[The C1155G mutation of the red blood cell glucose-6-phosphate dehydrogenase gene in a subject with severe hereditary chronic nonspherocytic anaemia]. / Mutacja C1155G genu dehydrogenazy 6-glukozo-fosforanowej krwinek czerwonych u chorego z ciezka wrodzona niesferocytowa niedokrwistoscia hemolityczna.

UNLABELLED: THE AIM of the study is a genetic analysis of hereditary chronic nonspherocytic anaemia in a case, caused by mutation in the glucose-6-phosphate dehydrogenase gene. MATERIALS AND METHODS: The activity of G6PD enzyme was established. PCR method and DNA sequencing were implemented for molecular studies. Bioinformatic methods were used to check the effect of the mutation on the enzyme structure. RESULTS: Direct sequencing of g6pd gene revealed the presence of 1155 C > G mutation which results in cysteine to tryptophan substitution at position 385. Bioinformatic analysis established that this mutation may be responsible for protein destabilization. CONCLUSIONS: 1. G6PD deficiency should be considered in patients with haemolytic anaemia of unknown etiology. 2. Molecular tests are necessary, especially in cases of suspected mutation carriers in G6PD gene.

[A case of hereditary over-hydrated stomatocytosis with stomatospherocytes and spherocytes in the blood]. / Przypadek diedzicznej uwodnionej stomatocytozy z obecnoscia stomatosferocytów i sferocytów we krwi.

UNLABELLED: A patient of 31 years of age with an atypical overhydrated hereditary stomatocytosis is described. The diagnosis was established on the basis of a markedly increased red cell volume with low MCHC, high osmotic fragility of red cells, but increased binding of eosin-5-maleimide (EMA) to red cells, presence of stomatospherocytes and large spherocytes in blood and a high sodium and low potassium concentration in erythrocytes. A double band 7 was found by SDS-PAGE of the erythrocyte membrane, but even when only one them was taken into account, the level of stomatin was normal. Expression of stomatospherocytes in patient's blood was erratic: in blood films prepared in 2005, both stomatospherocytes and large spherocytes were present but in those from 2008 large erythrocytes of spherocyte morphology predominated. Clinically, the disease symptoms were typical for haemolytic anemia. When heparinized blood of the patient was kept at 0 degrees Celsius for 24 h, the haemolysis of red cells amounted only to 2%. The patient's son, 5 years old, suffers from the same disease. CONCLUSION: In spite of its rarity, hereditary stomatocytosis and allied disorders should be taken into consideration in differential diagnosis of haemolytic anemia including newborns. The diagnosis is supported by finding increased binding of eosin-5-maleimide (EMA) dye to patients' erythrocytes associated with their elevated osmotic fragility. Absence of a significant count of stomatocytes in the blood does not exclude the diagnosis of overyhydrated hereditary stomatocytosis.

A novel mutation in the glucose-6-phosphate dehydrogenase gene in a subject with chronic nonspherocytic hemolytic anemia--characterization of enzyme using yeast expression system and molecular modeling.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common human enzymopathy. Human G6PD gene is highly polymorphic, with over 130 mutations identified, many of which cause hemolytic anemia. We studied a novel point mutation in the G6PD gene 1226 C-->G, predicting the proline 409 to arginine substitution (G6PD Suwalki). We expressed the human wild-type and mutated G6PD gene in yeast Saccharomyces cerevisiae which allowed the characterization of the Suwalki variant. We showed that human wild-type, as well as the mutated (1226 C-->G) G6PD gene, functionally complemented the phenotype displayed by the yeast strain with disruption of the ZWF1 gene (homologue of the human G6PD gene). Comparison of wild-type (wt) human G6PD purified from yeast and from blood shows no significant differences in the Km values for G6P and in the utilization rate for the substrate analogue, 2-deoxyG6P. The P409R substitution leads to drastic changes in G6PD kinetics. The specific activity as well as stability of mutated G6PD is also significantly reduced. Besides this, the effect of this mutation was analyzed using a model of the tertiary structure of the human enzyme. The localization of the P409R mutation suggests that it may influence the stability of the whole protein by changing tetramer interactions and disturbing the binding of structural NADP+.

Compound heterozygosity of two missense mutations in the NADH-cytochrome b5 reductase gene of a Polish patient with type I recessive congenital methaemoglobinaemia.

A case of type I methaemoglobinaemia observed in a Polish subject with compound heterozygosity for two mutations in the reduced nicotinamide adenine dinucleotide (NADH) cytochrome b5 reductase (b5R) gene is described. One is a novel mutation 647T-->C which leads to substitution of isoleucine by threonine at position 215 (I215T). This maternal mutation was found in several family members. A previously known mutation, 757G-->A, leads to the replacement of valine by methionine at position 252 (V252M). The latter mutation was found also in the father and one of the two brothers. The effects of these mutations were analysed on a model of the human b5R protein obtained by homology modelling. Although both amino acid substitutions are located in the NADH-binding domain, the whole protein structure, especially the region between the flavin adenine dinucleotide and NADH-binding domains, is disturbed. The structural changes in the I215T mutant are less prominent than those in the V252M mutant. We presume that the 647T-->C mutation is a type I mutation, however, it has not been observed in the homozygous state.