Congenital Disorders of Glycosylation

In: Glycans: Biochemistry, Characterization and Applications ISBN: 978-1-61942-541-5 Editor: Hector Manuel Mora-Montes, pp. 59-81 © 2012 Nova Science Publishers, Inc. No part of this digital document may be reproduced, stored in a retrieval system or transmitted commercially in any form or by any means. The publisher has taken reasonable care in the preparation of this digital document, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained herein. This digital document is sold with the clear understanding that the publisher is not engaged in rendering legal, medical or any other professional services.

Chapter III

Congenital Disorders of Glycosylation I. Martínez Duncker*1, C. Asteggiano2 and H. H. Freeze3 1

Human Glycobiology Laboratory, Science Faculty, Morelos State Autonomous University, Cuernavaca, Mexico. 2 Centro de Estudio de las Metabolopatías Congénitas, Facultad de Ciencias Médicas, Universidad Nacional de Córdoba y Facultad de Medicina, Universidad Católica de Córdoba, Argentina. 3 Sanford-Burnham Medical Research Institute, La Jolla, CA., U.S.A.

Abstract Glycans are highly diverse carbohydrate moieties that have been selected in evolution to convey dynamic structural and functional properties to the macromolecules to which they are attached. For this reason, correct glycan synthesis is essential for various developmental and physiological processes, particularly in multicellular organisms that use them as communication pathways. The disruption of glycan synthesis frequently results in multisystemic disease with neurological involvement. Congenital disorders of glycosylation (CDGs) have been and will likely remain a rapidly growing group of genetic human diseases that involve different defects in the synthesis or remodelling of N- and O-linked glycans as well as defects in glycosphingolipid and glycosylphosphatidylinositol (GPI) anchor glycosylation. The molecular and clinical characterization of CDGs has enormously contributed to understanding the physiologic roles of the glycosylation machinery and its interaction with other cellular machineries. More than 50 different types of defects have been described and although the first type was described in 1984, CDGs remain widely under-diagnosed or misdiagnosed. This review describes the genetic and biochemical basis of CDGs, as well as the clinical phenotypes and current methods to diagnose them that are ultimately required to establish corrective treatments that are also discussed.

Keywords: Glycosylation, congenital, glycan. *

Correspondence: Iván Martínez-Duncker, Av. Universidad 1001, Col. Chamilpa, Cuernavaca, Mor., Mexico 62209. Email: [email protected]

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I. Martínez Duncker, C. Asteggiano and H. H. Freeze

Introduction Glycosylation consists of the ubiquitous and dynamic modification of proteins and lipids with carbohydrate chains, also known as glycans, which mediate essential roles in glycoconjugate function. The synthesis of the different types of glycans is mediated by the function of hundreds of proteins that compose the “glycosylation machinery”. Mutations in proteins of this machinery, or in others that indirectly support its function, lead to aberrant glycosylation disorders known as Congenital Disorders of Glycosylation (CDGs) that usually manifest as multisystemic diseases. CDGs are autosomal recessive diseases with exception of EXT1/EXT2-CDG (autosomal dominant) and MAGT1-CDG (X-linked). CDGs should be suspected in all patients with an unexplained congenital syndrome with or without psychomotor impairment. In 1984, Jaeken et al. were the first to describe a new syndrome in identical twin sisters with a deficient sialylation of plasma and cerebrospinal transferrin [1], that 13 years later, was characterized as a defect in phosphomannomutase 2 (PMM2), the enzyme that converts mannose-1-phosphate (Man-1-P) to mannose-6-phosphate (Man-6-P) required for the synthesis of activated mannose substrate donors used by mannosyltransferases that synthesize mannosylated glycans [2]. Since then, the CDGs nomenclature [3] has expanded to include 50 disorders involving mutations in genes coding for proteins involved directly or indirectly in the biosynthesis of glycans, table 1. In this review we will address the molecular basis, clinical phenotypes, diagnostics and available corrective treatments for CDGs.

Defects in N-linked Glycosylation N-Linked glycans mediate important roles in glycoprotein function, including adequate folding, solubility, structural stability, protease resistance and as moieties that influence or determine ligand binding [4]. Multistep assembly of N-linked glycans by glycosyltransferases starts in the membrane of the rough endoplasmic reticulum (rER) with the 14-step synthesis of a lipid-linked oligosaccharide (LLO) composed of three glucose (Glc), nine mannose (Man) and two N-acetylglucosamine (GlcNAc) residues covalently linked to the lipid carrier dolichol pyrophosphate (Dol-PP), figure 1A. Seven of the 14 steps are carried in the cytosolic face of rER by glycosyltransferases (GTs) using nucleotide sugars as donor substrates, and synthesizing a Man5GlcNAc2-PP-Dol intermediate that is flipped from the cytosol to the rER lumen, where synthesis proceeds by GTs that use dolichol-linked sugars as donors, generating Glc3Man9GlcNAc2-PP-Dol, figure 1A. Once synthesized, the Glc3Man9GlcNAc2 moiety of the LLO is transferred en bloc by the oligosaccharyl transferase complex (OST) to asparagine residues by linkage to carboxamido nitrogens [5]. Asparagine (Asn) residues targeted for N-linked glycosylation are located with rare exceptions in the consensus sequence Asn-X-Ser/Thr (where X is any amino acid except proline) [6-7]. Not all consensus sequences are N-linked glycosylated; because this protein modification is a co-translational process, and thus other factors are involved in selecting consensus sequences, such as accessibility of OST to the consensus sequence during the unfolded state of the protein.

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Table 1. List of Congenital Disorders of Glycosylation. Current nomenclature includes the affected gene and protein, and the Online Mendelian Inheritance in Man (OMIM) database reference number

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Following assembly and transfer, the processing stage of N-linked glycan synthesis is initiated by the removal of the first two Glc residues, followed by the reversible removal/addition of the third Glc, figure 1B. In the ER and Golgi compartment, up to five Man units can be removed by α-mannosidases and several GlcNAc, galactose (Gal), sialic acid (Sia) and fucose (Fuc) residues are added by the action of GTs that use nucleotide sugars as donor substrates, and that are provided by a family of nucleotide-sugar transporters [8]. N-glycans are classified into three types: (1) oligomannose, in which only mannose residues are attached to the Man3GlcNAc2 core; (2) complex, in which “antennae” initiated by N-acetylglucosaminyltransferases (GlcNAcTs) are attached to the core; and (3) hybrid, in which only mannose residues are attached to the Manα1–6 arm of the core and one or two antennae are on the Manα1–3 arm [9]. The type of mature glycan depends on the specific target protein, together with the genetic background and regulation of the glycosylation machinery. The machinery regulation is cell type specific, and the assembly of structures is determined by enzyme competition for available donors and acceptor substrates depending on abundance, affinity or location, which favour or prevent the synthesis of selected glycans as proteins move through the Golgi compartments. Sixteen defects exclusive of N-linked glycan biosynthesis have been identified in CDGs (Table 1/Figure 1). It is important to note that among these defects MPI- and PMM2-CDG could affect other pathways, but to our knowledge only abnormal N-linked glycosylation has been reported in these patients. The identification of other 19 disorders that affect N-linked glycosylation, among other glycosylation pathways will be treated separately in this review. Table 2 summarizes the clinical phenotypes of CDG patients with defects in N-linked glycosylation. A common feature of these CDGs is the presence of multisystemic pathology and psychomotor impairment, with exception of MPI-CDG and some patients with ALG8CDG that show normal psychomotor development.

Diagnostic Approach N-glycan defects can be screened by isoelectric focusing (IEF) or mass spectrometry [10] of serum transferrin (N-glycoprotein) and serum Apolipoprotein C III (ApoCIII), a mucin type O-glycoprotein [11]. This approach will detect the majority of N-glycan defects and mixed defects involving mucin type O-glycosylation. Assembly defects of N-linked glycans can cause either their degradation or progression towards the secretory pathway, resulting in type 1 patterns characterized by absence of entire glycan chains. Type II patterns are characterized by glycans with abnormal structure and indicate a defect in N-glycan processing. These patterns are easy to see by IEF of transferrin as a biomarker, figure 2. Normal transferrin has two glycan chains, predominantly biantennary, each one with 2 sialic acids. Absence of 1 or 2 entire chains (Type I) produces additional bands with 2 or 0 sialic acids. Type II defects generate new bands with 0-3 sialic acids. However, none of these patterns provide any information on glycan structure or on the genetic or enzymatic defect. This is the main reason to complement the biochemical diagnosis of CDG assembly defects (type 1 patterns) with structural analysis of the intermediate Dol-PP-glycans, for which a cell culture, normally from patient’s fibroblasts, must be established [12]. The type of intermediate that accumulates in the rER of cultured cells often points out to the defective synthesis step allowing specific gene mutational analysis. Before analysis of intermediate


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Dol-PP-glycans, it is recommended to rule out PMM2-CDG or MPI-CDG, the two most frequent assembly defects. Also, if the analysis of intermediate Dol-PP-glycans results in normal or an inconclusive result, analysis of dolichol synthesis defects should be considered.

Figure 1. N-Linked-glycosylation pathway and associated CDGs. A. Assembly of Glc3Man9GlcNAc2PP-Dol in the rER and its transfer en bloc by OST complex to nascent N-glycoproteins. B. Nglycoprotein processing in the rER and Golgi complex that can give high mannose, hybrid or complex type N-linked glycans. CDG defects are marked with X and the indication of the gene-CDG affected. COG; oligomeric Golgi Complex; COP, coat protein complex II. MPI- and SLC35C1-CDG can be treated with oral mannose or oral fucose, respectively.

Table 2. Clinical features of specific N-linked glycan CDGs. HM, hypomyelination; IgA, immunoglobulin A; Immunoglobulin G; ND, not determined; ORL, otorhinolaryngological; PN, polyneuropathy; RF, respiratory failure

Table 3. Clinical features of mixed CDGs

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Sialic Acid

4 3 2 1 0

Control

CDG Type II

CDG Type I

Figure 2. Transferrin IEF patterns in CDG defects. Samples containing 6 μg of total serum protein from Control, CDG-Type II and CDG-Type I were separated by IEF and stained with anti-Transferrin antibody. Each indicated band corresponds to the presence or absence of sialic acid on transferrin. Type I patterns have loss of entire chains and show transferrin containing 0, 2 or 4 sialic acids. Type II patterns have truncated chains with transferrin showing 0,1,2,3,4 sialic acids.

In the case of defects in the processing stage (type II pattern), structural analysis of protein bound glycans can sometimes point out the defective processing step, and direct to specific biochemical and gene analysis. If a type II IEF transferrin pattern is detected it should be followed with IEF of ApoCIII to rule out defects known to involve multiple glycosylation pathways. The transferrin profile can be abnormal in patients with fructosemia, galactosemia, alcohol abuse and bacterial infections that should be discarded when confronting an abnormal IEF profile. Also, some assembly (TUSC3-CDG) and processing defects (GCS1-, SLC35C1and SLC35A1-CDG) show a normal transferrin IEF profile.

Defects in O-linked Glycosylation O-Linked glycosylation consists in the attachment and assembly of glycans at serine (Ser), threonine (Thr) or hydroxylysine (hLy) residues of proteins. The biosynthesis of Olinked glycans is initiated after protein folding and can start in the late rER or Golgi apparatus. There are 7 different types of O-linked glycans found in humans that are classified based on the first sugar attached to the amino acid residue: O-N-acetylglucosaminyl for GlcNAc, O-N-acetylgalactosaminyl for N-acetylgalactosamine (GalNAc), O-galactosyl for Gal, O-xylosyl for Xylose (Xyl), O-mannosyl for Man, O-glucosyl for Glc, and O-fucosyl for Fuc. CDGs caused by defects in the synthesis of all types of these O-linked glycans with exception of O-Glc and O-GlcNAc have been described, figure 3. In contrast to N-linked glycosylation, for most O-linked glycosylation types a consensus sequence for the attachment


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of the first sugar residue remains unknown, with exception of O-Fuc and O-Glc [13-14]; although there are available algorithms based on neural networks that predict putative Olinked glycosylation sites [15-16].

Figure 3. O-Linked glycosylation disorders. Diagnosis of O-linked glycosylation defects is more difficult to screen for because of the lack of specific serum markers, and also because it requires the clinician and hospital laboratory to have access to a wider range of research collaborations, in order to achieve molecular diagnosis of all O-linked glycosylation disorders. Most O-linked glycosylation disorders require establishment of clinical suspicion followed by gene sequencing.

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Defects in O-GalNAc Glycans The most common type of O-linked glycans are O-GalNAc glycans also known as mucin type glycans. Seven mucin-type core structures can be distinguished in humans, being the most common core type 1 (Galβ1-3GalNAcα1-O-Ser/Thr) and 2 (GlcNAcβ1-6(Galβ13)GalNAcαSer/Thr), and most of them are extended by polylactosamine sequences that can have Sia, Fuc, GlcNAc and GalNAc residues in the non reducing termini. Only one defect in O-GalNAc synthesis has been described, GALNT3-CDG and is phenotypically related to Familial Tumoral Calinosis (FTC), figure 1A. FTC represents a clinically and genetically heterogeneous group of inherited diseases that manifest with dermal and subcutaneous deposition of calcified materials and hyperphosphatemia. The major forms of the disease are now recognized and FTC has been shown to result from mutations in one of three genes: fibroblast growth factor-23 (FGF23), coding for a potent phosphaturic protein, KL encoding Klotho, which serves as a co-receptor for FGF23, and GALNT3, which encodes a glycosyltransferase responsible for FGF23 O-linked glycosylation and [17]. GALNT3-CDG is characterized by hyperphosphatemia and ectopic calcification, generally accompanied by painful calcified subcutaneous masses, that can be complicated by secondary infections and incapacitating mutilations [18]. These calcified masses are initially asymptomatic, but progressively reach large sizes (up to 1.5 kg), thereby interfering with joint movement. Ulceration is usually accompanied by intolerable pain and is occasionally associated with secondary infections, rarely reported as a cause of death [19]. The hyperphosphatemia is due to increased renal phosphate retention. GALNT3 selectively directs O-linked glycosylation of FGF23 in a subtilisin-like proprotein convertase (SPC) recognition sequence motif at Thr178, which blocks proteolytic processing of FGF23 [20]. These findings suggested a novel posttranslational regulatory model for FGF23, involving competition between O-linked glycosylation by GALNT3 and protease processing to produce intact FGF23. Mutations in GALNT3 result in a cleavage of intact FGF23 before secretion, leading to an accumulation of fragmented FGF23 and reduced intact active FGF23 [21]. New data are expanding the spectrum of mutations in GALNT3, and are contributing to a better understanding of the phenotypic manifestations in this pathology [22]. Diagnosis is made by gene sequencing.

Defects in O-Gal Glycans The hLy residues are essential for collagen stability by crosslinking the collagen fibers, and also are the acceptor amino acids for O-linked glycosylation by Gal and its extension to the disaccharide glucosylgalactose [23]. PLOD-CDG is caused by mutations in the PLOD gene that encodes for lysyl hydroxylase 1 (LH1), an enzyme that hydroxylates specific lysines in collagen, figure 1B. PLOD-CDG, or the Ehlers Danlos variant VIA, is characterized by hypermobile joints, soft hyperextensible skin, kyphoscoliosis and severe muscular hypotonia. PLOD-CDG can be diagnosed by determining low LH1 activity in cultured skin fibroblasts from the patients, or low hLy content in skin biopsies [24]. Subsequent gene sequencing is required for mutational analysis.


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Defects in O-Xyl Glycans The O-xylosylation, figure 1C, initiates the synthesis of a tetrasaccharide linker (GlcAGal-Gal-Xyl-R) that links glycosaminoglycan moieties like heparan sulphate (HS) or chondroitin sulphate (CS) with the protein backbone. The HS proteoglycans are proteins that contain one or more HS chains composed of polymers of alternating GlcNAc and Glucuronic Acid (GlcA). GlcNAc can be further N-de-acetylated, N-sulphated and O-sulphated. The GlcA can be C-5 epimerized to iduronic acid and O-sulphated. These complex chains are added to selected core proteins through an O-xylosyl linked tetrasaccharide. The HS synthesis begins with the synthesis of the repeating disaccharide polymer by proteins called exostosins that form a hetero-oligomeric glycosyltransferase complex in the Golgi. Mutations in genes EXT1 and EXT2 decrease HS synthesis, causing multiple hereditary exostosis (MHE) or EXT1/EXT2-CDG, which is characterized by cartilage capped tumors that develop from the growth plate of endochondral bone [25]. Diagnosis is established by gene sequencing. Mutations in the BGALT7 gene that encodes for a galactosyltransferase that adds the second Gal to the Xyl residues of the linker tetrasaccharide causes the progeria variant of Ehlers Danlos or BGALT7-CDG, that is characterized by premature aging, elastic skin, thin and curly hair, joint laxitude, osteopenia and psychomotor impairment [26-27]. Diagnosis is established by gene sequencing. SLC35D1-CDG or Schneckenbecken dysplasia (SBD) is a lethal skeletal dysplasia caused by mutations in the SLC35D1 gene which codes for an UDP-GlcA/UDP-GalNAc ER transporter [28]. Reduced transport of these nucleotide sugars into the ER causes defects in the synthesis of CS, a polymer of alternating GlcA and GalNAc. This disorder is characterized by iliac bone and thoracic hypoplasia with ossification defects in the posterior vertebral bodies. Diagnosis is established by gene sequencing.

Defects in O-Man Glycans of α-Dystroglycan Defects in O-Man glycans, figure 1D, are limited to the O-linked glycosylation of αdystroglycan (α-DG), an extracellular membrane-associated protein that is a central component of the dystrophin-associated glycoprotein complex that binds to various extracellular matrix proteins including laminin-α2. The complex is responsible of maintaining membrane integrity in skeletal muscle, as well as in other tissues, including the central nervous system, eye, heart and kidney. Defects in O-Man glycan synthesis in α-dystroglycan are present as muscular dystrophies in the context of multi-systemic disease. Five defects have been identified as specific for the synthesis of O-Man glycans of α-DG. Protein-OMannosyltransferase 1/2 (POMT1/POMT2)-CDG, POMGnT1-CDG, FKTN-CDG, Fukutin related protein-(FKRP)-CDG and LARGE-CDG. Other reviews explain the place of αdystroglycanopathy among muscular dystrophies and genotype/phenotype correlations [2930]. Diagnosis of dystroglycanopathies is established upon detection of hypoglycosylated αdystroglycan at the sarcolemma of skeletal muscle fibres by immunolabeling and/or on Western blot [30].

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Defects in O-Fuc Glycans LFNG-CDG is caused by defects in O-Fuc glycan synthesis of Notch receptors, and is associated to spondylocostal dysostoses (SCDO), a heterogeneous group of vertebral malsegmentation disorders that arise during embryonic development by a disruption of somitogenesis [31]. Somites are precursors of the axial skeleton and associated musculature. Other radiological and clinical findings in these patients are multiple vertebral segmentation defects, partial fusion of ribs, abnormal vertebral arches, left renal agenesis, and a 'Cooleylike' hand appearance radiologically [32]. Mutations causing autosomal recessive forms of SCDO have been identified in two genes involved in the Notch signaling pathway: DLL3 (SCDO1) and MESP2 (SCDO2) and in LFNG that codes for Lunatic Fringe, a Golgi Ofucose-specific β1,3-N-acetylglucosaminyltransferase that adds GlcNAc residues to O-Fuc on the EGF-like repeats of Notch receptors, figure 1E [33]. Diagnosis is established by gene sequencing. B3GALTL-CDG or Peters’-plus syndrome is characterized by peculiar eye malformations, a variety of anterior eye-chamber defects including corneal opacities and iridocorneal adhesions besides growth retardation and variable abnormalities in other organs. Other major symptoms are a disproportionate short stature, developmental delay, characteristic craniofacial features, and cleft lip and/or palate [34]. The gene affected is B3GALTL that codes for a β-1,3-glucosyltransferase involved in the synthesis of the unusual O-linked disaccharide glucosyl-beta-1,3-fucose-O- found on the thrombospondin type-1 repeats (TSRs) of many biologically important proteins [35]. Biosynthesis of glucosyl-beta1,3-fucose-O- is initiated by protein O-fucosyltransferase-2, which attaches the fucosyl residue to a Ser or Thr residue within the TSR. B3GALTL subsequently transfers the glucose onto TSR-fucose [36]. This disaccharide modification is specific to thrombospondin type 1 repeats, found in extracellular proteins that function in cell–cell and cell–matrix interactions. Diagnosis is established by gene sequencing.

Defects in Glycosylphosphatidylinositol (GPI) Anchor and Glycosphingolipid Glycan Synthesis More than 100 proteins in human cells are GPI anchored [37]. GPIs are enriched in lipid rafts that are key components of the cell membrane that serve functions that include signal transduction pathways, vesicular trafficking organization, anchored protein conformation, apical protein targeting in polarised cells, and selective solubilisation of membrane-associated proteins. Two defects in the GPI biosynthesis pathway have been described: PIGM-CDG and PIGV-CDG. GPI biosynthesis, figure 4, is a multi-step process that initiates in the cytoplasmic side of the ER. In Step 1, GlcNAc is transferred from UDP-GlcNAc to phosphatidylinositol (PI) to form GlcNAc-PI. This reaction is catalysed by a multi-subunit enzyme termed GPI-GlcNAc transferase (GPI-GnT) that comprises 6 proteins (PIG-A, PIG-C, PIG-H, PIG-P, PIG-Q, and PIG-Y). In step 2, GlcNAc-PI is de-N-acetylated by PIG-L, generating GlcN-PI which is flipped to the lumen of the ER (step 3). In step 4, GlcN-PI is acylated by PIG-W to form GlcN-(acyl)PI. Afterwards, in step 5 and 6, two Man residues are added sequentially by PIG-

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M and PIG-V V to form Maanα1,6Manα α1,4GlcN-(acyyl)PI. PIG-M M works in coomplex with PIGP X that stabilizes PIG-M. In step 7, Man-1 M is moddified with ethhanolamine pphosphate (E EtNP) by PIG-N to generate Maanα1,6-(EtNP P)Manα1,4-G GlcN-(acyl)PI. In Step 8, Man-3 M is addeed by PIG-B to forrm Manα1,2M Manα1,6(EtN NP)α1,6Manα α1,4GlcN-(accyl)PI. Finally, in step 9 EtNP E is added to terminal t Man n-3 by a com mplex of GPII-ETIII with PIG-O (cataalytic) and PIIG-F (stabilizer) too form the mature GPI strructure (EtNP P)Manα1,2Manα1,6 (EtNP P)Manα1,4G GlcN(acyl)PI [38]. In mamm mals, the GPI core structuure can be fuurther modified. EtNP cann be added to o the Man 2 residuue (step 10) by b the action of PIG-G/PIG G-F (GPI-ET TII complex) and after step p8a fourth Man ccan be added by the action n of PIG-Z, aan α1,2-mannnosyltransferaase. The resuulting alternative mature m GPI annchor structuure with fourr mannoses iss then modiffied with EtN NP at Man-3 [38]. Finally, in sttep 11, the multisubunit m G GPI transamiidase attachess a protein to o any of the possibble mature GP PI anchors thhrough an am midic bond bettween the ω amino acid in n the protein C-terrminus and thhe EtNP of thee terminal Man. PIGM-C CDG involvess defects in the additionn of Man-1 to t GlcN-(acyyl)PI, step thhat is catalyzed byy PIG-M, it is i clinically characterizedd by abdomiinal vein throombosis, abssence seizures, bonne marrow faailure and sevverely reducedd levels of GPI-anchored G CD59 and CD24 C in granulocyytes with errythrocytes being b almostt completely spared of CD59 reducction. Diagnosis is made by seqquencing. Muutations have been locatedd in the promoter region of o the PIGM gene [[39].

Figure 4. GPI anchor struccture and assoociated defectts. CDG defeccts are markeed with X and d the G affected. Nuumbers indicaate described steps s in the biosynthesis off GPI indication of the gene-CDG anchors.

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PIGV-CDG involves defects in the PIGV gene that codes for the second mannosyltransferase of the GPI-anchor biosynthesis pathway, and was found in a subgroup of patients with the hyperphosphatasia and mental retardation syndrome (Mabry syndrome) [40]. The patients were dysmorphic (hypertelorism, long palpebral fissures, a broad nasal bridge and tip, downturned corners of the mouth, thin upper lip) with brachytelephalangy, and neurological symptoms including epilepsy and hypotonia. Also increased serum alkaline phosphatases were identified. Diagnosis is established by gene sequencing. ST3GAL5-CDG or Amish infantile epilepsy is caused by mutations in the ST3GAL5 gene that codes for a lactosylceramide α-2,3 sialyltransferase (GM3 synthase) that synthesizes ganglioside GM3. GM3 is synthesized by the transfer of Sia from CMP-Sia to the nonreduced terminal galactose residue of lactosylceramide through the α-2,3 glycosyl bond. This disease is clinically characterized by infantile-onset epilepsy associated with developmental stagnation and blindness in the Old Order Amish of Geauga County, Ohio. Brain MRI at older ages can show diffuse atrophy [41].

Mixed Defects in O- and N-glycosylation It is important to note that some CDGs affect various glycosylation pathways because they are caused by mutations in common proteins (transporters, enzymes, kinases) or proteins involved in maintaining a normal cellular context for the adequate function of the glycosylation machinery, Table 3. B4GALT1-CDG is caused by mutations in the B4GALT1 gene that codes for a β1,4galactosyltransferase involved in galactosylation of terminal N- and O-linked glycans [42]. It is clinically characterized by macrocephaly, hydrocephaly caused by a Dandy-Walker malformation, myopathy characterized by muscular hypotonia and reduced muscle mass, elevated liver enzymes and coagulopathy. Transferrin IEF profile is abnormal but ApoCIII profile is normal because the defective enzyme is not involved in the biosynthesis of ApoCIII O-linked glycans [43 , 44].

The Dolichol and Dol-P-Man Synthesis Pathways Two dolichol synthesis defects (DK1-CDG, SRD5A3-CDG) have been identified. DK1CDG is caused by mutations in the TMEM15 gene that encodes the enzyme responsible for the final step of the de novo biosynthesis of dolichol phosphate which is involved in several glycosylation reactions, such as N-linked glycosylation, GPI-anchor biosynthesis, and C- and O-linked mannosylation. DK1-CDG is characterised by hyperkeratosis, minimal hair growth, seizures, muscular hypotonia, tetraplegia, bilateral nistagmus (nastigmus) and dilated cardiomyopathy. Patients showed loss of oligosaccharide structures on serum transferrin giving a type I profile on mass spectrometry, and although analysis of LLOs showed no structural abnormalities of the N-linked glycans assembled on dolichol, they did show a severely reduced amount of total LLOs that led to analyze the biosynthetic pathway of dolichol phosphate [45].


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SRD5A3-CDG is caused by mutations in the SRD5A3 gene that encodes an enzyme necessary for the conversion of the alpha-isoprene unit of polyprenol to form dolichol, and thus acts as a polyprenol reductase. It is characterized by psychomotor retardation, a cerebello-ophthalmo-cutaneous syndrome with cerebellar atrophy/vermis malformations, eye abnormalities, skin abnormalities, also there can be kyphosis and contracture of large joints [46]. Fibroblast LLOs show no structural abnormalities but a reduced amount of newly synthesized oligosaccharide is identified [47]. Three defects in Dol-P-Man synthesis have been identified involving mutations in genes that code for DPM-1, DPM-2 and DPM-3 that form a GT complex required for transferring Man from GDP-Man to Dol-P to form Dol-P-Man, the substrate donor for mannosyltransferases involved in N-linked glycosylation, O-linked mannosylation and GPIanchor synthesis. DPM1-CDG is clinically characterized by developmental delay, cortical blindness, hypotonia, seizures, acquired microcephaly, telangiectases, and hemangiomas. Patients present abnormal type I IEF transferrin patterns and LLO analysis shows accumulation of Man5-LLO [48]. DPM2-CDG patients usually present muscular dystrophy, severe mental disability, microcephaly, myoclonic epilepsy, and cerebellar hypoplasia [49-50]. DPM3-CDG is clinically characterized by mild muscular dystrophy, dilated cardiomyopathy and stroke like episodes with no associated brain or eye involvement. α-Dystroglycan is hypoglycosylated and IEF of transferrin was abnormal showing a type 1 profile. It has been shown that patient cells have reduction of Dol-P-Man synthase activity that affects mostly O-Man glycan synthesis, leading to muscular dystrophy [51]. MPDU1-CDG is caused by mutations in a chaperone involved in the use of Dol-P-Man and Dol-P-Glc. Patients show psychomotor retardation, severe encephalopathy, scaly erythematous skin disorder. One patient had dwarfism with growth hormone deficiency. IEF of transferrin was abnormal with fibroblasts accumulating Man5GlcNAc2-PP-dolichol as well as Man9GlcNAc2-PP-dolichol. Reduced levels of GPI anchored CD59 were observed in patients fibroblasts [52].

The Sialic Acid Pathway Two sialic acid pathway defects have been identified. SLC35A1-CDG is caused by mutations in the SLC35A1 transporter or Golgi CMP-Sialic acid transporter that transports CMP-Sia from the cytoplasm to the lumen of the Golgi apparatus, where all sialyltransferases use it as donor substrate. Loss of this transporter causes hyposialylation of N- and O-linked glycans and is clinically characterized by neutropenia, infections and thrombocytopenia [53]. IEF analysis is altered in ApoCIII but not in transferrin. GNE-CDG is caused by mutations in the UDP-GlcNAc 2 epimerase/N-acetylmannosamine kinase (GNE) enzyme that converts UDP-GlcNAc to N-acetyl-mannosamine (ManNAc-6-P), a committed precursor of Sia biosynthesis [54]. It is phenotypically characterized as hereditary inclusion body myopathy type 2 (IBM2) which is an adult onset distal myopathy with rimmed vacuoles that is progressive and often leads to severe physical disability. IEF studies of serum transferrin, which contains only N-GlcNAc glycans, and serum ApoCIII, which contains only O-GalNAc glycans, appears normal in all IBM patients tested so far [55]. This suggests normal N-GlcNAc and O-GalNAc glycosylation in

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hepatically-derived serum glycoproteins in individuals with IBM [56]; but hyposialylation of specific N- and O-glycosylated proteins in muscle has been reported [55]. Therapeutic trials of an oral time released form of sialic acid are underway in a set of these patients.

Golgi Trafficking COG1/4/5/6/7/8-CDG. Only recently, defects in genes involved in vesicular trafficking have been shown to cause CDG. Mutations in the genes encoding subunits of the conserved oligomeric Golgi (COG) complex were the first group of CDG defects to be found outside of the glycosylation pathway. The COG complex has been described as a cytosolic protein complex that is peripherally associated to the Golgi, serving as a tethering factor for retrograde vesicular transport [57]. To date, mutations in genes encoding six of the eight COG subunits, namely COG1, COG4, COG5, COG6, COG7 and COG8, have been described as causing CDG. COG7 remains the one with most cases described [58]. The clinical phenotypes are shown in Table 3. Diagnosis is initially made by abnormal IEF of both transferrin and Apo-CIII, with subsequent studies on patient’s fibroblasts showing a deficient intra-Golgi retrograde transport upon treatment with brefeldin-A (BFA). ATPV60A2-CDG is caused by defects in the α2 subunit of the vacuolar H+ ATPase (VATPase), and is clinically characterized by congenital or postnatal microcephaly, large anterior fontanel with delayed closure, dysmorphy, cutis laxa, hypotonia, psychomotor retardation, and joint laxity [59]. Serum transferrin IEF revealed a type 2 pattern, but in some patients this test was normal in the first months, and abnormal on later retesting. Plasma ApoC-III IEF was also abnormal and characterized by an increase in the monosialo-isoform and a decrease of the disialo-isoform [60]. SEC23A-CDG or craniolenticulosutural dysplasia is characterized by late-closing fontanels, sutural cataracts, facial dysmorphism, and skeletal defects caused by mutations in the SEC23A gene [61-62], which codes for an essential component of the COPII-coated vesicles that transport secretory proteins from the ER to the Golgi complex. The fibroblasts of these patients have gross dilatation of the ER due to accumulation of proteins and also cytoplasmic mislocalization of SEC31 an other essential component of the COPII coat [63]. A zebrafish animal model of this disease indicates that glycosylation of matrix proteins is absent in SEC23A mutants, probably because extracellular matrix proteins are retained in the ER and not able to be transported to the Golgi for posttranslational modifications and eventually to the extracellular matrix [64]. SEC23B-CDG is caused by defects in the SEC23B gene that codes for a COPII component a type of vesicle coat protein that transports proteins from the rER to the Golgi apparatus. It is clinically characterized as a congenital dyserythropoietic anemia type II and disease is limited to the erythroblastic lineage that shows truncation of N-linked glycans on band 3 of erythrocyte membranes [65].


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Current Treatments for CDGs MPI-CDG: This is the only routinely treatable CDG. The defect in MPI (Fructose-6P