Global serum glycoform profiling for the investigation of dystroglycanopathies & Congenital Disorders of Glycosylation

The Congenital Disorders of Glycosylation (CDG) are an expanding group of genetic disorders which encompass a spectrum of glycosylation defects of protein and lipids, including N- & O-linked defects and among the latter are the muscular dystroglycanopathies (MD). Initial screening of CDG is usually based on the investigation of the glycoproteins transferrin, and/or apolipoprotein CIII. These biomarkers do not always detect complex or subtle defects present in older patients, therefore there is a need to investigate additional glycoproteins in some cases. We describe a sensitive 2D-Differential Gel Electrophoresis (DIGE) method that provides a global analysis of the serum glycoproteome. Patient samples from PMM2-CDG (n = 5), CDG-II (n = 7), MD and known complex N- & O-linked glycosylation defects (n = 3) were analysed by 2D DIGE. Using this technique we demonstrated characteristic changes in mass and charge in PMM2-CDG and in charge in CDG-II for α1-antitrypsin, α1-antichymotrypsin, α2-HS-glycoprotein, ceruloplasmin, and α1-acid glycoproteins 1&2. Analysis of the samples with known N- & O-linked defects identified a lower molecular weight glycoform of C1-esterase inhibitor that was not observed in the N-linked glycosylation disorders indicating the change is likely due to affected O-glycosylation. In addition, we could identify abnormal serum glycoproteins in LARGE and B3GALNT2-deficient muscular dystrophies. The results demonstrate that the glycoform pattern is varied for some CDG patients not all glycoproteins are consistently affected and analysis of more than one protein in complex cases is warranted. 2D DIGE is an ideal method to investigate the global glycoproteome and is a potentially powerful tool and secondary test for aiding the complex diagnosis and sub classification of CDG. The technique has further potential in monitoring patients for future treatment strategies. In an era of shifting emphasis from gel- to mass-spectral based proteomics techniques, we demonstrate that 2D-DIGE remains a powerful method for studying global changes in post-translational modifications of proteins

A Randomized Controlled Trial of Caries Prevention in Dental Practice

We conducted a parallel group randomized controlled trial of children initially aged 2 to 3 y who were caries free, to prevent the children becoming caries active over the subsequent 36 mo. The setting was 22 dental practices in Northern Ireland, and children were randomly assigned by a clinical trials unit (CTU) (using computer-generated random numbers, with allocation concealed from the dental practice until each child was recruited) to the intervention (22,600-ppm fluoride varnish, toothbrush, 50-mL tube of 1,450 ppm fluoride toothpaste, and standardized, evidence-based prevention advice) or advice-only control at 6-monthly intervals. The primary outcome measure was conversion from caries-free to caries-active states. Secondary outcome measures were number of decayed, missing, or filled teeth (dmfs) in caries-active children, number of episodes of pain, and number of extracted teeth. Adverse reactions were recorded. Calibrated external examiners, blinded to the child’s study group, assessed the status of the children at baseline and after 3 y. In total, 1,248 children (624 randomized to each group) were recruited, and 1,096 (549 intervention, 547 control) were included in the final analyses. Eighty-seven percent of intervention and 86% of control children attended every 6-mo visit (P = 0.77). A total of 187 (34%) in the intervention group converted to caries active compared to 213 (39%) in the control group (odds ratio, 0.81; 95% confidence interval, 0.64–1.04; P = 0.11). Mean dmfs of those with caries in the intervention group was 7.2 compared to 9.6 in the control group (P = 0.007). There was no significant difference in the number of episodes of pain between groups (P = 0.81) or in the number of teeth extracted in caries-active children (P = 0.95). Ten children in the intervention group had adverse reactions of a minor nature. This well-conducted trial failed to demonstrate that the intervention kept children caries free, but there was evidence that once children get caries, it slowed down its progression (EudraCT No: 2009-010725-39; ISRCTN: ISRCTN36180119)

Early-onset fetal hydrops and muscle degeneration in siblings due to a novel variant of type IV glycogenosis

We report on 3 consecutive sib fetuses, presenting at 13, 12, and 13 weeks of gestation, respectively, with fetal hydrops, limb contractures, and akinesia, Autopsy of the first fetus showed subcutaneous fluid collections and severe degeneration of skeletal muscle. Histologic studies demonstrated massive accumulation of diastase-resistant periodic acid-Schiff-positive material in the skeletal muscle cells and epidermal keratinocytes of all 3 fetuses, Enzyme studies of fibroblasts from the 3rd fetus showed deficient activity of glycogen brancher enzyme, indicating that this is a new, severe form of glycogenosis type TV with onset in the early second trimester, (C) 1999 Wiley-Liss, Inc.</p