Imtiaz, Faiqa; (2002) Biochemical and genetic investigations on patients with congenital disorders of glycosylation. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

This study presents an overall review of the Congenital Disorders of Glycosylation (CDG) and describes genetic and enzymological investigations employed to identify and confirm the basic defect in 21 patients that were diagnosed as CDG-I on the basis of their clinical features and abnormal isoelectric focusing (IEF) pattern of serum transferrin. Fifteen patients from thirteen families were found to have CDG-Ia on the basis of markedly reduced phosphomannomutase (PMM) activity in fibroblasts in culture. Mutation analysis of the PMM2 gene demonstrated the presence of 8 missense mutations. All the patients were compound heterozygotes for these mutations. No correlation could be established between genotype and clinical/enzymological phenotypes in the CDG-Ia patients. Human PMM is encoded by two genes; PMM1 (22q13) and PMM2 (16p13), which are expressed in a tissue-specific manner. Patients with severe and milder forms of CDG-1a were analysed for any possible mutations in PMM1. No mutations were detected. Detailed enzyme kinetics experiments were performed to investigate the inhibition of PMM using synthetic analogues. One patient had reduced phosphomannose isomerase (PMI) activity in fibroblasts and genetic analysis of the MPI gene, encoding PMI showed a homozygous mutation, D131N, which confirmed the patient suffered from CDG-1b. Another patient who had normal PMM and PMI activities in fibroblasts was found to have missense mutations in the hALG6 gene encoding αl,3 glucosyltransferase and was classified as CDG-Ic. Four patients, classified as CDG-Ix, who had normal PMM and PMI activities but defective protein N-glycosylation as indicated by abnormal IEF patterns of serum transferrin, were investigated for plausible defects in the enzymes involved in the synthesis of precursor glucosyl donors for lipid-linked oligosaccharides, namely, GDP-mannose pyrophosphorylase, dolichol phosphate mannose synthase, and glutamine: fructose-6-phosphate amidotransferase, in fibroblasts. No significant difference was discernible in specific activities of these enzymes between control and patients' fibroblasts. These four patients were also tested for α-glucosidase I activity in fibroblasts (a marker for CDG-IIb), but showed no significant difference compared with controls.

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