Deficiency of Dol-P-Man Synthase Subunit DPM3 Bridges the Congenital Disorders of Glycosylation with the Dystroglycanopathies
Alpha-dystroglycanopathies such as Walker Warburg syndrome represent an important subgroup of the muscular dystrophies that have been related to defective O-mannosylation of alpha-dystroglycan. In many patients, the underlying genetic etiology remains unsolved. Isolated muscular dystrophy has not been described in the congenital disorders of glycosylation (CDG) caused by N-linked protein glycosylation defects. Here, we present a genetic N-glycosylation disorder with muscular dystrophy in the group of CDG type I. Extensive biochemical investigations revealed a strongly reduced dolichol-phosphate-mannose (Dol-P-Man) synthase activity. Sequencing of the three DPM subunits and complementation of DPM3-deficient CHO2.38 cells showed a pathogenic p.L85S missense mutation in the strongly conserved coiled-coil domain of DPM3 that tethers catalytic DPM1 to the ER membrane. Cotransfection experiments in CHO cells showed a reduced binding capacity of DPM3(L85S) for DPM1. Investigation of the four Dol-P-Man-dependent glycosylation pathways in the ER revealed strongly reduced O-mannosylation of alpha-dystroglycan in a muscle biopsy, thereby explaining the clinical phenotype of muscular dystrophy. This mild Dol-P-Man biosynthesis defect due to DPM3 mutations is a cause for alpha-dystroglycanopathy, thereby bridging the congenital disorders of glycosylation with the dystroglycanopathies.
Deﬁciency of Dol-P-Man Synthase Subunit DPM3
Bridges the Congenital Disorders of Glycosylation
with the Dystroglycanopathies
Dirk J. Lefeber,
Karin M. Huyben,
Frank W. Preijers,
Christa van den Elzen,
Lambert van den Heuvel,
and Ron A. Wevers
Alpha-dystroglycanopathies such as Walker Warburg syndrome represent an important subgroup of the muscular dystrophies that have
been related to defective O-mannosylation of alpha-dystroglycan. In many patients, the underlying genetic etiology remains unsolved.
Isolated muscular dystrophy has not been described in the congenital disorders of glycosylation (CDG) caused by N-linked protein glyco-
sylation defects. Here, we present a genetic N-glycosylation disorder with muscular dystrophy in the group of CDG type I. Extensive
biochemical investigations revealed a strongly reduced dolichol-phosphate-mannose (Dol-P-Man) synthase activity. Sequencing of
the three DPM subunits and complementation of DPM3-deﬁcient CHO2.38 cells showed a pathogenic p.L85S missense mutation in
the strongly conserved coiled-coil domain of DPM3 that tethers catalytic DPM1 to the ER membrane. Cotransfection experiments in
CHO cells showed a reduced binding capacity of DPM3(L85S) for DPM1. Investigation of the four Dol-P-Man-dependent glycosylation
pathways in the ER revealed strongly reduced O-mannosylation of alpha-dystroglycan in a muscle biopsy, thereby explaining the clinical
phenotype of muscular dystrophy. This mild Dol-P-Man biosynthesis defect due to DPM3 mutations is a cause for alpha-dystroglycan-
opathy, thereby bridging the congenital disorders of glycosylation with the dystroglycanopathies.
Congenital disorders of glycosylation (CDG) are inborn
errors of metabolism and various defects affect the
N-glycosylation of proteins. The complex N-glycan
biosynthesis follows a sequential and highly ordered
pathway in the cytoplasm, ER, and Golgi apparatus.
Because N-glycosylation is a very common cotranslational
modiﬁcation, genetic defects in this pathway generally
lead to a multisystem disease. Defects in the ER during
the assembly of the lipid-linked oligosaccharide and trans-
fer to nascent protein chains affect all N-linked proteins
and typically lead to such a multisystem presentation in
Alpha-dystroglycanopathies represent a separate group
of genetic glycosylation disorders, in which deﬁciency of
tissue-speciﬁc alpha-dystroglycan O-mannosylation has
been reported. These disorders present with a more
organ-restricted clinical phenotype, including muscular
dystrophy and a variable degree of structural brain and
eye anomalies. At the severe end, serious muscular
dystrophy, a complex brain development disorder, and
eye abnormalities can be found in the congenital muscular
dystrophies such as Walker-Warburg syndrome (WWS
[MIM 236670]). Within the same spectrum, subtle eye
abnormalities, clinically less signiﬁcant brain malforma-
tion, and a milder muscular dystrophy can be observed
in limb girdle muscular dystrophy (e.g., LGMD2I due to
[MIM 607155]). Several genes have
been discovered underlying the phenotypic spectrum of
the dystroglycanopathy syndromes (POMGnT1, POMT1,
POMT2, LARGE, FKTN, and FKRP). In a large number of
dystroglycanopathy patients, the underlying genetic
etiology remains unsolved.
Dolichol-phosphate-mannose (Dol-P-Man) synthase or
GDP-Man:Dol-P mannosyltransferase (EC 22.214.171.124), plays
an important role in four different glycosylation routes in
the ER. N-glycosylation,
and GPI-anchor formation
depend on Dol-P-Man
availability (Figure 1). In S. cerevisiae, the single-membrane-
spanning DPM1 protein comprises the Dol-P-Man synthase
activity, whereas in other yeasts and higher eukaryotes,
a Dol-P-Man synthase complex exists that is composed of
The catalytic subunit DPM1 is located in
the cytoplasm and transfers mannose from the nucleotide
sugar GDP-Man to membrane-embedded Dol-P, resulting
Laboratory of Pediatrics & Neurology, Institute for Genetic and Metabolic Disease,
Department of Pediatrics,
Department of Pathology,
Department of Human Genetics, Radboud University Nijmegen Medical Centre, 6500 HB Nijmegen, The Netherlands;
Regensburg, 93053 Regensburg, Germany;
Department of Pediatrics, Rethymnon General Hospital, Rethymnon 74100, Greece;
Fourth Pediatric Clinic
of the Aristotelian University of Thessaloniki, Papageorgiou Hospital, Thessaloniki GR-56403, Greece;
Department of Neurology, University of Athens,
Athens 11528, Greece;
Great Ormond Street Hospital, London WC1N 1EH, UK;
Department of Neurology, University of Crete, 71003 Heraklion-Crete,
Friedrich Miescher Institute, 4058 Basel, Switzerland;
Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan;
nology Frontier Research Center, Osaka University, Suita, Osaka 565-0871, Japan
These authors contributed equally to this paper
DOI 10.1016/j.ajhg.2009.06.006. ª2009 by The American Society of Human Genetics. All rights reserved.
76 The American Journal of Human Genetics 85, 76–86, July 10, 2009
in the formation of Dol-P-Man. DPM1 is anchored to the ER
membrane via the coiled-coil domain of DPM3 (Figure 1).
DPM2 is located in the ER membrane as well and is required
for stabilization of the complex. In addition, a role for DPM2
has been reported in GPI-anchor biosynthesis as the regu-
lator of GPI-N-acetylglucosaminyltransferase (GPI-GnT).
In the group of CDG-I, many subtypes have been found
with deﬁcient glycosyltransferases and identiﬁcation of
new disease variants with aberrant LLO ﬂipping from the
cytosol to the ER lumen,
and disturbed Dol-P biosyn-
has provided important insights in the complex
glycosylation processes in human disease. Here, we iden-
tify a defect in an accessory protein (DPM3) of a glycosyl-
transferase in the ER. The unique clinical phenotype of
a mild muscular dystrophy with dilated cardiomyopathy
may relate to aberrant protein N-glycosylation and O-man-
nosylation of alpha-dystroglycan.
Material and Methods
The use of CDG-Ie
ﬁbroblasts and serum was approved by
informed consent of parents and treating physician (M. Garcia-
Silva). DNA samples of Greek controls were obtained from A. Evan-
geliou. The use of ﬁbroblasts and serum of the current patient has
been approved by the parents and treating physician (Spilioti).
Transferrin isoelectric focusing was carried out as described
The clinical history did not show any indication for
the presence of alcohol abuse, fructosemia, or galactosemia as
a possible secondary cause for CDG-I transferrin isoelectric-
focusing proﬁles. A protein polymorphism was excluded by neur-
aminidase digestion of the samples and by the normal proﬁles of
both parents. Analyses of lipid-linked oligosaccharides, formation
elongation of Dol-PP-
by cytosolic mannosyltrans-
were performed in ﬁbro-
blasts as described.
N-glycan analysis was carried out essentially as described.
patient serum was denatured and N-glycans were released by over-
night incubation with PNGaseF (New England Biolabs). After puri-
ﬁcation (PGC-SPE, Supelco), the glycan mixture was permethylated
and puriﬁed on a SepPak C18 cartridge (Waters). Permethylated
N-glycans were analyzed by MALDI-LTQ mass spectrometry
(Thermo Finnigan). For molecular weight determination of serum
transferrin, immunopuriﬁcation and online desalting were fol-
lowed by ESI-MS detection as described.
Dol-P-Man Synthase in Fibroblasts and CHO Cells
Skin ﬁbroblasts were cultured in M199 medium (Life Technologies)
supplemented with 10% fetal calf serum and 1% penicillin þ strep-
tomycin in a humiﬁed atmosphere containing 5% CO
The reaction contained in a ﬁnal volume of 0.07 ml: 7 mM Tris-
HCl (pH 7.2), 7 mM MgCl
, 0.025 mCi GDP-[
(speciﬁc activity 303 mCi/ mmol), 2 mg dolichol-P, 0.1% Nonidet
P-40, and 0.05 mg membrane protein. Incubation was at 24
for the times indicated. The reaction was stopped by addition of
chloroform-methanol (3:1, by volume) and processed by phase
partitioning as described.
Radioactive glycolipids were separated
on silica gel 60 plates (Merck) developed in chloroform/methanol/
water (65:25:4, by volume). Radioactivity was detected by Phos-
Genetic Analysis of the DPM1, DPM2,
and DPM3 Genes
We designed intronic oligonucleotide primers in accordance with
public sequences (GenBank accession number NM_153741) to
Figure 1. Architecture of the Dol-P-Man Synthase Complex
The enzymatically active DPM1 subunit in the cytoplasm is anchored to subunits DPM2 and DPM3 in the ER membrane via the
C-terminal peptide of DPM3. Four biosynthetic pathways depend on Dol-P-Man: N-glycosylation (1), O-mannosylation (2), C-Manno-
sylation (3), and GPI-anchor biosynthesis (4).
The American Journal of Human Genetics 85, 76–86, July 10, 2009 77
ﬂank the coding sequences and exon-intron junctions. Primer
sequences are available on request. The exon fragments were
ampliﬁed in a reaction volume of 25 ml containing 100 ng DNA
template, 1 unit of Taq DNA polymerase (Invitrogen), 2.5 mlof
103 PCR buffer, 2.0 mM MgCl
, 50 ng of forward and reverse
primers, 0.25 mM dNTPs, and 0.75 ml DMSO. After an initial
step of 2 min at 94
C, PCR parameters were 35 cycles for 45 s dena-
turation at 94
C, 45 s annealing at Ta (between 53 and 58
1 min extension at 72
C. The cycles were followed by a ﬁnal exten-
sion step of 10 min at 72
C. We ran the amplimers on a 1.0%
agarose gel to check the speciﬁcity of the reaction. The same oligo-
nucleotides were used for direct sequencing with the BigDye
terminator cycle sequencing chemistry on an Applied Biosystems
ABI PRISM 3130xl Genetic Analyzer. The SIFT website was used for
Culture and Transfection of CHO Cells
CHO cells were maintained at 37
C under 5% CO
modiﬁed Eagle (DMEM; Invitrogen) containing 10% fetal calf
serum (Pan Biotech), penicillin (6 mg/L), and streptomycin
100 mg/L. For transfection, FuGENE HD transfection reagent
(Roche) was used in accordance with the manufacturer’s instruc-
tion. Growing cells were inoculated at about 25% density and
grown to ~75% conﬂuence, transfected with 18 mg DNA (plasmids
as indicated in Figure 4D and prepared according to Ashida et al.
and incubated for two days. Cells were harvested and resuspended
in 30 mM Tris/HCl, pH 7.5, containing 35% (v/v) glycerol, 3 mM
, 1 mM dithiotreitol, 1 mM PMSF, and 1 mM benzamidine
and centrifuged at 48 000 g for 20 min. The pellet was homoge-
nized in 10 mM Tris/HCl, pH 7.5, containing 1 mM MgCl
1 mM dithiotreitol, 1 mM PMSF, and 1 mM benzamidine and
centrifuged as above. The pellet was resuspended in 30 mM Tris/
HCl, pH 7.5, containing 35% (by volume) glycerol, 3 mM
, and 1 mM dithiotreitol and was used as the enzyme source.
Interaction Analysis of DPM1-DPM3
CHO K1 (WT) cells were transiently cotransfected with pME/
FLAG-DPM3 (WT, L85S, L74S/I78T/L85S or empty vector) and
pME/3HSV-DPM1. After 40 hr cultivation, cells were lysed with
0.5% digitonin, and the soluble fractions were subjected to immu-
noprecipitation with anti-FLAG M2-agarose beads (Sigma). From
unbound fractions, 3HSV-DPM1 was recovered with anti-HSV
(Novagen) and protein G-Sepharose beads (GE Healthcare).
Proteins were detected by western blotting with anti-FLAG M2
(Sigma) (31000) or anti-HSV (31000), and second antibody goat
anti-mouse IgG (H
L chain)-HRP (MBL) (34000). Detection was
carried out by LAS1000 (Fuji Film) with a West Pico Chemilumi-
nescent reagent (Pierce).
Immunohistochemistry on Muscle Biopsy
Immunohistochemical examination was performed on 10 mm
frozen sections of a biopsy from the m. rectus femoris, after
10 min ﬁxation in ice-cold acetone. One slide was stained with
hematoxylin-phloxin for morphological comparison. Anti-spec-
trin, anti-merosin (laminin-alpha2) and anti-b-dystroglycan (all
from Novocastra Laboratories Ltd, UK), and IIH6 (gift from K.
Campbell) were used as primary antibodies; Power Vision (Goat
a-Mouse/Rb HRP one component, Immunologic, Duiven) as was
used as a secondary antibody. Slides were stained with AEC (Scy-
Tek Laboratories) for detection.
Analysis of C-Mannosylation of Serum Properdin
Properdin was immunopuriﬁed from 100 ml of plasma or serum
from the DPM3-deﬁcient patient, a CDG type Ie patient, as well
as seven healthy individuals, as described.
of peptides T7 (residues 80–100; amino acid numbering as in Uni-
ProtKB/Swiss-Prot entry P27918) was analyzed by tandem LC–MS
in the multiple reaction monitoring mode.
for the two major glycoforms of peptide T7 were measured, as
well as those for the glycoforms expected when C-mannosylation
would be defective. For comparison of the properdin from different
individuals, the nonglycosylated peptide T28 was measured; the
measurement result reﬂected the amount of injected protein digest.
CD59 Expression on Fibroblasts
Cultured ﬁbroblast cells were incubated on ice for 15 min with
directly ﬂuorochrome-labeled antibodies CD45-ECD and with
CD59-PE directed against a GPI-anchored protein. CD45 was
purchased from Beckman Coulter (Miami, FL, USA), CD59 from
IQP, Groningen, The Netherlands). The cells were washed in PBS
with BSA. Fluorescence was determined on a FC500 ﬂow cytometer
(Beckman Coulter, Hileah, FL, USA). We used mouse IgG1 to detect
nonspeciﬁc binding and used cells incubated without antibodies to
determine autoﬂuorescence. Immunoﬂuorescence was compen-
sated for both background stainings. Data (collected in list mode)
were analyzed with the CXP software (Beckman Coulter).
Discovery of a Congenital Disorder of Glycosylation
Type I in a Patient with Muscular Dystrophy
This female patient, born to healthy Greek parents, was born
at term after an uneventful pregnancy by spontaneous
vaginal delivery. No consanguinity was known. Her birth
weight, length, and head circumference were normal. She
was nondysmorphic. Psychomotor development, growth,
and pubertal development were normal and the patient
was able to attend a regular primary school. A muscle biopsy
was performed at 11 years because of mild muscle weakness
and a waddling gait. Subtle structural changes were found
suggesting a mild myopathy. After Achilles tenotomy, no
further complaints were registered. At 20 years, dilated
cardiomyopathy was diagnosed after episodes of precordial
pain. No signs of cardiac muscle hypertrophy or outﬂow
obstruction were detected. Elevated CK was detected (1500
to 3000, normal < 280 IU/L) with mildly elevated transami-
nases (ASAT 80, ALAT 98, normal < 50 IU/L). A heart muscle
biopsy showed no structural or histological abnormalities. A
second open biopsy of the m. rectus femoris revealed
moderate muscular dystrophy (Figure 2) with ﬁber-size vari-
ation, multiple internal nuclei, necrotic ﬁbers, rimmed vacu-
oles, ﬁber splitting, and interstitial ﬁbrosis. At 21 years, the
patient experienced a stroke-like episode involving the right
temporo-parietal region.Cerebral angiography and ophthal-
mologic evaluation were normal. Metabolic investigations
were normal, but results of transferrin isoelectric focusing
showed an abnormal proﬁle suggesting a CDG type I pattern
(Figure 3A). In addition, isoelectric focusing of serum
thyroxine-binding globulin was slightly abnormal. Baseline
78 The American Journal of Human Genetics 85, 76–86, July 10, 2009
endocrine investigations, APTT, PTT, clotting factors, and
protein C and S were normal. However, on preventive anti-
coagulant therapy the coagulation was difﬁcult to control.
After recovery from the stroke-like episode and on long-
term antidiuretic therapy, the patient developed no further
progression of her cardiac disease. Currently, at the age of
27 years, the patient has a low normal IQ, and normal
head circumference, whereas her body length is below
the tenth centile and weight for height is at the 85
tile. The patient can walk without assistance, but needs
some support when climbing stairs. She has a mild prox-
Figure 2. Muscle Biopsy of m. rectus
femoris at 20 Years
Histochemistry with hematoxylin-phloxin
(HE) and immunohistochemistry with
IIH6 antibody against glycosylated alpha-
dystroglycan and with antibodies against
spectrin, merosin, and b-dystroglycan (b-
DG). A staining of a control muscle biopsy
with IIH6 is shown for comparison. The
scale bar represents 100 mm.
Figure 3. Determination of CDG Subtype
(A) Isoelectric focusing of serum transferrin. Proﬁles of patient and both parents are shown in lanes 3 and 4 þ 5, respectively. Controls are
presented in lane 1 (healthy control), lane 2 (CDG-Ia), lane 6 (CDG-II patient), and lane 7 (CDG-Ie). Numbers 0, 2, 3, and 4 indicate the
(B) Maldi mass spectrometry of permethylated N-glycans from serum expressed as relative percentage of the biantennary glycan at m/z
2794. Structures of the most abundant glycan isoforms are indicated.
(C) ESI-MS of immunopuriﬁed transferrin from serum. The increase of mass 77362 in the patient corresponds to an increase of mono-
glycosylated transferrin (GlcNAc, black square; Fuc, gray triangle; Man, gray circle; Gal, open circle; and NeuNAc, gray diamond).
imal muscle weakness, more promi-
nent on the left, and bilateral pes pla-
nus. There is no signiﬁcant alteration
in muscle mass, except for a mild
pseudohypertrophy of the calf musculature. The deep
tendon reﬂexes are decreased, except for the left extremity.
The Babinski sign is positive on the left; the Gower’s sign is
negative. There is no sign of central or peripheral ataxia.
Brain imaging showed no abnormalities that could be
related to either CDG or alpha-dystroglycanopathy.
In a repeat serum sample, transferrin isoelectric focusing
again showed a mildly abnormal proﬁle with increased
disialo- but also trisialotransferrin. Further mass spectro-
metric investigations of permethylated total serum
N-glycans of the second sample did not indicate marked
The American Journal of Human Genetics 85, 76–86, July 10, 2009 79
abnormalities apart from a very mild increase of m/z 2433
indicating a slight loss of sialic acid (Figure 3B). ESI-MS of
immunopuriﬁed transferrin showed an increase of mono-
glycosylated transferrin with a mass of 77362 Da and an
increased ratio of mono- versus diglycosylated transferrin
(0.21, normal < 0.07). No nonglycosylated transferrin
could be observed (Figure 3C). These results clearly indi-
cated the partial absence of complete biantennary N-
glycans, which is in agreement with a diagnosis CDG
type I and defective N-glycosylation in the ER.
Identiﬁcation of Reduced Dol-P-Man Synthase
Activity in Patient Fibroblasts
After exclusion of CDG-Ia and CDG-Ib by enzyme analysis
in ﬁbroblasts, analysis of lipid-linked oligosaccharides in
ﬁbroblast from the patient showed a normal proﬁle,
excluding additional CDG-Ic, -Id, -Ig, -Ih, and -Il. Formation
, as well as the elon-
gation of Dol-PP-GlcNAc
unremarkable, thereby also ruling out subtypes CDG-Ii, -Ij,
and -Ik. Oligosaccharyltransferase was normal as well.
Because the mass spectrometry studies were clearly indica-
tive for a CDG type I defect in this patient, more extended
investigations were performed, including Dol-P-Man syn-
thase activity measurement. Transfer of radioactive labeled
mannose from GDP-Man to Dol-P in patient ﬁbroblasts
clearly showed a lowered enzymatic activity to ~30% of
Figure 4. Dol-P-Man Synthase Activity
in Fibroblasts and CHO Cells
Microsomal membranes from ﬁbroblasts
or CHO cells were incubated with doli-
chol-P and radioactive GDP-Man and
then Dol-P-Man formation by TLC was
analyzed. (A) shows Dol-P-Man synthase
activity in human ﬁbroblasts. (B) shows
the time course of Dol-P-Man formation
in patient and control ﬁbroblasts. (C)
shows Dol-P-Man synthase activity in
CHO control, CHO2.38 (DPM3), and
lec15 (DPM2) cells. (D) shows Dol-P-Man
synthase activity in CHO2.38 cells after
transient transfection with different
DPM3 plasmids or an empty vector.
DPM3 plasmids used are indicated in
controls (Figures 4A and 4B). Using
this in vitro assay system, we found
the residual Dol-P-Man synthase
activity in ﬁbroblasts of a known
with DPM1 muta-
tions to be similar.
Mutation Analysis Indicated
a Homozygous Missense
Mutation in DPM3
To identify the causative genetic
defect, we subjected the coding
sequences of the three genes encoding subunits of the
Dol-P-Man synthase complex to DNA sequence analysis.
No mutations could be identiﬁed in catalytic subunit
DPM1 and accessory subunit DPM2, while sequencing of
accessory subunit DPM3 displayed a homozygous missense
mutation (c.254T>C [p.Leu85Ser]), present in heterozy-
gous state in both parents (Figure 5A). The missense muta-
tion was not detected in 240 control alleles from the same
The affected amino acid Leu85 is highly conserved
during evolution (Figure 5B) and a change to Ser was pre-
dicted to be intolerant with the SIFT database, which
uses conservation among species and amino acid charac-
teristics for predicting the tolerance level of a certain muta-
tion. Leucine 85 is present on the hydrophobic side of the
coiled-coil domain in DPM3, thereby forming the DPM1-
interacting region together with hydrophobic amino acids
Leu74, Ile78, and Ala81 (Figure 5B).
Conﬁrmation of the Pathogenicity of p.L85S Revealed
by Analyses with CHO2.38 Cells
Given that S. cerevisiae, often used for identifying genetic
defects by complementation of the corresponding ortholo-
does not contain DPM3, DPM3-deﬁcient
CHO2.38 cells were used as heterologous expression
system. Because of the importance of the coiled-coil
domain of DPM3 for DPM1 binding, we previously studied
80 The American Journal of Human Genetics 85, 76–86, July 10, 2009
the effect of mutated amino acids in this region on GPI-
anchor biosynthesis by FACS analysis of CD59 in CHO
Transient transfection of CHO2.38 cells with
DPM3 plasmids containing DeltaC (deletion of C-terminal
coiled-coil peptide), L74S/I78T/L85S triple, or L74S/L85S
double mutations showed no or very low CD59 expres-
sion, whereas L85S transfection restored CD59 expression
to wild-type levels. Protein expression was similar for all
mutants studied. However, the enzymatic activity of
Dol-P-Man synthase in these cells was not studied.
order to prove the pathogenicity of the p.L85S mutation
in our patient, we measured Dol-P-Man synthase activity
after transient transfection of CHO2.38 DPM3-deﬁcient
cells with different DPM3 plasmids (Figure 4D). No enzyme
activity was found in CHO2.38 cells and in DPM2-deﬁ-
cient Lec15 cells (Figure 4C). Transfection of CHO2.38 cells
with full-length DPM3 restored Dol-P-Man synthase
activity. In contrast, transfection with plasmids DeltaC
and the L74S/L85S double mutant did not show any
complementation, whereas L85S transfection resulted in
slight Dol-P-Man production. Previously,
capacity of DPM3 to DPM1 has been studied by cotransfec-
tion of CHO K1 cells with 3HSV-DPM1 and FLAG-DPM3,
followed by immunoprecipitation with anti-FLAG beads.
DeltaC and L74S/I78T/L85S triple mutants lost their capa-
bility of interacting with DPM1 resulting in cytoplasmic
degradation of DPM1 via ubiquitination in the protea-
some. In the present study, similar experiments were per-
formed with the L85S single mutant (Figure 6). The
amount of 3HSV-DPM1 bound to FLAG-DPM3(L85S) was
signiﬁcantly reduced compared to that bound to FLAG-
DPM3(WT), under 0.5% digitonin solubilization (lane 2
versus lane 1). Furthermore, the total expressed amount
of 3HSV-DPM1 (bound plus unbound) was equivalent
after transfection with FLAG-DPM3(L85S) and FLAG-
DPM3(L74S/I78T/L85S) (lane 3) or empty vector (lane 4),
whereas total 3HSV-DPM1 was much higher after transfec-
tion with FLAG-DPM3(WT). These results suggest that the
p.L85S mutation in DPM3 in our patient may cause a reduc-
tion in DPM1-binding capacity and DPM1 stability in vivo.
Investigation of the Four Dol-P-Man-Dependent
Glycosylation Pathways Reveals a Strong Effect
As indicated in Figure 1, four different glycosylation routes
are initiated by or depend on the transfer of mannose to
protein or lipid-linked glycans in the ER. To investigate
the O-mannosylation pathway, we performed immunohis-
tochemical staining of a patient muscle biopsy by using
the IIH6 antibody speciﬁc for O- mannosylation-depen-
dent Laminin-binding glycans on alpha-dystroglycan.
IIH6 staining was overall strongly reduced as compared
Figure 5. Mutation Analysis and DPM3 Plasmids Used
(A) Mutation analysis of the Dol-P-Man synthase genes DPM1-3 showed a c.254T>C (p.L85S) missense mutation in DPM3. Forward
sequences are shown.
(B) Location of leucine 85 in the coiled-coil domain of the C-terminal peptide of DPM3 and its conservation. Hydrophobic amino acids
important for DPM1 binding are shown in bold.
(C) Structure of the plasmids used for transient transfection of CHO2.38 cells as shown in Figure 3D and DPM1-DPM3-binding studies as
shown in Figure 5.
The American Journal of Human Genetics 85, 76–86, July 10, 2009 81
to staining of the sarcolemma in a control muscle biopsy.
Immunohistochemical analyses with antibodies against
beta-dystroglycan, merosin (laminin-alpha2), and spectrin
showed normal sarcolemmal staining of the patient biopsy
(Figure 2). As described above, N-glycosylation of the
serum protein transferrin was found to be mildly affected
as studied by isoelectric focusing and mass spectrometry.
C-mannosylation was investigated by mass spectrometric
analysis of serum properdin that can be C-mannosylated
In controls, the most abundant forms
of this peptide are MM00 (two C-mannosylated tryptho-
phans, not O-fucosylated) and MMFG (two C-linked
mannoses and an O-fucosylated threonine) as indicated
in Figure 7A. The peptides with a single mannose residue
(M0FG and M000) are present in much smaller amounts,
whereas the nonmannosylated forms were not detected.
In the DPM3-deﬁcient patient (Figure 7B) and the CDG-
Ie (MIM 608799) patient (data not shown), both doubly
C-mannosylated species were found, but no increase was
observed in the less-mannosylated species with no or
a single C-linked mannose residue. If reduced Dol-P-Man
levels would have affected the C-mannosylation of serum
properdin, an increase of less-mannosylated species would
have been expected. Interestingly, an increase in fucosyla-
tion state was observed in the mannosylated peptides in
the CDG-Ie and DPM3-deﬁcient patients. The results indi-
cate that C-mannosylation of peptide T7 from serum
properdin is unaltered in both patients as compared to
the control sample and that, for an as yet unknown reason,
O-fucosylation is signiﬁcantly more pronounced
(Figure 7B). The inﬂuence of DPM3 mutations on cell-
surface CD59 expression as an indicator of GPI-anchor
biosynthesis has been studied previously. Transfection of
CHO2.38 cells with DPM3 plasmid containing the L85S
mutation showed normal CD59 expression, whereas trans-
fection with equal levels of the more severe DeltaC and
L74S/I78T/L85S triple mutants clearly led to reduced
Analysis of CD59 expression on
patient ﬁbroblasts showed a similar result. DPM3-deﬁcient
ﬁbroblasts showed an equal expression of CD59 as
compared to control ﬁbroblasts, whereas ﬁbroblasts from
a CDG-Ie patient showed a slight reduction in CD59
expression (Figure 7C).
In this report, we present a genetic glycosylation disorder
in the group of alpha-dystroglycanopathies in a patient
with mild muscular dystrophy and biochemical features
of a congenital disorder of glycosylation. So far, CDG and
dystroglycanopathies have been regarded as two separate
groups of glycosylation disorders, each caused by deﬁ-
ciency of enzymes affecting a particular glycosylation
pathway: N-glycosylation in CDG and O-mannosylation
in the alpha-dystroglycanopathies. Here, we show that a
mild defect in the accessory protein DPM3 of Dol-P-Man
synthase, required for four different glycosylation routes,
can lead to an isolated phenotype of muscular dystrophy
most likely resulting from deﬁcient O-mannosylation of
Dol-P-Man is the glycosyl donor for all mannosylation
reactions taking place on the luminal side of the ER. Cell
models exist with a deﬁciency of the Dol-P-Man synthase
subunits or MPDU1, a protein required for Dol-P-Man and
Dol-P-Glucose utilization of which the speciﬁc biochemical
function remains to be determined. In the mouse Thy-1
CHO Lec15 cells (DPM2),
and Lec35 cells (MPDU1),
Dol-P-Man leads to Dol-P-P-GlcNAc
in the ER and to reduced protein N-glycosylation. The
absence of the expression of GPI-anchored proteins at the
plasma membrane indicates defective GPI-anchor biosyn-
thesis in the ER. C-mannosylation of RNase has been inves-
tigated in Lec35 cells
and was shown to be strongly
reduced. Because Dol-P-Man is required for C-mannosyla-
tion and O-mannosylation,
these types of glycosylation
can be expected to be abnormal in all Dol-P-Man syn-
thase-deﬁcient cell lines. The mild biochemical phenotype
in this DPM3-deﬁcient patient allowed us to investigate
subtle effects of reduced Dol-P-Man availability on the
different glycosylation pathways. O-mannosylation
seemed to be most affected, as revealed by strongly reduced
glycosylation-speciﬁc IIH6 staining of a muscle biopsy. It
can be expected that CDG-Ie patients with DPM1 mutations
suffer from a similar or even more severe deﬁciency of
Figure 6. Binding of DPM1 and DPM3
CHO K1 cells were transiently cotransfected with pME/3HSV-
DPM1 and either pME/FLAG-DPM3(WT) (lane 1), pME/FLAG-
DPM3(L85S) (lane 2), pME/FLAG-DPM3(L74S/I78T/L85S) (lane
3), or an empty vector (lane 4). After 40 hr culture, the cells were
lysed with 0.5% digitonin and the complex of FLAG-DPM3 and
3HSV-DPM1 was immunoprecipitated (IP) with anti-FLAG beads
(A). Unbound 3HSV-DPM1 was recovered from the supernatant
of FLAG IP (FLAG sup) with anti-HSV and protein G-beads (B).
Proteins were detected by western blotting (WB) with indicated
82 The American Journal of Human Genetics 85, 76–86, July 10, 2009
alpha-DG mannosylation. Analysis of O-mannosylation
has so far not been reported in the few known CDG-Ie
patients, although elevated CK was a common symptom
in all reported cases. The isoelectric-focusing result with a
mildly abnormal transferrin pattern indicates a slight effect
on protein N-glycosylation. In the analysis of lipid-linked
oligosaccharides, no abnormalities could be detected under
low-glucose conditions in cultured ﬁbroblasts of our pa-
tient, whereas in CDG-Ie ﬁbroblasts, Dol-PP-GlcNAc
Possibly, cell-culturing conditions for [
Man incorporation for LLO analysis prevented the detec-
tion of mild abnormalities as present in the cell line of our
patient. The abnormal N-glycosylation due to slightly
reduced Dol-P-Man levels could be the result of two effects,
i.e., reduced incorporation of mannose by the ER manno-
syltransferases and also a reduced stimulation of UDP-
GlcNAc:Dol-P GlcNAc-P-transferase by Dol-P-Man. This
ﬁrst enzyme in N-glycan biosynthesis is regulated by Dol-
Regulation of the other Dol-P-Man-depen-
dent glycosylation pathways by Dol-P-Man concentrations
is unknown. The normal GPI-anchor biosynthesis in
DPM3(L85S) mutant cells suggests that this mild mutation
is insufﬁcient for affecting GPI-anchor formation. CD59
has been found reduced in cultured ﬁbroblasts of a CDG-
indicating an effect on GPI-anchor formation
in the case of a more severe Dol-P-Man synthase deﬁciency.
Finally, the normal results for serum properdin C-mannosy-
lation indicate that the residual Dol-P-Man levels are sufﬁ-
cient for normal C-mannosylation in DPM3-deﬁcient and
CDG-Ie patients, whereas the severe Dol-P-Man deﬁciency
in Lec35 cells is detrimental for C-mannosylation.
increased fucosylation of serum properdin is not readily
explained by lowered Dol-P-Man levels. Possibly, an
increased expression of protein O-fucosyltransferase as
response to disease could explain this alteration. Similarly,
increased expression of a1,6-fucosyltransferase has been
postulated as cause for increased N-glycan core fucosylation
in CDG-I patients.
In addition, a potentially increased
pool of GDP-Man due to lowered Dol-P-Man levels could
increase the conversion of GDP-Man into GDP-Fucose.
Our results suggest that from all four glycosylation routes,
O-mannosylation of alpha-dystroglycan catalyzed by
POMT1 and POMT2 is most severely affected by decreased
Dol-P-Man synthase activity. A possible explanation could
be that the Km for this enzymatic reaction is higher than
that for the other glycosylation routes. A reduction in Dol-
P-Man levels would ﬁrst affect O-mannosylation with
a smaller effect on the other glycosylation routes. In
(MIM 600737) and Sialuria patients
269921), decreased or increased levels of the sialic acid
donor CMP-NANA mainly affected O-linked glycans and
to a lesser extent N-glycans. At least in the case of Sialuria,
Figure 7. Analysis of C-Mannosylation and GPI-Anchored Protein CD59
(A) Structure of the two main glycoforms of peptide T7 from properdin.
(B) Properdin was immunopuriﬁed from serum or plasma of patients and healthy controls. C-mannosylated tryptic peptides were exam-
ined by tandem LC-MS in the multiple reaction monitoring (MRM) mode. Traces for the MRM transitions (for the precursor/y9 pair) are
shown for a healthy control and patient. Peak identity was assigned on the basis of the presence of the other transitions speciﬁc for the
different glycoforms. The signal intensities were normalized relative to the nonglycosylated peptide T28, thereby allowing comparison
(C) CD59 expression on control and patient ﬁbroblasts by FACS analysis. A comparison is shown of a control ﬁbroblast with DPM3-
deﬁcient ﬁbroblasts (left panel) and CDG-Ie ﬁbroblasts (right panel).
The American Journal of Human Genetics 85, 76–86, July 10, 2009 83
this difference has been attributed to a higher Km value of
the mucin core 1 O-glycan a2,6-sialyltransferase compared
to other sialyltransferases. Although no experimental
evidence exists, a regulatory role of DPM3 for alpha-dystro-
glycan O-mannosylation could also explain the more severe
effect on O-mannosylation in comparison with the other
glycosylation pathways. For example, the regulatory role
of DPM2 in GPI-anchor biosynthesis
lead to different biochemical abnormalities and a different
clinical phenotype in DPM2-deﬁcient patients. In view of
the considerable residual enzymatic activity of Dol-P-Man
synthase in our patient, it may be anticipated that patients
with more severe mutations in DPM3 will present with
a more severe multisystem presentation already in child-
hood. The full phenotypic spectrum will evolve when
more patients with a DPM3 defect are identiﬁed.
The disease course in our patient with a slowly progres-
sive muscle disease and elevated CK, dilated cardiomyop-
athy, and stroke-like episode, but no congenital brain or
eye malformations, is signiﬁcantly milder than in WWS
or MEB (MIM 253280) disease and mimics dystroglycanop-
athy patients at the milder end of the spectrum similar to
LGMD2I caused by loss of fukutin-related protein. Direct
DNA sequencing of all coding exons and splice sites of
FKRP in our patient did not show any mutations, whereas
no homozygosity was identiﬁed for any of the six known
dystroglycanopathy genes with coupled microsatellite
markers (data not shown). In CDG-I patients, muscular
dystrophy has not been reported explicitly, although severe
muscle weakness was observed in dolichol kinase-deﬁcient
(CDG-Im [MIM 610768]). Progressive muscle
disease with elevated CK was found in a few cases of CDG-
Dilated cardiomyopathy has been described in
milder cases of alpha-dystroglycan-deﬁcient patients with
a phenotype of limb-girdle muscular dystrophy, demon-
strating an early-onset dilated cardiomyopathy already in
Interestingly, cardiomyopathy has been re-
ported in several children diagnosed with CDG, in most
reported cases with a congenital hypertrophic or obstructive
type, mostly associated with edema collection or hydrops.
The few CDG cases described so far with a dilated cardiomy-
opathy included the dolichol-kinase-deﬁcient patients.
The occurrence of a stroke-like episode has been observed
In our patient, a stroke-like episode was rather
peculiar; no cardiac thrombus or vascular anomaly was
observed, cardiac function and blood pressure were within
the normal range, and coagulation studies were normal as
well. Still, the challenge to sustain an adequate level of anti-
coagulation after the vascular incident suggested an imbal-
ance between different coagulants and anticoagulants. The
same phenomenon has been observed in CDG-Ia (MIM
212065) patients who have minor protein C and S abnor-
malities and normal APTT/PT values, and who are receiving
preventive anticoagulation therapy.
Recently, it has been discussed that the main clinical
features in CDG-Id (ALG3 [MIM 601110]),
(DPM1), and CDG-In ( RFT1 [MIM 612015])
explained by deﬁcient N-glycosylation because of consid-
erable overlap in the severity of symptoms. However, chil-
dren with DPM1 mutations had severe congenital visual
loss, optic atrophy, and seizures,
and one of the chil-
dren was diagnosed with congenital frontal cortical hypo-
These observations support a signiﬁcant overlap
with the dystroglycanopathies caused by POMGnT1,
POMT1, and Fukutin mutations and are not common in
N-glycosylation disorders. Children diagnosed with muta-
tions in the MPDU1 gene (CDG-If [MIM 609180])
have reduced Dol-P-Man levels. The phenotype in two
MPDU1-defective patients was reported as early-onset
visual loss and severe epilepsy and is comparable to the
presentation observed in children diagnosed with CDG-
Ie. So far, no muscular dystrophy, cardiomyopathy, or CK
elevation was described in CDG-If. The presence of skin
abnormalities (erythroderma, hyperkeratosis, or ichtiosis)
was absent in the so far described cases of Dol-P-Man deﬁ-
ciency. A recently discovered defect in GPI-anchor biosyn-
thesis caused by mutations in the PIGM gene (MIM
610293), a mannosyltransferase, presents with refractory
absence seizures and recurrent venous thrombosis.
estingly, in DPM1-deﬁcient patients, epilepsy is a promi-
nent clinical feature, even though epilepsy occurs in other
CDG subtypes, such as CDG-Ic. Our mild DPM3-deﬁcient
case did not present with epilepsy and we could not detect
a GPI deﬁciency. Although no genetic disorders are
currently associated with defective C-mannosylation, the
normal results of serum properdin suggest that abnormal
C-mannosylation does not contribute to the clinical
phenotype in CDG-Ie and our patient.
In conclusion, it seems that in the very mild end of the
clinical spectrum of Dol-P-Man-deﬁcient patients, deﬁ-
cient O-mannosylation of alpha-dystroglycan is the most
dominant factor in determining the clinical outcome,
whereas in the more severely affected patients, other
symptoms are present such as clotting abnormalities and
epilepsy that could be related to, e.g., N-glycosylation
and GPI synthesis defects. We have identiﬁed a genetic
deﬁciency that we propose to name
a patient with muscle dystrophy leading to a new cause
for the mild spectrum of alpha-dystroglycanopathy
patients, thereby bridging the congenital disorders of
glycosylation with the dystroglycanopathies.
Recently, it has been suggested to leave the alphabetical
numbers for assigning CDG subtype and include gene
names to indicate function.
We propose to keep CDG-I
because this indicates an ER glycosylation defect and to
include the gene name for adding function: CDG-
I(DPM3). According to current CDG nomenclature, this
new defect should be described as CDG-Io.
We would like to acknowledge the parents of the CDG-Ie patient
and M. Garcia-Silva for providing the CDG-Ie ﬁbroblast reference
cell line. We gratefully acknowledge K. Campbell for the kind gift
84 The American Journal of Human Genetics 85, 76–86, July 10, 2009
of IIH6 antibody. LC-MS experiments of immunopuriﬁed trans-
ferrin were performed by J. O’Brien. K. Huyben, K. Verrijp, and
E. van Kaauwen are acknowledged for technical assistance. This
work was supported by grants from the Deutsche Forschungsge-
meinschaft and the Ko
rber-Stiftung to L.L. and Euroglycanet
(LSHM-CT2005-512131) and Metakids to D.J.L. and R.A.W. The
work at the Friedrich Miescher Institute was supported by the
Novartis Research Foundation.
Received: April 3, 2009
Revised: June 4, 2009
Accepted: June 12, 2009
Published online: July 2, 2009
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