Park HD 1 , Ko AR 2 , Ki CS 1 , Lee SY 1 , Cho SY 2 , Kim SH 2 , Sohn YB 2 , Park SW 2 , Jin DK 2 1 Dept of Lab Med, Sungkyunkwan Univ, Seoul, Korea, Republic of 2 Dept of Pediatrics, Sungkyunkwan Univ, Seoul, Korea, Republic of Background: Mucopolysaccharidosis IVA (MPS IVA; OMIM #253000) is caused by the deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS). The GALNS gene encodes GALNS, a lysosomal enzyme involved in the catabolism of keratan and chondroitin sulfate. Herein, we examined biochemical and genetic aberrations from six Korean patients presenting with classic MPS IVA. Methods: GALNS activity was measured in peripheral blood leukocytes or skin fibroblasts. PCR-direct sequencing was performed to investigate the molecular defects of GALNS gene. We also investigated the mutational status for the family members of the patients as well as 50 healthy subjects. Results: The mean age of six female patients was 8.0±5.2 years (range: 2-17 years). Severe reduction of GALNS enzyme was found in all patients. Introduction: MPS IVA is characterized by deficient activity of Nacetyl-galactosamine6sulfatase (GALNS), causing excessive lysosomal storage of keratan sulfate (KS). Patients experience impaired endurance and respiratory function. Case Description: This 16 y/o male patient with genotype homozygous c.452>T (p.Pro151Leu) started ERT with weekly infusions of GALNS (BMN 110) on 10Jun2009. Medical history included aortic regurgitation, genu valgum, limited mobility and overall weakness. Baseline physical exam showed short stature, cardiac murmur, shortness of breath, and joint laxity. Before ERT, he could not sign the consent form, was completely non-ambulatory, and required full assistance for self-care and activities of daily living. During treatment, he became stronger, gaining ability to rise from wheelchair and use toilet without assistance. After 79 weeks, mobility, breathing, and strength improved. He could crawl on his knees, ascend 30 steps, and he was able to sign the consent form for the extension study. Screening urine KS of 24.5 ug/mg creatinine had decreased to 10.8 by Week 72. Conclusions: ERT is a potential new treatment option for MPS IVA, which is expected to reduce KS storage, leading to improvements in respiratory function and endurance. This patient's markedly improved strength and endurance during trial participation improved his daily function. Conflict of Interest declared. Background:Mucopolysaccharidosis IV A(MPS IVA) is a rare autosomal recessive disease caused by deficiency of the lysosomal enzyme Nacetylgalactosamine-6-sulfatase(GALNS),resulting in storage of keratansulfate in many tissues and organs.This accumulation causes a severe skeletal dysplasia with short stature,and affects the eye, heart and other organs. Clinical trials with ERT for this disease are in progress. We describe an innovative fluorometric method for the assay of GALNS in dried blood spots(DBS). Materials and Methods: We used DBS as the enzyme source and compared it with leukocytes,having studied 25 MPS IVA patients and 54 healthy controls.We optimized the assay conditions in DBS,including incubation time and stability on different storage temperatures and along time.Results in DBS were compared to the ones obtained in leucocytes using the standard technique. Results and conclusions: The described 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 transportation,and is especially useful for testing patients from more remote areas of large countries,and when samples need to cross country borders.We believe this assay could be easily incorporated by reference laboratories and play a role in the screening for MPS IVA, contributing to the earlier detection of affected patients. Mucopolysaccharidosis(MPS ) VI is caused by deficient arylsulfatase B. Albinism is characterized by the absence of pigment in the skin, hair, eyes due to production of melanin. Case: The patient was the first of consanguineous parents. She had sensorineural deafness, generalized albinism., coarse face at the age of eighteen months old. Dysostosis multiplex by skletal X-Ray, high excretion of mucopolysaccarides in the urine, reduced arylsulfatase B enzyme activity in leucocytes (73,75 nmol/hour/mgprotein (209±96)) confirmed the diagnosis of MPS VI. The other sulfatase enzyme levels were normal. ARSB gene sequencing revealed homozygous c.908 G>A change in exon 5. This variant, predicted to result in an amino acid change from glycine to glutamic acid (p.G303E), has neither been shown to be associated with MPS VI nor has it been listed in the Ensembl SNP database. Conclusion: MPS type VI is caused by mutations in the ARSB gene, located in chromosome 5 (5q13-5q14). Over 130 ARSB mutations have been reported, causing absent or reduced arylsulfatase B activity. On the other hand the gene accounting for oculocutaneous albinism type 4 is locatad in chromosome 5 (5p13.2). We speculated that, possible chromosomal rearrengements should be further investigated in this case because of this extremely rare co-existence. Mucopolysaccharidosis VI(MPS VI,or Maroteaux-Lamy syndrome)is caused by the deficiency of the lysosomal enzyme N-acetylgalactosamine 4-sulfatase (ARSB).This deficiency causes the storage of dermatan sulphate in tissues, leading to a progressive and severe bone dysplasia and to problems in many organs and systems.MPS VI is a very rare condition,which was a relatively high incidence(13 cases identified so far)in the county of Monte Santo (50,000 inhabitants),in Bahia state,Northeast region of Brazil, where a common mutation(H178L)was found in all cases.As MPS VI could be treated with ERT and as there are indications that a better outcome may be expected in early treated cases,a newborn screening program for MPS VI was set up on this specific area.To the program already in place for PKU,hypothyroidism and hemoglobin disorders,a screening for MPS VI was added by ARSB activity assay and by the detection of the common mutation, both performed on DBS. The standardization of the techniques of enzyme assay,DNA extraction and mutation detection were already completed and the test on newborn samples has started on January 1st,2011.The possibility of detecting carriers will help to calculate the expected frequency of this disease in the area, and will also be a tool to target genetic counseling to the more susceptible families. Mucopolysaccharidosis type VI or Maroteaux-Lamy syndrome is an autosomal recessive lysosomal storage disorder resulting from impaired function of the enzyme N-acetylgalactosamine-4-sulfatase (arylsulfatase B, ARSB). In this study, the mutation spectrum of ARSB gene in 19 Turkish patients from 15 different families diagnosed with MPS type VI was determined by using direct sequence analysis. In total, 6 different types of disease-causing mutations (c.1036delG, p.L321P, p.R160X, p.R191X, p. C192R and p.E390K) and 4 polymorphisms (p.V358M, p.V376M, IVS1-26 T>C, IVS5-28A>C) were identified in the patients with MPS type VI. One of the six mutations (p.E390K) is novel in Turkish patients. As an interesting result, the most frequent mutation in our screened cohort was p. L321P (c.961 T>C) with 53% frequency. Our findings are shown inconsistency with the knowledge of the literature, since p.L321P (c.961 T>C) is not one of the common mutation in Caucasians.The mutational analyses on ARSB gene will identify structure/function relationships of the enzyme, more accurate interpretation of genotype/ phenotype correlations and for choosing appropriate treatments. Background: Cardiac abnormalities are frequent in patients with MPS VI and are an important cause of morbidity and mortality. Case Report: A four months old girl was admitted due to failure to thrive, bronchilolitis and heart murmur. The diagnosis of congenital mitral anomaly with severe regurgitation and severe dilatation of left atrium and ventricle was defined. Symptomatic treatment was started with high doses of diuretics and ACE-inibithor, but patient maintained severe heart failure, several respiratory infections and poor weight gain. At the age of 18 months, heart surgery was proposed to valve repair but was refused by parents. Meanwhile it became apparent a phenotype suggestive of MPS VI; diagnosis was established by enzymatic assay and ERT-Galsulfase was started. Now she accomplished 18 months of treatment and clinically is greatly improved, but still maintains aggressive therapeutics of congestive heart failure. Conclusions: This case presents with an unusual early severe valvular involvement with few systemic signs of MPS VI misleading the etiologic diagnosis initially. We emphasize the value of identification of a systemic disease in the context of apparently isolate mitral anomaly in an infant to timely begin specific treatment. Adequate management of these patients is still a challenge to multidisciplinary teams. Mucopolysaccharidosis type VI (Maroteaux-Lamy syndrome) is an autosomal recessive disorder caused by a deficiency in the lysosomal enzyme N-acetylgalactosamine 4 sulfatase ( arylsulfatase B, or ASB). We reviewed 10 patients with MPS VI ( five males and five females; age range 1-15.5 years) treated with weekly intravenous infusions of rhASB (1.0 mg/ kg) for at least 6 months. Gestational and perinatal data were normal for all patients. During the course of the disease, all patients except one whom we diagnosed at 4 months because of his sibling with the diagnosis of MPS VI developed coarsened facial features, short stature, heart valve disease, eye problems, musculoskeletal problems, hepatosplenomegaly and neurological abnormalities. We assesed the biochemical and clinical response every 3 months. All patients received rhASB enzyme replacement therapy (ERT) and showed improvement or stabilisation in clinical manifestations after onset of therapy. The most frequently noted benefits were improvement of endurance, reduced hepatosplenomegaly and decrease in urinary glycosaminoglycan excretion. ERT was well tolerated by all patients. This treatment is thus beneficial and appears to be safe for treatment of MPS VI in Turkish patients. These results indicate that the earlier ERT is started, the greater the response. Two siblings with mucopolysaccharidosis type VI (Maroteaux-Lamy syndrome) have had seven years treatment with enzyme replacement therapy (ERT) using galsulfase (Naglazyme) weekly at a dose of1mg/kg starting from 8 weeks and 3.5 years of age respectively. Treatment has been well tolerated by both siblings with no infusion associated reactions recorded. In the younger sibling ERT has preserved joint movement, cardiac valves, liver and spleen size, height (10th centile) and facial morphology. He has mild scoliosis (23 degrees), stable mild corneal clouding but has recently developed evidence of carpal tunnel syndrome and has significant skeletal disease with a waddling gait from avascular necrosis of the hips. The older sibling initially had a marked improvement in joint and scoliosis mobility which have been maintained and has softening of her facial features and stabilised cardiac valve pathology and corneal clouding. Her height is 6 cms below the 1st centile after correcting for scoliosis which has progressed. Rodding of her spine, which was considered necessary prior to initiating ERT, has been deferred until completion of growth. She has required surgery for severe pes cavus. This paper adds further evidence that early initiation of ERT will slow or prevent the natural pathological progression of MPS VI. Mucopolysaccharidoses VI is an inherited lysosomal storage disease, associated with deficiency of arylsulphatase B. The accumulation of dermatan sulphate leads the symptoms of coarse face, short stature, dysostosis multiplex, contractures, cardiac valve disease, reduced pulmonary function, hepatosplenomegaly, hearing loss, sleep apnoea, corneal clouding, hernias, and generally normal intelligence. Diagnosis based on clinical findings, deficient enzyme activity and demonstration of normal activities of other sulphatase enzymes. With enzyme replacement therapy the prognosis is getting better. Case report: This 33 month-old-monozygotic-twin sisters are from a first degree cousin's marriage. They admitted with complaints of frequent respiratory infections, and limited mobility. Their older sister who had a similar facial appearance died from respiratory insufficiency at the age of 13. They have coarse face, short stature, corneal clouding, pectus carinatum, mitral valve insufficiency, claw hands, dysostosis multiplex, and normal intelligence. Enzyme analysis revealed undetectable levels of arylsulphatase B, with other sulphatases within normal limits. They have been on enzyme replacement therapy for one year. Discussion: With enzyme replacement therapy their effort capacity, and mobility have improved and the frequency and the severity of respiratory infections have decreased. To the best of our knowledge these patients are the first reported twins with mucopolysaccharidoses. Results: Ten patients were included. The median follow-up was 2.4 years (range 1.1-3.8 years). Galsulfase had a significant positive effect on the flexion of the right shoulder (p<0.001), liver size (p<0.001) and spleen size (p=0.017). Flexion of the left shoulder showed a positive trend (p=0.07). Two patients showed a significant decrease of left ventricular mass index (LVMI) Zscore. The one patient who showed a decreased forced vital capacity (FVC) at baseline, showed better FVC results during ERT (p=0.023). For the total group a mean decline of 84% in urinary glycosaminoglycan values was observed. ERT did not have an effect on cardiac valve regurgitation and hearing function. Background: Lysosomal storage disorders (LSDs) affect patient quality of life (QoL) but little is known about the disease impact on family life. We assessed the impact of LSDs on daily living of patients and their families and evaluated parent perceptions on the disease and enzyme replacement therapy (ERT). Patients and Methods: Adaptive behaviour, mental development, and cognitive function were assessed in 12 patients with LSDs (MPS I, MPS VI, Pompe, and Gaucher) over a 4-year period. Fifteen parents were evaluated for emotional well-being, satisfaction with life, perception on the severity and prognosis of the disease, disease impact on family QoL, and expectations on and satisfaction with ERT. Results: Adaptive behaviour, mental development and/or cognitive function were impaired in most patients. Depression and emotional disturbance was common among the mothers. Most parents considered their QoL as satisfactory. The disease impact and stress on the family was high, but appeared to decrease after onset of ERT. The high expectations of ERT before the start of treatment decreased to a more realistic level thereafter. Conclusions: LSDs greatly affect the lives of patients and their families. Psychological counselling of parents and providing realistic information about the expectable benefits from ERT is very important. Conflict of Interest declared. Background: Lysosomal storage disorders (LSDs) are a heterogeneous group of inherited metabolic diseases. The definitive diagnosis of these diseases includes the determination of enzyme activity in plasma, leukocytes, or cultured fibroblasts. In recent years, dried blood samples collected on filter paper (DBS) have been utilized. This technique offers a series of advantages, like the low transportation requirements which explain the increasing preference it enjoys currently. Objectives: To analyze the effect of storage conditions on activity of αgalactosidase A, arylsulfatase B and α-glucosidase. Material and Methods: Blood was collected and directly spotting on filter paper and stored at different temperatures (−20, 4, 25 and 37:C) and storage times (3, 10, 17 and 180 days) . The influence of filter paper size was also assessed (3.0 and 1.2 mm). Results: α-glucosidase A activity significantly decreased after the 10th day, while arylsulfatase B activity only differed significantly after the 180th day, and α-galactosidase A activity remained constant throughout this storage time. Excellent correlation coefficients were observed for the two filter paper sizes used. Conclusions: Both paper sizes may be employed. Filter paper specimens should be transported under refrigeration as soon as possible after blood collection. -Laboratory (1992 -Laboratory ( -2010 were reviewed to screen individuals for MPS and ML by using CTS and/or tarsal tunnel syndrome (CTS/TTS) as a screening indicator. All individuals underwent CTS release surgery were included. Results: 2254 individuals (age 0-18 years) underwent NCS. The reason for referral was CTS/TTS in 1.6%. 3% (68/2254) had CTS/TTS. There was history of trauma in 47% (32/68). 26% (18/68) had an underlying disease. In the latter group 38.8% (7/18) had diagnosis of MPS (3 with MPS I, 2 with MPS II, 1 with MPS IV and 1 with MPS VI). 17/68 individuals underwent carpal tunnel release surgery. 5/17 had diagnosis of MPS in the latter group. One additional patient with MPS IIIA had normal NCVat age 4.5 years who will be followed with periodic NCS. Background: Oligosaccharidoses are Lysosomal Storage Disorders (LSDs) caused by mutations in genes encoding exoglycosidases and lead to accumulation of N-linked oligosaccharides within lysosomes. Due to overlapping clinical symptoms, diagnosis of oligosaccharidoses is challenging, involving individual enzymatic assays or urinary oligosaccharide analysis. Here, we aim to determine a universal buffer for the development of an in vitro multiplex assay to measure exoglycosidase activities from human tissues using tandem mass spectrometry (MS/MS). Methods: Classical 4-methylumbelliferyl (4MU) fluorometric assays were compared to determine an optimal universal buffer used to develop a MS/ MS based multiplex assay involving commercial substrates targeting exoglycosidases from fibroblasts. Results: A 30 mM citrate-phosphate buffer with a pH at 4.3 was the universal condition. Additionally, a MS/MS multiplex assay was established for beta-galactosidase and beta-hexosaminidase using substrates composed of 4MU and paranitrophenol (PNP) linked to particular monosaccharides. Enzyme activities from fibroblasts were successfully assayed in one vial by simultaneous measurement of 4MU and PNP reaction products. Conclusions: A universal buffer allows investigation of several exoglycosidases in a single vial. This was demonstrated for beta-galactosidase and beta-hexosaminidase using distinctive starting substrates and MS/MS technology. Substrates giving mass differentiable products for other exoglycosidases will delineate a multiplex assay that can rapidly identify oligosaccharidoses. Background: MPS disorders are the result of primary defects in lysosomal enzymes. Depending on the specific gene defect, the catabolism of one or more of the MPS is blocked leading to accumulation of the corresponding substrate(s). Objectives: To develop a LC-MS/MS method for glycosaminoglycan quantification: dermatan sulfate (DS) and heparan sulfate (HS) using urine filter paper samples. Methods: A urine filter paper disc was eluted, evaporated under nitrogen. Methanolysis was performed using methanol-HCl•3 N with heating at 65°C for 75 min. Deuterated DS and HS internal standards were used. An Alliance 2795XE-LC coupled to a Quattro micro MS/MS (Waters) was used. A chromatographic gradient allowed good separation with an Atlantis T3 column (9 min run). Data were recorded in positive electrospray ionization. Results: Validation of the method provided good results: linearity (r2> 0.995), accuracy (Bias<20%) and precision (CV<15%). Levels of DS and HS in controls were below LOQs. We analyzed 28 MPS patients: 9 MPS I, 2 MPS IH; 6 MPS IH/S; 1 MPS IS, 5 MPS II and 7 MPS VI. In every case, we were able to differentiate MPS patients from controls. Conclusion: A robust methodology was devised to measure glycosaminoglycans using urine filter paper samples. Results: The frequency of hospitalization for respiratory infections decreased in the 1 year following start of home mechanical ventilation. We have encountered no severe ventilator-related issues.No patient died during this study Conclusion: Home mechanical ventilation therapy is very helpful ; this approach helps patients to spend their lives calmly with their family at home. However, gaining regional support is paramount as many local services, such as ambulance departments, schools and regional nursing centres need to provide support to these patients and their families for home mechanical ventilation to be possible. Families often also require financial and psychological support. Olkhovych N.V. 1 , Pichkur N.O 1 , Ivanova T.P 1 , Trofimova N.S. 1 , Nedoboy A.N. 1 , Kormoz S.N. 1 , Gorovenko N.G. 2 1 National Children Hospital "OHMATDET", Kyiv, Ukraine 2 National Medical Academy of Postdiploma, Kyiv, Ukraine Introduction: Chitinases are enzymes that hydrolyze chitin and have been found in a wide variety of non-vertebrate species; recently a human analog of chitinases, chitotriosidase (CT) has been identified. Extreme elevations of plasma CT activity are observed in patients with Gaucher's disease (GD). The 24 bp duplication in the CT gene, resulting in an inactive protein, has been reported. The carrier prevalence is as high as 30 to 40% and the CT activity is half that in individuals with the wild-type gene. However no systematic evaluation of plasma CT activity has been carried out in GD patients taking into account the status of the allele defective for CT and dose in patients on enzyme replacement therapy (ERT). Case report: A 63 year old patient presented mild clinical presentation of GD: splenomegaly, thrombocytopenia. "Gaucher-like cells were revealed by bone marrow biopsy. The glucocerebrosidase activity-3,7 nmol/h/mg protein ( normal 5,5-9,5 nmol/h/mg protein) and chitotriosidase activity-0 nmol/h/ml pl. Genotype was N370S/N370S. The duplication 24 bp in both alleles was detected in CT gene. Conclusion: For monitoring treatment efficacy GD in this patient should be used in other biochemical markers, such as tartrate-resistant acid phosphatase.