A splicing mutation in the alpha/beta GlcNAc-1-phosphotransferase gene results in an adult onset form of mucolipidosis III associated with sensory neuropathy and cardiomyopathy.
ABSTRACT A 47-year-old female who presented with a dilated cardiomyopathy and mild neuropathy was found to have pseudoHurler polydystrophy (mucolipidosis III). The serum lysosomal enzymes were strikingly elevated and GlcNAc-1-phosphotransferase activity in the patient's fibroblasts was 3% of normal. Sequence analysis of the patient's genomic DNA revealed a homozygous mutation of the last nucleotide of the 135-bp exon 7 of the phosphotransferase gene encoding the alpha/beta subunits, resulting in aberrant splicing and skipping of this exon. Remarkably, none of the skeletal and connective tissue anomalies characteristic of the disease were present. This case is the first example of mucolipidosis III presenting in an adult patient and further broadens the clinical spectrum of the disease.
- SourceAvailable from: Jules G Leroy[Show abstract] [Hide abstract]
ABSTRACT: Mucolipidosis (ML) II and ML IIIα/β are allelic autosomal recessive metabolic disorders due to mutations in GNPTAB. The gene encodes the enzyme UDP-GlcNAc-1-phosphotransferase (GNPT), which is critical to proper trafficking of lysosomal acid hydrolases. The ML phenotypic spectrum is dichotomous. Criteria set for defining ML II and ML IIIα/β are inclusive for all but the few patients with phenotypes that span the archetypes. Clinical and biochemical findings of the 'intermediate' ML in eight patients with the c.10A>C missense mutation in GNPTAB are presented to define this intermediate ML and provide a broader insight into ML pathogenesis. Extensive clinical information, including radiographic examinations at various ages, was obtained from a detailed study of all patients. GNPTAB was sequenced in probands and parents. GNPT activity was measured and cathepsin D sorting assays were performed in fibroblasts. Intermediate ML patients who share the c.10A>C/p.K4Q mutation in GNPTAB demonstrate a distinct, consistent phenotype similar to ML II in physical and radiographic features and to ML IIIα/β in psychomotor development and life expectancy. GNPT activity is reduced to 7-12% but the majority of newly synthesized cathepsin D remains intracellular. The GNPTAB c.10A>C/p.K4Q missense allele results in an intermediate ML II/III with distinct clinical and biochemical characteristics. This delineation strengthens the utility of the discontinuous genotype-phenotype correlation in ML II and ML IIIα/β and prompts additional studies on the tissue-specific pathogenesis in GNPT-deficient ML.European Journal of Human Genetics advance online publication, 18 September 2013; doi:10.1038/ejhg.2013.207.European journal of human genetics: EJHG 09/2013; · 3.56 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Multiple sulfatase deficiency (MSD), mucolipidosis (ML) II/III and Niemann–Pick type C1 (NPC1) disease are rare but fatal lysosomal storage disorders caused by the genetic defect of non-lysosomal proteins. The NPC1 protein mainly localizes to late endosomes and is essential for cholesterol redistribution from endocytosed LDL to cellular membranes. NPC1 deficiency leads to lysosomal accumulation of a broad range of lipids. The precise functional mechanism of this membrane protein, however, remains puzzling. ML II, also termed I cell disease, and the less severe ML III result from deficiencies of the Golgi enzyme N-acetylglucosamine 1-phosphotransferase leading to a global defect of lysosome biogenesis. In patient cells, newly synthesized lysosomal proteins are not equipped with the critical lysosomal trafficking marker mannose 6-phosphate, thus escaping from lysosomal sorting at the trans Golgi network. MSD affects the entire sulfatase family, at least seven members of which are lysosomal enzymes that are specifically involved in the degradation of sulfated glycosaminoglycans, sulfolipids or other sulfated molecules. The combined deficiencies of all sulfatases result from a defective post-translational modification by the ER-localized formylglycine-generating enzyme (FGE), which oxidizes a specific cysteine residue to formylglycine, the catalytic residue enabling a unique mechanism of sulfate ester hydrolysis. This review gives an update on the molecular bases of these enigmatic diseases, which have been challenging researchers since many decades and so far led to a number of surprising findings that give deeper insight into both the cell biology and the pathobiochemistry underlying these complex disorders. In case of MSD, considerable progress has been made in recent years towards an understanding of disease-causing FGE mutations. First approaches to link molecular parameters with clinical manifestation have been described and even therapeutical options have been addressed. Further, the discovery of FGE as an essential sulfatase activating enzyme has considerable impact on enzyme replacement or gene therapy of lysosomal storage disorders caused by single sulfatase deficiencies.Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 04/2009; · 5.30 Impact Factor
Chapter: Mucolipidosis III Gamma[Show abstract] [Hide abstract]
ABSTRACT: Mucolipidosis III gamma (ML III gamma) is a slowly progressive disorder characterized by childhood onset of radiographic evidence of mild to moderate dysostosis multiplex; joint stiffness and pain initially in the shoulders, hips, and fingers; and gradual mild coarsening of facial features. Cardiorespiratory complications (restrictive lung disease, thickening and insufficiency of the mitral and aortic valves, left ventricular hypertrophy) can be significant. A few affected individuals have mild cognitive impairment. Because ML III gamma has only recently been distinguished from the more common ML III alpha/beta, previously published descriptions of ML III may have inadvertently included both of these disorders. Thus, much is to yet be learned about the specific manifestations and natural history of ML III gamma. In ML III gamma the activity of nearly all lysosomal hydrolases is up to tenfold higher in plasma and other body fluids than in normal controls because of inadequate targeting to lysosomes. ML III gamma is caused by mutations in GNPTG, which encodes the gamma subunit of the enzyme UDP-N-acetylglucosamine: lysosomal hydrolase N-acetylglucosamine 1-phosphotransferase. (Of note, the alpha and beta subunits of this enzyme are encoded by GNPTAB, mutations in which cause ML III alpha/beta.) Clinically available molecular genetic testing of GNPTG detects two disease-causing mutations in more than 95% of individuals with ML III gamma. Treatment of manifestations: Low-impact physical therapy is usually well tolerated. Carpal tunnel signs may require tendon release. In late childhood or early adolescence relief of hip pain becomes important; in older adolescents and adults bilateral hip replacement has been successful. Later in the disease course management focuses on relief of general bone pain associated with osteoporosis. In severe cases, when significant valvular dysfunction disrupts ventricular function, valve replacement should be seriously considered. Prevention of secondary complications: Because of concerns about airway management, surgical intervention should be undertaken only in tertiary care settings with pediatric anesthesiologists and intensivists. Persons with valvular involvement should be given antibiotic prophylaxis before minor and major surgical procedures (including dental procedures) to prevent bacterial endocarditis. Surveillance: Yearly outpatient clinic visits unless cardiac and/or respiratory monitoring need more frequent attention; annual orthopedic assessment; annual ophthalmology evaluation to monitor for corneal opacities and the possibility of adult-onset retinal degeneration; ERG for those with suspected retinal abnormalities; annual monitoring by echocardiogram for progressive valvular insufficiency; DEXA scan every five years to monitor for metabolic bone disease. Agents/circumstances to avoid: Stretching exercises because they are ineffective, painful, and may damage the surrounding joint capsule and adjacent tendons. ML III gamma is inherited in an autosomal recessive manner. At conception, each sib of an affected individual has a 25% chance of being affected, a 50% chance of being an asymptomatic carrier, and a 25% chance of being unaffected and not a carrier. Carrier testing for at-risk relatives and prenatal diagnosis for pregnancies at increased risk are possible if the disease-causing mutations in the family are known.GeneReviews™, Edited by Roberta A Pagon, Thomas D Bird, Cynthia R Dolan, Karen Stephens, Margaret P Adam, 01/2010; University of Washington, Seattle.
American Journal of Medical Genetics 132A:369–375 (2005)
A Splicing Mutation in the a/b GlcNAc-1-
Phosphotransferase Gene Results in an Adult
Onset Form of Mucolipidosis III Associated
With Sensory Neuropathy and Cardiomyopathy
Richard A. Steet,1Roger Hullin,2Mariko Kudo,3Michele Martinelli,2Nils U. Bosshard,5
Thomas Schaffner,4Stuart Kornfeld,1* and Beat Steinmann5
1Department of Internal Medicine, Washington University School of Medicine, Saint Louis, Missouri
2Department of Cardiology, University Hospital, Bern, Switzerland
3Genzyme Corporation, Oklahoma City, Oklahoma
4Institute of Pathology, Medical School, University Bern, Bern, Switzerland
5Division of Metabolism and Molecular Pediatrics, University Children’s Hospital, Zurich, Switzerland
A47-year-old female who presented with adilated
cardiomyopathy and mild neuropathy was found
to have pseudoHurler polydystrophy (mucolipi-
dosis III). The serum lysosomal enzymes were
strikingly elevated and GlcNAc-1-phosphotrans-
ferase activity in the patient’s fibroblasts was 3%
of normal. Sequence analysis of the patient’s
genomic DNA revealed a homozygous mutation
of the last nucleotide of the 135-bp exon 7 of the
phosphotransferase gene encoding the a/b sub-
units, resulting in aberrant splicing and skipping
of this exon. Remarkably, none of the skeletal and
connective tissue anomalies characteristic of the
and further broadens the clinical spectrum of the
? 2005 Wiley-Liss, Inc.
KEY WORDS:mucolipidosis III; lysosomal sto-
rage disorder; GlcNAc-1-phospho-
I-cell disease (mucolipidosis II, ML II; MIM 252500) and
pseudoHurler polydystrophy (mucolipidosis III, ML III; MIM
252600) are autosomal recessive lysosomal storage disorders
defined by decreased activity of the N-acetylglucosamine
enzyme in the synthesis of the mannose 6-phosphate target-
ing signal for lysosomal enzymes [Kornfeld and Sly, 2001].
Unlike lysosomal storage disorders affecting single enzymes
in a catabolic pathway, these disorders are characterized by
impaired sorting of multiple enzymes to lysosomes and their
subsequent secretion from cells. ML II and ML III share
similar clinical features including skeletal abnormalities.
Mucolipidosis II is clinically more severe, often noted shortly
after birth, whereas mucolipidosis III has a later onset,
normally in early childhood, and a slower course.
The targeting of acid hydrolases to lysosomes requires the
synthesis of mannose 6-phosphate residues on the oligosac-
charides of these enzymes [Kornfeld, 1986]. This post-transla-
tional modification of high-mannose type oligosaccharides of
enzymes, GlcNAc-1-phosphotransferase and phosphodiester
glycosidase (uncovering enzyme). The first enzyme catalyzes
the transfer of GlcNAc-1-phosphate to mannose residues on
the oligosaccharides; the second enzyme removes the outer
GlcNAc residues to produce the phosphomonoester mannose-
6-phosphate [Kornfeld et al., 1982]. GlcNAc-1-phosphotrans-
ferase exists as a complex containing three distinct subunits
(a2, b2, g2) [Bao et al., 1996]. The a/b subunits, encoded by one
gene, contain the catalytic activity of the enzyme while the
regulatory gsubunit, encoded byaseparate gene, is thought to
be responsible for binding specifically to lysosomal enzymes.
with dilated cardiomyopathy and mild neuropathy but none of
asplicejunction ofthe a/bsubunit GlcNAc-1-phosphotransfer-
ase gene was identified in the patient that causes skipping of
exon 7 and results in deficient enzyme activity in patient
fibroblasts. Our findings suggest that genetic variants of ML
MATERIALS AND METHODS
Primary cell lines were established from patient and control
skinfibroblasts.Thefibroblasts weremaintainedat 378Cinan
atmosphere of 5% CO2in aMEM supplemented with 10% fetal
All reagents were obtained from Sigma, Saint Louis,
Missouri, USA unless otherwise noted. [32P]-UDP-GlcNAc
was kindly provided as a gift by Dewan Haque at Genzyme
(Oklahoma City, OK). Cathepsin D anti-serum was provided
by Walter Gregory (Kornfeld Laboratory, Washington Uni-
versity School of Medicine, Saint Louis, Missouri).
The activity of the following enzymes in serum, leukocytes,
and fibroblasts was determined fluorimetrically according
Richard A. Steet and Roger Hullin contributed equally to this
*Correspondence to: Stuart Kornfeld, Department of Internal
Medicine, Washington University School of Medicine, 660 S.
Euclid Avenue, Saint Louis, MO 63110.
Received 25 May 2004; Accepted 15 October 2004
? 2005 Wiley-Liss, Inc.
to Galjaard : a-iduronidase (EC 18.104.22.168), a-fucosidase
(EC 22.214.171.124), a-galactosidase (EC 126.96.36.199), b-galactosidase
188.8.131.52), and b-hexosaminidase (EC 184.108.40.206). Assay of acid
phosphatase (EC 220.127.116.11) was done according to Beutler et al.
GlcNAc-1-phosphotransferase activity was measured in
microsomal preparations from fibroblasts as described earlier
[Reitman and Kornfeld, 1981]. Briefly, cells were lysed by
sonication in Tris buffer pH 7.5 containing 0.25M sucrose and
microsomes prepared by ultracentrifugation. Reactions using
a-methylmannoside as an acceptor and [32P]-UDP-GlcNAc
as substrate were conducted in the following reaction buffer:
50 mM Tris pH 7.5, 0.3% Lubrol, 50 mM N-acetylglucosamine,
250 mM DTT, 20 mg/ml bovine serum albumin, 10 mM sodium
molybdate, 1 mM MgCl2, and 1 mM MnCl2. Activity units are
calculated as pmoles of GlcNAc-[32P] transferred/hr. Protein
concentration was measured using the micro BCA protein
assay kit (Pierce, Rockford, IL, USA).
Analysis of Phosphorylated
Fibroblasts cultures (80%–90% confluent) were labeled in
(ICN Radiochemicals, Irvine, CA) for 4 hr followed by a 1 hr
chase in complete media. Cell pellets were harvested and
extracted sequentially with 2?2 ml chloroform:methanol
(2:1), 50% ethanol, and chloroform:methanol:water (10:10:3).
The remaining protein pellet was solubilized in 50 mM Tris
pH 6.8, 0.5% SDS by boiling and incubated overnight at 378C
with 2 U of endoglycosidase H (New England Biolabs, Beverly,
MA, USA) following addition of citrate buffer, pH 5.5. Endo
H-sensitive oligosaccharides were separated from resistant
material using a Centricon10 filtration unit. The Endo H-
sensitive oligosaccharides were analyzed by QAE-sephadex
chromatography following mild acid hydrolysis (pH 2.0, 1008C
for 30 min) which removes any covering GlcNAc residues
[Cantor et al., 1992]. In some cases, samples were digested
overnight with 10 U of alkaline phosphatase prior to analysis.
Cathepsin D Sorting
Metabolic labeling and sorting of cathepsin D in control and
patient fibroblasts was done as previously described [Braulke
etal., 1987]. Briefly, cell monolayers at ?70%–80% confluency
were washed twice with cysteine/methionine-free DMEM
and labeled for 1 hr with 1 ml of 1 mCi/ml Tran35S-label
(ICN Radiochemicals) in the presence of 20 mM Hepes and
5 mM mannose 6-phosphate. Excess unlabeled methionine
(10 mM final concentration) was added to initiate a 4 hr chase.
The amount of cathepsin D sorted was determined by over-
night immunoprecipitation of equivalent aliquots of the lysed
cell and media high-speed supernatants at 48C with rabbit
anti-human cathepsin D antiserum and protein A-Sepharose
beads (Repligen, Cambridge, MA). After five washes with
buffer containing 1M KCl and 1% Triton X-100, immuno-
precipitates were subjected to 10% SDS–PAGE under non-
reducing conditions. Gels were treated with EN3HANCE
(PerkinElmer Life Sciences, Boston, MA, USA), dried, and
exposed to a Phosphoimager screen (Molecular Dynamics,
Piscataway, NJ, USA). Quantitation of the various bands was
performed using Phosphoimager software. The percentage of
cathepsin D sorted was calculated as the ratio of radioactivity
in the processed form to the sum of the processed and secreted
The sequence of GlcNAc-1-phosphotransferase g subunit
[Raas-Rothschild et al., 2000] with the exception of the
first primer. 50-CTGGCGCGGCTCCTGTTGCTC-30was used
instead. In all cases, total RNA was isolated using the Trizol
method and single-stranded cDNA generated using Super-
script RTII system (Invitrogen, Carlsbad, CA, USA). PCR
amplification and sequencing of the gene encoding GlcNAc-1-
phosphotransferase subunits a and b was done using primer
sets to be reported elsewhere (Kudo et al., submitted). The
identified by RT-PCR using primer sets specific for regions
exon 7 (EX7R: 50-GCATTAGCACTAATCCAGGG-30) as well as
8 (EX8R: 50-CTGCTTAGACTGGCTGATGG-30). Skipping of
exon 7 in the patient0s mRNA was confirmed by direct
sequencing of amplified PCR products following synthesis of
single-stranded cDNA (primers: 50-GTTGAAGATGCCCA-
GAAGC-30). The presence of the homozygous mutation in the
patient and identification of carriers for this mutation were
verified by Spe1 digestion of a PCR product spanning the
mutated base generated using the following primers: 50-
was prepared from blood cells using a Qiagen DNA extrac-
The sequence environment of all splice mutations was
analyzed using the SSPNN program, and a splice-site score
(SSS) was obtained (URL address: http://www.fruitfly.org/
Patient HE, a 47-year-old woman, was the second of three
children born to parents originating from the same Swiss
valley (Emmental) (Fig. 1); consanguinity of the parents could
not be ascertained positively. The patient’s childhood and
adolescence were normal. She finished secondary school,
married, and delivered two healthy boys. In 1997, the patient
was admitted for a non-familial subacute tetraspastic
syndrome in combination with hyposensibility and paresthe-
sias of the distal upper and lower extremities. Neurological
members; squares, male family members; symbols with a slash, deceased
problems. Carriers detected by genotyping are indicated with a þ/? symbol.
Pedigree of ML III patient. Circles denote female family
370Steet et al.
workup initially revealed a sensory neuropathy and months
later, a discrete lower extremity motor neuropathy was also
Sensory neuropathy was documented by orthodromic neu-
rography comparing left and right sural nerve. The affected
side was demonstrated by slower impulse propagation (39 vs.
45m/sec), reduced amplitude ofimpulse (1.18 vs.7.25mV), and
increased number of phases (14 vs. 4). Evoked motor nerve
potentials in upper and lower extremities were normal but
demonstrated a discrete asymmetry with respect to the
from interventricular septum; Sudan black (a), transmission electron
microscopy (TEM; b–e). Dark, coarse sudanophilic inclusions (arrow in a)
are rich in phospholipids resistant to solvent extraction by the embedding
procedure. The hemalum-eosin stain (not shown) revealed the same, often
ellipsoid paranuclear structures as yellow to tan inclusions, similar in color
to lipofuscin. This unusual finding prompted the study by TEM. The larger
inclusions consisted of aggregated whorls of osmiophilic membranes
Clinical features of the patient. a–e: Endomyocardial biopsy
reminiscent of myelin figures (arrow in b). Smaller structures comprised
degenerate lysosomal organelles (arrow in c) and aggregated or isolated
structures bound by a single membrane containing stacked electron-dense
membranes (arrows in d and e) similar in size to mitochondria (left in
panel e). f–h: Cardiac MRI. Arrows mark dilated right ventricle (f), dilated
right ventricular outflow tract (g), and thinned left ventricular apex (h).
(i) Unremarkable X-rays film of the right hand.
Adult Onset Form of Mucolipidosis III 371
anterior tibial muscle. Spinal cord and cerebral magnetic
resonance imaging was normal except for an old thromboem-
bolic cerebral infarction in the left frontal lobe that was not
Further workup revealed a biventricular cardiomyopathy
(Fig. 2f–h). The left ventricular ejection fraction was reduced
to 25%, but hemodynamic parameters were normal in the
pulmonary and systemic circulation. Over the next few years
theneurologic featuresremainedunchanged, butsymptoms of
heart failure progressed despite medical treatment. In 2001,
an endomyocardial biopsy demonstrated hypertrophied cardi-
omyocytes with birefringent inclusion bodies (Fig. 2a). Elec-
tron microscopy revealed prominent lysosomes containing
rolledlipid bilayers(Fig. 2b–e).Thisledtoaninvestigation for
Fabry disease. As shown in Table I, the activity of multiple
lysosomal enzymes was distinctly elevated in the patient’s
serum suggestive of mucolipidosis III.
Due to worsening heart failure, the patient underwent
ogical evidence of lysosomal storage disease in the allograft
at 1 year post-transplant. At the time of transplantation, no
obvious pathology of the cardiac valves was described (not
shown). The patient died in 2004 from intractable chronic
infections with pneumocystic carini and pseudomonas despite
relaxation of immunosuppressive therapy. At autopsy, neural
tissue was examined and shown to contain inclusions similar
to those observed in myocardial tissue (data not shown),
however, the number of inclusion bodies per cell was smaller.
No other manifestations of mucolipidosis apart from cardiac
and neural tissue were observed. Together, the similar histol-
ogical changes observed in the two organs of the patient
suggest a common underlying disease process.
The family history is remarkable in that the father died at
age 72 due to cardiac failure and that cardiac disease was also
noted in other members of the patient’s family (Fig. 1). Family
tion or presented with coronary heart disease.
Consistent with the diagnosis of mucolipidosis III, cultured
skin fibroblasts from the patient were found to be deficient in
a subset of lysosomal enzymes (Table II). Notably, the levels of
b-glucuronidase, b-galactosidase, and a-fucosidase were not
decreased to the same extent as usually observed in ML II/ML
III patients. Joint mobility and skin were normal. Radiological
examination revealed minor degenerative changes with osteo-
chondrosis in the cervical, thoracic, and lumbar spine, slight
osteoporosis of the pelvis, and unremarkable right hand films
(Fig. 2i). Analysis of bone tissue at autopsy, however, suggests
that the degenerative changes are not likely due to the diag-
nosed disease. Taken together, these findings indicate the
presence of a mild form of ML III with an atypical clinical
The activity of the GlcNAc-1-phosphotransferase enzyme
was measured in control and patient fibroblast microsomes
using a-methylmannoside as an acceptor. The activity of
the GlcNAc-1-phosphotransferase enzyme from the patient’s
TABLE I. Enzyme Activities in Serum of Patient, Controls, and Other ML II/III Patients
Activity (mU/ml serum)
Patient range (n)a
Average Range (n)
aNumber of determinations during 1.5 years, before, and after heart transplantation; there were no tendencies
towardsincreased ordecreasedenzyme activities during thisperiod.Threesubjects takingsimilar medications as
the patient had enzyme activities in the normal range.
bDifferent batches of substrate.
TABLE II. Enzyme Activities in Fibroblasts of Patient, Controls, and Other ML II/III Patients
Activity (mU/mg protein)
ML II/III (n¼5)
data not shown); note that acid phosphatase and b-glucosidase activity is normal in ML II/III patients.
372Steet et al.
fibroblast extracts was 3.2?2.7% that of control samples
(2.3?0.4 vs. 51.4?6.2 U/mg protein, respectively). The
deficient enzyme activity in this patient is consistent with
previously reported ML III cases (range: 2%–20% activity
relative to control) and warranted further genetic investiga-
tion of the two genes encoding GlcNAc-1-phosphotransferase.
Mutation Analysis of Phosphotransferase Gene
The sequence of the cDNA encoding the g subunit of phos-
photransferase was analyzed by RT-PCR. Sequencing of the
PCR products revealed no mutations in the g subunit of
phosphotransferase, consistent with the previous observation
that mutations in this subunit result in normal phosphotrans-
[Varki et al., 1981].
The sequence of the gene encoding the a/b subunit was
analyzed using genomic DNA isolated from patient’s fibro-
blasts. The complete sequence of this gene will be published
elsewhere (Kudo et al., submitted). Sequencing analysis of the
patient’s amplified PCR products revealed a homozygous
G!A substitution in the last nucleotide of exon 7 (Fig. 3A).
Since such mutations have been previously reported to cause
pathological splicing events such as exon skipping (see
‘‘Discussion’’), we examined this possibility using RT-PCR.
PCR products were amplified corresponding to regions that
either span exon 7 (Fig. 3B; EX6F/EX8R) or originate within
exon 7 (Fig. 3B; EX5F/EX7R) using cDNA prepared from
amplification of products corresponding to exons 5–7 and
exons 6–8 generated the expected fragment sizes of ?220 and
?370 bases, respectively (Fig. 3C, lanes 2 and 3). No fragment
was amplified, however, in the patient sample when a forward
primer that hydridizes to exon 7 was used (Fig. 3C, lane 4).
Furthermore, the amplified exon 6–8 fragment in the patient
sample migrated at a smaller size (?235 bases, lane 5) com-
135 bp likely corresponds to the 135-nt of exon 7 and strongly
indicated that this exon is skipped in the patient’s GlcNAc-
1-phosphotransferase mRNA. The skipping of exon 7 was
directly confirmed by sequencing of an amplified fragment of
1-phosphotransferase gene. A faint band of 150 bp in the
patient sample (lane 5) was sequenced but found not to cor-
respond to any sequence within the phosphotransferase gene.
Genomic DNA from the patient’s family was analyzed by
restriction enzyme digestion of amplified PCR products. The
the presence of a homozygous mutation in the patient was
confirmed following Spe1 digestion. Several family members
including the mother of the patient were found to be hetero-
zygous for this mutation (Figs. 1 and 4). This heterozygosity
correlated well with the elevation of serum lysosomal enzyme
activity above the normal range (Fig. 5) in the affected family
members, confirming and extending earlier findings [Varki
et al., 1982].
Effect of GlcNAc-1-Phosphotransferase Deficiency on
Lysosomal Enzyme Phosphorylation and Sorting
We next measured the total phosphorylated high mannose
whether this decrease in phosphotransferase activity cor-
related with a reduction in the synthesis of the lysosomal
enzyme targeting signals. To do so, confluent fibroblast cul-
tures were pulse-labeled with mannose, high mannose oligo-
saccharides released by Endo H treatment and analyzed by
skipping. A: G!A in the last base of exon 7 in the patient. B: Schematic
representation of cDNA regions around exons 5–8. Primers used are shown
with arrows. C: Agarose gel electrophoresis of RT/PCR fragments of normal
(lanes 2 and 3) and patient (lanes 4 and 5) fibroblast RNA. Lanes 2 and 4,
primers EX5F/EX7R; lanes 3 and 5, primers EX6F/EX8R; lanes 1 and 6,
DNA. The G!A mutation was confirmed by RFLP analysis using Spe1.
non-carrier (7/I in pedigree).
Restriction enzyme digestion of patient and relative genomic
Adult Onset Form of Mucolipidosis III 373
to uncover mannose 6-phosphate residues. Oligosaccharides
containing one Man-6-P residue eluted from the column with
70 mM NaCl and those with two Man-6-P residues eluted with
140 mM NaCl. In control cells, nearly 2% of the total radio-
activity bound to the QAE-sephadex column indicating the
presence of charged oligosaccharides. Alkaline phosphatase
abolished the ability of the released oligosaccharides to bind to
residues. In contrast, only 0.5% of the total radioactivity in
the patient’s samples bound to the column demonstrating a
substantial decrease in the synthesis of oligosaccharides
bearing mannose 6-phosphate residues. Furthermore, in the
patient the oligosaccharides containing two residues were
more reduced (20% of control) as compared with those con-
taining just one residue (30% of control). This decrease in the
amount of total phosphorylated high mannose oligosacchar-
ides is similar to those observed for a known ML III patient
(data not shown). These findings indicate that although the
patient’s fibroblasts contain only 3% normal phosphotransfer-
ase activity, they are still able to phosphorylate the high
mannose oligosaccharides of lysosomal enzymes to roughly
25% the level found in control fibroblasts.
Next, we looked at whether the decrease in phosphotrans-
ferase activity led to the missorting of a specific lysosomal
enzyme, cathepsin D. The processing of newly-synthesized
cathepsin D molecules was examined in patient and normal
fibroblasts by pulse-chase experiments (Fig. 6). Fibroblasts
from the patient missorted cathepsin D, indicating that the
targeting of this enzyme was strongly affected by the phospho-
transferase deficiency. Thismissorting wasnot due tochanges
in the levels of the cation-independent mannose 6-phosphate
receptor since levels of this protein were identical in patient
and control cell lysates as determined by Western blotting
(data not shown).
The ML III patient described in this report is unique in two
respects: age of onset and clinical presentation. At 47 years of
age, this is the oldest presentation for an ML III patient
described to date. The typical appearance of symptoms in ML
a few instances, the onset of symptoms is delayed until the
teenage years [Tylki-Szymanska et al., 2002].
Cardiac manifestations have been reported in both ML II
and ML III patients. Notably, cardiomyopathy has been
observed in ML II patients [Okada et al., 1985; Schulz et al.,
1987; Muller et al., 2000] and valvular heart disease detected
in ML III patients [Raas-Rothschild et al., 2004]. However,
sensory neuropathy and cardiomyopathy as the presenting
symptomsinanadult MLIIIpatient isanovelfinding. Inlight
ofourobservations, analysisofserum lysosomal enzyme activ-
ity in patients with cardiomyopathy or sensory neuropathy of
unknown etiology may prove fruitful.
are typical manifestations of ML III, although in rare cases
the involvement may be limited to specific joints, such as the
hip joint [Freisinger et al., 1992].
We have demonstrated that the homozygous mutation
(G!A) in the last nucleotide of exon 7 in the patient results
in the skipping of this exon and the production of a minimal
amount of functional enzyme. Although the splice-site muta-
tion does not result in a large change in a quantitative splice
score (1.00 vs. 0.91 SSPNN; see ‘‘Materials and Methods’’),
no normally spliced product was detected (Fig. 4). One
possibility to account for this observation is the existence of a
very long intron (?10,000 bases) following exon 7. Previous
reports have shown that intron length can substantially affect
splicing efficiency [Bell et al., 1998]. Therefore, even small
decreases in the theoretical splicing efficiency can result in
members. The mean enzyme activities of the non-carriers are expressed as
100%. The mean values for carriers are increased to 223% (P<0.0045;
Wilcoxon test) for b-glucuronidase (closed circles) and to 182% (P<0.0014)
for b-hexosaminidase AþB (closed triangles). The patient’s enzyme
Enzyme activities in the serum of the patient and family
cathepsin D molecules were immunoprecipitated from fibroblast cell
extracts (C) and culture media (M) and quantified. The sorting efficiency is
calculated with the intermediate and mature forms of cathepsin D from the
cell extracts (with int.) and the mature form only (without int.). In either
case, thepatient’scells hypersecrete cathepsinD.Theresultsshownare the
average of two independent experiments.
Cathepsin D sorting in fibroblasts. Newly synthesized labeled
374Steet et al.
literature where a G!A transition in the last base of an exon
results in exon skipping [Grandchamp et al., 1989; Weil et al.,
1989; Akli et al., 1990; Krawczak et al., 1992; Klima et al.,
We demonstrated that only 3% normal GlcNAc-1-phospho-
transferase activity in patient’s fibroblasts is sufficient to
phosphorylate 25% of the total lysosomal enzyme oligosac-
charides. It is likely, therefore, that increases in total enzyme
activity to as little as 6%–10% may be sufficient to phos-
phorylate lysosomal enzymes to a level that would avoid a
pathological outcome. In other lysosomal storage disorders
[Leinekugel et al., 1992] as well as other genetic disorders like
cystic fibrosis [Rowntree and Harris, 2003], it has been
demonstrated that minimal activity can avoid disease. There-
fore, stimulation of GlcNAc-1-phosphotransferase activity via
pharmacological intervention may represent a promising
The residual phosphotransferase activity in the fibroblasts
could arise by two mechanisms. The deletion of the exon could
result in a splicing isoform with a low level of residual activity.
Alternatively, the splicing isoform may be completely
inactive, and the 3% of activity observed is the result of a
small amount of proper splicing. Since some misfolding
such as glycerol or DMSO [Ohashi et al., 2003], we tested the
effect of these agents on the activity of phosphotransferase in
(data not shown). In our patient, organ damage was most
pronounced in the heart. It is known from the literature that
splicing efficiency within different tissues depends on numer-
ous factors including the relative level of various splicing
factors [Rio, 1993; Gallego et al., 1996; Black, 2003]. It is
plausible, therefore, that most tissues of the patient can
sufficiently splice phosphotransferase mRNA and produce
enough normal mRNA and enzyme to avoid disease in those
tissues. Likewise, the level of enzyme needed in different
tissues likely varies as well.
Akli S, Chelly J, Mezard C, Gandy S, Kahn A, Poenaru L. 1990. A ‘‘G’’ to
‘‘A’’ mutation at position-1 of a 50splice site in a late infantile form of
Tay–Sachs disease. J Biol Chem 265:7324–7330.
Bao M, Elmendorf BJ, Booth JL, Drake RR, Canfield WM. 1996. Bovine
phosphotransferase. II. Enzymatic characterization and identification
of the catalytic subunit. J Biol Chem 271:31446–31451.
Baum H, Dodgson KS, Spencer B. 1959. The assay of arylsulfatases A and B
in human urine. Clin Chim Acta 4:453–455.
Bell MV, Cowper AE, Lefranc MP, Bell JI, Screaton GR. 1998. Influence of
intron length on alternative splicing of CD44. Mol Cell Biol 18:5930–
Beutler E, Kuhl W, Matsumoto F, Pangalis G. 1976. Acid hydrolases
in leukocytes and platelets of normal subjects and in patients with
Gaucher’s and Fabry’s disease. J Exp Med 143:975–980.
Black DL. 2003. Mechanisms of alternative pre-messenger RNA splicing.
Annu Rev Biochem 72:291–336.
Braulke T,Geuze HJ, Slot JW, Hasilik A, von Figura K.1987. On the effects
of weak bases and monensin on sorting and processing of lysosomal
enzymes in human cells. Eur J Cell Biol 43:316–321.
Cantor AB, Baranski TJ, Kornfeld S. 1992. Lysosomal enzyme phos-
phorylation. II. Protein recognition determinants in either lobe of
carboxyl lobe oligosaccharides. J Biol Chem 267:23349–23356.
Freisinger P, Padovani JC, Maroteaux P. 1992. An atypical form of
mucolipidosis III. J Med Genet 29:834–836.
Galjaard H. 1980. Genetic metabolic diseases. Early diagnosis and pre-
natal analysis. Amsterdam/New York/Oxford: Elsevier/North Holland
Gallego ME, Sirand-Pugnet P, Durosay P, Clouet d’Orval B, d’Aubenton-
Carafa Y, Brody E, Expert-Bezancon A, Marie J. 1996. Tissue-specific
pre-mRNA: Positive and negative regulations. Biochimie 78:457–465.
Grandchamp B, Picat C, de Rooij F, Beaumont C, Wilson P, Deybach
JC, Nordmann Y. 1989. A point mutation G!A in exon 12 of the
porphobilinogen deaminase gene results in exon skipping and is re-
sponsible for acute intermittent porphyria. Nucleic Acids Res 17:6637–
KlimaH,UllrichK,Aslanidis C,FehringerP,Lackner KJ,Schmitz G.1993.
A splice junction mutation causes deletion of a 72-base exon from the
mRNA for lysosomal acid lipase in a patient with cholesteryl ester
storage disease. J Clin Invest 92:2713–2718.
Kornfeld S. 1986. Trafficking of lysosomal enzymes in normal and disease
states. J Clin Invest 77:1–6.
Kornfeld S, Sly WS. 2001. I-cell disease and pseudo-Hurler polydystrophy:
Disorders of lysosomal enzyme phosphorylation and localization. In:
disease, New York: McGraw-Hill. pp. 3469–3483.
Kornfeld S, Reitman ML, Varki A, Goldberg D, Gabel CA. 1982. Steps in the
phosphorylation of the high mannose oligosaccharides of lysosomal
enzymes. Ciba Found Symp 92:138–156.
Krawczak M, Reiss J, Cooper DN. 1992. The mutational spectrum of single
base-pair substitutions in mRNA splice junctions of human genes:
Causes and consequences. Hum Genet 90:41–54.
Leinekugel P, Michel S, Conzelmann E, Sandhoff K. 1992. Quantitative
correlation between the residual activity of beta-hexosaminidase A and
arylsulfatase A and the severity of the resulting lysosomal storage
disease. Hum Genet 88:513–523.
Muller P, Reichenbach H, Mockel A, Buhrdel P. 2000. I-cell disease
complicated by unusual dilatative cardiomyopathy. J Inherit Metab
Ohashi T, Uchida K, Uchida S, Sasaki S, Nihei H. 2003. Intracellular
mislocalization of mutant podocin and correction by chemical chaper-
ones. Histochem Cell Biol 119:257–264.
Okada S, Owada M, Sakiyama T, Yutaka T, Ogawa M. 1985. I-cell disease:
Clinical studies of 21 Japanese cases. Clin Genet 28:207–215.
Zeigler M, Mandel H, Toth S, Roe B, Munnich A, Canfield WM. 2000.
Molecular basis of variant pseudo-Hurler polydystrophy (mucolipidosis
IIIC). J Clin Invest 105:673–681.
Raas-Rothschild A, Bargal R, Goldman O, Ben-Asher E, Groener JE,
Toutain A, Stemmer E, Ben-Neriah Z, Flusser H, Beemer FA,
Penttinen M, Olender T, Rein AJ, Bach G, Zeigler M. 2004. Genomic
organization of the UDP-N-acetylglucosamine-1-phosphotransferase
gamma subunit (GNPTAG) and its mutations in mucolipidosis III.
J Med Genet 41:e52.
Reitman ML, Kornfeld S. 1981. Lysosomal enzyme targeting. N-acetylglu-
cosaminylphosphotransferase selectively phosphorylates native lysoso-
mal enzymes. J Biol Chem 256:11977–11980.
Rio DC. 1993. Splicing of pre-mRNA: Mechanism, regulation, and role in
development. Curr Opin Genet Dev 3:574–584.
Rowntree RK, Harris A. 2003. The phenotypic consequences of CFTR
mutations. Ann Hum Genet 67:471–485.
Schulz R, Vogt J, Voss W, Hanefeld F. 1987. Mucolipidosis type II
(I-cell disease) with unusually severe heart involvement. Monatsschr
variability in mucolipidosis III (pseudo-Hurler polydystrophy). Am J
Med Genet 108:214–218.
Varki AP, Reitman ML, Kornfeld S. 1981. Identification of a variant of
mucolipidosis III (pseudo-Hurler polydystrophy): A catalytically active
N-acetylglucosaminylphosphotransferase that fails to phosphorylate
lysosomal enzymes. Proc Natl Acad Sci U S A 78:7773–7777.
Varki A, Reitman ML, Vannier A, Kornfeld S, Grubb JH, Sly WS.
1982. Demonstration of the heterozygous state for I-cell disease and
pseudo-Hurler polydystrophy by assay of N-acetylglucosaminylpho-
sphotransferase in white blood cells and fibroblasts. Am J Hum Genet
Weil D, D’Alessio M, Ramirez F, Steinmann B, Wirtz MK, Glanville RW,
Hollister DW. 1989. Temperature-dependent expression of a collagen
splicing defect in the fibroblasts of a patient with Ehlers–Danlos
syndrome type VII. J Biol Chem 264:16804–16809.
Adult Onset Form of Mucolipidosis III 375