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The biosynthesis of asparagine (ASN)-linked oligosaccharides of glycoproteins proceeds along an extended pathway, spread over the cytosol, the ER, and the Golgi compartment. In the cytosol and the ER, numerous glycosyltransferases stepwise attach monosaccharides to a growing LLO. The sugar donors are either nucleotides or dolichol phosphate-linked sugars. The completed basic glycan (Glc3 Man9GlcNAc2) is transferred en bloc within the ER to an asparagine residue of a nascent protein, through the oligosaccharyltransferase complex (Fig. 1). In the ER and the Golgi compartment, several glycosidases and glycosyltransferases remodel the oligosaccharide chain into a more complex structure by removal of glucose and mannose residues and the addition of GlcNAc, galactose, fucose, and sialic acid residues (1).

Figure 1
figure 1

Simplified scheme of the cytosolic and ER part of the N-glycosylation pathway. The pathway begins in the cytoplasm with the synthesis from glucose of the activated sugar precursor GDP-Man. Key enzymes in this pathway are: (1) phosphoglucose isomerase; (2) MPI; (3) PMM; and (4) GDP-Man synthase. Dolichol phosphate mannose and dolichol phosphate glucose are synthesized from GDP-Man and UDP-Glc (reactions 5 and 15, respectively). The two dolichol phosphate sugar precursors are then flipped from the cytoplasmic side of the ER membrane to the luminal side. On the cytoplasmic side of the ER membrane, one molecule of GlcNAc-1-phosphate is transferred from UDP-GlcNAc to dolichol phosphate to form dolichol pyrophosphate GlcNAc, followed by addition of GlcNAc to form dolichol pyrophosphate (GlcNAc)2 (reaction 6). Five Man residues are then added from GDP-Man to form dolicholpyrophosphate(GlcNAc)2(Man)5 (reaction 7), which then flips to the luminal side (reaction 8) and serves as an acceptor for four Man residues derived from dolichol phosphate mannose (reaction 9). Three Glc residues are then added from dolichol phosphate glucose (reactions 10, 11, and 12). The Glc3Man9GlcNAc2 oligosaccharide is transferred from dolichol pyrophosphate to an asparagine residue of the nascent glycoprotein to initiate protein N-glycosylation by the action of oligosaccharyltransferase (reaction 13). The three Glc residues are removed by glycosidases, and the protein proceeds to the Golgi apparatus for further processing. CDG types Ia, Ib, Ic, Id, and Ie are caused by defective enzymes at steps 3, 2, 10, 9, and 5, respectively.

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Glycoconjugates play many critical roles in metabolism (2), e.g. in cell recognition and adhesion, cell migration, protease resistance, host defense, and antigenicity. Therefore it is obvious that hypoglycosylation of proteins leads to variable and in general severe and ubiquitous problems in affected individuals. In human, eight inherited diseases are known to date to be caused by defects in the synthesis of N-glycans. Following recent recommendations (3, 4), congenital disorders of glycosylation (CDG) are divided into two groups (Table 1). CDG-I comprises defects in the assembly of the LLO chain and its transfer to the protein. Known enzyme defects are located to the cytosol (CDG-Ia, CDG-Ib) and the ER (CDG-Ic, CDG-Id, and CDG-Ie). CDG-II refers to defects in the processing of the protein-bound glycans, either late in the ER (CDG-IIb) or in the Golgi compartment (CDG-IIa and CDG-IIc). It is convention that untyped cases are labeled CDG-x until they are fully characterized.

Table 1 Congenital disorders of N -linked glycosylation: classification (as of June 2001)
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IEF of serum transferrin remains the most powerful screening test for CDG, although not all types can be detected by this assay. A type 1 IEF pattern (observed in CDG-I) is characterized by a decrease of tetrasialotransferrin and an increase of di- and asialotransferrin bands, whereas a type 2 pattern (observed in CDG-II) shows in addition an increase of tri- and monosialotransferrin.

This review presents highlights of the known and newly discovered congenital disorders of N-linked glycosylation and summarizes diagnostic and therapeutic approaches.

Clinical Presentations

CDG-Ia (PMM deficiency).

An estimated 300 patients are known worldwide with this most frequent type of CDG. Often patients can be diagnosed in the neonatal or early infantile period on the basis of typical clinical features (inverted nipples and fat pads) in addition to strabismus, muscular hypotonia, failure to thrive, and elevated transaminases (5, 6). As a rule there is osteopenia. A very common feature is cerebellar hypoplasia, which can usually be documented at or shortly after birth. There is a substantial childhood mortality of approximately 25%, owing to severe infections or organ failure (7, 8).

At a later age, the impairment of the neurologic system becomes more evident with a variable degree of mental retardation, cerebellar dysfunction, and retinitis pigmentosa. Some children experience seizures or exhibit stroke-like episodes. In adults, nonprogressive ataxia, stable mental retardation, and peripheral neuropathy mainly characterize the disease. The large majority of patients are wheelchair bound. Adult female patients present as a rule with hypergonadotropic hypogonadism (9, 10). Thanks to the broadened screening for CDG, the number of patients with a less typical presentation is increasing, including children with nearly normal psychomotor development.

CDG-Ia is a result of PMM 2 (EC 5.4.2.8) deficiency (11), and the gene is localized on chromosome 16p13 (12, 13).

CDG-Ib (MPI deficiency).

Although MPI (EC 5.3.1.8) catalyzes the reaction one step upstream of PMM, the clinical presentation of this disease is very different from the PMM deficiency and the other CDG types. Some 20 patients are known. The classic clinical presentation is protein-losing enteropathy, congenital hepatic fibrosis, and coagulopathy without overt neurologic manifestations (14–18). Other presentations are persistent vomiting (15) and hypoglycemia (18). Early diagnosis is essential, because patients can be successfully treated with oral mannose (see “Treatment”).

CDG-Ic (ALG 6 deficiency).

ALG 6 deficiency (CDG-Ic) causes mainly a neurologic disorder that is in general milder than CDG-Ia (19, 20). Features of CDG-Ia, such as cerebellar hypoplasia, polyneuropathy, fat pads, and inverted nipples, are missing (21, 22). In two of 20 patients the disease had a fatal outcome because of severe coagulopathy and hormonal disturbances, combined with untreatable seizures (unpublished data). This type of CDG is probably underdiagnosed because of the absence of typical morphologic features and cerebellar hypoplasia. The enzyme, ALG 6, catalyzes the attachment of the first glucose to the LLO intermediate Man9GlcNAc2-PP-dolichol in the ER.

CDG-Id (ALG 3 deficiency).

Thus far, only one patient has been described with an ALG 3 deficiency (23). This child was first reported in 1995 as CDG “type IV”(24). The boy presented with microcephaly, severe epilepsy, and nearly absent psychomotor development. The IEF of serum transferrin (type 1 pattern) shows no increase of asialotransferrin. The defect is in the ALG 3 that transfers mannose from dolichol-phosphate mannose to the LLO intermediate Man5GlcNAc2-PP-dolichol.

CDG-Ie (dolichol-P-mannose synthase 1 deficiency).

Two groups have reported on four severely handicapped patients (in three families) with minimal psychomotor development, microcephaly, absent visual contact, and severe epilepsy. Brain imaging revealed delayed myelination in two of them. As in the previous types, IEF of serum transferrin showed a type 1 pattern with only little or no increase of asialotransferrin. This clinical picture is caused by a dolichol-P-mannose synthase deficiency (EC 2.4.1.83), and mutations in DPM1, the catalytic subunit of dolichol-P-mannose synthase, have been identified (25, 26).

CDG-IIa (N-acetyl-glucosaminyltransferase II deficiency).

When in the early 1990s an Iranian girl and a Belgian boy were reported with severe psychomotor retardation but without peripheral neuropathy or cerebellar hypoplasia, it became clear that CDGs are a heterogeneous group of disorders. Up to now, four children in three families have been diagnosed as CDG-IIa (27, 28). They showed craniofacial dysmorphy, stereotypic hands movement, and psychomotor retardation to a variable degree. Glycan structural studies revealed a similar monoantennary N-acetyllactosamine type glycan as found in patients with congenital dyserythropoietic anemia type II (29, 30). This suggested an N-acetylglucosaminyltransferase II (EC 2.4.1.143) deficiency, which was demonstrated in monocytes and fibroblasts (31, 32). Mutations have been found in the MGAT2 gene (33).

CDG-IIb (glucosidase I deficiency).

Very recently, a different type of CDG was identified in a child with severe developmental delay, muscular hypotonia, recurrent edema, seizures, and peculiar dysmorphy, including retrognathia, high arched palate, and overlapping of fingers. It results from defective glucosidase that, in the ER, reacts in the first step of the processing of the glycan structure, after its transfer to the protein. Interestingly, IEF of transferrin was normal, whereas the IEF of serum hexosaminidase was slightly abnormal. The defect was found after oligosaccharide analysis of urine revealed the presence of a tetrasaccharide [Glc(α1–2)Glc(α1–3)Glc (α1–3)Man](34).

CDG-IIc (GDP-fucose transporter deficiency).

Three patients have been reported (35, 36); they exhibited craniofacial dysmorphism, severe psychomotor retardation, hypotonia, and growth retardation. A typical feature was recurrent infections with marked leukocytosis. Neutrophils of these patients lack sialyl-Lewis x, a fucose-containing carbohydrate ligand of the selectin family of cell adhesion molecules. Sialyl-Lewis x is necessary for the recruitment of neutrophils to the infection focus. Other fucose-containing carbohydrate sequences, such as A, B, O, and Lewis A blood groups, are also absent. Mutations in the gene for a specific GDP-fucose transporter were identified as the underlying causes of this so-called leukocyte adhesion deficiency type II (37, 38).

CDG-x.

The number of patients with a CDG not belonging to the known types is steadily increasing. They undoubtedly harbor different defects in the numerous other steps of the N- and O-glycosylation pathways. Their clinical presentations are very variable: hydrops fetalis, dysmorphy, and early death after intractable seizures (39); severe hypotonia, cataracts, failure to thrive, absent psychomotor development, progressive microcephaly, and death in status epilepticus (40); oligohydramnion, dysmorphy, hypotonia, seizures, cerebellar hypoplasia, and severe thrombocytopenia (41); oligohydramnion, hypotonia, diarrhea, vomiting, ascites, demineralization of distal bones, and tubulopathia (42); dysmorphy, muscular hypotonia, and infantile spasms (43).

Table 2 summarizes either the most common or most characteristic symptoms of the known defects. Note that for most of the disorders only very few patients are known, making a comprehensive clinical description premature.

Table 2 Signs and symptoms in CDG
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DIAGNOSIS

In view of the extremely broad clinical spectrum of known CDG patients, it is recommended to consider CDG in any unexplained multisystem disorder. Figure 2 proposes a stepwise diagnostic flowchart for the detection and characterization of CDG patients.

Figure 2
figure 2

Proposed diagnostic flowchart for congenital disorders of N-linked glycosylation (CDG). *The use of EDTA plasma can cause artifacts as a result of iron chelation.

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IEF of serum transferrin.

Transferrin, one of the predominant serum glycoproteins, has two N-glycosylation sites. Hence, most transferrin molecules carry two biantennary chains with terminal sialic acid residues, and tetrasialotransferrin is the main serum sialotransferrin. Deficient synthesis of N-glycans results in a deficient incorporation of sialic acid, the terminal negatively charged sugar. The molecules acquire a more positive charge, which causes a cathodal shift in the IEF pattern of transferrin. In the so-called type 1 pattern (the most frequent), there is an increase of di- and asialotransferrin and a decrease of tetrasialotransferrin, whereas the type 2 pattern is a combination of the type 1 pattern and an increase of trisialotransferrin and, but not always, monosialotransferrin. One has to make sure that the abnormal pattern is not the result of a transferrin protein variant by performing IEF of the parents' transferrin. Preincubation of the sample with neuraminidase is another means to differentiate between CDG and protein variants. It is worthwhile performing IEF of other glycoproteins (e.g. hexosaminidase, thyroxine-binding globulin, or α1- antitrypsin) to document that there is a generalized glycosylation defect in the patients (44).

Because IEF is based on the deficiency of sialic acid, fucose defects cannot be picked up. In patients suspected of a fucose defect, the blood groups should be determined: they show the Bombay blood group (38). Also, the IEF of serum transferrin was normal in the one reported patient with CDG-IIb (34) and can be normal in patients with CDG-Ia (45).

Carbohydrate-deficient transferrins can also be observed in uncontrolled fructosemia and galactosemia and in chronic alcohol ingestion (46–50). The cathodal shift on IEF disappears with efficient treatment.

Enzymatic measurements.

PMM and MPI activity are most readily measured in fibroblasts or leukocytes (11). Regarding PMM activity, leukocytes seem to be more reliable than fibroblasts, because a high residual activity has been observed in the fibroblasts of some CDG-Ia patients, whereas the leukocyte values were always in the clearly abnormal range (51). Thus, patients with slightly decreased or low normal values of PMM in fibroblasts might still harbor mutations in PMM2. Especially in the case of a clinical picture that strongly suggests CDG-Ia, it is worthwhile to look for PMM2 mutations.

Mutation studies.

The PMM2 gene was cloned in 1997 (13), and more than 50 different mutations have been identified (13, 52–56). There is a clear predominance of missense mutations. The R141H mutation is found in approximately 40% of all patients. F119L is frequent in the Northern European countries owing to a founder effect (54, 57), whereas V231M and P113L are frequent all over Europe (Table 3) (13, 58). Most patients are compound heterozygous for two different mutations, and homozygosity is not observed for R141H or other mutations that severely impair the protein (52, 53, 59–61).

Table 3 Mutations in CDG-Ia, CDG-Ib, and CDG-Ic
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Mutations in the MPI gene have been found in most patients with an MPI deficiency. The gene is localized on chromosome 15p22 and again, mutations are generally of the missense type. Because a very limited number of patients are known, the spectrum of mutations is rather limited for the moment (Table 3) (14, 16, 18, 62).

Genetic studies in CDG-Ic patients revealed a prevalence of the A333V mutation in ALG 6, probably related to a founder effect in the Dutch population. Only a handful of other mutations has been described to date (Table 3) (21, 22, 63, 64).

Prenatal diagnosis.

Prenatal diagnosis has become reliable for CDG-Ia (54, 65) since the mapping of the disease locus on chromosome 16p13 (12), and the subsequent identification of the enzymatic defect (11) and the cloning of the PMM2 gene (13). Early attempts of prenatal diagnosis on the basis of transferrin IEF in fetal blood have failed and thus revealed that this method is not reliable (66, 67). Enzymatic measurements of PMM activities in cultured amniocytes or trophoblasts are useful but may give inconclusive data (65). As a result, preference is given to the direct mutation analysis in the fetus.

Prenatal diagnosis is possible in all other types of CDG for which the molecular defect is known, on condition that the diagnosis has been confirmed in the index patient or the mutations have been detected in the parents.

LLO analysis.

The N-glycosylation pathway in the ER is highly conserved in eukaryotic cells (19), and yeast genetic techniques have been instrumental in the identification of new N-glycosylation disorders in human. By comparing LLO structures of patients with those of yeast mutant strains, the defects of CDG-Ic, -Id, and -Ie have been elucidated (19, 20, 23, 25, 26). The power of the LLO analysis is of course limited to defects in the cytosol and ER.

Glycan structure analysis.

Structural studies of the complex asparagine-linked glycans have mainly been performed on serum transferrin, i.e. IEF, chromatofocusing, Western blotting, capillary zone electrophoresis, and anion exchange chromatography (68–74). Electrospray mass spectrometry of protein-bound oligosaccharide chains (glycoproteins) can separate the different glycoforms (75), and nuclear magnetic resonance spectroscopy determines the glycan structures and molecular mass of the glycovariants (76). Such glycan structure analysis will be instrumental for the elucidation of CDG-x cases, by pinpointing candidate enzymes and genes responsible for the abnormal N-glycan synthesis.

TREATMENT

CDG-Ia.

Unfortunately, an efficient treatment is still not available for the CDG-Ia patients. Although it was reported that incubation with mannose resulted in an increased incorporation of mannose in patients' fibroblasts (77), mannose administration to CDG-Ia patients did not improve the clinical or biochemical features (78, 79). Also fucose supplementation, with the aim of enhancing the GDP-mannose pool, was not successful (T. Marquardt, personal communication). Studies with a ketogenic diet in CDG-Ia are ongoing. The rationale for this treatment is the observation that glucose starvation improves N-glycosylation in fibroblasts from CDG-Ia patients (80).

As to symptomatic treatment, we have obtained efficient prevention of stroke-like events by using 0.5 mg/kg per day acetylsalicylic acid (C. Van Geet and J. Jaeken, in preparation). In patients with recurrent fractures, biphosphonates should be considered.

CDG-Ib.

In CDG-Ib patients, treatment with oral mannose (4–6 doses per day × 100–150 mg/kg per day) has shown significant improvement (81–83). Serum mannose levels should be greater than 200 μM. Because mannose supplementation is probably a lifelong therapy for MPI-deficient patients, side effects have to be monitored carefully. High mannose intake can cause osmotic diarrhea (81). Also a slight increase of the glycosylated Hb (HbA1c) has been observed (82).

CDG-IIc.

In one patient with a GDP-fucose transporter defect, fucose supplementation (25 mg/kg per day in three doses) was reported to improve the fucosylation of glycoproteins and to control the recurring infections (36).

CONCLUSION AND OUTLOOK

In the last 2 years, the field of CDG has been rapidly expanding, thanks to the increasing awareness of the clinical variability of CDG and the use of yeast genetic technology. The extremely wide clinical spectrum of CDG makes a broad screening for these disorders in children as well as in adults mandatory by using serum transferrin IEF. It is quite certain that many new glycosylation disorders are awaiting identification inasmuch as it is estimated that some 500 genes (approximately 0.5 to 1% of the translated human genome) participate in oligosaccharide synthesis and function (84). The development of animal models offers the potential to test therapeutic approaches and will be helpful in understanding the pathogenesis of these disorders.

ADDENDUM

Very recently two new CDG have been identified: CDG-If (an ER defect) and CDG-IId (a Golgi defect). CDG-If has been reported in four patients with a severe encephalopathy and, in three of them, a dry, scaling skin with erythroderma. Mutations were found in the Lec35/MPDU1 gene involved in the use of dolichylphosphomannose and dolichylphosphoglucose (85, 86). CDG-IId was described in a patient with moderate psychomotor retardation and macrocephaly as well as myopathy. A homozygous mutation (insertion of a single nucleotide) was found in the gene for UDP-Gal: N-acetyl-glucosamine β-1,4-galactosyltransferase I (87, 88).