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1.
Mol Genet Metab Rep ; 7: 55-62, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-27134828

RESUMO

The Congenital Disorders of Glycosylation (CDG) are an expanding group of genetic disorders which encompass a spectrum of glycosylation defects of protein and lipids, including N- & O-linked defects and among the latter are the muscular dystroglycanopathies (MD). Initial screening of CDG is usually based on the investigation of the glycoproteins transferrin, and/or apolipoprotein CIII. These biomarkers do not always detect complex or subtle defects present in older patients, therefore there is a need to investigate additional glycoproteins in some cases. We describe a sensitive 2D-Differential Gel Electrophoresis (DIGE) method that provides a global analysis of the serum glycoproteome. Patient samples from PMM2-CDG (n = 5), CDG-II (n = 7), MD and known complex N- & O-linked glycosylation defects (n = 3) were analysed by 2D DIGE. Using this technique we demonstrated characteristic changes in mass and charge in PMM2-CDG and in charge in CDG-II for α1-antitrypsin, α1-antichymotrypsin, α2-HS-glycoprotein, ceruloplasmin, and α1-acid glycoproteins 1&2. Analysis of the samples with known N- & O-linked defects identified a lower molecular weight glycoform of C1-esterase inhibitor that was not observed in the N-linked glycosylation disorders indicating the change is likely due to affected O-glycosylation. In addition, we could identify abnormal serum glycoproteins in LARGE and B3GALNT2-deficient muscular dystrophies. The results demonstrate that the glycoform pattern is varied for some CDG patients not all glycoproteins are consistently affected and analysis of more than one protein in complex cases is warranted. 2D DIGE is an ideal method to investigate the global glycoproteome and is a potentially powerful tool and secondary test for aiding the complex diagnosis and sub classification of CDG. The technique has further potential in monitoring patients for future treatment strategies. In an era of shifting emphasis from gel- to mass-spectral based proteomics techniques, we demonstrate that 2D-DIGE remains a powerful method for studying global changes in post-translational modifications of proteins.

2.
Ann Clin Biochem ; 51(Pt 6): 657-61, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24847134

RESUMO

BACKGROUND: In cerebrospinal fluid (CSF) spectrophotometry, if the net bilirubin absorbance (NBA) and net oxyhaemoglobin absorbance (NOA) are both raised with a visible oxyhaemoglobin peak, the revised national guidelines for analysis of CSF bilirubin advise interpreting the results as 'Consistent with subarachnoid haemorrhage (SAH)' regardless of the CSF total protein concentration of the specimen. We wanted to study the range of CSF total protein concentrations found in confirmed SAH to establish if the CSF total protein value can give further guidance on the likelihood of SAH. METHODS: Consecutive cases from five different hospital sites were included if the CSF NBA was greater than 0.007 AU and the NOA was greater than 0.02 AU with a visible oxyhaemoglobin peak. For the cases identified, the laboratory information management system and patient records were interrogated to identify the total protein concentration of the CSF specimen and whether SAH had ultimately been confirmed or excluded by other methods and supporting evidence. RESULTS: Results from 132 patients were included. The CSF total protein range in confirmed SAH was 0.23-3.08 g/L with a median concentration of 0.7 g/L (n = 51). In the SAH excluded group, the CSF total protein range was 0.43-29 g/L with a median concentration of 1.9 g/L (n = 81). CONCLUSIONS: Although confirmed SAH was not associated with the very highest concentrations of CSF total protein, a definite CSF protein cut-off concentration above which SAH could reliably be excluded cannot be recommended.


Assuntos
Bilirrubina/líquido cefalorraquidiano , Proteínas do Líquido Cefalorraquidiano/análise , Oxiemoglobinas/líquido cefalorraquidiano , Hemorragia Subaracnóidea/líquido cefalorraquidiano , Hemorragia Subaracnóidea/diagnóstico , Humanos , Estudos Retrospectivos , Sensibilidade e Especificidade
3.
J Proteome Res ; 12(7): 3471-9, 2013 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-23742123

RESUMO

The Congenital Disorders of Glycosylation (CDG) are a devastating group of genetic disorders that encompass a spectrum of glycosylation defects and are characterized by the underglycosylation of or the presence of abnormal glycans on glycoproteins. The N-linked CDG disorders (Type I and II) are usually diagnosed in chemical pathology laboratories by an abnormal serum transferrin isoelectric focusing (IEF) pattern. Transferrin has been the protein of choice for CDG analysis because it is well characterized, highly abundant, and easily detected in plasma. However, IEF provides limited information on the glycosylation defect and requires a separate and extensive glycan analysis to diagnose CDG Type II. We have therefore developed a simple bead-based immunoaffinity and mass spectrometry-based assay to address these issues. Our method uses immuno-purified transferrin and proteolytic digestion followed by a rapid 30 min mass spectral analysis and allows us to identify both micro- and macroheterogeneity of transferrin by sequencing of peptides and glycopeptides. In summary, we have developed a simple, rapid test for N-linked glycosylation disorders that is a significant improvement on existing laboratory tests currently used for investigating defective N-linked glycosylation.


Assuntos
Defeitos Congênitos da Glicosilação/diagnóstico , Glicoproteínas/sangue , Transferrina/química , Transferrina/isolamento & purificação , Cromatografia de Afinidade , Defeitos Congênitos da Glicosilação/metabolismo , Defeitos Congênitos da Glicosilação/patologia , Glicoproteínas/isolamento & purificação , Glicosilação , Humanos , Espectrometria de Massas , Peptídeos/química , Peptídeos/classificação , Polissacarídeos/sangue , Polissacarídeos/química , Polissacarídeos/classificação , Transferrina/classificação
5.
J Stroke Cerebrovasc Dis ; 20(6): 489-93, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20719531

RESUMO

The workup of patients with suspected subarachnoid hemorrhage (SAH) presenting late is complicated by a loss of diagnostic sensitivity of computed tomography (CT) brain imaging and cerebrospinal fluid (CSF) bilirubin levels. In this prospective longitudinal study of CSF ferritin levels in SAH, serial CSF samples from 14 patients with aneurysmal SAH requiring extraventricular drainage (EVD) were collected. The control group comprised 44 patients presenting with headache suspicious of SAH. Nine patients underwent a traumatic spinal tap. CSF ferritin levels were significantly higher in the patients with SAH compared with controls (P < .0001). The upper reference range of CSF ferritin is 12 ng/mL, and there was no significant difference between the traumatic and normal spinal taps (mean, 9.0 ng/mL vs 3.9 ng/mL; P = .59). CSF ferritin levels increased after SAH, from an average of 65 ng/mL on day 1 to 1750 ng/mL on day 11 (P < .01). Both the Fisher and Columbia CT scores were significantly correlated with CSF ferritin level. The increase in CSF ferritin level after SAH and possibly may provide additional diagnostic information in patients with suspected SAH who present late to the clinic.


Assuntos
Ferritinas/líquido cefalorraquidiano , Hemorragia Subaracnóidea/diagnóstico , Adulto , Idoso , Biomarcadores/líquido cefalorraquidiano , Estudos de Casos e Controles , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Modelos Lineares , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Estudos Prospectivos , Sensibilidade e Especificidade , Punção Espinal , Hemorragia Subaracnóidea/líquido cefalorraquidiano , Hemorragia Subaracnóidea/diagnóstico por imagem , Hemorragia Subaracnóidea/terapia , Fatores de Tempo , Tomografia Computadorizada por Raios X , Regulação para Cima , Adulto Jovem
6.
Proteomics ; 6(7): 2295-304, 2006 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16552784

RESUMO

A method for the diagnosis of the congenital disorders of glycosylation type I (CDG-I) by SELDI-TOF-MS of serum transferrin immunocaptured on protein chip arrays is described. The underglycosylation of glycoproteins in CDG-I produces glycoforms of transferrin with masses lower than that of the normal fully glycosylated transferrin. Immobilisation of antitransferrin antibodies on reactive-surface protein chip arrays (RS100) selectively enriched transferrin by at least 100-fold and allowed the detection of patterns of transferrin glycoforms by SELDI-TOF-MS using approximately 0.3 microL of serum/plasma. Abnormal patterns of immunocaptured transferrin were detected in patients with known defects in glycosylation (CDG-Ia, CDG-Ib, CDG-Ic, CDG-If and CDG-Ih) and in patients in whom the basic defect has not yet been identified (CDG-Ix). The correction of the N-glycosylation defect in a patient with CDG-Ib after mannose therapy was readily detected. A patient who had an abnormal transferrin profile by IEF but a normal profile by SELDI-TOF-MS analysis was shown to have an amino acid polymorphism by sequencing transferrin by quadrupole-TOF MS. Complete agreement was obtained between analysis of immunocaptured transferrin by SELDI-TOF-MS and the IEF profile of transferrin, the clinical severity of the disease and the levels of aspartylglucosaminidase activity (a surrogate marker for the diagnosis of CDG-I). SELDI-TOF-MS of transferrin immunocaptured on protein chip arrays is a highly sensitive diagnostic method for CDG-I, which could be fully automated using microtitre plates and robotics.


Assuntos
Erros Inatos do Metabolismo dos Carboidratos/sangue , Erros Inatos do Metabolismo dos Carboidratos/diagnóstico , Análise Serial de Proteínas , Sequência de Aminoácidos , Erros Inatos do Metabolismo dos Carboidratos/genética , Eletroforese em Gel Bidimensional , Glicoproteínas/sangue , Glicosilação , Humanos , Dados de Sequência Molecular , Polimorfismo Genético , Análise Serial de Proteínas/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Transferrina/análise , Transferrina/genética , Transferrina/metabolismo
7.
Mol Genet Metab ; 79(2): 142-5, 2003 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12809646

RESUMO

We identified a novel mutation in the glycogen phosphorylase gene (PGYL) in a Chinese patient with glycogen storage disease (GSD) type VI. The patient presented with gross hepatomegaly since the age of two without history of any hypoglycemic attack. Otherwise, he was largely asymptomatic. Liver tissue enzyme assays revealed a mild deficiency of total glycogen phosphorylase. Both PGYL and PHKA2 genes were sequenced. The patient was homozygous of a missense mutation G233D in PGYL. This location forms a hairpin turn secondary structure and the small glycine residue is completely conserved in all the orthologous proteins from Escherichia coli to mammals. This is the sixth reported mutation of this form of GSD.


Assuntos
Glicogênio Fosforilase Hepática/genética , Glicogênio Fosforilase Hepática/metabolismo , Doença de Depósito de Glicogênio Tipo VI/genética , Mutação , Sequência de Aminoácidos , Criança , Sequência Conservada , Éxons , Glicogênio Fosforilase Hepática/química , Doença de Depósito de Glicogênio Tipo VI/metabolismo , Doença de Depósito de Glicogênio Tipo VI/patologia , Hepatomegalia/genética , Hepatomegalia/patologia , Humanos , Masculino , Modelos Moleculares , Dados de Sequência Molecular , Fosforilase Quinase/genética , Conformação Proteica
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