RESUMEN
Mucopolysaccharidosis type IVA (MPS IVA) was described in 1929 by Luis Morquio from Uruguay and James Brailsford from England, and was later found as an autosomal recessive lysosomal storage disease. MPS IVA is caused by mutations in the gene encoding the enzyme, N-acetylgalactosamine-6-sulfate sulfatase (GALNS). Reduced GALNS activity results in impaired catabolism of two glycosaminoglycans (GAGs), chondroitin-6-sulfate (C6S) and keratan sulfate (KS). Clinical presentations of MPS IVA reflect a spectrum of progression from a severe "classical" phenotype to a mild "attenuated" phenotype. More than 180 different mutations have been identified in the GALNS gene, which likely explains the phenotypic heterogeneity of the disorder. Accumulation of C6S and KS manifests predominantly as short stature and skeletal dysplasia (dysostosis multiplex), including atlantoaxial instability and cervical cord compression. However, abnormalities in the visual, auditory, cardiovascular, and respiratory systems can also affect individuals with MPS IVA. Diagnosis is typically based on clinical examination, skeletal radiographs, urinary GAG, and enzymatic activity of GALNS in blood cells or fibroblasts. Deficiency of GALNS activity is a common assessment for the laboratory diagnosis of MPS IVA; however, with recently increased availability, gene sequencing for MPS IVA is often used to confirm enzyme results. As multiple clinical presentations are observed, diagnosis of MPS IVA may require multi-system considerations. This review provides a history of defining MPS IVA and how the understanding of the disease manifestations has changed over time. A summary of the accumulated knowledge is presented, including information from the International Morquio Registry. The classical phenotype is contrasted with attenuated cases, which are now being recognized and diagnosed more frequently. Laboratory based diagnoses of MPS IVA are also discussed.
Asunto(s)
Condroitinsulfatasas/genética , Glicosaminoglicanos/metabolismo , Mucopolisacaridosis IV/diagnóstico , Mucopolisacaridosis IV/genética , Fibroblastos/enzimología , Fibroblastos/metabolismo , Glicosaminoglicanos/genética , Humanos , Mucopolisacaridosis IV/patología , Mutación , FenotipoRESUMEN
Mucopolysaccharidosis IVA (MPS IVA), also known as Morquio A, is a rare, autosomal recessive disorder caused by a deficiency of the lysosomal enzyme N-acetylgalatosamine-6-sulfate-sulfatase (GALNS), which catalyzes a step in the catabolism of glycosaminoglycans (GAGs), keratan sulfate (KS) and chondroitin-6-sulfate (C6S). It leads to accumulation of the KS and C6S, mainly in bone and cornea, causing a systemic skeletal chondrodysplasia. MPS IVA has a variable age of onset and variable rate of progression. Common presenting features include elevation of urinary and blood KS, marked short stature, hypoplasia of the odontoid process, pectus carinatum, kyphoscoliosis, genu valgum, laxity of joints and corneal clouding; however there is no central nervous system impairment. Generally, MPS IVA patients with a severe form do not survive beyond the third decade of life whereas those patients with an attenuated form may survive over 70 years. There has been no effective therapy for MPS IVA, and care has been palliative. Enzyme replacement therapy (ERT) and hematopoietic stem cell therapy (HSCT) have emerged as a treatment for mucopolysaccharidoses disorders, including Morquio A disease. This review provides an overview of the clinical manifestations, diagnosis and symptomatic management of patients with MPS IVA and describes potential perspectives of ERT and HSCT. The issue of treating very young patients is also discussed.
Asunto(s)
Terapia de Reemplazo Enzimático/métodos , Trasplante de Células Madre Hematopoyéticas/métodos , Mucopolisacaridosis IV/diagnóstico , Mucopolisacaridosis IV/terapia , Animales , Humanos , Sulfato de Queratano/metabolismo , Mucopolisacaridosis IV/metabolismoRESUMEN
BACKGROUND: Quantification studies of mutated mRNAs have not been carried out on Morquio A patients. Such studies are very important for the determination of stability of premature termination codons (PTC) bearing transcripts in order to assess the appropriateness of introducing the newly developed therapeutic strategies such as "stop codon read-through therapy". METHODS: This paper focuses on the study of the GALNS gene and mRNAs in two severe forms of Morquio A patients' fibroblasts with development of a new and rapid real-time RT-PCR for detection and quantification of absolute mRNA copy number. RESULTS: We identified two new mutations c.385A>T (p.K129X) and c.899-1G>C) in Pt1 and a known splicing defect c.120+1G>A in Pt2. Using RT-PCR and real-time RT-PCR in Pt2 we detected low levels of mRNAs, suggesting its instability; in Pt1, we detected three aberrant mRNAs introducing premature stop codons, suggesting that both the c.385A>T and c.899-1G>C mutations produce mRNAs capable of escaping the nonsense-mediated decay (NMD) pathway. CONCLUSIONS: The development of a real-time RT-PCR assay allows to absolutely quantify the GALNS mRNAs carrying mutations that lead to PTCs bearing transcripts, which escape the NMD process and are potentially suitable for the new therapeutic approach.
Asunto(s)
Condroitinsulfatasas/genética , Fibroblastos/enzimología , Mucopolisacaridosis IV/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Alelos , Niño , Codón sin Sentido/genética , Exones/genética , Femenino , Perfilación de la Expresión Génica , Humanos , Masculino , Mutación , ARN Mensajero/análisis , ARN Mensajero/genéticaRESUMEN
Mucopolysaccharidosis IVA is an autosomal recessive disease caused by the deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS). Mutation screening of the GALNS gene was performed for seven MPS IVA patients with attenuated phenotypes from three unrelated families. Four of 5 missense mutations identified in this study (p.F167V, p.R253W, p.R380S, p.P484S) and two reported (p.F97V, p.N204K), associated with attenuated phenotypes, were characterized using in vitro stable expression experiments, enzyme kinetic study, protein processing and structural analysis. The stably expressed mutant enzymes defining the attenuated phenotype exhibited a considerable residual activity (1.2-36.7% of the wild-type GALNS activity) except for p.R380S. Enzyme kinetic studies showed that p.F97V, p.F167V and p.N204K have lower affinity to the substrate compared with other mutants. The p.F97V enzyme was the most thermolabile at 55 degrees C. Immunoblot analyses indicated a rapid degradation and/or an insufficiency in processing in the mutant proteins. Tertiary structure analysis revealed that although there was a tendency for 'attenuated' mutant residues to be located on the surface of GALNS, they have a different effect on the protein including modification of the hydrophobic core and salt-bridge formation and different potential energy. This study demonstrates that 'attenuated' mutant enzymes are heterogeneous in molecular phenotypes, including biochemical properties and tertiary structure.
Asunto(s)
Condroitinsulfatasas/genética , Mucopolisacaridosis IV/genética , Mutación Missense , Adolescente , Adulto , Animales , Células CHO , Condroitinsulfatasas/química , Condroitinsulfatasas/deficiencia , Condroitinsulfatasas/metabolismo , Cricetinae , Cricetulus , Análisis Mutacional de ADN , Estabilidad de Enzimas , Exones , Femenino , Predisposición Genética a la Enfermedad , Calor , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Italia , Japón , Cinética , Masculino , Persona de Mediana Edad , Modelos Moleculares , Mucopolisacaridosis IV/enzimología , Pakistán , Linaje , Fenotipo , Procesamiento Proteico-Postraduccional , Estructura Terciaria de Proteína , Índice de Severidad de la Enfermedad , Especificidad por Sustrato , TransfecciónRESUMEN
Mucopolysaccharidosis IVA (MPS IVA; Morquio A disease) is a lysosomal storage disorder caused by deficiency of N-acetylgalactosamine-6-sulfate sulfatase. The natural history of this disease is incompletely understood. To study which variables influence the clinical outcome, we conducted a study in which MPS IVA patients were asked to fill out a questionnaire with inquiries regarding family history, diagnosis, signs and symptoms, height, weight, surgical history, physical activity, and general complaints. A total of 326 patients (172 male, 154 female) from 42 countries enrolled in the Morquio A Registry programme. The mean age of patients enrolled was 14.9 years for males and 19.1 years for females, with a wide range of 1-73 years. Sixty-four per cent of the patients were under 18 years. Initial symptoms were recognized between 1 and 3 years of age (mean age 2.1 years) and mean age at diagnosis for the patients was 4.7 years. A progressive skeletal dysplasia was commonly observed among the MPS IVA patients. Fifty per cent of patients underwent surgical operations to improve their quality of life. The most frequent surgical sites include neck (51%), ear (33%), leg (26%) and hip (25%). The birth length for affected males and females was 52.2 +/- 4.7 cm and 52.2 +/- 4.5 cm, respectively. The final adult height for affected males and females was 122.5 +/- 22.5 cm and 116.5 +/- 20.5 cm, respectively. The results of this study provide a reference for assessment of efficacy for studies of novel therapies.
Asunto(s)
Internacionalidad , Mucopolisacaridosis IV/fisiopatología , Sistema de Registros , Adolescente , Adulto , Edad de Inicio , Anciano , Estatura , Peso Corporal , Niño , Preescolar , Progresión de la Enfermedad , Femenino , Humanos , Lactante , Masculino , Registros Médicos , Persona de Mediana Edad , Actividad Motora , Mucopolisacaridosis IV/epidemiología , Mucopolisacaridosis IV/genética , Mucopolisacaridosis IV/cirugía , FenotipoRESUMEN
Mucopolysaccharidosis II (Hunter disease), a lysosomal storage disorder caused by a deficiency of iduronate-2-sulfatase (IDS), has variable clinical phenotypes. Nearly 300 different mutations have been identified in the IDS gene from patients with Hunter disease, but the correlation between the genotype and phenotype has remained unclear. We studied the characteristics of 11 missense mutations, which were detected in the patients or artificially introduced, using stable expression experiments and structural analysis. The mutants found in the attenuated phenotype showed considerable residual activity (0.2-2.4% of the wild-type IDS activity) and those in the severe phenotype had no activity. In immunoblot analysis, both the 73-75 kDa precursor and processed forms were detected in the expression of 'attenuated' mutants (R48P, A85T and W337R) and the artificial active site mutants (C84S, C84T). The 73-75 kDa initial precursor was detected in the 'severe' mutants (P86L, S333L, S349I, R468Q, R468L). The truncated 68 kDa precursor form was synthesized in the Q531X mutant. The results of immunoblotting indicated rapid degradation and/or insufficiency in processing as a result of structural alteration of the IDS protein. A combination of analyses of genotype and molecular phenotypes, including enzyme activity, protein processing and structural analysis with an engineered reference protein, could provide an avenue to understanding the molecular mechanism of the disease and could give a useful tool for the evaluation of possible therapeutic chemical compounds.
Asunto(s)
Iduronato Sulfatasa/química , Iduronato Sulfatasa/genética , Mucopolisacaridosis II/genética , Mutación , Animales , Células CHO , Línea Celular , Cricetinae , Genotipo , Humanos , Immunoblotting , Modelos Moleculares , Mucopolisacaridosis II/metabolismo , Mucopolisacaridosis II/terapia , Fenotipo , Procesamiento Proteico-Postraduccional , Estructura Terciaria de ProteínaRESUMEN
Glycosaminoglycans are accumulated in both mucopolysaccharidoses (MPS) and mucolipidoses (ML). MPS I, II, III and VII and ML II and ML III patients cannot properly degrade heparan sulphate (HS). In spite of the importance of HS storage in the metabolic pathway in these diseases, blood and urine HS levels have not been determined systematically using a simple and economical method. Using a new ELISA method using anti-HS antibodies, HS concentrations in blood and urine were determined in MPS and ML II and ML III patients. HS concentrations were determined in 156 plasma samples from MPS I (n = 23), MPS II (n = 26), MPS III (n = 24), MPS IV (n = 62), MPS VI (n = 5), MPS VII (n = 5), ML II (n = 8) and ML III (n = 3), and 205 urine samples from MPS I (n = 33), MPS II (n = 33), MPS III (n = 30), MPS IV (n = 82), MPS VI (n = 7), MPS VII (n = 9), ML II (n = 8) and ML III (n = 3). The ELISA method used monoclonal antibodies against HS. MPS I, II, III and VII and ML II and III patients had significant elevation in plasma HS, compared to the age-matched controls (p < 0.0001). Eighty-three out of 89 (93.3%) of individual values in the above MPS types and ML were above the mean +2SD of the controls. In urine samples, 75% of individual values in patients with those types were above the mean +2SD of the controls. In contrast to the previous understanding of the HS metabolic pathway, plasma HS levels in all five MPS VI and 15% of MPS IV patients were elevated above the mean +2SD of the controls. These findings suggest that HS concentration determined by ELISA, especially in plasma, could be a helpful marker for detection of the most severe MPS I, II, III, VI and VII and ML II, distinguishing them from normal populations.
Asunto(s)
Ensayo de Inmunoadsorción Enzimática/métodos , Heparitina Sulfato/química , Mucolipidosis/diagnóstico , Mucopolisacaridosis/diagnóstico , Adolescente , Biomarcadores/metabolismo , Química Clínica/métodos , Niño , Preescolar , Cromatografía Líquida de Alta Presión , Relación Dosis-Respuesta a Droga , Glicosaminoglicanos/química , Heparina/química , Heparitina Sulfato/sangre , Heparitina Sulfato/orina , Humanos , Lactante , Recién Nacido , Mucolipidosis/sangre , Mucolipidosis/orina , Mucopolisacaridosis/sangre , Mucopolisacaridosis/orinaRESUMEN
The mucopolysaccharidoses (MPS) is characterized by accumulation of glycosaminoglycans (GAGs), and mucolipidosis (ML) by accumulation of GAGs and sphingolipids. Each type of MPS accumulates specific GAGs. The lysosomal enzymes N-acetylgalactosamine-6-sulphate sulphatase and beta-galactosidase involve the stepwise degradation of keratan sulphate (KS). Deficiency of these enzymes results in elevation of KS levels in the body fluids and in tissues, leading to MPS IV disease. In this study, we evaluated blood and urine KS levels in types of MPS and ML other than MPS IV. Eighty-five plasma samples came from MPS I (n = 18), MPS II (n = 28), MPS III (n = 20), MPS VI (n = 3), MPS VII (n = 5) and ML (n = 11) patients while 127 urine samples came from MPS I (n = 34), MPS II (n = 34), MPS III (n = 32), MPS VI (n = 7), MPS VII (n = 9) and ML (n = 11) patients. KS levels were determined using the ELISA method. Plasma KS levels varied with age in both control and patient populations. In all age groups, the mean values of plasma KS in MPS and ML patients were significantly higher than those in the age-matched controls. Plasma KS values in four newborn patients were above the mean + 2SD of the age-matched controls (mean, 41 ng/ml). Overall, 85.9% of individual values in non-type IV MPS and ML patients were above the mean + 2SD of the age-matched controls. For urine KS levels, 24.4% of individual values in patients were above the mean + 2SD of the age-matched controls. In conclusion, KS in blood is elevated in each type of non-type IV MPS examined, in contrast to the conventional understanding. This finding suggests that measurement of KS level provides a new diagnostic biomarker in a wide variety of mucopolysaccharidoses and mucolipidoses in addition to MPS IV.
Asunto(s)
Sulfato de Queratano/sangre , Sulfato de Queratano/orina , Mucolipidosis/metabolismo , Mucopolisacaridosis/metabolismo , Adolescente , Adulto , Factores de Edad , Anciano , Anciano de 80 o más Años , Especificidad de Anticuerpos , Biomarcadores , Niño , Preescolar , Reacciones Cruzadas , Ensayo de Inmunoadsorción Enzimática , Humanos , Lactante , Recién Nacido , Sulfato de Queratano/inmunología , Persona de Mediana Edad , Mucolipidosis/diagnóstico , Mucopolisacaridosis/diagnóstico , Sensibilidad y EspecificidadAsunto(s)
Condroitinsulfatasas/genética , Metilación de ADN , Análisis Mutacional de ADN/métodos , Mucopolisacaridosis/genética , Mutación/genética , Adolescente , Niño , Islas de CpG/genética , ADN/química , ADN/genética , Femenino , Pruebas Genéticas/métodos , Genoma Humano , Humanos , Masculino , Mucopolisacaridosis/diagnóstico , Mucopolisacaridosis/enzimología , Fenotipo , Reacción en Cadena de la Polimerasa/métodos , Polimorfismo Conformacional Retorcido-SimpleRESUMEN
Gene therapy may provide a long-term approach to the treatment of mucopolysaccharidoses. As a first step toward the development of an effective gene therapy for mucopolysaccharidosis type IVA (Morquio syndrome), a recombinant retroviral vector, LGSN, derived from the LXSN vector, containing a full-length human wildtype N-acetylgalactosamine-6-sulfate sulfatase (GALNS) cDNA, was produced. Severe Morquio and normal donor fibroblasts were transduced by LGSN. GALNS activity in both Morquio and normal transduced cells was several fold higher than normal values. To measure the variability of GALNS expression among different transduced cells, we transduced normal and Morquio lymphoblastoid B cells and PBLs, human keratinocytes, murine myoblasts C2C12, and rabbit synoviocytes HIG-82 with LGSN. In all cases, an increase of GALNS activity after transduction was measured. In Morquio cells co-cultivated with enzyme-deficient transduced cells, we demonstrated enzyme uptake and persistence of GALNS activity above normal levels for up to 6 days. The uptake was mannose-6-phosphate dependent. Furthermore, we achieved clear evidence that LGSN transduction of Morquio fibroblasts led to correction of the metabolic defect. These results provide the first evidence that GALNS may be delivered either locally or systematically by various cells in an ex vivo gene therapy of MPS IVA.
Asunto(s)
Condroitinsulfatasas/genética , Terapia Genética , Mucopolisacaridosis IV/terapia , Retroviridae/genética , Transducción Genética , Animales , Técnicas de Cocultivo , Humanos , Mucopolisacaridosis IV/patología , ConejosRESUMEN
In seven patients with mucopolysaccharidoses (1 Hurler, 1 Hurler-Scheie, 4 Hunter, 1 Sly), cranial (1)H-magnetic resonance spectroscopy was performed to evaluate the accumulation of mucopolysaccharides and biochemical changes in the CNS in vivo before and after bone marrow transplantation (BMT). In two of seven patients, (1)H-magnetic resonance spectroscopy was performed before and after BMT. Nuclear magnetic resonance spectra of dermatan sulfate and chondroitin sulfate-C and magnetic resonance spectroscopy of chondroitin sulfate-C and urine from patients with mucopolysaccharidoses showed resonance higher than the chemical shift of myoinositol in the brain (3.7 ppm). The resonance was considered to contain signals from mucopolysaccharide molecules. The resonance was measured as presumptive mucopolysaccharides (pMPS). In white matter lesions detected by magnetic resonance imaging, pMPS/creatine ratios and choline/creatine ratios were consistently higher than control ratios. In white matter without lesions, choline/creatine ratios were higher than control ratios. Patients with higher developmental quotient or intelligence quotient tended to show higher N:-acetylaspartate/creatine ratios and lower pMPS/creatine ratios in basal ganglia. After BMT, the pMPS/creatine ratio in white matter lesions of patient 3, with Hunter syndrome, was slightly decreased, but in none of the patients was the ratio ever below the control ratios, even 7 y after BMT. In white matter without lesions, the pMPS/creatine ratio in patient 3 was decreased to the control ratios after BMT, but although the choline/creatine ratios were gradually decreased, they remained higher than the control ratio, 2 y after BMT. These results suggest that evaluation of pMPS, choline, and N:-acetylaspartate by (1)H-magnetic resonance spectroscopy is an important technique that may provide useful biochemical information in vivo on the neurologic process and the efficacy of BMT in patients with mucopolysaccharidoses.
Asunto(s)
Trasplante de Médula Ósea , Encéfalo/metabolismo , Mucopolisacaridosis/metabolismo , Mucopolisacaridosis/terapia , Adolescente , Adulto , Encéfalo/diagnóstico por imagen , Niño , Preescolar , Glicosaminoglicanos/análisis , Humanos , Espectroscopía de Resonancia Magnética , RadiografíaRESUMEN
Mucopolysaccharidosis type VII (MPS VII; Sly syndrome) is an autosomal recessive lysosomal storage disorder due to an inherited deficiency of beta-glucuronidase. A naturally occurring mouse model for this disease was discovered at The Jackson Laboratory and shown to be due to homozygosity for a 1-bp deletion in exon 10 of the gus gene. The murine model MPS VII (gus(mps/mps)) has been very well characterized and used extensively to evaluate experimental strategies for lysosomal storage diseases, including bone marrow transplantation, enzyme replacement therapy, and gene therapy. To enhance the value of this model for enzyme and gene therapy, we produced a transgenic mouse expressing the human beta-glucuronidase cDNA with an amino acid substitution at the active site nucleophile (E540A) and bred it onto the MPS VII (gus(mps/mps)) background. We demonstrate here that the mutant mice bearing the active site mutant human transgene retain the clinical, morphological, biochemical, and histopathological characteristics of the original MPS VII (gus(mps/mps)) mouse. However, they are now tolerant to immune challenge with human beta-glucuronidase. This "tolerant MPS VII mouse model" should be useful for preclinical trials evaluating the effectiveness of enzyme and/or gene therapy with the human gene products likely to be administered to human patients with MPS VII.
Asunto(s)
Glucuronidasa/metabolismo , Tolerancia Inmunológica/genética , Mucopolisacaridosis VII/patología , Mutación , Transgenes , Animales , Secuencia de Bases , Sitios de Unión , Cartilla de ADN , Glucuronidasa/genética , Ratones , Ratones Transgénicos , Mucopolisacaridosis VII/enzimología , Mucopolisacaridosis VII/genética , Mucopolisacaridosis VII/inmunología , FenotipoRESUMEN
Hereditary hemochromatosis (HH) is a common disorder of iron metabolism caused by mutation in HFE, a gene encoding an MHC class I-like protein. Clinical studies demonstrate that the severity of iron loading is highly variable among individuals with identical HFE genotypes. To determine whether genetic factors other than Hfe genotype influence the severity of iron loading in the murine model of HH, we bred the disrupted murine Hfe allele onto three different genetically defined mouse strains (AKR, C57BL/6, and C3H), which differ in basal iron status and sensitivity to dietary iron loading. Serum transferrin saturations (percent saturation of serum transferrin with iron), hepatic and splenic iron concentrations, and hepatocellular iron distribution patterns were compared for wild-type (Hfe +/+), heterozygote (Hfe +/-), and knockout (Hfe -/-) mice from each strain. Although the Hfe -/- mice from all three strains demonstrated increased transferrin saturations and liver iron concentrations compared with Hfe +/+ mice, strain differences in severity of iron accumulation were striking. Targeted disruption of the Hfe gene led to hepatic iron levels in Hfe -/- AKR mice that were 2.5 or 3.6 times higher than those of Hfe -/- C3H or Hfe -/- C57BL/6 mice, respectively. The Hfe -/- mice also demonstrated strain-dependent differences in transferrin saturation, with the highest values in AKR mice and the lowest values in C3H mice. These observations demonstrate that heritable factors markedly influence iron homeostasis in response to Hfe disruption. Analysis of mice from crosses between C57BL/6 and AKR mice should allow the mapping and subsequent identification of genes modifying the severity of iron loading in this murine model of HH.
Asunto(s)
Hemocromatosis/metabolismo , Hierro/metabolismo , Animales , Modelos Animales de Enfermedad , Hemocromatosis/genética , Hígado/metabolismo , Hígado/patología , Ratones , Ratones Endogámicos AKR , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Noqueados , Especificidad de la Especie , Bazo/metabolismo , Bazo/patología , Transferrina/metabolismoRESUMEN
Mucopolysaccharidosis IVA (MPS IVA; OMIM#253000), a lysosomal storage disorder caused by a deficiency of N -acetylgalactosamine-6-sulfate sulfatase (GALNS), has variable clinical phenotypes. To date we have identified 65 missense mutations in the GALNS gene from MPS IVA patients, but the correlation between genotype and phenotype has remained unclear. We studied 17 missense mutations using biochemical approaches and 32 missense mutations, using structural analyses. Fifteen missense mutations and two newly engineered active site mutations (C79S, C79T) were characterized by transient expression analysis. Mutant proteins, except for C79S and C79T, were destabilized and detected as insoluble precursor forms while the C79S and C79T mutants were of a soluble mature size. Mutants found in the severe phenotype had no activity. Mutants found in the mild phenotype had a considerable residual activity (1.3-13.3% of wild-type GALNS activity). Sulfatases, including GALNS, are members of a highly conserved gene family sharing an extensive sequence homology. Thus, a tertiary structural model of human GALNS was constructed from the X-ray crystal structure of N -acetylgalacto-samine-4-sulfatase and arylsulfatase A, using homology modeling, and 32 missense mutations were investigated. Consequently, we propose that there are at least three different reasons for the severe phenotype: (i) destruction of the hydrophobic core or modification of the packing; (ii) removal of a salt bridge to destabilize the entire conformation; (iii) modification of the active site. In contrast, mild mutations were mostly located on the surface of the GALNS protein. These studies shed further light on the genotype-phenotype correlation of MPS IVA and structure-function relationship in the sulfatase family.
Asunto(s)
Condroitinsulfatasas/genética , Mucopolisacaridosis IV/genética , Mucopolisacaridosis IV/metabolismo , Mutación Missense , Secuencia de Aminoácidos , Sitios de Unión/genética , Western Blotting , Condroitinsulfatasas/química , Cristalografía por Rayos X , Fibroblastos/metabolismo , Genotipo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fenotipo , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , TransfecciónRESUMEN
BACKGROUND AND OBJECTIVE: Most patients with hereditary hemochromatosis are homozygous for a Cys282AETyr mutation in the HFE gene. This mutation has been shown to impair the association of the HFE gene product with b(2)-microglobulin and to prevent its cell surface presentation in transfected COS-7 and 293 cells. This study was performed to examine the expression of HFE protein in epithelial cells, macrophages, and circulating leukocytes obtained from normal subjects and patients with hereditary hemochromatosis. DESIGN AND METHODS: Antisera against two different peptides of the HFE protein were used to immunostain tissue sections and isolate granulocytes, lymphocytes and monocytes. RESULTS: Immunocytochemical staining showed that the HFE protein is expressed in gastric epithelial cells, tissue macrophages, and circulating monocytes and granulocytes. The cell surface associated signal, which was seen in normal gastric epithelial cells, monocytes and macrophages, was also present in C282Y mutant cells from patients with hereditary hemochromatosis, although at apparently reduced amounts in these cells. INTERPRETATION AND CONCLUSIONS: From these studies, it is clear that the C282Y mutation reduces but does not completely prevent presentation of the HFE protein on the cell surface of human monocytes, tissue macrophages, and gastric epithelial cells.
Asunto(s)
Antígenos HLA/biosíntesis , Antígenos de Histocompatibilidad Clase I/biosíntesis , Proteínas de la Membrana/metabolismo , Sustitución de Aminoácidos , Células Epiteliales/química , Células Epiteliales/patología , Genes MHC Clase I , Hemocromatosis/genética , Hemocromatosis/metabolismo , Hemocromatosis/patología , Proteína de la Hemocromatosis , Humanos , Inmunohistoquímica , Macrófagos/química , Macrófagos/patología , Monocitos/química , Monocitos/patología , Mutación Puntual , Coloración y Etiquetado , Estómago/patologíaAsunto(s)
Glicosaminoglicanos/orina , Recién Nacido/orina , Factores de Edad , Peso al Nacer , Creatinina/orina , Femenino , Estudios de Seguimiento , Edad Gestacional , Humanos , Lactante , Masculino , Mucopolisacaridosis/diagnóstico , Mucopolisacaridosis/orina , Mucopolisacaridosis II/orina , Mucopolisacaridosis III/orina , Mucopolisacaridosis VII/orinaRESUMEN
Deficiency of lysosomal enzyme N-acetylgalactosamine-6-sulfate sulfatase (GALNS) leads to mucopolysaccharidosis IV A (MPS IV A), for which there is no definitive treatment so far. Although a number of mutations of the GALNS gene of MPS IV A patients have been described, pathogenesis of the disorder still remains elusive. In order to facilitate in vivo studies using model animals for MPS IV A, we isolated and performed molecular characterization of the mouse homolog of human GALNS. The 2.3-kb cDNA contains a 1560-bp open reading frame encoding 520 amino acid residues. The coding region has 84% similarity to the human GALNS cDNA at amino acid level. The mouse Galns gene was mapped by interspecific backcross analysis to the distal region of chromosome 8 where it co-segregates with Aprt. Northern blot analysis showed a wide expression of a single-copy gene, being higher especially in liver and kidney. The Galns gene was isolated from S129vJ genomic library and its genomic organization was characterized. The mouse Galns gene was about 50-kb long and organized into 14 exons and 13 introns. All intron-exon splice junctions conformed to the GT/AG consensus sequence except exon 8/intron 8 junction. Primer extension shows multiple transcription initiation sites between -44 and -75 although major transcription initiation site was observed at -90 bp from the ATG codon. The 5'-flanking region lacks canonical TATA and CAAT box sequences, but is G+C rich with 10 GC boxes (potential Sp1 binding sites), characteristic of a housekeeping gene promoter.
Asunto(s)
Condroitinsulfatasas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Condroitinsulfatasas/deficiencia , Cromosomas Humanos Par 8 , Clonación Molecular , ADN Complementario/química , ADN Complementario/aislamiento & purificación , Femenino , Fibroblastos/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Mucopolisacaridosis IV/genética , ARN Mensajero/análisis , Mapeo Restrictivo , Alineación de Secuencia , TransfecciónRESUMEN
Hereditary hemochromatosis (HH) is a common autosomal recessive disorder characterized by excess absorption of dietary iron and progressive iron deposition in several tissues, particularly liver. Liver disease resulting from iron toxicity is the major cause of death in HH. Hepatic iron loading in HH is progressive despite down-regulation of the classical transferrin receptor (TfR). Recently a human cDNA highly homologous to TfR was identified and reported to encode a protein (TfR2) that binds holotransferrin and mediates uptake of transferrin-bound iron. We independently identified a full-length murine EST encoding the mouse orthologue of the human TfR2. Although homologous to murine TfR in the coding region, the TfR2 transcript does not contain the iron-responsive elements found in the 3' untranslated sequence of TfR mRNA. To determine the potential role for TfR2 in iron uptake by liver, we investigated TfR and TfR2 expression in normal mice and murine models of dietary iron overload (2% carbonyl iron), dietary iron deficiency (gastric parietal cell ablation), and HH (HFE -/-). Northern blot analyses demonstrated distinct tissue-specific patterns of expression for TfR and TfR2, with TfR2 expressed highly only in liver where TfR expression is low. In situ hybridization demonstrated abundant TfR2 expression in hepatocytes. In contrast to TfR, TfR2 expression in liver was not increased in iron deficiency. Furthermore, hepatic expression of TfR2 was not down-regulated with dietary iron loading or in the HFE -/- model of HH. From these observations, we propose that TfR2 allows continued uptake of Tf-bound iron by hepatocytes even after TfR has been down-regulated by iron overload, and this uptake contributes to the susceptibility of liver to iron loading in HH.
Asunto(s)
Hemocromatosis/metabolismo , Hígado/metabolismo , Receptores de Transferrina/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN Complementario , Modelos Animales de Enfermedad , Expresión Génica , Hemocromatosis/genética , Humanos , Hierro/metabolismo , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , ARN Mensajero/metabolismo , Receptores de Transferrina/genética , Homología de Secuencia de Aminoácido , Distribución TisularRESUMEN
Magnifying electronic endoscopes are frequently used to evaluate the pit patterns of the colorectal mucosa, but such endoscopes suffer from a number of problems. For example, they tend to have long, hard tips and heavy controller sections. In addition, the magnified endoscopic images obtained are often quite coarse due to the small number of pixels in the charge-coupled device (CCD). As a result, at higher magnification ratios, the orientation of the field of view is easily lost. A newly developed prototype colorectal electronic endoscope (Toshiba Corporation, Tokyo) overcomes these problems. The length of the hard tip of the scope and the weight of the controller section are comparable to those of the TCE-3680MH (Toshiba Corporation). High-resolution magnified images can be obtained, because a 410,000-pixel CCD is employed. Two magnification methods are available, optical magnification and electronic zooming, permitting images to be magnified by a factor of up to 90-120 without losing the orientation of the field of view. This newly developed magnifying electronic endoscope was found to be very useful, allowing us to observe the pit patterns of the colorectal mucosa in 82 small colorectal polyps measuring 7 mm or less in diameter.
RESUMEN
Lysosomal beta-glucuronidase shows a dual localization in mouse liver, where a significant fraction is retained in the endoplasmic reticulum (ER) by interaction with an ER-resident carboxyl esterase called egasyn. This interaction of mouse egasyn (mEg) with murine beta-glucuronidase (mGUSB) involves binding of the C-terminal 8 residues of the mGUSB to the carboxylesterase active site of the mEg. We isolated the recombinant human homologue of the mouse egasyn cDNA and found that it too binds human beta-glucuronidase (hGUSB). However, the binding appears not to involve the active site of the human egasyn (hEg) and does not involve the C-terminal 18 amino acids of hGUSB. The full-length cDNA encoding hEg was isolated from a human liver cDNA library using full-length mEg cDNA as a probe. The 1941-bp cDNA differs by only a few bases from two previously reported cDNAs for human liver carboxylesterase, allowing the anti-human carboxylesterase antiserum to be used for immunoprecipitation of human egasyn. The cDNA expressed bis-p-nitrophenyl phosphate (BPNP)-inhibitable esterase activity in COS cells. When expressed in COS cells, it is localized to the ER. The intracellular hEg coimmunoprecipitated with full-length hGUSB and with a truncated hGUSB missing the C-terminal 18-amino-acid residue when extracts of COS cells expressing both proteins were treated with anti-hGUSB antibody. It did not coimmunoprecipitate with mGUSB from extracts of coexpressing COS cells. Unlike mEg, hEg was not released from the hEg-GUSB complex with BPNP. Thus, hEg resembles mEg in that it binds hGUSB. However, it differs from mEg in that (i) it does not appear to use the esterase active site for binding since treatment with BPNP did not release hEg from hGUSB and (ii) it does not use the C terminus of GUSB for binding, since a C-terminal truncated hGUSB (the C-terminal 18 amino acids are removed) bound as well as nontruncated hGUSB. Evidence is presented that an internal segment of 51 amino acids between 228 and 279 residues contributes to binding of hGUSB by hEg.