Immunochemistry of Lysosomal Storage Disorders

Clinical Chemistry 52:9 1660 1668 (2006) Reviews Immunochemistry of Lysosomal Storage Disorders Emma Parkinson-Lawrence, Maria Fuller, John J. Hopwood, Peter J. Meikle, and Doug A. Brooks* Background: Lysosomal storage disorders are a group of genetic diseases, each with a broad spectrum of clinical presentation that ranges from attenuated to severe. The immunochemical analysis of patient samples is aimed at several key aspects of patient management, including early detection of the disorder, prediction of clinical severity, determining the most appropriate therapeutic regimen, and monitoring of patients on therapy. Methods: In this study, we review the current and emerging technology available to achieve these assessments. Results: Immune assays have direct practical application for the early detection, diagnosis and prognosis of lysosomal storage disorder patients. Multiplexing of these assays may provide a platform to allow newborn screening for multiple lysosomal storage disorders. Conclusions: We have reviewed the immunochemical techniques available for the analysis of lysosomal storage disorder patient samples and advise that these may be used in conjunction with other technologies for effective patient management. 2006 American Association for Clinical Chemistry Lysosomal storage disorders (LSDs) 1 are a group of more than 50 inherited diseases, which have a combined incidence of 1:7700 live births (1). Each disorder is caused by the dysfunction of either a lysosomal enzyme or a lysosome-associated protein involved in enzyme activation, enzyme targeting, or lysosomal biogenesis. These defects lead to the accumulation of substrate that would normally be degraded in the endosome lysosome system. In severely affected patients, this ultimately leads to the chronic and progressive deterioration of affected cells, Lysosomal Diseases Research Unit, Department of Genetic Medicine, Children, Youth and Women s Health Service, North Adelaide, South Australia, Australia, and Department of Paediatrics, University of Adelaide, Adelaide, South Australia, Australia. *Address correspondence to this author at: Lysosomal Diseases Research Unit, Department of Genetic Medicine, Children, Youth and Women s Health Service, 72 King William Rd., North Adelaide, South Australia 5006, Australia. Fax 61-8-8161-7100; e-mail douglas.brooks@adelaide.edu.au. Received December 5, 2005; accepted June 15, 2006. Previously published online at DOI: 10.1373/clinchem.2005.064915 tissues, and organs. Most LSDs display a broad spectrum of clinical manifestations, which have been previously identified as clinical subtypes [such as the Hurler/Scheie definition of mucopolysaccharidosis (MPS) I and the infantile-, juvenile-, and adult-onset forms of Pompe disease]. Some of the clinical symptoms that are observed in multiple LSDs (e.g., most of the MPSs) include bone abnormalities, organomegaly, coarse hair/facies, and central nervous system (CNS) dysfunction (2). At the severe end of the clinical spectrum, the onset of pathology tends to be rapid and progressive, whereas at the attenuated end, disease onset is later and progress less rapid. With the advent of molecular biology/genetics and the characterization of many of the genes associated with LSDs, it has now been recognized that the range of clinical severity may in part be ascribed to different disease-causing variations within the same gene. However, genotype phenotype correlations are not always informative (3). For example, in Gaucher disease, there are sometimes substantial differences in clinical manifestation between patients with the same genotype, and in some instances, one patient has been severely affected whereas another was virtually disease free (4). Other factors, including genetic background and environment, can also play a role in disease progression. The broad spectrum of clinical presentation in LSDs can make clinical diagnosis extremely difficult, taking months to years in some instances. Therapies, such as bone marrow or hematopoietic stem-cell transplantation and enzyme replacement therapy (ERT), are currently available for several LSDs, including MPS I (5, 6), MPS VI (7), Gaucher disease (8), and Fabry disease (9, 10). Furthermore, clinical trials of ERT for MPS II (11) and Pompe disease (12) are in progress. Other strategies being developed include substrate deprivation (13 17), gene replacement (18 22), premature stop codon read-through (23), and chemical chaperone (24 1 Nonstandard abbreviations: LSD, lysosomal storage disorder; CNS, central nervous system; ERT, enzyme replacement therapy; HRP, horseradish peroxidase; LAMP, lysosomal membrane glycoprotein; MPS, mucopolysaccharidosis. 1660

Clinical Chemistry 52, No. 9, 2006 1661 26) therapies. The success of these treatment strategies in some cases (e.g., CNS pathology) may rely on the commencement of therapy before the pathology becomes irreversible. Recent progress toward newborn screening for LSDs holds promise for early detection (27 29). However, when patients are identified presymptomatically, decisions on the best therapeutic approach to apply will be difficult in the absence of a clinical prognosis. Accurate and sensitive methods are therefore required for the prediction of clinical phenotype, particularly the predisposition to CNS pathology. This review discusses immunochemical approaches that have been used to address the need for early detection, phenotype prediction, and characterization of the disease process in patients with LSDs. development of immunochemical tools for lysosomal proteins A number of lysosomal proteins have been purified to homogeneity from human tissues such as the liver, placenta, and kidney (30, 31). However, amounts of specific lysosomal proteins that can be obtained from tissue sources is limited, and this approach has been largely replaced by expression systems using either prokaryotic or eukaryotic cell cultures (32 35). The production of large amounts of specific lysosomal proteins in culture has enabled the purification of these proteins for applications such as ERT and antibody production. With established methods, both monoclonal and polyclonal antibodies have been raised against a number of lysosomal proteins (Table 1) (36 44). Typically produced are antibodies against purified protein, purified recombinant protein, or synthetic peptides that represent specific linear sequence epitopes on the protein. Immunizing animals with either native or reduced and denatured protein can enhance the production of antibodies that respectively recognize discontinuous/conformational epitopes and linear sequence epitopes. These antibodies have different specificities and can be used to probe different protein structures. For example, antibodies to linear sequence/denatured epitopes have allowed the specific visualization of protein proteolytic processing, by detecting the molecular subunits of the protein after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (45). In contrast, antibodies that recognize discontinuous/conformational epitopes have been used for pulse-chase labeling and immune precipitation techniques to investigate protein synthesis and provide further information on the stepwise processing of lysosomal proteins (46). Immunoaffinity chromatography has been used to purify lysosomal proteins of interest (45) and, more recently, has formed the basis of many assay systems. A specific antibody can be immobilized to a solid phase (e.g., microtiter well) and used to bind the protein from a biological mixture. The immobilized protein can then be analyzed for activity by use of a substrate (e.g., radiolabeled or fluorogenic substrate as described below) or Table 1. Summary of protein and substrate markers in different studies, detected by immune assays for some lysosomal storage disorders. References Marker LSD Immune quantification (protein) Immune capture (activity) Histology Protein -L-Iduronidase MPS I 37 37, 60 Iduronate-2-sulfatase MPS II 42, 61, 74 61 Sulfamidase MPS IIIA 39 N-Acetyl-galactosamine-4-sulfatase MPS VI 38, 43, 72, 73 38, 43, 72, 73 -Galactosidase Fabry 41 41 Saposin C Fabry 41 Saposin C Multiple 69, 70 Acid -glucosidase Pompe 40 59 -Glucosidase Gaucher 44 44 LAMP-1 Multiple 68, 70 LAMP-2 Multiple 78 CCL18 Gaucher 79 Chitotriosidase a Gaucher Substrate G M2 MPS I, MPS II, MPS IIIA, MPS IIIB, MPS VII 50, 51 G M3 51 Heparan sulfate MPS I, MPS IIIA, MPS IIIB, MPS VII 50 Keratan sulfate MPS IVA 49 Trihexosylceramide Fabry 62, 63 a Enzyme activity assays have been developed for this protein (80).

1662 Parkinson-Lawrence et al.: Immunochemistry of Lysosomal Storage Disorders quantified by use of a secondary antibody (46). Antibodies produced against lysosomal proteins have been used to immunoquantify both activity and protein (Table 1) from dried-filter blood spots, plasma, cell extracts, and urine (37 46). For sandwich-based protein quantification, there are a number of signal detection systems. The 2 most commonly used for lysosomal proteins have been horseradish peroxidase (HRP) and the lanthanide element europium (Eu 3 ): the HRP system involves binding a specific primary antibody to the lysosomal protein and then an HRP-labeled species-specific 2nd antibody, which can then be detected by an HRP enzyme reaction; the 2nd system involves direct labeling of the primary antibody with Eu 3 chelated to N 1 -(p-isothiocyanatobenzyl)-diethylenetriamine-n 1,N 2,N 2,N 3 -tetraacetic acid. Lowering the ph releases the Eu 3 from the antibody, and the free Eu 3 then forms a complex with 2-napthoyltrifluoroacetone and tri-n-octylphosphine oxide present in the enhancement solution to produce a highly fluorescent molecule with a relatively long half-life, thereby enabling the use of time-resolved fluorescence detection. This approach has been less prone to problems with specificity and background reactivity because of the single antibody reagent. In addition, the Eu 3 label has a greater linear range and has proved more sensitive than enzyme-linked methods (47). Antibodies have also been generated against the substrate that accumulates in LSD patients (Table 1), including heparan sulfate (48), keratan sulfate (49), G M2 and G M3 gangliosides (50), and other glycosphingolipids (51). Antibodies have been used for the localization of heparan sulfate in murine models of the mucopolysaccharidoses (50). Similarly, anti-g M2 and anti-g M3 antibodies were also used to colocalize gangliosides in MPS IIIA mouse tissues by use of confocal microscopy. Subsequent observations demonstrated that although G M2 and G M3 gangliosides were in the same neurons, they were located in separate populations of cytoplasmic vesicles and only partially colocalized with the primary substrate heparan sulfate. Antibodies against gangliosides as well as other glycosphingolipids have been used to investigate the cellular processes in LSDs (51). Much of this work has focused on the altered trafficking and homeostatic control of these lipids within the endosomal-lysosomal system. These immunohistochemical studies have provided information on the disease process and in the future may be developed for use in LSD diagnosis/prognosis. A potential limitation is that immunohistochemistry techniques are not quantitative, but other technologies (e.g., mass spectrometry, gas chromatography, and HPLC) may allow the functional development of these disease markers. application of immune assays Diagnosis of LSDs. The majority of LSDs result from defects in enzymes involved in the digestion of specific substrates. Consequently, after clinical suspicion, most LSDs are diagnosed by either a decrease in or loss of enzyme activity, usually involving an artificial substrate with a fluorescent tag such as 4-methylumbelliferone, or a natural substrate in which a fragment of the biological substrate is radioactively or otherwise labeled. These assays are mostly performed on sera samples, leukocytes, or cultured skin fibroblasts. The collection and transport of cellular samples can be problematic, prompting the development of enzyme assays from dried blood spots on filter paper. This form of patient sample has the advantages of being easily collected and stored as well as being simple and inexpensive to transport. In 2001, Chamoles et al. reported a number of direct enzyme assays for LSDs performed on dried blood spots, including -l-iduronidase (deficient in MPS I) (52), -galactosidase (deficient in Fabry disease) (53), -d-galactosidase (deficient in G M1 gangliosidosis) (54), and a range of other enzymes capable of diagnosing LSDs that present with a Hurler-like phenotype (55). These reports were followed by the development of similar assays for -glucocerebrosidase and acid sphingomyelinase (deficient in Gaucher disease and Niemann-Pick disease types A and B, respectively) (56), -hexosaminidase (deficient in Tay Sachs and Sandhoff diseases) (57), and acid -glucosidase (deficient in Pompe disease) (58). Although many of these assays have proven to be reliable for the diagnosis of LSDs from dried blood spots, for some enzymes, direct assay can be problematic. With acid -glucosidase, for example, other -glucosidase activities, predominantly maltose glucoamylase, that are present in whole leukocytes can contribute to measured activity. A similar situation exists for some of the lysosomal sulfatases in which the fluorescent substrate 4-methylumbelliferyl sulfate can be hydrolyzed by a number of sulfatases. In direct assays, these problems have been partly addressed by selective inhibition of the unwanted activities (58). However, immunecapture activity assays (Table 1) circumvent the need for specific substrates, because the specificity resides with the capture antibody. Such assays have been developed for N-acetylgalactosamine-4-sulfatase (deficient in MPS VI) (43) and acid -glucosidase (59), enabling successful diagnosis in dried blood spots, without the problems associated with unrelated enzyme activities. Additional immune-capture activity assays have been reported for -l-iduronidase (60), -galactosidase (41), -glucosidase (44), and iduronate-2-sulfatase (61). Notably, the immune-binding step in the iduronate-2-sulfatase assay had the unusual effect of allowing this enzyme, once bound to polyclonal antibody, to catabolize the 4-methylumbelliferyl sulfate substrate, whereas uncaptured iduronate-2sulfatase did not. A major limitation of immunecapture methods is the availability of antibody reagents with appropriate specificity for the enzyme. In some cases, the availability of the appropriate fluorescencelabeled or alternatively labeled substrate can also be restrictive.

Clinical Chemistry 52, No. 9, 2006 1663 Identification of Heterozygous Individuals. For most LSDs, the identification of heterozygous individuals in the population is not a high priority because the disorders are relatively rare, with carrier rates between 1:120 and 1: 1 000 (1). Thus, the chance of a known carrier having an affected child with a partner from the general population is small. However, for X-linked disorders (Fabry disease, MPS II, and Danon disease) carrier females have a 1:2 chance of their male children being affected. In addition, Fabry heterozygotes often develop clinical pathology requiring therapeutic intervention. Direct enzyme analysis in leukocytes is not reliable for the detection of carriers of either Fabry disease or MPS II. Although carrier information can be obtained from enzyme analysis of individual hair roots, which develop from a very small number of progenitor cells, this method is labor-intensive and is rarely offered as a diagnostic service. Molecular techniques are becoming the main method for carrier detection. In the absence of a known variation, whole gene sequencing may be required, and it is also possible that mutations in noncoding regions may be missed. An alternative approach has used a monoclonal antibody to semiquantify trihexosylceramide in cultured fibroblasts from Fabry disease patients by use of laser scanning confocal imaging (62, 63): this method allowed the identification of Fabry heterozygotes and showed that 50% of fibroblasts had trihexosylceramide accumulation. As an alternative to carrier detection, immune-based techniques hold promise for screening broad sections of the community, for example, to screen renal clinic patients for Fabry disease. To this end, immune quantification of -galactosidase protein and activity from dried blood spots has been used to identify Fabry patients, and the combination of -galactosidase protein and saposin C protein has been used to discriminate heterozygotes from controls (41). Population/Newborn Screening for LSDs. Diagnosing LSD patients can be made difficult by the broad range of clinical presentation, particularly the late- or adult-onset forms, for which symptoms are less specific and often not indicative of an LSD. There have been a number of proposals for LSD screening in high risk groups, including screening for Fabry disease in patients from renal and cardiac clinics (64 66) and Danon disease in children with hypertrophic cardiomyopathy (67). The use of immune assays in these screening programs could facilitate rapid, early diagnosis of affected individuals. For many LSDs, the identification of affected individuals before the onset of what are often irreversible symptoms is likely to offer the best outcome for the patient and family. This is particularly important for those LSDs that affect the CNS or bone development. In the absence of a family history, the only practical way to diagnose individuals presymptomatically is through a newborn screening program. The immune-based and free activity assays described above have obvious application for newborn screening of LSDs with dried blood spot samples. However, the cost of screening for individual LSDs would, in most cases, be prohibitive, because the incidence rates range from 1:50 000 births to 1:4 000 000 births (1). Screening for multiple LSD disorders in a single test could address this problem. Immune quantification assays for lysosomal membrane glycoprotein LAMP-1 (68) and saposin C (69) have been reported, and these proteins were increased in multiple LSDs (Table 1). Although these markers were increased in the majority of patients, the amount of overlap between affected and control populations proved to be too great for a viable screening program (70). Other more promising approaches to detect multiple LSDs include the mass spectrometric detection of various analytes (71) and the multiplex detection of lysosomal proteins (27). Immune assays have been developed and applied to the detection of variant and wild-type protein for a number of LSDs, including MPS I (37, 60), MPS II (42, 61), MPS IIIA (39), MPS VI (38, 43), Pompe disease (40), Fabry disease (41), and Gaucher disease (44) (Table 1). These studies have shown that the amount of variant protein in the majority of LSD patient samples is low and is 0% 5% of that detected in unaffected control samples. These observations raised the possibility of using protein quantification rather than activity as the basis for a newborn screening program. The multiplexing of these protein assays has been achieved, and a recent report showed the detection of at least 11 different lysosomal proteins from a single 3-mm dried blood spot (27). The analysis of these proteins from blood spots allowed the detection of all patients with the following disorders: MPS I ( -l-iduronidase), MPS II (iduronate-2-sulfatase), MPS IIIA (sulfamidase), MPS VI (4-sulfatase), metachromatic leukodystrophy (arylsulfatase A), Niemann-Pick disease types A and B (acid sphingomyelinase), and multiple sulfatase deficiency. It also allowed the detection of most Fabry ( galactosidase), Pompe ( -glucosidase), and Gaucher disease ( -glucocerebrosidase) patients, as well as patients with I-cell disease. A different approach, the multiplexing of enzyme activity analysis for dried blood spots, has also been reported (28). Five separate enzyme reactions were performed ( glucosidase, -glucosidase, -galactosidase, -galactosidase, and acid sphingomyelinase) with 5 separate 2-mm dried blood spots and a cassette of synthetic substrates and internal standards. The enzyme reaction mixtures were then combined and the buffer salts removed by use of liquid liquid extraction followed by solid-phase extraction with silica gel. Acarbose served as an inhibitor for interfering -glucosidase present in neutrophils and allowed the lysosomal enzyme implicated in Pompe disease to be selectively analyzed. Electrospray ionization-tandem mass spectrometry was used to simultaneously quantify the enzyme products (28). Whether immune quantification or mass spectrometry will ultimately form the basis of a platform for newborn screening of LSDs will await further development and

1664 Parkinson-Lawrence et al.: Immunochemistry of Lysosomal Storage Disorders validation of these technologies. Of critical importance in the validation of such technology will be the sensitivity and specificity of the assays for each LSD. Because of the low incidence of LSDs, the number of false negatives will need to be low enough to ensure that cases are not missed. At the same time, the number of false positives must be restricted to keep the cost of the program within acceptable limits and to prevent additional testing and unnecessary counseling of the families. Pilot studies on populations with known LSD birth prevalence will be required to provide accurate details on these test limitations. In addition to the technical issues associated with screening for LSDs, there are a number of ethical issues that will need to be resolved before newborn screening for LSDs is accepted and implemented. For some LSDs, the evidence is clear that early diagnosis and intervention before the onset of irreversible pathology will provide a substantial benefit to the newborn; however, for many LSDs there is no currently recognized therapy. The issues concerned with screening for disorders for which there is no therapy are difficult and contentious. In these cases, there must be an evaluation of the balance between potential harm and good. What then are the other potential benefits to early diagnosis? Early diagnosis will enable genetic counseling of the parents and allow them to make informed reproductive choices. In LSD families, it is not uncommon to have 2 or more affected children before the first is diagnosed. Early diagnosis, as provided by a newborn screening program, would also avoid the prolonged (months to years) and stressful process of diagnosis that is the current situation for many patients and families. These benefits are primarily directed to the family rather than the newborn, but they are nonetheless important. The critical question is whether the benefits flow on to the newborn from a family that is better informed and prepared for the disease. On the other hand, the potential harm to the parent newborn relationship resulting from the knowledge of an incurable disorder and the concept of depriving families of a normal child for that period of time until the child presents clinically must be considered. This concern will be particularly relevant for those disorders for which there is an adultonset form of the disease, because the affected newborn may not present with disease for 30 years or more. These are difficult issues to define and quantify but must be addressed by the community before screening can commence. It is likely that the answers to these questions will be different for each LSD and may vary between countries and with time as new therapies are developed. Thus, the application of technology for newborn screening must be flexible and adaptable to meet these changing needs. Prediction of Disease Severity in LSD Patients. Although the early detection of LSD patients by a newborn screening program will offer the prospect of early intervention and better treatment outcomes, early detection also raises a number of potential problems for the patient, the family, and the clinician. Prediction of clinical severity, which is currently based on clinical presentation, will be difficult if not impossible for many patients. Consequently, selection of an appropriate therapeutic intervention will be uncertain and could put the patient at risk. For example, MPS I patients currently have the option of intravenous ERT, which is effective for somatic but probably not CNS pathology and has a low associated risk, or hematopoietic stem-cell transplantation, which has been shown to be effective in slowing or halting the progression of CNS pathology but is associated with a relatively high risk of morbidity and mortality. Selection of appropriate therapies will require an accurate prediction of disease severity and progression for each individual. When therapy is commenced, it may be necessary to assess the efficacy of treatment to tailor dose and regimen to individual patients. For most LSDs, patients have only 1% 5% of both variant protein and residual enzyme activity compared with unaffected controls (37 44). For pathogenesis to proceed, the level of enzyme activity in the endosome lysosome system must be limiting, leading to reduced efficiency of substrate hydrolysis and consequently substrate storage. In the search for markers of pathogenesis that correlate with the clinical presentation of the patient, the amounts of variant protein, residual enzyme activity, and substrate, present as primary markers for investigation. The correlations between patient genotype, amount of variant protein/residual activity, and clinical presentation have been investigated for many LSDs. Because all of these indicators are in dynamic balance, it is expected that they may be indicative of the disease process in LSD patients. For a number of LSDs, the amount of residual mutant protein in cell extracts has been shown to roughly correlate with the clinical severity of the patient. For example, immune-quantification assays developed for -l-iduronidase (deficient in MPS I; Hurler syndrome, Scheie syndrome) and 4-sulfatase (deficient in MPS VI; Maroteaux Lamy syndrome) were able to detect residual protein in fibroblast extracts, and the amount of mutant protein showed some correlation with clinical severity (37, 38, 72, 73). A possible limitation of using just a protein detection system is for those variations that give rise to typical amounts of dysfunctional protein. For example, by use of a monoclonal antibody based immune-quantification assay developed for the measurement of -liduronidase (deficient in MPS I; Hurler syndrome, Scheie syndrome), a severely affected patient with 6 times the amount of protein found in unaffected control patients was found to have no enzyme activity (73). Antibodies raised to specific structural regions of a protein can provide further insight into the structural abrogation of variant protein, potentially enabling the prediction of disease severity, and this approach has been investigated for MPS VI and MPS II patients (38, 74). The capacity of immune-capture methods to accurately

Clinical Chemistry 52, No. 9, 2006 1665 measure residual enzyme activity and protein and give a measure of specific activity provides another possible way of assessing clinical phenotype in many LSD patients. This can be illustrated with the most common LSD, Gaucher disease, in which the many disease-causing variations in the -glucocerebrosidase gene and complex genetics make it difficult to predict the severity of disease from patient genotype alone (75). Thus, LSD patients with apparently the same genotype can exhibit quite different phenotypes (3). The value of genotype phenotype correlations in Gaucher disease is primarily in distinguishing nonneuronopathic from neuronopathic disease by the presence of an N370S allele (76). Usually, the course of the disease is less progressive and has later onset in patients homozygous for N370S, although there are patients with this genotype who display severe disease. Furthermore, the L444P allele, in the absence of a mild mutation, is nominally associated with the neuronopathic form of the disease, but homozygosity for the L444P variation is alleged to include some type 1 patients. The accurate measurement of residual -glucocerebrosidase activity and variant protein in fibroblasts has been used to demarcate Gaucher patients with and without neuropathology (44), although this study included only a small sample set and therefore this approach will require further validation. In addition to residual protein and activity, there are examples in which the amount of stored substrate has been shown to correlate with disease severity. Recently, the use of an immune-based assay for the quantification of keratan sulfate in MPS IVA has been reported (49). An ELISA using a specific monoclonal antibody to keratan sulfate was developed for the analysis of blood and urine specimens from MPS IVA patients and age-matched controls. A 2 8-fold increase in the amount of circulating keratan sulfate was observed for MPS IVA patients compared with controls, as was a 6 7-fold increase in urine. Moreover, there was a correlation between disease severity and the amount of keratan sulfate in blood and urine (49, 77), suggesting that this method may be useful for longitudinal assessment of disease severity. The immune quantification of ceramide trihexoside in cultured fibroblasts with laser scanning confocal microscopy has also been used to differentiate the classical and variant forms of Fabry disease based on the degree of storage (63). To some extent, the analysis of substrates for certain disorders can be limited by tissue-restricted and intraorgan patterns of substrate accumulation. In addition, there are also only a limited number of available immunochemical markers for substrate. From the combined studies above, it is evident that there is a very narrow range of enzyme activity and, in most cases, immune-detected protein, which is responsible for the broad spectrum of clinical phenotypes in LSD patients. The correlation of clinical severity with these markers can therefore be useful but is often not absolute. Similarly, genotype can be informative in some cases, particularly for severe deletion or nonsense mutations, and the amount of stored substrate can also provide information on disease severity. However, the prediction of clinical severity with a single marker is, in practice, somewhat limited, and there is a need for combined marker analysis to discriminate between different patients within the spectrum of clinical phenotypes identified for each disorder. A combined marker study has recently been reported for MPS I patients, in which immunedetectable protein and activity were partially predictive of clinical severity, but the inclusion of substrate markers detected by mass spectrometry enabled further patient discrimination and was predictive of the onset of neuropathology (60). In many LSDs, a combination of markers will probably be needed to elucidate the complex pathophysiology that is characteristic of these disorders. characterization of the disease process The primary defect in most LSDs is a gene mutation leading to a deficiency of a specific lysosomal enzyme activity. However, additional cellular processes can have a major effect on the clinical phenotype within each disorder. Thus, the lysosomal dysfunction resulting from the primary enzyme defect and the subsequent lysosomal storage can lead to many secondary changes in cell metabolism as part of the pathogenic process. The aberrations in lysosomal biogenesis and function in LSDs offer additional markers that may not be directly related to the primary defect but may still be predictive of disease severity. To this end, analysis of lysosomal biogenesis may be useful for both specific patient characterization and the identification of markers that predict clinical severity and disease progression. Markers of Lysosomal Biogenesis. Lysosomal biogenesis involves the biosynthesis, processing, vesicular traffic, and incorporation of resident proteins into the endosome lysosome system, together with their continuous recycling and replacement. All points in the process of endosome lysosome biogenesis and function are potentially susceptible to the effects of mutations leading to genetic disease. Moreover, mutations in the coding sequence of a lysosomal hydrolase may alter the way it interacts with this complex processing machinery. The altered degradative capacity of the endosome lysosome system, as a result of a primary enzyme deficiency, has the capacity to impact both the dynamic flux between different vesicular compartments in the endosome lysosome network and the static composition of these compartments. Proof of the concept that markers of lysosomal biogenesis can be used to detect an LSD and are altered in response to lysosomal storage has been demonstrated for the lysosomal associated membrane proteins, LAMP-1 (68) and LAMP-2 (78). Increases in the amount of lysosomal membrane proteins are thought to reflect a common feature in LSDs, the distension of the endosome

1666 Parkinson-Lawrence et al.: Immunochemistry of Lysosomal Storage Disorders lysosome compartments by the accumulation of undegraded substrate. Pilot studies have shown that the amounts of LAMP are indicative of an LSD in many but not all patients (70). It is now evident that each LSD has a different effect on the composition of endosome lysosome compartments. Secondary Markers of Lysosomal Dysfunction. In Gaucher disease, several different markers have been evaluated to predict disease severity and monitor clinical progression. The chemokine CCL18, which reflects the amount of macrophage activation, and chitotriosidase are both indicative of disease severity and the response of Gaucher patients to ERT. Although chitotriosidase can be determined in an activity assay (79), the use of this marker is limited because of the presence of homozygosity for a null allele in 6% of patients (80). This limitation must be balanced against the availability of an immune-quantification assay for the chemokine CCL18 (79). A clinical evaluation of these markers has provided strong evidence of their value in patient assessment and monitoring in a therapeutic setting. Clearly, secondary markers for other LSDs are required, and immune-based methods provide one way of achieving this objective. From these studies, it is clear that biomarkers other than the defective enzyme/protein have the potential to reflect disease severity. The multiplexing of a range of lysosomal markers, including both the functional and structural components of the endosome lysosome system, could reflect the pathological impact of an LSD and allow the prediction of clinical severity. Although this approach is in its infancy, it may help to resolve some of the problems associated with clinical phenotype prediction. We have received funding support from the National Health and Medical Research Council of Australia as project and research fellowship grants. J.J.H., P.J.M., and D.A.B. hold a patent for the multiplex analysis of lysosomal proteins, and commercial funding has been received by J.J.H. and P.J.M. for the development of some of this technology. This article describes this and other available technology for the screening of LSDs. E.J.P. and M.F. declare no conflict of interest for this article. References 1. Meikle PJ, Hopwood JJ, Clague AE, Carey WF. Prevalence of lysosomal storage disorders. JAMA 1999;281:249 54. 2. Neufeld EF, Meunzer J. The mucopolysaccharidoses. In: Scriver CR, Beaudet AL, Sly WS, Vaile D, eds. The Metabolic and Molecular Basis of Inherited Disease, 8th ed. New York: McGraw- Hill, 2001;3421 52. 3. McCabe LL, McCabe ER. Complexity in genetic diseases: how patients inform the science by ignoring the dogma. Am J Med Genet A 2006;140:160 1. 4. Beutler E, Grabowski GA. Gaucher disease. In: Scriver CR, Beaudet AL, Sly WS, Vaile D, eds. The Metabolic and Molecular Basis of Inherited Disease, 8th ed. New York: McGraw-Hill, 2001;3635 68. 5. Hopwood JJ, Vellodi A, Scott HS, Morris CP, Litjens T, Clements PR, et al. Long-term clinical progress in bone marrow transplanted mucopolysaccharidosis type I patients with a defined genotype. J Inherit Metab Dis 1993;16:1024 33. 6. Wraith JE, Clarke LA, Beck M, Kolodny EH, Pastores GM, Muenzer J, et al. Enzyme replacement therapy for mucopolysaccharidosis I: a randomized, double-blinded, placebo-controlled multinational study of recombinant human -L-iduronidase (Laronidase). J Pediatr 2004;144:581 8. 7. Harmatz P, Whitley CB, Waber L, Pais R, Steiner R, Plecko B et al. Enzyme replacement therapy in mucopolysaccharidosis VI (Maroteaux-Lamy syndrome). J Pediatr 2004;144:574 80. 8. Barton NW, Brady RO, Dambrosia JM, Di Bisceglie AM, Doppelt SH, Hill SC, et al. Replacement therapy for inherited enzyme deficiency macrophage-targeted glucocerebroside for Gaucher s disease. N Engl J Med 1991;324:1464 70. 9. Beck M. Agalsidase alfa-a preparation for enzyme replacement therapy in Anderson-Fabry disease. Expert Opin Investig Drugs 2002;11:851 8. 10. Mehta A. Agalsidase alfa: specific treatment for Fabry disease. Hosp Med 2002;63:347 50. 11. Muenzer J, Lamsa JC, Garcia A, Dacosta J, Garcia J, Treco DA. Enzyme replacement therapy in mucopolysaccharidosis type II (Hunter syndrome): a preliminary report. Acta Paediatr Suppl 2002;91:98 9. 12. Van den Hout JM, Kamphoven JH, Winkel LP, Arts WF, De Klerk JB, Loonen MC, et al. Long-term intravenous treatment of Pompe disease with recombinant human -glucosidase from milk. Pediatrics 2004;113:e448 57. 13. Elstein D, Hollak C, Aerts JM, van Weely S, Maas M, Cox TM, et al. Sustained therapeutic effects of oral miglustat (Zavesca, N- butyldeoxynojirimycin, OGT 918) in type I Gaucher disease. J Inherit Metab Dis 2004;27:757 66. 14. Lachmann RH, Miglustat. Oxford GlycoSciences/Actelion [Review]. Curr Opin Investig Drugs 2003;4:472 9. 15. Moyses C. Substrate reduction therapy: clinical evaluation in type 1 Gaucher disease. Philos Trans R Soc Lond B Biol Sci 2003; 358:955 60. 16. Cox TM. Substrate reduction therapy for lysosomal storage diseases. Acta Paediatr Suppl 2005;94:69 75. 17. Butters TD, Dwek RA, Platt FM. Imino sugar inhibitors for treating the lysosomal glycosphingolipidoses. Glycobiology 2005;15:43R 52R. 18. Loannou YA, Enriquez A, Benjamin C. Gene therapy for lysosomal storage disorders. Expert Opin Biol Ther 2003;3:789 801. 19. Cheng SH, Smith AE. Gene therapy progress and prospects: gene therapy of lysosomal storage disorders. Gene Ther 2003;10: 1275 81. 20. McCown TJ. Adeno-associated virus (AAV) vectors in the CNS. Curr Gene Ther 2005;5:333 8. 21. Griffey M, Macauley SL, Ogilvie JM, Sands MS. AAV2-mediated ocular gene therapy for infantile neuronal ceroid lipofuscinosis. Mol Ther 2005;12:413 21. 22. Martino S, Marconi P, Tancini B, Dolcetta D, De Angelis MG, Montanucci P, et al. A direct gene transfer strategy via brain internal capsule reverses the biochemical defect in Tay-Sachs disease. Hum Mol Genet 2005;14:2113 23. 23. Hein LK, Bawden M, Muller VJ, Sillence D, Hopwood JJ, Brooks DA. -L-iduronidase premature stop codons and potential readthrough in mucopolysaccharidosis type I patients. J Mol Biol 2004;338:453 62. 24. Lin H, Sugimoto Y, Ohsaki Y, Ninomiya H, Oka A, Taniguchi M, et al. N-Octyl- -valienamine up-regulates activity of F213I mutant

Clinical Chemistry 52, No. 9, 2006 1667 -glucosidase in cultured cells: a potential chemical chaperone therapy for Gaucher disease. Biochim Biophys Acta 2004;1689: 219 28. 25. Sawkar AR, Cheng WC, Beutler E, Wong CH, Balch WE, Kelly JW. Chemical chaperones increase the cellular activity of N370S -glucosidase: a therapeutic strategy for Gaucher disease. Proc Natl Acad Sci U S A 2002;99:15428 33. 26. Matsuda J, Suzuki O, Oshima A, Yamamoto Y, Noguchi A, Takimoto K, et al. Chemical chaperone therapy for brain pathology in G(M1)-gangliosidosis. Proc Natl Acad Sci U S A 2003;100: 15912 7. 27. Meikle PJ, Dean CJ, Grasby D, Bockmann MR, Whittle AM, Lang DL, et al. Newborn screening for lysosomal storage disorders. Mol Genet Metab 2006 April 3 (Epub ahead of print). 28. Li Y, Scott CR, Chamoles NA, Ghavani A, Pinto BM, Turecek F, et al. Direct multiplex assay of lysosomal enzymes in dried blood spots for newborn screening. Clin Chem 2004;50:1785 96. 29. Wang D, Eadala B, Chamoles NA, Turecek F, Scott CR, Gelb MH, et al. Tandem mass spectrometric analysis of dried blood spots for screening of mucopolysaccharidosis I in newborns. Clin Chem 2005;51:898 900. 30. Clements PR, Brooks DA, Saccone GT, Hopwood JJ. Human -L-iduronidase. 1. Purification, monoclonal antibody production, native and subunit molecular mass. Eur J Biochem 1985;152: 21 8. 31. Bielicki J, Freeman C, Clements PR, Hopwood JJ. Human liver iduronate-2-sulphatase. Purification, characterization and catalytic properties. Biochem J 1990;271:75 86. 32. Anson DS, Taylor JA, Bielicki J, Harper GS, Peters C, Gibson GJ, et al. Correction of human mucopolysaccharidosis type-vi fibroblasts with recombinant N-acetylgalactosamine-4-sulphatase. Biochem J 1992;284:789 94. 33. Bielicki J, Hopwood JJ, Wilson PJ, Anson DS. Recombinant human iduronate-2-sulphatase: correction of mucopolysaccharidosis-type II fibroblasts and characterization of the purified enzyme. Biochem J 1993;289:241 6. 34. Bielicki J, Hopwood JJ, Melville EL, Anson DS. Recombinant human sulphamidase: expression, amplification, purification and characterization. Biochem J 1998;329:145 50. 35. Montfort M, Garrido E, Hopwood JJ, Grinberg D, Chabas A, Vilageliul L. Expression and functional characterization of human mutant sulfamidase in insect cells. Mol Genet Metab 2004;83: 246 51. 36. Zola H, Brooks DA. Techniques. In: Hurrell JGR, ed. Monoclonal hybridoma antibodies: techniques and applications. Boca Raton, FL: CRC Press, 1981:1 57. 37. Ashton LJ, Brooks DA, McCourt PA, Muller VJ, Clements PR, Hopwood JJ. Immunoquantification and enzyme kinetics of -Liduronidase in cultured fibroblasts from normal controls and mucopolysaccharidosis type I patients. Am J Hum Genet 1992; 50:787 94. 38. Brooks DA, McCourt PA, Gibson GJ, Ashton LJ, Shutter M, Hopwood JJ. Analysis of N-acetylgalactosamine-4-sulfatase protein and kinetics in mucopolysaccharidosis type VI patients. Am J Hum Genet 1999;48:710 9. 39. Perkins KJ, Byers S, Yogalingam G, Weber B, Hopwood JJ. Expression and characterization of wild type and mutant recombinant human sulfamidase. Implications for Sanfilippo (Mucopolysaccharidosis IIIA) syndrome. J Biol Chem 1999;274:37193 9. 40. Umapathysivam K, Whittle AM, Ranieri E, Bindloss C, Ravenscroft EM, van Diggelen OP, et al. Determination of acid -glucosidase protein: evaluation as a screening marker for Pompe disease and other lysosomal storage disorders. Clin Chem 2000;46:1318 25. 41. Fuller M, Lovejoy M, Brooks DA, Harkin ML, Hopwood JJ, Meikle PJ. Immunoquantification of -galactosidase: evaluation for the diagnosis of Fabry disease. Clin Chem 2004;50:1979 85. 42. Parkinson EJ, Muller V, Hopwood JJ, Brooks DA. Iduronate-2- sulphatase protein detection in plasma from mucopolysaccharidosis type II patients. Mol Genet Metab 2004;81:58 64. 43. Hein LK, Meikle PJ, Dean CJ, Bockmann MR, Auclair D, Hopwood JJ et al. Development of an assay for the detection of mucopolysaccharidosis type VI patients using dried blood-spots. Clin Chim Acta 2005;353:67 74. 44. Fuller M, Lovejoy M, Hopwood JJ, Meikle PJ. Immunoquantification of -glucosidase: diagnosis and prediction of severity in Gaucher disease. Clin Chem 2005;51:2200 2. 45. Brooks DA. The immunochemical analysis of enzyme from mucopolysaccharidoses patients. J Inherit Metab Dis 1993;16:3 15. 46. Brooks DA. Application of monoclonal antibodies to the study of molecules in solution. In: Zola H, ed. Introduction to biotechniques series. Oxford, United Kingdom: BIOS Scientific Publishers 2000;81 100. 47. Ryall RG, Gjerde EM, Gerace RL, Ranieri E. Modifying an enzyme immunoassay of immunoreactive trypsinogen to use time-resolved fluorescence. Clin Chem 1993;39:224 8. 48. David G, Bai XM, Van der Schueren B, Cassiman JJ, Van den Burgh H. Developmental changes in heparan sulfate expression: in situ detection with mabs. J Cell Biol 1992;119:961 75. 49. Tomatsu S, Okamura K, Taketani T, Orii KO, Nishioka T, Gutierrez MA, et al. Development and testing of new screening method for keratan sulfate in mucopolysaccharidosis IVA. Pediatr Res 2004; 55:592 7. 50. McGlynn R, Dobrenis K, Walkley SU. Differential subcellular localization of cholesterol, gangliosides, and glycosaminoglycans in murine models of mucopolysaccharide storage disorders. J Comp Neurol 2004;480:415 26. 51. Walkley SU. Secondary accumulation of gangliosides in lysosomal storage disorders. Semin Cell Dev Biol 2004;15:433 44. 52. Chamoles NA, Blanco M, Gaggioli D. Diagnosis of -L-iduronidase deficiency in dried blood spots on filter paper: the possibility of newborn diagnosis. Clin Chem 2001;47:780 1. 53. Chamoles NA, Blanco M, Gaggioli D. Fabry disease: enzymatic diagnosis in dried blood spots on filter paper. Clin Chim Acta 2001;308:195 6. 54. Chamoles NA, Blanco M, Iorcansky S, Gaggioli D Specola N, Casentini C. Retrospective diagnosis of GM1 gangliosidosis by use of a newborn-screening card. Clin Chem 2001;47:2068. 55. Chamoles NA, Blanco MB, Gaggioli D, Casentini C. Hurler-like phenotype: enzymatic diagnosis in dried blood spots on filter paper. Clin Chem 2001;47:2098 102. 56. Chamoles NA, Blanco M, Gaggioli D, Casentini C. Gaucher and Niemann-Pick diseases enzymatic diagnosis in dried blood spots on filter paper: retrospective diagnoses in newborn-screening cards. Clin Chim Acta 2002;317:191 7. 57. Chamoles NA, Blanco M, Gaggioli D, Casentini C. Tay-Sachs and Sandhoff diseases: enzymatic diagnosis in dried blood spots on filter paper: retrospective diagnoses in newborn-screening cards. Clin Chim Acta 2002;318:133 7. 58. Chamoles NA, Niizawa G, Blanco M, Gaggioli D, Casentini C. Glycogen storage disease type II: enzymatic screening in dried blood spots on filter paper. Clin Chim Acta 2004;347:97 102. 59. Umapathysivam K, Hopwood JJ, Meikle PJ. Determination of acid -glucosidase activity in blood spots as a diagnostic test for Pompe disease. Clin Chem 2001;47:1378 83. 60. Fuller M, Brooks DA, Evangelista M, Hein LK, Hopwood JJ, Meikle PJ. Prediction of neuropathology in mucopolysaccharidosis I patients. Mol Genet Metab 2005;84:18 24. 61. Dean CJ, Bockmann MR, Hopwood JJ, Brooks DA, Meikle PJ. Detection of mucopolysaccharidosis type II by measurement of

1668 Parkinson-Lawrence et al.: Immunochemistry of Lysosomal Storage Disorders iduronate-2-sulfatase in dried blood-spot and plasma samples. Clin Chem 2006; In Press. 62. Kotani M, Ozawa H, Kawashima I, Ando S, Tai T. Generation of one set of monoclonal antibodies specific for a-pathway ganglio-series gangliosides. Biochim Biophys Acta 1992;21:97 103. 63. Itoh K, Kotani M, Tai T, Suzuki H, Utsunomiya T, Inoue H, et al. Immunofluorescence imaging diagnosis of Fabry heterozygotes using confocal laser scanning microscopy. Clin Genet 1993;44: 302 6. 64. Bekri S, Enica T, Ghafari G, Plaza I, Champenois G, Choukroun R, et al. Fabry disease in patients with end-stage renal failure: the potential benefits of screening. Nephron Clin Pract 2005;101:9. 65. Nakao S, Kodama T, Takenaka A, Tanaka Y, Yasumoto A, Yoshida T, et al. Fabry disease: detection of undiagnosed hemodialysis patients and identification of a renal variant phenotype. Kidney Int 2003;64:801 7. 66. Sachdev B, Takenaka T, Teraguchi H, Tei C, Lee P, McKenna WJ, et al. Prevalence of Anderson-Fabry disease in male patients with late onset hypertrophic cardiomyopathy. Circulation 2002;105: 1407 11. 67. Yang Z, McMahon CJ, Smith LR, Bersola J, Adesina AM, Breinholt JP, et al. Danon disease as an underrecognized cause of hypertrophic cardiomyopathy in children. Circulation 2005;112:1612 7. 68. Meikle PJ, Brooks DA, Ravenscroft EM, Yan M, Williams RE, Jaunzems AE, et al. Diagnosis of lysosomal storage disorders: evaluation of a lysosome-associated membrane protein LAMP-1 as a diagnostic marker. Clin Chem 1997;43:1325 35. 69. Chang MH, Bindloss CA, Grabowski GA, Qi X, Winchester B, Hopwood JJ, et al. Saposins A, B, C, and D in plasma of patients with lysosomal storage disorders. Clin Chem 2000;46:167 74. 70. Meikle PJ, Ranieri E, Simonsen H, Rozaklis T, Ramsay SL, Whitfield PD, et al. Newborn screening for lysosomal storage disorders: clinical evaluation of a two-tier strategy. Paediatrics 2004;114:909 16. 71. Chace DH, Kalas TA, Naylor EW. Use of tandem mass spectrometry for multianalyte screening of dried blood specimens from newborns. Clin Chem 2003;49:1797 817. 72. Brooks DA, McCourt PA, Gibson GJ, Hopwood JJ. Immunoquantification of the low abundance lysosomal enzyme N-acetylgalactosamine 4-sulphatase. J Inherit Metab Dis 1990;13:108 20. 73. Brooks DA, Harper GS, Gibson GJ, Ashton LJ, Taylor JA, McCourt PA, et al. Hurler syndrome: a patient with abnormally high levels of -L-iduronidase protein. Biochem Med Metab Biol 1992;47:211 20. 74. Parkinson-Lawrence E, Turner C, Hopwood J, Brooks D. Analysis of normal and mutant iduronate-2-sulphatase conformation. Biochem J 2005;386:395 400. 75. Montfort M, Chabas A, Vilageliu L, Grinberg D. Functional analysis of 13 GBA mutant alleles identified in Gaucher disease patients: Pathogenic changes and modifier polymorphisms. Hum Mutat 2004;23:567 75. 76. Sibille A, Eng CM, Kim SJ, Pastores G, Grabowski GA. Phenotype/ genotype correlations in Gaucher disease type I: clinical and therapeutic implications. Am J Hum Genet 1993;52:1094 101. 77. Tomatsu S, Dieter T, Schwartz IV, Sarmient P, Giugliani R, Barrera LA, et al. Identification of a common mutation in mucopolysaccharidosis IVA: correlation among genotype, phenotype, and keratan sulfate. J Hum Genet 2004;49:490 4. 78. Hua CT, Hopwood JJ, Carlsson SR, Harris RJ, Meikle PJ. Evaluation of the lysosome-associated membrane protein LAMP-2 as a marker for lysosomal storage disorders. Clin Chem 1998;44: 2094 102. 79. Deegan PB, Moran MT, McFarlane I, Schofield JP, Boot RG, Aerts JM, et al. Clinical evaluation of chemokine and enzymatic biomarkers of Gaucher disease. Blood Cells Mol Dis 2005;35:259 67. 80. Fusetti F, von Moeller H, Houston D, Rozeboom HJ, Dijkstra BW, Boot RG, et al. Structure of human chitotriosidase: implications for specific inhibitor design and function of mammalian chitinaselike lectins. JBC Papers in Press. Published on April 17, 2002 as Manuscript M201636200.