The efficacy of intracerebroventricular idursulfase-beta enzyme replacement therapy in mucopolysaccharidosis II murine model: heparan sulfate in cerebrospinal fluid as a clinical biomarker of neuropathology.

Mucopolysaccharidosis II (MPS II) is caused by a deficiency of iduronate-2-sulfatase that results in accumulation of glycosaminoglycans (GAG), including heparan sulfate (HS), which is considered to contribute to neuropathology. We examined the efficacy of intracerebroventricular (ICV) enzyme replacement therapy (ERT) of idursulfase-beta (IDS-ß) and evaluated the usefulness of HS as a biomarker for neuropathology in MPS II mice. We first examined the efficacy of three different doses (3, 10, and 30 µg) of single ICV injections of IDS-ß in MPS II mice. After the single-injection study, its long-term efficacy was elucidated with 30 µg of IDS-ß ICV injections repeated every 4 weeks for 24 weeks. The efficacy was assessed by the HS content in the cerebrospinal fluid (CSF) and the brain of the animals along with histologic examinations and behavioral tests. In the single-injection study, the 30 µg of IDS-ß ICV injection showed significant reductions of HS content in brain and CSF that were maintained for 28 days. Furthermore, HS content in CSF was significantly correlated with HS content in brain. In the long-term repeated-injection study, the HS content in the brain and CSF was also significantly reduced and correlated. The histologic examinations showed a reduction in lysosomal storage. A significant improvement in memory/learning function was observed in open-field and fear-conditioning tests. ICV ERT with 30 µg of IDS-ß produced significant improvements in biochemical, histological, and functional parameters in MPS II mice. Furthermore, we demonstrate for the first time that the HS in the CSF had significant positive correlation with brain tissue HS and GAG levels, suggesting HS in CSF as a useful clinical biomarker for neuropathology.

A Liquid Chromatography-Quadrupole-Time-of-Flight Mass Spectrometric Assay for the Quantification of Fabry Disease Biomarker Globotriaosylceramide (GB3) in Fabry Model Mouse.

Fabry disease is a rare lysosomal storage disorder resulting from the lack of α-Gal A gene activity. Globotriaosylceramide (GB3, ceramide trihexoside) is a novel endogenous biomarker which predicts the incidence of Fabry disease. At the early stage efficacy/biomarker study, a rapid method to determine this biomarker in plasma and in all relevant tissues related to this disease simultaneously is required. However, the limited sample volume, as well as the various levels of GB3 in different matrices makes the GB3 quantitation very challenging. Hereby we developed a rapid method to identify GB3 in mouse plasma and various tissues. Preliminary stability tests were also performed in three different conditions: short-term, freeze-thaw, long-term. The calibration curve was well fitted over the concentration range of 0.042⁻10 µg/mL for GB3 in plasma and 0.082⁻20 µg/g for GB3 in various tissues. This method was successfully applied for the comparison of GB3 levels in Fabry model mice (B6;129-Glatm1Kul/J), which has not been performed previously to the best of our knowledge.

BGN Mutations in X-Linked Spondyloepimetaphyseal Dysplasia.

Spondyloepimetaphyseal dysplasias (SEMDs) comprise a heterogeneous group of autosomal-dominant and autosomal-recessive disorders. An apparent X-linked recessive (XLR) form of SEMD in a single Italian family was previously reported. We have been able to restudy this family together with a second family from Korea by segregating a severe SEMD in an X-linked pattern. Exome sequencing showed missense mutations in BGN c.439A>G (p.Lys147Glu) in the Korean family and c.776G>T (p.Gly259Val) in the Italian family; the c.439A>G (p.Lys147Glu) mutation was also identified in a further simplex SEMD case from India. Biglycan is an extracellular matrix proteoglycan that can bind transforming growth factor beta (TGF-ß) and thus regulate its free concentration. In 3-dimensional simulation, both altered residues localized to the concave arc of leucine-rich repeat domains of biglycan that interact with TGF-ß. The observation of recurrent BGN mutations in XLR SEMD individuals from different ethnic backgrounds allows us to define "XLR SEMD, BGN type" as a nosologic entity.

AAV8-mediated expression of N-acetylglucosamine-1-phosphate transferase attenuates bone loss in a mouse model of mucolipidosis II.

Mucolipidoses II and III (ML II and ML III) are lysosomal disorders in which the mannose 6-phosphate recognition marker is absent from lysosomal hydrolases and other glycoproteins due to mutations in GNPTAB, which encodes two of three subunits of the heterohexameric enzyme, N-acetylglucosamine-1-phosphotransferase. Both disorders are caused by the same gene, but ML II represents the more severe phenotype. Bone manifestations of ML II include hip dysplasia, scoliosis, rickets and osteogenesis imperfecta. In this study, we sought to determine whether a recombinant adeno-associated viral vector (AAV2/8-GNPTAB) could confer high and prolonged gene expression of GNPTAB and thereby influence the pathology in the cartilage and bone tissue of a GNPTAB knock out (KO) mouse model. The results demonstrated significant increases in bone mineral density and content in AAV2/8-GNPTAB-treated as compared to non-treated KO mice. We also showed that IL-6 (interleukin-6) expression in articular cartilage was reduced in AAV2/8-GNPTAB treated ML II mice. Together, these data suggest that AAV-mediated expression of GNPTAB in ML II mice can attenuate bone loss via inhibition of IL-6 production. This study emphasizes the value of the MLII KO mouse to recapitulate the clinical manifestations of the disease and highlights its amenability to therapy.

Effect of systemic high dose enzyme replacement therapy on the improvement of CNS defects in a mouse model of mucopolysaccharidosis type II.

BACKGROUND: Mucopolysaccharidosis type II (MPS II, Hunter syndrome), is caused by a deficiency of iduronate-2-sulfatase (IDS). Despite the therapeutic effect of intravenous enzyme replacement therapy (ERT), the central nervous system (CNS) defects persist because the enzyme cannot cross the blood-brain barrier (BBB). There have been several trials of direct infusion to the cerebrospinal space showing promising results; however, this approach may have limitations in clinical situations such as CNS infection. The objective of this study was to improve the CNS defect with systemic high-dose ERT. METHODS: Systemic ERT was performed using three doses (1, 5, and 10 mg/kg weekly) of IDS for three different durations (1, 3, and 6 months) in IDS knock out (KO) mice of two age groups (2 months, 8 months). GAG measurement in tissues, brain pathology, and behavioral assessment were analyzed. RESULTS: Brain IDS activities increased in parallel with the concentrations of IDS injected. The glycosaminoglycan (GAG) level and histopathology in the brains of the young mice improved in a dose- and duration-dependent manner; however, those were not improved in the old mice, even at higher doses of IDS. The spontaneous alternation behavior was recovered in young KO mice treated with ≥ 5 mg/kg IDS; however, no significant improvement was observed in old KO mice. CONCLUSIONS: These results suggest that high-dose ERT given to mice of earlier ages may play a role in preventing GAG accumulation and preventing CNS damage in IDS KO mice. Therefore, ERT above the present standard dose, starting in early childhood, could be a promising treatment regimen for reducing neurological impairment in Hunter syndrome.

Decreased performance in IDUA knockout mouse mimic limitations of joint function and locomotion in patients with Hurler syndrome.

BACKGROUND: Mucopolysaccharidosis type I (MPS I) is caused by the deficiency of alpha-L-iduronidase (IDUA), which is involved in the degradation of glycosaminoglycans (GAGs), such as heparan sulfate and dermatan sulfate in the lysosome. It has been reported that joint symptoms are almost universal in MPS I patients, and even in the case of attenuated disease, they are the first symptom that brings a child to medical attention. However, functional tests and biological markers have not been published for the evaluation of the limitations in joint and locomotion in animal model-mimicking MPS. METHODS: We generated IDUA knockout (KO) mice to observe whether they present impairment of joint function. KO mice were characterized phenotypically and tested dual-energy X-ray absorptiometry analysis (DEXA), open-field, rotarod, and grip strength. RESULTS: The IDUA KO mice, generated by disruption between exon 6 and exon 9, exhibited clinical and laboratory findings, such as high urinary GAGs excretion, GAGs accumulation in various tissues, and significantly increased bone mineral density (BMD) in both female and male mice in the DEXA of the femur and whole bone. Remarkably, we observed a decrease in grasp function, decreased performance in the rotarod test, and hypo-activity in the open-field test, which mimic the limitations of joint mobility and decreased motor performance in the 6-min walk test in patients with MPS I. CONCLUSIONS: We generated a new IDUA KO mouse, tested open field, rotarod and grip strength and demonstrated decrease in grip strength, decreased performance and hypo-activity, which may be useful for investigating therapeutic approaches, and studying the pathogenesis of joint and locomotion symptoms in MPS I.

Pharmacokinetics, Pharmacodynamics, and Efficacy of a Novel Long-Acting Human Growth Hormone: Fc Fusion Protein.

The current recombinant human growth hormone (rhGH) therapy requires daily subcutaneous (sc) injections, which results in poor patient compliance, especially in young children. To reduce the dosing frequency, we generated a chimeric protein of rhGH and the Fc-domain of immunoglobulin G (IgG) (rhGH-Fc). The pharmacokinetics and pharmacodynamics of sc-injected rhGH-Fc were assessed in male Sprague-Dawley rats and hypophysectomized rats, respectively. A single sc injection of rhGH-Fc at a dose of 0.2 mg/kg slowly reached a Cmax of 16.80 ng/mL and remained for 7 days with a half-life of 51.1 h. Conversely, a single sc injection of rhGH 0.2 mg/kg rapidly reached a Cmax of 46.88 ng/mL and declined with a half-life of 0.55 h to baseline values in 4 h. In the efficacy study, the sc-injected rhGH-Fc induced rapid weight gain and tibial width growth at a dose of 240 µg/animal. The effect of two injections of rhGH-Fc separated by 1 week was comparable to that of the same dose of 14 daily injections of rhGH. The rhGH-Fc is a novel candidate for long-acting rhGH therapy with more convenient weekly administration, as it reduces glomerular filtration and receptor-mediated clearance while allowing for the rapid reversal of potential adverse events.

Morphological, Molecular, and Biochemical Characterization of Monounsaturated Fatty Acids-Rich Chlamydomonas sp. KIOST-1 Isolated from Korea.

Microalgae hold promise as producers of sustainable biomass for the production of biofuels and other biomaterials. However, the selection of strains with efficient and robust production of desirable resources remains challenging. In this study, we isolated a green microalga from Korea and analyzed its morphological, molecular, and biochemical characteristics. Microscopic and phylogenetic analyses demonstrated that the isolate could be classified into the genus Chlamydomonas, and we designated the isolate Chlamydomonas s p. K IOST -1. Compositions of protein, lipid, and carbohydrate in the microalgal cells were estimated to be 58.8 ± 0.2%, 22.7 ± 1.2%, and 18.5 ± 1.0%, respectively. Similar to other microalgae belonging to Chlorophyceae, the dominant amino acid and monosaccharide in Chlamydomonas sp. KIOST-1 were glutamic acid and glucose. On the other hand, the proportions of saturated fatty acids, monounsaturated fatty acids, and polyunsaturated fatty acids clearly differed from other species in the genus Chlamydomonas, and monounsaturated fatty acids accounted for a large portion (41.3%) of the total fatty acids in the isolate. Based on these results, Chlamydomonas sp. KIOST-1 has advantageous characteristics for biomass production.

Morphological, molecular, and biochemical characterization of astaxanthin-producing green microalga Haematococcus sp. KORDI03 (Haematococcaceae, Chlorophyta) isolated from Korea.

A unicellular red microalga was isolated from environmental freshwater in Korea, and its morphological, molecular, and biochemical properties were characterized. Morphological analysis revealed that the isolate was a unicellular biflagellated green microalga that formed a non-motile, thick-walled palmelloid or red aplanospore. To determine the taxonomical position of the isolate, its 18S rRNA and rbcL genes were sequenced and phylogenetic analysis was performed. We found that the isolate was clustered together with other related Haematococcus strains showing differences in the rbcL gene. Therefore, the isolated microalga was classified into the genus Haematococcus, and finally designated Haematococcus sp. KORDI03. The microalga could be cultivated in various culture media under a broad range of pH and temperature conditions. Compositions of the microalgal cellular components were analyzed, and its protein, carbohydrate, and lipid compositions were estimated to be 21.1 ± 0.2%, 48.8 ± 1.8%, and 22.2 ± 0.9%, respectively. In addition, D-glucose and D-mannose were the dominant monosaccharides in the isolate, and its amino acids were composed mainly of aspartic acid, glutamic acid, alanine, and leucine. Moreover, several polyunsaturated fatty acids accounted for about 80% of the total fatty acids in Haematococcus sp. KORDI03, and the astaxanthin content in the red aplanospores was estimated to be 1.8% of the dry cell weight. To the best of our knowledge, this is the first report of an Haematococcus sp. isolated from Korea, which may be used for bioresource production in the microalgal industry.

Heterozygous mutations in cyclic AMP phosphodiesterase-4D (PDE4D) and protein kinase A (PKA) provide new insights into the molecular pathology of acrodysostosis.

Acrodysostosis without hormone resistance is a rare skeletal disorder characterized by brachydactyly, nasal hypoplasia, mental retardation and occasionally developmental delay. Recently, loss-of-function mutations in the gene encoding cAMP-hydrolyzing phosphodiesterase-4D (PDE4D) have been reported to cause this rare condition but the pathomechanism has not been fully elucidated. To understand the pathogenetic mechanism of PDE4D mutations, we conducted 3D modeling studies to predict changes in the binding efficacy of cAMP to the catalytic pocket in PDE4D mutants. Our results indicated diminished enzyme activity in the two mutants we analyzed (Gly673Asp and Ile678Thr; based on PDE4D4 residue numbering). Ectopic expression of PDE4D mutants in HEK293 cells demonstrated this reduction in activity, which was identified by increased cAMP levels. However, the cells from an acrodysostosis patient showed low cAMP accumulation, which resulted in a decrease in the phosphorylated cAMP Response Element-Binding Protein (pCREB)/CREB ratio. The reason for this discrepancy was due to a compensatory increase in expression levels of PDE4A and PDE4B isoforms, which accounted for the paradoxical decrease in cAMP levels in the patient cells expressing mutant isoforms with a lowered PDE4D activity. Skeletal radiographs of 10-week-old knockout (KO) rats showed that the distal part of the forelimb was shorter than in wild-type (WT) rats and that all the metacarpals and phalanges were also shorter in KO, as the name acrodysostosis implies. Like the G-protein α-stimulatory subunit and PRKAR1A, PDE4D critically regulates the cAMP signal transduction pathway and influences bone formation in a way that activity-compromising PDE4D mutations can result in skeletal dysplasia. We propose that specific inhibitory PDE4D mutations can lead to the molecular pathology of acrodysostosis without hormone resistance but that the pathological phenotype may well be dependent on an over-compensatory induction of other PDE4 isoforms that can be expected to be targeted to different signaling complexes and exert distinct effects on compartmentalized cAMP signaling.

A biochemical and physicochemical comparison of two recombinant enzymes used for enzyme replacement therapies of hunter syndrome.

Mucopolysaccharidosis II (MPS II, Hunter syndrome; OMIM 309900) is an X-linked lysosomal storage disease caused by a deficiency in the enzyme iduronate-2-sulfatase (IDS), leading to accumulation of glycosaminoglycans (GAGs). For enzyme replacement therapy (ERT) of Hunter syndrome, two recombinant enzymes, idursulfase (Elaprase(®), Shire Human Genetic Therapies, Lexington, MA) and idursulfase beta (Hunterase(®), Green Cross Corporation, Yongin, Korea), are currently available in Korea. To compare the biochemical and physicochemical differences between idursulfase and idursulfase beta, we examined the formylglycine (FGly) content, specific enzyme activity, mannose-6-phosphate (M6P) content, sialic acid content, and in vitro cell uptake activity of normal human fibroblasts of these two enzymes.The FGly content, which determines the enzyme activity, of idursulfase beta was significantly higher than that of idursulfase (79.4 ± 0.9 vs. 68.1 ± 2.2 %, P < 0.001). In accordance with the FGly content, the specific enzyme activity of idursulfase beta was significantly higher than that of idursulfase (42.6 ± 1.1 vs. 27.8 ± 0.9 nmol/min/µg protein, P < 0.001). The levels of M6P and sialic acid were not significantly different (2.4 ± 0.1 vs 2.4 ± 0.3 mol/mol protein for M6P and 12.3 ± 0.7 vs. 12.4 ± 0.4 mol/mol protein for sialic acid). However, the cellular uptake activity of the normal human fibroblasts in vitro showed a significant difference (Kuptake, 5.09 ± 0.96 vs. 6.50 ± 1.28 nM protein, P = 0.017).In conclusion, idursulfase beta exhibited significantly higher specific enzyme activity than idursulfase, resulting from higher FGly content. These biochemical differences may be partly attributed to clinical efficacy. However, long-term clinical evaluations of Hunter syndrome patients treated with these two enzymes will be needed to demonstrate the clinical implications of significant difference of the enzyme activity and the FGly content.

Improvement of cardiac function by short-term enzyme replacement therapy in a murine model of cardiomyopathy associated with Hunter syndrome evaluated by serial echocardiography with speckle tracking 2-D strain analysis.

Cardiac systolic function is significantly decreased in a proportion of patients with Hunter syndrome. This study was performed to evaluate the change in myocardial function associated with enzyme replacement therapy (ERT) in a mouse model of cardiomyopathy associated with Hunter syndrome. Thirty 9-week-old iduronate-2-sulfatase (IDS) knockout mice received either intravenous injection of human recombinant IDS (ERT group, N=15) or saline (control group, N=15) for 5 weeks. Echocardiography was performed at baseline and after treatment. Echocardiographic parameters of left ventricular (LV) systolic function and 2-dimensional radial and circumferential strain were assessed. At follow-up, there was a significant increase in LV fractional shortening and radial and circumferential strain in the ERT group only. Notable myocardial fibrosis was observed in the control group only. In the murine model of Hunter syndrome, ERT exerts beneficial effects on cardiac function, which can be evaluated by serial echocardiographic evaluation including 2-dimensional strain analysis.

Calpain-mediated proteolysis of polycystin-1 C-terminus induces JAK2 and ERK signal alterations.

Autosomal dominant polycystic kidney disease (ADPKD), a hereditary renal disease caused by mutations in PKD1 (85%) or PKD2 (15%), is characterized by the development of gradually enlarging multiple renal cysts and progressive renal failure. Polycystin-1 (PC1), PKD1 gene product, is an integral membrane glycoprotein which regulates a number of different biological processes including cell proliferation, apoptosis, cell polarity, and tubulogenesis. PC1 is a target of various proteolytic cleavages and proteosomal degradations, but its role in intracellular signaling pathways remains poorly understood. Herein, we demonstrated that PC1 is a novel substrate for µ- and m-calpains, which are calcium-dependent cysteine proteases. Overexpression of PC1 altered both Janus-activated kinase 2 (JAK2) and extracellular signal-regulated kinase (ERK) signals, which were independently regulated by calpain-mediated PC1 degradation. They suggest that the PC1 function on JAK2 and ERK signaling pathways might be regulated by calpains in response to the changes in intracellular calcium concentration.

Therapeutic effects of repetitive transcranial magnetic stimulation in an animal model of Parkinson's disease.

Repetitive transcranial magnetic stimulation (rTMS) is used to treat neurological diseases such as stroke and Parkinson's disease (PD). Although rTMS has been used clinically, its underlying therapeutic mechanism remains unclear. The objective of the present study was to clarify the neuroprotective effect and therapeutic mechanism of rTMS in an animal model of PD. Adult Sprague-Dawley rats were unilaterally injected with 6-hydroxydopamine (6-OHDA) into the right striatum. Rats with PD were then treated with rTMS (circular coil, 10 Hz, 20 min/day) daily for 4 weeks. Behavioral assessments such as amphetamine-induced rotational test and treadmill locomotion test were performed, and the dopaminergic (DA) neurons of substantia nigra pas compacta (SNc) and striatum were histologically examined. Expression of neurotrophic/growth factors was also investigated by multiplex ELISA, western blotting analysis and immunohistochemistry 4 weeks after rTMS application. Among the results, the number of amphetamine-induced rotations was significantly lower in the rTMS group than in the control group at 4 weeks post-treatment. Treadmill locomotion was also significantly improved in the rTMS-treated rats. Tyrosine hydroxylase-positive DA neurons and DA fibers in rTMS group rats were greater than those in untreated group in both ipsilateral SNc and striatum, respectively. The expression levels of brain-derived neurotrophic factor, glial cell line-derived neurotrophic factor, platelet-derived growth factor, and vascular endothelial growth factor were elevated in both the 6-OHDA-injected hemisphere and the SNc of the rTMS-treated rats. In conclusion, rTMS treatment improved motor functions and survival of DA neurons, suggesting that the neuroprotective effect of rTMS treatment might be induced by upregulation of neurotrophic/growth factors in the PD animal model.

High-dose enzyme replacement therapy attenuates cerebroventriculomegaly in a mouse model of mucopolysaccharidosis type II.

The natural progression of the severe form of mucopolysaccharidosis II in children is a rapid decline of neurodevelopmental function with hydrocephalus. Recombinant human iduronate-2-sulfatase enzyme replacement therapy (ERT) under a standard regimen seems to have limited effect. Therefore, we determined whether early, high-dose ERT attenuated ventriculomegaly and histologic abnormalities in the brains of IdS-knockout mice. IdS-knockout mice received saline or recombinant human IdS (0.5/1.0/2.0 mg kg(-1)) intravenously once weekly, starting at 4 weeks of age and continuing until 20 weeks. ERT with 2.0 mg kg(-1), but not 0.5 or 1.0 mg kg(-1), significantly attenuated enlarged ventricles, as confirmed by in vivo 7-teslar brain magnetic resonance image (MRI) at 20 weeks. However, neuronal cytoplasmic vacuolization and morphological alteration in the purkinje cells on brain histology and glycosaminoglycan (GAG) levels in brain homogenates were reduced in mice receiving ERT at lower dose than 2.0 mg kg(-1). Additionally, GAG levels significantly correlated with the percent volume ratio of ventricle to whole brain. These results suggested that high-dose systemic ERT started early in life could be a promising therapeutic modality for improving neurologic dysfunction including ventriculomegaly in children with severe Hunter syndrome.

Improvement of CNS defects via continuous intrathecal enzyme replacement by osmotic pump in mucopolysaccharidosis type II mice.

Mucopolysaccharidosis type II (MPS II), also known as Hunter syndrome (OMIM 309900), is a rare, X-linked lysosomal storage disorder caused by a deficiency of iduronate-2-sulfatase (IDS; EC 3.1.6.13), which is involved in the lysosomal degradation of glycosaminoglycans (GAG). Although intermittent intrathecal (IT) injection of the enzyme has been introduced as a method to overcome the blood-brain barrier, continuous IT infusion of the enzyme would be more physiologic. This study was performed to investigate responses in the brain of MPS II mice to varying doses of continuous IT infusion of recombinant human IDS (rh-IDS) in MPS II mice by osmotic pump in three different doses (2.4, 4.8, and 12 µg/day) of rh-IDS for 3 weeks. The results showed that the group treated with 12 µg/day doses of rh-IDS demonstrated decreased GAG concentrations compared to the untreated KO mice group (P = 0.003). After 3 weeks of continuous IT ERT, the brain tissues of the high-dose IT-treated KO mice showed a reduction of vacuolation in the cerebral cortex, thalamus and cerebellar cortex, which was not observed in the low- and medium-dose KO mice groups. Moreover, the anti-NeuN signal representing intact neuron was restored in the cortexes of the high-dose group. In conclusion, continuous IT infusion of the deficient enzyme was effective in improving CNS defects in the MPS II mice, and could be a valuable therapeutic method for treating neurological deterioration in patients with MPS II.

Phase I/II clinical trial of enzyme replacement therapy with idursulfase beta in patients with mucopolysaccharidosis II (Hunter syndrome).

BACKGROUND: Mucopolysaccharidosis II (MPS II, Hunter syndrome) is a rare X-linked lysosomal storage disorder caused by the deficiency of iduronate-2-sulfatase (IDS). In affected patients, glycosaminoglycan (GAG) accumulates in the lysosomes of many organs and tissues contributing to the pathology associated with MPS II. The objective of this phase I/II clinical study was to evaluate the efficacy and safety of recombinant human iduronate-2-sulfatase (idursulfase beta, Hunterase®) in the treatment of MPS II. METHODS: Thirty-one MPS II patients between 6 and 35 years of age were enrolled in a randomized, single-blinded, active comparator-controlled phase I/II trial for 24 weeks. Patients were randomized to active comparator infusions (N=11), 0.5 mg/kg idursulfase beta infusions (N=10), or 1.0 mg/kg idursulfase beta infusions (N=10). The primary efficacy variable was the level of urinary GAG excretion. The secondary variables were changes in the distance walked in 6 minutes (6-minute walk test, 6MWT), echocardiographic findings, pulmonary function tests, and joint mobility. RESULTS: Patients in all three groups exhibited reduction in urine GAG and this reduced GAG level was maintained for 24 weeks. Urine GAG was also significantly reduced in the 0.5 mg/kg and 1.0 mg/kg idursulfase beta groups when compared to the active comparator group (P = 0.043, 0.002, respectively). Changes in 6MWT were significantly greater in the 0.5 mg/kg and 1.0 mg/kg idursulfase groups than in the active comparator group (p= 0.003, 0.015, respectively). Both idursulfase beta infusions were generally safe and well tolerated, and elicited no serious adverse drug reactions. The most frequent adverse events were urticaria and skin rash, which were easily controlled with administration of antihistamines. CONCLUSIONS: This study indicates that idursulfase beta generates clinically significant reduction of urinary GAG, improvements in endurance as measured by 6MWT, and it has an acceptable safety profile for the treatment of MPS II.

Five novel mutations of GALNS in Korean patients with mucopolysaccharidosis IVA.

Mucopolysaccharidosis IVA (MPS IVA; OMIM #253000) is caused by the deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS), a lysosomal enzyme involved in the catabolism of keratan and chondroitin sulfate. In this study, we examined biochemical and genetic data from 6 Korean patients presenting with classic MPS IVA by measuring GALNS activity in peripheral blood leukocytes and skin fibroblasts. We initially identified Korean patients with MPS IVA by clinical, biochemical, and genetic analyses. We performed PCR-direct sequencing to identify molecular defects of the GALNS gene in patients and assessed the mutational statuses of family members as well as 50 healthy unrelated subjects. In silico analyses were performed to check for novel mutations. The mean age of the six female patients was 8.0 ± 5.2 years (range: 2-17 years), and were all found to have severe reductions of GALNS enzyme. A total of 12 mutant alleles were identified, corresponding to 7 different mutations. Five novel mutations were c.218A>G (p.Y73C), c.451C>A (p.P151T), c.725C>G (p.S242C), c.752G>A (p.R251Q), and c.1000C>T (p.Q334X). Two other mutations were c.1156C>T (p.R386C) and c.1243-1G>A. Two mutations, c.451C>A and c.1000C>T, accounted for 58% of all mutations in this sample.

LIN28B polymorphisms are associated with central precocious puberty and early puberty in girls.

PURPOSE: Single-nucleotide polymorphism (SNP) markers within LIN28B have been reported to be related to the timing of pubertal growth. However, no study has investigated the frequency of genetic markers in girls with precocious puberty (PP) or early puberty (EP). This study aimed to determine the frequency of putative genetic markers in girls with PP or EP. METHODS: Genomic DNAs were obtained from 77 and 109 girls that fulfilled the criteria for PP and EP, respectively. The controls in this study were 144 healthy volunteers between 20 and 30 years of age. The haplotypes were reconstructed using 11 SNPs of LIN28B, and haplotype association analysis was performed. The haplotype frequencies were compared. Differences in the clinical and laboratory parameters were analyzed according to the haplotype dosage. RESULTS: Eleven SNPs in LIN28B were all located in a block that was in linkage disequilibrium. The haplotype could be reconstructed using 2 representative SNPs, rs4946651 and rs369065. The AC haplotype was less frequently observed in the PP group than in the controls (0.069 vs. 0.144, P=0.010). The trend that girls with non-AC haplotypes tended to have earlier puberty onset (P=0.037) was illustrated even in the EP+PP patient group by Kaplan-Meier analysis. CONCLUSION: The results of the present study showed that non-AC haplotypes of LIN28B had a significant association with PP in girls.

Clinical, biochemical, and genetic analysis of two korean patients with trichorhinophalangeal syndrome type I and growth hormone deficiency.

Tricho-rhino-phalangeal syndrome type I (TRPSI) is a rare autosomal dominant hereditary disorder characterized by sparse hair, bulbous nose, long philtrum, thin upper lip, and skeletal abnormalities including cone-shaped epiphyses, shortening of the phalanges, and short stature. TRPSI is caused by mutations in the TRPS1 gene. Herein, we report two Korean cases of TRPSI. Although both patients (a 17-year-old-female and a 14-year-old male) had typical clinical findings, Patient 1 had an additional growth hormone (GH) deficiency. Treatment with recombinant human growth hormone (rhGH) 0.7 IU/kg/week led to an increase in growth velocity. Over 10 years of GH therapy, the mean growth velocity was 5.7 ± 0.9 cm/year. However, the patient 2 did not show apparent GH deficiency by GH stimulation test, had a poor response with rhGH therapy and GH therapy was discontinued after 6 months. Upon genetic analysis of the TRPS1 gene, two mutations were found. Patient 1 had a heterozygous mutation c.2520dupT (p.Arg841LysfsX3) which had not been previously reported. Patient 2 had a known nonsense mutation c.1630C>T (p.Arg544X). In summary, we were the first to report Korean patients with mutation of TRPS1.