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Articles |
1
Lysosomal Diseases Research Unit, Departments of Chemical Pathology and Histopathology, Women's and Children's Hospital, 72 King William Rd., North Adelaide, South Australia, 5006, Australia.
2
Department of Medical Biochemistry and Biophysics, Umea
University, Umea, S-901 87, Sweden.
a Author for correspondence. Fax 61 8–8204 7100;
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Abstract |
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Key Words: indexing terms: blood spot • Guthrie card • skin fibroblast • time-resolved fluorescence immunoassay
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Except for those cases with a family history of the disease, presymptomatic detection of LSD can only be achieved by newborn screening. Currently, even after the presentation of clinical symptoms, the diagnosis of a LSD is a complex process involving a range of assays performed on urine, blood, and in some disorders, skin fibroblasts. These assays are time consuming, expensive, and invasive, making them unsuitable for newborn screening applications. To justify the screening of the entire neonatal population for a given disorder or group of disorders, there are several criteria that need to be satisfied; these criteria can be summarized as two broad considerations. First, does neonatal diagnosis provide clear-cut benefits to the neonate and family? Second, are these benefits reasonably balanced by the total cost of screening?
The greatest benefit to the affected neonate will come from effective therapy. In recent years, treatment of some LSDs has become possible. Cystinosis is treated with cysteamine (2)(3), several LSDs including mucopolysaccharidosis (MPS) I and MPS VI have been responsive to bone marrow transplants (4)(5), and Gaucher disease is currently being treated by enzyme replacement therapy, which, like bone marrow transplantation, is theoretically applicable to a wide range of LSDs. Recombinant enzymes deficient in many LSDs have been characterized, and now numerous animal models are being used to evaluate enzyme replacement and gene therapies for these disorders. Animal models currently in use include dog models for fucosidosis (6) and MPS VII (7); cat models for MPS I and VI (7)(8); goat models of ß-mannosidosis (9) and MPS IIID (10); and mouse models for MPS VII (11), galactosialidosis (12), and Nieman–Pick disease (13). Within the next 5 to 10 years, effective therapies will probably be available for many of the LSDs. The effectiveness of these therapies, particularly for those LSDs involving CNS and bone pathologies, will rely heavily upon the early diagnosis and treatment of the disorder, before the onset of irreversible pathology. Animal studies involving bone marrow transplantation in a fucosidosis dog model (predominantly CNS pathology) (6) and enzyme replacement therapy studies in a MPS VI cat model (predominantly bone pathology) (8)(14) have shown a clear correlation between the age when treatment was commenced and efficacy. A further consideration, critical to bone marrow transplant therapy, is that early diagnosis of the LSD will allow clinicians to take advantage of the window of opportunity presented by the naturally suppressed immune system of the neonate to maximize the chances of a successful engraftment. Early detection of these disorders has the added advantage of permitting genetic counseling of the parents, with the option of prenatal diagnosis in subsequent pregnancies, and management of the affected child. Accurate techniques for monitoring progress of the treatment regimes are also required.
One common feature of these disorders is the accumulation and storage of material normally degraded within the lysosome and transported across the lysosomal membrane. This generally results in an increase in the number and size of lysosomes within the cell from >1% to as much as 50% of total cellular volume. We propose that the concentration of certain lysosomal proteins would be increased as a result of storage and that these proteins may prove to be useful diagnostic markers for the detection of all LSDs. The ability to detect all LSDs in a single procedure would be amenable to newborn screening for this group of disorders.
In this study we investigated the concentrations of several lysosomal proteins in skin fibroblasts from a range of LSDs and identified lysosome-associated membrane protein LAMP-1 as having the greatest increase associated with lysosomal storage. The concentration of LAMP-1 in plasma samples from both normal and LSD-affected individuals has been determined to evaluate the suitability of LAMP-1 as a diagnostic marker of LSD.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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cell culture
Human diploid fibroblasts were established from skin biopsies
submitted to the Women's and Children's Hospital for diagnosis
(15). Cell lines were maintained according to established
procedures in Eagle's basal medium, 100 mL/L fetal calf serum, and
antibiotics, in a 5% CO2 atmosphere incubator, unless
otherwise stated.
Normal and LSD-affected skin fibroblast cell lines used in this study
are listed in Table 1
. For experimental use, each skin fibroblast cell line was
plated in 6 x 75 cm2 flasks with Eagle's basal
medium and allowed to reach confluency, which was designated time point
t = 0 for the experiment. Once confluent, cells were
maintained for up to a further 6 weeks.
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preparation of cell extracts
Cells were washed twice with Dulbecco's PBS and removed from the
flask by trypsination [2 mL of trypsin–versene solution (CSL,
Melbourne, Australia) per flask for 5 min at 37 °C]. The cells were
immediately washed twice with cold PBS and cells from one flask from
each time point were resuspended in 200 µL of saline containing 10
mL/L Nonidet P40. Cell lysates were prepared by five freeze/thaw
cycles, clarified by microcentrifugation (1000g, 5 min), and
assayed for lysosomal enzymes and proteins. Cells from the second flask
of each time point were prepared for electron microscopy as described.
electron microscopy
Fibroblast cells were harvested and then fixed for 2–3 h with 20
mL/L formaldehyde and 20 mL/L glutaraldehyde in 0.1 mol/L cacodylate
buffer containing 5 mmol/L calcium chloride, pH 7.2. Specimens were
postfixed in 10 g/L osmium tetroxide in 0.1 mol/L cacodylate buffer and
5 mmol/L calcium chloride, pH 7.2. Specimens were dehydrated in a
graded series of aqueous ethanol and embedded in Spurr's low-viscosity
epoxy resin (TAAB, Berkshire, UK).
Semithin (1-µm thick) survey sections were obtained with an Ultracut Ultramicrotome (Leica, Vienna, Austria) and stained with 10 g/L toluidine blue in 10 g/L borax. For each block a correctly oriented area for sectioning was selected. Ultrathin sections with a silver interference color (60–90 nm thick) were cut and mounted on 100-mesh hexagonal copper (G 100 HEX) grids (Gilder Grids, Grantham, UK). Sections were stained with 20 g/L uranyl acetate in 500 mL/L aqueous ethanol followed by Reynolds lead citrate and examined with a Hitachi H-7000 transmission electron microscope (Hitachi, Tokyo, Japan) operating at an accelerating voltage of 75 kV.
Estimates for the volume density of vacuoles in each cell line were obtained by evaluating 10 randomly selected fibroblasts photographed in entirety at a magnification of 9000x. Area density measurements with a X/Y (180/720) point double square test grid were performed and the volume density calculated by summing the points that fell on vacuoles and dividing by the number of points lying over the fibroblast cell cytoplasm.
protein/enzyme assays
Protein was assayed by the bicinchoninic acid method with bovine
serum albumin as a calibrator (16). Acetyl coenzyme
A:
-glucosaminide N-acetyltransferase was determined by
the method described by Meikle et al. (17). The activities
of acid phosphatase, ß-hexosaminidase, -iduronidase, and
ß-glucosidase were determined by using the 4-methylumbelliferyl
fluorogenic substrates, 4-methylumbelliferyl phosphate for acid
phosphatase activity (18), 4-methylumbelliferyl
2-acetamido-2-deoxy-ß-D-glucopyranosidase for
ß-hexosaminidase activity (19),
4-methylumbelliferyl--L-iduronide for -iduronidase
activity (20), and
4-methylumbelliferyl-ß-glucopyranoside for ß-glucosidase activity
(21).
production of antibodies
Anti-LAMP-1 monoclonal antibody (clone BB6) and anti-LAMP-1
polyclonal antibody have been described previously
(22)(23). For the production of the
anti-LAMP-1 monoclonal antibody clone 4F5, mice were immunized with
lysosomal membranes purified from human placenta (17).
Membranes were denatured by boiling in 10 mL/L 2-mercaptoethanol for 5
min and the pelleted membranes extracted with chloroform:methanol
(2:1) x 2. Female Balb/C mice were immunized according to
the following schedule: 50 µg of antigen in 400 µL of PBS by
intrasplenic injection; 14 days later, 50 µg of antigen in 200 µL
of PBS/incomplete Freund's adjuvant emulsion by intraperitoneal
injection; 21 days later, 50 µg of antigen in 200 µL of PBS by
intraperitoneal injection. Four days later the spleen cells were
harvested and fused with P3.653 myeloma cells as described by Zola and
Brooks (24).
purification of lamp-1
Total membranes from human placenta were prepared as follows:
Fresh placenta (450 g) was dissected into 1–2-cm strips and washed
three times with cold 0.25 mol/L sucrose, 1 mmol/L EDTA, pH 7.0, and
then minced and homogenized [Omnimix (Sorvall, Newtown, CT) 1 min,
full speed] in 800 mL of the same buffer. The cell debris was pelleted
at 750g for 10 min and homogenized a further two times. The
supernatants were combined, filtered through cotton gauze, and made up
to 10 mmol/L CaCl2. After 1 h at 4 °C, the
placental membranes were pelleted at 10 000g for 90 min.
The membranes were resuspended in 1 mol/L NaCl (320 mL), frozen/thawed
three times, and then pelleted at 100 000g for 1 h.
The 1 mol/L NaCl wash was repeated and the membranes finally taken up
into 320 mL of solubilization buffer [50 mmol/L
3-(N-morpholino)propanesulfonic acid (MOPS), 1 mmol/L EDTA,
150 mmol/L NaCl, 100 mL/L glycerol, 10 g/L Thesit, pH 7.0] and stirred
at 4 °C for 16 h. The insoluble material was pelleted at
100 000g for 1 h and the supernatant recovered.
The supernatant was made up to 3 mmol/L CaCl2 and 3
mmol/L MgCl2, and then applied to a 70-mL column of
concanavalin A–Sepharose (Pharmacia Biotech, Uppsala, Sweden)
preequilibrated in solubilization buffer containing the
CaCl2 and MgCl2. The column was washed with the
same buffer, and the bound proteins including LAMP-1 were eluted by
solubilization buffer containing 100 g/L -methyl mannoside. The
eluate was applied to a 5-mL column of red dye no. 78 (Centre for
Protein and Enzyme Technology, LaTrobe University, Bundoora, Australia)
and the LAMP-1 recovered in the flow-through.
Anti-LAMP-1 monoclonal antibody 4F5 (20 mg) was coupled to Affigel (10 mL) and used for the affinity purification of LAMP-1. The red dye flow-through (120 mL) was mixed with the anti-LAMP-1 affinity gel and rocked gently for 16 h at 4 °C; the gel was then poured into a column and washed with PBS. The LAMP-1 was eluted from the column with 100 mmol/L triethylamine, pH 11.5, dialyzed against water, and lyophilized.
sodium dodecyl sulfate–polyacrylamide gel electrophoresis
(sds-page)
Purified LAMP-1 was run on 12.5% SDS-PAGE with the method of
Laemmli (25) and stained with Brilliant Blue G-colloidal
stain (Sigma-Aldrich, Castle Hill, Australia).
immunoquantification of lamp-1
Determination of LAMP-1 was performed with a time-resolved
fluorescence immunoassay. In this type of assay, the detecting antibody
is labeled with a lanthanide metal (usually europium) chelated into
N1-(p-isothiocyanatobenzyl)-diethylenetriamine-N1,
N2, N1
,
N1
-tetraacetic acid. Detection of the labeled
antibody is achieved by lowering the pH to release the Eu3+
from the antibody and the subsequent complex formation with
2-napthoyltrifluoroacetone and tri-n-octylphosphine
oxide. The complex formed is highly fluorescent with a relatively long
half-life, which enables the use of time-resolved fluorescence
detection to eliminate background interferences
[26, 27].
Anti-LAMP-1 monoclonal antibody (clone BB6) was labeled with Eu by using the Delfia® Eu3+-labeling kit (Wallac, North Ryde, Australia). The labeled antibody was purified from aggregated antibody and free Eu3+ label on a Pharmacia Superose 12 fast-phase liquid chromatography column (1.5 x 30 cm) eluting with 50 mmol/L Tris/HCl, pH 7.8, 9 g/L NaCl. The amount of Eu3+ conjugated to each antibody molecule was determined from protein and fluorescence values of the conjugate.
Samples were assayed for LAMP-1 by either a one- or two-step method. In the one-step method, microtiter plates (Immulon 4; Dynatech Labs., Chantilly, VA) were coated with anti-LAMP-1 polyclonal antibody at 5 mg/L for 4 h at 37 °C (100 µL/well diluted in 0.1 mol/L NaHCO3) and washed with Delfia wash buffer (x 6). Samples were diluted in Delfia assay buffer containing 200 µg/L Eu3+-labeled anti-LAMP-1 monoclonal antibody (100 µL/well) and incubated in wells overnight at 4°C. Plates were incubated at room temperature for 1 h and then washed (x 6). Delfia enhancement buffer (200 µL/well) was added, the plates shaken for 10 min at room temperature, and the fluorescence measured on a 1234 Delfia research fluorometer. In the two-step method, if samples contained chemicals that were incompatible with the Eu3+ label (e.g., EDTA, citrate), then the following modifications were made. The plates were coated with the polyclonal antibody and washed as described, samples were diluted in Delfia assay buffer without the Eu3+-labeled antibody and incubated overnight at 4 °C, and then incubated at room temperature for 1 h and washed (x 6). Assay buffer containing 200 µg/L Eu3+-labeled anti-LAMP-1 monoclonal antibody (100 µL) was added to each well and incubated at room temperature for 2 h. Plates were then washed (x 6), enhancement buffer was added, and the fluorescence measured as described.
For determination of LAMP-1 in blood spots, the one-step method was used with the following modifications: Blood spots were incubated with 200 µL of assay buffer containing 200 µg/L Eu3+-labeled anti-LAMP-1 monoclonal antibody. The plates were shaken for 1 h at room temperature before the overnight incubation at 4 °C, and then again for 1 h at room temperature before washing and addition of enhancement buffer.
fractionation of whole blood
Peripheral blood leukocytes and plasma were isolated from whole
blood collected in heparinized tubes by the method of Kampine et al.
(28), and the white cell pellet was resuspended in saline
containing 10 mL/L Nonidet P-40 (lysis buffer). Red cells isolated in
the same procedure were washed twice with saline before being
resuspended in lysis buffer. The saline washes were centrifuged to
pellet the white cells, which were combined with the original white
cell pellet. The supernatants were pooled with the plasma for
determination of LAMP-1 protein.
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Results |
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). To determine if there was any increase in the concentration
of lysosomes in normal or affected cells after reaching confluency,
cells were grown for either 0, 3, or 6 weeks after confluency before
harvesting. Lysosomal volume density showed little change over this
period, whereas the specific activities of acid phosphatase,
ß-glucosidase, and -iduronidase all showed slight increases and
ß-hexosaminidase showed up to a 50% increase over the same period.
Table 2
shows data only from the third harvest at 6 weeks after
confluency. Eight affected cell lines showed between 1.8- and 3.3-fold
increase in lysosomal volume density, notably SF1594 (Salla), SF3420
(cystinosis), SF3223 (MPS VI), and SF3421 (Pompe). Whereas some cell
lines showed an increase in the concentration of lysosomal enzymes, the
results were not always consistent with the volume density
measurements, although cell lines that showed little or no increase in
lysosomal volume density generally showed little or no increase in any
of the lysosomal marker enzymes examined.
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purification of lamp-1
To obtain a pure preparation of LAMP-1 for the calibration of the
LAMP-1 immunoquantification, we purified the protein with a combination
of affinity, dye-binding, and immunoaffinity chromatography. The
preparation of total placental membranes resulted in a LAMP-1 yield of
only 25% of the total LAMP-1 present in the placenta, despite the fact
that >60% of the lysosomal membrane enzyme acetyl coenzyme
A:-glucosaminide N-acetyltransferase was associated with
these membranes. However, solubilization of the membranes,
chromatography on concanavalin A–Sepharose, and subsequent red dye
chromatography all gave >80% recovery of LAMP-1. From a single
placenta we recovered ~1 mg of LAMP-1 in the red dye column
flow-through. The successive immunoprecipitation of the LAMP-1 from
this sample resulted in the recovery of ~100 µg per precipitation.
The purified LAMP-1 appeared as a homogeneous band on Coomassie
Blue-stained SDS-PAGE (not shown), was quantified by the bicinchoninic
acid method, and subsequently used as a calibrator for the
immunoquantification of LAMP-1 protein.
immunoquantification of lamp-1
Labeling of the BB6 monoclonal antibody with Eu3+
resulted in ~5 Eu3+ atoms per antibody molecule. When
used in the one-step immunoquantification assay as described, this gave
a linear response over the range 0.1–12.5 ng/well LAMP-1. A lower
response was obtained with plates coated with 5 mg/L polyclonal
antibody as compared with 10 mg/L (Fig. 1
). The two-step assay gave ~50% of the signal of the one-step
assay with a linear range up to 25 ng/well. A linear response was also
observed when whole blood or plasma from either unaffected or
LSD-affected individuals was assayed (1–50 µL). The intraassay CV
was <9%. The interassay variation, as determined from the CVs of the
calibration curve points over nine assays performed on four different
days, ranged from 2% to 9% across the linear range of the assay. The
calibration curves were subject to linear regression analysis and gave
values for Sy|x of between 0.14 and
0.45 ng/well with an average of 0.26 ng/well; the intercept values had
an average of 0.08 ng/well with a standard deviation of 0.16 ng/well.
Precision studies were also performed on plasma samples. Five plasma
samples ranging in LAMP-1 concentration from 300 to 1200 µg/L were
assayed in triplicate on 10 separate occasions. The intraassay
variation was <6%, whereas the interassay variation was <8%.
Analytical recovery studies were performed by adding a known amount of
purified LAMP-1 to various amounts of plasma in the two-step assay; the
results showed an inverse relation between the concentration of plasma
and the recovery of exogenously added LAMP-1. When 10 mL of plasma was
included per well (the largest volume assayed), recovery of exogenous
LAMP-1 was 68%.
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The effect of the concentration of Eu3+-labeled
antibody on the assay was investigated and we observed that the
increase in signal showed an almost linear correlation with antibody
concentration up to 400 µg/L (Fig. 2
). In all experimental assays performed, 200 µg/L labeled
antibody resulted in suitable sensitivity.
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lamp-1 concentrations in skin fibroblasts
In a separate experiment, the concentration of LAMP-1 was
determined over a 2-week period in four unaffected and four
LSD-affected cell lines, including MPS VI, MPS II, Pompe, and Salla
(see Table 1
for cell lines used). LAMP-1 concentrations showed no
increase over this time period in any cell line, but were increased in
all of the affected cell lines relative to unaffected control cells.
The control cell lines had an average LAMP-1 concentration of 2.8
± 0.4 ng/mg cell protein, whereas the affected cell lines had 4.2,
14.4, 8.7, and 16 ng/mg cell protein for MPS VI, MPS II, Pompe, and
Salla cell lines respectively. All assays were performed in triplicate.
Electron microscopy indicated storage in all four affected cell lines
compared with normal controls (Fig. 3
).
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lamp-1 concentrations in blood
To determine the suitability of LAMP-1 as a marker for newborn
screening for LSDs, we immunoquantified the LAMP-1 present in blood
spot samples taken from 186 unaffected newborns. We observed a
characteristic skewed distribution with a median of 1.3 ng/spot and the
5th and 95th percentiles at 0.76 and 3.3 ng/spot respectively
(Fig. 4
). There was no correlation between LAMP-1 concentration and
age, sex, or birth weight of the newborns. To investigate the
concentrations of LAMP-1 in LSD-affected individuals, we retrieved
plasma samples from the Departmental archives of 320 LSD-affected
individuals, representing 25 disorders and 152 age-matched (median
= 7, range = 0–66) unaffected individuals. LAMP-1 concentrations
in these samples (Fig. 5
) showed a tight distribution in the normal population with a
median of 303 µg/L and the 5th and 95th percentiles at 175 and 448
µg/L respectively. The majority of the LSD-affected individuals had
LAMP-1 concentrations that were above the normal range (72% above the
95th percentile of the control population), with some individuals
having up to 10 times the median concentration of the control
population. When taken by individual disorder, we observed that 17 of
the 25 disorder groups tested had >88% of individuals above the 95th
percentile of the control population, with 12 groups having 100% above
the 95th percentile (Table 3
). We observed a significant correlation between LAMP-1
concentrations and age in the normal population, with a Pearson
correlation coefficient of -0.37 and a significance level <0.001. Of
the affected groups, only Fabry and Gaucher had sufficient numbers and
age range to test for a correlation; the Fabry group showed a Pearson
correlation coefficient of -0.49 with a significance level of 0.02,
whereas the Gaucher group showed no significant correlation between
LAMP-1 concentrations and age.
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The finding of LAMP-1, a membrane protein, in plasma led us to investigate the proportion of LAMP-1 present in the various fractions of whole blood. Samples of whole blood from six unaffected individuals were fractionated and the proportion of LAMP-1 present in white cells, red cells, and plasma was determined. Whole-blood samples had an average of 226 ± 31 µg/L LAMP-1, with the distribution being 53% ± 7% in the plasma, 32% ± 5% in the red blood cell pellet, and 15% ± 5% in the white cell pellet.
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Discussion |
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We have started with the basic premise that one feature common to all LSDs is the formation and accumulation of lysosomal storage vacuoles and an associated increase in some lysosomal proteins. We have observed that, for many LSDs associated with the deficiency of one enzyme, there is a secondary increase of other lysosomal enzymes. In the first part of this study we attempted to identify lysosomal enzymes that are increased and therefore may be suitable markers of lysosomal storage. However, of those cell lines that displayed storage, there was no single enzyme that was uniformly increased. The relatively small increases of lysosomal enzymes seen in affected cell lines clearly do not correlate with the increases observed in the lysosomal volume; presumably the bulk of the increase in lysosomal volume results from the storage of substrate and the increased osmotic presure resulting from that storage. A number of the LSD-affected cell lines showed minimal or no increase in lysosomal storage relative to the unaffected controls despite coming from severely affected patients. This may relate to the metabolism of skin fibroblasts, particularly in culture, being different from the affected tissues in vivo, and so not being required to metabolize large quantities of the stored substrates. For example, keratan sulfate, the major stored substrate in MPS IVA, is not produced in large amounts by skin fibroblasts and hence is not stored. Consequently, skin fibroblasts may not be suitable models to study lysosomal storage in all LSDs.
In our evaluation of LAMP-1 as a potential marker, we selected four of
the fibroblast cell lines that showed significant concentrations of
lysosomal storage under the electron microscope (Fig. 3
). In these cell
lines LAMP-1 appeared to be a potentially useful marker of lysosomal
storage, showing up to fivefold increase relative to normal controls.
The greater increase of LAMP-1 compared with the soluble lysosomal
enzymes in affected cell lines may relate to the fact that LAMP-1 is a
membrane protein thought to have a role in the stabilization of the
lysosomal membrane against hydrolytic degradation (31),
and as such would be essential for the structural integrity of the
storage vacuoles.
Immunoquantification with time-resolved fluorescence has been reported for numerous proteins and low-molecular-mass analytes (32)(33). This technology offers several advantages over conventional ELISA-type assays, including increased sensitivity and extended dynamic range. We have used this technology to develop an immunoassay that will allow the determination of LAMP-1 concentrations in 3-mm blood spot samples from Guthrie cards and in plasma samples. The decreased recovery of exogenously added LAMP-1 to plasma samples suggests that there is some interaction of LAMP-1 with plasma proteins. The nature of this interaction is unclear at this stage and further studies are in progress. However, the linear response observed for blood and plasma from both unaffected and LSD-affected individuals indicates that the decreased recovery of LAMP-1 from plasma will have little effect on the ability of the assay to distinguish affected from unaffected individuals.
For a diagnostic assay to be suitable for newborn screening, the assay
must be inexpensive and ideally should be performed on a blood spot
taken from a Guthrie card, as this negates the requirement for the
collection of an additional sample. In a blind study we investigated
the concentration of LAMP-1 in blood spots from 186 newborns by using
the immunoquantification assay. The histogram (Fig. 4
) shows a typical
skewed distribution, as seen for other analyses in the newborn period.
On the limited population studied, one individual had a LAMP-1
concentration of 7 ng per blood spot. This concentration could possibly
relate to the concentration of white cells present in the blood.
Newborns generally have a high leukocyte count, up to
25 000–35 000/mm1
within the first day after birth;
this decreases rapidly to a mean of only 12 000/mm1
at 2
weeks of age. Other factors such as inflammation or infection may also
contribute to increased LAMP-1 concentrations. Interestingly, we
observed that a significant proportion of the LAMP-1 in whole blood was
present in the plasma (an average of 53% in unaffected controls). The
mechanism by which a lysosomal membrane protein is solubilized and
released from the cells is unclear; however, this has important
implications for the use of LAMP-1 as a diagnostic marker. If LAMP-1 is
secreted from cells into circulation or alternatively results from the
turnover of fragile storage-laden cells, then one might expect that,
regardless of the site of pathology where LAMP-1 is increased, this
increase will be detectable in a sample of whole blood, serum, or
plasma.
Within the Department of Chemical Pathology we have archives of blood spots collected over the past 15 years. However, attempts to retrospectively test blood spots taken from LSD-affected individuals were unsuccessful, as the age of the blood spots was found to be critical for the recovery and determination of LAMP-1 (data not shown). Three-year-old blood spots taken from the archives gave only 30% of the LAMP-1 relative to fresh blood spots. It is unclear whether this is the result of incomplete elution of the LAMP-1, as the older blood spots appear to not elute as effectively, or a result of the degradation of the LAMP-1 in the blood spot to an extent that is no longer recognized by the immunoquantification assay. To determine the potential of LAMP-1 as a LSD screening marker we investigated the concentrations of LAMP-1 in plasma samples from LSD-affected individuals taken at the time of diagnosis. These samples were collected and stored at -20 °C in the departmental archives. We determined the concentrations in 320 affected individuals representing 25 different LSDs and compared these with 152 unaffected age-matched control samples.
When considered as a group, ~70% of LSD-affected individuals had
increased LAMP-1 concentrations. This would suggest that additional
markers will be required for a complete screening procedure. The
increase of LAMP-1 observed appears to be specific for certain
disorders (Table 3
). Interestingly, the disorders that did not show an
increase in LAMP-1 were those that stored predominantly sphingolipids
or sphingolipid derivatives. Gaucher disease is an exception to this
observation, storing glucoceramide but with a mean LAMP-1 concentration
of 956 µg/L. The Gaucher disease group also showed a relatively wide
range of LAMP-1 concentrations and considerable overlap with the
control population. Investigation of the clinical presentation of the
Gaucher-affected patients with low LAMP-1 concentrations showed that
all but one were mild or asymptomatic at the time of diagnosis. The
exception to this was a 9-day-old infant with severe (type II)
presentation, although the two other neonates diagnosed with Gaucher
disease had LAMP-1 concentrations above the control population range.
In this study, the classification of patients into disorder groups was based on the biochemical defect present; however, most of these groups had a range of clinical severity. For those disorders in which <50% of patients showed an increased LAMP-1 concentration, the increased concentrations were generally seen in the younger and presumably more severe patients, in particular the metachromatic leukodystrophy, Fabry, Nieman–Pick (A/B), Nieman–Pick (C), Tay–Sachs disease, and GM-1 gangliosidosis groups. In other disorders, in particular I-cell, even the clinically less severe patients had increased LAMP-1 concentrations as evidenced by a 25-year-old individual with a LAMP-1 concentration of 1019 µg/L.
For most disorders in which LAMP-1 was increased, the storage material was glycosaminoglycan and (or) oligosaccharides. However, Pompe disease, which stores glycogen, showed an increase in only one of four samples. This may reflect the low number tested as we also measured a threefold increase in the concentration of LAMP-1 in skin fibroblasts from a different Pompe-affected individual. All Pompe disease patients showed a classical severe infantile presentation. As a group, the MPS disorders were clearly identified, with 112 of the 115 individuals tested having LAMP-1 concentrations above the 95th percentile of the control group. These disorders all store either heparan sulfate, dermatan sulfate, keratan sulfate, or a combination of these. The degree of LAMP-1 increase observed in the different disorders presumably relates to a number of factors that may include not only the type of stored substrate and the amount of storage, but also the site(s) of storage. In general one could say that the disorders affecting mesenchymal tissues are characterized by high LAMP-1 concentrations and those affecting primarily neurologic, epithelial, or endothelial do not have high LAMP-1 concentrations. However, the variability of phenotype and severity in many disorders makes these types of generalizations difficult, and there will be numerous exceptions. Further studies on the nature and source of the plasma form of LAMP-1 will be required to resolve the question of relative concentrations in the different disorders.
The plasma samples we examined in this study were, for the most part,
from individuals ranging in age from 0 to 10 years (Table 3
), although
in a few disorders the median age at diagnosis was higher, in
particular Fabry, Gaucher, and Neiman–Pick type (A/B), which were 27,
12, and 22 years respectively. The control group was selected to
reflect the age range of affected individuals; the median age was 7
years and the range covered was 0–66 years. A significant, inverse
correlation was observed between LAMP-1 concentrations and age in both
the control population and the Fabry-affected group. This correlation
was not present in the Gaucher-affected group, presumably as a result
of the high LAMP-1 concentrations in most of these patients. The
question of whether LAMP-1 concentrations will also be high in newborn
infants, when Guthrie cards are collected, must also be considered. Six
of the plasma samples examined were from LSD-affected infants <30 days
old, in which an increased LAMP-1 concentration would be expected, and
five of these individuals had LAMP-1 concentrations above the 95th
percentile of the normal population. Other evidence that newborns would
be expected to have high LAMP-1 concentrations comes from electron
microscopy studies on a 14-week MPS IVA-affected human fetus that
clearly showed storage vacuoles in fibroblasts and chondrocytes (S.
Byers, Women's and Children's Hospital, Adelaide, Australia,
unpublished observations), indicating that the storage process begins
early in gestation and so could be well advanced by birth.
Visualization of storage vacuoles by electron microscopy has also been
proposed as a method for prenatal diagnosis of mucolipidosis
(34). Studies on a MPS VI cat model have shown storage
vacuoles present in aorta, liver, cartilage cornea, and skin in a
mid-term fetus and in 2-day-old kittens (14). There are
numerous other reports of storage vacuoles present in lymphocytes and
other cells of affected newborns (35). It appears that for
the majority of LSDs, storage occurs prenatally, possibly commencing
from conception, and is well advanced in the neonate, although in most
cases clinical symptoms are not apparent at this stage.
We have demonstrated that the concentration of LAMP-1 in fibroblasts correlates to the concentration of lysosomal storage vacuoles and that LAMP-1 is increased in the plasma of ~70% of LSD-affected individuals. The immunoquantification assay developed for LAMP-1 with time-resolved fluorescence technology can determine the concentration in a blood spot taken from a Guthrie card and therefore is amenable to newborn screening for these disorders. Current spot test methods would require the collection of additional urine samples and do not cover a wide range of LSDs, and are therefore impractical for newborn screening. We propose that LAMP-1 may be a useful diagnostic marker for the detection of most LSD-affected individuals at birth. We have identified several disorders that do not show a significant increase in LAMP-1 and we are in the process of evaluating additional markers that may be necessary for the development of an effective screening program. Chitotriosidase, recently reported to be increased in plasma from Gaucher disease patients as well as 10 of 23 other lysosomal disorders (36), could be one such marker. We propose that this type of assay would be the first tier in a newborn screening program and would serve to identify a group of individuals who are at increased risk of being LSD affected. This group, which may represent the top 1–5% of the population, would then be further examined (with the same Guthrie card) with a panel of second-tier diagnostic assays designed to detect the storage product for the particular disorder involved. This would effectively identify the affected individuals from the false positives identified in the first-tier screen. We are currently developing the second-tier screening assays for this procedure.
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) or 10 mg/L (
) with the one-step quantification
method as described in Materials and Methods.
