Dysostosis Multiplex in Human Mucopolysaccharidosis Type 1 H and in Animal Models of the Disease.

Long-term outcomes of enzyme replacement therapy for Taiwanese patients with Mucopolysaccharidosis I

Mucopolysaccharidosis (MPS) includes a group of lysosomal storage diseases. Among them, type III (Sanfilippo) disease is rarely described in Taiwan because of their complexity. With accurate quantifying and precise separation of urinary glycosaminoglycans (GAGs), and specific enzyme measurements, two cases of MPS IIIB disease were able to be described. They both had mild-to-moderate degrees of mental retardation, facial dysmorphism and dysostosis multiplex which do not differ from other types of MPS. Total amounts of GAGs in the urine were only mildly elevated but, among them, heparan sulfate was the highest. Skin fibroblasts alpha-N-acetyl-glucosaminidase activities were low in both cases. Therefore, analysis of GAGs and enzyme assays are important for the diagnosis of patients suspected to have MPS, especially type III disease.

Lysosomal Storage Disease Factors: the Work of Elizabeth F. Neufeld

To evaluate the radiological features of mucopolysaccharidoses (MPS), 15 cases were collected for review in this hospital, retrospectively (1985-1995). Eight cases of Hurler syndrome, two cases of Hunter syndrome, two cases of Sanfilippo syndrome and three cases of Morquio syndrome were classified. Varying severity of dysostosis multiplex is the general bony manifestation of MPS, but special appearance may occur in particular types. Hurler syndrome is the prototype of MPS. The main findings were as follows: "J" shaped sella turcica, paddle-like ribs, anterior inferior beaking (hook-like) of lower thoracic-upper lumbar hypoplastic vertebral bodies, flared iliac wings, constrictive iliac bodies, diaphyseal expansion of long bones, distal ulna and radius tilt toward each other, bullet-like proximal phalanges and central pointing of proximal metacarpals. Hunter and Sanfilippo syndromes had the appearance of moderate to mild dysostosis multiplex. Morquio syndrome had distinctive bony changes as vertebral plana and tongue-like protrusion in the anterior part of the lower thoracic-upper lumbar vertebral bodies, particularly short of the distal deformed ulna and poor ossification of the proximal lateral tibial epiphyses. Although clinical presentations and the hallmarks of bony changes helped possible classification of MPS, definite diagnosis depends on enzyme analysis.

Abstract: Brain cellular fractions were prepared in bulk from four non‐neurological patients and from five patients with mucopolysaccharidosis (MPS). Glycosaminoglycans and lipids were isolated and chemically analyzed. Results of the present study: in the normal controls glycosaminoglycans as μg per mg protein (mean) were 2.2 in neuronal perikarya, 2.0 in astroglia, 2.1 in oligodendroglia, 3.3 in neuropile from gray matter and 3.2 in a mixed fraction from white matter. In the partially myelinated axons from gray and white matter of an 8‐month‐old infant, the concentration was 6.9 and 2.6 μg per mg protein, compared with 2.8 and 0.8 μg per mg protein, respectively, in the adult patients. It was estimated that chondroitin sulfates constituted more than one‐half of the total glycosaminoglycan. Hyaluronic acid, heparan sulfate and dermatan sulfate were also present in all cell types and fractions. Cholesterol, phospholipids, cerebrosides, sulfatide and gangliosides were present in all cell types and fractions, but differed widely in concentration. There was a four‐ to sixfold increase in the concentration of total glycosaminoglycans in the neuronal perikarya of patients with MPS IH, II and IIIA. The increased glycosaminoglycans were heparan sulfate in MPS IIIA and dermatan sulfate plus heparan sulfate in MPS IH and II. Similar changes were found in the astroglia and in the other brain fractions of those patients. The concentration of the gangliosides Gm2, Gm3, Gd3 and ceramide dihexoside was markedly increased in the neurons and other brain fractions of the same patients. The quantities of Gm3, Gm2 and Gd3 together amounted to 65% of the total gangliosides of the neurons, indicating changes of the same magnitude seen in the gangliosidoses. All these patients exhibited mental retardation. The concentration and composition of glycosaminoglycans, gangliosides and neutral hexosyl ceramides in the neuronal perikarya of the patient with MPS IS was normal. There was only a small increase of dermatan sulfate content in the neuropile, mixed fraction and myelinated axons from the white matter and some increase of ceramide dihexoside content in the myelinated axons. This patient was an adult of normal intelligence.

856. Neonatal Liver-Directed Gene Therapy Markedly Reduces Bone Disease in MPS I

Mucopolysaccharidosis type VI (MPS VI) or Maroteaux-Lamy syndrome is an autosomal recessive lysosomal storage disease caused by a deficiency of arylsulfatase B(ARSB), which is required in the degradation of dermatan sulfate and chondroitin sulfate. The deficiency of ARSB leads to an accumulation of dermatan sulfate and chondroitin sulfate in lysosomes and gross excretion in the urine.Few articles about clinical study and ARSB gene mutation analysis of Chinese MPS VI patients were published. This study aimed to explore the clinical features and characteristics of ARSB gene in Chinese children with MPS VI. Thirteen children were diagnosed as MPS VI by ARSB enzyme activity determination during the period from 2009 to 2013. Their clinical features, radiological findings and urine glycosaminoglycan (GAG) levels were retrospectively reviewed. Direct sequencing was used to identify any mutation in the ARSB gene. Thirteen children were diagnosed at the average age of (3.9 ± 2.2) years with 6 male and 7 female. All of these children presented with severe form and onset at an early age of (1.5 ± 0.8) years.Other clinical features included coarse facies, short stature, skeleton deformity, corneal clouding, hepatosplenomegaly with normal intelligence. The radiological findings in all children were characteristic of dysostosis multiplex, like abnormal development of vertebral bodies of the spine, campylorrhachia and paddle-shaped widened ribs. The MRI in case 2 showed cervical cord compression and multiple cysts degeneration in the corona radiate, cella lateralis and callosum.High urine GAG levels were detected, (307.10 ± 112.14) mg/L (Normally below 70 mg/L) and (722.28 ± 245.68) µg/mg creatinine. The ARSB enzyme activity in leukocytes was low, (13.29 ± 6.22) nmol/(mg×h) [Normal range (47-169) nmol/(mg×h)] by fluorogenic assay and (0.24 ± 0.18) U/g [Normal range (1.01-11.47) U/g] by colorimetric assay. A total of 11 mutations were identified by molecular analysis, including seven previously reported mutations (p.L72R, p.G167R, p.G303E, p.F399L, p. T442M, p.Y255X and p.R327X) and four novel mutations (p.Y175D, p.S403X, p.S464X and large deletion including ex. 2, 3). The c.1197C>G (p.F399L) mutation was the most common mutation in this study (31%). The severe form of MPS VI is characterized by early onset and rapid illness progression. Both the radiological findings and increased urine GAG are important clues to diagnose MPS VI.Large decrease or absence of ARSB activity is diagnostic for MPS VI.Four novel mutations of ARSB gene were identified. The reported mutation c.1197C>G (p.F399L) was the hot-spot mutation in this study.

BACKGROUND AND OBJECTIVE: Mucopolysaccharidosis I (MPS I) is a rare, recessively inherited, lysosomal storage disorder caused by deficiency on the enzyme α-L-iduronidase. This defect results in accumulation of heparan and dermatan sulfate in different tissues and organs due to a deficiency in the catabolism of glycosaminoglycans. The overall incidence of MPS I is 0.99-1.99/100,000 live births. Enzyme replacement therapy (ERT) with recombinant α-L-iduronidase (laronidase) has shown to significantl improve the quality of life in children. To describe clinical characteristics, enzyme activity and genetic finding in the first Vietnamese patient with MPS type I with aldurazyme replacement therapy. METHODS: Clinical features, biochemical finding, enzyme activity, mutation analysis and management in a 4 years 6-month-old girl was study. Based on analysis of a patient’s clinical symptoms associated with enzyme α-L-iduronidase activity measurement in leukocyte, the diagnosis of MPS type I was therefore made. Genomic DNAs were extracted from peripheral blood leukocytes from the patient and identify mutation of IDUA gene, 14 exons and their intronic boundaries of the IDUA gene were sequenced using genomic DNA from the patient. The patient has been treated with aldurazyme infusion every week with the dose of 0.58 mg/kg/week. RESULTS: A 4 years 6-month-old girl was presented with joint stiffness at 2 years old. She was admitted with the features of short status, coarse facial, corneal clouding, carpel tunnel syndrome and joint stiffness, kyphosis, abdominal distension, palpable liver at 3 cm below the costal margin, sleep disturbances/snoring. Laboratory showed: hearing lost at right ear in acoumetry, hepatosplenomegaly in ultrasound with right liver length of 117 mm, spleen length of 89 mm, a 6-minute walk test distance of 158.6 m, α-I-duronidase 0.43 nmoL/mg Prot/hrs (normal: 41.8±15.9), urine glycosaminoglycan (GAG) of 508.83 mg/g creatinine (normal: 10.74-112.02). PCR sequencing of IDUA gene showed a novel heterozygous sequence variant c.1046A>G (p.Asp349Gly). After 6 months: more active, decreasing of wheezing in sleep, a 6-minute walk test distance of 252 m, ultrasound showed normal live and spleen size, urine GAGs of 61.18 mg GAGs/g Creatinine. CONCLUSIONS: Enzyme therapy can improve of clinical manifestation which will lead to improvements in life expectancy and quality of life in MPS I patients.

Mucopolysaccharidosis type I (MPS-I), caused by a deficiency of α-l-iduronidase (IDUA; EC 3.2.1.76) activity, can manifest as three major phenotypes, usually defined by clinical criteria: Hurler (severe), Scheie (mild), and Hurler–Scheie (intermediate) syndromes. IDUA is crucial for degradation of glycosaminoglycans such as dermatan and heparan sulfate. Failure to break down these polysaccharides causes physical changes such as joint stiffness, skeletal abnormalities, and corneal clouding. Hurler syndrome is characterized by valvular heart disease, mental deterioration, and death in childhood. Enzyme replacement therapy has been developed for MPS-I, and bone marrow transplantation is beneficial if performed early (1). Because early detection is necessary for optimum clinical response to therapy, the need for newborn screening of MPS-I is under active discussion. Lysosomal enzymes can be measured in rehydrated dried blood spots (DBS) (2)(3)(4)(5)(6)(7)(8). Fluorometric, radiometric, and electrospray ionization tandem mass spectrometry (ESI-MS/MS) assays have been developed. The latter offer the capability of assaying the products of several enzymes simultaneously (multiplexing) (8). In this report, we describe an ESI-MS/MS assay that directly measures the reaction velocity of IDUA in rehydrated DBS for the newborn screening of MPS-I. We also show that the assay can be combined with ESI-MS/MS assays of Niemann–Pick type A/B, Krabbe, Gaucher, Pompe, and Fabry diseases (8) for the simultaneous analysis of six lysosomal storage diseases. All experiments with DBS were conducted in compliance with Institutional Review Board review. All MPS-I-affected patients had been diagnosed previously with established clinical and biochemical procedures. DBS were kept at ambient temperature during shipment (<10 days) and then stored at −20 °C in zip-lock plastic bags (one bag sealed inside of a second bag). Zip-lock bags were kept in a sealed plastic box containing desiccant (anhydrous CaSO4 granules). We developed …

Type I mucopolysaccharidoses (MPS I) include three autosomal recessive disorders (Hurler, MPS IH; Scheie, MPS IS; and Hurler-Scheie, MPS IH-S) caused by the deficient activity of the lysosomal hydrolase α-L-iduronidase with the consequent accumulation of dermatan and heparan sulfate in the lysosomes of several cell types [1]. MPS IH-S is an attenuated disease and the patients show minor facial and skeletal dysmorphism, regular intelligence, mild cardiac and respiratory disease, hepatosplenomegaly, and a normal lifespan. The most common feature is corneal opacification [2], whose morphological basis was not studied in detail. In this work we performed a structural and ultrastructural analysis of the cornea in a patient with MPS IH-S. The patient underwent penetrating keratoplasty and the corneal button was immediately processed for light and electron microscopy. From the micrographs a morphometric analysis was also performed. The corneal epithelium showed superficial cells with few microfolds and evident intercellular spaces. The wing cell layer was formed either by cells with well-evident tonofilaments and small peripheral clear vesicles, or with bilobed nucleus and large paranuclear vesicles filled with granular material. The basal cells showed polygonal shape, with many small vesicles, placed generally in the supranuclear cytoplasm: the intercellular space was enlarged by granular material. The Bowman’s layer was either normal in thickness and structure, or thinner and formed by granular material of variable electron density. The stroma was formed by irregular lamellae of differently oriented collagen, by a large number of keratocytes filled with vesicles, and by intercellular granular material. The corneal endothelium showed degenerative changes. The morphometric analysis of the collagen fibrils diameter provided a mean diameter of 21.71±2.09 nm. Hemidesmosomes were less numerous in the basal cells when compared to the normal cornea. Stromal keratocytes were reduced in their number, particularly in the anterior stroma. Our data showed in MPS IH-S patient pronounced changes of the epithelium, of the Bowman’s layer and of the stroma, consistent with the corneal opacity. As the etiology of the disease is a deficiency of α-L-iduronidase and the consequent accumulation of glycosaminoglycans, we are of the opinion that the stromal keratocytes are the first cells to be involved in the pathogenesis of the corneal disease. The accumulation of the aberrant products seems able to induce morphological changes of both the Bowman’s layer and the corneal epithelium.

Abstract— Lipids and certain lysosomal enzymes were measured in the cerebral gray and white matter and in the liver of unaffected controls and six patients with mucopolysaccharidosis (MPS). Three of the patients had MPS Type I (Hurler), one Type II (Hunter), one Type IIIA (Sanfilippo A) and one Type V (Scheie). The glycosaminoglycans (GAG) of those tissues have been fully characterized previously (Constantopouloset al., 1976).Results of the present study: the normally minor brain monosialogangliosides GM2 and GM3 were markedly increased in the gray and to a lesser extent in the white matter of all the patients, except the patient with MPS Type V. On an average GM2 comprised 8.2 and 6.3, and GM3 11.8 and 6.0% of the total ganglioside neuraminic acid of the gray and white matter respectively in all patients with MPS I, II, and IIIA (normal subjects had less than 1).Ceramide dihexoside was also increased in the gray matter of the patients with MPS I, MPS II and MPS IIIA.The sphingolipid abnormalities were found only in tissues containing excessive amounts of partially degraded dermatan and heparan sulfates or heparan sulfate alone.Of the six acid hydrolases assayed, the activity of /f‐glucosaminidase was increased in both brain and liver, while that of α‐galactosidase and β‐galactosidase was diminished, particularly in the liver.These results suggest that the partially degraded heparan sulfate (and perhaps the dermatan sulfate) which accumulate in the tissues of the patients with MPS may inhibit catabolic enzymes of various sphingolipids. In turn, accumulation of sphingolipids could be responsible at least for some of the brain damage and the mental retardation in MPS I, II and IIIA.

The present paper describes 3 out of a total of 9 siblings, aged 9, 17, and 18, with the following symptoms: gargoyle-like facial features, clouding of the cornea in both eyes, dysostosis multiplex, slightly impaired intelligence, hepatosplenomegaly, umbilical hernia, and increased secretion of mucopolysaccharides in the urine, in particular dermatan and heparan sulfate. Some of the symptoms are mid-way between those of Hurler's and Scheie's syndromes, both having the same deficiency of the enzyme alpha-1-iduronidase. McKusik developed the theory that the genes responsible for the clinical pictures of Hurler's and Scheie's syndromes are alleles and hence cases such as those described here should be considered as allelomorphic compounds.

Mutations of the iduronate-2-sulfatase (IDS) gene is the ultimate cause of Hunter syndrome. Clarification of the nature of mutations will create a necessary premise for prenatal gene diagnosis. A mucopolysaccharidosis (MPS) type II patient and his parents from an ethnic minority in Yunnan province were studied to identify their possible mutation in IDS gene to establish the basis for prenatal gene diagnosis. The patient was a boy, 6 years and 10 months old. Urine glycosaminoglycans (GAGs) assay was used for preliminary diagnosis of the patient and his parents with the disease. The three related persons' DNA was extracted and the concentration and purity of the DNA were measured after the urine test results confirmed the diagnosis. Polymerase chain reaction-denaturing high performance liquid chromatography (PCR-DHPLC) analysis was performed to detect the position of the mutation around the hot spots of mutation in exon 9, 3, 8 of the IDS gene. DNA bidirectional direct sequencing was applied to analyze the mutation detected by PCR-DHPLC. The results of GAGs test showed that in the child with MPS, dermatan sulfate (DS) was positive (+++), heparan sulfate (HS) (+++), chondroitin sulfate (CS) and keratan sulfate (KS) were negative (-); while in his parents none of DS, HS, CS and KS was positive. Abnormal peaks in exon 9 of IDS gene shown by PCR-DHPLC were found in the patient. His mother had heterozygotic peaks. A new frame-mutation (1343-TT) in exon 9 of IDS gene of this patient was confirmed by DNA sequencing. The position where mutation occurred was inside codon 407 (TTT), that means two "T" deleted at position 1343 base pair (1343-TT) in cDNA of the IDS gene, caused a new frame-mutation. It caused elongation of the amino acid chain to a terminal codon TGA at position 429. Thus the peptide chain was shortened from 550 to 428 amino acids. The patient is a hemizygote of the mutation and his mother is a heterozygote. A new frame-mutation (1343-TT) on the IDS gene was identified in this study. The patient is a hemizygote and his mother is a heterozygote. The mutation (1343-TT) resulted in loss of 122 amino acids, which probably caused seriously decreased enzyme activity of IDS, and the authors speculate that this mutation may be the pathological basis of the disease. So, if the mother becomes pregnant again, a prenatal gene diagnostic test for the same mutation should be performed. Furthermore, PCR-DHPLC followed by DNA sequencing are effective methods for diagnosis, including prenatal diagnosis of MPS II.

Correction of Clinical Manifestations of Canine Mucopolysaccharidosis I with Neonatal Retroviral Vector Gene Therapy

Mucopolysaccharidosis I Hurler (MPSI-H) is a pediatric lysosomal storage disease caused by genetic deficiencies in IDUA, coding for α-l-iduronidase. Idua(-/-) mice share similar clinical pathology with patients, including the accumulation of the undegraded glycosaminoglycans (GAGs) heparan sulfate (HS), and dermatan sulfate (DS), progressive neurodegeneration, and dysostosis multiplex. Hematopoietic stem cell transplantation (HSCT) is the most effective treatment for Hurler patients, but reduced intensity conditioning is a risk factor in transplantation, suggesting an underlying defect in hematopoietic cell engraftment. HS is a co-receptor in the CXCL12/CXCR4 axis of hematopoietic stem and progenitor cell (HSPC) migration to the bone marrow (BM), but the effect of HS alterations on HSPC migration, or the functional role of HS in MPSI-H are unknown. We demonstrate defective WT HSPC engraftment and migration in Idua(-/-) recipient BM, particularly under reduced intensity conditioning. Both intra- but especially extracellular Idua(-/-) BM HS was significantly increased and abnormally sulfated. Soluble heparinase-sensitive GAGs from Idua(-/-) BM and specifically 2-O-sulfated HS, elevated in Idua(-/-) BM, both inhibited CXCL12-mediated WT HSPC transwell migration, while DS had no effect. Thus we have shown that excess overly sulfated extracellular HS binds, and sequesters CXCL12, limiting hematopoietic migration and providing a potential mechanism for the limited scope of HSCT in Hurler disease.