Prediction of neuropathology in mucopolysaccharidosis I patients

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Abstract

Mucopolysaccharidosis I is a lysosomal storage disorder caused by a deficiency of the lysosomal hydrolase α-l-iduronidase, which is required for the degradation of heparan sulphate and dermatan sulphate. Given the wide spectrum of disease severity in mucopolysaccharidosis I patients, one of the challenges for managing the disorder is to accurately predict clinical phenotype. Enzyme replacement therapy by intravenous infusion is unlikely to make a significant impact on central nervous system pathology and patients displaying this clinical manifestation may respond better to bone marrow transplantation. In order to predict whether mucopolysaccharidosis I patients are going to develop central nervous system pathology, we investigated a number of biochemical parameters in cultured skin fibroblasts from patients of different genotype/phenotype. Residual levels of α-l-iduronidase activity and protein were determined using sensitive immune-quantification assays and fibroblast cell extracts from patients with central nervous system pathology generally had lower levels of α-l-iduronidase than patients with no evidence of central nervous system disease. A total of 15 oligosaccharides, derived from heparan sulphate and dermatan sulphate, was measured in fibroblast extracts using electrospray-ionisation tandem mass spectrometry and all were shown to discriminate mucopolysaccharidosis I from controls. Of these, two trisaccharides were able to group patients based on the presence/absence of central nervous system disease. Moreover, a ratio of α-l-iduronidase activity to these trisaccharides provided clear discrimination between mucopolysaccharidosis I patients with and without central nervous system pathology. We suggest that this type of analysis may be very useful for predicting disease severity in mucopolysaccharidosis I patients.

Introduction

The mucopolysaccharidoses (MPS) are a group of genetic disorders characterised by a deficiency in one of a series of lysosomal enzymes, required to degrade glycosaminoglycans (GAGs). MPS I (Hurler syndrome; Scheie syndrome; McKusick 25280) is the most common of the MPS disorders in Caucasians and has an incidence of 1:120,000 live births [1]. MPS I is caused by a deficiency of α-l-iduronidase (IDUA; EC 3.2.1.76), which is required for the sequential degradation of the GAGs heparan sulphate (HS) and dermatan sulphate (DS). An IDUA deficiency results in the failure to remove α-l-iduronic acid residues from the non-reducing end of these GAGs, causing the accumulation of these substrates in the lysosomes of affected cells. This lysosomal storage leads to the chronic and progressive deterioration of cells, tissues and organs, and the urinary secretion of partially degraded GAGs [2].

MPS I patients display a wide spectrum of clinical presentation, ranging in gradations from the archetypal severe Hurler syndrome to the more attenuated Scheie syndrome [2]. Classically, three clinical descriptions have been documented for MPS I patients, with Hurler–Scheie syndrome representing an intermediate clinical presentation between the two extremes listed above. Hurler syndrome patients are usually diagnosed within the first year of life and, if untreated, usually die before 10 years of age. The disease process is progressive with symptoms that include skeletal and joint deformities, coarse facial features, corneal clouding, hernia, cardiac disease, hepatosplenomegaly, fatigue, hydrocephalus, enlarged tongue, and mental retardation. Scheie syndrome patients have a more attenuated clinical phenotype, with variable presentation and delayed onset of the latter clinical symptoms, but these patients can have normal intelligence, stature, and lifespan. Nonetheless, there are many Hurler–Scheie patients whose clinical presentation is broad and lies between the two extremes of Hurler and Scheie.

Therapies are available for MPS I patients. Bone marrow transplantation has some value in treating both somatic tissue and central nervous system (CNS) pathology, however this procedure carries significant risk [3]. For effective treatment of Hurler syndrome patients via bone marrow transplantation, early diagnosis and treatment, before the onset of irreversible pathology, is essential. Enzyme replacement therapy has recently become available for MPS I patients, but as intravenous infusion of enzyme is not effective in accessing sites of brain pathology, due to the blood–brain barrier, it is currently limited to the treatment of patients without CNS disease [4], [5].

When patients are identified early in the disease process, as a result of astute clinicians or potentially following the introduction of newborn screening programs, the decision of therapy options will be arduous in the absence of methods to predict phenotype, in particular CNS pathology. There are currently no accurate methods for assessing the clinical phenotype of the patient or the rate of disease progression following treatment, compared to that expected had the patient remained untreated. A suitable set of biomarkers is required to effectively predict disease severity and progression in presymptomatic MPS I patients to decide on the most effective therapeutic strategy, and additionally to monitor patients receiving therapy.

Approximately 89 IDUA gene mutations have been reported1 in MPS I patients and mutation analysis has enabled the prediction of clinical severity in some patients, mainly at the severe end of the clinical spectrum [6], [7], [8]. For example, two relatively common missense mutations W402X and Q70X have been associated with a severe Hurler syndrome presentation. Collectively missense alleles account for over three-quarters of the MPS I mutant alleles in Caucasians, but in most cases, clinical criteria are still used to define the patient phenotype [9]. This reflects the overall conclusion that genotype is often not informative for predicting clinical severity and disease progression in MPS I patients.

In this paper, we report on the use of specific substrates, in combination with genotype, level of mutant protein and residual IDUA activity to provide a coordinated biochemical picture in relation to disease severity in MPS I. We have measured DS- and HS-derived oligosaccharides by electrospray ionisation-tandem mass spectrometry (ESI-MS/MS) and used sensitive immune-quantification assays to measure IDUA activity and protein in cultured skin fibroblasts from MPS I patients. The ability to evaluate patients using biochemical parameters will be important in deciding on therapeutic alternatives, such as bone marrow transplantation and/or enzyme replacement therapy for MPS I and for monitoring patients receiving therapy.

Section snippets

Materials

Cell culture materials were from Gibco BRL (Glen Waverley, Vic) and JRH Biosciences (Lenexa, KS). Recombinant human IDUA was prepared from a CHO-K1 expression system as previously described [10]. Sheep anti-IDUA polyclonal antibody was produced against recombinant human IDUA and affinity purified against the same protein as described for the anti-α-glucosidase polyclonal antibody [11]. The affinity purified polyclonal antibody was labelled with Eu3+ as described previously [12]. Microtitre

Cultured human skin fibroblasts

Thirteen human skin fibroblast cell lines were used in this study, representing a selection of different genotypes as shown in Table 1. Not all fibroblast cell lines were available at one- and 10-weeks post-confluence. Fibroblasts were cultured from eight MPS I patients known to develop CNS pathology and five MPS I patients with no evidence of CNS disease at the time of diagnosis. The MPS I patients with CNS pathology were all diagnosed at a younger age compared to the MPS I patients with no

Discussion

The primary disease markers for MPS I include genotype, level of mutant protein, enzyme catalytic capacity, and level of storage product. The different MPS I clinical phenotypes that have been recognised are not easily distinguished using conventional biochemical markers. In theory, the severity of clinical presentation for MPS I (and other lysosomal storage disorder) patients can be explained by a balance between the residual IDUA protein/activity and its impact on substrate load. It has been

Acknowledgments

We thank Miriam Harkin for assistance with cell culture, Alison Whittle for the purification of the sheep polyclonal antibody and Melanie Lovejoy for the Eu3+ labelling of ID1A. This work was supported by the NH&MRC (Australia) and the Wellcome Trust (UK), Grant reference number 060104Z/00/Z.

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