Effects of temporary low-dose galactose supplements in children aged 5–12 y with classical galactosemia: a pilot study

“…Serum was prepared from freshly spun blood samples and immediately frozen at −20 °C. IgG isolation was performed on a robotized glycomics platform as described previously, using Protein G 96-well filter plates (Fisher Scientific, Pittsburgh, PA) (7,37). The purity of the isolated IgG was assessed using 10 % reducing SDS-PAGE in an Xcell SureLock Mini-Cell (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions (data not shown).…”

“…N-glycans were released from IgG using the automated method and peptide N-glycanase F (1,000 U/ml) as described elsewhere (7,22). Released N-glycans were then labeled by reductive amination with the fluorophore 2-AB 41,42 using a Ludger Tag 2-AB glycan labeling kit as described by the manufacturer (Ludger, Oxford, UK).…”

“…Our group recently described glycosylation abnormalities in IgG N-glycans of patients with galactosemia along with the potential application of IgG N-glycans as biomarkers for patients (6,7). Glycosylation is a widespread post-translational modification of proteins and deregulation of glycosylation is associated with a wide range of diseases (8).…”

“…Glycosylation is a widespread post-translational modification of proteins and deregulation of glycosylation is associated with a wide range of diseases (8). Along these lines, we have previously described galactose incorporation ratios as a method of studying the presence of N-glycan processing defects in children with galactosemia (7). In earlier and ongoing studies, we have identified that galactosylation is abnormal in patients with galactosemia and may play a role in the neurological manifestations by means of impaired galactosylation of cerebrosides and glycoproteins in the brain (5,7 and unpublished data).…”