BackgroundClassical Galactosaemia (CG) (OMIM #230400) is a rare inborn error of galactose metabolism caused by deficiency of the enzyme galactose-1-phosphate uridylyltransferase (GALT). Long-term complications persist in treated patients despite dietary galactose restriction with significant variations in outcomes suggesting epigenetic glycosylation influences. Primary Ovarian Insufficiency (POI) is a very significant complication affecting females with follicular depletion noted in early life. We studied specific glycan synthesis, leptin system and inflammatory gene expression in white blood cells as potential biomarkers of infertility in 54 adults with CG adults (27 females and 27 males) (age range 17–51 yr) on a galactose-restricted diet in a multi-site Irish and Dutch study. Gene expression profiles were tested for correlation with a serum Ultra-high Performance Liquid Chromatography (UPLC)-Immunoglobulin (IgG)-N-glycan galactose incorporation assay and endocrine measurements.ResultsTwenty five CG females (93%) had clinical and biochemical evidence of POI. As expected, the CG female patients, influenced by hormone replacement therapy, and the healthy controls of both genders showed a positive correlation between log leptin and BMI but this correlation was not apparent in CG males. The strongest correlations between serum leptin levels, hormones, G-ratio (galactose incorporation assay) and gene expression data were observed between leptin, its gene and G-Ratios data (rs = − 0.68) and (rs = − 0.94) respectively with lower circulating leptin in CG patients with reduced IgG galactosylation. In CG patients (males and females analysed as one group), the key glycan synthesis modifier genes MGAT3 and FUT8, which influence glycan chain bisecting and fucosylation and subsequent cell signalling and adhesion, were found to be significantly upregulated (p < 0.01 and p < 0.05) and also the glycan synthesis gene ALG9 (p < 0.01). Both leptin signalling genes LEP and LEPR were found to be upregulated (p < 0.01) as was the inflammatory genes ANXA1 and ICAM1 and the apoptosis gene SEPT4 (p < 0.01).ConclusionsThese results validate our previous findings and provide novel experimental evidence for dysregulation of genes LEP, LEPR, ANXA1, ICAM1 and SEPT4 for CG patients and combined with our findings of abnormalities of IgG glycosylation, hormonal and leptin analyses elaborate on the systemic glycosylation and cell signalling abnormalities evident in CG which likely influence the pathophysiology of POI.

Background Classical galactosaemia (OMIM #230400) is a rare disorder of carbohydrate metabolism caused by deficiency of the galactose-1-phosphate uridyltransferase enzyme. The pathophysiology of the long-term complications, mainly cognitive, neurological and female fertility problems, remains poorly understood. Current clinical methods of biochemical monitoring lack precision and individualization with an identified need for improved biomarkers for this condition. Methods We report the development and detailed validation of an automated ultraperformance liquid chromatography N-glycan analytical method of high peak resolution applied to galactose incorporation into human serum IgG. Samples are prepared on 96-well plates and the workflow features rapid glycoprotein denaturation, enzymatic glycan release, glycan purification on solid-supported hydrazide, fluorescent labelling and post-labelling clean-up with solid-phase extraction. Results This method is shown to be accurate and precise with repeatability (cumulative coefficients of variation) of 2.0 and 8.5%, respectively, for G0/G1 and G0/G2 ratios. Both serum and processed N-glycan samples were found to be stable at room temperature and in freeze-thaw experiments. Conclusions This high-throughput method of IgG galactose incorporation is robust, affordable and simple. This method is validated with the potential to apply as a biomarker for treatment outcomes for galactosaemia.

Background Classical galactosemia (CG) (OMIM #230400) is a rare disorder of carbohydrate metabolism, due to deficiency of galactose‐1‐phosphate uridyltransferase (EC 2.7.7.12). The pathophysiology of the long‐term complications, mainly cognitive, neurological, and female infertility remains poorly understood. Objectives This study investigated (a) the association between specific IgG N‐glycosylation biomarkers (glycan peaks and grouped traits) and CG patients (n = 95) identified from the GalNet Network, using hydrophilic interaction ultraperformance liquid chromatography and (b) a further analysis of a GALT c.563A‐G/p.Gln188Arg homozygous cohort (n = 49) with correlation with glycan features with patient Full Scale Intelligence Quotient (FSIQ), and (c) with galactose intake. Results A very significant decrease in galactosylation and sialylation and an increase in core fucosylation was noted in CG patients vs controls (P < .005). Bisected glycans were decreased in the severe GALT c.563A‐G/p.Gln188Arg homozygous cohort (n = 49) (P < .05). Logistic regression models incorporating IgG glycan traits distinguished CG patients from controls. Incremental dietary galactose intake correlated positively with FSIQ for the p.Gln188Arg homozygous CG cohort (P < .005) for a dietary galactose intake of 500 to 1000 mg/d. Significant improvements in profiles with increased galactose intake were noted for monosialylated, monogalactosylated, and monoantennary glycans. Conclusion These results suggest that N‐glycosylation abnormalities persist in CG patients on dietary galactose restriction which may be modifiable to a degree by dietary galactose intake.

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