CDGS Type

CDG-type Affected protein Gene Chromosome OMIM... 

Fig. 4.5.2 Actual strategies for CDG diagnosis. Initial investigations on CDG patients are routinely carried out by isoelectric focusing (IEF) of serum transferrin. With a CDG type I pattern, subsequent analysis should imply determination of phosphomannomutase (PMM) and phos-phomannose isomerase (PMI) activities. Further studies, like analysis of the lipid-linked- and protein-bound-oligosaccharides, determination of enzyme or sugar transporter activities and molecular biology studies often have to be performed in more specialised laboratories. HPLC High-performance liquid chromatography, TLC thin-layer chromatography...

Fig. 4.5.3 IEF patterns of serum transferrin. Sera from a control (lane 1), three CDG-I (CDG-Ia, CDG-Ic and CDG-Id lanes 2-4) and three CDG-II patients (CDG- , CDG-IId and CDG-IIx lanes 5-7) were analysed by IEF. In case of a control person, the main form of the protein carries four negatively charged sialic acid residues, even though small amounts of penta- and trisialo-transferrin are detectable. Additional disialo- and asialotransferrin forms indicate CDG-type I (left side). In some cases of CDG type II, additional trisialo- and monosialotransferrin forms may occur, which are due to the loss of either one or three sialic acid residues (right side). Isoforms of transferrin that are independent of a pathological phenotype and that cause double bands in IEF are visible in lanes 4 and 6. CDG-IIx indicates a transferrin pattern that is caused by a so far unknown molecular defect from the CDG-II type...

Calibration of IEF is performed with sera from healthy control persons as well as sera derived from patients with defined CDG types. 

Data analysis is carried out by comparing IEF patterns of controls and defined CDG types with patients in suspicion of CDG. IEF of transferrin from controls show predominantly the tetrasialo form of the protein, whereas in case of CDG-I patients additional di- and asialo bands appear (Fig. 4.5.3). 

To compare sera from patients suffering from unclear CDG types, sera of healthy persons and patients with already defined CDG are used as controls for neuraminidase treatment studies. 

Data analysis is carried out by comparing IEF patterns from patients suspected of having a CDG with healthy controls and already defined CDG types. Following neuraminidase treatment, controls and CDG patients will predominantly present with the asialo form of transferrin. In the case of mutations that affect the protein backbone of transferrin, additional bands appear (Fig. 4.5.4). 

Metabolic labelling studies on LLO with [2-3H] mannose are carried out in fibroblasts of patients who present with a characteristic CDG-type IIEF transferrin pattern but normal PMM and PMI activities, thereby excluding CDG-Ia and CDG-Ib. Investigations require the extraction and analysis of LLO by HPLC and TLC. 

Metabolic labelling studies require primary skin fibroblasts from patients and healthy controls. It is also helpful to have an internal control from CDG-type I patients, who accumulate shortened LLO, like Man5GlcNAc2 (CDG-Id, CDG-Ie) or Man7GlcNAc2 (CDG-Ig Fig. 4.5.6). 

Metabolic labelling is carried out in primary skin fibroblasts. It is helpful to have an internal control from a CDG-type I patient with an early N-glycosylation deficiency like CDG-Ii or CDG-Ik. 

The LLO profile of the patient under investigation is compared to profiles of controls and patients with known CDG types. 

Several sugar-nucleotide pool defects have been identified Type I carbohydrate-deficient glycoprotein syndrome (CDGs type I), a defect in phosphomannomu-tase, one of the enzymes responsible for converting glucose to GDP-mannose, results in an absence of... 

GDP-mannose that is essential for glycoprotein synthesis. This defect is autosomal recessive and has been mapped to human chromosome 16pl3.3-pl3.12. All infants with CDGs have neurological abnormalities, failure to thrive, developmental delay, and dysmorphic features. Female patients with CDGs type 1 have hypergonadotropic hypogonadism and do not attain secondary sexual characteristics. 

CDG-type I syndrome [264,266,267] is due to a deficiency in the oligosaccharidyltrans-ferase which transfers en bloc onto the nascent protein the oligosaccharide linked to dolichol diphosphate. Later, van Schaftingen and Jaeken [1995, FEES Lett. 377, 318-320] demonstrated that the syndrome was due in fact to a phosphomannomutase deficiency, an enzyme which provides the mannose-1-phosphate required for the initial steps of protein glycosylation. This leads to four transferrin isoforms non-glycosylated, glycosylated in Asn-413 or in Asn-611 and in both Asn-413 and 611. 

CDG-type II syndrome [265,268] is a separate variant since it is characterized by a severe decrease in the activity of A-acetylglucosaminyltransferase II (UDP-GlcNAc a6-D-mannoside (3-l,2-A-acetylglucosaminyltransferase). As a consequence, the serotransferrin isoforms contain two truncated monoantennary glycans of which the primary structures are described in Fig. 19. 

Fig. 19. Primary structure of the glycan from human serotransferrin isolated from a patient with carbohydrate-deficient syndrome (CDG) type II [265,268], R, GIcNAc(pi-4)GIcNAc(pi-N)Asn.

The lEF pattern may be inspected visually and most patients will be picked up by an increase of asialo- and disialotransferrin (CDG type 1 pattern). Some patients may display a so-called CDG type 2 pattern, with additional increase of monosialo- and trisialotransferrin. A better evaluation stems from densito-metric scanning of the lEF gels. The reference values for the various transferrins are shown in Table D.2 (G. de Jong, manuscript in preparation). 

It should be noted that there are several patients with CDG type 1 syndrome having a virtually normal transferrin lEF pattern. 

The major population of CDGSs is identified as type I. Infant patients of CDGS type I show neurologic abnormalities including hypotonia and hyporeflexia, growth... 

Other enzymes involved in synthesis of oligosaccharide precursors are also found as causative genes for CDGS type I. Mutations on phosphomannoisomerase (PMI),... 

Interestingly, phenotypes of type Ib and Ic are reported to be milder than type la CDGS [14], Especially, the phenotype of PMI mutation (type Ib) does not show neurological symptoms. Because PMI mutation in CDGS type Ib does not inhibit dolichol-phospho-mannose synthesis from mannose, oral administration of mannose has been shown to be effective in type Ib [15]. 

So far only two patients have been reported as CDGS type II [16]. Both patients showed coarse facial features, low-set ears, widely spaced nipples, ventricular septal defects, generalized hypotonia and limb weakness. They had a severe psychomotor retardation but no peripheral neuropathy and a normal cerebellum on nuclear magnetic resonance imaging. 

Indeed, GnT-II activity was greatly reduced in fibroblast extracts from CDGS type II patients [16]. Direct sequence of GnT-II coding region from CDGS type II patients identified point mutations on the catalytic domain of GnT-II [17]. These mutation were inherited consistently with CDGS type II phenotypes, cause cell extracts expressing these mutant GnT-II proteins, which have the same missense... 

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