Clinical Chemistry 46:9
1318 –1325 (2000)
Enzymes and Protein
Markers
Determination of Acid ␣-Glucosidase Protein:
Evaluation as a Screening Marker for Pompe
Disease and Other Lysosomal Storage Disorders
Kandiah Umapathysivam,1 Alison M. Whittle,1 Enzo Ranieri,2 Colleen Bindloss,1
Elaine M. Ravenscroft,1 Otto P. van Diggelen,3 John J. Hopwood,1 and
Peter J. Meikle1*
Background: In recent years, there have been significant advances in the development of enzyme replacement and other therapies for lysosomal storage disorders (LSDs). Early diagnosis, before the onset of
irreversible pathology, has been demonstrated to be
critical for maximum efficacy of current and proposed
therapies. In the absence of a family history, the presymptomatic detection of these disorders ideally can be
achieved through a newborn screening program. One
approach to the development of such a program is the
identification of suitable screening markers. In this
study, the acid ␣-glucosidase protein was evaluated as a
marker protein for Pompe disease and potentially for
other LSDs.
Methods: Two sensitive immunoquantification assays
for the measurement of total (precursor and mature) and
mature forms of acid ␣-glucosidase protein were used to
determine the concentrations in plasma and dried blood
spots from control and LSD-affected individuals.
Results: In the majority of LSDs, no significant increases above control values were observed. However,
individuals with Pompe disease showed a marked decrease in acid ␣-glucosidase protein in both plasma and
whole blood compared with unaffected controls. For
plasma samples, this assay gave a sensitivity of 95%
with a specificity of 100%. For blood spot samples, the
sensitivity was 82% with a specificity of 100%.
1
Lysosomal Diseases Research Unit and 2 State Screening Services, Department of Chemical Pathology, Women’s and Children’s Hospital, 72 King
William Rd., North Adelaide, South Australia 5006, Australia.
3
Department of Clinical Genetics, Erasmus University, PO Box 1738, 3000
DR Rotterdam, The Netherlands.
*Author for correspondence. Fax 61-8-8204-7100; e-mail pmeikle@
medicine.adelaide.edu.au.
Received March 20, 2000; accepted May 31, 2000.
Conclusions: This study demonstrates that it is possible
to screen for Pompe disease by screening the concentration of total acid ␣-glucosidase in plasma or dried blood
spots.
© 2000 American Association for Clinical Chemistry
Pompe disease, or glycogen storage disease type II, is an
autosomal recessive disorder of glycogen metabolism
caused by a deficiency of lysosomal acid ␣-glucosidase.
Patients with Pompe disease are unable to degrade glycogen stored in the lysosome, leading to the accumulation
of this substrate in lysosomal storage vacuoles. Morphologically, this produces an increase in the size and number
of lysosomes in the cell. Pompe disease can present as
infantile, juvenile, or adult onset forms. The infantile
onset form is characterized by massive cardiomegaly,
macroglossia, progressive muscle weakness (including
respiratory muscles), and marked hypotonia, with death
occurring within the first 2 years of life. The juvenile and
adult onset forms manifest as slower progressive muscular disorders that are limited to skeletal muscle, with
death usually occurring from respiratory failure (1 ). The
heterogeneous presentation of Pompe disease results, at
least in part, from the occurrence of different mutations in
the lysosomal acid ␣-glucosidase gene, which can lead to
variable effects on the functional capacity of the mutant
enzyme.
According to Wisselaar et al. (2 ), lysosomal acid ␣-glucosidase (EC 3.2.1.3) is synthesized as a precursor protein
with molecular mass of 110 kDa, which is then transported from the endoplasmic reticulum to the trans Golgi
network. A large proportion of precursor molecule is
transported to the lysosomes where it is proteolytically
converted to 76- and 70-kDa forms via a long-lived
intermediate molecule of 95 kDa. A small amount of
precursor protein is transported to the plasma membrane
and secreted.
1318
Clinical Chemistry 46, No. 9, 2000
Pompe disease is one of ⬎40 distinct genetic diseases
known collectively as lysosomal storage disorders (LSDs).
LSDs have a combined incidence of ⬃1 in 5000 births (3 ).
On the basis of clinical diagnosis, Pompe disease has a
reported incidence of 1 in 201 000 births in the Australian
population (4 ); however, recent studies based on carrier
detection in the general population have indicated that
the incidence is much greater, at ⬃1 in 40 000 births, in
both the US (5 ) and The Netherlands (6 ).
Definitive treatment for Pompe disease is not currently
available. However, two main treatment strategies are
being developed. Correction of the enzyme deficiency by
enzyme replacement therapy is well advanced (7, 8 ), and
gene therapy using viral vectors is also under development (9 –12 ). In the quail and mouse animal models,
enzyme replacement therapy using the precursor form of
acid ␣-glucosidase (7, 13 ) has led to the clinical and
metabolic correction of Pompe disease. It is anticipated
that this type of therapy will be available for human use
in the near future. Similar treatment strategies are being
developed for many LSDs (3 ), and it is well recognized
that early diagnosis and treatment will provide a substantial improvement in the efficacy of these therapies for this
group of disorders. However, in the absence of a family
history, the only practical way to achieve early diagnosis
is through a newborn screening program.
The clinical diagnosis of Pompe disease is confirmed
by the virtual absence (in infantile onset) or markedly
reduced (in juvenile and adult onset) activity of acid
␣-glucosidase in muscle biopsies and cultured fibroblasts.
Prenatal diagnosis can be made by determining the acid
␣-glucosidase activity in cultured amniotic cells and/or in
chorionic villus biopsies (14, 15 ) and also by mutation
analysis (15 ). However, current diagnostic tools are not
suitable for large-scale screening. Immunoquantification
of the acid ␣-glucosidase protein can be adapted for
large-scale screening; however, the protein concentration
may not be diminished in all individuals with Pompe
disease. Normal amounts of enzyme, which appear to be
catalytically inactive, have been reported to be common in
infantile patients in China (16 ).
We previously evaluated lysosomal membrane glycoprotein-1 (LAMP-1) (17 ) and LAMP-2 (18 ) as effective
screening markers for LSDs. Although these proteins
were determined to be useful markers for many LSDs,
they showed only marginal specificity and sensitivity for
Pompe disease. In this study, we proposed to evaluate
acid ␣-glucosidase as a marker for Pompe disease and
other LSDs. Accordingly, we developed two sensitive
immunoquantification assays for the determination of
either the total (precursor and mature forms) or mature
form only of acid ␣-glucosidase protein in dried blood
spots and plasma. We used these assays to determine the
concentrations of these proteins in plasma and dried
blood spot samples taken from unaffected and LSDaffected individuals.
1319
Materials and Methods
patient samples
Dried blood spots used in this study were deidentified
and were part of the routine samples collected between 48
and 72 h after birth, by the Neonatal Screening Laboratory
at the Women’s and Children’s Hospital, Adelaide, South
Australia. Blood spots were also obtained from previously
diagnosed Pompe patients.
Plasma samples used in this study were from samples
submitted to the National Referral Laboratory for the
Diagnosis of Lysosomal, Peroxisomal and Other Genetic
Disorders and samples processed for routine biochemistry in the Department of Chemical Pathology, Women’s
and Children’s Hospital. Additional plasma samples from
Gaucher patients were obtained from Dr. Allan Cooper
(Willink Biochemical Institute, Manchester, UK).
reagents
Acid ␣-glucosidase proteins. Pharming BV (The Netherlands) provided recombinant precursor and mature forms
of acid ␣-glucosidase proteins purified from rabbit (8 )
and mouse milk, respectively (19 ). Purity of the proteins
was established by sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis carried out on 12.5%
acrylamide gels using the method of Laemmli (20 ) and
staining with silver (21 ). Protein calibrators were quantified by the bicinchoninic acid method of Smith et al. (22 )
using bovine serum albumin as a calibrator.
Polyclonal antibodies. Sheep anti-acid ␣-glucosidase polyclonal antibody was produced against the recombinant
precursor form of the protein. A sheep received subcutaneous injections containing 2 mg of protein in 1 mL of an
emulsion of phosphate-buffered saline (pH 7.4) and complete Freund’s adjuvant, followed by four booster injections (2 mg each) with incomplete Freund’s adjuvant,
each 3 weeks apart. One week after the last injection, the
sheep was bled out and serum collected.
Monoclonal antibodies. The hybridoma cell lines producing
monoclonal antibodies that recognize both the precursor
and mature forms (43D1) (23 ) and the mature form only
(43G8) (24 ) of the acid ␣-glucosidase protein were provided by Pharming BV (The Netherlands).
Purification of antibodies. Sheep polyclonal antibody was
purified on a 5-mL HitrapTM protein G affinity column
(Pharmacia Biotech) followed by an acid ␣-glucosidase
affinity column. The acid ␣-glucosidase affinity column
was prepared by coupling 5 mg of the precursor form of
acid ␣-glucosidase protein to 2.5 mL of Affi-Prep Hz
support (Bio-Rad) according to the manufacturer’s instructions.
Briefly, 5 mL of sheep serum was diluted with 5 mL of
phosphate-buffered saline (pH 7.4) and centrifuged at
2200g for 10 min at 4 °C. The centrifuged serum was
passed through a 0.2 ␮m filter, and then loaded onto the
1320
Umapathysivam et al.: Acid ␣-Glucosidase as a Marker for LSDs
protein G column at a flow rate of 0.5 mL/min. The
column was washed with phosphate-buffered saline (pH
7.4), and the antibody was eluted with 0.1 mol/L H3PO4/
NaH2PO4 (pH 2.5) and immediately neutralized by adding 1.0 mol/L Na2HPO4 (1:10, by volume). The protein
content was estimated by the absorbance at 280 nm
(absorbance ⫽ 1.4 for 1.0 g/L protein). The eluate was
diluted fourfold and then loaded onto the acid ␣-glucosidase affinity column at the same flow rate. The column
was washed and eluted as described for the protein G
column.
Monoclonal antibodies 43D1 and 43G8 were purified
from cell culture supernatants by ammonium sulfate
precipitation (25 ) followed by affinity purification on the
acid ␣-glucosidase and protein G affinity columns, respectively.
Europium labeling of monoclonal antibodies. Purified monoclonal antibodies 43D1 and 43G8 were labeled with Eu3⫹
chelate, using the DELFIA® labeling kit (EG&G Wallac),
and purified on a Pharmacia Superose 12 fast-phase liquid
chromatography column (1.5 ⫻ 30 cm) as described by
Meikle et al. (17 ). The coupling efficiencies were determined from protein mass and fluorescence output of the
conjugate.
immunoquantification of acid ␣-glucosidase
Total (precursor and mature) acid ␣-glucosidase protein
was determined with a polyclonal/monoclonal (43D1)
sandwich immunoassay, whereas the mature acid ␣-glucosidase protein was specifically determined using a
monoclonal (43D1)/monoclonal (43G8) sandwich immunoassay. Each assay was performed as either a one-step
assay for the determination of protein in dried blood spots
or a two-step assay for the determination of protein in
plasma samples.
One-step assay. Microtiter plates (Immulon 4; Dynatech
Technologies) were coated overnight at 4 °C with 100
␮L/well of affinity purified anti-acid ␣-glucosidase polyclonal antibody (2 mg/L) or monoclonal antibody 43D1 (4
mg/L). Coated plates were prewashed once in DELFIA
wash buffer (EG&G Wallac). Dried blood spots were
placed in microtiter wells with 200 ␮L of assay buffer
containing 200 ␮g/L of either Eu3⫹-labeled 43D1 (determination of total ␣-glucosidase) or 43G8 (determination of
mature ␣-glucosidase) monoclonal antibody. The microtiter plates were shaken (at 20 °C for 60 min) and incubated overnight at 4 °C. The microtiter plates were again
shaken at 20 °C for 60 min, blood-spot filters were removed by suction, and the plates were washed six times
with DELFIA wash buffer. This was followed by the
addition of 200 ␮L of DELFIA enhancement solution
(EG&G Wallac). The microtiter plates were shaken at
20 °C for 15 min, and the fluorescence was read on a
DELFIA 1234 Research Fluorometer (EG&G Wallac).
Two-step assay. Immunoquantification of both the total
and the mature forms of acid ␣-glucosidase in plasma
samples collected with EDTA or citrate as the anticoagulant was performed using the two-step assay. Plates were
coated with antibodies as described for the one-step
assay. Samples were diluted with DELFIA assay buffer
(100 ␮L/well). The plates were shaken at 20 °C for 60 min
and incubated for 5 h at 20 °C. The plates were washed six
times, and 100 ␮L of assay buffer containing 200 ␮g/L of
either Eu3⫹-labeled 43D1 or 43G8 monoclonal antibody
was added to each well. The plates were shaken at 20 °C
for 15 min and incubated overnight at 4 °C. The plates
were washed six times, and DELFIA enhancement solution (200 ␮L) was added to each well. The plates were
shaken at 20 °C for 15 min, and the fluorescence was read
on a DELFIA 1234 Research Fluorometer.
The concentrations of the total and the mature form of
acid ␣-glucosidase in both the blood spots and the plasma
were calculated using Multicalc Data Analysis software
(EG&G Wallac).
preparation of calibrators and quality-control
samples
The precursor form of acid ␣-glucosidase was used as a
calibrator for immunoquantification of the total acid
␣-glucosidase, whereas the mature form was used for the
immunoquantification of the mature protein.
Blood spot calibrators were used to determine the total
and mature forms of acid ␣-glucosidase protein in dried
blood spots. The purified precursor and mature acid
␣-glucosidase proteins were diluted in buffer (40 g/L
human serum albumin, 25 g/L human ␥-globulin, 20
mmol/L Tris-HCl, 150 mmol/L NaCl, pH 7.8). These
proteins were further diluted threefold with washed
sheep red blood cells to give final concentrations of 200,
100, 50, 25, and 12.5 ␮g/L. Similarly, two blood spot
controls containing low (60 ␮g/L) and high (180 ␮g/L)
concentrations of acid ␣-glucosidase protein were also
prepared as described above. Aliquots (50 ␮L) of the
calibrators and controls were spotted on Whatman 180
BFC filter paper and were air dried overnight at room
temperature.
Liquid calibrators were used to determine the total and
mature forms of acid ␣-glucosidase protein in plasma.
Liquid calibrators were prepared by diluting the precursor and mature forms of acid ␣-glucosidase protein in
DELFIA assay buffer to give final concentrations of 5, 2.5,
1.25, 0.625, and 0.313 ␮g/L for the precursor form and 10,
5, 2.5, 1.25, and 0.625 ␮g/L for the mature forms of the
protein. Three quality-control samples containing 0.1, 1.0,
and 2.0 ␮g/L precursor protein or 1.0, 2.0, and 8.0 ␮g/L
mature protein were prepared.
Dried blood spot calibrators and controls were stored
in sealed bags containing silica gel at ⫺70 °C. Liquid
calibrators and controls were stored at ⫺70 °C. Both dried
blood spot and liquid calibrators were assayed in duplicate at the beginning of each plate. Single estimations of
Clinical Chemistry 46, No. 9, 2000
1321
the quality-control samples were made for each analytical
assay. Dried blood spots were assayed singly, and any
repeat samples were assayed in duplicate. All plasma
calibrators and samples were assayed in duplicate.
Results
immunoquantification of acid ␣-glucosidase
The acid ␣-glucosidase calibrators were ⬎99% pure based
on sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis. After silver staining, the precursor form
showed a single band at 112 kDa, and the mature formed
a single band at 81 kDa. These corresponded to the
published precursor (110 kDa) and mature (76 kDa) forms
of acid ␣-glucosidase, respectively (2 ).
The immunoquantification assay for acid ␣-glucosidase was optimized by standard procedures to achieve
the appropriate assay precision required for concentrations seen in blood spots and plasma. In addition, the
specificities of the acid ␣-glucosidase antibodies for the
precursor and the mature forms of the protein were
determined (Fig. 1). The degree of cross-reactivity for the
precursor form of the protein by the monoclonal antibody
43G8 was ⬍3% (Fig. 1B). To monitor assay performance,
three quality-control samples (low, medium, and high) for
plasma and two (low and high) for dried blood spots were
included within each analytical run. Precision profiles for
the analysis of total and mature forms of acid ␣-glucosidase using liquid calibrators and blood spot calibrators
showed CVs ⬍7.5%. Precision studies for plasma study
were conducted over 60 days with 28 observations performed. Blood spot precision studies were conducted
with 36 observations over 30 days. Assays of dried blood
spots stored for different periods of time (1, 4, 12, and 52
weeks) demonstrated that the dried blood spots were
stable for at least 3 months when stored at room temperature. However, a significant decrease in the medium
concentration (⬃29%) was found after storage for 12
months.
acid ␣-glucosidase concentrations in plasma
The total (precursor and mature) and mature forms of
acid ␣-glucosidase protein were determined in plasma
samples from 195 control individuals and 404 LSD-affected individuals, representing 26 different disorders
(Table 1 and Fig. 2). In the control population, the total
concentration of acid ␣-glucosidase protein had a skewed
distribution with a median of 17.1 ␮g/L and the 5th and
95th percentiles at 5.6 and 34.7 ␮g/L, respectively. The
concentration of the mature form of the acid ␣-glucosidase protein in the control population was very low, with
a median value of 0.2 ␮g/L and 5th and 95th percentiles
at 0 and 3.66 ␮g/L, respectively. Among the LSD-affected
individuals, all individuals with acid lipase deficiency
and 86%, 60%, and 58% of individuals with mucolipidosis
II/III, Niemann-Pick (A/B) disease, and Gaucher disease,
respectively, had total acid ␣-glucosidase concentrations
higher than the 95th percentile of the control group (Table
Fig. 1. Calibration curves for precursor and mature forms of acid
␣-glucosidase in plasma using the two-step assay.
Acid ␣-glucosidase liquid calibrators for the precursor (䡺) and mature (E) forms
of the protein were assayed for total protein (A) using monoclonal antibody 43D1
and for mature protein (B) using monoclonal antibody 43G8 as described in
Materials and Methods. Under standard assay conditions, a linear response was
observed over the range 0 –20 ␮g/L for both the precursor and mature forms of
acid ␣-glucosidase. Comparison of precursor and mature forms of the protein
with each of the antibody combinations demonstrated that monoclonal antibody
43D1 recognizes both the precursor and the mature forms of acid ␣-glucosidase,
whereas the monoclonal antibody 43G8 recognizes only the mature form of the
protein.
1). In the Pompe disease group, the concentrations of both
the total and mature forms of acid ␣-glucosidase were
significantly reduced (P ⬍0.001) based on nonparametric
statistical analysis (Mann–Whitney test). Only 1 of 22
plasma samples from Pompe patients had a total acid
␣-glucosidase concentration within the control range.
distribution of acid ␣-glucosidase protein in
blood spots
The distribution of total acid ␣-glucosidase in blood spots
from the newborn population showed a characteristic
skewed distribution (Fig. 3). The median concentration of
the 1951 blood spots was 59.0 ␮g/L with 5th and 95th
percentiles of 28.6 and 112.0 ␮g/L, respectively. The
Umapathysivam et al.: Acid ␣-Glucosidase as a Marker for LSDs
1322
Table 1. Acid ␣-glucosidase concentrations in plasma from control and LSD-affected individuals.
Disorder
n
Total acid
␣-glucosidasea
␮g/L
Control
Acid lipasec
Fabryc
Galactosialidosis
Gaucherc
GM Id
Mucolipidosis II/IIIc
Krabbe
Mannosidosis
MLD
MSDc
MPS I
MPS IIc
MPS IIIAc
MPS IIIB
MPS IIIC
MPS IIID
MPS IVAc
MPS VI
N-P (A/B)c
N-P (C)c
Pompee
Sandhoff
SASc
TSDc
TSD (A/B)c
195
2
27
1
87
14
14
14
9
34
4
24
23
24
19
3
3
16
10
10
13
22
6
18
23
2
17 (2–61)
76 (65–87)
10 (5–46)
14
40 (0–737)
16 (7–29)
149 (7–273)
17 (5–31)
10 (6–25)
17 (1–55)
27 (22–102)
19 (3–27)
26 (13–46)
23 (2–40)
20 (10–38)
18 (13–22)
25 (6–29)
14 (6–21)
12 (5–65)
39 (39–84)
24 (15–84)
0.7 (0–3.3)
22 (17–32)
27 (18–40)
19 (5–48)
32 (29–35)
Mature acid
␣-glucosidasea
␮g/L
0.2 (0–14)
5 (3–7)
0.1 (0–3.6)
2
19 (0–768)
4 (2–11)
7 (0–19)
0.4 (0–1.6)
1.5 (1–3)
0.6 (0–4.5)
2.9 (1–78)
1 (0–5.8)
2 (0–6.5)
1.2 (0–2.5)
1 (0–3)
0.6 (0.3–0.8)
0.3 (0.2–0.5)
1 (0.1–1.3)
0.2 (0–11)
0.6 (3–31)
3 (0–11)
0 (0–0.22)
0.8 (0–3.5)
1 (0–11)
0.2 (0–9)
1.4 (1–2)
% >95th
percentileb
100
14.8
0
57.5
0
86
7.1
0
2.9
25
0
17.4
25
5.3
0
0
0
10
60
15.4
0
27.8
21.7
0
a
Median value (range).
Percentage of each disorder group with total acid ␣-glucosidase concentrations above the 95th percentile of the control population (34.7 ␮g/L).
c
Represents those LSDs in which the total acid ␣-glucosidase concentrations were significantly different from the control population (nonparametric statistical
analysis).
d
GM I, GM I gangliosidosis; MLD, metachromatic leukodystrophy; MSD, multiple sulfatase deficiency; MPS, mucopolysaccharidosis; N-P, Niemann-Pick disease;
SAS, sialic acid storage disease; TSD, Tay-Sachs disease.
e
Of Pompe-affected individuals, 95.5% had total acid ␣-glucosidase concentrations below the control population range.
b
median concentration of the mature form in blood spots
was 53 ␮g/L with 5th and 95th percentiles of 24 and 110
␮g/L, respectively. The correlation between the total
(precursor and mature forms) and mature forms of the
protein in blood spots was 0.83 (Pearson correlation) and
was significant at P ⫽ 0.01 (two-tailed). In a separate
study, the concentration of total acid ␣-glucosidase was
determined in blood spots from 12 juvenile and 12 adult
controls and compared with 20 newborn samples. Nonparametric statistical analysis (Kruskal–Wallis test) indicated no significant differences among these groups. The
total and mature forms of acid ␣-glucosidase were measured in dried blood spots from 20 individuals with
Pompe disease and 2 carriers (Table 2). The concentration
of the total acid ␣-glucosidase protein was lower than the
0.2 percentile of newborn population in 16 of the 17
Pompe patients, whereas the concentration in all carriers
tested was below the 0.6 percentile. In addition, the
concentration of both the total and mature forms of acid
␣-glucosidase protein were measured in the correspond-
ing plasma from three Pompe patients and one carrier
(Table 2). The concentrations of the protein were also
reduced in the plasma from these patients, supporting the
low values observed in the dried blood spots.
Discussion
The LSDs are a large family of rare genetic disorders
involving the lysosome that can lead to the manifestation
of severe clinical symptoms. The incidence of LSD disorders (⬃1 in 5000 births in Australia) is comparable to
other intensively studied genetic disorders such as cystic
fibrosis and phenylketonuria (1 in 2500 and 1 in 14 000
births in Australia, respectively). In most LSDs, the pathology of the disease is not apparent at birth and
manifests in the first few years of life. If current and
proposed therapies are to achieve maximum efficacy, it
will be required that these disorders are detected early,
before the onset of irreversible pathology, particularly
central nervous system and/or bone pathology. Except in
cases where a family history is available, presymptomatic
1323
Clinical Chemistry 46, No. 9, 2000
Fig. 2. Box plots of total acid ␣-glucosidase concentrations in plasma from 195 control and 404 LSD-affected individuals.
The concentrations of the total (precursor and mature forms) and mature form only of acid ␣-glucosidase in plasma were determined using 5-␮L samples in the two-step
immunoquantification assay as described in Materials and Methods. N, number of samples in each group. Center bars show the median ␣-glucosidase concentration
for each disorder, shaded areas shown the 25th and 75th percentiles, and top and bottom bars show the limits of the range. 䡺 and ⴱ represent outliers and extreme
outliers, respectively. GM1, GM1 gangliosidosis; MLD, metachromatic leukodystrophy; MSD, multiple sulfatase deficiency; MPS, mucopolysaccharidosis; N-P,
Niemann-Pick disease; SAS, sialic acid storage disease; TSD, Tay-Sachs disease.
diagnosis of LSDs can be achieved only by a mass
screening program.
In this study, we investigated acid ␣-glucosidase as a
potential screening marker for Pompe disease in particular and for LSDs in general. Earlier studies showed that
LAMP-1 (17 ) and a related protein, LAMP-2 (18 ), were
increased in the majority of LSDs (17 ) but were not
increased in ⬃35% of patients representing several specific LSDs.
To determine the usefulness of the acid ␣-glucosidase
protein as a screening marker for LSD, we measured the
Fig. 3. Histogram of the distribution of total acid ␣-glucosidase in dried
blood spots from newborns.
Concentrations of the total acid ␣-glucosidase protein in dried blood spots from
1951 newborns were determined with the one-step quantification method as
described in Materials and Methods. The median value of the 1951 dried blood
spots was 59.0 ␮g/L with 5th and 95th percentiles of 28.6 and 112.0 ␮g/L,
respectively. The inset is the expanded histogram of the distribution of the total
acid ␣-glucosidase in newborns that was less than the 3rd percentile (25 ␮g/L).
concentrations of both the total and mature forms of the
protein in plasma from LSD-affected individuals and
compared these with concentrations in plasma from control individuals. The majority of acid ␣-glucosidase in
plasma from the control population was the precursor
Table 2. Acid ␣-glucosidase concentrations in blood spots
and plasma from Pompe-affected individuals and carriers.
Sample ID
Disorder
Onset
GC3
GC4
GC19
GC20
GC25
GC13
GC14
GC15
GC16
GC17
GC21
GC22
GC23
GC24
GC27
GC28
GC94
GC5
GC7
GC26
Pompe
Pompe
Pompe
Pompe
Pompe
Pompe
Pompe
Pompe
Pompe
Pompe
Pompe
Pompe
Pompe
Pompe
Pompe
Pompe
Pompe
Carrier
Carrier
Carrier
Infantile
Infantile
Juvenile
Juvenile
Juvenile
Adult
Adult
Adult
Adult
Adult
Adult
Adult
Adult
Adult
Adult
Adult
Adult
Adult
Total acid
␣-glucosidasea
Mature acid
␣-glucosidasea
1.5
36.6
10.1
0.0
0.0
0.0
0.0
0.0
0.8
0.0
1.0
1.9
0.0
0.0
0.6 (0.6)
0.0 (0.0)
10.8 (0.5)
13.6
10.5
16.6 (6.7)
0.2
56.4
12.9
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
2.1
0.0
0.0
0.0 (0.1)
0.0 (0.0)
4.5 (0.2)
13.4
8.4
13.1 (0.2)
a
Expressed as ␮g/L of whole blood for Guthrie cards and ␮g/L (in parentheses) for plasma.
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Umapathysivam et al.: Acid ␣-Glucosidase as a Marker for LSDs
form. The acid ␣-glucosidase protein was increased in
individuals with acid lipase deficiency, mucolipidosis
II/III, Gaucher disease, and Niemann-Pick disease (type
A/B). The observed increase in plasma from mucolipidosis II/III patients was identified as the precursor form of
the protein. This results from the mistargeting of newly
synthesized protein in these patients because of the absence of the mannose-6-phosphate moiety on the enzyme.
The increases observed in patients deficient for acid lipase
also represented an increase in the concentration of precursor protein. However, the increases observed in patients with Gaucher disease and Niemann-Pick disease
(type A/B) resulted primarily from an increase in the
mature form of the protein. This implies a release of
protein from the lysosome in these disorders. This may
result from cell death and subsequent breakdown or
alternatively, from specific exocytosis of lysosomal contents from affected cells. As a general marker for LSDs,
acid ␣-glucosidase has limited application because it is
significantly increased in relatively few disorders.
Plasma samples from 22 patients with Pompe disease
showed a significant decrease in the concentration of the
acid ␣-glucosidase protein. Only one Pompe disease patient had protein concentrations within the control range.
Individuals with significant concentrations of acid ␣-glucosidase protein may represent different mutations in the
gene that lead to the absence or marked reduction in
catalytic capacity of the mutant enzyme but normal
processing (1 ).
In dried blood spots, 14 of 17 patients had concentrations below the range of the control population (based on
1951 samples) and 16 of 17 patients were below the 0.4
percentile. One infantile patient had significant concentrations of acid ␣-glucosidase as determined from the dried
blood spot. The majority of this protein was the mature
form as determined by the assay specific for the mature
form, which indicates lysosomal processing. No mutational data were available on this patient. A percentage of
Pompe patients are reported to have mutations that lead
to significant concentrations of ␣-glucosidase protein with
reduced activity. One such mutation is Asp 6453 Glu,
which is reported to be the most common mutation in the
Chinese population in Taiwan, accounting for 36% of
mutations (26 ). This mutation has been reported to lead to
the mistargeting and misprocessing of the acid ␣-glucosidase protein (27 ), so that only the precursor form of the
protein is present in the cells. Clearly this is not the
mutation present in our high-protein patients. However, a
potential approach to the identification of patients expressing only the precursor form is to screen for the
mature form of the acid ␣-glucosidase protein. This approach would be limited to whole blood or blood spot
samples because the concentration of mature protein in
plasma is low in the unaffected population.
We have demonstrated that it is feasible to reliably
measure the total acid ␣-glucosidase protein (precursor
and mature forms) and the mature form only in either
dried blood spots or plasma. In addition, we have shown
a good correlation between the absence or reduced concentrations of total acid ␣-glucosidase protein and the
incidence of Pompe disease. For plasma samples, when
we used a cutoff concentration of 2.0 ␮g/L, the total acid
␣-glucosidase assay gave a sensitivity of 95% with a
specificity of 100% (21 of 22 patient samples were below
the control range based on 195 unaffected, age-matched
control subjects). Analysis of the blood spot samples using
a cutoff concentration of 6 ␮g/L gave a sensitivity of 82%
with a specificity of 100% (14 of 17 patients were below
the range of the control population). Increasing the cutoff
to 11 ␮g/L increased the sensitivity (94%) but decreased
the specificity (99.8%). A specificity of 99.8% would
produce a large number of false positives that may also
include many carriers, although the numbers of carriers in
this study are insufficient to determine this value accurately. Although these studies were based on a comparison of infantile, juvenile, and adult patients with a control
population of 1951 newborns, comparison of smaller
control groups of newborns, juveniles, and adults showed
no statistically significant difference in the concentration
of acid ␣-glucosidase, indicating no age correlation.
On the basis of this study, it is feasible to screen for
Pompe disease by determining the concentration of total
acid ␣-glucosidase protein in plasma or dried blood spots.
However, additional work needs to be done to increase
the sensitivity without a corresponding decrease in the
specificity. One possible approach is the development of a
two-tiered screening strategy involving an initial protein
determination followed by an enzyme activity determination made on a second blood spot from the same Guthrie
card. The second-tier assay would be performed on the
top 0.5% of the population as determined from the
first-tier assay. Alternative approaches may involve mutation analysis or determination of lysosomal substrate
storage as a second tier of the screen. Additional work in
these areas is ongoing.
This work was supported by Pharming BV (The Netherlands) and the National Health and Medical Research
Council of Australia. We thank Dr. Allan Cooper (Willink
Biochemical Institute, Manchester, UK) for providing additional plasma samples from Gaucher patients, Dr. Martina Baethmann (University of Essen, Essen, Germany) for
blood spot samples from some Pompe patients, and Dr.
Arnold Reuser (Department of Clinical Genetics, Erasmus
University, Rotterdam, The Netherlands) for helpful comments in the preparation of the manuscript. We also
gratefully acknowledge Rosemarie Gerace, Bronwen Bartlett, and Kerry Barnard from the State Screening Service of
South Australia for assisting with the screening of blood
spots.
Clinical Chemistry 46, No. 9, 2000
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