A Multiplex Assay for the Diagnosis of Mucopolysaccharidoses and Mucolipidoses

Introduction

Diagnosis of the mucopolysaccharidoses (MPSs) generally relies on an initial analysis of total glycosaminoglycan (GAG) excretion in urine. Often the dimethylmethylene blue dye-binding (DMB) assay is used, although false-negative results have been reported. We report a multiplexed diagnostic test with a high sensitivity for all MPSs and with the potential to identify patients with I-cell disease (ML II) and mucolipidosis III (ML III).

Methods

Urine samples of 100 treatment naive MPS patients were collected and analyzed by the conventional DMB assay and a multiplex assay based on enzymatic digestion of heparan sulfate (HS), dermatan sulfate (DS) and keratan sulfate (KS) followed by quantification by LC-MS/MS. Specificity was calculated by analyzing urine samples from a cohort of 39 patients suspected for an inborn error of metabolism, including MPSs.

Results

The MPS cohort consisted of 18 MPS I, 16 MPS II, 34 MPS III, 10 MPS IVA, 3 MPS IVB, 17 MPS VI and 2 MPS VII patients. All 100 patients were identified by the LC-MS/MS assay with typical patterns of elevation of HS, DS and KS, respectively (sensitivity 100%). DMB analysis of the urine was found to be in the normal range in 10 of the 100 patients (sensitivity 90%). Three out of the 39 patients were identified as false-positive, resulting in a specificity of the LS-MS/MS assay of 92%. For the DMB this was 97%. All three patients with MLII/MLIII had elevated GAGs in the LC-MS/MS assay while the DMB test was normal in 2 of them.

Conclusion

The multiplex LC-MS/MS assay provides a robust and very sensitive assay for the diagnosis of the complete spectrum of MPSs and has the potential to identify MPS related disorders such as MLII/MLIII. Its performance is superior to that of the conventional DMB assay.     

Mucopolysaccharidoses--biochemical mechanisms of diseases and therapeutic possibilities

Mucopolysaccharidoses (MPS) are inherited metabolic diseases from the group of lysosomal storage disorders (LSD). They are caused by genetic defects resulting in the absence or severe deficiency in one of lysosmal hydrolases involved in degradation of glycosaminoglycans (GAG). Partially degraded GAGs are accumulated in lysosomes, causing dysfunction of cells, tissues and organs. Last years did bring some breakthrough discoveries, which were important to understand biochemical mechanisms of MPS appearance and course, as well as to develop therapeutic procedures for these inherited metabolic disorders.     

Changes in cellular processes occurring in mucopolysaccharidoses as underestimated pathomechanisms of these diseases

Mucopolysaccharidoses (MPS) are a group of genetic disorders belonging to lysosomal storage diseases. They are caused by genetic defects leading to a lack or severe deficiency of activity of one of lysosomal hydrolases involved in degradation of glycosaminoglycans (GAGs). Partially degraded GAGs accumulate in lysosomes, which results in dysfunctions of cells, tissues, and organs. Until recently, it was assumed that GAG accumulation in cells is the major, if not the only, mechanism of pathogenesis in MPS, as GAGs may be a physical ballast for lysosomes causing inefficiency of cells due to a large amount of a stored material. However, recent reports suggest that in MPS cells there are changes in many different processes, which might be even more important for pathogenesis than lysosomal accumulation of GAGs per se. Moreover, there are many recently published results indicating that lysosomes not only are responsible for degradation of various macromolecules, but also play crucial roles in the regulation of cellular metabolism. Therefore, it appears plausible that previous failures in treatment of MPS (i.e., possibility to correct only some symptoms and slowing down of the disease rather than fully effective management of MPS) might be caused by underestimation of changes in cellular processes and concentration solely on decreasing GAG levels in cells.     

Comparison of Rebound Tonometry,Perkins Applanation Tonometry and Ocular Response Analyser in Mucopolysaccharidosis Patients

Aims

To investigate the feasibility and to compare three devices measuring intraocular pressure (IOP) in mucopolysaccharidosis patients (MPS): iCare rebound tonometer (RT), Perkins applanation tonometer (PAT) and ocular response analyzer (ORA)

Methods

MPS patients who underwent at least two examinations out of: RT, PAT and ORA at the same visit were identified and retrospectively analyzed in this study.

Results

17 patients fulfilled the inclusion criterion. In all 17 patients IOP measurements were performed with RT (34 eyes) and ORA (33 eyes), while PAT measurement was possible in only 12 (24 eyes) patients. The RT, corneal-compensated intraocular pressure (IOPcc) and Goldmann-correlated intraocular pressure (IOPg) differed relevantly from IOP assessed with PAT. Corneal clouding in MPS patients correlated positively with PAT, RT and IOPg (r = 0.3, 0.5, and 0.5 respectively), but not with IOPcc (r = 0.07). The MPS-related corneal clouding correlated positively with biomechanical corneal parameters assessed with ORA: corneal hysteresis (r = 0.77) and corneal resistance factor (r = 0.77) either.

Conclusions

RT and ORA measurements were tolerated better than applanation tonometry in MPS patients. IOP measurements assessed with RT and ORA differed relevantly from PAT. Corneal-compensated IOP assessed with ORA seems to be less affected by the MPS-related corneal clouding than applanation or rebound tonometry. RT and ORA measurements should be preferred for IOP assessment in patients with MPS.     

Biochemical,machine learning and molecular approaches for the differential diagnosis of Mucopolysaccharidoses

This study was aimed to construct classification and regression tree (CART) model of glycosaminoglycans (GAGs) for the differential diagnosis of Mucopolysaccharidoses (MPS). Two-dimensional electrophoresis and liquid chromatography–tandem mass spectrometry (LC–MS/MS) were used for the qualitative and quantitative analysis of GAGs. Specific enzyme assays and targeted gene sequencing were performed to confirm the diagnosis. Machine learning tools were used to develop CART model based on GAG profile. Qualitative and quantitative CART models showed 96.3% and 98.3% accuracy, respectively, in the differential diagnosis of MPS. The thresholds of different GAGs diagnostic of specific MPS types were established. In 60 MPS positive cases, 46 different mutations were identified in six specific genes. Among 31 different mutations identified in IDUA, nine were nonsense mutations and two were gross deletions while the remaining were missense mutations. In IDS gene, four missense, two frameshift, and one deletion were identified. In NAGLU gene, c.1693C?>?T and c.1914_1914insT were the most common mutations. Two ARSB, one case each of SGSH and GALNS mutations were observed. LC–MS/MS-based GAG pattern showed higher accuracy in the differential diagnosis of MPS. The mutation spectrum of MPS, specifically in IDUA and IDS genes, is highly heterogeneous among the cases studied.

     

Patients with left bundle branch block pattern and high cardiac risk myocardial SPECT: does the current management suffice?

Introduction

Myocardial perfusion SPECT (MPS) is frequently used for cardiovascular risk stratification. The significance of MPS in patients with abnormal electrical ventricular activation is often questionable. This review assesses the value of MPS for risk stratification of patients with intrinsic left bundle branch block or that due to right ventricular apical pacing.

Methods

We reviewed the literature by a search of the MEDLINE database (January 1980 to September 2010). The terms prognosis or prognostic value were combined with SPECT and LBBB or pacing or pacemakers. MPS was categorised as low and high risk according to the original definitions.

Results

We identified 11 studies suitable for review. A low-risk MPS is associated with a low risk of cardiac events whereas high-risk MPS carries a 4.8-fold increased risk, 95% CI [3.2 – 7.2] (p < 0.0001). Despite secondary prevention and an improved medical and interventional care, these figures have hardly changed over time.

Conclusion and clinical implications

A low-risk MPS permits a policy of watchful waiting whereas a high-risk MPS requires further analysis and treatment. The persistent high cardiac death and acute myocardial infarction rate after a high-risk MPS suggest that the current management of these patients does not suffice and needs reconsideration.     

Tissue distribution of DNA-Hsp65/TDM-loaded PLGA microspheres and uptake by phagocytic cells

Background

Mucopolysaccharidosis type IIIA (MPS IIIA) is the most common of the mucopolysaccharidoses. The disease is caused by a deficiency of the lysosomal enzyme sulphamidase and results in the storage of the glycosaminoglycan (GAG), heparan sulphate. MPS IIIA is characterised by widespread storage and urinary excretion of heparan sulphate, and a progressive and eventually profound neurological course. Gene therapy is one of the few avenues of treatment that hold promise of a sustainable treatment for this disorder.

Methods

The murine sulphamidase gene cDNA was cloned into a lentiviral vector and high-titre virus produced. Human MPS IIIA fibroblast cultures were transduced with the sulphamidase vector and analysed using molecular, enzymatic and metabolic assays. High-titre virus was intravenously injected into six 5-week old MPS IIIA mice. Three of these mice were pre-treated with hyperosmotic mannitol. The weight of animals was monitored and GAG content in urine samples was analysed by polyacrylamide gel electrophoresis.

Results

Transduction of cultured MPS IIIA fibroblasts with the sulphamidase gene corrected both the enzymatic and metabolic defects. Sulphamidase secreted by gene-corrected cells was able to cross correct untransduced MPS IIIA cells. Urinary GAG was found to be greatly reduced in samples from mice receiving the vector compared to untreated MPS IIIA controls. In addition, the weight of treated mice became progressively normalised over the 6-months post-treatment.

Conclusion

Lentiviral vectors appear promising vehicles for the development of gene therapy for MPS IIIA.     

Ultrasonographic Features of Hip Joints in Mucopolysaccharidoses Type I and II

Objectives

The primary aim of this study was to assess the ultrasonographic features of hip joints in patients with mucopolysaccharidosis (MPS) type I and II in comparison with healthy population. The secondary aims were to correlate these features with clinical measures and to evaluate the utility of ultrasound in the diagnosis of MPS disease.

Materials and Methods

Sixteen MPS I (n = 3) and II (n = 13) patients were enrolled in the present study and underwent clinical and radiological evaluation, and bilateral high-resolution ultrasonography (US) of hip joints. The distance from the femoral neck to joint capsule (synovial joint space, SJS), joint effusion, synovial hyperthrophy, and local pathological vascularization were evaluated. The results were compared to the healthy population and correlated with clinical and radiological measures.

Results

1. There was a difference in US SJS between children with MPS disease and the normative value for healthy population (7mm). Mean values of SJS were 15.81 ± 4.08 cm (right hip joints) and 15.69 ± 4.19 cm (left joints). 2. No inflammatory joint abnormalities were detected in MPS patients. 3. There was a clear correlation between US SJS and patients’ age and height, while no clear correlation was observed between SJS and disease severity.

Conclusions

1. Patients with MPS I and II present specific features in hip joint ultrasonography. 2. The data suggests that ultrasonography might be effective in the evaluation of hip joint involvement in patients with MPS and might present a valuable tool in facilitating the diagnosis and follow up of the disease.     

Short stature as a presenting symptom of attenuated Mucopolysaccharidosis type I: case report and clinical insights

Background

Mucopolysaccharidosis type I (MPS I) results in significant disease burden and early treatment is important for optimal outcomes. Recognition of short stature and growth failure as symptoms of MPS I among pediatric endocrinologists may lead to earlier diagnosis and treatment.

Case presentation

A male patient first began experiencing hip pain at 5 years of age and was referred to an endocrinologist for short stature at age 7. Clinical history included recurrent respiratory infections, sleep apnea, moderate joint contractures, mild facial dysmorphic features, scoliosis, and umbilical hernia. Height was more than ??2 SD below the median at all time points. Growth velocity was below the 3rd percentile. Treatment for short stature included leuprolide acetate and recombinant human growth hormone. The patient was diagnosed with MPS I and began enzyme replacement therapy with laronidase at age 18.

Conclusions

The case study patient had many symptoms of MPS I yet remained undiagnosed for 11 years after presenting with short stature. The appropriate path to MPS I diagnosis when patients present with short stature and/or growth failure plus one or more of the common signs of attenuated disease is described. Improved awareness regarding association of short stature and growth failure with attenuated MPS I is needed since early identification and treatment significantly decreases disease burden.

     

Effect of genistein and coenzyme Q10 in oxidative damage and mitochondrial membrane potential in an attenuated type II mucopolysaccharidosis cellular model

Mucopolysaccharidosis type II (MPS II) is an inborn error of the metabolism resulting from several possible mutations in the gene coding for iduronate-2-sulfatase (IDS), which leads to a great clinical heterogeneity presented by these patients. Many studies demonstrate the involvement of oxidative stress in the pathogenesis of inborn errors of metabolism, and mitochondrial dysfunction and oxidative stress can be related since most of reactive oxygen species come from mitochondria. Cellular models have been used to study different diseases and are useful in biochemical research to investigate them in a new promising way. The aim of this study is to develop a heterozygous cellular model for MPS II and analyze parameters of oxidative stress and mitochondrial dysfunction and investigate the in vitro effect of genistein and coenzyme Q10 on these parameters for a better understanding of the pathophysiology of this disease. The HP18 cells (heterozygous c.261_266del6/c.259_261del3) showed almost null results in the activity of the IDS enzyme and presented accumulation of glycosaminoglycans (GAGs), allowing the characterization of this knockout cellular model by MPS II gene editing. An increase in the production of reactive species was demonstrated (p < .05 compared with WT vehicle group) and genistein at concentrations of 25 and 50 µm decreased in vitro its production (p < .05 compared with HP18 vehicle group), but there was no effect of coenzyme Q10 in this parameter. There was a tendency for lysosomal pH change in HP18 cells in comparison to WT group and none of the antioxidants tested demonstrated any effect on this parameter. There was no increase in the activity of the antioxidant enzymes superoxide dismutase and catalase and oxidative damage to DNA in HP18 cells in comparison to WT group and neither genistein nor coenzyme q10 had any effect on these parameters. Regarding mitochondrial membrane potential, genistein induced mitochondrial depolarization in both concentrations tested (p < .05 compared with HP18 vehicle group and compared with WT vehicle group) and incubation with coenzyme Q10 demonstrated no effect on this parameter. In conclusion, it is hypothesized that our cellular model could be compared with a milder MPS II phenotype, given that the accumulation of GAGs in lysosomes is not as expressive as another cellular model for MPS II presented in the literature. Therefore, it is reasonable to expect that there is no mitochondrial depolarization and no DNA damage, since there is less lysosomal impairment, as well as less redox imbalance.     

Macrolide use in the previous years is associated with failure to eradicate Helicobacter pylori with clarithromycin‐containing regimens

Background

There is some evidence that prior use of macrolide antibiotics is a useful predictor of the likelihood of standard triple therapy failure in Helicobacter pylori eradication. In this study, we have evaluated whether previous intake of macrolides correlates with failure to eradicate H. pylori using two different first‐line clarithromycin‐containing regimens.

Materials and Methods

Retrospective study of 212 patients with H. pylori infection treated with one of two first‐line clarithromycin‐containing regimens: 108 patients treated with triple therapy for 10 days and 104 patients treated with concomitant therapy for 10 days. The intake of macrolides (clarithromycin, azithromycin, and other macrolides) prior to the eradication therapy was obtained from the electronic medical record, which contains information regarding all the medication prescribed to the patients since the year 2004.

Results

One hundred of 212 patients (47.2%) had received at least one treatment with macrolides during the years prior to the eradication therapy. H. pylori eradication rates were significantly lower in patients with previous use compared to patients without previous use of macrolides, both with triple therapy (60.8% vs 92.9%; P < .0001) and with concomitant therapy (85.7% vs 98.2%; P = .024).

Conclusions

Previous use of macrolides correlates with a low H. pylori eradication rate with triple and concomitant clarithromycin‐containing regimens. In addition, our study shows that in patients without previous use of macrolides, triple therapy achieves per‐protocol eradication rates over 90%.     

Anti-TNF-alpha therapy enhances the effects of enzyme replacement therapy in rats with mucopolysaccharidosis type VI

Background

Although enzyme replacement therapy (ERT) is available for several lysosomal storage disorders, the benefit of this treatment to the skeletal system is very limited. Our previous work has shown the importance of the Toll-like receptor 4/TNF-alpha inflammatory pathway in the skeletal pathology of the mucopolysaccharidoses (MPS), and we therefore undertook a study to examine the additive benefit of combining anti-TNF-alpha therapy with ERT in a rat model of MPS type VI.

Methodology/Principal Findings

MPS VI rats were treated for 8 months with Naglazyme® (recombinant human N-acetyl-galactosamine-4-sulfatase), or by a combined protocol using Naglazyme® and the rat-specific anti-TNF-alpha drug, CNTO1081. Both protocols led to markedly reduced serum levels of TNF-alpha and RANKL, although only the combined treatment reduced TNF-alpha in the articular cartilage. Analysis of cultured articular chondrocytes showed that the combination therapy also restored collagen IIA1 expression, and reduced expression of the apoptotic marker, PARP. Motor activity and mobility were improved by ERT, and these were significantly enhanced by combination treatment. Tracheal deformities in the MPS VI animals were only improved by combination therapy, and there was a modest improvement in bone length. Ceramide levels in the trachea also were markedly reduced. MicroCT analysis did not demonstrate any significant positive effects on bone microarchitecture from either treatment, nor was there histological improvement in the bone growth plates.

Conclusions/Significance

The results demonstrate that combining ERT with anti-TNF- alpha therapy improved the treatment outcome and led to significant clinical benefit. They also further validate the usefulness of TNF-alpha, RANKL and other inflammatory molecules as biomarkers for the MPS disorders. Further evaluation of this combination approach in other MPS animal models and patients is warranted.     

Autonomic nervous system dysfunction predicts poor prognosis in patients with mild to moderate tetanus

Background  

Autonomic nervous system (ANS) dysfunction is present in up to one third of patients with tetanus. The prognostic value of ANS dysfunction is known in severe tetanus but its value is not well established in mild to moderate tetanus.     

Characterization of two heparan sulphate-binding sites in the mycobacterial adhesin Hlp

Background  

The histone-like Hlp protein is emerging as a key component in mycobacterial pathogenesis, being involved in the initial events of host colonization by interacting with laminin and glycosaminoglycans (GAGs). In the present study, nuclear magnetic resonance (NMR) was used to map the binding site(s) of Hlp to heparan sulfate and identify the nature of the amino acid residues directly involved in this interaction.     

Anti‐Helicobacter pylori therapy in localized gastric mucosa‐associated lymphoid tissue lymphoma: A prospective,nationwide, multicenter study in Japan

Background

Helicobacter pylori eradication therapy was approved in Japan for the first‐line, standard treatment of H. pylori‐positive gastric mucosa‐associated lymphoid tissue (MALT) lymphoma. Although several retrospective studies or small‐scale single‐center studies have been reported, a prospective, large‐scale, nationwide, multicenter study has not been reported from Japan.

Materials and Methods

We conducted a prospective, nationwide, multicenter study to evaluate the clinical efficacy of rabeprazole‐based triple H. pylori eradication therapy for patients with localized gastric MALT lymphoma in practice‐based clinical trial. A total of 108 H. pylori‐positive patients with stage I/II₁ gastric MALT lymphoma underwent H. pylori eradication therapy. The primary endpoints were complete remission (CR) rate and the rate of transfer to secondary treatment. The secondary endpoints were CR maintenance duration and overall survival (OS).

Results

CR of lymphoma was achieved in 84 of 97 patients (86.6%), during the period 2.0‐44.7 months (median, 5.3 months) after starting H. pylori eradication treatment. CR was maintained in 77 of 81 patients (95.1%) for 0.4‐53.2 months (median, 33.1 months). Secondary treatments (radiotherapy, rituximab, or gastrectomy) for gastric MALT lymphoma were needed in 10 of the 97 patients (10.31%). During follow‐up, OS rate was 96.9% (94/97) and the causes of 3 deaths were not related to lymphoma.

Conclusions

Rabeprazole‐based H. pylori eradication therapy demonstrated a high CR rate, long CR maintenance, and a good OS for patients with localized gastric MALT lymphoma in this prospective, practice‐based, multicenter study.     

Illness Perception and Clinical Treatment Experiences in Patients with M. Maroteaux-Lamy (Mucopolysaccharidosis Type VI) and a Turkish Migration Background in Germany

Introduction

Mucopolysaccharidosis VI (MPS VI) is an inherited lysosomal storage disease caused by a mutation of the gene for arylsulfatase B (ASB). Of the thirty-one patients registered in Germany, almost fifty percent have a Turkish migration background. MPS VI is treated by enzyme replacement therapy (ERT), which is time-consuming and expensive.

Methods

This interdisciplinary study explored the illness perceptions and clinical treatment experiences among ten MPS VI patients with a Turkish migration background in two centers for metabolic diseases (Berlin and Mainz, Germany). The clinical treatment situation was observed and semi-structured interviews were conducted with patients and health care personnel, in addition to participatory observation in four patients'' everyday environments in Berlin. The data from the interviews, patient records, and personal field notes were encoded, cross-related, and analyzed.

Results

Patients'' acknowledgement of the disease and coping strategies are influenced predominantly by the perception of their individual health status and the handling of the disease within their family. Patients'' willingness to cooperate with treatment strategies is further modified by their knowledge of the disease and the relationships with their health care providers. In this analysis, cultural factors turned out to be marginally relevant.

Conclusion

As with other chronic and debilitating diseases, effective treatment strategies have to reach beyond delivering medication. Health care providers need to strengthen the support for patients with a migration background. In this regard, they should respect the patients'' cultural and social background and their personal perception of the disease and the therapy. Yet structural and social aspects (clinical setting, family and educational background) may be more crucial here than “cultural barriers.”     

Mucopolysaccharidosis type II: European recommendations for the diagnosis and multidisciplinary management of a rare disease

?

Mucopolysaccharidosis type II (MPS II) is a rare, life-limiting, X-linked recessive disease characterised by deficiency of the lysosomal enzyme iduronate-2-sulfatase. Consequent accumulation of glycosaminoglycans leads to pathological changes in multiple body systems. Age at onset, signs and symptoms, and disease progression are heterogeneous, and patients may present with many different manifestations to a wide range of specialists. Expertise in diagnosing and managing MPS II varies widely between countries, and substantial delays between disease onset and diagnosis can occur. In recent years, disease-specific treatments such as enzyme replacement therapy and stem cell transplantation have helped to address the underlying enzyme deficiency in patients with MPS II. However, the multisystem nature of this disorder and the irreversibility of some manifestations mean that most patients require substantial medical support from many different specialists, even if they are receiving treatment. This article presents an overview of how to recognise, diagnose, and care for patients with MPS II. Particular focus is given to the multidisciplinary nature of patient management, which requires input from paediatricians, specialist nurses, otorhinolaryngologists, orthopaedic surgeons, ophthalmologists, cardiologists, pneumologists, anaesthesiologists, neurologists, physiotherapists, occupational therapists, speech therapists, psychologists, social workers, homecare companies and patient societies.

Take-home message

Expertise in recognising and treating patients with MPS II varies widely between countries. This article presents pan-European recommendations for the diagnosis and management of this life-limiting disease.