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Diagnosis of lysosomal storage disorders: evaluation of lysosome-associated membrane protein LAMP-1 as a diagnostic marker.

INDEXING TERMS: blood spot * Guthrie card * skin fibroblast * time-resolved fluorescence immunoassay

Lysosomal storage disorders (LSDs) represent a group of 39 distinct genetic diseases, each one resulting from a deficiency of a particular lysosomal protein or, in a few cases, from nonlysosomal proteins that are involved in lysosomal biogenesis. The importance of these disorders to healthcare becomes obvious when the group incidence rate for LSD (1:5000 births) is compared with well-known and intensively studied genetic disorders for which newborn screening is currently performed, such as phenylketonuria (1:14 000) and cystic fibrosis (1:2500). LSDs generally affect young children and have a devastating impact on the child and the family involved. Affected individuals can present with a wide range of clinical symptoms depending upon the specific disorder and the particular genotype involved. Central nervous system (CNS) dysfunction, from behavioral problems to severe mental retardation, is characteristic of many LSDs. In the mucopolysaccharidoses, other symptoms may include skeletal abnormalities, organomegaly, corneal clouding, and dysmorphic features [1]. In severe cases, the child requires constant medical management of the disorder but dies before adolescence.

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 [beta]-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.

Materials and Methods


Blood spots used in this study were part of the routine samples collected from neonates from the South Australian population. Plasma samples used were from samples submitted to the National Referral Laboratory for LSD screening and samples processed for routine biochemistry.


Human diploid fibroblasts were established from skin biopsies submitted to the Women's and Children's Hospital for diagnosis [151. Cell lines were maintained according to established procedures in Eagle's basal medium, 100 mL/L fetal calf serum, and antibiotics, in a 5% C[O.sub.2] 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 [cm.sup.2] 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.


Cells were washed twice with Dulbecco's PBS and removed from the flask by trypsination [2 mL of trypsinversene solution (CSL, Melbourne, Australia) per flask for 5 min at 37 [degrees]C]. The cells were immediately washed twice with cold PBS and cells from one flask from each time point were resuspended in 200 [micro]L 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.


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-[micro]m 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 was assayed by the bicinchoninic acid method with bovine serum albumin as a calibrator [16]. Acetyl coenzyme A:[alpha]-glucosaminide N-acetyltransferase was determined by the method described by Meikle et al. [17]. The activities of acid phosphatase, [beta]-hexosaminidase, a-iduronidase, and [beta]-glucosidase were determined by using the 4-methylumbelliferyl fluorogenic substrates, 4-methylumbelliferyl phosphate for acid phosphatase activity [18], 4-methylumbelliferyl 2-acetamido-2-deoxy-[beta]-D-glucopyranosidase for [beta]-hexosaminidase activity [19], 4-methylumbelliferyl-[alpha]-L-iduronide for [alpha]-iduronidase activity [20], and 4-methylumbelliferyl-[beta]-glucopyranoside for [beta]-glucosidase activity [21].


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 [micro]g of antigen in 400 [micro]L of PBS by intrasplenic injection; 14 days later, 50 [micro]g of antigen in 200 [micro]L of PBS/incomplete Freund's adjuvant emulsion by intraperitoneal injection; 21 days later, 50 [micro]g of antigen in 200 [micro]L 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].


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 7508 for 10 min and homogenized a further two times. The supernatants were combined, filtered through cotton gauze, and made up to 10 mmol/L Ca[Cl.sub.2]. After 1 h at 4 [degrees]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 [degrees]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 Ca[Cl.sub.2], and 3 mmol/L Mg[Cl.sub.2] and then applied to a 70-mL column of concanavalin A--Sepharose (Pharmacia Biotech, Uppsala, Sweden) preequilibrated in solubilization buffer containing the Ca[Cl.sub.2] and Mg[Cl.sub.2]. The column was washed with the same buffer, and the bound proteins including LAMP-1 were eluted by solubilization buffer containing 100 g/L [alpha]-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 [degrees]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.


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).


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 [N.sup.l]-(p-isothiocyanatobenzyl)- diethylenetriamine-[N.sup.l],[ [N.sup.2], [N.sup.3], [N.sup.3]-tetraacetic acid. Detection of the labeled antibody is achieved by lowering the pH to release the [Eu.sup.3+] 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[R] [Eu.sup.3+]-labeling kit (Wallac, North Ryde, Australia). The labeled antibody was purified from aggregated antibody and free [Eu.sup.3+] label on a Pharmacia Superose 12 fast-phase liquid chromatography column (1.5 X 30 cm) eluting with 50 mmol/L Tris/HCI, pH 7.8, 9 g/L NaCl. The amount of [Eu.sup.3+] 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 [degrees]C (100 [micro]L/well diluted in 0.1 mol/L NaHC[O.sub.3]) and washed with Delfia wash buffer (X 6). Samples were diluted in Delfia assay buffer containing 200 [micro]g/L [Eu.sup.3+]-labeled anti-LAMP-1 monoclonal antibody (100 [micro]L/well) and incubated in wells overnight at 4 [degrees]C. Plates were incubated at room temperature for 1 h and then washed (X 6). Delfia enhancement buffer (200 [micro]L/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 [Eu.sup.3+] 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 [Eu.sup.3+]-labeled antibody and incubated overnight at 4 [degrees]C, and then incubated at room temperature for 1 h and washed (X 6). Assay buffer containing 200 [micro]g/L [Eu.sup.3+]-labeled anti-LAMP-1 monoclonal antibody (100 [micro]L) 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 [micro]L of assay buffer containing 200 [micro]g/L [Eu.sup.3+]-labeled anti-LAMP-1 monoclonal antibody. The plates were shaken for 1 h at room temperature before the overnight incubation at 4 [degrees]C, and then again for 1 h at room temperature before washing and addition of enhancement buffer.


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.



In an attempt to identify a suitable marker of lysosomal proliferation, we investigated the concentrations of several lysosomal enzymes in a range of LSD-affected cell types (Table 2). 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, [beta]-glucosidase, and [alpha]-iduronidase all showed slight increases and [beta]-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.


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:[alpha]-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 [micro]g 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.


Labeling of the BB6 monoclonal antibody with [Eu.sup.3+] resulted in ~5 [Eu.sup.3+] 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 [micro]L). 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 Syl, 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 [micro]g/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%.


The effect of the concentration of [Eu.sup.3+]-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 [micro]g/L (Fig. 2). In all experimental assays performed, 200 [micro]g/L labeled antibody resulted in suitable sensitivity.


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 NIPS VI, NIPS 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 [+ or -] 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 NIPS VI, NIPS 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).



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 [micro]g/L and the 5th and 95th percentiles at 175 and 448 [micro]g/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.

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 [+ or -] 31 [micro]g/L LAMP-1, with the distribution being 53% [+ or -] 7% in the plasma, 32% [+ or -] 5% in the red blood cell pellet, and 15% [+ or -] 5% in the white cell pellet.



LSDs are a large group of rare genetic disorders. Incidence levels vary depending upon the disorder and the population being studied. Values as high as 1:18 500 for aspartylglucosaminuria in the Finnish population [29] and 1:24 000 for Sanfillipo syndrome (types A, B, C, and D) in The Netherlands [30] have been reported. However, for the most part the incidence levels are of the order of 1:50 000 to 1:100 000 or less. If newborn screening for lysosomal storage disorders is to be practical and economically viable, it will be necessary to screen for multiple disorders simultaneously.


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 [311, 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/[mm.sup.3] within the first day after birth; this decreases rapidly to a mean of only 12 000/[mm.sup.3] 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 [degrees]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 [micro]g/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 [micro]g/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.

We gratefully acknowledge Vivienne Muller and Bill Carey for supplying plasma samples from LSD-affected individuals. We thank Enzo Ranierri for advice on the immunoquantification of LAMP-1 with time-delayed fluorescence. This work was supported by the Channel 7 Children's Research Foundation, the Australian Research Council, and the National Health and Medical Research Council of Australia.

Received December 23, 1996; revised and accepted April 18, 1997.


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Lysosomal Diseases Research Unit, Departments of Chemical Pathology and (1) 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.

*Author for correspondence. Fax 61 8-8204 7100; e-mail

(3) Nonstandard abbreviations: LSD, lysosomal storage disorder; MPS, mucopolysaccharidosis; CNS, central nervous system; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Table 1. LSD-affected skin fibroblast cell lines used in this study.

Cell lines Disorder

SF1594 (a) Salla
SF1779 (a) MPS II
SF850; SF1770 MPS IVA
SF3420; SF3215 Cystinosis
SF3168 (a); SF913; SF3223 MPS VI
SF2451; SF3823; SF2469 Gaucher
SF2925; SF613 [alpha]-Mannosidosis
SF2182 Fucosidosis
SF2512; SF3425 Tay-Sachs
SF3960 (a); SF3847; SF3421 Pompe
SF2551; SF3390 MSD (e)

Cell lines Enzyme deficiency

SF1594 (a) Sialic acid transporter
SF1779 (a) Iduronate-2-sulfatase
SF850; SF1770 Galactose-6 sulfatase
SF3420; SF3215 Cystine transporter
SF486; SF737 Sulfamidase
SF3168 (a); SF913; SF3223 N-Acetylgalactosamine-4 sulfatase
SF2451; SF3823; SF2469 [beta]-Glucosidase
SF2925; SF613 [alpha]-Mannosidase
SF2182 [alpha]-Fucosidase
SF2512; SF3425 [beta]-Hexosaminidase A
SF3960 (a); SF3847; SF3421 [alpha]-Glucosidase
SF2551; SF3390 Sulfatases

Cell lines Stored material

SF1594 (a) Sialic acid
SF1779 (a) DS (b)/HS(c)
SF850; SF1770 KS (a)
SF3420; SF3215 Cystine
SF486; SF737 HS
SF3168 (a); SF913; SF3223 DS
SF2451; SF3823; SF2469 Glucocerebroside
SF2925; SF613 Oligosaccharides
SF2182 Sphingolipids/oligosaccharides
SF2512; SF3425 GM2-ganglioside
SF3960 (a); SF3847; SF3421 Glycogen
SF2551; SF3390 Sulfatide/HS/DS/steroid sulfate

(a) Denotes cell lines used for LAMP-1
quantification and electron microscopy studies (Fig. 3).

(b) Dermatan sulfate.

(c) Heparan sulfate.

(d) Keratan sulfate.

(e) Multiple sulfatase deficiency.

Table 2. Lysosomal enzyme concentrations and volume density
determinations of LSD-affected and control cell lines at
6 weeks postconfluency.

Cell IInes Disorder VD (a) [beta]-Hex (b)

 Normal (n = 10) (f) 9 .0(3.1) 241(45)
SF 1594 Salla 30.1 593
SF 850 MPS IVA 9.6 304
SF 1770 MPS IVA 9.5 247
SF 3420 Cystinosis 20 504
SF 3215 Cystinosis 11.3 283
SF 486 MPS IIIA 8.6 353
SF 737 MPS IIIA 12.5 213
SF 913 MPS VI 11.5 285
SF 3223 MPS VI 21.0 381
SF 2451 Gaucher nd 323
SF 3823 Gaucher 7.7 199
SF 2469 Gaucher 12.1 265
SF 2925 [alpha]-Mannosidosis 18.8 345
SF 613 [alpha]-Mannosidosis 12.8 263
SF 2182 Fucosidosis 2.9 300
SF 2512 Tay-Sachs (1) 5.9 284
SF 3425 Tay-Sachs (1) 10.3 355
SF 3847 Pompe 21 279
SF 3421 Pompe 20.7 362
SF 2551 MSD (g) 17.4 172
SF 3390 MSD 16.2 159

Cell IInes ACP (c) [beta]-Glu (d) [alpha]-Ida (e)

 121(54) 6.8(4.6) 11.1 (8.7)
SF 1594 380 11.9 18.8
SF 850 98 4.2 29.8
SF 1770 40 4.5 3.0
SF 3420 188 7.1 21.3
SF 3215 157 3.4 4.0
SF 486 44 5.6 7.1
SF 737 173 3.4 9.9
SF 913 96 6.6 5.3
SF 3223 123 3.9 10.0
SF 2451 140 0.1 6.7
SF 3823 83 0.1 19.9
SF 2469 106 0.1 20.3
SF 2925 107 3.0 13.0
SF 613 83 10.2 51.5
SF 2182 117 4.2 21.1
SF 2512 136 2.9 19.4
SF 3425 219 10.8 13.4
SF 3847 223 6.3 2.0
SF 3421 99 4.9 4.5
SF 2551 86 4.2 32.1
SF 3390 47 4.6 14.0

(a) Percentage volume density of lysosomes calculated from
electron micrographs.

(b) [beta]-Hexosaminidase activity expressed as nmol/min
per mg of cell protein.

(c) Acid phosphatase activity expressed as nmol/min per mg
of cell protein.

(d) [beta]-Glucosidase activity expressed as nmol/min per mg
of cell protein.

(e) [alpha]-Iduronidase activity expressed as nmol/min per
mg of cell protein.

(f) Values shown are means (SD) of the 10 control cell lines.

(g) Multiple sulfatase deficiency.

Table 3. Detection of LSD on the basis of increased
LAMP-1 concentrations in plasma.

Disorder Stored substrates

Fabry [alpha]-Galactosylsphingolipids;
Galactosialidosis Oligosaccharides
Gaucher Glucoceramide
GM1-gangliosidosis GM1-gangliosides; oligosaccharides;
 glycolipids; keratan sulfate
I-cell Glycolipids; oligosaccharides
Krabbe Gal actocerarn ides

[alpha]-Mannosidosis [alpha]-Mannosides
Metachomatic leukodystrophy Sulfatides
MPS I Dermatan sulfate; heparan sulfate
MPS II Dermatan sulfate; heparan sulfate
MPS IIIA Heparan sulfate
MPS IIIB Heparan sulfate
MPS HIC Heparan sulfate
MPS HID Heparan sulfate
MPS IVA Keratan sulfate
MPS VI Dermatan sulfate
Multiple sulfatase deficiency Sulfatides; glycolipids;
Niemann-Pick (A and B) Sphingomyelin
Niemann-Pick (C) Cholesterol; sphingomyelin
Pompe Glycogen
Sandhoff GM2-gangliosides; oligosaccharides
Sialic acid storage Sialic acid; glucuronic acid
Tay-Sachs (1) GM2-gangliosides; oligosaccharides
Tay-Sachs (AB) GM2-gangliosides
Wolman Cholesterol esters

Disorder n Age (a)

Control 152 7(0-66)
Fabry 24 27(4-47)

Galactosialidosis 1 16
Gaucher 51 12(0-68)
GM1-gangliosidosis 12 1 (0-15)

I-cell 15 3(0-25)
Krabbe 12 0.4(0 -1)
[alpha]-Mannosidosis 4 4(3-15)
Metachomatic leukodystrophy 32 3(0-30)
MPS I 22 1 (0-29)
MPS II 23 3(0-11)
MPS IIIA 20 4(1-17)
MPS IIIB 16 3(2-21)
MPS HIC 3 11(6-20)
MPS HID 3 3(0 -3)
MPS IVA 16 3(0-12)
MPS VI 12 4(0-16)
Multiple sulfatase deficiency 2 5(3 -7)

Niemann-Pick (A and B) 9 22(1-44)
Niemann-Pick (C) 10 12(0-41)
Pompe 4 0.4(0 -1)
Sandhoff 6 1
Sialic acid storage 2 2(0 -3)
Tay-Sachs (1) 17 1 (0-27)
Tay-Sachs (AB) 2 7(6 -8)
Wolman 2 1(0 -1)

Disorder Median (b) % > 95 (c)

Control 308 5
Fabry 359 25

Galactosialidosis 1653 100
Gaucher 956 92
GM1-gangliosidosis 1071 92

I-cell 1815 100
Krabbe 407 17
[alpha]-Mannosidosis 1181 100
Metachomatic leukodystrophy 379 19
MPS I 1470 100
MPS II 1406 100
MPS IIIA 1039 100
MPS IIIB 880 100
MPS HIC 787 100
MPS HID 1010 100
MPS IVA 699 88
MPS VI 1018 92
Multiple sulfatase deficiency 507 100

Niemann-Pick (A and B) 385 33
Niemann-Pick (C) 391 20
Pompe 462 25
Sandhoff 565 100
Sialic acid storage 1035 100
Tay-Sachs (1) 425 41
Tay-Sachs (AB) 489 100
Wolman 496 50

(a) Median age (range) of patients in each group.

(b) Median value of plasma LAMP-1 expressed as [micro]g/L.

(c) Percentage of each disorder group with LAMP-1
concentrations above the 95th percentile of the control
population (448 [micro]g/L).
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Title Annotation:Molecular Pathology
Author:Meikle, Peter J.; Brooks, Doug A.; Ravenscroft, Elaine M.; Yan, Miao; Williams, Ruth E.; Jaunzems, A
Publication:Clinical Chemistry
Date:Aug 1, 1997
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