N-Glycan Abnormalities in Children with Galactosemia Karen P. Coss,† Colin P. Hawkes,‡ Barbara Adamczyk,§ Henning Stöckmann,§ Ellen Crushell,‡ Radka Saldova,§ Ina Knerr,‡ Maria E. Rubio-Gozalbo,∥ Ardeshir A. Monavari,‡ Pauline M. Rudd,§ and Eileen P. Treacy*,‡,⊥ †

University College Dublin (UCD), Clinical Research Centre, Mater Misericordiae University Hospital, Eccles Street, Dublin, Ireland National Centre for Inherited Metabolic Disorders (NCIMD), Children’s University Hospital, Temple Street, Dublin, Ireland § National Institute for Bioprocessing Research and Training (NIBRT), GlycoScience Group, Mount Merrion, Blackrock, Dublin, Ireland ∥ Maastricht University Medical Centre, Minderbroedersberg 4, Maastricht, The Netherlands ⊥ Trinity College, College Green, Dublin, Ireland ‡

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 ultraperformance 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). KEYWORDS: galactosemia, children, HILIC-UPLC, IgG, glycan analysis, N-glycan processing abnormalities

INTRODUCTION Classical galactosemia (OMIM 230400) is a rare, autosomal recessive disorder of carbohydrate metabolism caused by mutations in the galactose-1-phosphate uridyltransferase (GALT) gene. Deficiency of GALT (EC results in a toxic build-up of intermediates of the galactose metabolism (Leloir) pathway, such as galactose-1-phosphate (gal-1-p) and galactitol.1 Galactose-intoxicated galactosemia neonates exhibit high levels of gal-1-p, which decrease upon the introduction of dietary restriction of galactose. However, beyond the neonatal stage, biochemical measures of monitoring red blood cell (RBC) gal-1-p and urinary galactitol are not informative biomarkers in determining the response to treatment2−5 and individual galactose tolerance. The exact pathophysiology determining the long-term complications of galactosemia remains unclear. 1,2,6 The complications noted are not related to diet control or birth order.3,7 Several large-scale multicenter studies have shown that long-term complications persist in all populations regardless of early diagnosis and initiation of treatment.8−12 © 2013 American Chemical Society

The galactose-intoxicated galactosemia neonate experiences endoplasmic reticulum (ER) stress likely as a result of increases in gal-1-p, which in turn causes disruptions to the inositol monophosphatase pathway,6,13,14 which may also be evident in other intoxication-like hereditary metabolic disorders.15 It is of great importance for treatment approaches to this condition to determine if long-term outcomes are related predominantly to early pre- and postnatal galactose intoxication effects or whether they may also be modified by ongoing glycosylation abnormalities. Abnormalities in the levels of UDP-hexose uptake, particularly UDP-galactose and UDP-glucose, have been reported in galactosemia.1,16,17 A number of studies have investigated UDP-galactose and UDP-glucose levels in galactosemia erythrocytes and dermal fibroblast cultures with varying results, with some studies suggesting decreased UDP-hexose levels, particularly UDP-galactose,16−19 while others do not support Received: April 23, 2013 Published: December 12, 2013 385 | J. Proteome Res. 2014, 13, 385−394

Journal of Proteome Research


Table 1. Patient Biochemical and Clinical Characteristics






1 2 3 4b


11 months 4 years 9 years 6 years

Q188R/Q188R S135L/S135L Q188R/Q188R Q188R/Q188R

Irish African Irish Traveler Irish-American

5b 6 7 8 9 10


4 years 6 years 4 years 7 years 6 years 9 years

Q188R/Q188R Q188R/Q188R Q188R/Q188R Q188R/Q188R Q188R/Q188R Q188R/Q188R

Irish-American Irish Irish Traveler Irish Traveler Irish Irish Traveler



1 years 5 months

PMM2-CDG R141H/V231M




2 years

PMM2-CDG A108 V/R123Q




15 years

PMM2-CDG T237R/C241S


complications FSIQ < 70, neonatal cataracts FSIQ 40, autistic, speech delay FSIQ 74, ataxia, speech delay developmental delay FSIQ 80 FSIQ 91, speech delay FSIQ 80 FSIQ 70−79, speech difficulties FSIQ 78, verbal dyspraxia FSIQ 73, learning difficulties and verbal dyspraxia dysmorphic, failure to thrive, psychomotor retardation, liver dysfunction and coagulopathy, hypogonadism hypotonia, developmental delay, cerebellar atrophy psychomotor retardation, ataxia



neonate 18 months neonate neonate

N-glycan abnormalities in children with galactosemia.

Galactose intoxication and over-restriction in galactosemia may affect glycosylation pathways and cause multisystem effects. In this study, we describ...
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