Amniotic fluid α-fetoprotein microheterogeneity in the prenatal diagnosis of congenital disorders of glycosylation type Ia.

BACKGROUND: Congenital disorders of glycosylation are a group of clinically and biochemically diverse defects. The current screening method (based on analysis of transferrin), which is used postnatally for the most frequent types, is however not suitable for prenatal diagnosis. The aim of the study was to investigate whether alterations in the microheterogeneity of α-fetoprotein would provide more reliable results. METHODS: During the 14th-19th weeks of gestation, 140 amniotic fluid samples were obtained by amniocentesis and tested for fetal developmental abnormalities. α-Fetoprotein was analyzed using isoelectric focusing on Immobiline DryPlate pH 4-7, rehydrated in urea (8 mol/L), and molecular forms of the glycoprotein were detected by immunofixation and silver staining. RESULTS: A difference in the relative proportion of individual α-fetoprotein bands (particularly increase of band II density) was found in a case where a congenital disorder of glycosylation was diagnosed postnatally, and in two other samples from pregnancies which resulted in termination, without further examination. CONCLUSIONS: Our potential for further testing is limited; thus far, no other congenital disorders of glycosylation-positive samples have been available. Verification of our results in another laboratory with the exclusion of several potentially pertinent variables is advisable.

Transferrin D protein variants in the diagnosis of congenital disorders of glycosylation (CDG).

Congenital disorders of glycosylation are a rapidly growing group of inherited (neuro)metabolic disorders characterized by defects in glycosylation of proteins and lipids. This study discusses an analytical problem in the differentiation between hypoglycosylation and transferrin (Tf) protein variants. Analysis of serum Tf by isoelectric focusing is used as a common method suitable for screening 19 out of a total of 22 types of glycosylation defects identified so far. In three members of a family, several indicators showed evidence of a Tf protein variant, however, routine neuraminidase-based demonstration failed to confirm this result. On the assumption that we should be able to exclude Tf protein variants at the screening-level of the diagnostic algorithm, our concern is a possible cause of our failure to confirm some of the Tf D variants (in contrast to the other C, B and D allelic combinations that are commonly well identified). Several explanations are discussed.

Screening and diagnosis of congenital disorders of glycosylation.

The aim of this paper is to review the diagnostics of congenital disorders of glycosylation (CDG), an ever expanding group of diseases. Development delay, neurological, and other clinical abnormalities as well as various non-specific laboratory changes can lead to the first suspicion of the disease. Still common screening test for most CDG types, including CDG Ia, is isoelectric focusing/polyacrylamide gel electrophoresis (IEF). IEF demonstrates the hypoglycosylation of various glycoproteins, usually serum transferrin. Other methods, such as agarose electrophoresis, capillary electrophoresis, high-performance liquid chromatography, micro-column separation combined with turbidimetry, enzyme-(EIA) and radioimmunoassay (RIA) have also been used for screening. However, these methods do not recognize all CDG defects, so other approaches including analysis of membrane-linked markers and urine oligosaccharides should be taken. Confirmation of diagnosis and detailed CDG subtyping starts with thorough structure analysis of the affected lipid-linked oligosaccharide or protein-(peptide)-linked-glycan using metabolic labeling and various (possibly mass-spectrometry combined) techniques. Decreased enzyme activity in peripheral leukocytes/cultured fibroblasts or analysis of affected transporters and other functional proteins combined with identification of specific gene mutations confirm the diagnosis. Prenatal diagnosis, based on enzyme assay or mutation analysis, is also available. Peri-/post-mortem investigations of fatal cases are important for genetic counseling. Evaluation of various analytical approaches and proposed algorithms for investigation complete the review.

HPLC profiling of Trp-related metabolites in humans.

Screening for metabolic abnormalities of Trp has been introduced using SPE pre-treatment, TLC and/or two HPLC procedures. The excretory pattern in urine (occasionally also plasma and CSF levels) has been followed in a group of 390 children showing various symptoms of a metabolic defect and in 195 patients with skin diseases, namely those associated with photosensitivity, such as porphyria, vitiligo, alopecia, psoriasis, erythematodes, and others. Excretory abnormalities of either indican, kynurenine, 3-hydroxyanthranilic acid or indolylacryloylglycine have been occasionally combined with myopathy, seizures, liver and intestinal symptoms. Several indoles and kynurenine derivatives present changes in the alopecia group and the vitiliginous patients.

Churg-Strauss syndrome in childhood: a case report.

Churg-Strauss syndrome is a rare form of small-vessel vasculitis. In the current report, we describe the case of a 17-year-old Czech girl predominantly characterized by peripheral neuropathy, the presence of cardiac and pulmonary involvement, hypereosinophilia, asthma, and sinusitis that led to the diagnosis of Churg-Strauss syndrome.

Microheterogeneity of some serum glycoproteins in neurodegenerative diseases.

BACKGROUND: Participation of protein polymorphism is often considered in the pathogenesis of various diseases. Aberrant protein glycosylation has been recognized to play major roles in human disorders, including neurodegenerative diseases. OBJECTIVE: The aim of the study was to examine possible involvement of protein genetic variants and degree of glycosylation of some serum glycoproteins in the manifestation of neurodegenerative disorders in a Czech population sample. METHODS: Apolipoprotein (Apo) E and three main serum markers of glycosylation defects (transferrin, Tf, alpha1-antitrypsin, aAT and ApoCIII) in patients with Alzheimer's dementia (AD), Parkinson's disease (PD) and vascular dementia (n=62, 139 and 44, respectively) were analyzed by isoelectric focusing. Children with serious neurological symptoms (n=55) and three age-matched control groups (n=45, 45 and 42) were examined for comparison. RESULTS: Of the supposedly pathognomonic protein variants Tf C2, aAT ZM and ApoE e4 only the latter was detected with higher frequency in AD patients; significant synergy of the C2/e4 allelic combination was not confirmed. The most prominent finding among PD adults was an increased appearance of Tf C2 allele and significant mean hypoglycosylation of ApoCIII, besides a C2/e4 positive correlation in PD seniors. Laboratory signs of Tf hypoglycosylation and a pattern of Tf/ApoCIII hyperglycosylation have been occasionally found.

Pitfalls and drawbacks in screening of congenital disorders of glycosylation.

Congenital disorders of glycosylation include a group of diseases, each of them caused by different protein (mostly enzyme) impairment due to a specific gene defect. The many subtypes are classified according to clinical features, enzymology and molecular genetic analyses. Problems in diagnostics arise from the great diversity in clinical presentation, usually age-related, and different severities of individual types of these, by far underdiagnosed, diseases. Also the biochemical findings tend to vary, even within a single type. No one screening test, common for all types, is available so far. Several methods of choice may be used in the first approach; other procedures must follow for detailed typing of the defect. Possible drawbacks and pitfalls in the diagnostics from the viewpoint of our 3-year studies and practical screening experience are presented.