Eur J Pediatr (2003) 162: 359–379

DOI 10.1007/s00431-002-1136-0

R EV IE W

T. Marquardt Æ J. Denecke

Congenital disorders of glycosylation: review of their molecular bases,

clinical presentations and specific therapies

Received: 14 March 2002 / Revised: 6 November 2002 / Accepted: 7 November 2002 / Published online: 15 March 2003

Springer-Verlag 2003

Abstract Congenital disorders of glycosylation (CDG,

formerly named carbohydrate-deficient glycoprotein
Introduction
syndromes) are a rapidly growing family of inherited
Congenital disorders of glycosylation (CDG) are inher-
disorders affecting the assembly or processing of glycans
ited metabolic diseases caused by defects in the biosyn-
on glycoconjugates. The clinical spectrum of the differ-
thesis of glycoconjugates. These disorders, formerly
ent types of CDG discovered so far is variable, ranging
known as carbohydrate-deficient glycoprotein syn-
from severe multisystemic disorders to disorders
dromes (CDG-syndromes, CDGS), were recently
restricted to specific organs. This review deals with
renamed due to limitations of the old nomenclature
clinical, diagnostic, and biochemical aspects of all
[13, 14].
characterized CDGs, including a disorder affecting the
In CDG, hypoglycosylation of different glycoproteins
N-glycosylation of erythrocytes, congenital dyserythro-
and occasionally of other glycoconjugates leads to a
poietic anemia type II (CDA II/HEMPAS), and the first
variety of symptoms affecting multiple systems. In most
disorders affecting O-glycosylation. Since the clinical
cases, statomotor and mental retardation is present.
spectrum of symptoms in CDG is variable and may be
Although about 1% of the human genome codes for
unspecific, a generous selective screening for the pres-
genes involved in glycosylation processes [37], it was not
ence of CDG is recommended.
until 1995 that a disorder in the biosynthesis of N-gly-
cans was first characterized [137]. This review focuses on
Keywords CDG Æ LAD II Æ CDA II Æ HEMPAS Æ
the clinical presentation of the different CDG types,
Glycosylation
their biochemical and molecular basis, and the specific
treatment options known to date. Online information
Abbreviations aa Amino acid Æ CDA Congenital
can be found at our homepage (http://cdg.uni-
dyserythropoietic anemia Æ CDG Congential disorder of
muenster.de/) and a mutation database at http://
glycosylation Æ HEMPAS Hereditary erythroblastic
www.med.kuleuven.ac.be/cdg/.
multinuclearity with positive acidified serum test Æ LLO
Lipid-linked oligosaccharides Æ MRI Magnetic
resonance imaging Æ OST Oligosaccharyltransferase Æ N-glycosylation of proteins
PDO Protein-derived oligosaccharides Æ RER Rough
endoplasmic reticulum Æ Sia Sialic acid Most types of CDG that have been discovered so far
were identified by demonstrating defects in the N-gly-
cosylation pathway of glycoproteins. Although O-gly-
cosylation or the glycosylation of glycolipids is affected
in some of them, the main alterations concern the N-
glycosylation pathway. Understanding the processes
Further information on congenital disorders of glycosylation
(CDG) can be found at: http://cdg.uni-muenster.de/
involved is therefore essential to an understanding of the
biochemical basis of known CDG as well as of types still
T. Marquardt (&) Æ J. Denecke to be discovered.
Klinik und Poliklinik für Kinderheilkunde, Protein translation occurs on ribosomes either in the
Albert-Schweitzer-Str. 33, 48149 cytoplasm or on the membrane of the rough endoplas-
Münster, Germany
E-mail: marquat@uni-muenster.de mic reticulum (RER). Whereas proteins synthesized in
Tel.: +49-251-8358519 the cytoplasm generally remain unglycosylated, the
Fax: +49-251-8356085 majority of proteins that are secreted or destined to be
360
361
362
b tensive research in the past. One of the most important
Fig. 1 Synthesis of dolichol, assembly and processing of N-linked
glycans and molecular defects of known CDG types. Dolichol is a
polyisoprenyl containing 18–21 isoprene units. It serves as an
precursor monosaccharide donor and carrier of the assembled
oligosaccharide in the ER membrane prior to its transfer to the
nascent protein. Enzymes catalyze the conversion of isopentenyl-
pyrophosphate and farnesylpyrophosphate to polyprenol and
finally dolichol phosphate (upper part). Assembly of N-glycans is
initiated on the cytoplasmic side of the ER membrane by
transferring two GlcNAc residues (blue squares) to Dol-P using
UDP-GlcNAc as GlcNAc donor. This process is catalyzed by
GlcNAc 1-P transferase and chitobiose synthase. The product
GlcNAc2-P-P-Dol, is extended by different mannosyltransferases
using GDP-mannose (mannose: red dots) as the donor substrate.
After assembly of a biantennary Man5GlcNAc2 structure on the
cytoplasmic side of the ER-membrane, the oligosaccharide is
translocated to the lumenal side of the organelle (light grey),
catalyzed by a flipping enzyme. For the subsequent enzymatic
steps, mannose and glucose attached to Dol-P must be translocated
into the lumen of the ER by a Dol-P-sugar flipping mechanism.
ER-localized mannosyl- and glucosyltransferases elongate the
branches to a Glc3Man9GlcNAc2 oligosaccharide (glucose: yellow
triangle). This oligosaccharide is transferred by the multi-subunit
enzyme OST to glycosylation consensus sites of the nascent
protein. After removal of the glucose residues and one mannose
from the a1,6 branch, the glycoprotein is transferred to the Golgi
by vesicular transport. In the Golgi, mannosidase I cleaves
mannose, enabling the transfer of GlcNAc onto the a1,3 arm by
the GlcNAc-transferase I. Mannosidase II cleaves 2 mannose
residues from the a1,6 arm, followed by the transfer of GlcNAc by
GlcNAc-transferase II. Following this step, the structure of
N-glycans is characterized by a broad heterogeneity. Fucosyltrans-
ferase 8 may transfer fucose (grey triangle) in a1,6 linkage to the
core GlcNAc and multiple galactosyltransferases, GlcNAc-trans-
ferases, sialyltransferases and fucosyltransferases spread over cis,
median, and trans-Golgi catalyze the formation of complex
oligosaccharides. Green rhomb, galactose; pink rhomb, sialic acid.
The mouse symbol designates the availability of a knock-out mouse,
in red are cell lines generated from yeast or from CHO cells and
expressing specific enzymatic defects
membrane proteins are glycosylated. The latter are
synthesized on ribosomes attached to the outer mem-
brane of the RER. Upon the start of translation, the
N-terminal part of these proteins is translocated across
the membrane. At the lumenal side of the membrane, a
multimeric enzymatic complex, oligosaccharyl transfer-
ase (OST), recognizes the motif Asn-X-Ser/Thr (gly-
cosylation consensus site; X cannot be proline) and
attaches a preassembled oligosaccharide to the aspara-
gine. There is only a short time frame for the OST to
transfer the oligosaccharide. The translocation speed is
not constant, and pauses during translocation might
facilitate the glycosylation of these sites.
The transferred oligosaccharide is a branched struc-
ture of 14 monosaccharides consisting of two N-acetyl-
glucosamines (GlcNAc), nine mannoses (Man) and three
glucose residues (Glc). Biosynthesis of this structure
occurs at the membrane of the rough endoplasmic re-
ticulum (RER) in a step-by-step process on a lipid an-
chor, dolichol (Fig. 1). Dozens of enzymes and
transporters are required for the synthesis of this lipid-
linked oligosaccharide (LLO). The assembly steps of the
oligosaccharide on dolichol have been the focus of in-
summaries is the review by Kornfeld & Kornfeld [85] but
other more recent reviews give an extensive overview as
well [59]. Assembly and processing reactions are sum-
marized in Fig. 1.
Biosynthesis of the oligosaccharide starts at the cy-
toplasmic site of the RER membrane. Dolichol phos-
phate (Dol-P) is the acceptor for the first assembly
reaction catalyzed by a GlcNAc 1-P transferase that uses
UDP-GlcNAc as GlcNAc donor. The product, Glc-
NAc-P-P-Dol, is then extended by chitobiose synthase
that adds a second GlcNAc in b1,4-linkage, again using
UDP-GlcNAc as donor. This structure is further
elongated by different mannosyltransferases that use
GDP-mannose as donor substrate. The first mannose is
attached in b1,4-linkage to the GlcNAc and sequentially
the oligosaccharide branches into an a1,3 mannose and
an a1,6 mannose branch. The a1,3 branch is extended by
two more mannoses in order to build up a biantennary
Man5GlcNAc2 structure. At this stage, the oligosac-
charide is translocated from the cytoplasmic side of the
RER membrane to the lumen where further elongation
takes place. A flipping enzyme catalyzing this reaction
has recently been identified in yeast [58]. The protein,
Rft1, catalyzes the flipping of Man5GlcNAc2-PP-Dol
but not of Dol-P-Man [58]. Further elongation steps use
Dol-P-Man and Dol-P-Glc as substrates since the
membrane of the RER is impermeable to UDP-GlcNAc
and GDP-mannose. Dolicholphosphate sugars are syn-
thesized on the cytoplasmic side and have to flip to the
lumen, a step that is probably enzyme-catalyzed. The
gene product of the human MPDU1 gene was recently
established as being essential for the flipping of Dol-
P-Man and Dol-P-Glc from the cytoplasmic to the
lumenal side of the ER, although MPDU1 seems not to
be the flippase itself [140]. ER-localized mannosyl- and
glucosyltransferases then elongate the oligosaccharide to
its final structure. The a1,6-branch is further elongated
with mannoses generating another a1,3/ a1,6-branching,
whereas the a1,3-branch is elongated with two glucose
residues in a1,3-linkage and a terminal glucose in
a1,2-linkage.
This preassembled oligosaccharide, Glc3Man9Glc-
NAc2, is subsequently transferred by oligosaccharyl-
transferase (OST) to glycosylation consensus sites of the
nascent chain. 9 subunits of OST have been found in
yeast and four corresponding subunits have been iden-
tified in humans. OST has a high substrate affinity with
the fully assembled oligosaccharide whereas shorter
species are transferred inefficiently. Immediately after
the transfer, the oligosaccharide is trimmed by the
membrane-bound glucosidase I that removes the termi-
nal a1,2-linked glucose, followed by removal of the two
a1,3-linked glucose residues by soluble a-glucosidase II.
High-mannose oligosaccharides with a single glucose
residue interact with ER chaperones calnexin and cal-
reticulin, an interaction that is essential for the so-called
quality control of newly folded glycoproteins in the
RER [110]. After the removal of the first a1,2 mannose

from the branched a1,6 arm by one of two ER-based
a-mannosidases, the glycoproteins travel to the Golgi by
vesicular transport.
In the Golgi, heterogeneity of carbohydrate struc-
tures is generated by a variety of enzymes. Mannosidases
I generate a Man5 structure followed by elongation of
the a1,3 arm with GlcNAc in b1,2-linkage by GlcNAc-
transferase I (GnT I). Mannosidase II cleaves the two
mannose residues from the a1,6 arm, and GlcNAc-
transferase II (GnT II) adds GlcNAc in b1,2-linkage to
the other branch. Galactosyltransferase and sialyltrans-
ferase then elongate both branches to the final carbo-
hydrate structure (Sia2Gal2GlcNAc2Man3GlcNAc2).
Fucosyltransferase 8 (FuT8) catalyzes the a1,6 core fu-
cosylation present at some glycoproteins [105]. a1,6-FuT
activity requires an unsubstituted GlcNAc residue of the
a1,3-antennae of N-glycans [125] and the asialo-, aga-
lacto-triantennary structure is the most suitable sub-
strate [77, 105]. Besides biantennary oligosaccharides of
the structure described above, a large variety of different
glycans is generated in the Golgi apparatus.
Defects in the early biosynthesis pathway lead to a
uniform hypoglycosylation phenotype of glycoproteins.
This is caused by a distinct substrate specificity of OST:
whereas Glc3Man9GlcNAc2 is efficiently transferred
by OST, most of the intermediates are not. For this
reason, many different defects leading to a truncation
of the LLO will result in a lack of the complete oli-
gosaccharide on the glycoprotein at some glycosylation
consensus sites. There are conflicting data on whether
the non-occupation of certain consensus sites is random
[146] or not (Winchester et al., Glycobiology meeting,
Boston 2000). Mutations in one of the OST subunits
have been shown to have the same effect [123, 147], but
no corresponding human disorders have been found so
far.
O-glycosylation of proteins
O-glycosylation is a widespread type of glycosylation in
eukaryotic and in part also in prokaryotic cells. Whereas
N-glycosylation starts cotranslationally in the ER and
further processing takes place in the Golgi, O-glycosy-
lation occurs posttranslationally and is generally initi-
ated in the cis-Golgi [118]. Hydroxyl groups of the
amino acids serine (Ser), threonine (Thr), and sometimes
proline are the acceptor sites of the protein. No con-
sensus sequences of amino acids determining O-gly-
cosylation sites could be elucidated, although there is
evidence that some structures are glycosylated prefer-
entially [135].
The first step in the most common type of O-gly-
cosylation, mucin-type O-glycosylation, is the transfer of
N-acetylgalactosamine (GalNAc) to Ser/Thr of the
protein in the Golgi, forming the so-called Tn-antigen.
Addition of Gal, GalNAc or GlcNAc onto this first
GalNAc builds up a panel of eight different core struc-
tures known so far in mucin-type O-glycans (for review
363
see [51]). Further elongation and modification by
polylactosamine extension, sialylation, fucosylation,
sulfatation and acetylation produce a broad variety of
glycans. Major antigens such as AB0 blood groups and
Lewis antigens are often present on N- and O-glycans
[10].
Many glycosyltransferases, especially for the synthe-
sis of the glycan core structures, are specific to O-gly-
cosylation, but there are overlapping enzyme patterns
for N- and O-glycosylation as well [10, 33, 51, 135].
Besides these prevailing types of O-glycosylation,
there are some deviating pathways for O-glycans: 1)
addition of b-O-GlcNAc to Ser/Thr of various nuclear
and cytoplasmatic proteins by a specific and highly
conserved GlcNAc-transferase, often regulating the ac-
tivity of transcription factors by competition with
phosphorylation [22, 55, 89], 2) direct glucosylation,
fucosylation and galactosylation of specific proteins [5,
53], 3) O-mannosylation [33].
There are four glycosylation disorders concerning
O-glycosylation known so far. The progeria variant of
Ehlers Danlos syndrome, the hereditary multiple exos-
toses disease, the muscle eye brain, and the Walker
Warburg disease. The latter two impair the assembly of
O-mannosyl glycans [148]. O-mannosylation is a fre-
quent and essential type of glycosylation in yeast but
only a few proteins of mammals, mainly of brain and
muscle, carry O-mannosyl glycans [33, 47].
The pathway of O-mannosylation in yeast is well
known. The first and second mannose residues are at-
tached to the proteins in the ER using Dol-P-Man as
substrate donor. Further elongation is performed in the
Golgi resulting in linear chains of 1–7 mannose residues
[33]. In mammals, the synthesis pathway of O-man-
nosylation is not fully understood, but there is evidence
that the first step of the O-mannosylation in mammals,
catalyzed by the protein-O-mannosyl transferase
(POMT1), is located in the ER, whereas elongation of
this glycan is located in the Golgi. In contrast to yeast O-
mannosyl glycans, O-glycans of mammals may carry
other sugars besides mannose.
CDG types
Eleven different human disorders in the N-glycosylation
pathway and three in O-glycosylation are known today.
These disorders have different clinical phenotypes and it
is important to realize that some of these diseases have
little more in common than their names and biochemical
bases. Whereas the former nomenclature was based
mainly on the isoelectric focusing pattern of serum
transferrin, the new nomenclature is based on the in-
tracellular localization of the molecular defect. All bio-
synthesis defects up to and including OST are named
type I, whereas defects in the processing of the oligo-
saccharide or O-glycosylation defects are named type II.
The different enzymatic defects are characterized by
small letters [13, 14].
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Table 1 CDG types characterized to date

New name Old name Affected protein Affected gene Reference

CDG-I
CDG-Ia CDGS Ia Phosphomannomutase 2 PMM 2 [137]
CDG-Ib CDGS Ib Phosphomannose Isomerase PMI [106]
CDG-Ic CDGS Ic/V Dol-P-Glc: hALG6 [63, 82]
Man9GlcNAc2-Glucosyltransferase
CDG-Id CDGS IV Dol-P-Man: hALG3/Not56L [83]
Man5GlcNAc2-Mannosyltransferase
CDG-Ie CDGS IV Dol-P-Man Synthase 1 DPM 1 [65, 79]
CDG-If – MPDU1/Lec35 [86, 121]
CDG-Ig – Dol-P-Man: hALG12 [134]
Man7GlcNAc2PP-Dol mannosyltransferase
CDG-II
CDG-IIa CDGS II GlcNAc-Transferase 2 (GnT II) MGAT2 [74, 133]
CDG-IIb – Glucosidase I HSAGLUCIE [30]
CDG-IIc LAD II GDP-Fucose Transporter [92, 93]
CDG-IId – b1,4 galactosyltransferase [52]

The previously described CDG syndrome type III
[127] now belongs to the CDG-x group since no
underlying defect is known. Since hardly any protein is
hypoglycosylated in this disorder, it is doubtful whether
it is a primary glycosylation disorder at all. More than
20% of the CDG patients identified still cannot be as-
cribed to one of the known enzyme defects and are
named CDG-x. CDG-Ia to CDG-Ig and CDG II-a to
CDG-IId are currently characterized (Table 1).
CDG-Ia
CDG-Ia (McKusick 212065) was first observed in ho-
mozygous twin sisters and published in an abstract by
Jaeken and coworkers in 1980 [71]. It is the most frequent
and best known CDG with more than 500 patients
worldwide and is caused by a deficiency of phospho-
mannomutase, a cytosolic enzyme that converts mannose
6-P to mannose 1-P [137] (Fig. 1). As a consequence, the
GDP-Man pool in CDG-Ia fibroblasts is reduced to 10%
of normal [119]. Whereas normal fibroblasts mainly
synthesize Glc3Man9GlcNAc2 oligosaccharides on their
dolichol, CDG-Ia fibroblasts predominantly make
truncated forms, mainly Man5GlcNAc2 [109].
Many different mutations have been found in the
corresponding gene, PMM2, located on chromosome
16p13 [99]. The most frequent one, R141H, accounts for
40% of the disease alleles of CDG-Ia patients [122]. It
has a high carrier frequency of 1/70 in the general
population, but has never been found in a homozygous
form, suggesting that this combination may be lethal [81,
100, 122]. The second most frequent mutation, F119L,
has a much lower carrier frequency, estimated at 1/1000
[122]. PMM with the R141H mutation has virtually no
activity (0.4% of normal) [81, 114]. Whereas wild-type
PMM has a normal activity at 40C, many mutant forms
have low thermal stability [81, 114]. V231M has normal
activity at 30C, but none at 40C [114]. Although these
data are from in vitro experiments, fever might endanger
CDG-Ia children by decreasing the low PMM activity in
their cells even further. PMM2 is widely distributed in
tissues, with the highest activity in intestinal mucosa
[113]. In contrast, a second PMM, PMM1, was found in
rats to be expressed only in lung and brain [113]. Sur-
prisingly, PMM1 accounts for 66% of the total PMM
activity in rat brain [113]. Under the assumption that
this is similar in humans, it is still unclear why the loss of
PMM2 activity in the human brain is not compensated
for by PMM1.
Inverted nipples and abnormal subcutaneous fat pads
are characteristics of CDG-Ia and are already present at
birth [21] (Fig. 2b,c). Fat pads disappear with increasing
age [88]. Axial hypotonia, psychomotor retardation and
internal strabismus (Fig. 2 a) are common symptoms.
Ataxia on the basis of marked cerebellar atrophy is
usually present and some children develop epilepsy [70,
88]. Tendon reflexes have commonly disappeared by
1 year of age [70, 88] and nerve conduction velocity es-
pecially at the lower limbs is already decreased by the
age of 6–8 months [69, 88]. Joint contractures and
skeletal deformities may occur. Most children do not
learn to walk without support. Feeding problems and
failure to thrive often make tube feeding necessary [112].
Impaired night vision and retinitis pigmentosa have been
reported [130]. Stroke-like episodes, usually with com-
plete recovery can occur mainly during feverish infec-
tions [69, 88]. Sometimes hypoventilation makes
artificial ventilation necessary early on [21]. Hypertro-
phic obstructive cardiomyopathy (HOCM) can develop
very rapidly after birth and cardial effusion may be de-
tectable [21]. Nonimmune hydrops fetalis is a possible
presentation of CDG-Ia in newborns [27]. Ultrasound
c
Fig. 2a–d Phenotype of different CDG types. CDG-Ia: internal
strabism (a), inverted nipples (b) and abnormal fat pads (c) as
characteristics of infants suffering from CDG-Ia. CDG-IIc: short
limbs and a flat face with a broad and depressed nasal bridge are
features of patients suffering from CDG-IIc (d). CDG-If: ichthyosis
and erythema of the skin in a CDG-If patient (e)
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investigations show hyperechogenic kidneys due to
multiple microcysts; sometimes nephrotic syndrome is
present early on, while renal tubular function is usually
normal [129]. In the first years of life, mortality is
around 20% [70], but the clinical condition stabilizes
with increasing age and life-threatening events rarely
occur after the first decade. Hypoglycosylation of co-
agulation factors is frequent but only rarely results in
thrombosis or hemorrhage.
As in other CDGs, serum transaminases are often
elevated [107]. Absolute concentrations of transferrin
and a1-antitrypsin in the serum are normal [107].
Hypoproteinemia, low cholesterol levels and low chol-
inesterase levels are common findings [107]. Anti-
thrombin III, factor XI and protein C show reduced
activities [136].
Hypoglycosylation of serum transferrin in CDG-Ia
was first discovered in 1984 [72]. Based on this observa-
tion, the isoelectric focusing (IEF) of serum transferrin
became the commonly used diagnostic test for most
CDG types (Fig. 3). The IEF of serum transferrin of
CDG patients shows a cathodal shift towards a higher
isoelectric point caused by the absence of the terminal
sialic acid residues [126]. Not only transferrin but many
different serum proteins visualize hypoglycosylation on
one-dimensional IEF [60, 61, 107], SDS-PAGE gels
Fig. 3 Isoelectric focusing (IEF) of serum transferrin. The IEF of
serum transferrin is the commonly used screening test for CDG. In
controls, the major band is tetrasialotransferrin. In CDG-I patients
(shown here: CDG-Ia), additional bands are seen, migrating at the
position of disialo- and asialotransferrin. As seen in the diagrams
of possible structures on the right, the IEF does not reveal the
structural change of the attached oligosaccharide. Since only the
terminal sialic acid residues are charged, abnormal migration can
be due to different structural alterations. In addition, an amino acid
change in the protein core of the transferrin molecule can mimic
CDG. Blue square, GlcNAc; red circle, mannose; green rhomb,
galactose; pink rhomb, sialic acid; grey bar, protein core
[145], or two dimensional gels [54]. Haptoglobin is nor-
mally absent [54]. It has been clearly demonstrated that
the carbohydrates released from the transferrin of CDG-
Ia patients are not truncated and that either one or both
oligosaccharides are missing in about 50% of the trans-
ferrin population [138, 139, 145]. Non-glycosylation has
been reported to occur randomly at either of the two
glycosylation sites [146], but other authors have found a
preference for one of the sites (Winchester et al., Gly-
cobiology Meeting, Boston 2000). The IEF pattern of
different glycoproteins is normal during intrauterine life
and directly after birth; hypoglycosylation becomes ap-
parent in the 2nd–3rd postnatal week [20].
It is still unclear whether dietary supplementation
with mannose has any beneficial effects in CDG-Ia
children. In CDG-Ia fibroblasts, mannose can correct
the size of the truncated lipid-linked oligosaccharides
(LLO) [84, 109]. Addition of 1 mM mannose to the
culture medium has no effect on the GDP-Man pool in
normal fibroblasts, but nearly normalizes the reduced
pool in CDG-Ia fibroblasts [109, 119]. The mechanism
of this correction is unknown. Based on these experi-
ments, mannose therapy has been tried in CDG-Ia pa-
tients [80, 96, 102, 103]. Mannose given orally in a dose
of 0.2 g/kg b.w. leads to peak mannose concentrations
of more than 200 lmol/l after 1 h in healthy subjects,
with a clearing half-time of 4 h [3]. Mannose levels in
CDG-Ia children after oral incorporation are often
lower (T.Marquardt, manuscript in preparation). No
clear effect of mannose therapy in CDG-Ia has been
reported so far. A 3-week continuous intravenous
mannose infusion with serum mannose levels up to
2 mmol/l followed by 6 months of oral mannose treat-
ment failed to correct the hypoglycosylation of different
glycoproteins [102, 103]. However, no study has yet in-
vestigated the glycosylation status of non-liver-derived
proteins during therapy.

CDG-Ib
CDG-Ib (McKusick 602579) and its therapy were first
described in 1998 by our study group [106] and inde-
pendently by two other groups in the same year [26, 75].
Our group also proposed a therapeutic approach with
mannose [106]. The disorder is caused by a deficiency of
phosphomannose isomerase that affects the endogenous
production of mannose 6-P (Fig. 1). Several summaries
on the first patients with CDG-Ib have been published
[28, 38] and approximately 20 patients are known to date.
In contrast to CDG-Ia, mental and motor develop-
ment is normal [26, 106]. The predominant symptom of
CDG-Ib is chronic diarrhea, commonly starting during
the first year of life [75]. Cyclic vomiting can be a leading
symptom [6, 26]. Failure to thrive and protein-losing
enteropathy can occur [106]. Partial villus atrophy can be
present in duodenal biopsies and might lead to suspect
coeliac disease [75, 106]. Hypoglycemia occurs frequently
[6, 75] and inadequately elevated insulin levels have been
found to be causative in some patients but not in others
[6, 29]. Some patients developed hepatic fibrosis [6, 28,
75]. Hypoalbuminemia, elevated aminotransferases and
low AT III activity are common findings in CDG-Ib
patients [6]. Thrombotic episodes and severe bleeding
complications may complicate the course [106]. Several
children died [75] or were in life-threatening conditions.
CDG-Ib was the first disorder of glycosylation where
a specific therapy was available. Symptoms can be ef-
fectively treated with the oral uptake of mannose [106].
Mannose supplementation increases the depleted man-
nose 6-P pool, thereby normalizing the hypoglycosyla-
tion of proteins. Mannose normalizes hypoproteinemia
and blood coagulation and effectively treats the symp-
toms of CDG-Ib like protein-losing enteropathy and
hypoglycemia. An oral dose of 1 g mannose per kg body
weight per day divided into five doses is used. Higher
doses can induce osmotic diarrhea. Whereas the clinical
symptoms disappear rapidly, significant improvement of
the transferrin IEF pattern during mannose therapy
takes several months of treatment to occur [29, 106].
Considering the short biological half-life of transferrin
of approximately 1 week, it is unclear why it takes so
long for the IEF pattern to normalize.
CDG-Ic
CDG-Ic (McKusick 603147) is caused by a defect in the
a1,3-glucosyltransferase and is characterized by accu-
mulation of Man9GlcNAc2-P-P-Dol [12, 63]. The cor-
responding gene has 14 exons [64], codes for a 507 aa
membrane protein, and was named hALG6, derived
from the name of its ortholog in Saccharomyces cerevi-
siae. A333 V is a common mutation [50] but other mu-
tations have also been described [141]. The disorder was
formerly also called type V [82].
Psychomotor retardation is the main clinical symp-
tom of the more than 30 CDG-Ic patients known so far.
367
The clinical presentation is milder than in CDG-Ia.
Muscular hypotonia is a common symptom and nearly
all patients have recurrent seizures [50]. In contrast to
CDG-Ia, there are no characteristic physical stigmata.
Brainstem auditory-evoked potentials, tendon reflexes
and nerve conduction velocities are normal and there are
no obvious cerebral abnormalities in MRI [50]. In early
childhood, internal strabism (as observed in CDG-Ia)
can occur. At 2–3 years of age, the children are able to
sit and they usually start walking at 3–4 years of age
[50]. Speech development is delayed.
The transferrin IEF pattern looks similar to the one
found in CDG-Ia. Activities of AT III and factor XI are
considerably reduced. The hypoglycosylation of b-CSF
trace protein, a CSF glycoprotein with two biantennary
N-linked sugars and a molecular weight of 27 kDa, is
less severe than in CDG-Ia patients [49].
CDG-Id
CDG-Id (McKusick 601110) is caused by a deficiency of
the first Dol-P-Man-dependent mannosyltransferase
that adds mannose to Man5GlcNAc2-P-P-Dol in the
endoplasmic reticulum [83]. It was formerly termed
CDGS IV [83, 128]. Truncated LLOs of the structure
Man5GlcNAc2 accumulate. In the three known patients
with this disorder, an ortholog gene to the Sacchar-
omyces cerevisiae hALG3 gene, the human Not (neighbor
of tid) 56-like protein gene, had a homozygous missense
mutation that reduced the enzymatic activity.
Only one patient has been described in the literature,
another one can be found in the Leuven mutation
database, and the third one will be described shortly
(Denecke and Marquardt, manuscript in preparation).
The described patient was suffering from tetraspastic
paresis, microcephaly, severe and intractable seizures,
minimal psychomotor development, and atrophy of the
optic nerve. Dysplastic ears, abnormalities of the uvula,
a high-arched palate and coloboma of the iris were
described. Hypoplasia of the cerebellum similar to
CDG-Ia was present [128]. Transferrin IEF showed an
increase in disialotransferrin whereas only minimal
amounts of asialotransferin were detected.
CDG-Ie
CDG-Ie (McKusick 603503) is caused by a defect in the
dolichol-phosphate-mannose synthase 1 gene (DPM 1),
that codes for the cytosolic subunit of the Dol-P-Man
synthase multimer (Dol-P-Man synthase) [65, 79]. It was
formerly also termed CDGS IV, a group that was found
to consist of two subtypes based on different molecular
defects [79, 128]. Four patients with this disorder have
been described so far [65, 79]. In all patients there is
some residual enzymatic activity: approximately 5%.
Considering the severity of the clinical phenotype of
these patients, mutations causing a complete loss of
368
enzymatic activity might very well be lethal. The affected
gene codes for the catalytic subunit of Dol-P-Man syn-
thase, has nine exons, and is located on chromosome
20q13. No mutations have so far been identified in the
gene DPM2, which encodes for the second membrane-
anchored subunit of the enzyme, or in DPM3 another
subunit of Dol-P-Man synthase. LLO analysis is char-
acterized by an accumulation of Man5GlcNAc2-P-P-Dol
[65, 79]. It has to be proven if CDG-Ie can be caused by
heterogenous molecular defects.
Severe psychomotor retardation combined with
muscular hypotonia and seizures starting in the 1st year
of life are predominant symptoms in children suffering
from CDG-Ie [65, 79]. An absence of visual fixation was
observed in several patients and cortical blindness was
diagnosed in some of them [65, 79]. Social contact failed
to develop. Gothic palate, hypertelorism, dysplastic nails
and knee contractures are features of the disorder [65].
Body weight, length and head circumference might be
normal at birth, but later on microcephaly is typical of
CDG-Ie. Magnetic resonance imaging reveals cerebral
atrophy. Transferrin IEF shows a significant amount of
disialotransferrin, but hardly any asialotransferrin [79].
b-CSF trace protein is hypoglycosylated in CDG-Ie
patients.
There are conflicting results concerning the effect of
mannose supplementation in CDG-Ie fibroblasts:
whereas a correction of LLO size was described by one
group [79], no effect was recorded by others [65].
CDG-If
CDG-If is based on alterations in an RER-located
transmembrane protein called MPDU1/Lec35
(McKusick 604041) [86, 121]. MPDU1 is the abbrevia-
tion for mannose-P-dolichol utilization defect 1 and
Lec35 CHO cells have the same molecular defect. The
product of MPDU1 is a 27 kDa protein with two putative
membrane spanning regions and a cytosolic C-terminal
ER retention signal (KKXX). The N-terminal half of the
protein is predicted to have a cytosolic orientation [141].
The function of this protein is unknown, but it seems to
play a crucial role in the translocation of mannose and
glucose into the lumen of the RER. Man5GlcNAc2 as
well as Man9GlcNAc2 accumulate on dolichol.
The four patients identified so far have shown psy-
chomotor retardation, muscular hypotonia, seizures,
absence of speech development, short stature, failure to
thrive, feeding problems, impaired vision and retinitis
pigmentosa [86, 121]. Two of them have shown ichthy-
osis of the skin (Fig. 2e).
CDG-Ig
The first patient suffering from CDG-Ig was described
by Thiel et al. [134], closely followed by descriptions by
other groups [15, 48]. The first patient, a girl of Indian
origin, suffered from delayed psychomotor development,
convulsions starting at the age of 14 months, micro-
cephaly, muscular hypotonia, supragluteal fat pats,
dysplastic ears and a short filtrum. The partial throm-
boplastin time was prolonged. Transferrin IEF showed a
pattern similar to the one seen in CDG-Ia but with less
asialotransferrin. Magnetic resonance imaging of the
brain showed enlarged lateral ventricles.
The prevailing oligosaccharide structure on the doli-
chol of the patient was Man7GlcNAc2 whereas the ma-
ture GlcNAc2Man9Glc3 was reduced to about 5% of
total dolichol-linked oligosaccharides, thus indicating a
defect in the gene of the Dol-P-Man:Man7GlcNAc2PP-
Dol mannosyltransferase. This enzyme transfers a
mannosyl residue to Dol-PP-GlcNAc2Man7 using Dol-
P-Man as substrate [11]. The activity of the patient’s
enzyme was severely reduced and the coding gene, the
human ortholog of the yeast ALG12 gene, showed two
alleles with different point mutations, one resulting in a
stop codon with a loss of the C-terminal 74 amino acids
and one causing a substitution of leucine with proline.
CDG-IIa
CDG-IIa (McKusick 212066) is caused by mutations in
the MGAT2 gene on chromosome 14q21 that codes for
N-acetylglucosaminyltransferase II (GnT II) localized in
the Golgi membrane [133]. The entire coding region is
on one single exon.
Compared to CDG-Ia children, more profound psy-
chomotor retardation but no peripheral neuropathy and
normal deep-tendon reflexes are present in the four
CDG-IIa patients known so far. A dysmorphic coarse
face, large low-set ears and widely spaced nipples have
been described [117]. CDG-IIa patients show generalized
hypotonia, limb weakness and stereotypical behavior.
Epilepsy developed in one patient. MRI of the brain
revealed cortical atrophy, focal white matter lesions and
delayed myelinization but no cerebellar atrophy. De-
creased AT III, factor IX and XII activities are present
[73]. Transferrin IEF shows a characteristic pattern with
disialotransferrin as the most prominent band [73, 117].
CDG-IIb
CDG-IIb (McKusick 606056) is caused by a deficiency
of glucosidase I, the first enzyme to act on the oligo-
saccharide after its transfer to the nascent chain. The
activity in cultured skin fibroblasts was reduced to less
than 3% of control values. The enzyme has a molecular
mass of 92 kDa and its gene is localized on chromosome
2p12–13.
Only one patient with CDG-IIb, a girl of consan-
guineous parents, has been identified so far [30]. Dys-
morphic features such as broad nose, retrognathism,
high-arched palate and overlapping fingers were de-
scribed. Muscular hypotonia was present and motor

nerve conduction velocity was reduced. Hypoventilation
and apnea made artificial ventilation necessary. Seizures
developed at 3 weeks of age. Increasing hepatomegaly
developed and histological findings of liver tissue revealed
proliferation and dilatation of the bile ducts, fibrosis, fat
accumulation, iron storage and multilamellar inclusion
bodies in hepatocytes. Following a rapid decline and a
stuporous state, the girl died at 2.5 months of age.
A prominent abnormal tetrasaccharide band (Glc-
Glc-Glc-Man) found upon thin-layer chromatography
of the urine was the pathognomonic finding in the CDG-
IIb patient. It is important to note that the IEF patterns
of serum transferrin and b-CSF trace protein were
normal in the patient. Thus, the standard screening test
for CDG fails to detect CDG-IIb patients. Most likely, a
cleavage of the unprocessed oligosaccharides by an
endo-a1,2-mannosidase in the Golgi [59] releases the
tetrasaccharide found in the urine, allowing further
processing of the carbohydrate.
Interestingly, no hypoglycosylation of serum glyco-
proteins occurs in CDG-IIb. It is intriguing to speculate
that the absence of monoglucosylated oligosaccharide
intermediates in CDG-IIb might disable ER quality
control, which is normally responsible for the retention
of misfolded glycoproteins [110].
CDG-IIc
CDG-IIc (McKusick 266265) was discovered by Etzioni
in 1992 and named leukocyte adhesion deficiency type II
(LAD II) [35]. Fucosylated glycoconjugates are severely
diminished in this disorder, due to a defect of GDP-
fucose import into the Golgi [93]. The absence of fu-
cosylated selectin ligands on the surface of neutrophil
granulocytes causes the leukocyte adhesion deficiency
[35, 97]. Addition of fucose to the culture medium re-
stores the expression of fucosylated ligands in LAD II
cells [78, 98].
In two of the patients, a defect of the GDP-fucose
transporter in the membrane of the Golgi apparatus was
found [93, 131]. Both children have mutations in puta-
tive transmembrane regions of the transporter [93].
Short limbs and stature, a flat face with a broad and
depressed nasal bridge, long eyelashes and broad palms
are dysmorphic features of the five known patients [35,
39, 97] (Fig. 2d). Moderate to severe psychomotor re-
tardation is present. Increased peripheral leukocyte
counts are the predominant finding and are already
present in newborns. Leucocytosis and immune
deficiency are due to the absence of fucosylated selectin
ligands, decreasing the adhesion of leukocytes to endo-
thelial cells and migration of neutrophils to infection
focuses [35, 97]. Since a1,2 fucose is a component of the
H-antigen, patients lack a detectable ABO blood group
on their erythrocytes (Bombay blood group) [35].
Etzioni described a 5-year follow-up of a 10-year-old
boy with LAD II [36]. The height and weight of the child
were below the 3rd percentile and speech development
369
was absent. In early infancy he had had severe episodes
of pneumonia, but later on there was no increased
incidence of infections. Nevertheless, his leukocyte
counts were constantly elevated to around 40,000/ll.
The major clinical problem was persistent periodontitis.
In two patients, leukocyte adhesion deficiency could
be effectively treated with oral fucose [62, 95, 98]. Oral
fucose supplementation did not correct all fucosylation
processes. H-antigen was still absent on the erythrocyte
surface after 2 years of treatment in one of the patients
[95, 98]. Depending on the mutation in the GDP-fucose
transporter, fucose therapy was thought to be ineffective
in some CDG-IIc patients [131], but a patient with a
severe truncation of the GDP-fucose transporter also
responded to fucose therapy [62].
CDG-IId
CDG-IId (McKusick 607091) is caused by a deficiency
of b1,4 galactosyltransferase I. Only one patient with
this disorder is known to date. In this child, a frameshift
in the coding region caused by an insertion led to a
protein truncated at the C-terminal end by 50 amino
acids. This protein mislocalized to the ER instead of to
the Golgi [52].
The patient, a 2.5-year-old boy, was suffering from
muscular hypotonia with increased serum creatinine
kinase levels. He presented a Dandy-Walker malforma-
tion with macrocephalus at birth and progressive hy-
drocephalus later on. CDG-IId was detected with the
standard transferrin screening test showing an unusual
pattern where asialo-, monosialo- and to a lesser extent
disialotransferrin were the major isoforms [111].
Other glycosylation defects
Walker-Warburg syndrome and muscle–eye–brain
disease
Recently two disorders of O-glycosylation have been
described [8, 148] (Fig. 4). A defect in the gene of the
protein O-mannosyl b-1,2-N-acetylglucosaminyltrans-
ferase (POMGnT1) causes a disease that has been re-
ferred to as muscle–eye–brain (MEB) disease (McKusick
253280) [116]. The gene responsible for MEB disease
had previously been mapped to chromosome 1p32–34
[23] and now POMGnT1 could be located to the same
region. POMGnT1 is a type II membrane protein similar
to other Golgi glycosyltransferases and catalyzes the b-
1,2 linkage of GlcNAc to O-linked mannose using UDP-
GlcNAc as donor substrate. The gene consists of 22
exons coding for a protein of approximately 80 kDa.
Northern blot analysis revealed a broad distribution in
several tissues. Patients showed homozygous and com-
pound heterozygous point mutations in the affected gene
[148]. The other disorder, Walker-Warburg syndrome
(McKusick 236670), affects the first enzyme in the same
370

b
Fig. 4 O-mannosylation. O-mannosylation is a posttranslational
protein modification occurring predominantly in nervous tissue
and in the muscle. Mannosylation of a serine or threonine occurs
probably in the RER and further elongation takes place in the
Golgi apparatus. Symbols are the same as in Fig. 1

glycosylation pathway, protein O-mannosyl transferase

I (POMT1), at least in 30% of the patients [8].
The so-called O-mannosyl glycans are common in
fungi but rare in mammals and are limited to few gly-
coproteins, which are located mainly in brain, nerve and
skeletal muscle [33, 132]. An important O-mannosylated
protein, the dystroglycan complex, consists of a b-dys-
troglycan subunit, a transmembrane glycoprotein, and
a-dystroglycan, an extracellular membrane glycoprotein.
a-dystroglycan is heavily glycosylated. The predicted
weight of the protein core is approximately 74 kDa
whereas the glycosylated protein has an apparent mo-
lecular mass of 156 kDa, judged by SDS-PAGE. N-
glycosylation plays a minor role in a-dystroglycan while
sialylated O-glycans with the structure Siaa2–3Galb1–
4GlcNAcb1–2Man seem to play a crucial role in the
binding of a-dystroglycan to the extracellular matrix
protein laminin [17, 34]. Impairment of a-dystroglycan
function due to the lack of O-mannosyl glycans, neces-
sary for neuronal migration and muscular function,
seems to be the crucial mechanism causing MEB disease.
Although a molecular defect of MEB is now character-
ized, the biosynthetic pathway of O-mannosyl glycans in
mammals is not fully understood. The initiation step of
mammalian O-mannosylation is catalyzed by POMT1,
which seems to be located in the ER (Fig. 4). Further
elongation takes place in the Golgi.
The clinical presentation of MEB disease consists of
congenital glaucoma and myopia, retinal hypoplasia,
pallor of the optic discus, congenital muscular dystro-
phy, severe muscular hypotonia, myoclonic jerks,
mental retardation, hydrocephalus, and electroenceph-
alographic abnormalities. MRI shows pachygyria, a flat
brainstem and cerebellar hypoplasia. The clinical pre-
sentation of Walker-Warburg syndrome is more severe
with lissencephaly, encephaloceles, microphthalmos,
and death usually within the first few months of life
[24].

Progeria variant of Ehlers-Danlos syndrome

The progeria variant of Ehlers-Danlos syndrome

(McKusick 130070) was reported in one patient showing
a clinical picture partially overlapping with Ehlers-
Danlos syndrome and progeria showing hypermobile
joints and loose but elastic skin in addition to aged ap-
pearance and scanty scalp hair [87]. Further symptoms
such as short stature, osteopenia of all bones, defective
deciduous teeth, delayed wound healing, hypotonic
muscle, craniofacial disproportion and developmental
delay were clearly distinct from Ehlers-Danlos syndrome
and progeria.

Hypoglycosylation of a small proteoglycan resulting
in a core protein carrying only one xylose O-linked to
serine was the biochemical characteristic implying a
defect of a b-galactosyltransferase. A defect of the cod-
ing gene was shown by Quentin et al. [115]. The
B4GalT7 catalyzes the second step of the glycosamino-
glycan synthesis initiated by the addition of xylose to
serine of the protein. The activity of B4GalT7 was
markedly reduced and the enzyme was abnormally
thermolabile. Molecular characterization of the
B4GalT7 gene [2, 108] allowed the screening for muta-
tions in the B4GalT7 gene in the patient described by
Kresse et al. This patient was compound heterozygote
for one mutation, resulting in a non-functional protein
mislocalized in the cytoplasm (L206P) and another
mutation in the same exon resulting in an enzyme with
markedly reduced activity (A186D) while the parents
were heterozygous for either of the mutations [108].
Hereditary multiple exostoses
Hereditary multiple exostoses (HME) (McKusick
133700 and 133701) is the most frequent type of benign
bone tumor. The incidence is about 1 to 50,000–100,000
in Western populations, with an autosomal dominant
inheritance. Patients suffer from multiple cartilage-cap-
ped tumors located primarily at the long bones, often a
short stature and various orthopedic deformities [124].
The risk of developing malignancies of the cartilage or
bone is significantly increased in HME patients. For-
mation of exostoses starts in early infancy and continues
until the cessation of growth.
Mapping studies revealed that the vast majority of
HME patients are linked to chromosome 8q24.1 [94] and
chromosome 11p11-p12 [143]. Subsequently candidate
genes for HME could be cloned and were found to be
exostosin-1 (EXT1) and exostosin-2 (EXT2) [143, 144],
coding for two homolog and well-conserved type-II
transmembrane glycoproteins expressed ubiquitously in
human tissue. Biochemical studies confirmed that EXT1
and EXT2 possess glycosyltransferase activity necessary
for the formation of heparan sulfate by adding single D-
glucoronic acid and N-acetylglucosamine. EXT1 and
EXT2 are part of a hetero-oligomeric complex with
higher glycosyltransferase activity than the single pro-
teins [91, 104]. Thus the mutation of either EXT1 or
EXT2 results in the same clinical phenotype. A knock-
out mouse of the EXT1 gene showed embryonic lethality
whereas heterozygous mice with a 50% reduction of
heparan sulfate had no obvious abnormalities although
patients with HME develop exostoses in an autosomal
dominant trait [90]. The development of exostoses is
proposed to be mediated by a lack of heparan sulfate
proteoglycans that play a crucial role in the negative
feedback loop regulating chondrocyte proliferation and
maturation [32]. There are no causal therapeutic options
for HME; exostoses and deformities of bones are treated
symptomatically.
371
CDA II (HEMPAS)
Congenital dyserythropoietic anemias (CDAs) are
inherited disorders of erythropoiesis. They were first
subdivided into three different types by Heimpel et al. in
1968 [56]. CDA type II is the most common form, with
more than 250 cases described. It is also known as he-
reditary erythroblastic multinuclearity with positive
acidified-serum test (HEMPAS) because of hemolysis of
CDA-II erythrocytes by sera of about 30% of healthy
ABO-compatible donors in a slightly acid pH optimized
for complement action [25]. Mild to severe normocytic
anemia is the predominant symptom; jaundice, spleno-
megaly (Fig. 5a) and gallbladder disease are common.
Hemochromatosis and liver cirrhosis rarely complicate
the course of the disease [57, 142]. Due to the often mild
clinical presentation, the median age of diagnosis was
15.9 years in a study covering 98 CDA II patients [66].
In CDA II patients, 10–40% of erythroblasts are bi- and
multinucleated (Fig. 5b), an observation that serves as a
major diagnostic criterion [56]. Endoplasmic reticulum
membranes are found close to the cell membrane in
erythroblasts and erythrocytes [9, 66, 142] (Fig. 5d). The
observation that band 3 (anion exchange protein 1) and
band 4.5 (Glut1), two prevailing membrane proteins of
red blood cells, show a narrower aspect and faster mi-
gration in sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) [4, 7, 76] revealed that a
defect in glycosylation of erythrocyte membrane pro-
teins is a common biochemical characteristic of all pa-
tients suffering from CDA II and may be the cause of the
disorder [41, 101, 120]. Glycosylation abnormalities are
limited to erythroblasts [76]. Although a report de-
scribing hypoglycosylation of serum transferrin in one
patient exists [45], glycoproteins synthesized in the liver
and glycoproteins of lymphocytes are generally not
affected [76].
Band 3 has one N-glycosylation site normally carry-
ing a branched oligosaccharide with many lactosamine
repeats (Galb1,4GlcNAcb1,3) [40]. Western blot analy-
sis of erythrocyte membranes probed with tomato lectin,
a lectin specific for polylactosamines, showed severely
reduced signals in CDA II patients [76] (Fig. 5c). Mass
spectrometry demonstrated a truncation of polylactos-
amines and the presence of incompletely processed high
mannose and hybrid glycans [66, 76]. Structural data of
N-glycans suggested a possible defect of a-mannosidase
II affecting the trimming of the a1,6-branch in early
Golgi processing. An a-mannosidase II knock-out
mouse developed dyserythropoietic anemia whereas
glycan formation in other tissues was not affected due to
the presence of an additional mannosidase [18]. In two
CDA II patients, a-mannosidase II was shown to have
lower activity and a reduced Northern blot signal; one
patient had truncated transcripts of the a-ManII gene.
However, even in these patients, no mutation in the
a-ManII gene could be found [44]. Two further enzymes
were proposed to be affected in other CDA II patients,
N-acetylglucosaminyltransferase II (activities in two
372

b negative charges. Any modification of the carbohydrate
Fig. 5a–d HEMPAS. a The clinical picture of CDA II presenting
with anemia, icterus and splenomegaly. b Normal, bi- and
multinucleated erythroblasts in the bone marrow of CDA II
patients. c Western blot of CDA II erythrocyte membrane
preparations probed with tomato-lectin, specific for polylactosam-
ines, showing a virtual absence of binding in CDA II. d Electron
microscopy of CDA II erythrocytes showing a double membrane
due to the presence of ER close to the cell membrane (arrow). ER
organelle loss seems to be incomplete in these cells
patients 11% and 30% of normal) [42] and galactosyl-
transferase (activity of the membrane-bound form 24%
of normal in 1 patient) [43]. The patient with reduced
galactosyltransferase activity exhibited many severe
symptoms which are not typical of HEMPAS, and in the
coding gene of the galactosyltransferase no mutation
could be found. Thus, reduced activity is likely to be a
secondary effect (M. Fukuda, personal communication).
A defect of the N-acetylglucosaminyltransferase II could
be assigned to CDG-IIa described in detail above. The
activity of these three enzymes, the expression levels and
the sequences of the coding genes revealed no abnor-
malities in other patients.
Linkage analysis excluded the genes of a-mannosi-
dase, a-mannosidase IIx and N-acetylglucosaminyl-
transferase II and located the CDA II gene to
chromosome 20q11.2. However, the same group re-
ported that 5–10% of CDA II patients could not be
linked to this locus, suggesting a genetic heterogeneity of
CDA II [46, 67, 68].
Although a truncation of N-glycans is the common
characteristic of CDA II, the molecular basis of this
disorder still remains an enigma.
Secondary glycosylation disorders
In patients with classic galactosemia (galactose-1-phos-
phate uridyltransferase deficiency) before dietary treat-
ment and thus under galactose exposure, truncated
glycans are formed mimicking CDG in transferrin IEF.
The majority of carbohydrates released from serum
transferrin of these patients are asialo-agalactose struc-
tures [16]. Hypoglycosylation of transferrin can also
occur in fructose intolerance [1].
Diagnostic procedures
Screening test: isoelectric focusing of transferrin
The common diagnostic test for CDG is isoelectric fo-
cusing (IEF) of serum transferrin (Fig. 3). Before IEF is
performed, transferrin has to be saturated with iron.
Transferrin has two biantennary N-linked carbohydrate
side-chains attached to amino acids 413 and 611. In
CDG, the carbohydrate chains are either completely
missing or are truncated. In both cases there is a loss of
terminal a2,6-bound sialic acids, resulting in a loss of
373
that affects the terminal sialic acid residues therefore
results in a change in the isoelectric point of the trans-
ferrin. The IEF pattern does not allow a distinction
between truncated or completely missing oligosaccha-
rides.
In healthy controls, the major form of transferrin
contains two carbohydrate side-chains with two terminal
sialic acids each (tetrasialo-transferrin, isoelectric point
5.4, 80 kDa). Due to triantennary structures, some
higher sialylated isoforms are present (Fig. 3, left lane).
In CDG-Ia, a subpopulation of transferrin carries either
only one carbohydrate side-chain (disialo-transferrin) or
no carbohydrate at all (asialo-transferrin) (Fig. 3, right
lane). The resulting IEF pattern has been described as
type I pattern. All CDG-I entities described so far show
the same pattern (although the amount of abnormal
isoforms varies). Thus, the IEF pattern of transferrin
does not distinguish between the different subtypes of
CDG.
In CDG-II, the number of glycans is normal, but
processing defects of the oligosaccharide lead to trun-
cated carbohydrate structures. In CDG-IIa, truncation
of one branch of the diantennary carbohydrate occurs.
Nearly the whole population of transferrin molecules
shows an isoelectric shift, an IEF pattern called the type
II pattern.
Although the IEF of transferrin is still the best
screening test for CDG, not all CDG types can be de-
tected in this way. CDG-IIb, CDG-IIc, muscle–eye–
brain disease, Walker-Warburg syndrome, hereditary
multiple exostoses, progeria variant of Ehlers-Danlos
syndrome, and HEMPAS are not detectable by trans-
ferrin IEF. Whereas thin-layer chromatography of urine
oligosaccharides is the method of choice in the case of
CDG-IIb, CDG-IIc and HEMPAS can be detected by a
loss of lectin binding to specific glycoconjugates. Even
some CDG-Ia patients might be missed by the IEF test.
One of our CDG-Ia patients with a severe phenotype
and common mutations in the PMM2 gene was followed
up for 6 years and serum samples for transferrin IEF
were drawn at regular intervals. The typical type I hy-
poglycosylation pattern was observed in the first 4 years
of follow-up, but since then the IEF pattern as well as
AT III and factor XI activities have normalized. Phos-
phomannomutase activity in leukocytes is still below 5%
of normal. The reason for this phenomenon is unknown.
Currently, there is no single screening test for the de-
tection of all forms of CDG.
Functional antithrombin III (AT III) tests often show
reduced activities but there are known CDG patients
with PMM deficiency or other defects who show normal
AT III activities. Therefore, transferrin IEF should be
performed in all patients where CDG is suspected.
Children with different types of CDG and especially the
group of children with not yet identified molecular de-
fects (CDG-x) show a broad spectrum of clinical
symptoms, making it difficult to decide who should be
screened for CDG. There are known CDG children with
374

dilative cardiomyopathy as the only symptom. Others Table 2 Clinical symptoms, findings of diagnostic procedures, and
have either cutis laxa, ichthyosis, unexplained throm- laboratory investigations that might be associated with CDG
boembolic events or hyperthermia caused by anesthetics. Neurology
Although the majority of children have multisystem Psychomotor retardation (normally present
disease with psychomotor retardation, there are several in all CDG but NOT in CDG-Ib)
children who show no neurological involvement at all. Epilepsia
Stroke-like episodes (CDG-Ia)
Knock-out mice for mannosidase II or GnT V [19, 31] Ataxia
suggest that some forms of autoimmune disease in hu- Decreased or missing tendon reflexes (CDG-Ia)
mans might also be caused by a primary glycosylation Tetraspastic paresis
defect. Currently, it is impossible to devise a general Microcephaly
guideline about who should be screened for CDG and Cerebellar atrophy (CDG-Ia)
Cerebral atrophy
who should not. Nevertheless it is advisable to screen Abnormal eye movements
any child with psychomotor retardation, unexplained Muscular hypotonia
multisystem disease, autoimmune disorders and chronic Decreased nerve conduction velocity (CDG-Ia)
inflammatory disease, cardiomyopathy, protein-losing Eyes
Strabism (CDG-Ia, -Ic)
enteropathy, cyclic vomiting, anemia with reduced os- Retinitis pigmentosa
motic resistance of the erythrocytes, persistent leukocy- Optical atrophy
tosis or low AT III levels for CDG. Symptoms present in Coloboma
patients with inherited glycosylation disorders are sum- Skeletal/Growth
Failure to thrive
marized in Table 2. Growth retardation
There are some pitfalls to the transferrin IEF test. A Short limbs (CDG-IIc)
number of protein variants of transferrin are known and Joint contractures
some of them affect the isoelectric point of the molecule Delayed puberty
[107]. Often, double bands are an indication of a protein Hypogonadism
Gastrointestinal
variant. If an abnormal IEF pattern is detected, IEF be- Chronic diarrhea (CDG-Ib)
fore and after neuraminidase treatment [107] is necessary Protein-losing enteropathy (CDG-Ib)
in order to rule out protein variants. In addition, SDS- Cyclic vomiting (CDG-Ib)
polyacrylamide gel electrophoresis (SDS-PAGE) or sep- Hepatic fibrosis (CDG-Ib)
Heart
aration of the serum glycoproteins on two-dimensional Cardiomyopathy (dilated or hypertrophic; CDG-Ia, CDG-x)
gels gives valuable supplementary information. Glycosy- Cardial effusion
lation of plasma proteins of a fetus suffering from CDG-Ia Hydrops fetalis
was normal during intrauterine life and became abnormal Renal
Congenital nephrotic syndrome (CDG-Ia)
3 weeks after delivery [20]. We additionally found normal Skin
glycosylation of serum proteins in a fetus suffering from Inverted nipples (CDG-Ia)
CDG-Id (Denecke and Marquardt, manuscript in prep- Abnormal fat pads (CDG-Ia)
aration). Thus diagnostic IEF for CDG should not be Ichthyosis (CDG-If)
performed before 3 weeks of age to avoid false-negative Coagulation
Thrombosis
results. Furthermore, other disorders, including untreat- Bleeding tendency
ed fructose intolerance and galactosemia as well as alco- Decreased AT III activity (characteristic for CDG)
hol abuse might lead to functional inhibition of enzymes Decreased clotting factor XI activity
involved in the carbohydrate biosynthesis or processing, Decreased protein C
Laboratory
thus mimicking CDG. Elevated liver enzymes
In all patients where an abnormal IEF test is present, Hypoproteinemia
the specific enzyme defect has to be determined. Enzyme Low cholesterol levels
activities for CDG-Ia and -Ib can be determined in Low cholinesterase levels
Elevated FSH, LH and prolactin
leukocytes; for all other CDG types, cultured skin fi- Anemia
broblasts from the patient are necessary. Leukocytosis (CDG-IIc)
Bombay blood group (CDG-IIc)

In parenthesis CDG types are listed where the symptom is char-

Specific analyses
In order to investigate CDG patients where the clinical
phenotype does not clearly suggest a certain type, analysis
of lipid-linked and protein-derived oligosaccharides syn-
thesized in the patients’ fibroblasts is the method of
choice. The method has been described in detail [109] and
the principle can be summarized as follows: fibroblasts are
labeled for 30 min in the presence of 3H-mannose. The
cells are extracted with chloroform/methanol 2:1 to re-
acteristic. Please note that only the most common CDG types are
listed and that these symptoms can also occur in other CDG types.
Strabism, for instance, is nearly always present in CDG-Ia, can be
present in CDG-Ic, but is also seen in other CDG types
lease Dol-P-Man into the supernatant and with water in
order to release free oligosaccharides, mannose phos-
phates and sugar nucleotides. The remaining pellet is ex-
tracted with chloroform/methanol/water in order to
release the lipid-linked oligosaccharides (LLO) (being

more polar because of their oligosaccharide). The protein-
derived oligosaccharides (PDO) are released by treatment
of the final pellet with PNGase F. The oligosaccharides
are then analyzed by HPLC.
The majority of known CDG types have been discov-
ered on the basis of an abnormal LLO profile. If the LLO
profile in fibroblasts of a CDG-x patient is normal, ra-
dioactivity incorporated into LLO should be normalized
to the amount of total cellular protein to check the
amount of LLO that has been made. Double-labeling with
3
H-mannose and 35S-methionine and determination of
the amounts of the different isotopes incorporated into
newly made proteins is important for the detection of
oligosaccharyltransferase defects. Analysis of PDO
structures by HPLC should be performed in order to
check for processing defects. Enzymatic release of glycans
from proteins separated on one- or two-dimensional gels
with subsequent MALDI-TOF analysis of the glycan is
another valuable tool for the detection of type II CDG.
Future
The current knowledge of the clinical spectrum of CDG
is certainly limited and most likely biased by the fact
that it is mainly children with neurological phenotypes
that are screened for CDG. The recent discovery of new
CDG types has proven that CDG may cause clinical
phenotypes very different from the known types of this
disorder. Since more than 20% of all children already
known to have CDG have none of the known molecular
defects, it seems likely that many more disorders will be
discovered in the coming years.
Acknowledgements Supported by grants MA 1229/3-1/-2 from the
Deutsche Forschungsgemeinschaft to T.M. and from Innovative
Medizinische Forschung (IMF) Münster to T.M. and J.D. S.
Bushuven is acknowledged for initial help with the graphic work.
This review is dedicated to Prof. Erik Harms for his ongoing
support that made our contribution to this rapidly expanding field
possible.If CDG is suspected, a transferrin IEF test should be
performed. Many laboratories in Europe offer this service. For
analysis in our laboratory, 0.1 ml serum should be sent by regular
mail to: Stoffwechsellabor der Kinderklinik, Domagkstr. 3b,
48129 Münster, Germany.
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