Download Congenital disorders of glycosylation type Ia and Ib

DOCTOR OF MEDICAL SCIENCE
Congenital disorders of
glycosylation type Ia and Ib
Genetic, biochemical and clinical studies
Susanne Kjærgaard
This review has been accepted as a thesis together with eight previously published papers, by the University of Copenhagen, March 31, and defended on
August 20, 2004.
H:S Rigshospitalet, Department of Clinical Genetics, Copenhagen.
Correspondence: Susanne Kjærgaard, John F. Kennedy Instituttet, Gl.
Landevej 7, 2600 Glostrup, Denmark.
Official opponents: Professor, PhD Niels A. Jensen and Associate Professor,
Doctor of Dental Surgery Henrik Clausen.
Dan Med Bull 2004;51:350-63.
1. INTRODUCTION
Congenital disorders of glycosylation (CDG), formerly known as
carbohydrate-deficient glycoprotein syndromes, are a rapidly expanding group of inherited multisystem disorders resulting from
defects in N-linked carbohydrate side-chains (glycans) of proteins.
The molecular basis of several types of CDG has been elucidated
during the past few years, and 12 types have been recognized by February 2003. CDG type Ia (CDG-Ia, OMIM #212065), by far the
most common, is the focus of this thesis, and less emphasis is placed
on CDG type Ib (CDG-Ib, OMIM #602579). CDG-Ia is caused by
mutations in PMM2 and the resulting deficiency of the enzyme
phosphomannomutase (PMM). Clinical manifestations include
psychomotor retardation, hypotonia, failure to thrive, ataxia, liver
dysfunction and dysmorphic features. CDG-Ib is due to mutations
in MPI, which lead to deficiency of the enzyme phosphomannoisomerase (PMI). Manifestations of CDG-Ib include hepatic fibrosis, protein-losing enteropathy, thrombosis and hypoglycemia, but
no primary neurological impairment.
PMM and PMI both play a role in maintaining the intracellular
supply of mannose derivatives required for glycosylation of proteins,
and deficiency of one of the enzymes leads to hypoglycosylation.
lular compartments: the cytosol, the rough endoplasmic reticulum
(rER) and the Golgi apparatus (Figure 1). In the first part of this
pathway PMM and PMI participate in the synthesis of guanosine
diphosphomannose (GDP-mannose) from mannose-6-P. The nucleotide sugars donate sugars attached to the dolichol-pyrophosphate to form the lipid-linked oligosaccharide (LLO) precursor. Two
N-acetylglucosamines and five mannoses are added to the LLO precursor, which is anchored to the cytosolic membrane of the rER.
Subsequently, the LLO precursor is flipped to the luminal side of
rER, where more sugars are added sequentially using dolichol-Pmannose/glucose as donors. When the branched oligosaccharide
moiety contains two N-acetylglucosamines, nine mannoses and
three glucoses, it is linked to selected asparagine residues of the nascently translated polypeptide. Next, three glucoses and one mannose are removed sequentially, and the glycoprotein is transferred to
the Golgi apparatus for final modification. Six additional mannoses
are removed and replaced by two or more each of N-acetylglucosamine and galactose, and the biosynthesis of the oligosaccharide
branches are terminated by the addition of sialic acid residues. Certain glycoproteins are further modified by fucosylation or phosphorylation. Finally, the glycoproteins are secreted from the Golgi to
their sites of action. Secretory glycoproteins are secreted into the
body fluids, membrane glycoproteins are incorporated in cell membranes, and lysosomal enzymes are taken up by lysosomes (Varki et
al, 1999, Jaeken et al, 2001).
Glycans participate in the folding of glycoproteins in the rER
and modify their stability, action and clearance. Glycans are essential for the function of cell surface receptors, as signals for protein
targeting, mediators of cell-to-cell interaction, mediators of protein turnover and as protection of polypeptides from proteases
(Varki et al, 1999).
2.2. CDG TYPES AND NOMENCLATURE
Several human disorders due to defects in the N-glycosylation pathway have recently been characterised. The disorders fall into two
groups based on the localisation of the enzymatic defect (Participants, First International Workshop on CDGS 1999). Group I CDG
(CDG-I) refers to defects in the initial steps of N-linked protein
glycosylation. These defects affect the assembly of LLO and/or its
transfer to asparagine residues on nascent polypeptides. Group II
CDG (CDG-II) refers to defects in the processing of protein-bound
glycans or the addition of other glycans to the polypeptides. Untyped patients are assigned to CDG-x until their genetic defect is
established. Currently known CDG types, the corresponding enzyme deficiencies, and the genes are listed in Table 1.
The aims of the studies were:
1. to characterise Danish CDG-Ia patients biochemically and molecularly
2. to study the effect of PMM2 mutants on phosphomannomutase
function in vitro
3. to investigate the origin of the predominant PMM2 mutations
R141H and F119L by haplotype analysis
4. to characterise the clinical phenotype of CDG-Ia due to the predominant R141H/F119L PMM2 genotype
5. to study the effect of mannose therapy in patients with CDG-Ia
and CDG-Ib.
2. BACKGROUND
2.1 BIOSYNTHESIS AND FUNCTIONS OF N-GLYCANS
The widespread post-translational glycosylation of proteins affect
their properties and functions. Most secreted proteins and membrane proteins (receptors and transporters) as well as some intracellular proteins (e.g., lysosomal enzymes) have glycan side chains,
which are added to asparagine (N-glycosylation) or serine/threonine polypeptide residues (O-glycosylation). Involving 30-40 enzymatic and transport steps, N-glycosylation runs through three cel350
2.3. ISOELECTRIC FOCUSING OF TRANSFERRIN
– A SCREENING TEST FOR CDG
An individual glycoprotein has a heterogeneous but consistent
population of N-linked glycans, which can be separated and visualised by isoelectric focusing (IEF) and Western blotting/immunoprecipitation (Stibler et al, 1998, Niehues et al, 1998). Various glycoforms of the protein are each represented by a band in the IEF pattern. Transferrin is only one of many hypoglycosylated proteins in
CDG, but its microheterogeneity on IEF has turned out to be a sensitive biochemical marker of most N-glycosylation defects (Stibler et
al, 1998). Normally, the predominant glycoform of transferrin contains four oligosaccharide branches (tetrasialotransferrin) on two
N-linked glycans. Hypoglycosylation of proteins results in partial
deficiency of the terminal, negatively charged sialic acid, and hence
in a cathodal shift of the IEF pattern. In patients with CDG-I the
proportions of glycoforms with zero (asialo-) and two (disialotransferrin) oligosaccharide branches are increased with a corresponding
decrease in tetrasialotransferrin – a “type I” pattern (Figure 2) (Stibler et al, 1991b).
Dried blood spots and serum can be used for IEF (Stibler and
Cederberg 1993). A false-positive IEF pattern may be seen when
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Man
Man
Glc
Glc
Man
Man
Glc
Glc
PMI
Cytosol
Ib
Fru-6-P
Fru
-6 -P
Man
Man-6-P
-6 -P
Glc-6-P
Glc
-6 -P
PMM Ia
GDP-Man
GDP
- Man
Man-1-P
Man
-1 -P
Man
Man
P2
P2
P2
Man
P
P
Ie
If
Man55
Man
Man6
P2
P2
Man77 Man
Man88
Man
P2
P2
Id
P2
Ig
Man
Man 55
Glc
Glc
Glc 3 3
Glc22 Glc
Glc
Man 99 Man
Man 99 Man 9 9
Man
Man 99
Man
P2
P2
Ic
Golgi
IIc
GDP-fuc
rER
P
Man
Man
GDP-fuc
P2
Glc
Glc 33
Man
Man 99
Glc
Glc 22
Man
Man 99
Glc
Glc
Man
Man 99
Man 99
Man
Man88
Man
Man 33
Man
Man 55
Man
Man 55
Man
Man88
Man
P2
Ih
IIb
Sia
Sia 22
Gal
Gal 22
Gal 2 2
Man
Man 33
Man 33
Man
Man33
Man
IId
IIa
Man: Mannose; Glc: Glucose; Fru: Fructose; Gal: Galactose; Fuc: Fucose; Sia: Sialic acid
Dolichol
N-Acetylglucosamine
Transporter
Figure 1. N-linked protein glycosylation pathway (simplified). The currently known defects are indicated by red.
using EDTA-anticoagulated plasma, and abnormal IEF pattern not
related to CDG may be seen in serum from patients with untreated
galactosemia, untreated hereditary fructose intolerance or alcohol
abuse (Jaeken et al, 1996, Stibler 1991a, Stibler et al, 1997). Amino
acid polymorphisms of transferrin can alter its IEF pattern (Stibler
et al, 1988). IEF of serum transferrin from parents of a CDG-suspected individual will differentiate between a transferrin protein
variant and abnormal glycosylation.
Normal IEF pattern has been reported in a few cases with enzymatically and genetically proven CDG-Ia (Fletcher et al, 2000,
Dupre et al, 2001). Abnormal glycosylation of transferrin (and other
glycoproteins) tends to improve slightly with increasing age in
CDG-Ia, which might obscure the diagnosis in a minority of older
patients (Dupre et al, 2001, Stibler et al, 1998). It is remarkable, and
still unexplained, that abnormal glycosylation of serum transferrin
(and other glycoproteins) is not detectable by IEF in CDG-Ia fetuses
and preterm newborns until the 36th gestational week (Clayton et
al, 1993, Stibler and Skovby 1994a). IEF analysis cannot differentiate
types of CDG, and additional biochemical and genetic analyses are
needed to reach a specific diagnosis. Quantitative determination of
Carbohydrate-Deficient Transferrin (CDT) is an alternative screening method, which measures the amount of di-, mono- and asialotransferrin (Stibler et al, 1991a). However, elevated values need to be
confirmed by IEF.
2.4. PATIENTS
By February 2003, 32 patients from 26 unrelated Danish families
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had been diagnosed with CDG-Ia, 17 of whom were born in 19901998. This corresponds to an incidence of 1:41 000 newborns. We
know of two additional, deceased patients with clinical manifestations highly suggestive of CDG-Ia, but biological materials were not
available for confirmation. So far only two sibs with CDG-Ib and
one patient with CDG-Ig have been identified. All these patients had
a “type I“ pattern IEF of serum transferrin (Figure 2) (Kjaergaard et
al, 1998a, Westphal et al, 2001b, Grubenmann et al, 2002).
3. CONGENITAL DISORDER OF GLYCOSYLATION TYPE IA
3.1. PHOSPHOMANNOMUTASE DEFICIENCY
Phosphomannomutase deficiency was established as the cause of
CDG-Ia in 1995 by the detection of PMM activities ≤ 10% in fibroblasts/leukocytes/liver tissue from patients and of intermediate
activities in their parents (Van Schaftingen and Jaeken 1995). PMM
is a cytosolic enzyme that catalyses the conversion of mannose-6-P
to mannose-1-P (Figure 1). PMM requires mannose-1,6-biphosphate or glucose-1,6-biphosphate as a cofactor, which serves to
phosphorylate the catalytic site of PMM. Mannose-1-P is required
for the synthesis of GDP-mannose, the donor of mannose to the
growing LLO precursor. PMM deficiency results in depletion of the
mannose-1-P and GDP-mannose pools in fibroblasts (Körner et al,
1998a, Rush et al, 2000). Hence, the incorporation of mannose into
LLO is reduced, and glycoproteins lacking complete N-linked
glycans are synthesised (Wada et al, 1992, Yamashita et al, 1993,
Powell et al, 1994, Krasnewich et al, 1995). Fibroblasts with PMM
deficiency do not accumulate mannose-6-P, which suggests that
351
Table 1. Currently known CDG
types, corresponding enzyme
deficiencies, and genes.
Type
Deficiency
Gene
Reference
CDG-Ia
Phosphomannomutase
PMM2
Van Schaftingen 1995
Matthijs 1997
CDG-Ib
Phosphomannoisomerase
MPI
Niehues 1998
Jaeken 1998
CDG-Ic
Dolichyl-P-Glc:Man9GlcNAc2-PP-dolichyl
α-1,3-glucosyltransferase
ALG6
Körner 1998
Imbach 1999
CDG-Id
Dolichyl-P-Man:Man5GlcNAc2-PP-dolichyl
α-1,3-mannosyltransferase
ALG3
Körner 1999
CDG-Ie
Dolichol-P-Man synthase 1
DPM1
Imbach 2000
Kim 2000
CDG-If
Dolichol-P-Man utilisation defect
MPDU1
Schenk 2001
Kranz 2001
CDG-Ig
Dolichyl-P-Man:Man7GlcNAc2-PP-dolichyl
α-1,6-mannosyltransferase.
ALG12
Chantret 2002a
Grubenmann 2002
CDG-Ih
Dolichyl-P-Glc:Glc1Man9GlcNAc2-PP-dolichyl
α-3-glucosyltransferase
ALG8
Chantret 2003
CDG-IIa
UDP-GlcNAc:α-6-D-mannoside β-1,2-Nacetylglucosaminyltransferase II (GlcNAcT II)
MGAT2
Jaeken 1994
Tan 1996
CDG-IIb
α-1,2-glucosidase I
GCS1
De Praeter 2000
CDG-IIc
GDP-fucose transporter
FUCT1
Lübke 2001
CDG-IId
N-acetylglucosamine β-1,4-galactosyltransferase I
B4GALT1
Hansske 2002
CDG-x
Genetic basis unknown
PMI maintains an equilibrium between mannose-6-P and fructose6-P (Figure 1) (Körner et al, 1998a).
PMM activities in extracts of cultured fibroblasts from Danish
CDG patient have been measured by the assay described by Van
Schaftingen and Jaeken (1995): the reduction of NADP to NADPH
was measured spectrophotometrically following coupled enzymatic reactions catalysed by PMI, phosphoglucoisomerase and glucose-6-P dehydrogenase, and mannose-1-P was used as substrate
(Figure 3). PMM activities in cultured fibroblasts ranged from undetectable to 15% of controls, thereby documenting PMM deficiency in all patients (Kjaergaard et al, 1998a).
Grünewald et al, (2001) pointed out a possible pitfall in the diagnosis of PMM deficiency after observing CDG-Ia patients with high
residual PMM activity overlapping control values in cultured fibroblasts. This problem was not encountered in assays of fresh leukocytes and liver tissue. The variability of PMM activity among different cell types points to a cell-specific effect, as yet unexplained. One
possibility is that mutant PMM or the isozyme (see below) become
Control
overexpressed in rapidly dividing fibroblasts with high protein synthesis as opposed to fully differentiated leukocytes with little protein
synthesis (Grünewald et al, 2001). The use of fresh leukocytes does
not always circumvene difficulties of interpretation of results since
PMM values of some carriers overlap with control or patient values
(Grünewald et al, 2001).
PMM is a homodimer of 30 kDa subunits, whose tertiary structure is not yet known (Kepes and Schekman 1988, Pirard et al,
1997). Two isozymes of PMM with 66% sequence homology have
been identified: PMM1 and PMM2. They catalyse the same reaction
at approximately equal rates, but PMM2 is more substrate-specific
(Pirard et al, 1999a). PMM2 is the defective enzyme in CDG-Ia. The
role of PMM1 is still a puzzle, and no disorder has so far been associated with defects of PMM1. Studies in normal rat on the tissue distribution/expression of PMM1 and PMM2 by Western/Northern
blot analyses showed that PMM2 is the only isozyme detectable in
most tissues except brain and lung, where PMM1 accounts for approximately 66% and 13% of the total activities, respectively (Pirard
CDG
Ia
CDG
Ib
CDG
Ig
+
Tetrasialotransferrin
Figure 2. IEF pattern of serum transferrin from a normal control, CDG-Ia-,
CDG-Ib- and CDG-Ig patient. The
number of sialic acid residues (negative charges) is given at the right.
The anode and catode are given at
the left. The duplicate bands in the
CDG-Ig lane are probably due to a
genetic variant of transferrin at the
polypeptide level.
352
Disialotransferrin
Asialotransferrin
–
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Man
Man
Glc
Glc
Hexokinase
Hexokinase
Man-6-P
Man
-6 -P
Phosphoglucoisomerase
PMM
66-P-gluconate
- P - gluconate
GlcGlc-6-P
-6 -P
FruFru-6-P
-6 -P
PMI
NADP NADPH
Glucose-6-Pdehydrogenase
Man-1-P
Figure 3. Enzymatic reactions involved in the assays for determination
of PMM activity in fibroblasts and of
serum mannose.
Man: Mannose; Glc: Glucose; Fru: Fructose
et al, 1999a). In contrast, Northern blot analysis of normal human
tissues showed widespread expression of both PMM1 and PMM2
(Matthijs et al, 1997a and b). The highest expression of PMM2 was
seen in pancreas and liver, organs with a predominant role in glycoprotein synthesis. PMM2 was weakly expressed in brain, unlike
PMM1. How these observations correlate with the severe CNS involvement of CDG-Ia is not known. However, the low PMM activity
measured in liver, fibroblasts and leukocytes of CDG-Ia patients
indicates that PMM1 contributes little to the total PMM activity in
these cell types, and that it does not rescue PMM2 deficiency.
3.2. THE PMM2 GENE
CDG-Ia is inherited as an autosomal recessive trait, and a locus was
assigned to chromosome 16p13 by linkage analysis (Jaeken et al,
1991, Martinsson et al, 1994). Two genes both encoding a PMM
have been cloned based on their homology to yeast PMM (SEC53)
(Kepes and Schekman 1988). The first gene, PMM1, is localised on
chromosome 22q13, which excludes its involvement in CDG-Ia
(Matthijs et al, 1997a, Wada et al, 1997). The second gene, PMM2, is
localised on 16p13, and the detection of mutations in this gene
showed conclusively that PMM deficiency is responsible for CDG-Ia
(Matthijs et al, 1997b). PMM2 contains 8 exons, and its cDNA has
an open reading frame of 738 base pairs encoding a protein of 246
amino acids. The high homology (57%) at both the nucleotide and
the amino acid level between yeast PMM (SEC53) and PMM2 (and
PMM1) indicates a strong conservation from yeast to man, reflecting a fundamental role of PMM (Matthijs et al, 1997b).
A processed pseudogene, PMM2ψ, located on chromosome 18p,
has also been identified. Caution is therefore required when designing primers for mutation analysis, as identical mutations have been
reported in PMM2 and PMM2ψ (Schollen et al, 1998).
Figure 4. Position of the reported
PMM2 mutations. Mutations underlined have been identified in
Danish patients with CDG-Ia.
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24delC
C9Y
F11C
G15R
G15E
1
3.3. PMM2 MUTATIONS
Molecular analysis has revealed a large number of mutations in
CDG-Ia patients worldwide. Seventy-one PMM2 mutations have
been reported in patients from 24 countries, including Denmark
(Figure 4) (Kjaergaard et al, 1998a and 1999, Kondo et al, 1999,
Matthijs et al, 2000, Grünewald et al, 2001, http://www.med.kuleuven.ac.be/cdg/Mutation/cdg-Ia.htm). The mutations are scattered
over the gene with some clustering in exon 5 and 8. Sixty-four of 71
different mutations are of the missense type, the others being three
single base pair deletions, one four base pair deletion, two splice site
mutations and one nonsense mutation. The mutations in the Danish CDG-Ia patients were identified by Single Strand Conformation
Analysis (SSCA) on genomic DNA after PCR amplification with intronic primers followed by direct DNA sequencing and confirmation by restriction analysis (Kjaergaard et al, 1998a and 1999). The
seven mutations identified so far are all of the missense type and result in replacement of amino acids that are strictly conserved among
phosphomannomutases of yeast (SEC53), Candida albicans and
human phosphomannomutases (PMM1 and PMM2), suggesting
that these amino acids are important for the catalytic activity of the
enzyme. Two mutations accounted for 88% of the mutations: 44%
of the CDG-Ia alleles harboured the R141H mutation, 44% of the
CDG-Ia alleles harboured the F119L mutation, and 83% of the patients were compound heterozygous for these mutations (Kjaergaard et al, 1998a). Surprisingly, we did not observe homozygosity
for either of the two predominant mutations. The absence of homozygotes expected on the basis of Hardy-Weinberg equilibrium was
statistically highly significant. Subsequently, we and others have detected homozygosity for F119L, but homozygosity for R141H has
been conspicuously absent in results of genotype analysis (Matthijs
et al, 1998a and 2000, Kjaergaard et al, 1998a and 1999, Bjursell et al,
2000, Imtiaz et al, 2000, De Lonlay et al, 2001). These observations
IVS1-1G>A
L32R
G42R
V44A
Y64C
D65Y
V67M
P69S
Y76C
IVS3+2T>C
E93A
N101K
C103F
L104V
Y106C
A108V
324delG
P113L
2
3
4
G117R
F119L
I120T
R123Q
R123X
V129M
P131A
I132T 451 -454delGAAA
E151G
I132N
I153T
389delC
F157S
E139K
R141H
R162W
F144L
F172V
D148N
G175R
5
6
F183S
D185G
D188G
C192G
H195R
E197A
F206T
G208A
G214S
N216I
N216S
D217E
H218L
D223E
D223N
T226S
G228R
G228C
Y229S
V231M
A233T
T237R
T237M
R238G
R238P
R239W
C241S
7
8
3’
353
suggest that homozygosity for R141H is not compatible with life or,
less likely, leads to another clinical phenotype.
3.4. RELATIONSHIP BETWEEN PMM2 GENOTYPE
AND RESIDUAL PMM ACTIVITY
The extensive allelic heterogeneity worldwide and the somewhat
variable residual PMM activity in different cell types from the same
patient complicate the study of the correlation between genotype
and residual PMM activity. Furthermore, PMM activity is low in extracts of control fibroblasts, and differentiation of PMM activities
below 0.5 nmol/mg/min is not reliable, which make it difficult to
compare residual activities of various mutant fibroblast cell lines.
We and others observed profoundly decreased PMM activity in
fibroblasts from all patients (Kjaergaard et al, 1998a, Imtiaz et al,
2000). In general, there was no clear correlation between genotype
and residual PMM activity except for the R141H/F119L genotype,
which invariably was associated with very low PMM activity (Kjaergaard et al, 1998a, Grünewald et al, 2001). Certain genotypes
(R141H/C241S, F157S/C241S, R141H/T237M) were associated with
a relatively high residual PMM activity in fibroblasts (30-70% of
controls) (Grünewald et al, 2001).
3.5. IN VITRO EXPRESSION STUDIES
To investigate the impact of PMM2 mutations on the catalytic activity of the enzyme, wild type and mutant PMM2 were expressed in E.
coli (Kjaergaard et al, 1999, Pirard et al, 1999b). We cloned PMM2cDNA into a pET3a expression vector, and the six different mutations identified at the time (R141H, F119L, C192G, G117R, T237R
and D223E) were each introduced into PMM2-cDNA by site-directed mutagenesis. Specific activities of recombinant PMM2 suggested the presence of two types of mutations. One type resulted in
virtually null activity, and the other type resulted in PMM2 with decreased activity and/or altered properties such as affinity for the
substrate and stability. The R141H protein had no residual PMM2
activity, and the F119L protein had approximately 25% of normal
activity, higher Km for mannose-1-P and less thermostability (Kjaergaard et al, 1999, Pirard et al, 1999b). The G117R and T237R proteins also had virtually no residual PMM2 activity, whilst the D223E
and C192G proteins had activities in the normal range but higher
Km’s for mannose-1-P and less thermostability (Kjaergaard et al,
1999). The recombinant V231M protein has been shown to retain
residual PMM2 activity (Pirard et al, 1999b).
Each of our patients has at least one mutation that retains residual
PMM2 activity when expressed in E. coli (Table 2). However, this
activity is not always reflected by activities depending on two mutant PMM2 alleles found in cultured fibroblasts from the patients
(Kjaergaard et al, 1999, Pirard et al, 1999b). The activity in cultured fibroblast of some but not all genotypes was less than expected from the activity of the recombinant protein, which might
indicate poor stability of the mutant PMM2 and/or a dominantnegative effect of the null activity polypeptide. Even fibroblasts homozygous for the F119L had very low or undetectable PMM activ-
Table 2. PMM2 genotypes identified in 26 unrelated Danish CDG-Ia patients.
PMM2 genotype
No. of
patients
R141H/F119L . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
R141H/C192G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
R141H/V231M . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
F119L/F119L . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
F119L/G117R . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
F119L/V231M . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
T237R/D223E . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
The recombinant proteins corresponding to the mutations underlined have
no detectable PMM activity.
354
ity (Kjaergaard et al, 1999). The lack of correlation may also be explained by difficulties in comparing activities due to the relatively
low sensitivity of the PMM assay and the low PMM activity in control fibroblasts.
So far, no CDG-Ia patient has been found to have two PMM2 mutations conveying virtually no PMM2 activity when expressed in
vitro (Kjaergaard et al, 1999, Pirard et al, 1999b, Vuillaumier-Barrot
et al, 1999 and 2000, Westphal et al, 2001a). Even though one should
be cautious applying the results of in vitro studies in E. coli to the in
vivo situation in man, the expression studies support the hypothesis
that homozygosity for R141H (or another “severe” mutation) impairs PMM2 activity to a degree incompatible with life, whilst F119L
(or another “mild” mutation) conveys a phenotype compatible with
survival due to residual PMM2 activity. The fate of zygotes homozygous for R141H is unknown. The absence of patients harbouring
two null alleles points to an essential role of PMM2 in normal cellular functions, and suggests that such zygotes are lost in early pregnancy. An absolute requirement for residual activity is compatible
with the glycosylation, however deficient, of circulating proteins in
CDG-Ia patients and with the apparent lack of other biopathways to
synthesize mannose-1-P and GDP-mannose.
Within one of the three extremely conserved protein sequences
(motifs) delineated in PMM, the phosphorylation site (see p. 7) has
been assigned to an aspartate residue at position 12 (Aravind et al,
1998, Collet et al, 1998). The motifs may be involved in the catalytic
site of PMM, but none of the mutations identified in Danish
patients affects the amino acids within these motifs. Indeed, mutations distant to the presumed catalytic site may influence conformation and thereby activity of the enzyme. More knowledge of the
structure of PMM is needed to predict the impact of a mutation on
the enzymatic function of PMM2.
3.6. GENETIC EPIDEMIOLOGY
CDG-Ia occurs worldwide. The reported incidence is highest in
Denmark (1:41 000 newborns) followed by Sweden (1:80 000), the
Netherlands and Belgium (Matthijs et al, 2000, Kjaergaard et al,
2001). R141H, by far the most frequent PMM2 mutation, is present
in 73% of Caucasian patients (Matthijs et al, 2000). R141H and
F119L make up the vast majority of mutations of Scandinavian patients (Kjaergaard et al, 1998a, Bjursell et al, 2000). Genotypes are
much more heterogeneous in other Western European countries
(Matthijs et al, 2000). The predominance of two mutations among
Danish patients reflects the genetic homogeneity of the Danish
population also seen in other diseases, e.g., cystic fibrosis (Schwartz
et al, 1993) and galactosemia (M. Schwartz, unpublished data).
Prior to cloning of the PMM2 gene one specific haplotype was
shown to be markedly overrepresented among patients from Southwestern Sweden, Southern Norway and Eastern Denmark, indicating one shared ancestral mutation (Bjursell et al, 1997). In order to
investigate the origin of the two predominant mutations, haplotype
analysis of the Danish CDG-Ia families was performed using markers defining the Scandinavian founder haplotype and one additional
closely linked marker (D16S3020, located within 20kb of PMM2)
(Kjaergaard et al, 1998a). R141H and F119L were each associated
with a specific haplotype. Our suggestion that the Scandinavian
founder mutation was F119L was subsequently confirmed (Kjaergaard et al, 1998a, Bjursell et al, 1998). Haplotype analysis of CDGIa patients with the R141H mutation from other Western European
countries also revealed a clear linkage disequilibrium for the same
haplotype as seen in the Danish patients (Schollen et al, 2000a).
These haplotypes indicated that R141H and F119L originated from
two ancestral mutations. R141H showed more recombinations with
some of the more distant markers than F119L, suggesting that
R141H is older than F119L. The frequency of F119L was highest
among Scandinavian patients, and it gradually decreased when
moving southward in Europe (Matthijs et al, 2000). The F119L mutation may well have arisen in a Scandinavian ancestor. R141H was
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frequent among patients from all Western European countries and
the US compatible with an ancient origin (Matthijs et al, 2000).
The lack of patients homozygous for R141H was further explored
by determining the frequency of R141H in two normal populations.
The carrier frequency in Dutch neonates and in Danish blood donors was 1:79 and 1:60, respectively (Schollen et al, 2000a). These
frequencies were not significantly different, and pooled data gave a
frequency of 1:72. Under Hardy-Weinberg equilibrium an incidence
of R141H homozygotes of approximately 1:20 000 would be expected. These observations further support that R141H in the
homozygous state is not compatible with life.
Both populations were also analysed for the second most frequent
mutation, F119L. No F119L carriers were identified, indicating a
much lower carrier frequency of this mutation than of R141H
(Schollen et al, 2000a).
3.7. PRENATAL DIAGNOSIS
Prior to the identification of PMM2 as the gene involved in CDG-Ia,
prenatal diagnosis was based on linkage analysis and measurement
of PMM activity in amniotic and chorionic villus cells (Bjursell et al,
1998, Charlwood et al, 1998, Matthijs et al, 1998b). Problems with
the risk of error due to recombination and with interpretation of
enzyme activity were avoided when mutation analysis became available.
Prenatal diagnosis by mutation analysis of chorionic villus cells is
now routinely performed and widely requested by affected families
in Denmark. So far, we have performed 18 prenatal analyses: seven
fetuses were compound heterozygotes, and these pregnancies were
terminated. Seven fetuses were heterozygote carriers, and four were
homozygous normal (unpublished data). Seven compound heterozygotes out of 18 at-risk pregnancies is a high proportion of affected
fetuses compared to the expected 25%, but statistically not significant. We are in the process of pooling results of prenatal mutation
analyses with other diagnostic centres to estimate the recurrence
risk in a larger sample of CDG-Ia families.
3.8. CLINICAL PRESENTATION
CDG-Ia was first described by Jaeken et al, in 1980. The clinical features vary with age, and usually go through four stages: the infantile
alarming multisystem stage, the childhood ataxia-mental retardation stage, the teenage leg atrophy stage and the adult hypogonadic
stage (Table 3) (Jaeken et al, 1991). The first stage is variable in severity, but often dramatic, requiring frequent hospitalisations. The
child becomes more stable with increasing age, and loss of acquired
skills is unusual. Most patients are wheelchair-bound, but motor
and communicative skills vary. The overall mortality rate is approximately 20%, mostly occurring in the first years of life due to infection, multiple organ failure or heart tamponade (Jaeken et al, 1991
and 2001).
The clinical spectrum of CDG-Ia has expanded since the initial
description. Moderate to severe neurological dysfunction, variable
dysmorphic features and variable involvement of other organs are
now recognised, and a phenotype with only borderline cognitive
impairment has been reported recently (Jaeken et al, 2001, Van Ommen et al, 2000, Drouin-Garraud et al, 2001, Enns et al, 2002). Rare
presenting features, such as hyperinsulinemic hypoglycemia, congenital nephrotic syndrome and obstructive cardiomyopathy, have
also been reported (Van der Knaap et al, 1996, Imtiaz et al, 2000,
Böhles et al, 2001).
3.9. BIOCHEMICAL FEATURES
A large number of serum glycoproteins have been shown to have
abnormal IEF pattern and/or altered concentration or enzymatic
activity, including transport proteins, glycoprotein hormones, coagulation and anticoagulation factors, lysosomal enzymes and
enzyme inhibitors (Jaeken et al, 2001). Clinically important biochemical features include:
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Table 3. Clinical stages of CDG-Ia.
Infantile alarming multisystem stage 1
Failure to thrive
Floppiness
Psychomotor retardation
Abnormal subcutaneous fat pads
Inverted nipples
Hepatomegaly and liver dysfunction
Cerebellar atrophy
Esotropia
Pericardial effusions
Multisystemic failure
Childhood ataxia mental retardation stage 2
Mental retardation (IQ: 40-60)
Motor disability
Peripheral neuropathy
Cerebellar ataxia
Tapetoretinal degeneration
Stroke-like episodes
Seizures
Teenage leg atrophy stage 3
Atrophy of lower limbs
Stable cerebellar ataxia
Deformity of thorax and spine
Osteopenia
Absence of puberty in females
(hypergonadotropic hypogonadism)
Adult stable disability stage 4
Stable neurological impairment
Short stature
Kyphoscoliosis and long thin extremities
Premature aging of the skin in females
Retinitis pigmentosa
1. Decreased activity of factor XI and decreased activities of the coagulation inhibitors ATIII and protein C are present in the vast
majority of CDG-Ia patients, and abnormal glycoforms of the
latter two have been shown (Van Geet and Jaeken 1993, Stibler et
al, 1996, Kjaergaard et al, 2001). A complex coagulopathy may be
responsible for stroke-like episodes, thromboembolic events and
hemorrhages seen in some patients.
2. Adolescent females have elevated levels of luteinising hormone
and follicle-stimulating hormone (De Zegher and Jaeken 1995).
Absence of puberty and hypergonadotropic hypogonadism are
consistent features of CDG-Ia girls with few exceptions. Kristiansson et al, (1995) were not able to demonstrate hypoglycosylation of gonadotropins. Oestradiol secretion in response to
exogenous gonadotropins has suggested biological inactivity of
FSH, but the lack of response in one Danish patient confirmed
that primary ovarian failure might also be responsible for absence of puberty (De Zegher and Jaeken 1995, Kristiansson et al,
1995, Kjaergaard et al, 2001). Oestradiol therapy induced growth
of breast tissue and pubic hair in two Danish females (Kjaergaard
et al, 2001).
3. Lysosomal enzyme activities in serum are frequently elevated
(e.g., arylsulphatase A, β-galactosidase, β-glucuronidase and βhexosaminidase). In contrast, lysosomal enzyme activities are
frequently low in leukocytes (e.g., α-fucosidase, β-glucuronidase,
α-iduronidase, α- and β-mannosidase) (Barone et al, 1998).
These observations may be explained by deficiency of the mannose-6-P targeting signal necessary for their uptake in lysosomes.
Abnormal glycoforms of N-acetylglucosaminidase and fucosidase have been demonstrated (Jaeken et al, 1991). Findings of
slightly to moderately increased lysosomal enzyme activities in
plasma and/or moderately decreased activities in leukocytes from
a patient with neurological impairment should alert the clinician
to the possibility of CDG.
4. Abnormal glycosylation of serum transferrin, thyroid-binding
globulin and α1-antitrypsin are consistently observed (Stibler et
355
al, 1998). These glycoproteins were used as parameters in our
mannose therapy study (see p. 12).
5. Consistent biochemical abnormalities of non-glycosylated proteins include raised liver transaminases and hypoalbuminemia
(Jaeken et al, 1991, Kjaergaard et al, 2001). Liver transaminases
usually rise during infections, but tend to normalise with
increasing age. Hypoalbuminemia may contribute to the extravascular fluid accumulations, e.g., pericardial effusion and ascites, seen in some patients.
3.10. CLINICAL PHENOTYPE IN RELATION TO GENOTYPE
Most reports on the clinical manifestations of CDG-Ia predate the
availability of PMM2 mutation analysis (Kristiansson et al, 1989,
Jaeken et al, 1991, Petersen et al, 1993, Stibler et al, 1994b). A recent
mutation update revealed 73 different PMM2 genotypes among 249
CDG-Ia patients, including Danish patients, and the R141H/F119L
genotype accounted for 27% of the patients (Matthijs et al, 2000).
The extensive genetic heterogeneity has complicated attempts to
study genotype-phenotype correlation (Imtiaz et al, 2000, De Lonlay et al, 2001). The predominance of the R141H/F119L genotype in
our large series of CDG-Ia patients allowed us to study its clinical
phenotype and outcome (Kjaergaard et al, 2001). The features of 23
patients are listed in Table 4. All patients had an early, uniform presentation with severe feeding problems, hypotonia, inverted nipples,
abnormal subcutaneous fat pads and developmental delay obvious
before age 6 months. Motor ability ranged from none to walking
with a rolator, and vocabulary ranged from none to comprehensible
speech. The overall mortality ascribed to CDG was 18%.
Brain imaging showed cerebellar atrophy in all but two patients.
Cerebral atrophy was frequently present, and serial imaging indicated that the cerebellar and cerebral atrophy ran a progressive
course in some patients, as previously observed (Jensen et al, 1995).
Table 4. Clinical features of CDG-Ia patients with R141H/F119L genotype.
Consistent features (present in >90% of the patients)
Neonatal presentation
Severe feeding difficulties
Severe failure to thrive
Severe hypotonia
Developmental delay obvious before age 6 months
Hepatic dysfunction (raised liver transaminases and
coagulation abnormalities)
Inverted nipples
Abnormal subcutaneous fat pads
Esotropia
Delayed visual maturation
Mental retardation
Cerebellar atrophy
Ataxia
Muscular atrophy
Peripheral neuropathy
Febrile seizures
Absence of puberty in females
Frequent manifestations (present in ≥ 50% of the patients)
Pericardial effusions (asymptomatic)
Afebrile seizures
Stroke-like episode
Acquired microcephaly
Congenital joint restriction
Hernia/hydrocele/retentio testis in males
Thorax deformity
Myopia
Decreased vision
Less frequent (present in < 50% of the patients)
Neurogenic hearing impairment
Thromboembolic event
Episode of prolonged bleeding
Episode of ascites
Kyphoscoliosis
Fracture due to minimal trauma
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The mean head circumference standard deviation score declined
gradually from normal at birth to –1.9 at age 5 (Kjaergaard et al,
2002).
Eye manifestations were evaluated during another Danish survey
(Jensen et al, 2003). Patients with the R141H/F119L genotype had
congenital esotropia, delayed visual maturation and a reduced rod
response by electroretinography. The vast majority also had low
visual acuity and progressive myopia.
Failure to thrive is a frequent feature of CDG-Ia, although the onset and degree in individual patients have been variable (Jaeken et al,
1991, Petersen et al, 1993, Kristiansson et al, 1998). Feeding problems and failure to thrive were prominent in all our patients, and we
analysed longitudinal data of weight and length to delineate the pattern of prepubertal growth (Kjaergaard et al, 2002). Anthropometric
data of patients with identical CDG genotypes were remarkably
similar. Measurements of weight and length were within normal
range at birth. During the first 7 months of life, standard deviation
scores of mean weight and length declined markedly, 2.7 and 2.4, respectively, and there was no prepubertal catch up. Our patients fed
poorly, and the majority had frequent vomiting and diarrhoea during the first year of life. Although data on their protein and caloric
intake and gastrointestinal losses were not available, inadequate nutrition is the most likely major explanation of the severe failure to
thrive. Hormones and other factors involved in growth are likely to
be affected by defective glycosylation, but previous studies have not
been able to hold growth hormone or thyroid hormones responsible
(Macchia et al, 1995, De Zegher and Jaeken. 1995). Controlled
dietary and hormonal interventional studies as well as studies of
intestinal protein loss are required to further explore the causes of
growth failure.
Patients with the R141H/F119L genotype have an early “classical”
presentation, including severe failure to thrive, but with variable
functional outcome, and they may well represent the severe end of
the broad clinical spectrum (Kjaergaard et al, 2001, Grünewald et al,
2001). Although few patients share other genotypes, some of these
seem to be associated with severe disease and high mortality
(R141H/D188G, R141H/V231M) or mild disease (R141H/C251S,
R141H/F183S, F157S/C241S) (Matthijs et al, 2000, Grünewald et al,
2001, Erlandson et al, 2001, Drouin-Garraud et al, 2001). In general,
the PMM2 genotype does not seem to be a good predictor of clinical/functional outcome in individual CDG-Ia patients.
Clinical variability, especially of functional outcome, was evident
among patients with identical PMM2 genotypes and even among
affected sibs and monozygotic twins (Kjaergaard et al, 2001). The
lack of any clear genotype-phenotype correlation parallels other diseases, e.g., Gaucher disease (Dipple and McCabe 2000). The interand intrafamilial variability in CDG-Ia suggests that additional environmental factors and/or genes that are inherited independently
of PMM2 have an impact on the clinical phenotype. Apart from unknown environmental stresses, polymorphisms/mutations in other
genes, e.g., of the N-glycosylation pathway, may contribute to the
severity of the disease. In fact, severely affected patients have an
increased frequency of a polymorphism (F304S) in the ALG6 gene
compared to moderately and mildly affected CDG-Ia patients
(Westphal et al, 2002). ALG6 encodes the glycosyltransferase that
adds the first glucose to the growing LLO in the rER (Figure 1)
(Körner et al, 1998c). ALG6 is the gene mutated in CDG type Ic
(Table 1), and the polymorphism compromises glycosylation when
expressed in yeast under certain conditions (Westphal et al, 2002).
Clearly, this is not an explanation of clinical variability in a highly
complex disorder. Several biochemical steps with possible modifying influences occur between a mutated gene and its clinical ourcome. The importance of the genetic background has been demonstrated in animal models in which phenotypic differences among
strains were obvious, e.g., the survival of the CDG type IIa (CDGIIa) mice. Wang et al, (2001) introduced a deletion into the mouse
germline that reproduced the glycan biosynthetic defect responsible
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for human CDG-IIa (see p. 13). Mice lacking the Mgat2 gene had
deficiency of N-acetylglucosaminyltransferase II activity and severe
gastrointestinal, hematologic, and skeletal abnormalities. All Mgat2null mice died in early postnatal life. However, crossing the Mgat2
mutation into another genetic background (strain) resulted in
Mgat2-null mice surviving for several months.
3.11. MANNOSE THERAPY
Mannose is presumed to enter human fibroblasts by a mannose-specific transporter, supplying most of the mannose for N-glycosylation of proteins (Figure 1). Previously, glucose was thought to be the
major source of mannose in this process (Pannerselvam and Freeze
1996b, Panneerselvam et al, 1997a). In vitro studies have demonstrated that addition of exogenous mannose (≥ 0.25 mM) to the culture medium of CDG-Ia fibroblasts can correct both the deficient
mannose incorporation and the size of lipid- and protein-linked
oligosaccharides (Panneerselvam and Freeze 1996a, Körner et al,
1998b).
Furthermore, CDG-Ia patients had low blood mannose levels
(average: 25 µM) compared to those of normal subjects (average: 55
µM) (Panneerselvam et al, 1997b).
These observations encouraged us to perform a short-term study
of dietary mannose therapy in CDG-Ia patients. Information about
mannose absorption, clearance and tolerance in man was sparse. We
demonstrated that both normal controls and CDG-Ia patients had
at least a two- or threefold elevation in blood mannose at a dose of
0.1 g/kg body weight given at 3 h intervals (Alton et al, 1997). Mannose in serum was determined by an enzymatic assay described by
Etchison and Freeze (1997): glucose was selectively removed from
the sample by glucokinase and glucose-6-P-dehydrogenase, whereupon anionic reaction products and excess substrates were removed
by anion-exchange chromatography. Mannose in the effluent was
then assayed spectrophotometrically by the reduction of NADP to
NADPH following coupled enzymatic reactions catalysed by hexokinase, phosphoglucoisomerase and glucose-6-P-dehydrogenase in
the presence or absence of PMI (Figure 3).
Our study established the feasibility of testing mannose as a therapy of CDG-Ia patients. Next step was to determine whether shortterm supplementation of mannose in CDG-Ia patients could improve their abnormal glycosylation of circulating glycoproteins
(Kjaergaard et al, 1998b). Five CDG-Ia patients completed the protocol of enteral supplementation with mannose 0.1 g/kg body
weight every 3 h for 9 days. A higher dose had caused gastrointestinal distress (Alton et al, 1997). Unfortunately, the mannose levels
obtained in the patients were lower than the concentration shown to
correct abnormal glycosylation in fibroblasts. However, since the in
vitro correction was dose-dependent, a lower concentration therefore could be at least partial beneficial. Surprisingly, the mean concentrations of the glycoproteins tended to decline during continued
mannose supplementation, and CDT increased as did the abnormal
glycoforms, with the most pronounced changes observed after cessation of mannose supplementation. Physiological fluctuation of
blood levels of glycoproteins or methodological variations could explain these observations. However, it seemed unlikely that the same
trend in glycosylation of all glycoproteins analysed in five patients
should be caused by random variation. Since the aim of our study
was to test for correction of glycosylation in order to justify longterm mannose supplementation, we did not attempt to include a
hard-to-obtain group of normal children or a group of CDG-Ia patients given another substance than mannose, which might have
been able to illuminate the unexpected observations. Our results
suggest an effect of mannose on the glycosylation of circulating
glycoproteins in CDG-Ia, but it does not seem to be a beneficial one
in the short-term. More knowledge of mannose metabolism in man
is required to interpret the results correctly.
Mannose supplementation has not been effective biochemically
or clinically in other studies of CDG-Ia. Continuous intravenous
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mannose infusion for three weeks with serum levels up to 2 mM followed by oral mannose for six months did not improve the glycosylation of various glycoproteins (Mayatepek et al, 1997, Mayatepek
and Kohlmüller 1998).
Apparently, low PMM activity cannot be rescued by increasing
the amount of its substrate in CDG-Ia patients in vivo. The biochemical basis for the in vitro correction of protein glycosylation is
still not clear. So far the trials on mannose supplementation in
CDG-Ia have been small and uncontrolled, and a long-term trial has
not been reported.
Providing PMM-deficient cells with mannose-1-P may be a way
to increase the GDP- mannose pool, but mannose-1-P is not able to
penetrate cell membranes due to its high polarity. Attempts to develop membrane-permeant derivatives of mannose-1-P are in process (Rutschow et al, 2002). However, therapy of CDG-Ia remains
problematic. One reason is the prenatal onset of CDG-Ia demonstrated by the presence of dysmorphic features and neurological
dysfunction at birth. On the other hand, the normal fetal growth
and the failure to detect hypoglycosylation of transferrin in CDG-Ia
prenatally (see p. 6) suggest that maternal compensation and/or a
developmentally regulated alternate pathway may bypass PMM2 deficiency in utero. Presently, treatment offered patients with CDG-Ia
remains supportive.
4. CONGENITAL DISORDER OF GLYCOSYLATION TYPE IB
4.1. PHOSPHOMANNOISOMERASE DEFICIENCY
CDG-Ib is due to mutations in the MPI gene resulting in deficiency
of PMI (Niehues et al, 1998, Jaeken et al, 1998, De Koning et al,
1998a). PMI is a cytosolic Zn2+-dependent enzyme catalysing the interconversion of fructose-6-P and mannose-6-P (Figure 1). As previously outlined, mannose-6-P is the precursor of GDP-mannose,
which is required for glycosylation. PMI deficiency leads to a reduction of the GDP-mannose pool, and hence to incompletely glycosylated glycoproteins. PMI is widely distributed in human tissues,
and low PMI activities, ranging from 3% to 18% of controls, have
been demonstrated in leukocytes, cultured fibroblasts and liver
tissue from CDG-Ib patients (Niehues et al, 1998, Jaeken et al, 1998,
De Koning et al, 1998a, De Lonlay et al, 1999, Babovic-Vuksanovic
et al, 1999, Westphal et al, 2001b).
Human MPI has been cloned based on its homology to the gene
in yeast. MPI, located on chromosome 15q22, consists of eight exons
encoding a protein of 423 amino acids (Proudfoot et al, 1994, Schollen et al, 2000b). Fourteen different MPI mutations in 13 families
have been reported, and 12 of these mutations are of the missense
type, mostly affecting conserved amino acids (Schollen et al, 2000b,
Westphal et al, 2001b, Vuillaumier-Barrot et al, 2002).
4.2. CLINICAL PRESENTATION
Clinical manifestations of CDG-Ib include hepatic fibrosis, proteinlosing enteropathy, cyclic vomiting, thromboses, bleeding episodes
and hypoglycemia, the latter sometimes due to hyperinsulinemia
(Niehues et al, 1998, Jaeken et al, 1998, De Koning et al, 1998a, De
Lonlay et al, 1999, Babovic-Vuksanovic et al, 1999, Westphal et al,
2001b). The absence of both neurological involvement and dysmorphic features sets the clinical phenotype quite apart from those of
other types of CDG. Transferrin IEF pattern is identical to that of
patients with other type I CDG (Figure 2). Albumin is low, liver
transaminases are frequently raised, and the coagulopathy seen in
CDG-Ib is indistinguishable from that of CDG-Ia with low levels of
factor XI, ATIII and protein C.
Two Danish sibs with CDG-Ib have been diagnosed by detection
of “type I” transferrin IEF pattern, decreased PMI activity in cultured fibroblasts and identification of missense mutations in the
MPI gene (Westphal et al, 2001b). The patients were originally reported in 1980 by Pedersen and Tygstrup because of their unusual
clinical manifestations of recurrent venous thromboses, protein-losing enteropathy and hepatic fibrosis. No diagnosis was reached at the
357
time, and one sib died due to complications of the disease at 5 years
of age. The good long-term outcome of the surviving sib, now age
35, demonstrates that serious manifestations in childhood may subside and allow for a normal adult life. Higher metabolic demands in
infancy and childhood may overwhelm the cellular capacity for glycosylation, but with increasing age the patients become more stable as
observed in other metabolic diseases, e.g., defects in branched chain
amino acid catabolism. Symptoms, age at onset, severity of disease
and outcome of CDG-Ib vary among reported patients. The Danish
sibs presented later (2 1/2 y) than previously described CDG-Ib patients (0-12 months), and their clinical manifestations were clearly
episodic and occurred in association with fever. Environmental
stresses (e.g., infections) and genetic background may be important
factors in severity and outcome of the disease.
4.3. MANNOSE THERAPY
Mannose-6-P is normally derived from two sources: from fructose6-P via PMI or from free mannose entering the fibroblasts by a specific transporter, whereupon mannose is converted to mannose-6-P
by a hexokinase (Figure 1). The latter pathway bypasses PMI deficiency, and oral mannose supplementation has been beneficial both
clinically and biochemically to PMI-deficient patients. Daily oral
mannose supplements have substantially reversed their proteinlosing enteropathy, coagulopathy, and hypoglycemia as well as
improved their glycosylation of transferrin (Niehues et al, 1998, De
Lonlay et al, 1999, Babovic-Vuksanovic et al, 1999). Mannose supplementation in our patient normalised the ATIII level and improved her glycosylation of transferrin, which, as expected, returned
to the presupplementation state after cessation (Westphal et al,
2001b). Cellular import of mannose appeared to be much more efficient in our patient than in controls since her base-line serum
mannose was lower and blood clearance of oral mannose threefold
higher. These observations may be explained by upregulation of the
mannose-specific transporter. Mannose clearance studies have not
been reported in other CDG-Ib patients. Free mannose in man presumably comes from endogenous glycoprotein catabolism and dietary sources, but little is known about the availability of mannose in
the human diet. Differences in dietary mannose intake may also
contribute to the variable and episodic manifestations of CDG-Ib.
Although the metabolic defect in CDG-Ib is localised in the same
biosynthetic pathway and in the step immediately preceding the enzymatic step blocked in CDG-Ia, the clinical manifestations of the
two disorders are quite dissimilar. Why is the clinical presentation of
PMI deficiency mainly hepato-intestinal? Why is the central nervous
system spared in PMI deficiency as opposed to other types of CDG?
One possible explanation is that hexokinase I expressed in brain has
a high affinity for mannose (Km 5 mM), thereby bypassing PMI and
its deficiency. In contrast, glucokinase, the major hexokinase of
hepatocytes, has a very low affinity for mannose (Km 33 mM), which
may explain the liver involvement in CDG-Ib (Jaeken et al, 1998).
Other possible explanations include the existence of different PMI
isozymes and differences in expression of the mannose-specific
transporter.
5. CONGENITAL DISORDERS OF GLYCOSYLATION
– OTHER TYPES
Several other defects in N-glycosylation have recently been elucidated (Table 1 and Figure 1). Comprehensive descriptions are not
yet available as few patients of each type have been reported. In general, clinical manifestations are less specific than in CDG-Ia and Ib
and include variable psychomotor retardation, hypotonia, epilepsy,
variable dysmorphic features, and, frequently, coagulopathy and
raised liver transaminases. It is important to emphasize that patients
with other CDG types do not have the characteristics of CDG-Ia,
i.e., inverted nipples, abnormal subcutaneous fat pads or cerebellar
atrophy. Descriptions beyond the following summary of other CDG
types are outside the scope of this thesis.
358
CDG-Ic is due to deficiency of the glycosyltransferase, which adds
the first glucose to the growing LLO in the rER (Figure 1) (Körner et
al, 1998c, Imbach et al, 1999). The clinical presentation includes
moderate psychomotor retardation, hypotonia, epilepsy, feeding
problems, esotropia and coagulopathy (Grünewald et al, 2000). The
IEF of serum transferrin shows a “type I” pattern.
CDG-Id is due to deficiency of the mannosyltransferase, which
adds the sixth mannose to the growing LLO in the rER (Figure 1)
(Körner et al, 1999). Clinical features are spastic paresis, severe
psychomotor retardation, epilepsy, microcephaly and dysmorphism. ATIII may be reduced, and serum transferrin IEF shows a
“type I” pattern.
CDG-Ie is due to deficiency of the cytosolic subunit of dolichol-Pmannose synthetase, the enzyme required to generate dolichol-Pmannose, which is the donor of mannose for the growing LLO on
the luminal side of the rER (Figure 1) (Imbach et al, 2000, Kim et al,
2000). Clinical manifestations include severe psychomotor retardation, hypotonia, epilepsy, microcephaly, cortical blindness, hepatosplenomegaly, dysmorphic features and coagulopathy. Liver transaminases are raised, and serum transferrin IEF pattern shows a
“type I” pattern.
CDG-If is due to defects in the MPDU1 gene, which is required
for utilisation of the donor substrates, dolichol-P-mannose and
dolichol-P-glucose, but not their synthesis (Figure 1) (Kranz et al,
2001, Schenk et al, 2001). The exact function of the gene product is
not known. The clinical phenotype includes hypotonia, severe psychomotor retardation, seizures and variable skin affection. ATIII
may be reduced, but liver transaminases are normal. The serum
transferrin IEF shows a “type I” pattern.
CDG-Ig. Recently, transferrin IEF screening (“type I” pattern,
Figure 2) of a Danish patient with psychomotor retardation, hypotonia, failure to thrive, low immunoglobulins and low ATIII led to
the identification of a novel type of CDG, type Ig (Grubenmann et
al, 2002). CDG-Ig is due to mutations in the ALG12 gene encoding
the mannosyltransferase, which adds the eighth mannose to the
growing LLO in the rER (Figure 1) (Chantret et al, 2002a, Grubenmann et al, 2002).
CDG-Ih, the most recently characterised type of CDG, is due to
deficiency of the glycosyltransferase adding the second glucose onto
the growing LLO in the rER (Figure 1) (Chantret et al, 2003). The
only reported patient had hypoalbuminemia, protein-losing enteropathy, hepatomegaly and coagulopathy, but no CNS involvement – a
clinical presentation close to that of CDG-Ib. Serum transferrin IEF
showed a “type I” pattern.
CDG-IIa is caused by deficiency of N-acetylglucosaminyltransferase II, the enzyme adding a glucosamine residue to the oligosaccharide of the newly synthesised glycoprotein in the Golgi apparatus
(Figure 1) (Jaeken at al. 1994). Clinical features are severe psychomotor retardation, dysmorphism, hypotonia, epilepsy and stereotyped gestures. Raised liver transaminases and reduced ATIII activity are present. The serum transferrin IEF pattern is different from
that seen in CDG-I with markedly decreased or absent
tetrasialotransferrin and increased mono-, di-and trisialotransferrin
(“type II” pattern).
CDG-IIb is caused by deficiency of glucosidase I, the enzyme
removing the terminal glucose from the oligosaccharide after its
transfer to the polypeptide in the rER (Figure 1) (De Praeter et al,
2000). The only reported patient had severe hypotonia, dysmorphic
features, hypoventilation, seizures, severe infections, hepatomegaly
and coagulopathy. Serum transferrin IEF pattern was normal. The
diagnosis was based on structural analysis of an abnormal tetrasaccharide found by thin layer chromatography of urine from the patient in question.
CDG-IIc, usually called Leukocyte Adhesion Deficiency type II, is
caused by reduced transport of GDP-fucose into the Golgi, resulting
in hypofucosylation of glycoconjugates, including selectin ligands
(Lübke et al, 2001). Features of CDG-IIc are psychomotor retardation
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and dysmorphic features combined with recurrent severe non-suppurative bacterial infections and high blood leukocyte counts. The
immunodeficiency is due to defective selectin-dependent leukocyte
trafficking to the sites of infection. IEF of serum transferrin is normal.
Notably, oral fucose supplementation has proven beneficial biochemically and clinically in one CDG-IIc patient (Marquardt et al, 1999).
CDG-IId is due to deficiency of galactosyltransferase, the enzyme
adding galactose to the oligosaccharide of the newly synthesised
glycoprotein in the Golgi apparatus (Figure 1) (Hansske et al, 2002).
The only reported patient had myopathy, Dandy-Walker malformation, psychomotor retardation, raised liver transaminases and coagulopathy. IEF of serum transferrin showed a “type II” pattern.
CDG-x. Several cases with not yet characterised glycosylation defects detected by transferrin IEF have been reported. Their diverse
clinical presentations included psychomotor retardation, hypotonia,
dysmorphic features and seizures as well as hydrops fetalis, severe
neonatal diarrhoea and Budd-Chiari syndrome (Huemer at al. 2000,
Mention et al, 2001, De Koning et al, 1998b).
Within the last few years defects in proteoglycan synthesis and αdystroglycan synthesis have been identified, and the range of phenotypes related to glycosylation defects has been extended to previously known clinical diagnoses such as hereditary multiple exostoses, a progeroid variant of Ehlers-Danlos syndrome, muscleeye-brain disease and Walker-Warburg syndrome (Duncan et al,
2001, Okajima et al, 1999, Yoshida et al, 2001, Beltran-Valero De
Bernabe et al, 2002). I-cell disease (or mucolipidosis II), which is
due to deficiency of N-acetylglucosamine-1-phosphotransferase, required for synthesis of the mannose-6-P targeting signal, is usually
assigned to the group of lysosomal storage diseases, but it is in fact a
glycosylation defect. These disorders are not detected by IEF screening of serum transferrin.
6. DIAGNOSTIC STRATEGY
CDG (except type Ib) should be considered in a patient with unexplained psychomotor retardation, isolated or associated with the
clinical and biochemical features mentioned in Table 3 and p. 10. Inverted nipples, abnormal subcutaneous fat pads, cerebellar atrophy,
failure to thrive, pericardial effusions and liver dysfunction suggest
CDG-Ia.
Early diagnosis of CDG-Ib is crucial as the disorder is potentially
lethal, but curable with mannose supplementation. CDG-Ib should
be considered in unexplained liver disease and in protein-losing
enteropathy, isolated or in association with thrombosis, recurrent
vomiting and hypoglycemia in otherwise normal children. Low
ATIII activity and raised liver transaminases are common but not
consistent findings in CDG.
IEF of serum transferrin is a useful screening test, and it will identify the vast majority of patients with CDG (N-glycosylation defects), but not all (e.g., CDG-IIb and CDG-IIc). Protein variants of
transferrin and causes not related to CDG should be excluded when
an abnormal IEF pattern is detected (see p. 6). IEF analysis cannot
distinguish between different types of CDG, and further analyses are
needed to reach a specific diagnosis (Figure 5). Patients of Danish
origin with a “type I” transferrin IEF pattern are analysed for the
two most frequent PMM2 mutations, R141H and F119L. If not
found, all exons of PMM2 are sequenced. If normal, the next step is
to assay PMM activity in cultured fibroblasts or leukocytes, since
PMM deficiency could be due to large deletions or mutations in the
promoter not detected by routine sequencing. When CDG-Ia is excluded, an oligosaccharide profile of cultured fibroblasts is done to
detect accumulation and/or depletion of oligosaccharide structures
suggesting possible enzymatic defects and responsible genes. Profiling of oligosaccharides is based on HPLC analysis of LLO extracted
from cultured fibroblasts following incubation with [3H] mannose
(Powell et al, 1994). Next, the suspected enzyme is assayed and the
responsible gene screened for mutations.
The N-glycosylation pathway in the rER is highly conserved
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IEF of serum transferrin
Abnormal pattern
“Type I”
Abnormal pattern
“Type II”
PMM2, MPI analysis
PMM, PMI assay
N-glycan structure
analysis
LLO analysis
Enzymatic assay
Mutation analysis
Normal pattern
CDG not excluded
Enzymatic assay
Mutation analysis
Figure 5. Diagnostic strategy for CDG (N-glycosylation defects).
among eukaryotes, and yeast mutants have been valuable in characterisation of novel types of CDG-I. For example, fibroblasts from
the Danish patient who led to the identification of CDG-Ig accumulated lipid-linked GlcNA2Man7 oligosaccharides, as do alg12 mutant
yeast cells protein (Grubenmann et al, 2002). The sequence information obtained from the yeast alg12 locus made it possible to identify the orthologous human ALG12 gene, and mutations at the locus
were subsequently detected in patient DNA. In order to know if the
mutations compromised the function of the ALG12-encoded
mannosyltransferase, normal and mutant cDNA were expressed in
alg12-deficient yeast. Indeed the mutant human ALG12 protein was
unable to complement alg12 deficiency in yeast, as opposed to the
normal ALG12. The same approach has been used to characterise
CDG-Ic, CDG-Id and CDG-Ie.
The absence of psychomotor retardation in CDG-Ib sets the clinical phenotype apart from those of other CDG types. The diagnosis
is confirmed by demonstration of PMI deficiency in fibroblasts or
leukocytes and identification of MPI mutations.
“Type II” serum transferrin IEF pattern points to a N-glycosylation defect localised after the transfer of oligosaccharides to the
protein, and a structural analysis of protein-bound glycans is performed to suggest an affected enzymatic step. Then, the suspected
enzyme is assayed and the responsible gene screened for mutations.
CDG-IIb may be picked up by analysis of urine oligosaccharides
as described in the only case reported so far (De Praeter et al, 2000).
CDG-IIc is detected by determination of the blood groups as the
fucose-containing blood groups A, B, O and Lewis A are absent in
these patients, followed by mutation screening of the responsible
gene (Lübke et al, 2001).
7. CONCLUSIONS
CDG-Ia was diagnosed in 32 Danish patients by detection of abnormal serum transferrin IEF pattern, profoundly decreased PMM activity in fibroblasts and mutations in the PMM2 gene. The incidence
was 1:41 000 newborns, the highest reported so far. Two of seven
missense mutations predominated, R141H and F119L, and 83% of
the patients were compound heterozygous for these two mutations.
Unexpectedly, we did not observe homozygosity for the R141H mutation.
In vitro expression studies on the impact of mutations on PMM
activity suggested the presence of two types of mutations. One type
resulted in virtually null activity, and the other type resulted in
PMM2 with decreased activity and/or altered properties. Each of
our patients had at least one mutation that retained residual PMM2
activity when expressed in E. coli. The studies supported that homozygosity for R141H (or another “severe” mutation) impairs PMM2
activity to a degree incompatible with life, whilst F119L (or another
359
“mild” mutation) conveys a phenotype compatible with survival
due to residual PMM2 activity.
R141H and F119L were associated with separate specific haplotypes suggesting that each is derived from an ancestral mutation.
R141H is by far the most frequent mutation in Caucasian patients
with a carrier frequency of 1:60 in Denmark. F119L is a Scandinavian founder mutation with a much lower carrier frequency.
Patients with the R141H/F119L genotype have an early uniform
presentation with severe feeding problems, severe growth failure,
hypotonia, inverted nipples, abnormal subcutaneous fat pads and
developmental delay obvious before age 6 months but variable functional outcome. Patients with this genotype present with the “classical” phenotype and belong to the severe end of the broad clinical
spectrum of CDG-Ia.
Short-term mannose supplementation was not able to improve
glycosylation in CDG-Ia patients. In contrast, mannose supplementation in an adult CDG-Ib patient proved beneficial biochemically.
The reasons for the clinical variability seen among both CDG-Ia
and CDG-Ib patients with identical molecular defect are unknown,
but environmental factors and genetic background may have an
impact on the clinical phenotype. The genotype does not seem to be
a good predictor of clinical outcome in the individual patient.
Prenatal diagnosis by mutation analysis of chorion villus sample
has been performed in 18 at-risk pregnancies.
8. FUTURE PERSPECTIVES
Efforts should be made to raise awareness among physicians of CDG
as a large group of highly variable multi-system disorders, some of
which are treatable. Increased awareness and broader serum transferrin IEF screening will increase the number of CDG patients with
less typical presentations, and novel CDG types will be identified.
The many biochemical steps/genes involved in the glycosylation
pathways suggest a large number of potential defects leading to
pathological conditions. More detailed clinical characterisations by
compiling clinical data of novel CDG types is another way to promote their diagnosis and to improve their management.
How abnormal glycosylation leads to the plethora of symptoms
and signs in CDG-Ia and Ib is currently unexplained. Animal
models may be helpful in unravelling the pathophysiology and, ultimately, in exploring possible treatments. The availability of the
sequence and structure of each of the mouse Pmm1, Pmm2 and Mpi
genes is the initial step in development of mouse models with deficiency of one of the enzymes (Heykants et al, 2001, Davis et al,
2002). Based on the lethality of R141H homozygotes in humans, it is
unlikely that mice with complete ablation of PMM2 will produce
viable offspring. Introducing appropriate mutations in the gene
conveying residual PMM2 activity is more likely to have a viable
outcome.
Patients and the pharmaceutical industries will continue to benefit from increased knowledge of the impact of glycans on the stability, targeting, action and clearance of bioactive molecules. One
example is Gaucher disease due to deficiency of the lysosomal
enzyme glucocerebrosidase resulting in accumulation of glucocerebroside. During normal biosynthesis of lysosomal enzymes Nglycans become modified with man-6-P residues to target them to
the man-6-P receptor of lysosomes. Consequently, recombinant
glucocerebrosidase produced for enzyme replacement therapy in
Gaucher disease is modified to contain the targeting signal. The recombinant enzyme is thereby targeted to the correct site of action.
Collaboration with other centres dedicated to CDG is necessary
for several reasons. The diagnosis of and basic research in CDG
require diverse technologies, and gathering of patients is needed to
increase the number required for clinical studies. EUROGLYCAN, a
European collaboration financed by the EU, is an excellent forum
for these purposes. Research in CDG will most likely provide more
knowledge of the roles of N-glycans in physiology.
360
ABBREVIATIONS
ATIII
Antithrombin III
CDG
Congenital Disorders of Glycosylation
CDG-Ia Congenital Disorder of Glycosylation type Ia
CDG-Ib Congenital Disorder of Glycosylation type Ib
GDP
Guanosine diphosphate
CDT
Carbohydrate-Deficient Transferrin
IEF
Isoelectric focusing
LLO
Lipid-linked oligosaccharide
MPI
Gene encoding PMI
NADPH Nicotinamide adenine dinucleotide phosphate
PMI
Phosphomannoisomerase
PMM Phosphomannomutase (both isozymes)
PMM1 Phosphomannomutase 1 (isozyme 1)
PMM1 Gene encoding phosphomannomutase 1
PMM2 Phosphomannomutase 2 (isozyme 2)
PMM2 Gene encoding phosphomannomutase 2
rER
rough endoplasmic reticulum.
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