Rudolph's Pediatrics, 22nd Ed.

CHAPTER 163. Congenital Disorders of Glycosylation

Jaak Jaeken

Glycosylation is an important posttranslational protein modification occurring in the cytoplasm, the endoplasmic reticulum, and the Golgi apparatus. A rapidly growing family of genetic diseases is due to defects in protein glycosylation (congenital disorders of glycosylation [CDG]). Most CDG are severe, multisystem diseases with important neurological involvement. Some 30 CDG have been identified. CDG due to an N-glycosylation defect (there are 18 disorders) comprise two groups: CDG-I (with absence of one or more glycans; CDG-Ia through CDG-IL) and CDG-II (with incomplete glycans; CDG-IIa through CDG-IIf). Six disorders have been identified in O-glycosylation, including some long-known diseases such as hereditary multiple exostoses; another six disorders have a combined N- and O-glycosylation defect. Important tools in the diagnosis are transferrin isoelectric focusing, analysis of lipid-linked oligosaccharides and of protein-linked glycans, and mutation analysis.

PROTEIN GLYCOSYLATION

Congenital disorders of glycosylation (CDG) are a rapidly growing family of genetic diseases caused by defects in the synthesis of the glycan moiety of glycoconjugates (glycoproteins and gly-colipids). There are two main types of protein glycosylation: N-glycosylation and O-glycosylation. N-glycosylation (N-glycans attached to an amino group of asparagine of proteins) comprises an assembly part and a processing part and extends over three cellular compartments: the cytosol, the endoplasmic reticulum (ER), and the Golgi.

The assembly part of the N-glycosylation starts on the cytosolic side of the ER, with the transfer of N-acetylglucosamine (GlcNAc) phosphate from UDP-GlcNAc to membrane bound dolichyl monophosphate (Dol-P), forming GlcNAc-pyrophosphate-dolichol (GlcNAc-PP-Dol). One GlcNAc and five mannose (Man) residues are subsequently attached to this lipid-linked monosaccharide in a stepwise manner (Fig. 163-1). The donor of these mannoses is a nucleotide-activated sugar, GDP-Man, which is synthesized from fructose 6-phosphate, an intermediate of the glycolytic pathway (Fig. 163-2). The lipid-linked hep-tasaccharide Man5 GlcNAc2 is translocated by a flippase across the ER membrane and is elongated at the lumenal side by the attachment of four mannose residues and subsequently of three glucose residues. The four mannosyl-transferases and three glucosyltransferases involved require dolichyl-phosphate-bound monosaccharides (Dol-P-Man and Dol-P-Glc). The completed Glc3 Man9 GlcNAc2 oligosaccharide is then transferred to selected asparagine residues of the nascent proteins by the oligosaccharyltransferase complex.

FIGURE 163-1. Scheme of the endoplasmic reticulum part of the N-glycosylation pathway (see text for explanation). The black bar beside ALG6 indicates the defect in CDG-Ic (ALG6 or glucosyltransferase I defect).

The processing part of the N-glycosylation starts in the ER by trimming the glucoses (catalyzed by glucosidases I and II) and one mannose (catalyzed by α-mannosidase I). The residual glycoprotein intermediate is directed to the cis-Golgi, where the processing pathway branches. A minor branch targets glycoproteins to the lysosomes (after the action of a GlcNAc-phosphotransferase and removal of the GlcNAc residues, leaving high-mannose glycoproteins capped with Man 6-P). The main branch leads to further trimming man-noses (leaving a trimannosyl core) and the addition of GlcNAc, galactose, and eventually, sialic acid, in the medial- and trans-Golgi. Another modification of many N-glycoproteins in the Golgi is the attachment of fucose to the GlcNAc residue that is linked to asparagine.

FIGURE 163-2. Scheme of the synthesis of guan-osine diphosphate (GDP)-mannose from fructose 6-phosphate. Vertical red bars indicate defects in CDG-Ia (PMM2 defect) and in CDG-Ib (PMI defect).

O-glycosylation (O-glycans attached to the hydroxyl group of threonine or serine of proteins) has no processing part and thus consists only of assembly. Unlike N-glycosylation, this assembly mainly occurs in the Golgi. O-glycan structures show a greater diversity than N-gly-cans. Examples of important O-glycans are O-N-acetylgalactosaminylglycans (mucin-type glycans), O-xylosylglycans (glycosaminylglycans), O-mannosyl glycans, and O-fucosylglycans.

GENETIC DISEASES OF PROTEIN N-GLYCOSYLATION

Eighteen diseases are known in protein N-glycosylation: 14 assembly defects (CDG-I group), designated CDG-Ia through CDG-In, and four processing defects (CDG-II group), designated CDG-IIa through CDG-IId. We discuss only CDG-Ia, CDG-Ib, and CDG-Ic in some detail, since all the other diseases are very rare.

PHOSPHOMANNOMUTASE2 DEFICIENCY (CDG-IA)

CLINICAL PRESENTATION

CDG-Ia is by far the most frequent protein N-glycosylation disorder (some 600 patients known). The clinical spectrum is very broad: The nervous system is affected in all patients, and most other organs are involved in a variable way. The neurological picture comprises alternating internal strabism and other abnormal eye movements, axial hypotonia, psychomotor retardation, ataxia, and hyporeflexia. After infancy, symptoms include retinitis pigmentosa, stroke-like episodes, and sometimes epilepsy. During the first year(s) of life, there are variable feeding problems (anorexia, vomiting, diarrhea) that can result in severe failure to thrive. Other features are a variable dysmorphy (large, hypoplastic/dysplastic ears, abnormal subcutaneous adipose-tissue distribution [fat pads, inverted nipples]), mild to moderate hepatomegaly, skeletal abnormalities (including atlantoaxial subluxation), and hypogonadism. Some infants develop pericardial effusion or cardiomyopathy. At the other end of the clinical spectrum are patients with a very mild phenotype (no dysmorphy, slight psychomotor retardation). Patients often have an extroverted and happy appearance.

METABOLIC DERANGEMENT

Phosphomannomutase2 (PMM2) deficiency is a (cytosolic) defect in the second step of the mannose pathway (transforming mannose-6-phosphate into mannose-1-phosphate), which normally leads to the synthesis of guanosine diphosphate (GDP)-mannose (Fig. 163-2). This nucleotide sugar is the donor of mannose used in the ER to assemble the dolichol-pyrophosphate oligosaccharide precursor. Deficiency of GDP-mannose causes hypoglycosylation of numerous glycoproteins, including serum proteins, lysosomal enzymes, and membranous glycoproteins.

GENETICS

CDG-Ia (OMIM 212065) is an autosomal-recessive disease due to mutations of PMM2 on chromosome 16p13. At least 80 mutations have been identified (mainly missense mutations), the most frequent being the R141H mutation. Prenatal diagnosis is possible by enzymatic analysis of amniocytes and chorionic villus cells; this should be combined with mutation analysis of the PMM2 gene.

DIAGNOSTIC TESTS

The diagnosis of CDG-Ia (and of congenital disorders of N-glycosylation in general) is usually made by isoelectrofocusing (IEF) and immunofixation of serum transferrin (Fig. 163-1). Normal serum transferrin is mainly composed of tetrasialotransferrin and small amounts of mono-, di-, tri-, penta-, and hexasialotransferrins. The partial deficiency of sialic acid (a negatively charged and end-standing sugar) in CDG causes a cathodal shift. Two main types of cathodal shift can be recognized: Type 1 is characterized by an increase of both disialo- and asialotransferrin and a decrease of tetrasialotransferrin; in type 2 there is also an increase of the tri- and/or monosial-otransferrin bands (Fig. 163-3). In PMM2 deficiency, a type 1 pattern is found. Very recently, capillary zone electrophoresis of total serum has been introduced for the diagnosis of CDG (Fig. 163-1). In addition to the previously mentioned serum glycoprotein abnormalities, laboratory findings include elevation of serum transaminase levels, hypoalbuminemia, hypocholesterolemia, and tubular proteinuria. To confirm the diagnosis, the activity of PMM2 should be measured in leukocytes or fibroblasts.

FIGURE 163-3. Isoelectrofocusing of serum transferrin showing controls (C), a type 1 pattern, and a type 2 pattern (see text for explanation). The figures on the left indicate the number of sialic acid residues on each sialotransferrin fraction.

TREATMENT

No efficient treatment is available. There is a substantially increased mortality in the first years of life due to vital organ involvement or severe infection.1-4

PHOSPHOMANNOSE-ISOMERASE DEFICIENCY (CDG-IB)

CLINICAL PRESENTATION

Some 20 patients have been reported with this mainly hepatic-intestinal disease. Together with CDG-Ih, it is the only known N-linked CDG without (or with only minor) neurological involvement. Symptoms start between ages 1 and 11 months and consist of various combinations of recurrent vomiting, abdominal pain, protein-losing enteropathy, recurrent thromboses, gastrointestinal bleeding, liver disease, and symptoms of hypoglycemia. Several patients have died.

METABOLIC DERANGEMENT

The defect is in the first step in the biosynthesis of the nucleotide sugar GDP-mannose (Fig. 163-2). The substrate of the enzyme, fructose 6-phosphate, does not accumulate, since it is an intermediate of the glycolytic pathway. The blood biochemical abnormalities are indistinguishable from those found in CDG-Ia.

GENETICS

Inheritance of CDG-Ib (OMIM number 602579) is autosomal recessive. The gene (PMI) has been localized to chromosome 15q22, and several (missense) mutations have been identified. Prenatal diagnosis is possible.

DIAGNOSTIC TESTS

The diagnosis is confirmed by finding a decreased activity of phosphomannose isomerase in leukocytes or fibroblasts and/or mutation(s) in PMI.

TREATMENT

This is the only known CDG that is efficiently treatable. The treatment is simple and consists of oral mannose (1 g/kg body weight per day, divided in 4—6 doses). The rationale for this treatment is that hexokinases phosphorylate mannose to mannose 6-phosphate, thus bypassing the defect.5-7

GLUCOSYL TRANSFERASEI DEFICIENCY (CDG-IC)

CLINICAL PRESENTATION

This is the second most common protein N-glycosylation disease (some 30 patients identified). As in CDG-Ia, patients show hypotonia, strabismus, and seizures, but psychomotor development is less retarded, there is less dysmor-phy, and there is usually no retinitis pigmentosa or cerebellar hypoplasia. In one patient, idio-pathic intracranial hypertension and optic atrophy were reported.

For an unknown reason, some of the glyco-proteins have unusually low blood levels (particularly factor XI and coagulation inhibitors such as antithrombin and protein C). The reason why the clinical picture in these patients is much milder than that of PMM-deficient patients may be because a deficiency in gluco-sylation of the dolichol-linked oligosaccharides does not affect the biosynthesis of GDP-man-nose; therefore, it does not affect the biosynthesis of GDP-fucose or of glycosylphosphatidylinos-itol-anchored glycoproteins.

METABOLIC DERANGEMENT

Glucosyltransferase I deficiency is a defect in the attachment of the first glucose (of three) to the dolichol-linked mannose9-N-acetylglucosamine2 ER intermediate. It causes hypoglycosylation of serum glycoproteins, because nonglucosylated oligosaccharides are a suboptimal substrate for oligosaccharyltransferase.

GENETICS

CDG-Ic (OMIM 603147) is an autosomal recessive disease. The gene (hALG6) has been localized to chromosome 1p22.3. Prenatal diagnosis is possible.

DIAGNOSTIC TESTS

This disease illustrates that even in cases of mild psychomotor retardation without any specific dysmorphy, IEF of serum sialotransferrins has to be performed. When a type 1 pattern is found, PMM 2 and PMI deficiency must be considered first. If these enzymes show normal activities, the next step is analyzing the dolichol-linked oligosaccharides in fibroblasts. In CDG-Ic, the major fraction of these oligosaccharides consists of nine mannose and two N-acetylglucosamine residues without the normally present three glucose residues. Glucosyltransferase I activity has to be measured in fibroblasts, or mutation analysis must be performed.

TREATMENT AND PROGNOSIS

No efficient treatment is available. Long-term outcome is unknown, since all reported patients have been children.8,9

GENETIC DISEASES OF PROTEIN O-GLYCOSYLATION

Six genetic diseases have been identified in protein O-glycosylation. The most frequent is hereditary multiple exostoses, which is the only known CDG with autosomal dominant inheritance.

HEREDITARY MULTIPLE EXOSTOSES

CLINICAL PRESENTATION

This disease is characterized by osteochondromas of the ends of long bones. These tumors are often present at birth; their growth slows during adolescence and stops in adulthood. Malignant degeneration is present in only a small percentage of the lesions. Complications may result from compression of peripheral nerves and blood vessels.

METABOLIC DERANGEMENT

The basic defect is in the Golgi-localized EXT1/EXT2 complex, which has both glucuronyltrans-ferase and N-acetyl-D-hexosaminyltransferase activities involved in the polymerization ofheparan sulfate.

GENETICS

Transmission of hereditary multiple exostoses (OMIM 608177 and 608210) is autosomal dominant. The genes EXT1 and EXT2 have been localized to chromosomes 8q24.11 and 11p 12, respectively.

DIAGNOSTIC TESTS

The diagnosis is based on mutation analysis of the previously mentioned genes.

TREATMENT AND PROGNOSIS

Treatment is only necessary where there are complications or (rare) malignant degeneration. Prognosis is usually good.10

GENETIC DISEASES OF PROTEIN N- AND O-GLYCOSYLATION

Six genetic diseases have been identified with a combined N- and O-glycosylation defect. The most frequent is hereditary inclusion body myopathy.

RECESSIVE HEREDITARY INCLUSION BODY MYOPATHY

CLINICAL PRESENTATION

The disease is characterized by adult-onset, progressive distal and proximal muscle weakness; remarkably, the quadriceps muscles are spared.

METABOLIC DERANGEMENT

The defect is in the first two steps of the sialic acid synthesis catalyzed by UDP-GlcNAc epi-merase/kinase.

GENETICS

Inheritance of recessive hereditary inclusion body myopathy (OMIM 600737) is autosomal recessive. The gene (GNE) is located on chromosome 9p12.

DIAGNOSTIC TESTS

The diagnosis is confirmed by mutation analysis.

TREATMENT AND PROGNOSIS

No effective treatment is available. Patients eventually become wheelchair-bound in two to three decades, but, remarkably, the quadriceps muscles are spared.11 There have been recent reviews on congenital disorders of glycosylation (CDG).12,13

REFERENCES

See references on DVD.