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Purpose: To describe an efficient and effective multiplex screening strategy for sulfatide degradation disorders and mucolipidosis type II/III (MLII/III) using 3 mL of urine., Methods: Glycosaminoglycans were analyzed by liquid chromatography-tandem mass spectrometry. Matrix assisted laser desorption/ionization-time of flight tandem mass spectrometry was used to identify free oligosaccharides and identify 22 ceramide trihexosides and 23 sulfatides, which are integrated by 670 calculated ratios. Collaborative Laboratory Integrated Reports (CLIR; https://clir.mayo.edu) was used for post-analytical interpretation of the complex metabolite profile and to aid in the differential diagnosis of abnormal results., Results: Multiplex analysis was performed on 25 sulfatiduria case samples and compiled with retrospective data from an additional 15 cases revealing unique patterns of biomarkers for each disorder of sulfatide degradation (MLD, MSD, and Saposin B deficiency) and for MLII/III, thus allowing the formulation of a novel algorithm for the biochemical diagnosis of these disorders., Conclusions: Comprehensive and integrated urine screening could be very effective in the initial workup of patients suspected of having a lysosomal disorder as it covers disorders of sulfatide degradation and narrows down the differential diagnosis in patients with elevated glycosaminoglycans., (Copyright © 2019 Elsevier Inc. All rights reserved.)

To explore the correlation between glycosaminoglycan (GAG) levels and mucopolysaccharidosis (MPS) type, we have evaluated the GAG levels in blood of MPS II, III, IVA, and IVB and urine of MPS IVA, IVB, and VI by tandem mass spectrometry. Dermatan sulfate (DS), heparan sulfate (HS), keratan sulfate (KS; mono-sulfated KS, di-sulfated KS), and the ratio of di-sulfated KS in total KS were measured. Patients with untreated MPS II had higher levels of DS and HS in blood while untreated MPS III had higher levels of HS in blood than age-matched controls. Untreated MPS IVA had higher levels of KS in blood and urine than age-matched controls. The ratio of blood di-sulfated KS/total KS in untreated MPS IVA was constant and higher than that in controls for children up to 10 years of age. The ratio of urine di-sulfated KS/total KS in untreated MPS IVA was also higher than that in age-matched controls, but the ratio in untreated MPS IVB was lower than controls. ERT reduced blood DS and HS in MPS II, and urine KS in MPS IVA patients, although GAGs levels remained higher than the observed in age-matched controls. ERT did not change blood KS levels in MPS IVA. MPS VI under ERT still had an elevation of urine DS level compared to age-matched controls. There was a positive correlation between blood and urine KS in untreated MPS IVA patients but not in MPS IVA patients treated with ERT. Blood and urine KS levels were secondarily elevated in MPS II and VI, respectively. Overall, measurement of GAG levels in blood and urine is useful for diagnosis of MPS, while urine KS is not a useful biomarker for monitoring therapeutic efficacy in MPS IVA., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Objective: The aim of this study was to quantify glycosaminoglycans (GAGs) in amniotic fluid (AF) from an MPS VII fetus compared with age-matched fetuses obtained from normal pregnancies., Method: Disaccharides were measured by liquid chromatography tandem mass spectrometry, compared to age-matched controls. Enzyme assay was performed in AF supernatant or cultured amniocytes. GUSB was analyzed by next generation sequencing using Ion Torrent Personal Genome Machine with a customized panel., Results: No activity of β-glucuronidase was detected in fetal cells. The pregnancy was spontaneously terminated in the third trimester. Genetic studies identified a homozygous mutation of p.N379D (c.1135A > G) in the GUSB gene. Liquid chromatography tandem mass spectrometry showed that chondroitin sulfate, dermatan sulfate, heparan sulfate, and keratan sulfate levels were markedly increased in the MPS VII AF, compared to those in age-matched control AF (dermatan sulfate, heparan sulfate, and chondroitin-6-sulfate more than 10 × than age-matched controls; chondroitin-4-sulfate and keratan sulfate more than 3 times higher)., Conclusion: This is the first report of specific GAG analysis in AF from an MPS VII fetus, indicating that GAG elevation in AF occurs by 21 weeks of gestation and could be an additional tool for prenatal diagnosis of MPS VII and potentially other MPS types. © 2017 John Wiley & Sons, Ltd., (© 2017 John Wiley & Sons, Ltd.)

Mucopolysaccharidoses (MPSs) and mucolipidoses (ML) are groups of lysosomal storage disorders in which lysosomal hydrolases are deficient leading to accumulation of undegraded glycosaminoglycans (GAGs), throughout the body, subsequently resulting in progressive damage to multiple tissues and organs. Assays using tandem mass spectrometry (MS/MS) have been established to measure GAGs in serum or plasma from MPS and ML patients, but few studies were performed to determine whether these assays are sufficiently robust to measure GAG levels in dried blood spots (DBS) of patients with MPS and ML., Material and Methods: In this study, we evaluated GAG levels in DBS samples from 124 MPS and ML patients (MPS I=16; MPS II=21; MPS III=40; MPS IV=32; MPS VI=10; MPS VII=1; ML=4), and compared them with 115 age-matched controls. Disaccharides were produced from polymer GAGs by digestion with chondroitinase B, heparitinase, and keratanase II. Subsequently, dermatan sulfate (DS), heparan sulfate (HS-0S, HS-NS), and keratan sulfate (mono-sulfated KS, di-sulfated KS, and ratio of di-sulfated KS in total KS) were measured by MS/MS., Results: Untreated patients with MPS I, II, VI, and ML had higher levels of DS compared to control samples. Untreated patients with MPS I, II, III, VI, and ML had higher levels of HS-0S; and untreated patients with MPS II, III and VI and ML had higher levels of HS-NS. Levels of KS were age dependent, so although levels of both mono-sulfated KS and di-sulfated KS were generally higher in patients, particularly for MPS II and MPS IV, age group numbers were not sufficient to determine significance of such changes. However, the ratio of di-sulfated KS in total KS was significantly higher in all MPS patients younger than 5years old, compared to age-matched controls. MPS I and VI patients treated with HSCT had normal levels of DS, and MPS I, VI, and VII treated with ERT or HSCT had normal levels of HS-0S and HS-NS, indicating that both treatments are effective in decreasing blood GAG levels., Conclusion: Measurement of GAG levels in DBS is useful for diagnosis and potentially for monitoring the therapeutic efficacy in MPS., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: Mucopolysaccharidoses (MPS) are a group of inborn errors of metabolism that are progressive and usually result in irreversible skeletal, visceral, and/or brain damage, highlighting a need for early diagnosis., Methods: This pilot study analyzed 2862 dried blood spots (DBS) from newborns and 14 DBS from newborn patients with MPS (MPS I, n = 7; MPS II, n = 2; MPS III, n = 5). Disaccharides were produced from polymer GAGs by digestion with chondroitinase B, heparitinase, and keratanase II. Heparan sulfate (0S, NS), dermatan sulfate (DS) and mono- and di-sulfated KS were measured by liquid chromatography tandem mass spectrometry (LC-MS/MS). Median absolute deviation (MAD) was used to determine cutoffs to distinguish patients from controls. Cutoffs were defined as median + 7× MAD from general newborns., Results: The cutoffs were as follows: HS-0S > 90 ng/mL; HS-NS > 23 ng/mL, DS > 88 ng/mL; mono-sulfated KS > 445 ng/mL; di-sulfated KS > 89 ng/mL and ratio di-KS in total KS > 32 %. All MPS I and II samples were above the cutoffs for HS-0S, HS-NS, and DS, and all MPS III samples were above cutoffs for HS-0S and HS-NS. The rate of false positives for MPS I and II was 0.03 % based on a combination of HS-0S, HS-NS, and DS, and for MPS III was 0.9 % based upon a combination of HS-0S and HS-NS., Conclusions: Combination of levels of two or more different GAGs improves separation of MPS patients from unaffected controls, indicating that GAG measurements are potentially valuable biomarkers for newborn screening for MPS.

Mucopolysaccharidosis type II (MPS II) is a lysosomal storage disease caused by a deficient activity of iduronate-2-sulfatase, leading to abnormal accumulation of glycosaminoglycans (GAG). The main treatment for MPS II is enzyme replacement therapy (ERT). Previous studies described potential benefits of six months of ERT against oxidative stress in patients. Thus, the aim of this study was to investigate oxidative, nitrative and inflammatory biomarkers in MPS II patients submitted to long term ERT. It were analyzed urine and blood samples from patients on ERT (mean time: 5.2years) and healthy controls. Patients presented increased levels of lipid peroxidation, assessed by urinary 15-F2t-isoprostane and plasmatic thiobarbituric acid-reactive substances. Concerning to protein damage, urinary di-tyrosine (di-Tyr) was increased in patients; however, sulfhydryl and carbonyl groups in plasma were not altered. It were also verified increased levels of urinary nitrate+nitrite and plasmatic nitric oxide (NO) in MPS II patients. Pro-inflammatory cytokines IL-1β and TNF-α were increased in treated patients. GAG levels were correlated to di-Tyr and nitrate+nitrite. Furthermore, IL-1β was positively correlated with TNF-α and NO. Contrastingly, we did not observed alterations in erythrocyte superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase activities, in reduced glutathione content and in the plasmatic antioxidant capacity. Although some parameters were still altered in MPS II patients, these results may suggest a protective role of long-term ERT against oxidative stress, especially upon oxidative damage to protein and enzymatic and non-enzymatic defenses. Moreover, the redox imbalance observed in treated patients seems to be GAG- and pro-inflammatory cytokine-related., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Since we previously observed that in patients with mucopolysaccharidosis (MPS) the storage of undegraded glycosaminoglycans (GAG) occurs from birth, in the present study we aimed to compare normal, untreated MPS I mice (knockout for alpha-l-iduronidase-IDUA), and MPS I mice treated with enzyme replacement therapy (ERT, Laronidase, 1.2mg/kg every 2 weeks) started from birth (ERT-neo) or from 2 months of age (ERT-ad). All mice were sacrificed at 6 months. Both treatments were equally effective in normalizing GAG levels in the viscera but had no detectable effect on the joint. Heart function was also improved with both treatments. On the other hand, mice treated from birth presented better outcomes in the difficult-to-treat aortas and heart valves. Surprisingly, both groups had improvements in behavior tests, and normalization of GAG levels in the brain and IDUA injection resulted in detectable levels of enzyme in the brain tissue 1h after administration. ERT-ad mice developed significantly more anti-IDUA-IgG antibodies, and mice that didn't develop antibodies had better performances in behavior tests, indicating that development of antibodies may reduce enzyme bioavailability. Our results suggest that ERT started from birth leads to better outcomes in the aorta and heart valves, as well as a reduction in antibody levels. Some poor vascularized organs, such as the joints, had partial or no benefit and ancillary therapies might be needed for patients. The results presented here support the idea that ERT started from birth leads to better treatment outcomes and should be considered whenever possible, a observation that gains relevance as newborn screening programs are being considered for MPS and other treatable lysosomal storage disorders., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Mucopolysaccharidosis IVA (MPS IVA; Morquio A syndrome) is an autosomal recessive lysosomal storage disorder resulting from a deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS) activity. Diagnosis can be challenging and requires agreement of clinical, radiographic, and laboratory findings. A group of biochemical genetics laboratory directors and clinicians involved in the diagnosis of MPS IVA, convened by BioMarin Pharmaceutical Inc., met to develop recommendations for diagnosis. The following conclusions were reached. Due to the wide variation and subtleties of radiographic findings, imaging of multiple body regions is recommended. Urinary glycosaminoglycan analysis is particularly problematic for MPS IVA and it is strongly recommended to proceed to enzyme activity testing even if urine appears normal when there is clinical suspicion of MPS IVA. Enzyme activity testing of GALNS is essential in diagnosing MPS IVA. Additional analyses to confirm sample integrity and rule out MPS IVB, multiple sulfatase deficiency, and mucolipidoses types II/III are critical as part of enzyme activity testing. Leukocytes or cultured dermal fibroblasts are strongly recommended for enzyme activity testing to confirm screening results. Molecular testing may also be used to confirm the diagnosis in many patients. However, two known or probable causative mutations may not be identified in all cases of MPS IVA. A diagnostic testing algorithm is presented which attempts to streamline this complex testing process.

The mucopolysaccharidoses (MPSs) are inherited lysosomal storage disorders caused by the absence of functional enzymes that contribute to the degradation of glycosaminoglycans (GAGs). The progressive systemic deposition of GAGs results in multi-organ system dysfunction that varies with the particular GAG deposited and the specific enzyme mutation(s) present. Cardiac involvement has been reported in all MPS syndromes and is a common and early feature, particularly for those with MPS I, II, and VI. Cardiac valve thickening, dysfunction (more severe for left-sided than for right-sided valves), and hypertrophy are commonly present; conduction abnormalities, coronary artery and other vascular involvement may also occur. Cardiac disease emerges silently and contributes significantly to early mortality.The clinical examination of individuals with MPS is often difficult due to physical and, sometimes, intellectual patient limitations. The absence of precordial murmurs does not exclude the presence of cardiac disease. Echocardiography and electrocardiography are key diagnostic techniques for evaluation of valves, ventricular dimensions and function, which are recommended on a regular basis. The optimal technique for evaluation of coronary artery involvement remains unsettled.Standard medical and surgical techniques can be modified for MPS patients, and systemic therapies such as hematopoietic stem cell transplantation and enzyme replacement therapy (ERT) may alter overall disease progression with regression of ventricular hypertrophy and maintenance of ventricular function. Cardiac valve disease is usually unresponsive or, at best, stabilized, although ERT within the first few months of life may prevent valve involvement, a fact that emphasizes the importance of early diagnosis and treatment in MPS.

We analyzed placental tissue in one fetus with MPS II (iduronate sulphatase deficiency) and another with MPS VI (arylsulfatase B deficiency). Both were diagnosed prenatally, but families decided to continue pregnancies and placentas were collected at birth. We were able to demonstrate early storage of GAGs in both diseases by GAG measurement and microscopy analysis. Our results suggest that some alterations related to MPS storage, although not pronounced, may be observed in placental tissue of patients affected by MPS II and MPS VI., (Copyright © 2011 Elsevier Inc. All rights reserved.)

A cross-sectional survey in individuals affected with the lysosomal storage disease Mucopolysaccharidosis VI (MPS VI) was conducted to establish demographics, urinary glycosaminoglycan (GAG) levels, and clinical progression of the disease. The survey evaluated 121 bona fide MPS VI-affected individuals over the age of 4 years from 15 countries across the Americas, Europe, and Australasia representing greater than 10% of the estimated world prevalence of the disease. A medical history, complete physical exam, urinary GAG determination, and assessment of several clinical measures related to physical endurance, pulmonary function, joint range of motion, strength, and quality of life were completed for each participant. Although a wide variation in clinical presentation was observed, several general findings were obtained reflecting progression of the disease. Impaired physical endurance, as measured by the distance achieved in a 6-min walk, could be demonstrated across all age groups of MPS VI-affected individuals. High urinary GAG values (>200 mug/mg creatinine) were associated with an accelerated clinical course comprised of age-adjusted short stature and low body weight, impaired endurance, compromised pulmonary function, and reduced joint range of motion. An unexpected result was the predominance of urinary GAG values <100 mug/mg creatinine for those participants over the age of 20 years. Pending the collection of longitudinal data, these results suggest that urinary GAG levels predict clinical morbidity, and longer-term survival is associated with urinary GAG levels below a threshold of 100 mug/mg creatinine., ((c) 2005 Wiley-Liss, Inc.)

Mucopolysaccharidoses (MPSs) are rare inherited lysosomal storage diseases (LSDs) caused by deficient activity in one of the enzymes responsible for glycosaminoglycans lysosomal degradation. MPS II is caused by pathogenic mutations in the IDS gene, leading to deficient activity of the enzyme iduronate-2-sulfatase, which causes dermatan and heparan sulfate storage in the lysosomes. In MPS VI, there is dermatan sulfate lysosomal accumulation due to pathogenic mutations in the ARSB gene, leading to arylsulfatase B deficiency. Alterations in the immune system of MPS mouse models have already been described, but data concerning MPSs patients is still scarce. Herein, we study different leukocyte populations in MPS II and VI disease patients. MPS VI, but not MPS II patients, have a decrease percentage of natural killer (NK) cells and monocytes when compared with controls. No alterations were identified in the percentage of T, invariant NKT, and B cells in both groups of MPS disease patients. However, we discovered alterations in the naïve versus memory status of both helper and cytotoxic T cells in MPS VI disease patients compared to control group. Indeed, MPS VI disease patients have a higher frequency of naïve T cells and, consequently, lower memory T cell frequency than control subjects. Altogether, these results reveal MPS VI disease-specific alterations in some leukocyte populations, suggesting that the type of substrate accumulated and/or enzyme deficiency in the lysosome may have a particular effect on the normal cellular composition of the immune system. [ABSTRACT FROM AUTHOR]

Background: Mucopolysaccharidoses (MPS) are a group of rare illnesses caused by deficient activity of enzymes required for degradation of glycosaminoglycans (GAGs). Each type of MPS is caused by mutations in one of the genes that encode the 11 acid hydrolases involved in this degradation process, which are present in the lysosomes. Progressive accumulation of GAGs in the lysosomes result in cellular dysfunction and multisystemic clinical signs, with consequent decrease in quality of life and lifespan of the affected patients. The objective of the present work is to report a case of MPS type I in a dog. Case: A mixed-breed male dog of approximately 2-month-old weighing 2.5 kg was referred to Hospital Veterinário de Uberaba with a distended abdomen. At the clinical examination, the patient exhibited a regular nutritional status, pale mucous membranes, 7% dehydration, an arterial pulse rate of 120 beats per minute, a respiratory rate of 40 breaths per minute, and a heart rate of 120 beats per minute. There were increased abdominal volume and tension, and hepatosplenomegaly. The abdominal percussion exam produced a dull tone. Additional findings included muscular atrophy, increased volume in the metaphyseal areas of the thoracic and pelvic limbs, valgus limb deformity in the thoracic limbs, and instability of the hip joint. Radiographic examination revealed a series of bone alterations such as reduced vertebral bodies, a generalized decrease in radiopacity, thin cortical areas in long bones, narrowing of the pelvic canal, and marked deformation and irregularities in acetabular and epiphyseal (both proximal and distal) areas of the femurs and tibias. Ankylosis of the tibiotarsal and tarsometatarsal joints was also observed. There was also loss of trabecular structure and irregularities on the surfaces of all epiphyses of the bones, epiphyseal lines markedly open, and bones that were shorter and thicker than normal. The suspected diagnoses were pseudoachondroplasia and mucopolysaccharidosis. In view of the clinical and radiographic findings, tests were performed to investigate the clinical suspicion of MPS. Consequently, qualitative and quantitative tests of GAGs in the urine, as well as a blood enzymatic essay, were requested; results confirmed the diagnosis of MPS type I. Intensive treatment allowed the patient to reach adulthood. Whenever new clinical signs emerged, they were treated palliatively. As the disease became more severe, the patient died at the age of 3 years. Discussion: Mucopolysaccharidosis type I is a rare disease that exhibits variable clinical signs and for which there is no specific treatment in dogs; these characteristics hinder diagnosis and treatment of patients as the one described in this report. The major clinical signs observed in this case are in agreement with those reported in the literature, according to which the disease can cause severe alterations such as bone defects, increased volume and deformities in the joints of the limbs, corneal opacity, and enlargement of abdominal organs such as the liver and spleen. In considering diagnostic methods for MPS, the main screening test is quantification of GAGs in the urine. The confirmatory test for MPS consists of analysis of the activity of specific lysosomal enzymes in a blood sample; this test allowed the establishment of a diagnosis in this case. Enzyme replacement therapy, in which a recombinant enzyme is used, have yielded good results in humans and dogs. However, this treatment does not cure the disease - it only attenuates the clinical signs and enables the patient to reach adulthood. Access to enzyme replacement therapy was not possible in the present case. As a conclusion, MPS should be included in the differential diagnosis of developmental diseases in puppies. This highlights the importance of further studies and reports on this disease. [ABSTRACT FROM AUTHOR]

Objective: Mucopolysaccharidosis (MPS) VI is a rare disorder caused by an autosomal recessive mutation in the short arm of chromosome 5 (5q12-13) leading to an N-acetylgalactosamine-sulfatase lysosomal enzyme deficiency and numerous systemic clinical changes. The oral and maxillofacial complex may exhibit tooth eruption anomalies, macroglossia, gingival hypertrophy, mouth breathing, increased lower facial height, open bite, retrognathia, and progressive TMJ arthrosis. This report describes craniofacial growth changes in two MPS VI patients, sisters and daughters of outbred parents, who were longitudinally monitored from 11 to 15 years of age.Study Design: Skull lateral teleradiography and cephalometric tracings were performed. The measurements were assessed in the anteroposterior and vertical directions based on protocols by McNamara and Usp/Unicamp and compared to the normal reported ranges.Results: A similar skeletal class III malocclusion was observed in both patients. The jaw was retruded, the anterior skull base decreased, and the mandibular body was normal or larger than normal. The vertical growth direction differed between the patients; one was hyperdivergent, while the other was hypodivergent.Conclusions: By understanding the craniofacial growth changes in MPS VI patients, new treatment options may be developed for affected patients. [ABSTRACT FROM AUTHOR]

Mucopolysaccharidosis type I (MPS I) is caused by a deficiency of α-L-iduronidase (IDUA), resulting in accumulation of glycosaminoglycans (GAG) in lysosomes. Microencapsulation of recombinant cells is a promising gene/cell therapy approach that could overcome the limitations of the current available treatments. In the present study we produced alginate-poly-L-lysine-alginate (APA) microcapsules containing recombinant cells overexpressing IDUA, which were implanted in the subcutaneous space of MPS I mice in order to evaluate their potential effect as a treatment for this disease. APA microcapsules enclosing genetically modified Baby Hamster Kidney cells overexpressing IDUA were produced and implanted in the subcutaneous space of 4-month-old MPS I mice ( Idua ). Treatment was performed using two cell concentrations: 8.3 × 10 and 8.3 × 10 cells/mL. Untreated MPS I and normal mice were used as controls. Microcapsules were retrieved and analyzed after treatment. Increased IDUA in the liver, kidney and heart was detected 24 h postimplantation. After 120 days, higher IDUA activity was detected in the liver, kidney and heart, in both groups, whereas GAG accumulation was reduced only in the high cell concentration group. Microcapsules analysis showed blood vessels around them, as well as inflammatory cells and a fibrotic layer. Microencapsulated cells were able to ameliorate some aspects of the disease, indicating their potential as a treatment. To achieve better performance of the microcapsules, improvements such as the modulation of inflammatory response are suggested. Graphical Abstract: [ABSTRACT FROM AUTHOR]

Mucopolysaccharidosis type III (MPS III) is characterized by progressive neurological deterioration, behavioral abnormalities, a relatively mild somatic phenotype, and early mortality. Because of the paucity of somatic manifestations and the rarity of the disease, early diagnosis is often difficult. Therapy targeting the underlying disease pathophysiology may offer the greatest clinical benefit when started prior to the onset of significant neurologic sequelae. Here we review current practices in the laboratory diagnosis of MPS III in order to facilitate earlier patient identification and diagnosis. When clinical suspicion of MPS III arises, the first step is to order a quantitative assay that screens urine for the presence of glycosaminoglycan biomarkers using a spectrophotometric compound (e.g., dimethylmethylene blue). We recommend testing all patients with developmental delay and/or behavioral abnormalities as part of the diagnostic work-up because quantitative urine screening is inexpensive and non-invasive. Semi-quantitative urine screening assays using cationic dyes on filter paper (e.g., spot tests) have relatively high rates of false-positives and false-negatives and are obsolete. Of note, a negative urinary glycosaminoglycan assay does not necessarily rule out MPS because, in some patients, an overlap in excretion levels with healthy controls may occur. All urine samples that test positive for glycosaminoglycans with a quantitative assay should be confirmed by electrophoresis, thin layer chromatography, or tandem mass spectrometry, which further improves the sensitivity and specificity. The gold standard for diagnosis remains the enzyme activity assay in cultured skin fibroblasts, leukocytes, plasma, or serum, which can be used as a first-line diagnostic test in some regions. Molecular genetic analysis should be offered to all families of patients to allow genetic counseling for informed family planning. For a small number of variants, genotype–phenotype correlations are available and can offer prognostic value. Prenatal testing via enzyme activity assay in chorionic villi or amniotic fluid cells is available at a limited number of centers worldwide, but whenever possible, a molecular genetic analysis is preferred for prenatal diagnosis. To conclude, we discuss the development of newborn screening assays in dried blood spots and high-throughput methods for sequencing the protein-coding regions of the genome (whole exome sequencing) and their relevance to future changes in the MPS III diagnostic landscape. [ABSTRACT FROM AUTHOR]

Mucopolysaccharidosis I (MPS I) is an autosomal recessive lysosomal storage disease due to deficient α-L-iduronidase (IDUA) activity. It results in the accumulation of the glycosaminoglycans (GAGs) heparan and dermatan sulfate and leads to several clinical manifestations. Available treatments are limited in their efficacy to treat some aspects of the disease. Thus, new approaches have been studied for the treatment of MPS I. Here, we tested the ability of recombinant baby hamster kidney cells transfected with human IDUA cDNA in correcting skin fibroblasts from MPS I patients in vitro. Our results showed an increase in IDUA activity in MPS I fibroblasts after 15, 30 and 45 days of coculture with the capsules. Cytological analysis showed a marked reduction in GAG storage within MPS I cells. Enzyme uptake by the fibroblasts was blocked in a dose-dependent manner with mannose-6-phosphate (M6P), indicating that cells use the M6P receptor to internalize the recombinant enzyme. Capsules were effective in correcting MPS I cells even after a 12-month period of cryopreservation. Taken together, our results indicate that cell encapsulation is a potential approach for treatment of MPS I. This approach becomes particularly interesting as a complementary approach, since the capsules could be implanted in sites which current treatments available are not able to reach. Future studies will focus on the efficacy of this approach in vivo. Copyright © 2011 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Mucopolysaccharidosis type II (MPS II or Hunter syndrome) is a rare, inherited disorder caused by deficiency of the lysosomal enzyme iduronate-2-sulfatase. As a result of this deficiency, glycosaminoglycans accumulate in lysosomes in many tissues, leading to progressive multisystemic disease. The cardiopulmonary and neurological problems associated with MPS II have received considerable attention. Orthopedic manifestations are common but not as well characterized. This study aimed to characterize the prevalence and severity of orthopedic manifestations of MPS II and to determine the relationship of these signs and symptoms with cardiovascular, pulmonary and central nervous system involvement. Orthopedic manifestations of MPS II were studied using cross-sectional data from the Hunter Outcome Survey (HOS). The HOS is a global, physician-led, multicenter observational database that collects information on the natural history of MPS II and the long-term safety and effectiveness of enzyme replacement therapy. As of January 2009, the HOS contained baseline data on joint range of motion in 124 males with MPS II. In total, 79% of patients had skeletal manifestations (median onset, 3.5 years) and 25% had abnormal gait (median onset, 5.4 years). Joint range of motion was restricted for all joints assessed (elbow, shoulder, hip, knee and ankle). Extension was the most severely affected movement: the exception to this was the shoulder. Surgery for orthopedic problems was rare. The presence of orthopedic manifestations was associated with the presence of central nervous system and pulmonary involvement, but not so clearly with cardiovascular involvement. Orthopedic interventions should be considered on an individual-patient basis. Although some orthopedic manifestations associated with MPS II may be managed routinely, a good knowledge of other concurrent organ system involvement is essential. A multidisciplinary approach is required. [ABSTRACT FROM AUTHOR]

Abstract: Recombinant human α-l-iduronidase (Aldurazyme®, laronidase) is approved as an enzyme replacement therapy to treat the lysosomal storage disorder, mucopolysaccharidosis type I (MPS I) at a dose of 0.58mg/kg by once-weekly intravenous infusion. To assess whether alternate dosing regimens might provide a better reduction in lysosomal storage, a 26-week, randomized, open-label, multinational dose-optimization trial was conducted. The pharmacodynamic effect and safety of the approved laronidase dose was compared to three alternative regimens (1.2mg/kg every 2 weeks; 1.2mg/kg every week; 1.8mg/kg every 2 weeks) among 33 MPS I patients. The four treatment regimens showed no significant differences in the reduction of urinary glycosaminoglycan excretion or liver volume. Laronidase had an acceptable safety profile in all dose regimen groups. Infusion-associated reactions were the most common drug-related adverse events across dose regimens (by patient incidence), and included pyrexia (21%), vomiting (15%), rash (15%), and urticaria (12%). Patients in the approved dose group had the lowest incidence of drug-related adverse events (38% vs. 63–75%) and infusion-associated reactions (25% vs. 25–63%). There was one death: a patient with acute bronchitis died of respiratory failure 6h after completing the first laronidase infusion. The approved 0.58mg/kg/week laronidase dose regimen provided near-maximal reductions in glycosaminoglycan storage and the best benefit-to-risk ratio. The 1.2mg/kg every 2 weeks regimen may be an acceptable alternative for patients with difficulty receiving weekly infusions, but the long-term effects of this regimen are unknown. [Copyright &y& Elsevier]

Abstract: The objective of this study was to evaluate the long-term clinical benefits and safety of recombinant human arylsulfatase B (rhASB) treatment of mucopolysaccharidosis type VI (MPS VI: Maroteaux-Lamy syndrome), a lysosomal storage disease. Fifty-six patients derived from 3 clinical studies were followed in open-label extension studies for a total period of 97–260 Weeks. All patients received weekly infusions of rhASB at 1mg/kg. Efficacy was evaluated by (1) distance walked in a 12-minute walk test (12MWT) or 6-minute walk test (6MWT), (2) stairs climbed in the 3-minute stair climb (3MSC), and (3) reduction in urinary glycosaminoglycans (GAG). Safety was evaluated by compliance, adverse event (AE) reporting and adherence to treatment. Results: A significant reduction in urinary GAG (71–79%) was sustained. For the 12MWT, subjects in Phase 2 showed improvement of 255±191m (mean±SD) at Week 144; those in Phase 3 Extension demonstrated improvement from study baseline of 183±26m (mean± SE) in the rhASB/rhASB group at Week 96 and from treatment baseline (Week 24) of 117±25m in the placebo/rhASB group. The Phase 1/2 6MWT and the 3MSC from Phase 2 and 3 also showed sustained improvements through the final study measurements. Compliance was 98% overall. Only 560 of 4121 reported AEs (14%) were related to treatment with only 10 of 560 (2%) described as severe. Conclusion: rhASB treatment up to 5 years results in sustained improvements in endurance and has an acceptable safety profile. [Copyright &y& Elsevier]

Hunter syndrome is a rare genetic lysosomal storage disease that is caused by a deficiency, or absence, of iduronate-2-sulphatase, an enzyme needed to break down specific glycosaminoglycans (GAGs). As a result, GAGs build up in various tissues throughout the body leading to adverse neurological and non-neurological effects. This literature review focuses on the neurological findings. Although few magnetic resonance imaging studies have been conducted, those done have shown that patients with Hunter syndrome generally exhibit brain atrophy, enlarged periventricular spaces and ventriculomegaly. Similar findings have been reported in other mucopolysaccharide disorders. Enzyme replacement therapy is a novel treatment which has had success in treating peripheral disease in mice and humans. Conclusion: Future studies should focus on how structural and chemical signatures in the brain of Hunter patients are altered before and after enzyme replacement therapy, and how those alterations correlate with clinical outcome. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

Abstract Mucopolysaccharidosis type I (MPS I) is a multisystemic disorder caused by the deficiency of alpha-L-iduronidase (IDUA) that leads to intracellular accumulation of glycosaminoglycans (GAG). In the present study we aimed to use cationic liposomes carrying the CRISPR/Cas9 plasmid and a donor vector for in vitro and in vivo MPS I gene editing, and compare to treatment with naked plasmids. The liposomal formulation was prepared by microfluidization. Complexes were obtained by the addition of DNA at +4/−1 charge ratio. The overall results showed complexes of about 110 nm, with positive zeta potential of +30 mV. The incubation of the complexes with fibroblasts from MPS I patients led to a significant increase in IDUA activity and reduction of lysosomal abnormalities. Hydrodynamic injection of the liposomal complex in newborn MPS I mice led to a significant increase in serum IDUA levels for up to six months. The biodistribution of complexes after hydrodynamic injection was markedly detected in the lungs and heart, corroborating the results of increased IDUA activity and decreased GAG storage especially in these tissues, while the group that received the naked plasmids presented increased enzyme activity especially in the liver. Furthermore, animals treated with the liposomal formulation presented improvement in cardiovascular parameters, one of the main causes of death observed in MPS I patients. We conclude that the IDUA production in multiple organs had a significant beneficial effect on the characteristics of MPS I disease, which may bring hope to gene therapy of Hurler patients. Graphical abstract Unlabelled Image Highlights • Liposomes are efficient carriers for CRISPR/Cas9 system. • Liposomal CRISPR/Cas9 complexes showed efficient gene editing of MPS I fibroblasts. • Treatment of newborn MPS I mice promoted high serum IDUA levels for up to 6 months. • Treatment with liposomal complexes was more efficient than Naked CRISPR/Cas9. • Liposomal CRISPR/Cas9-treated mice improved GAG accumulation and cardiac function. [ABSTRACT FROM AUTHOR]

Measurement of enzymatic activity in newborn dried blood spots (DBS) is the preferred first-tier method in newborn screening (NBS) for mucopolysaccharidoses (MPSs). Our previous publications on glycosaminoglycan (GAG) biomarker levels in DBS for mucopolysaccharidosis type 1 (MPS-I) and MPS-II demonstrated that second-tier GAG biomarker analysis can dramatically reduce the false positive rate in NBS. In the present study, we evaluate two methods for measuring GAG biomarkers in seven MPS types and GM1 gangliosidosis. We obtained newborn DBS from patients with MPS-IIIA-D, -IVA, -VI, -VII, and GM1 gangliosidosis. These samples were analyzed via two GAG mass spectrometry methods: (1) The internal disaccharide biomarker method; (2) The endogenous non-reducing end (NRE) biomarker method. This study supports the use of second-tier GAG analysis of newborn DBS by the endogenous NRE biomarker method, as part of NBS to reduce the false positive rate. [ABSTRACT FROM AUTHOR]

The mucopolysaccharidosis (MPS) disorders are a group of lysosomal storage diseases caused by lysosomal enzyme deficits that lead to glycosaminoglycan accumulation, affecting various tissues throughout the body based on the specific enzyme deficiency. These disorders are characterized by their progressive nature and a variety of somatic manifestations and neurological symptoms. There are established treatments for some MPS disorders, but these mostly alleviate somatic and non-neurological symptoms and do not cure the disease. Patients with MPS I, II, III, and VII can present with neurological manifestations such as neurocognitive decline and behavioral problems. Treatment of these neurological manifestations remains challenging due to the blood-brain barrier (BBB) that limits delivery of therapeutic agents to the central nervous system (CNS). New therapies that circumvent this barrier and target brain disease in MPS are currently under development. They primarily focus on facilitating penetration of drugs through the BBB, delivery of recombinant enzyme to the brain by gene therapy, or direct CNS administration. This review summarizes existing and potential future treatment approaches that target brain disease in MPS. The information in this review is based on current literature and presentations and discussions during a closed meeting by an international group of experts with extensive experience in managing and treating MPS. [ABSTRACT FROM AUTHOR]

There is limited information regarding the long-term outcomes of hematopoietic stem cell transplantation (HSCT) for mucopolysaccharidosis II (MPS II). In this study, clinical, biochemical, and radiologic findings were assessed in patients who underwent HSCT and/or enzyme replacement therapy (ERT). Demographic data for 146 HSCT patients were collected from 27 new cases and 119 published cases and were compared with 51 ERT and 15 untreated cases. Glycosaminoglycan (GAG) levels were analyzed by liquid chromatography tandem mass spectrometry in blood samples from HSCT, ERT, and untreated patients as well as age-matched controls. Long-term magnetic resonance imaging (MRI) findings were investigated in 13 treated patients (6 ERT and 7 HSCT). Mean age at HSCT was 5.5 years (range, 2 to 21.4 years) in new patients and 5.5 years (range, 10 months to 19.8 years) in published cases. None of the 27 new patients died as a direct result of the HSCT procedure. Graft-versus-host disease occurred in 8 (9%) out of 85 published cases, and 9 (8%) patients died from transplantation-associated complications. Most HSCT patients showed greater improvement in somatic features, joint movements, and activity of daily living than the ERT patients. GAG levels in blood were significantly reduced by ERT and levels were even lower after HSCT. HSCT patients showed either improvement or no progression of abnormal findings in brain MRI while abnormal findings became more extensive after ERT. HSCT seems to be more effective than ERT for MPS II in a wide range of disease manifestations and could be considered as a treatment option for this condition. [ABSTRACT FROM AUTHOR]

Mucopolysaccharidosis type I (MPS I) is a lysosomal disorder characterized by a deficiency of alpha-L-iduronidase and storage of undegraded glycosaminoglycans (GAGs). Clinical findings of the disease include heart failure, and patients often need valve replacement. It has been shown that, later in life, MPS I mice develop those abnormalities, but to date, there have not been studies on the progression and pathogenesis of the disease. Therefore, in the present study, we evaluated heart function in normal and MPS I male mice from 2 to 8 months of age. Echocardiographic analysis showed left ventricular enlargement with progressive reduction in ejection fraction, fractional area change, and left ventricular fractional shortening in the MPS I hearts at 6 and 8 months of age and a reduction in acceleration time/ejection time ratio of the pulmonary artery starting at 6 months of age, which suggests pulmonary vascular resistance. Histological and biochemical analysis confirmed progressive GAG storage from 2 months of age and onwards in the myocardium and heart valves, which had also increased in thickness. Additionally, macrophages were present in the MPS I heart tissue. Collagen content was reduced in the MPS I mouse valves. Cathepsin B, an enzyme that is known to be able to degrade collagen and is involved in heart dilatation, displayed a marked elevation in activity in the MPS I mice and could be responsible for the heart dilatation and valves alterations observed. Our results suggest that the MPS I mice have progressive heart failure and valve disease, which may be caused by cathepsin B overexpression. [ABSTRACT FROM AUTHOR]

Mucopolysaccharidosis type IVA (MPS IVA) is an inborn error of glycosaminoglycan (GAG) catabolism due to the deficient activity of N-acetylgalactosamine-6-sulfate sulfatase that leads to accumulation of the keratan sulfate and chondroitin 6-sulfate in body fluids and in lysosomes. The pathophysiology of this lysosomal storage disorder is not completely understood. The aim of this study was to investigate oxidative stress parameters, pro-inflammatory cytokine and GAG levels in MPS IVA patients. We analyzed urine and blood samples from patients under ERT ( n = 17) and healthy age-matched controls ( n = 10–15). Patients presented a reduction of antioxidant defense levels, assessed by a decrease in glutathione content and by an increase in superoxide dismutase activity in erythrocytes. Concerning lipid and protein damage, it was verified increased urine isoprostanes and di-tyrosine levels and decreased plasma sulfhydryl groups in MPS IVA patients compared to controls. MPS IVA patients showed higher DNA damage than control group and this damage had an oxidative origin in both pyrimidine and purine bases. Interleukin 6 was increased in patients and presented an inverse correlation with GSH levels, showing a possible link between inflammation and oxidative stress in MPS IVA disease. The data presented suggest that pro-inflammatory and pro-oxidant states occur in MPS IVA patients even under ERT. Taking these results into account, supplementation of antioxidants in combination with ERT can be a tentative therapeutic approach with the purpose of improving the patient's quality of life. To the best of our knowledge, this is the first study relating MPS IVA patients with oxidative stress. [ABSTRACT FROM AUTHOR]

Patients with mucopolysaccharidoses (MPS) have accumulation of glycosaminoglycans in multiple tissues which may cause coarse facial features, mental retardation, recurrent ear and nose infections, inguinal and umbilical hernias, hepatosplenomegaly, and skeletal deformities. Clinical features related to bone lesions may include marked short stature, cervical stenosis, pectus carinatum, small lungs, joint rigidity (but laxity for MPS IV), kyphoscoliosis, lumbar gibbus, and genu valgum. Patients with MPS are often wheelchair-bound and physical handicaps increase with age as a result of progressive skeletal dysplasia, abnormal joint mobility, and osteoarthritis, leading to 1) stenosis of the upper cervical region, 2) restrictive small lung, 3) hip dysplasia, 4) restriction of joint movement, and 5) surgical complications. Patients often need multiple orthopedic procedures including cervical decompression and fusion, carpal tunnel release, hip reconstruction and replacement, and femoral or tibial osteotomy through their lifetime. Current measures to intervene in bone disease progression are not perfect and palliative, and improved therapies are urgently required. Enzyme replacement therapy (ERT), hematopoietic stem cell transplantation (HSCT), and gene therapy are available or in development for some types of MPS. Delivery of sufficient enzyme to bone, especially avascular cartilage, to prevent or ameliorate the devastating skeletal dysplasias remains an unmet challenge. The use of an anti-inflammatory drug is also under clinical study. Therapies should start at a very early stage prior to irreversible bone lesion, and damage since the severity of skeletal dysplasia is associated with level of activity during daily life. This review illustrates a current overview of therapies and their impact for bone lesions in MPS including ERT, HSCT, gene therapy, and anti-inflammatory drugs. [ABSTRACT FROM AUTHOR]

Mucopolysaccharidoses (MPS) are caused by deficiency of one of a group of specific lysosomal enzymes, resulting in excessive accumulation of glycosaminoglycans (GAGs). We previously developed GAG assay methods using liquid chromatography tandem mass spectrometry (LC–MS/MS); however, it takes 4–5 min per sample for analysis. For the large numbers of samples in a screening program, a more rapid process is desirable. The automated high-throughput mass spectrometry (HT-MS/MS) system (RapidFire) integrates a solid phase extraction robot to concentrate and desalt samples prior to direction into the MS/MS without chromatographic separation; thereby allowing each sample to be processed within 10 s (enabling screening of more than one million samples per year). The aim of this study was to develop a higher throughput system to assay heparan sulfate (HS) using HT-MS/MS, and to compare its reproducibility, sensitivity and specificity with conventional LC–MS/MS. HS levels were measured in the blood (plasma and serum) from control subjects and patients with MPS II, III, or IV and in dried blood spots (DBS) from newborn controls and patients with MPS I, II, or III. Results obtained from HT-MS/MS showed 1) that there was a strong correlation of levels of disaccharides derived from HS in the blood, between those calculated using conventional LC–MS/MS and HT-MS/MS, 2) that levels of HS in the blood were significantly elevated in patients with MPS II and III, but not in MPS IVA, 3) that the level of HS in patients with a severe form of MPS II was higher than that in an attenuated form, 4) that reduction of blood HS level was observed in MPS II patients treated with enzyme replacement therapy or hematopoietic stem cell transplantation, and 5) that levels of HS in newborn DBS were elevated in patients with MPS I, II or III, compared to those of control newborns. In conclusion, HT-MS/MS provides much higher throughput than LC–MS/MS-based methods with similar sensitivity and specificity in an HS assay, indicating that HT-MS/MS may be feasible for diagnosis, monitoring, and newborn screening of MPS. [ABSTRACT FROM AUTHOR]

Abstract: Mucopolysaccharidosis VII (MPS VII) is due to the deficient activity of β-glucuronidase (GUSB) and results in the accumulation of glycosaminoglycans (GAGs) in lysosomes and multisystemic disease with cardiovascular manifestations. The goal here was to determine the pathogenesis of mitral valve (MV) disease in MPS VII dogs. Untreated MPS VII dogs had a marked reduction in the histochemical signal for structurally-intact collagen in the MV at 6months of age, when mitral regurgitation had developed. Electron microscopy demonstrated that collagen fibrils were of normal diameter, but failed to align into large parallel arrays. mRNA analysis demonstrated a modest reduction in the expression of genes that encode collagen or collagen-associated proteins such as the proteoglycan decorin which helps collagen fibrils assemble, and a marked increase for genes that encode proteases such as cathepsins. Indeed, enzyme activity for cathepsin B (CtsB) was 19-fold normal. MPS VII dogs that received neonatal intravenous injection of a gamma retroviral vector had an improved signal for structurally-intact collagen, and reduced CtsB activity relative to that seen in untreated MPS VII dogs. We conclude that MR in untreated MPS VII dogs was likely due to abnormalities in MV collagen structure. This could be due to upregulation of enzymes that degrade collagen or collagen-associated proteins, to the accumulation of GAGs that compete with proteoglycans such as decorin for binding to collagen, or to other causes. Further delineation of the etiology of abnormal collagen structure may lead to treatments that improve biomechanical properties of the MV and other tissues. [Copyright &y& Elsevier]

Abstract: Hunter disease or mucopolysaccharidosis type II (MPS II) is an X-linked recessive lysosomal disorder caused by the deficit of the enzyme iduronate-2-sulfatase (IDS), involved in the catabolism of the glycosaminoglycans heparan and dermatan sulfate. Our aim was to search for molecular defects in the promoter region of the IDS gene in patients with previous biochemical diagnosis of MPS II and after we sequenced the whole IDS coding region and the exon/intron boundaries without detecting any pathogenic mutations. Screening of the promoter region of four patients detected in two of them a 178bp deletion and in the other two a single nucleotide substitution 818bp upstream of the coding region. The latter had never been described before in MPS II patients and it turned out to be a polymorphism. Our experience suggests that MPS II patients with no mutations detected in the IDS coding region should be screened in the promoter region of the gene. Findings will hopefully help to clarify the relationship between genotype and phenotype and will be useful for the correct molecular diagnosis of Hunter patients and the identification of female carriers, the latter particularly important for genetic counseling. [Copyright &y& Elsevier]

Abstract: Mucopolysaccharidosis type VII (MPS VII) is characterized by deficient β-glucuronidase (GUSB) activity, which leads to accumulation of chondroitin, heparan and dermatan sulfate glycosaminoglycans (GAGs), and multisystemic disease. MPS VII patients can develop kypho-scoliotic deformity and spinal cord compression due to disease of intervertebral disks, vertebral bodies, and associated tissues. We have previously demonstrated in MPS VII dogs that intervertebral disks degenerate, vertebral bodies have irregular surfaces, and vertebral body epiphyses have reduced calcification, but the pathophysiological mechanisms underlying these changes are unclear. We hypothesized that some of these manifestations could be due to upregulation of destructive proteases, possibly via the binding of GAGs to Toll-like receptor 4 (TLR4), as has been proposed for other tissues in MPS models. In this study, the annulus fibrosus of the intervertebral disk of 6-month-old MPS VII dogs had cathepsin B and K activities that were 117- and 2-fold normal, respectively, which were associated with elevations in mRNA levels for these cathepsins as well as TLR4. The epiphyses of MPS VII dogs had a marked elevation in mRNA for the cartilage-associated gene collagen II, consistent with a developmental delay in the conversion of the cartilage to bone in this region. The spine obtained at autopsy from a young man with MPS VII exhibited similar increased cartilage in the vertebral bodies adjacent to the end plates, disorganization of the intervertebral disks, and irregular vertebral end plate morphology. These data suggest that the pathogenesis of destructive changes in the spine in MPS VII may involve upregulation of cathepsins. Inhibition of destructive proteases, such as cathepsins, might reduce spine disease in patients with MPS VII or related disorders. [Copyright &y& Elsevier]

Abstract: Mucopolysaccharidosis (MPS) type I (Hurler syndrome) is a lysosomal storage disorder characterized by deficiency of alpha-l-iduronidase (IDUA), intracellular storage of glycosaminoglycans (GAGs) and progressive neurological pathology. The MPS I mouse model provides an opportunity to study the pathophysiology of this disorder and to determine the efficacy of novel therapies. Previous work has demonstrated a series of abnormalities in MPS I mice behavior, but so far some important brain functions have not been addressed. Therefore, in the present study we aimed to determine if MPS I mice have motor abnormalities, and at what age they become detectable. MPS I and normal male mice from 2 to 8 months of age were tested in open-field for locomotor activity, hindlimb gait analysis and hang wire performance. We were able to detect a progressive reduction in the crossings and rearings in the open field test and in the hang wire test in MPS I mice from 4 months, as well as a reduction in the gait length at 8 months. Histological examination of 8-month old mice cortex and cerebellum revealed storage of GAGs in Purkinje cells and neuroinflammation, evidenced by GFAP immunostaining. However TUNEL staining was negative, suggesting that death does not occur. Our findings suggest that MPS I mice have a progressive motor dysfunction, which is not caused by loss of neuron cells but might be related to a neuroinflammatory process. [Copyright &y& Elsevier]

Abstract: Background: Mucopolysaccharidosis IVA (MPS IVA), or Morquio Syndrome type A, is an autosomal recessive disease caused by deficiency of the lysosomal enzyme N-acetylgalactosamine-6-sulfatase (GALNS), resulting in excessive lysosomal storage of keratan sulfate in many tissues and organs. This accumulation causes a severe skeletal dysplasia with short stature, and affects the eye, heart and other organs, with many signs and symptoms. Morquio A syndrome is estimated to occur in 1 in 200,000 to 300,000 live births. Clinical trials with enzyme replacement therapy for this disease are in progress, and it is probable that the treatment, when available, would be more effective if started early. We describe an innovative fluorometric method for the assay of GALNS in dried blood spots (DBS). Methods: We used dried blood spots (DBS) as the enzyme source and compared it with leukocytes samples, having studied 25 MPS IVA patients and 54 healthy controls. We optimized the assay conditions, including incubation time and stability of DBS samples. To eppendorf type tubes containing a 3-mm diameter blood spot we added elution liquid and substrate solution. After 2 different incubations at 37°C, the amount of hydrolyzed product was compared with a calibrator to allow the quantification of the enzyme activity. Results in DBS were compared to the ones obtained in leukocytes using the standard technique. Results: The fluorescent methodology was validated in our laboratory and the assay was found sensitive and specific, allowing reliable detection of MPS IVA patients. The use of DBS simplifies the collection and transport steps, and is especially useful for testing patients from more remote areas of large countries, and when samples need to cross country borders. Conclusion: This assay could be easily incorporated into the protocol of reference laboratories and play a role in the screening for MPS IVA, contributing to earlier detection of affected patients. [Copyright &y& Elsevier]

Abstract: Mucopolysaccharidosis type II (MPS II) is a lysosomal storage disorder caused by deficiency of the enzyme iduronate-2-sulfatase, responsible for the degradation of glycosaminoglycans dermatan and heparan sulfate. Once the generation of free radicals is involved in the pathogenesis of many diseases, including some inborn errors of metabolism, the aim of this study was to evaluate blood oxidative stress parameters in MPS II patients, before and during 6months of enzyme replacement therapy. We found significantly increased levels of malondialdehyde and carbonyl groups in plasma as well as erythrocyte catalase activity in patients before treatment compared to the control group. Plasma sulfhydryl group content and total antioxidant status were significantly reduced before treatment, while superoxide dismutase enzyme was not altered at this time when compared to controls. During enzyme replacement therapy, there was a significant reduction in levels of malondialdehyde when compared to pretreatment. Sulfhydryl groups were significantly increased until three months of treatment in MPS II patients in comparison to pretreatment. There were no significant alterations in plasma total antioxidant status and carbonyl groups as well as in catalase and superoxide dismutase activities during treatment in relation to pretreatment. The results indicate that MPS II patients are subject to lipid and protein oxidative damage and present reduction in non-enzymatic antioxidants, suggesting a possible involvement of free radicals in the pathophysiology of this disease. Also, the results may suggest that enzyme replacement therapy seems to protect against lipid peroxidation and protein damage in these patients. [Copyright &y& Elsevier]

Abstract: Mucopolysaccharidosis type II (MPS II) is an X-linked recessive disease caused by deficiency of the lysosomal enzyme iduronate-2-sulfatase, leading to progressive accumulation of glycosaminoglycans in nearly all cell types, tissues and organs. Enzyme replacement therapy reduces the storage of these substances in the lysosomes. Oxidative stress is related to the pathophysiology of many disorders, including inborn errors of metabolism. Oxidative damage to protein and lipid has been described in MPS types I and III. The aim of this study was to analyze DNA damage, as determined by the alkaline comet assay using silver staining, in peripheral leukocytes from MPS II patients before treatment and during the first six months of enzyme replacement therapy. We also correlated DNA damage with lipid and protein oxidative damages, analyzed by plasma malondialdehyde levels and carbonyl group content, respectively. We found a significant increase in lipid and protein damage in MPS II patients before treatment when compared to controls. Also, our results showed greater DNA damage in terms of damage index (DI) in pretreatment MPS II patients (DI=18.0±2.4) when compared to controls (DI=66.0±2.0). Enzyme replacement therapy led to a significant decrease in levels of malondialdehyde and DNA damage when compared to pretreatment, but did not reach control values. Significant positive correlations between DNA damage and malondialdehyde levels, as well as carbonyl group content, were observed. Our findings indicate that MPS II patients are subject to DNA damage and that enzyme replacement therapy is able to protect against this process. [Copyright &y& Elsevier]

Abstract: In mucopolysaccharidosis VI, or Maroteaux–Lamy syndrome, deficiency of N-acetylgalactosamine 4-sulfatase leads to storage of glycosaminoglycans (GAGs) and MPS VI patients often develop spinal cord compression during the course of the disease due to GAG storage within the cervical meninges, requiring neurosurgical intervention, as intravenous (IV) enzyme replacement therapy (ERT) is not expected to cross the blood–brain barrier. We report the use of intrathecal (IT) recombinant human N-acetylgalactosamine 4-sulfatase (arylsulfatase B, or ASB) in a MPS VI child with spinal cord compression whose parents initially refused the surgical treatment. Assessments were performed at baseline, with clinical, neurological and biochemical evaluations, urodynamic studies and MRI of the CNS. Changes on these parameters were evaluated after IT infusions of ASB administered monthly via lumbar puncture (LP) in a IV ERT naive patient. To our knowledge, this was the first MPS VI patient who received IT ERT. Despite significant urodynamic improvement and some neurological amelioration, the patient developed worsening of walking capacity. After IV ERT was started, the patient presented with a generalized hypotonia and a life-saving surgical fixation of the neck was then performed. The results observed on this MPS VI patient suggest that instability of the cervical vertebrae could be unmasked by IV ERT as joint storage is reduced, and the decrease in neck stiffness and stability could confound the expected improvement of SCC manifestations following IT ERT. The study of further patients, if possible in a clinical trial setting, is needed to evaluate the potential of a non-surgical IT ERT treatment of SCC for MPS VI. [Copyright &y& Elsevier]

The mucopolysaccharidoses (MPS) include 11 rare disorders caused by deficiency of specific lysosomal enzymes resulting in the accumulation of undegraded glycosaminoglycans (GAGs) and several clinical consequences. The combined incidence for all MPS subtypes is approximately 1:25,000 live births. Clusters of these diseases have been identified in areas with high consanguinity and/or founder effect associated to endogamy. The MPS Brazil Network, associated to the Brazilian Institute of Population Medical Genetics, identified several MPS clusters in Latin America. Three clusters were confirmed in Brazil, of MPS IIIC (state of Paraíba), MPS IVA (state of Paraíba) and MPS VI (state of Bahia). Two clusters were identified in Ecuador: MPS IIIB (state of Manabi) and MPS IVA (state of Pastaza). A cluster of MPS VI was also identified in the Dominican Republic. Other clusters are being investigated in Haiti (MPS VI), Panamá (MPS IVA), and Brazil (MPS IIIB, Minas Gerais state). Haplotype analyses are underway to better characterize mutation profiles, and results already available for the clusters of MPS IVA and MPS VI in Brazil indicate founder effects with common ancestors. As one example of the benefits of cluster identification, a newborn screening program for MPS VI was implemented in the Brazilian region in order to provide early identification and treatment of new patients. In conclusion, examples of MPS clusters were identified in Latin America and it is likely that several others are still unreported. We aim to properly identify and characterize MPS clusters in Latin America, to better understand how they were originated and to be able to offer targeted preventive and management measures to the affected communities (Acknowledgements to the MPS Brazil Network, especially to Fernanda Bender, Fernanda Bittencourt, Diana Rojás Malaga, Aline Nemetz Bochernitsan, Jurema de Mari, and Franciele Barbosa Trapp). [ABSTRACT FROM AUTHOR]