Oral D-galactose supplementation in PGM1-CDG

Abstract

PurposePhosphoglucomutase-1 deficiency is a subtype of congenital disorders of glycosylation (PGM1-CDG). Previous casereports in PGM1-CDG patients receiving oral D-galactose (D-gal) showed clinical improvement. So far no systematic in vitro and clinical studies have assessed safety and benefits of D-gal supplementation. In a prospective pilot study, we evaluated the effects of oral D-gal in nine patients.MethodsD-gal supplementation was increased to 1.5 g/kg/day (maximum 50 g/day) in three increments over 18 weeks. Laboratory studies were performed before and during treatment to monitor safety and effect on serum transferrin-glycosylation, coagulation, and liver and endocrine function. Additionally, the effect of D-gal on cellular glycosylation was characterized in vitro.ResultsEight patients were compliant with D-gal supplementation. No adverse effects were reported. Abnormal baseline results (alanine transaminase, aspartate transaminase, activated partial thromboplastin time) improved or normalized already using 1 g/kg/day D-gal. Antithrombin-III levels and transferrin-glycosylation showed significant improvement, and increase in galactosylation and whole glycan content. In vitro studies before treatment showed N-glycan hyposialylation, altered O-linked glycans, abnormal lipid-linked oligosaccharide profile, and abnormal nucleotide sugars in patient fibroblasts. Most cellular abnormalities improved or normalized following D-gal treatment. D-gal increased both UDP-Glc and UDP-Gal levels and improved lipid-linked oligosaccharide fractions in concert with improved glycosylation in PGM1-CDG.ConclusionOral D-gal supplementation is a safe and effective treatment for PGM1-CDG in this pilot study. Transferrin glycosylation and ATIII levels were useful trial end points. Larger, longer-duration trials are ongoing.

Figure 1

Effect of D-gal supplementation on (a) aspartate transaminase (AST) and (b) alanine transaminase (ALT), on (c) anti-coagulation and (d) (e) (f) coagulation, on (g) TSH, (h) TBG, and (i) IGFBP-3. The shadowed area represents the reference range.

Figure 2

High-resolution mass spectrometry of intact serum transferrin. Baseline profiles of patient 2 (a) and patient 9 (b) show characteristic PGM1 glycoforms with truncated glycans and lack of whole glycans. Patient 9 shows a milder profile than patient 2; Spectra of patient 2 (c) and patient 9 (d) show large improvement through the reduction of abnormal glycosylation peaks upon 18 weeks of galactose treatment, as is highlighted by the green arrows.

Figure 3

High-resolution transferrin glycosylation analysis in PGM1-CDG patients before and after D-gal supplement. Ratios of a-, mono- and tri-sialo over tetra-sialo transferrin were calculated and compared with references ranges (Repeated measures of ANOVA). A-Glyco: <= 0,01040, Mono-glyco: <=0,02700 Trisyalo-Glyco: <=0,031900.

Figure 4

Lipid linked oligosaccharide analysis in 4 PGM1 deficient cell lines showing reduced LLO at Baseline and Improvement Following D-gal Supplementation To investigate whether PGM1 deficiency disrupts the formation of LLO, which is a required precursor for the synthesis of nascent N-linked glycoproteins in the ER, we performed lipid linked oligosaccharide (LLO) and protein linked oligosaccharide (PLO) analyses in the skin fibroblasts of patients 1, 2, 8, and cell-line 2013Y. The cells were deprived of glucose while the culture media was supplemented without or with 10mM D-gal (for one hour. The level of full-length LLO (Glc₃Man₉GlcNAc₂-PP-Dol, G3) or sugar moieties bound to newly synthesized PLO (mainly Glc₁Man₉ and Man₉), remained fairly unchanged in control cells (Figure 4, left column), indicating a high degree of metabolic fitness in these cells. In contrast, cells from all four patients showed a large amount of shortened LLO (Man₉GlcNAc₂-PP-dolichol) (Figure 4, LLO top row). In contrast, the PLO profile was indistinguishable from the control cells (Figure 4, PLO top row). Interestingly, galactose supplementation led to the reduction of shortened LLO (Man₉GlcNAc₂-PP-dolichol) in patients 1 2, and cell-line 2013Y, resulting in a LLO profile that is similar to control. No improvement was observed in patient 8 (Figure 4, LLO bottom row). D-Gal supplementation had no effect on the PLO profile in all patients (Figure 4, PLO bottom row).