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1.
Acta Histochem ; 123(2): 151678, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33434858

RESUMO

Mucopolysaccharidosis type I (MPS I) is a lysosomal storage disorder characterized by alpha-L-iduronidase (IDUA) deficiency, an enzyme responsible for glycosaminoglycan degradation. Musculoskeletal impairment is an important component of the morbidity related to the disease, as it has a major impact on patients' quality of life. To understand how this disease affects bone structure, morphological, biomechanical and histological analyses of femurs from 3- and 6-month-old wild type (Idua +/+) and MPS I knockout mice (Idua -/-) were performed. Femurs from 3-month-old Idua -/- mice were found to be smaller and less resistant to fracture when compared to their age matched controls. In addition, at this age, the femurs presented important alterations in articular cartilage, trabecular bone architecture, and deposition of type I and III collagen. At 6 months of age, femurs from Idua -/- mice were more resistant to fracture than those from Idua +/+. Our results suggest that the abnormalities observed in bone matrix and articular cartilage in 3-month-old Idua -/- animals caused bone tissue to be less flexible and more likely to fracture, whereas in 6-month-old Idua -/- group the ability to withstand more load before fracturing than wild type animals is possibly due to changes in the bone matrix.


Assuntos
Iduronidase/metabolismo , Mucopolissacaridose I/metabolismo , Mucopolissacaridose I/patologia , Animais , Fenômenos Biomecânicos/fisiologia , Colágeno/metabolismo , Modelos Animais de Doenças , Feminino , Fêmur/enzimologia , Fêmur/metabolismo , Fêmur/patologia , Iduronidase/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mucopolissacaridose I/enzimologia
2.
Protein J ; 40(1): 68-77, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33389473

RESUMO

Mucopolysaccharidosis type I is a rare autosomal recessive genetic disease caused by deficient activity of α-L-iduronidase. As a consequence of low or absent activity of this enzyme, glycosaminoglycans accumulate in the lysosomal compartments of multiple cell types throughout the body. Mucopolysaccharidosis type I has been classified into 3 clinical subtypes, ranging from a severe Hurler form to the more attenuated Hurler-Scheie and Scheie phenotypes. Over 200 gene variants causing the various forms of mucopolysaccharidosis type I have been reported. DNA isolated from dried blood spot was used to sequencing of all exons of the IDUA gene from a patient with a clinical phenotype of severe mucopolysaccharidosis type I syndrome. Enzyme activity of α-L-iduronidase was quantified by fluorimetric assay. Additionally, a molecular dynamics simulation approach was used to determine the effect of the Ser633Trp mutation on the structure and dynamics of the α-L-iduronidase. The DNA sequencing analysis and enzymatic activity shows a c.1898C>G mutation associated a patient with a homozygous state and α-L-iduronidase activity of 0.24 µmol/L/h, respectively. The molecular dynamics simulation analysis shows that the p.Ser633Trp mutation on the α-L-iduronidase affect significant the temporal and spatial properties of the different structural loops, the N-glycan attached to Asn372 and amino acid residues around the catalytic site of this enzyme. Low enzymatic activity observed for p.Ser633Trp variant of the α-L-iduronidase seems to lead to severe mucopolysaccharidosis type I phenotype, possibly associated with a perturbation of the structural dynamics in regions of the enzyme close to the active site.


Assuntos
Anormalidades Múltiplas/genética , Dermatan Sulfato/química , Heparitina Sulfato/química , Iduronidase/química , Mucopolissacaridose I/genética , Mutação Puntual , Anormalidades Múltiplas/enzimologia , Anormalidades Múltiplas/patologia , Anormalidades Múltiplas/terapia , Domínio Catalítico , Cristalografia por Raios X , Dermatan Sulfato/metabolismo , Terapia de Reposição de Enzimas/métodos , Expressão Gênica , Heparitina Sulfato/metabolismo , Humanos , Iduronidase/genética , Iduronidase/metabolismo , Lactente , Masculino , Simulação de Dinâmica Molecular , Mucopolissacaridose I/enzimologia , Mucopolissacaridose I/patologia , Mucopolissacaridose I/terapia , Análise de Componente Principal , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Especificidade por Substrato
3.
Cells ; 11(1)2021 12 31.
Artigo em Inglês | MEDLINE | ID: mdl-35011691

RESUMO

Deficit of the IDUA (α-L-iduronidase) enzyme causes the lysosomal storage disorder mucopolysaccharidosis type I (MPS I), a rare pediatric neurometabolic disease, due to pathological variants in the IDUA gene and is characterized by the accumulation of the undegraded mucopolysaccharides heparan sulfate and dermatan sulfate into lysosomes, with secondary cellular consequences that are still mostly unclarified. Here, we report a new fruit fly RNAi-mediated knockdown model of a IDUA homolog (D-idua) displaying a phenotype mimicking some typical molecular features of Lysosomal Storage Disorders (LSD). In this study, we showed that D-idua is a vital gene in Drosophila and that ubiquitous reduction of its expression leads to lethality during the pupal stage, when the precise degradation/synthesis of macromolecules, together with a functional autophagic pathway, are indispensable for the correct development to the adult stage. Tissue-specific analysis of the D-idua model showed an increase in the number and size of lysosomes in the brain and muscle. Moreover, the incorrect acidification of lysosomes led to dysfunctional lysosome-autophagosome fusion and the consequent block of autophagy flux. A concomitant metabolic drift of glycolysis and lipogenesis pathways was observed. After starvation, D-idua larvae showed a quite complete rescue of both autophagy/lysosome phenotypes and metabolic alterations. Metabolism and autophagy are strictly interconnected vital processes that contribute to maintain homeostatic control of energy balance, and little is known about this regulation in LSDs. Our results provide new starting points for future investigations on the disease's pathogenic mechanisms and possible pharmacological manipulations.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Mucopolissacaridose I/enzimologia , Mucopolissacaridose I/patologia , Sequência de Aminoácidos , Animais , Autofagossomos/metabolismo , Autofagia , Modelos Animais de Doenças , Regulação para Baixo/genética , Proteínas de Drosophila/química , Drosophila melanogaster/genética , Genes Essenciais , Glicólise , Lipogênese , Locomoção , Longevidade , Lisossomos/metabolismo , Músculos/metabolismo , Especificidade de Órgãos , Fenótipo , Interferência de RNA
4.
Mol Genet Metab ; 130(4): 255-261, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32563631

RESUMO

BACKGROUND: Mucopolysaccharidoses (MPS) are a group of rare, inherited metabolic diseases that result from a deficiency in one of several lysosomal enzymes essential for stepwise glycosaminoglycan (GAG) degradation, leading to GAG accumulation and widespread cellular pathology and clinical disease. Although disease presentation is heterogeneous, the clinical hallmarks are largely comparable across several MPS subtypes. Extensive data have shown that the level of urinary GAG (uGAG) excretion above normal is strongly correlated with disease severity and clinical outcomes in MPS diseases. Thus, change in uGAG excretion may have significant value as a potential primary endpoint in clinical trials of MPS diseases that are too rare to study using traditional clinical endpoints. METHODS: A retrospective medical chart review was undertaken of patients with MPS I, II, and VI who had been treated long term with enzyme replacement therapy (ERT). The relationship between uGAG reduction and clinical outcomes relevant to the major clinical manifestations of these MPS diseases was evaluated. A multi-domain responder index (MDRI) score was calculated, measuring the following 4 domains: 6-min walk test, pulmonary function, growth rate, and Clinician Global Impression of Change. For each domain, a minimal important difference (MID) was defined based on published information of these outcome measures in MPS and other diseases. RESULTS: Of the 50 patients evaluated, 18 (36%) had MPS I, 23 (46%) had MPS II, and 9 (18%) had MPS VI. Forty-two were clinical practice patients and 8 had participated in clinical trials. Across all MPS subtypes, the mean (± SD) uGAG level at baseline was 66.0 ± 51.5 mg/mmol creatinine (n = 48) and there was a mean reduction of 54.6% following ERT. Analysis of the MDRI score based on the MID defined for each domain showed a greater magnitude of improvement in patients with increased uGAG reduction when compared with those patients with lower uGAG reduction for all assessed uGAG thresholds, and a trend toward a higher likelihood of positive mean MDRI score in patients with a uGAG reduction ≥40%. CONCLUSIONS: In this retrospective study, uGAG reduction was associated with long-term clinical outcomes as assessed by a number of approaches, supporting the use of uGAG reduction as a biomarker primary endpoint.


Assuntos
Biomarcadores/urina , Terapia de Reposição de Enzimas/métodos , Glicosaminoglicanos/urina , Mucopolissacaridose II/patologia , Mucopolissacaridose I/patologia , Mucopolissacaridose VI/patologia , N-Acetilgalactosamina-4-Sulfatase/uso terapêutico , Criança , Pré-Escolar , Feminino , Seguimentos , Humanos , Lactente , Recém-Nascido , Masculino , Mucopolissacaridose I/enzimologia , Mucopolissacaridose I/terapia , Mucopolissacaridose I/urina , Mucopolissacaridose II/enzimologia , Mucopolissacaridose II/terapia , Mucopolissacaridose II/urina , Mucopolissacaridose VI/enzimologia , Mucopolissacaridose VI/terapia , Mucopolissacaridose VI/urina , Prognóstico , Estudos Retrospectivos
5.
Mol Genet Metab ; 130(3): 197-208, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32439268

RESUMO

Mucopolysaccharidosis type I (MPS-I), a lysosomal storage disorder caused by a deficiency of alpha-L-iduronidase enzyme, results in the progressive accumulation of glycosaminoglycans and consequent multiorgan dysfunction. Despite the effectiveness of hematopoietic stem cell transplantation (HSCT) and enzyme replacement therapy (ERT) in correcting clinical manifestations related to visceral organs, complete improvement of musculoskeletal and neurocognitive defects remains an unmet challenge and provides an impact on patients' quality of life. We tested the therapeutic efficacy of combining HSCT and ERT in the neonatal period. Using a mouse model of MPS-I, we demonstrated that the combination therapy improved clinical manifestations in organs usually refractory to current treatment. Moreover, combination with HSCT prevented the production of anti-IDUA antibodies that negatively impact ERT efficacy. The added benefits of combining both treatments also resulted in a reduction of skeletal anomalies and a trend towards decreased neuroinflammation and metabolic abnormalities. As currently there are limited therapeutic options for MPS-I patients, our findings suggest that the combination of HSCT and ERT during the neonatal period may provide a further step forward in the treatment of this rare disease.


Assuntos
Remodelação Óssea , Modelos Animais de Doenças , Terapia de Reposição de Enzimas/métodos , Transplante de Células-Tronco Hematopoéticas/métodos , Iduronidase/fisiologia , Mucopolissacaridose I/terapia , Animais , Animais Recém-Nascidos , Terapia Combinada , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mucopolissacaridose I/enzimologia , Mucopolissacaridose I/patologia
6.
Pediatr Res ; 87(1): 104-111, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31434105

RESUMO

BACKGROUND: Mucopolysaccharidosis I (MPS IH) is a lysosomal storage disease treated with hematopoietic cell transplantation (HCT) because it stabilizes cognitive deterioration, but is insufficient to alleviate all somatic manifestations. Intravenous laronidase improves somatic burden in attenuated MPS I. It is unknown whether laronidase can improve somatic disease following HCT in MPS IH. The objective of this study was to evaluate the effects of laronidase on somatic outcomes of patients with MPS IH previously treated with HCT. METHODS: This 2-year open-label pilot study of laronidase included ten patients (age 5-13 years) who were at least 2 years post-HCT and donor engrafted. Outcomes were assessed semi-annually and compared to historic controls. RESULTS: The two youngest participants had a statistically significant improvement in growth compared to controls. Development of persistent high-titer anti-drug antibodies (ADA) was associated with poorer 6-min walk test (6MWT) performance; when patients with high ADA titers were excluded, there was a significant improvement in the 6MWT in the remaining seven patients. CONCLUSIONS: Laronidase seemed to improve growth in participants <8 years old, and 6MWT performance in participants without ADA. Given the small number of patients treated in this pilot study, additional study is needed before definitive conclusions can be made.


Assuntos
Terapia de Reposição de Enzimas , Transplante de Células-Tronco Hematopoéticas , Iduronidase/administração & dosagem , Mucopolissacaridose I/terapia , Administração Intravenosa , Adolescente , Desenvolvimento do Adolescente , Criança , Desenvolvimento Infantil , Pré-Escolar , Esquema de Medicação , Terapia de Reposição de Enzimas/efeitos adversos , Feminino , Estado Funcional , Humanos , Iduronidase/efeitos adversos , Masculino , Mucopolissacaridose I/diagnóstico , Mucopolissacaridose I/enzimologia , Mucopolissacaridose I/fisiopatologia , Projetos Piloto , Fatores de Tempo , Resultado do Tratamento
7.
J Genet ; 982019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31544795

RESUMO

Mucopolysaccharidosis type I (MPSI) is a rare autosomal recessive disorder caused by mutations in alpha-L-iduronidase (IDUA) gene. IDUA contributes to the degradation of the glycosaminoglycans, including heparan sulphate and dermatan sulphate. Deficient activity of IDUA generates accumulation of glycosaminoglycans in lysosomes leading to MPS I. Here, we identified two boys with MPS I caused by a compound heterozygote of a reported c.265C > T (p.R89W) missense mutation in exon 2 and a novel c.1633G > T (p.E545*, 109) nonsense mutation in exon 11 of IDUA gene in a Chinese family. R89 is close to the active site and its replacement will affect the structure and function of IDUA. Besides, termination from E545 deletes one of the prominent domains and alters the spatial structure of IDUA. In conclusion, our study demonstrates a previously unrecognized mutation in IDUA gene and this report adds to the mutational spectrum observed.


Assuntos
Iduronidase/genética , Mucopolissacaridose I/genética , Povo Asiático , Criança , Pré-Escolar , Códon sem Sentido , Éxons , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Iduronidase/sangue , Masculino , Mucopolissacaridose I/sangue , Mucopolissacaridose I/enzimologia , Mutação de Sentido Incorreto , Domínios Proteicos/genética , Estrutura Terciária de Proteína/genética
8.
J Clin Lab Anal ; 33(8): e22963, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31386236

RESUMO

BACKGROUND: Mucopolysaccharidosis type I (MPSI) is a rare autosomal recessive disorder caused by a deficiency of α-l-iduronidase (IDUA) encoded by the IDUA gene. We examined the mutation spectrum of the IDUA gene to explain the clinical, biochemical, and molecular features in 21 Iranian patients with MPSI. METHODS: Sanger sequencing was used to measure the IDUA gene sequence in the coding region and exon-intron boundaries. We recorded the clinical findings of studied patients at the first diagnosis of disease and then during the treatment and follow-up. RESULTS: Five different missense disease-causing mutations were determined in our patient groups, indicating 90.48% of detection rate. The most widespread mutation was the p.Y109H, occurring in 15.625% of all alleles, which was reported for the first time in our study. Other frequent mutations were as follows: p.Ser157Pro (12.5%), p.Gly84Arg (12.5%), p.Asp257His (9.375%), and p.Asp301Glu (9.375%). Three ones of them were new missense mutations: p.Ser157Pro, p.Asp257His, and p.Asp301Glu. DISCUSSION: The results of this study explain the different spectrum of IDUA gene mutations in our patients with MPSI. We introduced here 32 different variants including four new variants: p.Y109H (15.625%), p.S157P (12.5%), p.D257H (9.375%), and p.D301E (9.375%). In this series, there was no relationship between the happening of clinical features and genotype variations and biochemical findings.


Assuntos
Biomarcadores/análise , Iduronidase/genética , Mucopolissacaridose I/genética , Mutação de Sentido Incorreto , Adolescente , Criança , Pré-Escolar , Análise Mutacional de DNA , Feminino , Seguimentos , Genótipo , Humanos , Lactente , Masculino , Mucopolissacaridose I/enzimologia , Mucopolissacaridose I/patologia , Fenótipo , Prognóstico
9.
Mol Genet Genomic Med ; 7(9): e00615, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31319022

RESUMO

BACKGROUND: Mucopolysaccharidosis type I (MPS I) is a rare, recessively inherited lysosomal storage disorder, characterized by progressive multi-systemic disease. It is caused by a reduced or absent alpha-l iduronidase (IDUA) enzyme activity secondary to biallelic loss-of-function variants in the IDUA. Over 200 causative variants in IDUA have been identified. Nevertheless, there is a fraction of MPS I patients with only a single mutated IDUA allele detectable. METHODS: As genetic testing of MPS I is usually based on sequencing methods, copy number variations (CNVs) in IDUA can be missed and therefore presumably remain underdiagnosed. The aim of this study was the detection of CNVs using an IDUA-specific in house multiplex ligation-dependent probe amplification (MLPA) assay. RESULTS: A total of five unrelated MPS I patient samples were re-analyzed after only a single heterozygous IDUA mutation c.979G>C (p.A327P), c.1469T>C (p.L490P), c.1598C>G (p.P533R), c.1205G>A (p.W402X), c.973-7C>G (p.?) could be identified. We detected a novel splice site variant c.973-7C>G (p.?), as well as two novel CNVs, a large deletion of IDUA exon 14 and 3'UTR c.(1828 + 1_1829-1)_(*1963_?)del, and a large duplication extending from IDUA exon 2 to intron 12 c.(157 + 1_158-1)_(1727 + 1_1728-1)dup. CONCLUSION: Together with the CNVs we previously identified, a total of four pathogenic IDUA CNVs have now been reported.


Assuntos
Variações do Número de Cópias de DNA , Iduronidase/genética , Mucopolissacaridose I/genética , Mutação , Feminino , Humanos , Reação em Cadeia da Ligase , Masculino , Mucopolissacaridose I/enzimologia
10.
Hum Mol Genet ; 27(20): 3612-3626, 2018 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-30052969

RESUMO

Mucopolysaccharidosis type I (MPS I) is caused by deficiency of α-l-iduronidase (IDUA), a lysosomal enzyme involved in the breakdown and recycling of glycosaminoglycans (GAGs). Although enzyme replacement therapy is available, the efficacy of the treatment for neuropathic manifestations is limited. To facilitate drug discovery and model disease pathophysiology, we generated neural stem cells (NSCs) from MPS I patient-derived induced pluripotent stem cells (iPSCs). The NSCs exhibited characteristic disease phenotypes with deficiency of IDUA, accumulation of GAGs and enlargement of lysosomes, in agreement with the severity of clinical subgroups of MPS I. Transcriptome profiling of NSCs revealed 429 genes that demonstrated a more extensive change in expression in the most severe Hurler syndrome subgroup compared to the intermediate Hurler-Scheie or the least severe Scheie syndrome subgroups. Clustering and pathway analysis revealed high concordance of the severity of neurological defects with marked dysregulation of GAG biosynthesis, GAG degradation, lysosomal function and autophagy. Gene ontology (GO) analysis identified a dramatic upregulation of the autophagy pathway, especially in the Hurler syndrome subgroup. We conclude that GAG accumulation in the patient-derived cells disrupts lysosomal homeostasis, affecting multiple related cellular pathways in response to IDUA deficiency. These dysregulated processes likely lead to enhanced autophagy and progressively severe disease states. Our study provides potentially useful targets for clinical biomarker development, disease diagnosis and prognosis, and drug discovery.


Assuntos
Iduronidase/genética , Mucopolissacaridose I/enzimologia , Células-Tronco Neurais , Fenótipo , Linhagem Celular , Criança , Pré-Escolar , Perfilação da Expressão Gênica , Glicosaminoglicanos/metabolismo , Humanos , Iduronidase/metabolismo , Células-Tronco Pluripotentes Induzidas , Lisossomos , Masculino , Mucopolissacaridose I/genética , Mucopolissacaridose I/metabolismo , Mutação
11.
Mol Genet Metab ; 123(2): 105-111, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29198892

RESUMO

Mucopolysaccharidosis type I (MPS I) is a lysosomal disease resulting from deficiency in the α-L-iduronidase (IDUA) hydrolase and subsequent accumulation of glycosaminoglycan (GAG). Clinically, enzyme replacement therapy (ERT) with IDUA achieves negligible neurological benefits presumably due to blood-brain-barrier (BBB) limitations. To investigate the plant lectin ricin B chain (RTB) as a novel carrier for enzyme delivery to the brain, an IDUA:RTB fusion protein (IDUAL), produced in N. benthamiana leaves, was tested in a murine model of Hurler syndrome (MPS I). Affect mice (n=3 for each group) were intravenously injected with a single dose of IDUAL (0.58, 2 or 5.8mgIDUAequivalents/kg) and analyzed after 24h. IDUA activities in liver, kidney and spleen increased significantly, and liver GAG levels were significantly reduced in all three groups. Plasma IDUA levels for all treated groups were high at 1h after injection and decreased by 95% at 4h, indicating efficient distribution into tissues. For long-term evaluations, IDUAL (0.58 or 2mg/kg, 8 weekly injections) was intravenously injected into MPS I mice (n=12 for each group). Thirteen days after the 8th injection, significant IDUA activity was detected in the liver and spleen. GAG levels in tissues including the brain cortex and cerebellum were significantly reduced in treated animals. Treated MPS I mice also showed significant improvement in neurocognitive testing. ELISA results showed that while there was a significant antibody response against IDUAL and plant-derived IDUA, there was no significant antibody response to RTB. No major toxicity or adverse events were observed. Together, these results showed that infusion of IDUAL allowed for significant IDUA levels and GAG reduction in the brain and subsequent neurological benefits. This RTB-mediated delivery may have significant implications for therapeutic protein delivery impacting a broad spectrum of lysosomal, and potentially neurological diseases.


Assuntos
Doenças do Sistema Nervoso Central/terapia , Terapia de Reposição de Enzimas , Iduronidase/administração & dosagem , Lisossomos/enzimologia , Mucopolissacaridose I/terapia , Lectinas de Plantas/química , Animais , Doenças do Sistema Nervoso Central/enzimologia , Modelos Animais de Doenças , Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos , Feminino , Humanos , Iduronidase/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mucopolissacaridose I/enzimologia , Lectinas de Plantas/administração & dosagem , Nicotiana/química
12.
J Cell Biochem ; 119(1): 555-565, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28608934

RESUMO

Mucopolysaccharidosis type I is a lysosomal genetic disorder caused due to the deficiency of the α-L-iduronidase enzyme (IDUA). Mutations associated with IDUA lead to mild to severe forms of diseases characterized by different clinical features. In the present study, we first performed a comprehensive analysis using various in silico prediction tools to screen and prioritize the missense mutations or nonsynonymous SNPs (nsSNPs) associated with IDUA. Subsequently, statistical analysis was empowered to examine the predictive ability and accuracy of the in silico prediction tool results supporting the disease phenotype ranging from mild to severe. Till date, no study has been carried out in IDUA in analyzing the impact of the nsSNPs at the structural level. In this context with the aid of pathogenic and stability prediction in silico tools, we identified nsSNPs R89Q, R89W, and P533R to be most deleterious and disease-causing having impact on the function of the protein. Extensive molecular dynamics analysis was performed using Gromacs to understand the deleterious nature of the mutants. Variations observed between the trajectory files of native and mutants R89Q, R89W, and P533R using Gromacs utilities enabled us to measure the adverse effects on the protein and could be the underlying reasons for the disease pathogenesis. These findings may be helpful in understanding the genotype-phenotype relationship and molecular basis of the disease to design drugs for better treatment. J. Cell. Biochem. 119: 555-565, 2018. © 2017 Wiley Periodicals, Inc.


Assuntos
Iduronidase/genética , Mucopolissacaridose I/genética , Mutação , Polimorfismo de Nucleotídeo Único , Análise Mutacional de DNA/métodos , Humanos , Mucopolissacaridose I/enzimologia
13.
BMC Med Genet ; 17(1): 58, 2016 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-27520059

RESUMO

BACKGROUND: Mucopolysaccharidosis I (MPS I) is an autosomal recessive lysosomal storage disorder caused by a lack of the lysosomal enzyme α-L-iduronidase (IDUA). To date, more than 200 IDUA mutations have been reported. However, only a few types of mutations are recurrent and the frequencies of mutations differ from country to country. METHODS: We performed the IDUA mutation analysis in seven patients who were biochemically diagnosed with MPS I in the Department of Pediatrics, Samsung Medical Center, from 2009 to 2014. Here, we describe the results of the IDUA mutation analysis in seven patients with MPS I and the IDUA mutational spectrum in Korean patients with MPS I, including previous data. RESULTS: The IDUA mutations were found in all 14 alleles of 7 patients, and 11 kinds of IDUA mutations were identified. The detected mutations were five missense mutations (p.A79V, p.L346R, p.T388K, p.P496R, and p.C577Y), two nonsense mutations (p.Y618* and p.R628*), two deletions (c.683delC and c.1591delC), one splice site mutation (c.972+1G>A), and one duplication (c.613_617dup). Among these, p.T388K, p.C577Y, c.683delC, c.1591delC, and c.972+1G>A were novel mutations that have not previously been reported. After taking everything into consideration, including IDUA mutation analysis of the previously reported 10 unrelated Korean patients with MPS I, p.L346R and c.704ins5 were most commonly found in Korean patients with MPS I. However, p.W402* and p.Q70*, which have mainly been found in Caucasian patients, were not found. CONCLUSION: As a result, p.L346R and c.704ins5, which were the most common in Korea, which is geographically situated midway between China and Japan, were some of the most common mutations in China and Japan, respectively. These results are especially worthy of notice.


Assuntos
Povo Asiático/genética , Iduronidase/genética , Mucopolissacaridose I/enzimologia , Mucopolissacaridose I/genética , Mutação , Alelos , China , Hibridização Genômica Comparativa , Análise Mutacional de DNA , Éxons , Feminino , Genótipo , Humanos , Japão , Masculino , Mucopolissacaridose I/patologia , Fenótipo , Polimorfismo Genético , República da Coreia
14.
Sci Rep ; 6: 22131, 2016 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-26899286

RESUMO

Although cord blood transplantation has significantly extended the lifespan of mucopolysaccharidosis type 1 (MPS1) patients, over 95% manifest cornea clouding with about 50% progressing to blindness. As corneal transplants are met with high rejection rates in MPS1 children, there remains no treatment to prevent blindness or restore vision in MPS1 children. Since MPS1 is caused by mutations in idua, which encodes alpha-L-iduronidase, a gene addition strategy to prevent, and potentially reverse, MPS1-associated corneal blindness was investigated. Initially, a codon optimized idua cDNA expression cassette (opt-IDUA) was validated for IDUA production and function following adeno-associated virus (AAV) vector transduction of MPS1 patient fibroblasts. Then, an AAV serotype evaluation in human cornea explants identified an AAV8 and 9 chimeric capsid (8G9) as most efficient for transduction. AAV8G9-opt-IDUA administered to human corneas via intrastromal injection demonstrated widespread transduction, which included cells that naturally produce IDUA, and resulted in a >10-fold supraphysiological increase in IDUA activity. No significant apoptosis related to AAV vectors or IDUA was observed under any conditions in both human corneas and MPS1 patient fibroblasts. The collective preclinical data demonstrate safe and efficient IDUA delivery to human corneas, which may prevent and potentially reverse MPS1-associated cornea blindness.


Assuntos
Cegueira/terapia , Doenças da Córnea/terapia , Dependovirus/genética , Terapia Genética/métodos , Iduronidase/genética , Mucopolissacaridose I/terapia , Apoptose/genética , Cegueira/enzimologia , Cegueira/genética , Western Blotting , Células Cultivadas , Córnea/metabolismo , Córnea/patologia , Doenças da Córnea/enzimologia , Doenças da Córnea/genética , Fibroblastos/citologia , Fibroblastos/metabolismo , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Iduronidase/metabolismo , Microscopia Confocal , Mucopolissacaridose I/enzimologia , Mucopolissacaridose I/genética , Técnicas de Cultura de Órgãos , Transfecção/métodos
15.
Clin Biochem ; 49(6): 458-462, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26592960

RESUMO

OBJECTIVES: Mucopolysaccharidoses (MPSs) are a group of rare, inherited metabolic disorders which result from the lack of one of the lysosomal enzymes responsible for the degradation of glycosaminoglycans. Early recognition of MPS is important as it enables prompt implementation of enzyme replacement therapy (ERT). Dipeptidyl peptidase-IV (DPP-IV) is a ubiquitous ectopeptidase which activity has been associated with the cell surface protein CD26. Our aims were to investigate plasma DPP-IV activity in untreated patients with MPS type II in comparison to control individuals and to evaluate changes of DPP-IV during ERT in MPS I or II patients. DESIGN AND METHODS: One MPS I and five MPS II patients were treated with ERT for up to 19 months. DPP-IV activity was measured in plasma with a colorimetric method using Gly-Pro-p-nitroanilide as a substrate. The reference intervals were observed in 17 healthy donors and in 9 MPS II individuals before ERT implementation. RESULTS: DPP-IV activity ranged from 557 to 1959 nmol/ml/h (median and interquartile range: 1453 [955­ 1554], n = 17) in plasma of control samples. In 9 untreated MPS II individuals, DPP-IV activity was higher and ranged from 2565 to 5968 nmol/ml/h (median and interquartile range: 4458 [4031­5161]). In 6 MPS patients receiving ERT, DPP-IV activity ranged from 2984 to 8628 nmol/ml/h. No declining tendency was observed during the treatment. CONCLUSIONS: DPP-IV activity is a good, newa nd valuable biomarker distinguishing between MPS and healthy individuals. However, it is not a useful marker of treatment efficacy and is unsuitable for monitoring.


Assuntos
Dipeptidil Peptidase 4/metabolismo , Terapia de Reposição de Enzimas , Mucopolissacaridose II/enzimologia , Mucopolissacaridose I/enzimologia , Colorimetria , Dipeptidil Peptidase 4/administração & dosagem , Dipeptidil Peptidase 4/sangue , Humanos , Mucopolissacaridose I/tratamento farmacológico , Mucopolissacaridose II/tratamento farmacológico , Projetos Piloto
16.
Orphanet J Rare Dis ; 10: 121, 2015 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-26407983

RESUMO

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.


Assuntos
Iduronidase/deficiência , Artropatias/diagnóstico por imagem , Artropatias/enzimologia , Locomoção/fisiologia , Mucopolissacaridose I/diagnóstico por imagem , Mucopolissacaridose I/enzimologia , Animais , Feminino , Força da Mão/fisiologia , Humanos , Iduronidase/genética , Artropatias/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mucopolissacaridose I/genética , Radiografia
17.
Curr Protoc Hum Genet ; 84: 17.17.1-17.17.8, 2015 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-25599668

RESUMO

Mucopolysaccharidosis type I (MPS I) is a lysosomal storage disorder due to deficiency of alpha iduronidase (IDUA). Progressive storage of dermatan and heparan sulfate throughout the body lead to a multiorgan presentation including short stature, dysostosis multiplex, corneal clouding, hearing loss, coarse facies, hepatosplenomegaly, and intellectual disability. Diagnosis of MPS I is based on IDUA enzyme analysis in leukocytes or dried blood spots (DBS) followed by molecular confirmation of the IDUA gene mutations in individuals with low enzyme activity. The advent of mass spectrometry methods for enzyme analysis in DBS has enabled high-throughput screening for MPS I in symptomatic individuals and newborn infants. The following unit provides the detailed analytical protocol for measurement of IDUA activity in DBS using tandem mass spectrometry.


Assuntos
Teste em Amostras de Sangue Seco/métodos , Iduronidase/deficiência , Leucócitos Mononucleares/química , Mucopolissacaridose I/diagnóstico , Dermatan Sulfato/biossíntese , Teste em Amostras de Sangue Seco/instrumentação , Terapia de Reposição de Enzimas , Expressão Gênica , Heparitina Sulfato/biossíntese , Humanos , Iduronidase/genética , Iduronidase/uso terapêutico , Lactente , Recém-Nascido , Leucócitos Mononucleares/enzimologia , Mucopolissacaridose I/tratamento farmacológico , Mucopolissacaridose I/enzimologia , Mucopolissacaridose I/genética , Mutação , Triagem Neonatal , Espectrometria de Massas em Tandem
18.
Pharm Res ; 32(3): 941-54, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25208876

RESUMO

PURPOSE: Mucopolysaccharidosis I is a genetic disorder caused by alpha-L-iduronidase deficiency. Its primary treatment is enzyme replacement therapy (ERT), which has limitations such as a high cost and a need for repeated infusions over the patient's lifetime. Considering that nanotechnological approaches may enhance enzyme delivery to organs and can reduce the dosage thereby enhancing ERT efficiency and/or reducing its cost, we synthesized laronidase surface-functionalized lipid-core nanocapsules (L-MLNC). METHODS: L-MLNCs were synthesized by using a metal complex. Size distributions were evaluated by laser diffraction and dynamic light scattering. The kinetic properties, cytotoxicity, cell uptake mechanisms, clearance profile and biodistribution were evaluated. RESULTS: Size distributions showed a D[4,3] of 134 nm and a z-average diameter of 71 nm. L-MLNC enhanced the Vmax and Kcat in comparison with laronidase. L-MLNC is not cytotoxic, and nanocapsule uptake by active transport is not only mediated by mannose-6-phosphate receptors. The clearance profile is better for L-MLNC than for laronidase. A biodistribution analysis showed enhanced enzyme activity in different organs within 4 h and 24 h for L-MLNC. CONCLUSIONS: The use of lipid-core nanocapsules as building blocks to synthesize surface-functionalized nanocapsules represents a new platform for producing decorated soft nanoparticles that are able to modify drug biodistribution.


Assuntos
Terapia de Reposição de Enzimas , Fibroblastos/efeitos dos fármacos , Iduronidase/química , Lipídeos/química , Mucopolissacaridose I/tratamento farmacológico , Nanocápsulas , Animais , Área Sob a Curva , Transporte Biológico , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Química Farmacêutica , Terapia de Reposição de Enzimas/efeitos adversos , Fibroblastos/metabolismo , Fibroblastos/patologia , Iduronidase/administração & dosagem , Iduronidase/genética , Iduronidase/farmacocinética , Iduronidase/toxicidade , Injeções Intravenosas , Taxa de Depuração Metabólica , Camundongos Knockout , Mucopolissacaridose I/enzimologia , Nanomedicina , Tamanho da Partícula , Tecnologia Farmacêutica/métodos , Distribuição Tecidual
19.
J Mol Graph Model ; 54: 107-13, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25459762

RESUMO

Human α-L-iduronidase (IDUA) is a member of glycoside hydrolase family and is involved in the catabolism of glycosaminoglycans (GAGs), heparan sulfate (HS) and dermatan sulfate (DS). Mutations in this enzyme are responsible for mucopolysaccharidosis I (MPS I), an inherited lysosomal storage disorder. Despite great interest in determining and studying this enzyme structure, the lack of a high identity to templates and other technical issues have challenged both bioinformaticians and crystallographers, until the recent publication of an IDUA crystal structure (PDB: 4JXP). In the present work, four alternative IDUA models, generated and evaluated prior to crystallographic determination, were compared to the 4JXP structure. A combined analysis using several viability assessment tools and molecular dynamics simulations highlights the strengths and limitations of different comparative modeling protocols, all of which are based on the same low identity template (only 22%). Incorrect alignment between the target and template was confirmed to be a major bottleneck in homology modeling, regardless of the modeling software used. Moreover, secondary structure analysis during a 50ns simulation seems to be useful for indicating alignment errors and structural instabilities. The best model was achieved through the combined use of Phyre 2 and Modeller, suggesting the use of this protocol for the modeling of other proteins that still lack high identity templates.


Assuntos
Iduronidase/química , Humanos , Iduronidase/genética , Iduronidase/metabolismo , Modelos Moleculares , Mucopolissacaridose I/enzimologia , Mutação , Estrutura Secundária de Proteína
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