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Risultati finali
N-linked protein glycosylation in the endoplasmic follows a defined pathway in all eukaryotic cells. A lipid-linked oligosaccharide is assembled at the membrane of the ER and then transferred to nascent polypeptide chains that enter the lumen of the ER. This complex pathway has been characterized at a molecular level in the model organism Saccharomyces cerevisiae. Biochemical analysis of the pathway in higher eukaryotic cells and the annotation of the human genome have revealed a high conservation of the pathway in all eukaryotic cells. Based on these observations, a strategy to identify the molecular cause of novel types of CDG I was successfully applied and resulted in the characterisation of different types of CDG I.
Identification of patients with altered glycosylation of the serum glycoprotein transferrin.
a) In case of a novel type of disease: analysis of lipid-linked oligosaccharide potentially accumulating in patient-derived fibroblasts.
b) Comparison of the accumulation-phenotype with the one observed in defined mutant strains of yeast. This comparison resulted in a hypothesis concerning the potential deficiency leading to the disease in a particular patient.
c) Based on the sequence similarity of the yeast and the homologous human protein, the corresponding human locus was identified in the human genome and corresponding cDNA was isolated by standard technologies.
d)In case mutant alleles were identified in the patient genome, the cDNA was expressed in appropriate yeast strains to test if the mutations lead to reduced activity of the corresponding protein. By this way, a functional assay for the human activity could be performed using yeast genetic techniques.
This strategy led to the identification of 8 novel types of CDG-I and the characterisation at a molecular level. It is one of the main results of the EUROGLYCAN network that a collaborative effort of clinical oriented and basic science research teams working with model organisms contributed in a short time to the identification of the molecular basis of congenital disorders.
In a joint effort, the EUROGLYCAN consortium has defined, at the clinical, genetic and molecular level, the following novel types of CDG within the project period 2000-2003. CDG-ICDG-Ie Dol-P-Man Synthase 1 DPM 1 CDG-If MPDU1/Lec35 CDG-Ig Dol-P-Man:Man7GlcNAc2-PP-Dol a6mannosyltransferase hALG12 CDG-Ii GDP- Man: Man1GlcNAc2-PP-Dol a3mannosyltransferase ( hALG2) CDG-Ik Dol-P-Man: GlcNAc2-PP-Dol ß4mannosyltransferase h ALG1 CDG-Il Dol-P-Man:Man8GlcNAc2-PP-Dol a2mannosyltransferase hALG9 CDG-IICDG IIc LAD II GDP Fucose TransporterCDG-IId - beta4 galactosyltransferase. All these types have been published except for types Ik and Il, which are in press or in preparation.
While 17 types of N-glycosylation disorders have been defined thus far, five were known before the establishment of the EUROGLYCAN consortium and four have been discovered by groups not affiliated with EUROGLYCAN. Thus, the joint effort in gathering clinical, molecular, genetic and cell biological expertise has been outstandingly rewarding.
The general procedure which led to the above mentioned list was:
1) referral of a newly diagnosed cases to the reference centre by the clinical expert centres across Europe;
2) exclusion of the common types, and referral of the unsolved cases to the research laboratories for biochemical and genetic work-up. In cases of type I defects, the groups took advantage of the well-characterized yeast mutants to localize the ER-associated defect.
Novel methods for the rapid, sensitive and reliable analysis of the carbohydrate chains (glycans) attached to serum/plasma proteins have been developed. This preliminary test gives a broad picture of the ability of an individual to produce carbohydrate chains of the appropriate structure and in the appropriate amount.
It can indicate whether a patient has a defect in the pathway for the biosynthesis of the carbohydrate chains (CDG) and provide very useful clues about the precise block in the pathway. This information will shorten considerably the time taken to make a definitive diagnosis of CDG-II. As well as detecting primary defects in glycosylation, the methodology will also be useful in detecting secondary defects in glycosylation such as galactosaemia, hereditary fructose intolerance and chronic alcoholism and other causes of cirrhosis of the liver. Analysis of the total proteins in serum/plasma obviates the costly and time-consuming need to separate individual proteins for analysis, as necessary previously.
Typically an analysis can be carried out on a drop of blood (a few microlitres) and it should be possible to fully automate the procedure, thereby providing a fast mass screening procedure. This test can be added to an existing repertoire of tests with enormous clinical and cost benefit.
A database has been established, that contains data on more than 400 patients with CDG. The database software has been developed specifically for the use of the database by the clinical and research centres, and the database can be consulted by the members of the network via internet.
The patient database is one of the most valuable deliverables of the project. It will allow epidemiological studies, and will be a register for patients eligible for a particular treatment. A long-term follow-up of the patients will also lead to a better recognition of the syndrome at an adult age. It will provide a basis for early prevention of disease-associated complications, by hormone supplementation, etc. It will also be useful for setting up trials protocols and for evaluating therapeutic effects.
EUROGLYCAN has supported 5 national centres, with an interest and a history in the research and diagnostics of rare, metabolic disease, to introduce and/or consolidate their activities in the field of CDG. They have set-up adequate screening techniques. The relevant equipment was bought through the network, and the collaborators and staff from the subcontractors have been trained. These national referral centres have all started the screening and have implemented this in their daily routine procedure. All 5 centres have actually been able to diagnose CDG cases in their country, thus serving the purpose of national reference and referral centre in these countries. Awareness about CDG has been raised considerably in these countries. The European reference centres in Nijmegen, Barcelona and Leuven serve as back up for advice and further work-up of positive cases.
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