Ziad Albahri

Genetic variants of transferrin in the diagnosis of protein hypoglycosylation

SummaryHuman transferrin (Tf) shows genetic polymorphisms, which may interfere in the screening of congenital disorders of glycosylation (CDG). Isoelectric focusing followed by direct immunofixation was used for Tf analysis in controls and several groups of patients. Equivocal results in one case have been recognized as a rare Tf CD variant. A higher incidence of some genetic variants has been reported in connection with certain diseases; of the seven Tf phenotypes detected in our set of samples, an apparently higher frequency of Tf C1C2 variant found in some groups of patients was not significant.

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 Alzheimers dementia (AD), Parkinsons 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.

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. J. Clin. Lab. Anal. 23:77–81, 2009.

Genetic variants of transferrin in cystic fibrosis

SummaryBackground:Genetic polymorphism of serum transferrin (Tf) was studied in order to differentiate between protein genetic variants and congenital disorders of glycosylation (CDG), further focusing on unusual findings.Methods:Screening of Tf hypoglycosylation was carried out by isoelectric focusing with direct immunofixation and Coomassie blue staining in 100 healthy controls and a group of 1247 patients with various symptoms and diagnoses.Results:Of the seven different genotypes detected, a significantly higher (p = 0.004) frequency of Tf C1C2 was found among 92 patients with cystic fibrosis; only the most severe DF508 mutation (in either homozygous or heterozygous form) was regularly present in the carriers of this Tf genotype, in contrast to those with the Tf C1C1 variant.Conclusions:Association of Tf C2 allele with various malfunctions has been noticed before, but is so far unresolved. This is the a report on increased frequency of Tf C1C2 genotype found in cystic fibrosis. Analysis of larger samples and independent confirmation of our results are needed.

Pitfalls and drawbacks in screening of congenital disorders of glycosylation.

Abstract 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.

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.

CDG: a new case of a combined defect in the biosynthesis of N- and O-glycans.

We report on a 5.5-year-old boy with a multiple malformation syndrome and biochemical abnormalities suggesting a combined defect in the biosynthesis of Nand O-glycans. The patient was followed prenatally for intrauterine growth retardation and a finding of intestinal obstruction (double bubble sign). Born full term as the first child of healthy, non-related Czech parents without remarkable family history, his Apgar scores were 6, 6, and 7; birth weight 2,310 g; body length 45 cm; and head circumference 32.5 cm. He presented with dysmorphic features, including dolichocephaly, low hairline, high nasal bridge, thin lips, small chin, gothic palate, blepharophimosis and epicanthus, in addition to other malformations, such as arachnodactyly, unusual palmar creases, sinus sacralis, pes equinovarus, hypospadias, inguinal hernia, cryptorchidism, vesicoureteral reflux, peripheral pulmonary stenosis, and patent foramen ovale. Shortly after birth a surgical correction of intestinal malrotation and duodenal stenosis (Ladd’s syndrome) was done. Neonatal brain ultrasonography, ophthalmologic examination, a skeletal survey and an electrocardiogram were normal. Chromosome analysis was normal. Laboratory tests including serum lactate, ammonia, and immunological investigations were within normal limits. Serum biotinidase, plasma and urinary amino acids, organic acids, purines and pyrimidines, sugars, oligoand mucopolysaccharides did not reveal any pathological changes. At age 4 months, the brain ultrasonography showed hypogenesis of the posterior part of the corpus callosum (later confirmed by MRI), and abnormal EEG delta waves were noted occipitally, without epilepsy. In addition to anemia (90 g/l, normal 105–135), elevated LDH (12.7 μkat/l, normal< 4.4) and elevated transaminases (1.1–2.2 μmol/l, normal<0.8), prolonged values of APTT (39.3–45.6, normal range 31.8–35.0) and PT (13.3, normal 11.7) were found; antithrombin III and CK were within the physiological range. At 3.5 years, the boy was admitted to hospital with severe laryngopharyngitis, and at that time a screening for congenital disorders of glycosylation (CDG) was performed. Isoelectric focusing (IEF) and HPLC of serum transferrin (Tf) showed a slight increase in asialo-Tf (4.0%; reference interval, 0.0–3.0%), a marked increase in disialoTf (18.7%; reference interval, 2.5–9.8%), increased trisialo-Tf (17.8%; reference interval, 3.4–13.7%) and decreased tetrasialo-Tf (38.8%; reference interval, 43.7– 68.1%). Abnormalities of alpha1-antitrypsin (aAT) and thyroxin-binding globulin pointed to a generalised defect Z. Albahri . E. Marklova . P. Dědek Department of Pediatrics, Faculty of Medicine, Charles University, Hradec Kralove, Czech Republic

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.

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

Abstract 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. Clin Chem Lab Med 2010;48:1281–5.