Iduronate-2-sulphatase Research Articles

Multifaceted Approach for Quantification and Enzymatic Activity of Iduronate-2-Sulfatase to Support Developing Gene Therapy for HunterSyndrome.

Mucopolysaccharidosis type II, commonly called Hunter syndrome, is a rare X-linked recessive disease caused by the deficiency of the lysosomal enzyme iduronate-2-sulphatase (I2S). A deficiency of I2S causes an abnormal glycosaminoglycans accumulation in the body's cells. Although enzyme replacement therapy is the standard therapy, adeno-associated viruses (AAV)-based gene therapy could provide a single-dose solution to achieve a prolonged and constant enzyme level to improve patient's quality of life. Currently, there is no integrated regulatory guidance to describe the bioanalytical assay strategy to support gene therapy products. Herein, we describe the streamlined strategy to validate/qualify the transgene protein and its enzymatic activity assays. The method validation for the I2S quantification in serum and method qualification in tissues was performed to support the mouse GLP toxicological study. Standard curves for I2S quantification ranged from 2.00 to 50.0 μg/mL in serum and 6.25 to 400 ng/mL in the surrogate matrix. Acceptable precision, accuracy, and parallelism in the tissues were demonstrated. To assess the function of the transgene protein, fit-for-purpose method qualification for the I2S enzyme activity in serum was performed. The observed data indicated that the enzymatic activity in serum increased dose-dependently in the lower I2S concentration range. The highest I2S transgene protein was observed in the liver among tissue measured, and its expression level was maintained up to 91 days after the administration of rAAV8 with a codon-optimized human I2S. In conclusion, the multifaceted bioanalytical method for I2S and its enzymatic activity were established to assess gene therapy products in Hunter syndrome.

Multiplexing Iduronate-2-Sulphatase (MPS-II) into a 7-Plex Lysosomal Storage Disorder MS/MS Assay Using Cold-Induced Phase Separation.

Mucopolysaccharidosis type II (MPS-II, Hunter syndrome, OMIM:30990) is a lysosomal storage disorder (LSD) that results in iduronate 2-sulphatase (I2S) enzyme deficiency. MPS-II was added to the Recommended Uniform Screening Panel (RUSP) in August 2022; thus, there is an increased demand for multiplexing I2S into existing LSD screening assays. After incubation with LSD synthetic substrates, extracts are cleaned using liquid-liquid extraction with ethyl acetate or protein precipitation using acetonitrile (ACN). We investigated cold-induced water ACN phase separation (CIPS) to improve the combination of 6-plex and I2S extracts to create a 7-plex assay, and compared it to room temperature ACN and ethyl acetate liquid-liquid extraction. The extracts were dried and resuspended in the mobile phase, and then analyzed using an optimized 1.9 min injection-to-injection liquid chromatography method coupled with tandem mass spectrometry (LC-MS/MS). The combination of ACN and CIPS improved the detection for I2S products without significant detriment to other analytes, which is attributable to a more complete coagulation and separation of heme, proteins, and extracted residual salts. Using CIPS for sample cleanup in dried blood spots (DBS) appears to represent a promising and straightforward way of achieving cleaner sample extracts in a new 7-plex LSD screening panel.

Diagnosis and Management of Mucopolysaccharidosis Type II (Hunter Syndrome) in Poland.

Mucopolysaccharidosis type II (MPS II; also known as Hunter syndrome) is a rare, inherited lysosomal storage disease. The disease is caused by deficiency of the lysosomal enzyme iduronate-2-sulphatase (I2S) due to mutations in the IDS gene, which leads to accumulation of glycosaminoglycans (GAGs). Deficiency of I2S enzyme activity in patients with MPS II leads to progressive lysosomal storage of GAGs in the liver, spleen, heart, bones, joints, and respiratory tract. This process disturbs cellular functioning and leads to multisystemic disease manifestations. Symptoms and their time of onset differ among patients. Diagnosis of MPS II involves assessment of clinical features, biochemical parameters, and molecular characteristics. Life-long enzyme replacement therapy with idursulfase (recombinant human I2S) is the current standard of care. However, an interdisciplinary team of specialists is required to monitor and assess the patient's condition to ensure optimal care. An increasing number of patients with this rare disease reach adulthood and old age. The transition from pediatric care to the adult healthcare system should be planned and carried out according to guidelines to ensure maximum benefit for the patient.

Examining the Effect of Polymer Extension on Protein-Polymer Interactions That Occur during Formulation of Protein-Loaded Poly(lactic acid-co-glycolic acid)-polyethylene Glycol Nanoparticles.

Protein therapeutics have the potential to treat a wide range of ailments due to the high specificity in their function and their ability to replace missing or mutated genes that encode for key cellular processes. Despite these advantages, protein drugs alone can cause adverse effects, such as the development of cross-reactive neutralizing antibodies. Through the encapsulation of proteins into nanoparticles, adverse effects and protein degradation can be minimized, thus improving protein delivery to sites of interest in the body. Nanoparticles comprised of poly(lactic acid-co-glycolic acid)-polyethylene glycol (PLGA-PEG) diblock copolymer are promising protein delivery systems as they are well characterized, non-toxic, and biocompatible. Desirable nanoparticle characteristics, such as neutral surface charge and uniformity in size and dispersity, can be achieved but often require the iterative manipulation of formulation parameters. Chain conformations in the formulation process are very important, and determining whether or not an extended or semi-collapsed polymer chain in the presence of a protein results in more favorable binding has yet to be investigated experimentally. Therefore, this work used atomistic molecular dynamics to examine the role of polymer extension on protein binding and its impact on the encapsulation process within PLGA-PEG nanoparticles. Three polymers (PLGA-PEG, PLGA, and PEG) were evaluated and iduronate-2-sulphatase (ID2S) was used as a model protein. We found highly expanded PLGA-PEG conformations led to more favorable binding with ID2S. Furthermore, PEG oligomers were observed to undergo transient binding with ID2S that was generally less favorable when compared to the other polymer types. The results also suggest that the relaxation times of the PLGA homopolymer and the PLGA-PEG copolymer at different molecular weights in relevant solvent mediums should be considered.

Diagnosis of Hunter Syndrome (Mucopolysaccharidosis Type II) in a Resource­ Limited Setting: A Case Report from Zambia

Abstract
 Hunter syndrome is one of the Mucopolysaccharidosis (MPS), type II. It is a rare genetic disorder due to a deficiency in the enzyme Iduronate 2-sulphatase. This deficiency leads to the accumulation of glycosaminoglycans (GAGs) dermatan sulphate and heparan sulphate. The GAGs accumulate both intracellularly and extracellularly, leading to abnormalities in different organ systems in the body. The definitive diagnosis of Hunter syndrome requires biochemical methods which can be a challenge in resource-limited settings, Zambia included. Presented here is a case of Hunter Syndrome in a 12-year-old male child and highlight clinical acumen as the main ingredient in making the diagnosis and distinguishing different types.

Diagnosis of Hunter Syndrome (Mucopolysaccharidosis Type II) in a Resource­ Limited Setting: A Case Report from Zambia

Abstract
 Hunter syndrome is one of the Mucopolysaccharidosis (MPS), type II. It is a rare genetic disorder due to a deficiency in the enzyme Iduronate 2-sulphatase. This deficiency leads to the accumulation of glycosaminoglycans (GAGs) dermatan sulphate and heparan sulphate. The GAGs accumulate both intracellularly and extracellularly, leading to abnormalities in different organ systems in the body. The definitive diagnosis of Hunter syndrome requires biochemical methods which can be a challenge in resource-limited settings, Zambia included. Presented here is a case of Hunter Syndrome in a 12-year-old male child and highlight clinical acumen as the main ingredient in making the diagnosis and distinguishing different types.

In vitro pharmacological profiling of selected small molecules compound using recombinant human iduronate-2-sulphatase

Background: Mucopolysaccharidoses type II (MPS II) is an X-linked lysosomal storage disease (LSD). It is due to mutation in IDS gene encoding iduronate-2-sulphatase (IDS) involved in the catabolism of dermatan sulphate and heparan sulphate. Currently, the treatments for MPS II patients are enzyme replacement therapy (ERT) and bone marrow transplantation (BMT). However, ERT is not effectively reducing the central nervous system manifestation and finding the suitable donor maybe quite challenging in BMT. Over the past decades, pharmacological chaperone has been an alternative approach for management of MPS II patient. Here, we described the in vitro profiling of small molecules in group of chondroitin/dermatan (CD) sulphate disaccharide, heparin oligosaccharides, unsaturated heparin disaccharides and 6-O-desulphated heparin oligosaccharide, using recombinant human iduronate-2-sulphatase (rhIDS). Twenty-one small molecule compounds with several concentrations were each screened by inhibition and thermal stability assays.
 Results: Our study revealed that condroitin dermatan trisulphate (CD3S), heparin tetrasaccharide (H4Sac), heparin octasaccharide (H8Sac) and heparin octadecasaccharide (H18Sac) showed high inhibition constant, Ki and low inhibition concentration, IC50 in comparison to others. In the thermal stability study, only rhIDS incubated with CD3S was found to preserve enzyme activity (20%) after incubated at 67oC.
 Conclusion: Overall, our experiments discovered that CD3S was able to bind, inhibit and chaperone rhIDS. These features suggest a potential pharmacological chaperone for MPS II.

Gene Therapy for Mucopolysaccharidosis Type II-A Review of the Current Possibilities.

Mucopolysaccharidosis type II (MPS II) is a lysosomal storage disorder based on a mutation in the IDS gene that encodes iduronate 2-sulphatase. As a result, there is an accumulation of glycosaminoglycans—heparan sulphate and dermatan sulphate—in almost all body tissues, which leads to their dysfunction. Currently, the primary treatment is enzyme replacement therapy, which improves the course of the disease by reducing somatic symptoms, including hepatomegaly and splenomegaly. The enzyme, however, does not cross the blood–brain barrier, and no improvement in the function of the central nervous system has been observed in patients with the severe form of the disease. An alternative method of treatment that solves typical problems of enzyme replacement therapy is gene therapy, i.e., delivery of the correct gene to target cells through an appropriate vector. Much progress has been made in applying gene therapy for MPS II, from cellular models to human clinical trials. In this article, we briefly present the history and basics of gene therapy and discuss the current state of knowledge about the methods of this therapy in mucopolysaccharidosis type II.

Mucopolysaccharidosis Type II: One Hundred Years of Research, Diagnosis, and Treatment.

Mucopolysaccharidosis type II (MPS II, Hunter syndrome) was first described by Dr. Charles Hunter in 1917. Since then, about one hundred years have passed and Hunter syndrome, although at first neglected for a few decades and afterwards mistaken for a long time for the similar disorder Hurler syndrome, has been clearly distinguished as a specific disease since 1978, when the distinct genetic causes of the two disorders were finally identified. MPS II is a rare genetic disorder, recently described as presenting an incidence rate ranging from 0.38 to 1.09 per 100,000 live male births, and it is the only X-linked-inherited mucopolysaccharidosis. The complex disease is due to a deficit of the lysosomal hydrolase iduronate 2-sulphatase, which is a crucial enzyme in the stepwise degradation of heparan and dermatan sulphate. This contributes to a heavy clinical phenotype involving most organ-systems, including the brain, in at least two-thirds of cases. In this review, we will summarize the history of the disease during this century through clinical and laboratory evaluations that allowed its definition, its correct diagnosis, a partial comprehension of its pathogenesis, and the proposition of therapeutic protocols. We will also highlight the main open issues related to the possible inclusion of MPS II in newborn screenings, the comprehension of brain pathogenesis, and treatment of the neurological compartment.

Modelling the neuropathology of lysosomal storage disorders through disease-specific human induced pluripotent stem cells

Mucopolysaccharidosis II (MPS II) is a lysosomal storage disorder (LSD), caused by iduronate 2-sulphatase (IDS) enzyme dysfunction. The neuropathology of the disease is not well understood, although the neural symptoms are currently incurable. MPS II-patient derived iPSC lines were established and differentiated to neuronal lineage. The disease phenotype was confirmed by IDS enzyme and glycosaminoglycan assay. MPS II neuronal precursor cells (NPCs) showed significantly decreased self-renewal capacity, while their cortical neuronal differentiation potential was not affected. Major structural alterations in the ER and Golgi complex, accumulation of storage vacuoles, and increased apoptosis were observed both at protein expression and ultrastructural level in the MPS II neuronal cells, which was more pronounced in GFAP + astrocytes, with increased LAMP2 expression but unchanged in their RAB7 compartment. Based on these finding we hypothesize that lysosomal membrane protein (LMP) carrier vesicles have an initiating role in the formation of storage vacuoles leading to impaired lysosomal function. In conclusion, a novel human MPS II disease model was established for the first time which recapitulates the in vitro neuropathology of the disorder, providing novel information on the disease mechanism which allows better understanding of further lysosomal storage disorders and facilitates drug testing and gene therapy approaches.

Mucopolysaccharidosis type II detection by Naïve Bayes Classifier: An example of patient classification for a rare disease using electronic medical records from the Canadian Primary Care Sentinel Surveillance Network.

Identifying patients with rare diseases associated with common symptoms is challenging. Hunter syndrome, or Mucopolysaccharidosis type II is a progressive rare disease caused by a deficiency in the activity of the lysosomal enzyme, iduronate 2-sulphatase. It is inherited in an X-linked manner resulting in males being significantly affected. Expression in females varies with the majority being unaffected although symptoms may emerge over time. We developed a Naïve Bayes classification (NBC) algorithm utilizing the clinical diagnosis and symptoms of patients contained within their de-identified and unstructured electronic medical records (EMR) extracted by the Canadian Primary Care Sentinel Surveillance Network (CPCSSN). To do so, we created a training dataset using published results in the scientific literature and from all MPS II symptoms and applied the training dataset and its independent features to compute the conditional posterior probabilities of having MPS II disease as a categorical dependent variable for 506497 male patients. The classifier identified 125 patients with the highest likelihood for having the disease and 18 features were selected to be necessary for forecasting. Next, a Recursive Backward Feature Elimination algorithm was employed, for optimal input features of the NBC model, using a k-fold Cross-Validation with 3 replicates. The accuracy of the final model was estimated by the Validation Set Approach technique and the bootstrap resampling. We also investigated that whether the NBC is as accurate as three other Bayesian networks. The Naïve Bayes Classifier appears to be an efficient algorithm in assisting physicians with the diagnosis of Hunter syndrome allowing optimal patient management.

HUNTER SYNDROME: FIRST ITALIAN CASE TREATED WITH ENZYME-REPLACEMENT THERAPY. TEN YEARS OF FOLLOW-UP

The Hunter syndrome (Mucopolysaccharidosis II, MPS II) is a severe genetic disease (X-linked recessive). Its incidence is about 0.3-0.71 per 100 000 live births. It is caused by the deficiency in the lysosomal enzyme iduronate-2-sulphatase (I2S), which catalyses the degradation of the glycosaminoglycans (GAG). This leads to a widespread accumulation of the GAG dermatan and heparan sulphate in many organs with a final progressive multi-system disease. The enzyme-replacement therapy (ERT) with idursulfase, a recombinant human I2S enzyme, is now available (since 2006) in many countries. We described the first case treated in Italy with this method. A 13-month-old male arrived to our Clinic and was diagnosed with MPS II. He was treated with ELAPRASE (0.5 mg/kg intravenously administered every week) since 32 months of age. We monitored the urinary GAG content, the patient’s detailed anthropometric (growth, weight, phenotypic aspects of the face, as well as chest, limbs and whole body), joint range of motion and skeletal radiographs, ultrasound studies of liver and spleen volumes, the distance covered in the 6-minute walk test and respiratory symptoms, echocardiography and heart valvulopathies, otorinolaryngological symptoms, audiological examinations, pain, neurological involvement and psychological tests. The patient was treated for 10 years and he is still in treatment. He now presents i) significant reduction in GAG levels in the biological fluids, ii) important improvements in lung function, in the ability of walking, in other visceral organs function and iii) a significant reduction of the liver and spleen volumes. We can conclude that an early diagnosis is fundamental for an efficient therapy of the Hunter syndrome disease. Indeed the treatment of patients with MPS II before the onset of clinical symptoms may significantly improve the quality of life and the survival. The ELAPRASE treatment is a good option for these patients, but not all patients with MPS II may be elected for this type of treatment. An accurate selection is fundamental also based on high cost of medication.

Identification and characterization of 20 novel pathogenic variants in 60 unrelated Indian patients with mucopolysaccharidoses type I and type II.

Mucopolysaccharidoses (MPS), a subgroup of lysosomal storage disorders, are caused due to deficiency of specific lysosomal enzyme involved in catabolism of glycosaminoglycans. To date more than 200 pathogenic variants in the alpha-l-iduronidase (IDUA) for MPS I and ∼500 pathogenic variants in the iduronate-2-sulphatase (IDS) for MPS II have been reported worldwide. The mutation spectrum of MPS type I and MPS type II disorders in Indian population is not characterized yet. In this study, we carried out clinical, biochemical, molecular and in silico analyses to establish the mutation spectrum of MPS I and MPS II in the Indian population. We conducted molecular analysis for 60 MPS-affected patients [MPS I (n = 30) (Hurler syndrome = 17, Hurler-Scheie syndrome = 13), and MPS II (n = 30) (severe = 18, attenuated = 12)] and identified a total of 44 [MPS I (n = 22) and MPS II (n = 22)] different pathogenic variants comprising missense, nonsense, frameshift, gross deletions and splice site variants. A total of 20 [MPS I (n = 14), and MPS II (n = 6)] novel pathogenic sequence variants were identified in our patient cohort. We found that 32% of pathogenic variants detected in IDUA were recurrent and 25% in MPS II. This is the first study revealing the mutation spectrum of MPS I and MPS II patients in the Indian population.

Dried blood spots allow targeted screening to diagnose mucopolysaccharidosis and mucolipidosis.

As patients with different types of mucopolysaccharidosis (MPS) and mucolipidosis (ML) may present with overlapping clinical features - including coarse face, hepatosplenomegaly, bone dysplasia and claw-hand deformities, collectively also called 'MPS-like phenotype', enzymatic and/or molecular genetic analyses are indispensable for accurate diagnosis and applying specific therapy. In this prospective study, we screened patients with symptoms compatible with MPS for MPS I, II (males) and VI. Dried blood spots/specimens (DBS) were collected from 200 patients with an MPS-like phenotype and analysed for activities of α-iduronidase (IDUA), iduronate-2-sulphatase (IDS), and arylsulphatase B (ARSB), the enzymes deficient in mucopolysaccharidosis (MPS) type I, II and VI, respectively. For the samples with pathologic enzyme activity, mutational analysis was carried out using the same DBS. Based on enzymatic analysis of 200 DBS samples, a total of 45 (22.5%) showed low activity; 17 for MPS I (8.5%), 11 for MPS II (5.5%) and 9 for MPS VI (4.5%). Enzyme activities were suggestive for ML II/III in 8 (4.0%) cases. For 41 (91.1%) samples, DNA could be extracted from the filter paper. Mutations were identified in 11 (64.7%), 11 (100%), 9 (100%) and 5 (62.5%) patients putatively diagnosed biochemically with MPS I, II, VI, and ML II/III, respectively. DBS enzymatic analysis can be used to diagnose MPS/ML. Initial results should be confirmed by a second enzyme assay and/or by molecular genetic testing. Given the advantages of DBS over other sample types in terms of ease of collection, storage and transportation, DBS are particularly useful for screening patients with an MPS-like phenotype in regions lacking specialised laboratories. In order to ascertain the diagnosis in a large number of cases, patients should be assessed in parallel for at least MPS I, II and VI.

A highly secreted sulphamidase engineered to cross the blood‐brain barrier corrects brain lesions of mice with mucopolysaccharidoses type IIIA

Mucopolysaccharidoses type IIIA (MPS-IIIA) is a neurodegenerative lysosomal storage disorder (LSD) caused by inherited defects of the sulphamidase gene. Here, we used a systemic gene transfer approach to demonstrate the therapeutic efficacy of a chimeric sulphamidase, which was engineered by adding the signal peptide (sp) from the highly secreted iduronate-2-sulphatase (IDS) and the blood-brain barrier (BBB)-binding domain (BD) from the Apolipoprotein B (ApoB-BD). A single intravascular administration of AAV2/8 carrying the modified sulphamidase was performed in adult MPS-IIIA mice in order to target the liver and convert it to a factory organ for sustained systemic release of the modified sulphamidase. We showed that while the IDS sp replacement results in increased enzyme secretion, the addition of the ApoB-BD allows efficient BBB transcytosis and restoration of sulphamidase activity in the brain of treated mice. This, in turn, resulted in an overall improvement of brain pathology and recovery of a normal behavioural phenotype. Our results provide a novel feasible strategy to develop minimally invasive therapies for the treatment of brain pathology in MPS-IIIA and other neurodegenerative LSDs.

Severe phenotype in MPS II patients associated with a large deletion including contiguous genes

Hunter disease or mucopolysaccharidosis type II (MPS II) is an X-linked recessive lysosomal disorder caused by the deficiency of iduronate-2-sulfatase, which is involved in the catabolism of the glycosaminoglycans (GAGs) heparan and dermatan sulphate. Our aim was to analyze three patients with severe Hunter syndrome that showed a total deletion of the iduronate-2-sulphatase (IDS) gene, after exon by exon PCR. DNA was used as a template for PCR synthesis of IDS, FRAXA, FRAXE, and DXS1113 specific amplicons. The DNA analysis for all three patients demonstrated a complete deletion of IDS, FRAXA, and FRAXE contiguous genes. We further performed SNP-array to delineate the deletion breakpoints and to characterize the deletion extension in the different patients. The results indicated a ∼9.4 Mb deletion in Patient 1, a ∼3.9 Mb deletion of the Xq27.3-Xq28 and a ∼3.1 Mb duplication of the X q28 region in Patient 2 and a ∼41.8 Kb deletion in Patient 3. SNP-array was shown to be important to map for deletion breakpoints. A comprehensive molecular analysis in patients with Hunter syndrome, especially in the ones presenting the severe form, is important to the understanding of the genetic determinants of the phenotype and for the genetic counseling to be provided to the families.

The improvement of iduronate 2-sulphatase (IDS) productivity by addition of sodium butyrate with temperature shift in serum-free suspension culture of recombinant cho cells

The improvement of iduronate 2-sulphatase (IDS) productivity by addition of sodium butyrate with temperature shift in serum-free suspension culture of recombinant cho cells

Hunter syndrome: Case report and review of literature

Hunter syndrome is one of the mucopolysaccharidosis, which are a rare group of genetic diseases. It is due to a deficiency in the enzyme Iduronate 2-sulphatase. This in turn leads to the accumulation of glycosaminoglycans, dermatan and heparan sulphate. The intra and extracellular accumulation of this substances lead to multisystemic organ abnormality. We present a patient with Hunter syndrome who presented with involvement of the skin, cardiovascular system, the eyes and musculoskeletal system. We have also included a literature review. As far as we know, it is the first case reported in a Nigerian.

Mucopolysaccharidosis type II: an update on mutation spectrum

Mucopolysaccharidosis type II (MPS II; Hunter disease) is caused by deficiency of the enzyme iduronate-2-sulphatase (IDS) and patients present with a wide range of clinical signs and symptoms. The level of activity of IDS, however, does not allow prediction of phenotype. In our study of unrelated individuals with MPS II, alterations in the IDS gene could be identified in all 155 patients. Investigations in families in which the occurrence of MPS II was sporadic revealed mosaicism in the mothers of a small number of patients and a high frequency of de novo mutations occurring preferentially during male meiosis. Mutations identified in our patients include 27 large alterations and 128 small gene alterations (96 different alterations). These data further confirm the extreme heterogeneity of IDS gene alterations, as more than 330 have been reported to date. This genetic heterogeneity may explain the high degree of clinical heterogeneity in MPS II. Therefore, attempts have been made to establish genotype-phenotype correlations in order to provide an indication of the likely prognosis and a basis on which to evaluate treatment. To date, some progress has been made in predicting the clinical phenotype from the genotype although it remains difficult in a few individual cases. However, as the crystallographic 3D structure of IDS is yet to be determined, evaluation of the impact of mutations on IDS activity is often time consuming. Furthermore, if a given mutation is recurrent, some patients carrying the same change may present with different phenotypes, suggesting that factors other than the IDS gene (e.g. other genes, environmental factors) can modulate the clinical phenotype. Although genotype-phenotype correlations may be difficult to establish, they will be of increasing importance for choosing the most appropriate therapy for an individual patient, as new therapeutic strategies may be targeted according to phenotype.

Iduronate-2-sulphatase protein detection in plasma from mucopolysaccharidosis type II patients

The lysosomal storage disorder mucopolysaccharidosis type II (MPS II) is caused by a deficiency in the activity of the lysosomal exohydrolase iduronate-2-sulphatase (IDS). MPS II patients present within a spectrum of clinical phenotypes, which reflects the dynamic balance between the level of mutant protein, its residual enzyme activity and the resultant level of storage product. In this study, we have developed an immunoquantification assay for the accurate detection of iduronate-2-sulphatase protein and applied this methodology to the analysis of mutant iduronate-2-sulphatase protein in plasma samples from MPS II patients. The detection limit for the assay was defined as 20 ng/ml for wild type iduronate-2-sulphatase, but could be extended to a detection limit of 0.3 ng/ml by heat denaturation of the protein/plasma sample. The mutant protein detected in plasma from MPS II patients displayed similar properties to heat denatured wild type iduronate-2-sulphatase, suggesting an altered protein conformation. The ratio of heat denatured to native ELISA reactivity could be used to confirm the diagnosis of MPS II (i.e., a ratio of >1 for normal protein and ⩽1 for mutant protein). Notably, four of the 20 patients tested had either normal or higher than normal levels of iduronate-2-sulphatase protein, but this protein also showed evidence of conformation change. The iduronate-2-sulphatase protein level detected in plasma from MPS II patients showed little or no direct correlation with the severity of the clinical phenotype observed in these patients.