Biomarkers for the mucopolysaccharidoses: Discovery and clinical utility

Molecular Genetics and Metabolism 106 (2012) 395–402

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Biomarkers for the mucopolysaccharidoses: Discovery and clinical utility☆ Lorne A. Clarke a,⁎, Bryan Winchester b, Roberto Giugliani c, d, Anna Tylki-Szymańska e, Hernan Amartino f a

Department of Medical Genetics, Child and Family Research Institute, University of British Columbia, Vancouver, British Columbia, Canada Biochemistry Research Group, UCL Institute of Child Health at Great Ormond Street Hospital, University College London, London, UK c Medical Genetics Service, HCPA, Department of Genetics, UFRGS, Porto Alegre, RS, Brazil d INAGEMP, Porto Alegre, RS, Brazil e Department of Metabolic Diseases, Children's Memorial Health Institute, Warsaw, Poland f Department of Pediatric Neurology, Hospital Universitario Austral Argentina Pilar, Buenos Aires, Argentina b

a r t i c l e

i n f o

Article history: Received 22 March 2012 Received in revised form 8 May 2012 Accepted 8 May 2012 Available online 14 May 2012 Keywords: Lysosomal storage disease Mucopolysaccharidoses Biological markers

a b s t r a c t The mucopolysaccharidoses (MPSs), a group of inherited lysosomal storage diseases, are complex, progressive, multisystem disorders with extreme clinical heterogeneity. The introduction of therapies that target the underlying enzyme deficiency in a number of the MPSs has brought to light the need for biomarkers that would aid in the evaluation of disease burden and as a means to objectively measure therapeutic response in individual patients. It is increasingly recognized that due to the extraordinarily complex pathogenesis of the MPSs, achieving these goals with a single analyte, such as urinary glycosaminoglycans, is unlikely. This recognition has created an impetus for the search for clinically useful biomarkers that reflect the disease pathogenesis and that are stage- or organ-specific. In this review, the current state of MPS biomarker research is discussed, with a focus on clinical utility in the MPSs. © 2012 Elsevier Inc. All rights reserved.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biochemical biomarkers for MPS in human studies . . . . . . . . . . . . . . . . . . . . . 2.1. Primary markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.1. Urinary glycosaminoglycan determination . . . . . . . . . . . . . . . . . 2.1.2. Determination of urinary oligosaccharides derived from GAGs . . . . . . . . 2.1.3. Serum/plasma glycosaminoglycan determination . . . . . . . . . . . . . . 2.1.4. Determination of plasma oligosaccharides derived from GAGs . . . . . . . . 2.1.5. Cerebral spinal fluid glycosaminoglycan determination . . . . . . . . . . . 2.2. Secondary markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.1. Heparin cofactor II-thrombin complex . . . . . . . . . . . . . . . . . . . 2.2.2. Dipeptidyl peptidase IV . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.3. Secondary changes in lipid composition and concentration . . . . . . . . . 2.2.4. Bone and joint disease . . . . . . . . . . . . . . . . . . . . . . . . . . Clinical and observational biomarkers for MPS in human studies . . . . . . . . . . . . . . 3.1. Hair morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Brain magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS)

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Abbreviations: 6MWT, six-minute walk test; BMP, bis(monoacylglycero)phosphate; CLN, ceroid lipofuscinosis; CSF, cerebral spinal fluid; DPP-IV, dipeptidyl peptidase IV; ESIMS/MS, electrospray ionization-tandem mass spectrometry; ERT, enzyme replacement therapy; GAG, glycosaminoglycan; GSLs, glycosphingolipids; Gb3, globotriaosylceramide; HCII-T, heparin cofactor II-thrombin complex; HSCT, hematopoietic stem cell transplantation; LSD, lysosomal storage disease; lyso-Gb3, globotriaosylsphingosine; MMPs, matrix metalloproteinases; MRI, magnetic resonance imaging; MRS, magnetic resonance spectroscopy; MPS, mucopolysaccharidosis; MPSs, mucopolysaccharidoses; NRE, non-reducing ends; SELDI-TOF, surface-enhanced laser desorption/ionization time of flight; TLR4, Toll-like receptor 4; TNF-α, tumor necrosis factor α; uGAGs, urinary glycosaminoglycans. ☆ Financial disclosures: Dr. Lorne Clarke has received speaker honoraria and travel support from Sanofi/Genzyme. Dr. Bryan Winchester has received travel or research grants and honoraria from Genzyme, Biomarin Pharmaceutical Inc., and Shire Human Genetic Therapies (HGT). Dr. Prof. Roberto Giugliani has received travel grants and/or speaker honoraria and/or investigator fees from Actelion, Amicus, BioMarin Pharmaceutical Inc., Genzyme and Shire HGT. Dr. Tylki-Szymańska has received honoraria and travel grants from Genzyme, BioMarin Pharmaceutical Inc., and Shire HGT. Dr. Hernan Amartino has received travel and research grants from Genzyme and Shire HGT. ⁎ Corresponding author at: Department of Medical Genetics, University of British Columbia, Child and Family Research Institute, Room C234, 4500 Oak Street, Vancouver, BC, Canada V6H 3N1. Fax: + 1 604 875 2376. E-mail address: [email protected] (L.A. Clarke). 1096-7192/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.ymgme.2012.05.003

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4. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction The term “disease biomarker” is used broadly in medical practice but most commonly connotes an analyte or measurable disease feature that can be used to objectively quantify the presence, extent, and clinical progress of a disease. Disease biomarkers are the focus of much research and have been shown to be useful in the field of oncology as a means to provide clinicians with the tools necessary for early diagnosis, accurate disease prognostication, optimization of therapy, and monitoring of therapeutic responsiveness [1]. The recent introduction of enzyme replacement therapy (ERT) for a number of the MPSs and the extreme clinical heterogeneity that is characteristic of these disorders have prompted the search for biomarkers useful for these diseases. The MPSs, a subgroup of the lysosomal storage diseases (LSDs), are progressive, multisystem disorders that are caused by genetic defects in the catabolism of glycosaminoglycans (GAGs). Each MPS disorder results from a deficiency in the activity of a specific lysosomal enzyme required for GAG catabolism [2]. The clinical heterogeneity seen in each of the MPSs presents a challenge for diagnosis and management. The clinical heterogeneity manifests as variability in the age of onset of symptoms as well as the rate of disease progression in each organ system involved. This heterogeneity is observed in all of the MPSs and is best illustrated in MPS I, in which patients fit into a broad disease spectrum ranging from Hurler syndrome (i.e., presentation within the first year of life with progressive multisystem disease and death by age 10 years if untreated), to Scheie syndrome (i.e., presentation in late childhood with slower multisystem disease progression and near normal life expectancy). The term Hurler–Scheie syndrome has been used to denote patients who fit between these extremes. Other than the progressive neurological degeneration that is characteristic of Hurler syndrome, there is no clear delineation of the boundaries between Hurler–Scheie and Scheie syndromes as such. A more useful nosology whereby patients are described as either severe or attenuated is now preferred. Each of the MPSs demonstrates this extensive clinical heterogeneity, and thus this dichotomous classification is likely more useful for all of the MPSs. The identification and validation of biomarkers for the MPSs would provide the clinician with the requisite tools to facilitate diagnosis, but more importantly would provide precision in the evaluation of disease burden. This would aid in disease prognostication after diagnosis as well as provide an objective measure of response to treatment [3]. The traditional methods of measuring urinary GAG excretion or enzyme activity do not achieve these objectives because the methodologies used are both non-specific and insensitive. In addition, the recent appreciation of the complexity underlying the pathophysiology of disease symptoms and complications in the MPSs underscores the limitations of these simple measurements. Although historically it was believed that the primary storage of GAGs and their deposition in tissues and the extracellular network [4–6] were solely responsible for the signs and symptoms associated with the MPSs, recent advances suggest that the pathophysiology of disease is more complex [7]. Defective catabolism of GAGs leads to perturbations of cellular, tissue, and organ homeostasis through the activation of secondary pathogenic cascades (reviewed in [8–10]). Within the cell, storage of GAGs has been shown to alter the endosomal network, impacting intracellular targeting pathways, endocytosis, and autophagy [11,12]. GAG storage also appears to inhibit other lysosomal enzymes, leading to secondary storage of a variety of molecules [13–15]. Extracellularly, GAGs are involved in signal transduction, the regulation of extracellular cytokines and inflammatory mediators, and the modulation of cross-talk between cells, so their accumulation potentially affects many key signaling pathways [9–11].

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This review will discuss the most recent data from human and animal studies, focusing on biomarkers that have potential clinical utility as well as future research that will likely identify additional MPS biomarkers (Table 1). 2. Biochemical biomarkers for MPS in human studies 2.1. Primary markers Primary biomarkers are analytes that represent the primary storage material. Since the primary block in the MPSs is in the catabolism of GAGs, the primary storage materials are GAG fragments. The hydrolases that are deficient in the majority of the MPS disorders are exo-hydrolases, and as such, they metabolize GAGs only from the non-reducing end (NRE) of the molecule. Therefore, the primary GAGs that accumulate in a specific disease are enriched with GAGs that all have the same NRE. For example, in MPS II, the primary defect in iduronate-2-sulfatase leads to the accumulation of GAGs that each end with a 2-sulfated iduronate residue. One critical concept to consider in the development and interpretation of methods to measure GAGs in bodily fluids or tissues is the extreme heterogeneity in the species of GAGs within normal and affected tissues [7,8]. In addition, it is critical to appreciate that each GAG fragment has biological activity that is dependent on its structure. Although GAGs are broadly classified into heparan sulfate, dermatan sulfate, keratan sulfate, chondroitin sulfate, or hyaluronan, these designations do not reflect the extreme molecular heterogeneity conferred by chain length (10–150 disaccharide units), N-sulfation, O-sulfation, N-acetylation and epimerization of saccharide units within the chains. The current methods used to measure GAGs Table 1 Biomarkers under investigation in the mucopolysaccharidoses. Biomarker Primary biochemical biomarkers Urine glycosaminoglycans [19–28]

Serum/plasma glycosaminoglycans [31–33] Cerebral spinal fluid glycosaminoglycans [6,34,35] Urinary/plasma oligosaccharides [29,30,32,33]

Secondary biochemical biomarkers Heparin cofactor II-thrombin complex [36–39] Dipeptidyl peptidase IV [40] Gangliosides GM2 and GM3 [41–46]

Bis(monoacylglycero)phosphate [47,48] Matrix metalloproteinases [49–52] TNF-α and other inflammatory markers [53,54] Clinical/observational biomarkers Hair morphology [56,57] Brain MRI images [58–61]

Potential use

Type of studies

Diagnosis Newborn screening Response to therapy Diagnosis Newborn screening Response to therapy Response to therapy

Human

Diagnosis Response to therapy Burden of disease (CNS)

Human

Diagnosis Response to therapy Antibody status/effect Response to therapy Burden of disease (CNS) Response to therapy

Human

Newborn screening Burden of disease (bone) Response to therapy

Response to therapy Burden of disease (CNS) Response to therapy

Human

Human

Human Mouse dog cat Human Mouse Human

Human Human

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in fluids or tissues consist of a non-specific dye binding assay, simple chromatographic separation, or measurement of specific disaccharides after depolymerization of the GAGs. Importantly, none of these methods reflects the exact structure of the accumulated GAG chains. Dye-binding assays rely on a positively charged reporter molecule (Alcian blue or dimethylmethylene blue) to bind to the negatively charged GAG molecules. The amount of reporter bound by GAGs in the test sample is compared with the amount of reporter bound by a known quantity of purified GAGs. This dye-binding method is clearly reflective of the amount of negatively charged molecules within a sample and is thus not specific for GAGs, nor does it differentiate between different GAG molecules [16]. Various chromatographic methods have been reported, each of which relies on visualization and semi-quantification of separated GAGs after dye binding [17]. Although this method is an improvement over dye-binding assays in that it is specific for GAGs, it still does not reflect the exact structure of the accumulated GAG chains. Alternative methods using GAG depolymerization and separation by liquid chromatography followed by mass spectrometry measurement of the liberated disaccharides can reflect a degree of specificity for GAG species within a sample [18,19]. Although both of these methods allow for an estimate of total GAG content, they do not reflect differences in the molecular weight or distribution of molecular weights of GAG chains, nor do they reflect the specific linear saccharide sequence or modifications of the chains. Thus, these methods are insensitive to increases in specific GAG oligosaccharides, particularly if they make up a very small portion of total GAG within a tissue (illustrated in Fig. 1). The panels in Fig. 1 illustrate that the total GAG content is similar in all of the samples; however, the figure does not illustrate that differences may also exist in the distribution of the molecular weights of the GAG chains, the specific linear saccharide sequences within the chains, and/or the chemical modifications to the saccharides within the chains. This is well illustrated in murine MPS I brain and liver, where Holley et al. demonstrated that affected mice showed accumulation of heparan sulfate that was highly sulfated at the N, 2-O and 6-O sites in brain and liver [20]. These issues become very important when one considers the possibility that certain specific GAG fragments may be the primary mediators of disease symptoms and complications in the MPSs [9]. 2.1.1. Urinary glycosaminoglycan determination The urinary GAG (uGAG) level as measured by various dye binding assays was initially established as a screening tool [21], and it is the most common biomarker now used for the MPSs. The uGAG level has been used extensively in clinical trials of ERT in MPS I, II, and VI as a pharmacodynamic marker of in vivo enzyme activity and a surrogate marker for response to treatment. It is clear that a rapid and significant drop in uGAG levels has been observed in all clinical trials following ERT treatment [22–27]. In one trial of ERT for MPS I patients under the age of 5 years, those who received double the standard dose had a more robust average decline in uGAG levels than did those who received the standard dose [28]. The lack of significant changes thereafter, however, limits the value of uGAG in long-term monitoring of disease progression. Use of the dermatan:chondroitin sulfate ratio (DS/CS) to monitor response to treatment may offer some improvements over the total uGAG level, as the ratio is not affected by age or hydration status and provides a relative proportion of a stored GAG to a non-stored GAG without the confounding effects of standardizing to creatinine. The DS/CS ratio, as determined by two-dimensional chromatography followed by densitometry-based semiquantification of extracted urinary GAGs, has been shown to positively respond to ERT or Hematopoietic stem cell transplantation (HSCT) in MPS I, II and VI patients [29]. Interestingly, the DS/CS ratio also shows correlation with leucocyte levels of αL-iduronidase in MPS I patients post HSCT [30]. There is some controversy as to whether the uGAG level can be used as a measure of the total body burden of disease or whether it

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2S 6S

2S 6S

2S

6S

2S

2S 2S 6S

2S 2S

6S

2S

Iduronic acid (I) Glucuronic acid (G)

2S N-acetylglucosamine

S Sulfate

2S 2S

2S 6S

2S 6S

2S

6S

6S

2S

2S 2S

2S

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Fig. 1. A simplified illustration representing the total glycosaminoglycan (GAG) content within a hypothetical sample of fluid or tissue from an unaffected individual, an individual affected with MPS I, and an individual affected with MPS II. The GAGs in the sample from the unaffected individual are heterogeneous in structure, whereas those in the samples from the MPS patients are enriched in GAGs that terminate with saccharides that cannot be cleaved. In MPS I, iduronidase deficiency results in a sample enriched in terminal iduronate-containing GAGs, and in MPS II, iduronate 2-sulfatase deficiency results in a sample enriched in terminal iduronate-2-sulfate-containing GAGs. Note that, although they differ in composition, each hypothetical sample would have the same absolute GAG content when assayed by dye binding analytical methods.

is only reflective of renal involvement. A study of 35 South American MPS II patients reported that a higher uGAG level was seen among the 18 patients with cognitive involvement (p = 0.042) than among the 17 without cognitive involvement [31]. A study attempting to correlate somatic disease severity with uGAG levels was less straightforward. Among 118 patients with MPS VI, a uGAG level over 200 μg/mg creatinine was associated with shorter stature and reduced forced vital capacity, but no reliable associations between uGAG level and mean shoulder

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flexion or distance walked on the six-minute walk test (6MWT) were seen [32]. Part of the problem is that empirically measuring somatic disease severity is not yet possible for the MPSs because there are no validated severity scales. The 6MWT is a useful tool in patients who are cognitively intact, but is impractical in patients with cognitive impairment or in those below the age of 5 years, and it cannot be corrected for altered growth patterns. The same is true of pulmonary function testing [23]. These factors complicate correlations between change-from-baseline uGAG data and somatic burden of disease. The other part of the problem is that uGAG levels naturally fall as a function of age, being very high in the neonatal period [33]. The development of age-specific reference ranges has facilitated interpretation of uGAG results, but the variance is certainly greatest in early childhood, the time point at which the diagnosis of MPS is made. In addition, uGAG levels are also influenced by height, body mass, and renal functional status [34]. Thus, the use of uGAG levels as a biomarker for total body disease burden or for long-term response to ERT is not as straight forward as its use as a biomarker for in vivo enzyme activity or as a screening tool. In lieu of another useful biomarker, however, it is commonly measured, as urine is easy to collect and relatively simple to process [35]. Looking forward, other biomarkers are being studied and developed that will hopefully clarify and supplement the role of uGAGs in the MPSs. 2.1.2. Determination of urinary oligosaccharides derived from GAGs Fuller et al. have reported using an electrospray ionization-tandem mass spectrometry (ESI-MS/MS) method to detect the presence of specific di- to pentasaccharide oligosaccharides in urine, which is potentially useful for diagnosis, prognosis, and monitoring response to therapy. They compared 26 distinct oligosaccharides in urine samples from 68 patients with MPS with those seen in control samples [36]. Of the 26 oligosaccharides identified, a panel of 10 was shown to be sufficient for the diagnosis of MPS and discrimination of MPS subtypes. An extension of this approach to 18 patients with MPS II of varying clinical severity showed complete discrimination of MPS II patients from unaffected controls [37]. Of greater interest was the finding that certain types of oligosaccharides were specifically and selectively elevated in patients with cognitive involvement (i.e., severe phenotype) but not in those with an attenuated phenotype. Urine samples from three MPS patients (MPS I, IVA, and VI) who had been treated by bone marrow transplantation have also been analyzed [36]. The level of a number of urinary oligosaccharides was seen to decrease after bone marrow transplantation in these patients, and by 3 years after transplantation, all oligosaccharide levels were normal. Further studies are needed to evaluate this approach, particularly in patients receiving ERT.

with MPS I before and during treatment with ERT [39]. The group included 1 Hurler (severe phenotype), 11 Hurler/Scheie (intermediate phenotype), and 2 Scheie (attenuated phenotype) patients. Disaccharide levels were found to decrease rapidly on ERT, and the decreases occurred regardless of age or clinical severity at baseline. The degree of decrease was not correlated with clinical improvement. These results suggest that the measurement of GAG-derived disaccharide levels is a useful biomarker for screening and may potentially be helpful in monitoring an initial response to therapy. Refinements in the methodology of serum/ plasma GAG measurement may eventually allow it to be used for more specific diagnostic purposes as well as for monitoring and tailoring long-term response to therapy. A novel NRE-based assay has recently been reported to reflect specific GAGs that accumulate secondary to the underlying deficient enzyme in MPS patients [40]. After GAG depolymerization, the specific NRE fragments of the GAGs that are unique in each MPS are quantified. As such this assay reflects the specific GAG species that are accumulating in each of the MPSs. The method is currently undergoing validation. 2.1.5. Cerebral spinal fluid glycosaminoglycan determination GAGs are present in the cerebral spinal fluid (CSF) of MPS patients [6,41]. As intrathecal enzyme replacement therapy is being developed for those MPS disorders with a neurodegenerative phenotype, quantitative analytical methods that can be used to measure the GAG concentration in small volumes of CSF have recently been developed. It is hoped that the CSF GAG concentration may prove to be a useful biomarker to measure the response to intrathecal ERT. Zhang et al. conducted a CSF GAG analysis on samples from 7 patients with MPS I and 22 control pediatric patients using ultra-performance liquid chromatography followed by ESI-MS/MS [42]. GAGs were quantified in the CSF after depolymerization to disaccharides using methanolysis, and the concentration of specific disaccharides was used to infer the levels of the parent GAG. At baseline, the concentrations of heparan and dermatan sulfates in the MPS I CSF samples were found to be at least 6-fold greater than the maximum level seen in the control patient CSF samples. All of the MPS I patients then went on to receive treatment with intrathecal and intravenous ERT followed by allogeneic transplantation. CSF samples from 100 days post treatment were available for 4 patients. CSF dermatan sulfate concentrations were reduced by more than 50%, and the heparan sulfate concentrations were reduced by between 17.5% and 58.6% after treatment, providing preliminary evidence that CSF GAG concentration could be used to monitor the response to intrathecal ERT. 2.2. Secondary markers

2.1.3. Serum/plasma glycosaminoglycan determination Serum GAGs have been proposed as a putative biomarker in MPS [2,4]. In one study, Tomatsu and colleagues analyzed dermatan and heparan sulfate levels in plasma samples from 120 patients with MPS I, II, III, or VI and from 112 healthy controls by digesting plasma samples with heparitinase and chondroitinase B to obtain disaccharides, followed by liquid chromatography-tandem mass spectrometric analysis [38]. All of the MPS patients, including 3 infants, had significantly higher levels of all disaccharides derived from dermatan and heparan sulfate than did controls (p b 0.0001). The relationship between plasma heparan and dermatan sulfate-derived disaccharide levels and the clinical severity of disease was not studied, however. Of note, the finding that derivatives of keratan sulfate were elevated in the plasma of patients with MPS I, II, III, and VI (subtypes that do not have keratan sulfate as the primary storage material) supports the concept that primary storage of one type of GAG can lead to secondary storage of other lysosomal substrates. 2.1.4. Determination of plasma oligosaccharides derived from GAGs A second study by the same group examined the levels of dermatan and keratan sulfate-derived disaccharides in plasma by liquid chromatography-tandem mass spectrometry in 14 patients

As biomarker research in the MPSs evolves, a growing body of evidence supports the idea that the search for a “global” MPS disease biomarker may be somewhat naïve in the face of such complex, progressive diseases. The multisystemic nature of the MPSs and the complexity of the underlying disease pathophysiology would suggest that secondary markers, i.e., those analytes or features that are a reflection of altered cellular and tissue homeostasis, are likely to be informative. These biomarkers may be specific to one disease stage or organ system. A number of secondary markers for the MPSs are being studied in human patients and/or are under investigation in vitro or in animal models. A selection of the best characterized is discussed below. 2.2.1. Heparin cofactor II-thrombin complex The concentration of heparin cofactor II-thrombin complex (HCII-T) has been shown to be elevated in the serum of patients with MPS I, II, III, and VI. This biomarker was initially discovered using a proteomics approach in a murine model of MPS I [43]. In a study of serum samples from 41 patients with MPS I, II, III, IV, and VI, HCII-T concentrations were 3 to 112 times the mean of the control group, and all patients had values higher than those of controls [44]. The greatest elevations

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were seen in patients with MPS that store dermatan sulfate (MPS I, II, and VI). Two separate longitudinal studies of serum HCII-T levels in patients with MPS have been reported [45,46]. Both studies reported that the serum HCII-T concentration in patients with MPS I, II, and VI showed significant fluctuations but decreased during ERT or after hematopoietic stem cell transplantation. The study by Clarke et al. also showed that many treated patients had serum HCII-T levels above the normal range despite normalization of uGAG levels [46]. In addition, that study showed for the first time that antibodies to recombinant idursulfase may have a biological effect. The authors found significant and persistent elevations in serum HCII-T concentration in antibody-positive MPS II patients receiving ERT, and these elevations reached higher levels than those seen in pretreatment. The elevations were only minimally reflected in uGAG levels. These data indicate that serum HCII-T may represent an MPS biomarker that is rapidly sensitive to changes in patients' clinical status. HCII-T has also been demonstrated to be measurable using newborn blood dot filter paper, although reported studies are restricted to samples obtained from MPS I mice. Nevertheless these studies indicate a potential use of this biomarker in newborn screening [47].

of brain GM2 and GM3 gangliosides, which correlated with reduction in lysosomal distention in mice [52] and storage lesions in dogs [53]. Similar correlations were found in studies of bone marrow transplantation in the feline MPS I model [54]. A major limitation of these types of studies is that they require tissue analysis. It would be of interest to study the levels of these lipids in cerebrospinal fluid of human MPS patients and healthy subjects. Another example is bis(monoacylglycero)phosphate (BMP), which is located primarily within the endosomal/lysosomal membranes of cells. Within the lysosome, BMP is found almost exclusively in the internal vesicles, where it helps promote the degradation of GSLs [55]. BMP also appears to regulate cholesterol transport by acting as a collection and distribution device. A study published in 2008 demonstrated that the composition of BMP species in cultured skin fibroblasts, as analyzed by ESI-MS/MS, was altered in patients with LSDs, including MPS I, II, and IIIA [56]. The authors also noted elevations in the concentration of the C(18:1)/C(18:1) species of BMP in plasma from patients with LSDs with visceral storage, particularly in macrophages (i.e., Gaucher disease and Niemann–Pick disease). No elevations were seen in patients with LSDs where storage occurs primarily in the central nervous system (i.e., gangliosidoses).

2.2.2. Dipeptidyl peptidase IV Dipeptidyl peptidase IV (DPP-IV) was identified by Beesley and colleagues [48] during a proteomic biomarker search strategy that analyzed plasma from patients with MPS I, IIIA, or IIIB using surfaceenhanced laser desorption/ionization time of flight (SELDI-TOF) mass spectrometry. Plasma samples were obtained from 34 patients with MPS I, 24 patients with MPS IIIA, 24 patients with MPS IIIB, and 66 controls. SELDI-TOF analysis revealed that the ratio between ApoCI (6630 Da) and ApoCI' (6432 Da), a truncated version of ApoCI lacking the N-terminal dipeptide, was consistently altered in the samples from MPS patients. Because the enzyme DPP-IV is responsible for the conversion, the investigators examined DPP-IV activity in plasma from the following numbers of patients: 34 MPS I, 8 MPS II, 24 MPS IIIA, 24 MPS IIIB, 6 MPS IIIC, 1 MPS IIID, 2 MPS IVA, 4 MPS VI, 6 Pompe, 15 Fabry, 3 Gaucher, 1 ceroid lipofuscinosis (CLN) type 1 (CLN1), and 4 CLN type 2. The median plasma DPP-IV activity for all of the MPS patients was found to be approximately three-fold greater than the median for the controls. The plasma DPP-IV activity was not appreciably elevated in Pompe or Gaucher disease but was slightly elevated in Fabry disease and CLN1 and CLN2 diseases, but much less so than in MPS. The authors next tried to determine if the plasma DPP-IV activity could be used to monitor MPS patients' response to ERT. DPP-IV activity was measured at weekly intervals in the plasma of 6 MPS I patients undergoing ERT over a 7 to 10 week period from the start of treatment. Activity levels decreased on treatment over time for all patients, although to a greater extent in some patients than in others. The authors were not able to draw any strong correlations between response to ERT and DPP-IV levels due to lack of certain baseline samples and short follow up, but future studies are planned.

2.2.4. Bone and joint disease The MPSs are characterized by severe and progressive joint involvement. Simonaro and colleagues have extensively studied the molecular, biochemical, and cellular changes occurring in these tissues and have presented compelling data suggesting that primary GAG storage induces innate inflammation in connective tissue, leading to altered growth and to bone destruction [57–59]. Their studies in animal models of MPS VI have linked GAG storage to activation of the Toll-like 4 pathway within cartilage and synovial cells. They hypothesize that activation of the synoviocyte Toll-like receptor 4 (TLR4) by extracellular GAGs leads to the release of proinflammatory cytokines into the synovial fluid, and this is supported by their findings of elevated levels of tumor necrosis factor (TNF)-α, ceramide, and interleukin (IL)-1β within the synovial fluid from MPS VII dogs [59] and serum from MPS VI rats [60]. Among other functions, proinflammatory cytokines can recruit and activate macrophages. Activated macrophages secrete matrix metalloproteinases (MMPs), which play a pivotal role in cartilage destruction. Elevated levels of MMPs have been seen in MPS VI rat synovial tissue [59] and serum [60]. The utility of cytokines as biomarkers for bone and joint disease in the MPS is supported by a recent study from the same group showing that MPS VI rats treated with ERT plus anti-TNF-α therapy displayed greater reductions in the levels of ceramide and TNF-α in tissue and serum than did rats treated with ERT alone. Rats in the combination therapy group also displayed significantly greater improvements in motor activity and mobility [61]. These findings have yet to be confirmed in humans. Although one study has shown evidence of increased TNF-α gene expression in RNA from peripheral blood of a patient with MPS VI that subsequently decreased during ERT, direct measurement of serum levels was not performed [62]. It should be noted that increased levels of circulating cytokines or other inflammation-based markers could potentially reflect more broadly upon MPS disease burden rather than being biomarkers of only bone and joint disease.

2.2.3. Secondary changes in lipid composition and concentration Secondary lysosomal storage of several types of lipids, including glycosphingolipids (GSLs), phospholipids, and cholesterol, has been demonstrated in tissues from patients and animal models of MPS [49,50]. These secondary storage materials may play key roles in disease pathogenesis. GSLs, particularly the gangliosides GM2 and GM3, are stored intraneuronally in the MPS and other LSDs, and the intraneuronal accumulation of GM2 has been correlated with ectopic dendritogenesis [51]. The secondary increase in brain gangliosides GM2 and GM3 has been used as a potential biomarker in gene therapy studies in murine and canine models of MPS I. Intracerebral injection of an adeno-associated virus coding for the defective enzyme in animal models of MPS1 resulted in a reduction or normalization in the levels

3. Clinical and observational biomarkers for MPS in human studies Clinical and observational features of a disease can also serve as biomarkers and offer the advantage of being noninvasive. This is particularly important in the MPSs, as most patients are at increased risk of anesthesia complications [63]. 3.1. Hair morphology Malinowska and colleagues first published evidence that hair morphology may be useful as a biomarker for response to treatment

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in patients with certain MPS subtypes [64]. The authors conducted a scanning electron micrograph analysis of hair samples from 14 controls and from the following MPS patients: 8 MPS I, 13 MPS II, 21 MPS IIIA, 9 MPS IIIB, 5 MPS IVA, 1 MPS IVB, and 3 MPS VI. Characteristic changes were observed in the samples from patients with MPS I, II, and III, and these changes normalized in all patients with MPS I after 1 year of treatment with ERT. Of note, the severity of the morphological changes at baseline did not correlate with disease severity, urinary GAG level, or residual enzyme activity. Hair morphology was subsequently used as a biomarker for response to treatment in a clinical trial of genistein, an agent under study for substrate reduction therapy in MPS III. After 12 months of treatment, hair morphology was significantly improved in 63% of 16 patients; however, none of the patients in the study showed cognitive improvements [65]. 3.2. Brain magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS) In studies examining MRI and MRS data from patients with MPS II, Vedolin et al. found that white matter lesions were most prominent in patients with cognitively involved MPS II [66], and that lesions became more extensive as disease duration increased [67]. Fan et al. later investigated the utility of brain MRI findings as a biomarker to discriminate between cognitively involved and non-cognitively involved MPS II [68]. Annual brain MRI imaging and neurodevelopmental assessments were obtained for 16 male patients with MPS II, 10 of whom had cognitive impairment. A decreased brain tissue/intracranial volume ratio and an increased lateral ventricle/intracranial volume ratio were found to be correlated with cognitive impairment. Recently, Davison et al. analyzed quantitative in vivo MRS data from patients with MPS II [69]. Eleven MRS studies from 7 male patients with MPS II (5 with moderate/severe cognitive impairment) were assessed and compared with those from controls. Multiple enlarged perivascular spaces and variable cerebral atrophy were seen in all the MPS II patients. Ventricular dilatation was seen in the 5 patients with moderate/ severe cognitive impairment. There were significantly decreased levels of total N-acetylaspartate as well as choline, gamma-aminobutyric acid, and glutamate in the white matter of MPS II patients as compared with the control group. Serial MRS studies from 4 patients showed that total N-acetylaspartate levels did not decrease over time in the 2 patients with no/slight cognitive impairment but did decrease significantly in the 2 patients who had moderate/severe impairment. Future studies that combine formal serial neurocognitive assessment with serial metabolite determinations are planned. 4. Discussion Biomarkers for the MPSs are desirable to facilitate early diagnosis and to gauge disease burden, allowing for the optimization of therapy and monitoring of therapeutic responsiveness. It is even possible to envision a scenario in which biomarkers in the MPSs could be used for individual customization of the ERT dose. Within the LSD family, primary and secondary accumulating metabolites or proteins specifically secreted by storage cells are generally considered good candidates for biomarkers. The classic example of such a clinically useful biomarker in an LSD is chitotriosidase, an enzyme specifically produced by Gaucher disease macrophages [70,71]. Because common tissue macrophages and dendritic cells do not produce chitotriosidase, the plasma activity level of this biomarker reflects the sum of secreted enzyme by Gaucher cells and, therefore, the total body burden of Gaucher cells [70]. Patients with high clinical severity scores always have chitotriosidase levels above 15,000 nmol/ml/h, and most have levels above 20,000 nmol/ml/h. Patients with less severe disease tend to have lower values. Changes in chitotriosidase levels also appear to be useful for monitoring treatment efficacy [72].

While very useful, the chitotriosidase level is not a perfect biomarker. About one-third of the population of most ethnic groups carries a 24-base pair duplication in the chitotriosidase gene that prevents the formation of chitotriosidase protein [73]. Gaucher patients heterozygous for this mutation have approximately half the amount of plasma chitotriosidase activity found in individuals with a wild-type chitotriosidase genotype. In these cases, the level must be multiplied by 2 to obtain the correct plasma chitotriosidase activity level [74]. About one in every 20 individuals is homozygous for this mutation and makes no chitotriosidase, rendering this biomarker useless when that individual is also a Gaucher patient [75]. Another primary metabolite that appears to be clinically useful as a biomarker in an LSD is globotriaosylsphingosine (lyso-Gb3) for patients with Fabry disease. Although the primary storage material globotriaosylceramide (Gb3) is excreted in the urine of Fabry patients, it is also present in the urine of normal, healthy controls at levels that overlap with those of affected patients, limiting its usefulness as a biomarker. Such is not the case for lyso-Gb3. Recent work has shown that urine from healthy controls contains no detectable lyso-Gb3 and that urine concentrations of this metabolite correlate with ERT status and a number of indicators of disease severity [76]. Preliminary work has also correlated plasma lyso-Gb3 levels with ERT status as well [77]. Ideal biomarkers for the MPSs would be similarly sensitive, specific, and easy to measure. As we have described, however, biomarker research in the MPSs is moving beyond the paradigm that a single analyte or feature can serve as a comprehensive biomarker for phenotype prediction, disease progression, and response to therapy. It has become clear that the disease symptoms and complications of the MPSs are not simply a result of primary GAG storage but also involve broad alterations of cellular and tissue homeostasis that occur as secondary pathogenic cascades are activated (Fig. 2). Based on the published evidence from human studies as well as biochemical research in animal models, it seems likely that in the future, organ- or pathway-specific biomarkers, rather than a single “one-stop-shop” biomarker, will play an increasing role in monitoring the MPSs, with the possibility of the use of a panel of biomarkers tailored to the individual patient. Identification of these pathways is being pursued with gene expression studies, proteomic analyses, imaging studies, morphology studies, and metabolomics studies. Once identified, these markers will require validation, with particular emphasis on their ability to reflect disease burden and progression. As such, it will be critical for biomarker studies to be performed in parallel or at least closely allied to the various longitudinal disease registries that are currently in use. Indeed, biomarkers might also help identify alternative pathogenic pathways that may serve as therapeutic targets in the MPSs. There is urgency to initiate methods and protocols to prospectively collect blood and/or fluid samples from MPS patients that are linked to clinical information that could ultimately be used to validate biomarkers. Access to biological samples obtained from MPS patients prior to the commencement of therapy is of particular importance in

Extracellular matrix MMPs Cathepsin GAGs

Altered cell signaling TGF-B pathway TLR4

GAG accumulation Vesicular network autophagy other lysosomal substrates endocytosis

cytokines chemokines

Fig. 2. Primary GAG accumulation in the MPSs produces a cascade of secondary pathogenic events. Biomarker research has focused on many of these secondary pathways.

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