N-linked Glycan Biosynthesis

Biosynthesis of the lipid precursor oligosaccharide involves a series of steps and enzymes that add sugars in specific linkages and in a specific order [Freeze, 2001a]. It begins with P-Dol + UDP-GlcNAc forming GlcNAc-P-P-Dol on the cytosolic face of the endoplasmic reticulum. Another UDP-GlcNAc donates a second GlcNAc using a different GlcNAc transferase, and this is followed by the addition of five Man units derived from GDP-Man. At this point, a “flippase” reorients the entire molecule from the cytosolic face into the endoplasmic reticulum lumen. A series of Man transferases use Man-P-Dol to add four more Man units, making a three-branched structure. Three glucosyltransferases sequentially add Glc from Glc-P-Dol to one branch to complete the sugar chain. This 14-sugar unit molecule is the optimal substrate for the multisubunit oligosaccharyl transferase (OST) complex that recognizes the Asn-X-Thr/Ser consensus sequence on the protein and adds the sugar chain to Asn. Recent studies indicate that some Asn-X-Cys sites can also be glycosylated [Zielinska et al., 2010; Ostasiewicz et al., 2010]. Truncated glycans are poorly transferred and sometimes fail to occupy normal glycosylation sites. Following glycan transfer, P-P-Dol is converted back to P-Dol and then to Dol. Recycling of these carriers is extensive.

Within a few minutes of transfer to protein, the sugar chain is processed (Figure 35-2; see also Figure 35-1). A set of two glucosidases removes the Glc units. For some proteins, processing stops here, but for the great majority, a mannosidase in the endoplasmic reticulum removes a single Man unit. The proteins are carried to the Golgi, where another mannosidase called Golgi mannosidase I removes up to three more Man units. At this point, processing starts to add sugars. UDP-GlcNAc donates a GlcNAc to one branch, making the sugar chain an acceptable substrate for Golgi mannosidase II, which removes an additional two Man units. This process clears the way for the addition of a second GlcNAc to a Man on the remaining branch. Depending on the protein, up to three more GlcNAc transferases add GlcNAc units to the Man residues in a specific order to initiate up to five branches. These branches are then extended with Gal, derived from UDP-Gal, and sialic acid (Sia), derived from cytidine monophosphate (CMP)-Sia. In some cases, GDP-Fuc donates a Fuc to one or more of the branches or to the GlcNAc linked to the protein. In other cases, sulfates, phosphates, or glucuronic acids are added to selected sugars. Individual branches may accumulate several repeating units of Galβ1,4GlcNAc before being capped by Sia. These reactions occur in selected cisternae of the Golgi, and each nucleotide sugar donor must be translocated from its origin in the cytoplasm or nucleus to the Golgi by a substrate-selective transporter [Gerardy-Schahn et al., 2001].

Fig. 35-2 Processing of N-linked glycans in the Golgi membrane.

Mannose trimming of the protein-bound sugar chains may stop or continue, as shown at the extreme left. Addition of a single N-acetylglucosamine or continued mannose trimming next leads to the build-up of sugar chains with 2–5 branches containing N-acetylglucosamine, galactose, and sialic acid. Fucose may be added to some chains, and sulfate may be attached to several other sugars using specific transferases. All of these reactions require delivery of the nucleotide sugar into the Golgi by specific transporters. Congenital disorder of glycosylation (CDG) defects (indicated by a circled “X”) occur in one of the trimming glycosidases, in a glycosyltransferase, and in two transporters. A series of mutations in 6 of the 8 COG subunits and a vacuolar H⁺/ATPase disturb N-glycan processing and other biosynthetic pathways by disrupting Golgi homeostasis. CMP, cytidine monophosphate; GDP, guanosine diphosphate; PAPS, 3′-phosphoadenosine–5′-phosphosulfate; UDP, uridine diphosphate.

PMM2 (CDG-Ia)

PMM2 (CDG-Ia) is the best-known and most frequently recognized form of CDGs, first reported by Jaeken and co-workers in 1980 [Jaeken et al., 1980]. The defective gene was identified in 1995 as PMM2, which encodes the phosphomannomutase (PMM) that converts Man-6-P → Man-1-P. This defect results in insufficient production of lipid-linked oligosaccharide, leading to empty glycosylation sites. More than 800 patients are known worldwide, and more than 100 mutations have been cataloged [Barone et al., 2008; Jaeken, 2003; Matthijs et al., 2000; Haeuptle and Hennet, 2009].

Hagberg and associates described four stages of the typical (severe) phenotype [Hagberg et al., 1993]. The first is the infantile phase, marked by various combinations of dysmorphism, abnormal fat distribution (supragluteal and vulval fat pads, focal lipoatrophy), inverted nipples, cryptorchidism, esotropia, recurrent infections, cardiomyopathy or pericardial effusions, coagulopathies, nephrotic syndrome, hypothyroidism, life-threatening episodes of hepatic failure, and unexplained coma. As many as 20 percent of infants with CDG-Ia succumb in this phase [Jaeken and Matthijs, 2001]. In the second phase (comprising the remainder of the first decade), children experience seizures and strokelike episodes, often precipitated by intercurrent infections. The third phase (in the second decade of life) is marked by slowly progressive cerebellar ataxia and limb wasting, and by progressive visual loss secondary to pigmentary retinopathy. Adult survivors have moderate mental retardation with severe ataxia and hypogonadism, with or without skeletal deformities. Presentations are highly variable. In one girl with CDG-Ia, findings on computed tomography (CT) of the head were normal at 9 months, but subsequent imaging studies demonstrated progressive atrophy [Mader et al., 2002]. The investigators concluded that the cerebellar hypoplasia reported in infancy in most children with CDG-Ia likely reflects atrophy of antenatal onset, rather than hypoplasia.

More extensive testing for CDGs has led to the identification of milder CDG-Ia phenotypes [Briones et al., 2002; de Lonlay et al., 2001; Grünewald, 2009]. The patients often have high residual levels of PMM2 activity [Drouin-Garraud et al., 2001; Grünewald et al., 2001; Westphal et al., 2001b]. Some patients have only borderline cognitive impairment, but strabismus persists in these very mild cases. Few adult CDG-Ia patients are employed. A longitudinal study of eight Spanish patients confirmed the wide range of clinical manifestations, ranging from neonatal hemorrhage, non-immune hydrops, and death, through mental retardation and motor impairment without acute decompensation in patients in their 20s, to one individual with normal development and only gastrointestinal dysfunction in childhood [Perez-Duenas et al., 2009].

The carrier frequency of the most common mutant allele is about 1 in 70 in the northern European population [Schollen et al., 2000]. It is believed to be lethal in the homozygous state. Genotype-phenotype correlations have not been informative, but some evidence suggests that frequent polymorphisms in other glycosylation-related genes may influence the severity of the phenotype. No effective specific therapy for CDG-Ia exists. Experiments using CDG-Ia cells suggested that increasing dietary mannose might improve glycosylation in patients, but clinical trials demonstrated no benefit [Kjaergaard et al., 1998; Marquardt et al., 1997; Mayatepek et al., 1997].

Population studies find the risk of having a second child with CDG-Ia to be close to 1 in 3, rather than the expected mendelian ratio of 1 in 4, suggesting that reduced glycosylation may have some selective advantage [Schollen et al., 2004b]. At-risk couples should be counseled appropriately.

MPI-CDG (Ib)

MPI-CDG (Ib) is caused by mutations in MPI, the gene encoding phosphomannose isomerase, which interconverts Man-6-P and Fructose (Frc)-6-P. This reaction produces most of the mannose for glycoprotein synthesis [de Koning et al., 1998; Niehues et al., 1998]. About 25 patients have been identified since its discovery in 1998 [de Lonlay and Seta, 2009]. This phenotype is not associated with any primary neurologic symptoms. Gastrointestinal and hepatic pathology, with hypoglycemia, coagulopathy, and protein-losing enteropathy, is characteristic.

MPI-CDG (Ib) is unique in that simple dietary mannose therapy corrects the abnormalities, except for liver fibrosis [de Lonlay and Seta, 2009; Durand et al., 2003; Niehues et al., 1998]. The dietary mannose is taken up through transporters and converted to Man-6-P, thereby bypassing the metabolic block. Mannose has no known side effects at the concentrations used. Nonenzymatic protein glycation occurs at high concentrations of mannose, which is considerably more reactive than glucose, and can raise hemoglobin A1c (HbA1c) levels [Harms et al., 2002]. Some patients have received such treatment for longer than 10 years without complications [Westphal et al., 2001a; de Lonlay and Seta, 2009]. Twenty percent of the patients who were subsequently confirmed to have phosphomannose isomerase deficiency, however, died before discovery of the disorder [Freeze, 2001a]. The potentially fatal outcome and availability of simple, effective treatment mandate investigation for CDGs in suspected cases. One adult patient with MPI deficiency was not able to tolerate oral mannose, but her protein-losing enteropathy appeared to respond to heparin therapy [Liem et al., 2008].

ALG6-CDG (Ic)

ALG6-CDG (Ic) resembles, but is less severe than PMM2-CDG. It is characterized by moderate psychomotor retardation, hypotonia, esotropia, seizures, and ataxia. Nevertheless, at least five children have died of CDG-related complications [Marquardt and Denecke, 2003; Newell et al., 2003; Westphal et al., 2000]. The defect is in a glycosyltransferase, hALG6, that results in production of a truncated lipid-linked oligosaccharide sugar chain, which is inefficiently transferred to proteins. Patients sometimes experience life-threatening protein-losing enteropathy during bouts of gastroenteritis [Westphal et al., 2000]. Skeletal dysplasia, including a unique form associated with brachytelephalangy has been described in a compound heterozygote for ALG6 [Drijvers et al., 2010]. An adult woman has been identified in whom ALG6 deficiency was associated with mental retardation, skeletal anomalies, virilization, and deep vein thrombosis [Sun et al., 2005]. ALG6 deficiency was first identified in 1998 and subsequently in more than 35 patients [Haeuptle and Hennet, 2009; Burda et al., 1998; Grünewald et al., 2000; Imbach et al., 2000a], making it the second most common form of CDG.

NOT56L-CDG (Id)

Patients with NOT56L-CDG have mutations in NOT56L (ALG3), which encodes the mannosyltransferase used to synthesize Man₆GlcNAc₂ [Körner et al., 1999]. The index patient had microcephaly, optic nerve atrophy, iris colobomas, epilepsy, spastic quadriparesis, and profound psychomotor delay. Another patient had similar features plus Dandy–Walker malformation, with agenesis of the cerebellar vermis and corpus callosum. She also had recurrent hypoglycemia, thrombocytopenia, hypoalbuminemia, and coagulopathy. A total of eight children with NOT56L-CDG have now been described; all have a severe phenotype, with 7 of the 8 manifesting neurological impairments, including seizures, visual impairment, psychomotor retardation, and cerebral or cerebellar hypoplasia; there were varying combinations of hepatic, hematologic, endocrine, and dysmorphic manifestations, as well [Kranz et al., 2007b].

ALG12-CDG (Ig)

Eight cases of ALG12-CDG (Ig) have been reported [Chantret et al., 2002; Grubenmann et al., 2002; Thiel et al., 2002; Eklund et al., 2005; Kranz et al., 2007c]. The patients had typical CDG abnormalities, including hypotonia, generalized developmental delay, cerebellar hypoplasia, and decreased coagulation factors. Dysmorphic features included a long thin face, flat nasal bridge, and epicanthal folds. Frequent infections probably reflect reduced immunoglobulin G (IgG) concentrations. Affected males have genital hypoplasia, a feature not reported in other types of CDGs. More recent reports have emphasized features of skeletal dysplasia. These patients have mutations in hALG12, which encodes dolichol-P-mannose: Man₇GlcNAc₂-PP-dolichyl mannosyltransferase. Patients accumulate the truncated lipid-linked oligosaccharide, which is inefficiently transferred to proteins.

ALG8-CDG (Ih)

Eight patients have been described with ALG8-CDG (Ih), caused by a deficiency in hALG8, the gene encoding the second glucosyltransferase in lipid-linked oligosaccharide synthesis [Chantret et al., 2003; Schollen et al., 2004a; Stolting et al., 2009; Vesela et al., 2009]. Three patients had mild clinical presentations, including two siblings with pseudogynecomastia, epicanthus, hypotonia [Stolting et al., 2009], mental retardation, and ataxia, whereas the others all had severe multi-organ failure. Most died within a few months, but one patient with severe developmental delay survived a year before succumbing to renal failure. The others experienced hepatointestinal symptoms; one girl had cortical, cerebellar, and optic atrophy and intractable seizures before her death from systemic complications at 2 months [Vesela et al., 2009].

ALG2-CDG (Ii)

The only child with ALG2-CDG (Ii) recognized to date was normal at birth, except for an iris coloboma. Seizures, hepatomegaly, and coagulation abnormalities emerged in the first year. Imaging studies revealed cerebral hypomyelination. Pathologic mutations were found in hALG2, the enzyme that adds the second mannose to the growing lipid-linked oligosaccharide sugar chain [Thiel et al., 2003].

DPAGT1-CDG (Ij)

DPAGT1-CDG (Ij) is caused by a deficiency in UDP-GlcNAc:dolichol phosphate N-acetylglucosamine-1 phosphate transferase (GPT) activity encoded by DPAGT1. Two patients had severe hypotonia, intractable seizures, mental retardation, microcephaly, and exotropia [Wu et al., 2003].

ALG1/HMT1-CDG (Ik)

Ten children with HMT1-CDG (Ik) have been described [Kranz et al., 2004; Schwarz et al., 2004; Dupre et al., 2010]. Fifty percent had complications during pregnancy, and several had postnatal complications. Eighty percent were hypotonic, and all had at least one seizure, most being intractable; 8 of the 10 were dysmorphic, 7 of the 10 had visual impairment, and 5 of the 10 were microcephalic. Fifty percent had a fatal outcome. Patients with HMT1-CDG are deficient in GDP-Man:GlcNAc₂-P-P-dolichol mannosyltransferase, encoded by the hALG1 gene, which adds the first Man to the lipid-linked oligosaccharide chain.

ALG9/DIBD1-CDG (Il)

Two patients with ALG9/DIBD1-CDG (Il) have been described. Abnormalities in the first case included severe microcephaly, central hypotonia, seizures, developmental delay, hepatomegaly, and bronchial asthma. This patient was found to carry a homozygous point mutation in human hALG9, whose product catalyzes the addition of both the seventh and ninth mannose units to the lipid-linked oligosaccharide chain [Frank et al., 2004]. The second patient was a girl with delayed development, epilepsy, hypotonia, failure to thrive, pericardial effusion, cystic renal disease, hepatosplenomegaly, esotropia, and inverted nipples. Neither lipodystrophy nor dysmorphic facial features were present. Magnetic resonance imaging (MRI) of the brain reflected cerebral and cerebellar atrophy and delayed myelination. Antithrombin III, factor XI, and cholesterol levels were low [Weinstein et al., 2005].

RFT1-CDG (In)

Six children have been described with RFT1-CDG (In) [Haeuptle et al., 2008; Clayton and Grunewald, 2009; Vleugels et al., 2009; Jaeken et al., 2009]. Most children have typical manifestations of the CDG spectrum: feeding problems, failure to thrive, severe developmental delay, poor to absent visual contact, epilepsy, and hypotonia, and variable respiratory, gastrointestinal, and coagulation abnormalities. All, however, have sensorineural deafness, which has only rarely been reported in other forms of CDG, and which is an important clue to this diagnosis. RFT1 is thought (but not absolutely proven) to be the flippase that transfers Man₅GlcNAc₂-PP-Dol from the cytoplasmic to the luminal face of the endoplasmic reticulum membrane.

MGAT2-CDG (IIa)

Three children with MGAT2-CDG (IIa) have been described [Cormier-Daire et al., 2000; Jaeken et al., 1994; Tan et al., 1996]. They were of Belgian, Iranian, and French descent, respectively, and all had severe psychomotor delay, acquired microcephaly and growth retardation, and variable combinations of hypotonia, ventricular septal defects, craniofacial dysmorphism (thin lips, hooked nose, large ears, hypertrophied gums, short neck), stereotypies, and coagulation defects. All were found to have impaired activity of the processing enzyme N-acetylglucosaminyltransferase II (MGAT2), which decreases the formation of multibranched N-linked glycans. A targeted disruption of this gene in the mouse faithfully recapitulates the human disorder [Wang et al., 2001].

GLS1-CDG (IIb)

Only one patient with GLS1-CDG (IIb) has been described [De Praeter et al., 2000]. Her parents were consanguineous. She had dysmorphic features, hypotonia, reduced nerve conduction velocity, seizures, and hepatomegaly with abnormal bile duct proliferation and fibrosis. The patient died at 2.5 months. This disorder was caused by mutations in α-glucosidase I, which trims the outermost glucose from oligosaccharides just after their transfer to nascent proteins. Although activity of the enzyme was severely decreased in this patient’s fibroblasts, transferrin glycosylation remained normal. This patient would be missed by the standard diagnostic assays. An endo-α-mannosidase apparently bypasses the glucosidase defect to permit normal oligosaccharide processing. Abnormal accumulation of fully glycosylated sugar chains may overwhelm the capacity of endoplasmic reticulum chaperone lectins.

TUSC3-CDG

Nine patients have been described with TUSC3-CDG. Seven were members of four sibships in a large consanguinous Iranian kindred, and all had nonsyndromic, moderate to severe mental retardation [Garshasbi et al., 2008]. Two were sibs from a small French family [Molinari et al., 2008]. TUSC3 codes for a subunit of the oligosaccharyltransferase complex (OST). It is not clear why these patients have no other systemic manifestations of hypoglycosylation, but it is theorized that differential tissue expression of another subunit associated with the OST (IAP) might compensate for TUSC3 deficiency in non-neurologic tissues [Garshasbi et al., 2008].

IAP-CDG

Screening of samples from 250 families with X-linked nonsyndromic mental retardation led to the identification of one patient who carried a c.932T/G, p.V311G mutation in the IAP gene [Molinari et al., 2008]. IAP (implantation-associated protein) is an ortholog to yeast Ost 6p. Like TUSC3, IAP is expressed in all tissues, including adult and fetal brain.

ALG11-CDG (Ip)

A brother and sister born to consanguineous Turkish parents were found to carry homozygous c.T257C mutations in the hALG11 gene; this disorder was designated ALG11-CDG, or CDG-Ip [Rind et al., 2010]. The proband was a girl who presented in the neonatal period with dysmorphism, vomiting, and hypotonia. She had visual and hearing impairment, intractable seizures, lipodystrophy, and elevated lactate without another metabolic explanation. She died at 2 years. Subsequently, a brother was born, who presented with hypotonia and vomiting at 6 weeks, and who was found to carry the same mutation. ALG11 encodes GDP-Man:Man₃GlcNAc₂-PP-dolichol mannosyltransferase. Deficiency of this enzyme impairs the elongation of lipid-linked oligosaccharides at the outer leaflet of the endoplasmic reticulum.

O-Xylosylglycan Synthesis

Glycosaminoglycans are very large glycan chains that usually are built on selected core proteins [Esko, 1999]. These molecules are located in the extracellular matrix, where they provide mechanical support and help to organize the matrix. At the cell surface, they bind growth factors and act as signaling molecules that enhance or inhibit cell proliferation. They are the most diverse and complex glycans. The protein-bound glycosaminoglycan molecules include heparan sulfate, chondroitin sulfate, dermatan sulfate, and keratan sulfate. All but the last are linked to protein through O-xylose, which is extended with two galactose units and a glucuronic acid. Each of these glycosaminoglycan chains is extended by one of several alternating disaccharides composed of GlcNAcα1,4GlcAβ1,4) (heparan sulfate) or GalNAcβ1,3GlcAβ1,3 (chondroitin sulfate and dermatan sulfate) or GlcNAcβ1,3Galβ1,4 (keratan sulfate). All of these glycosaminoglycans are partially sulfated. Hyaluronon, GlcNAcβ1,4GlcAβ1,3, is the only glycosaminoglycan that exists as a free glycan chain and is not sulfated.

Three known defects in glycosaminoglycan synthesis cause clinical disorders, but none has neurologic manifestations. Mutations in the gene B4GALT7 cause abnormal synthesis of dermatan sulfate and are associated with the progeroid variant of Ehlers–Danlos syndrome [Quentin et al., 1990; Faiyaz-Ul-Haque et al., 2004]. This gene encodes an enzyme, xylosylprotein 4-β-galactosyltransferase, that adds the first galactose residue to xylose in the core of glycosaminoglycan chains. Patients exhibit multiple abnormalities in connective tissue, leading to short stature, diffuse osteopenia, loose but elastic skin, hypermobile joints, and hypotonia. Cognitive impairment has not been reported.

The condition multiple hereditary exostoses is inherited as an autosomal-dominant trait and characteristically affects the metaphyses of long bones [Zak et al., 2002]. Patients with multiple hereditary exostoses have mutations in the genes EXT1 and EXT2, both of which encode co-polymerase components that participate in the assembly of alternating residues of GlcNAc and GluA that form the backbone of the heparan sulfate glycosaminoglycan chains. Because heparan sulfate binds to many types of growth factors, a partial decrease in heparan sulfate is thought to upset the delicate regulation of chondrocyte proliferation. This situation in turn leads to enhanced chondrocyte growth and the formation of exostoses. Surprisingly, a significant proportion of patients fall within the autistic spectrum. Rigorous experiments in Ext-deficient mouse models indicate molecular alterations corresponding to dramatic behavioral changes that correlate with reduced heparan sulfate content of selected tissues.

Macular corneal dystrophy (types I and II) is caused by a deficiency in a specific sulfotransferase (CHST6) called corneal N-acetylglucosamine-6-sulfotransferase (GlcNAc6ST), which is responsible for the sulfation of corneal keratan sulfate [Akama et al., 2000]. The unsulfated keratan chains are poorly soluble, and their eventual precipitation disrupts the collagen network, leading to thinning and loss of transparency of the corneal stroma. This progressive disorder manifests between ages 5 and 9 with very small, punctate corneal opacities. Erosions, painful photophobia, and sensation of an ocular foreign body develop subsequently.

O–N-acetylgalactosamine Synthesis

These mucinous types of molecules are located at the surface of epithelial cells or in their secretions, and typically subserve barrier functions [Marth, 1999]. They usually are composed of large clusters of O-galactose N-acetylglucosamine (O-GalNAc)-linked sugar chains, each containing 2–10 sugars. Only a few are known to serve specific functions: for example, in lymphocyte recirculation and leukocyte extravasation [Lowe, 2003]. Few biosynthetic defects are known, and none has obvious neurologic manifestations.

O-mannosylglycan Synthesis: Congenital Muscular Dystrophy and Limb-Girdle Spectrum

Overview

The dystrophin glycoprotein complex assembles on the sarcolemma of skeletal muscle cells. Mutations that affect the integrity of this complex cause congenital muscular dystrophies by compromising the integrity of the basement membrane [Martin, 2003]. Alpha-dystroglycan is a major component of this complex and contains O-mannose-linked glycans (i.e., those with an O linkage between Ser/Thr and mannose residues). Mutations in genes encoding enzymes involved in this O-linked glycosylation cause a group of rare congenital muscular dystrophies with an autosomal-recessive inheritance pattern and a variable degree of brain involvement [Martin and Freeze, 2003]. Additional types of congenital muscular dystrophy likely will be found to result from as yet unrecognized defects in this glycosylation pathway (Figure 35-3).

Fig. 35-3 O-mannose glycan biosynthesis.

Biosynthesis of O-mannose-linked glycans on molecules such as α-dystroglycan begins with the addition of mannose from Man-P-Dol in the lumen of the endoplasmic reticulum. Defects in its addition are responsible for some cases of Walker–Warburg syndrome. An N-acetylglucosamine is added to the O-linked mannose using a specific transferase and a transporter-delivered UDP donor. Mutations in this transferase cause muscle-eye-brain disease. Extension can occur using GalNAc or by galactose and followed by sialic acid. All of these require transporter-delivered donors. A novel pathway converts O-Man to O-Man-6-P, and then an additional reaction involving LARGE forms a phosphodiester of unknown composition on GalNAc-GlcNAc-Man-Ser/Thr glycans. CMP, cytidine monophosphate; S/T, serine/threonine; UDP, uridine diphosphate.

Functional Defects

Defects in this pathway are depicted in Table 35-1. Deficiencies in POMT1 and POMT2 impair the addition of the linkage Man unit to the protein, preventing further elongations at that site. These mutations account for about 30 percent of the diagnosed cases of Walker–Warburg syndrome [Beltran-Valero de Bernabe et al., 2002; Muntoni et al., 2004]. Defects in POMGNT1 impair addition of GlcNAc to Man and cause muscle-eye-brain disease [Zhang et al., 2003].

Other congenital muscular dystrophies also have been linked to glycosylation abnormalities, but the specific molecular mechanisms are not known. These proteins include fukutin, fukutin-related protein, and LARGE. Mutations in FCMD cause Fukuyama congenital muscular dystrophy, common in Japan. Mutations in the fukutin-related protein gene, FKRP, cause CMD1C and limb-girdle muscular dystrophy [Muntoni et al., 2004]. The LARGE gene causes a form of murine muscular dystrophy, and mutations in its human homolog were found in a patient with a congenital muscular dystrophy and mental retardation [Longman et al., 2003]. The clinical presentations of congenital muscular dystrophies reflect the manifestations associated with specific mutations and may overlap. Mutations in POMT1, FKRP, and FCMD all have been associated with a Walker–Warburg syndrome phenotype [Muntoni et al., 2004]. FCMD, FKRP, and LARGE all have glycosyltransferase-like domains and characteristic catalytic residues, but transferase activity has not been demonstrated. Sequence homologies usually are insufficient to predict the specific transferase reactions because sugar-specific signatures are few, and the acceptor substrate may require both peptide and glycan recognition. Overexpression of LARGE can functionally bypass multiple glycosylation defects in α-dystroglycan, offering a potentially broad therapeutic approach [Barresi et al., 2004].

Clinical Features

The European Neuromuscular Center proposed a set of diagnostic criteria for congenital muscular dystrophies. These include hypotonia beginning in the first 6 months, early multiple contractures, diffuse weakness and muscular atrophy with sparing of extraocular muscles, normal mental development (in many), variable course, early elevation of serum creatine kinase, myopathic changes on electromyography (EMG), and necrotic-regenerative changes on muscle biopsy [Dubowitz, 1997].

The three forms of congenital muscular dystrophy associated with impaired O-glycosylation have considerable phenotypic overlap, as mentioned earlier. Fukuyama congenital muscular dystrophy is characterized by cortical dysgenesis and simple myopia. Affected children have global developmental delay, with regression in motor skills in the latter half of the first decade and death in adolescence [Messina et al., 2010]. Almost all of the patients with Fukuyama congenital muscular dystrophy have seizures. Fukuyama congenital muscular dystrophy is the most common form of congenital muscular dystrophy in Japan, in contrast with Western populations, in which merosin deficiency predominates.

Muscle-eye-brain disease is characterized by more severe pathologic changes than in Fukuyama congenital muscular dystrophy, including cobblestone lissencephaly, midline defects, and brainstem flattening associated with progressive myopia, retinal degeneration, and cataracts.

Walker–Warburg syndrome also features type II lissencephaly, in combination with ventriculomegaly, cerebellar malformations, retinal and anterior chamber malformations, and congenital cataracts. Walker–Warburg syndrome represents the most severe congenital muscular dystrophy phenotype, with congenital blindness, hypotonia, profound developmental delay, and failure to thrive. Most affected children die in the first 6 months of life [Messina et al., 2010].

Biochemical and Genetic Tests

Several monoclonal antibodies recognize some feature of the O-Man chain on α-dystroglycan, and their binding is greatly reduced or eliminated in all of these disorders [Michele et al., 2002]. Antibody against the peptide portion binds normally, but the loss of the glycan chains reduces the apparent size of the protein. Specific enzymatic assays can be used to diagnose muscle-eye-brain disease [Zhang et al., 2003], and for POMT1 [Lommel et al., 2010]. Sequencing of the genes is needed to identify specific mutations. It is likely that additional biosynthetic genes will be identified soon.

DPM1-CDG (Ie)

DPM1-CDG is another severe phenotype, characterized by psychomotor delay, profound hypotonia, microcephaly, cortical blindness, and intractable seizures [Imbach et al., 2000b; Kim et al., 2000]. Laboratory testing revealed elevated creatine kinase and depressed antithrombin III. Some children have dysmorphic features, including downslanting palpebral fissures, flat occiput and nasal bridge, hemangiomas of the occiput and sacrum, a high narrow palate, and mild limb shortening. Dolichol phosphate mannose synthase (DPM1) activity is markedly diminished in all reported cases.

DPM3-CDG (Io)

A 27-year-old woman has been reported with DPM3 deficiency, whose phenotype includes short stature, dilated cardiomyopathy, a strokelike episode, and a myopathy characterized by mild proximal weakness and absent glycosylated α-dystroglycan on muscle biopsy [Lefeber et al., 2009]. DPM activity is required to provide Man-P-Dol precursors for N-, C-, and O-linked glycosylation and for glycophosphatidylinositol anchor biosynthesis. In this case, a primary defect in the N-glycosylation pathway has led to an O-linked disorder – an α-dystroglycanopathy.

MPDU1-CDG (If)

Four children with MPDU1-CDG have been described. Abnormalities included severe psychomotor retardation and variable features, including growth retardation, optic nerve atrophy, icthyosis, dysmorphism (parietal bossing, thin lips), hypo- or hypertonia, enlarged extra-axial spaces, thrombocytopenia, transient deficiency of growth hormone and insulin-like growth factor 1, and mild elevations of creatine kinase [Kranz et al., 2001; Schenk et al., 2001b]. All have mutations in MPDU1/Lec35, leading to deficient function of the Lec35 protein. Lec35 may act as a chaperone for P-Dol in the endoplasmic reticulum membrane, ensuring appropriate lateral spacing of Man-P-Dol and Glc-P-Dol. In the absence of such spacing, these compounds may form rafts that alter local concentration gradients, impairing synthesis of the lipid-linked oligosaccharide and its accessibility to the oligosaccharyltransferase complex.

B4GALT1-CDG (IId)

A single patient with B4GALT1-CDG (IId) has been described. Abnormalities included psychomotor retardation, a Dandy–Walker malformation, progressive hydrocephalus, hypotonia, and myopathy [Hansske et al., 2002]. CDG-IId was caused by mutations in B4GALT1, encoding one of the isozymes that add β-1,4-galactose units to N-linked glycans during processing. The other isozymes do not compensate for this transferase deficiency.

GNE-CDG

Mutations in GNE, which encodes UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase, cause both hereditary inclusion body myopathy (IBM2) and distal myopathy with rimmed vacuoles [Grandis et al., 2010]. IBM2 presents with a distal myopathy that later spreads proximally, but characteristically spares the quadriceps. Creatine kinase is mildly elevated. The disorder is most frequent among Iranian Jews, who carry an M712 T mutation in GNE. Nonaka myopathy, or distal myopathy with rimmed vacuoles, has a similar presentation, but is usually associated with different mutations.

SLC35A1-CDG (IIf)

The sole reported patient with SLC35A1-CDG (IIf) had severe thrombocytopenia and complete loss of the sialyl-Lewis-X antigen on leukocytes [Willig et al., 2001; Martinez-Duncker et al., 2005]. This disorder was associated with mutations in the gene encoding the Golgi cytidine monophosphate (CMP)-sialic acid transporter [Martinez-Duncker et al., 2005]. Although platelet membrane proteins exhibited altered glycosylation, transferrin and other serum glycoproteins were normal. This finding underscores the limitations of transferrin analysis.

SLC35A1-CDG (IIc)

SLC35A1-CDG (IIc) was originally described as leukocyte adhesion deficiency II and is characterized by moderate to severe mental retardation, rhizomelic short stature, a broad flat nasal bridge, microcephaly, elevated leukocytes, frequent infections, persistent marked neutrophilia, and periodontitis. The disorder is caused by mutations in the GDP-fucose transporter, which limits the synthesis of fucosylated glycans [Lübke et al., 2001; Lühn et al., 2001]. One of these is sialyl Lewis-X (sLeX), a glycan essential for leukocyte rolling before extravasation. Oral fucose supplements effectively reduced leukocytosis in two patients by allowing synthesis of sufficient sLeX [Hidalgo et al., 2003; Marquardt et al., 1999]. These patients also lack fucosylated H-antigen, the precursor for the ABO blood group. Fucose supplements have not provoked antigen synthesis or immunologic reactions. Transferrin glycosylation is normal in this type.

COG complex