Anna Adamowicz-Salach

Erythrocyte membranes from a patient with congenital dyserythropoietic anaemia type I (CDA‐I) show identical, although less pronounced, glycoconjugate abnormalities to those from patients with CDA‐II (HEMPAS)

Congenital dyserythropoietic anaemias (CDAs) are rare hereditary disorders characterized by ineffective erythropoiesis and multinuclearity of erythroblasts. Three main types of the disease have been described. Glycoconjugate abnormalities in erythrocyte membrane glycoconjugates, consisting of hypoglycosylation of band 3 and accumulation of certain glycosphingolipids including lactotriaosylceramide, neolactotriaosylceramide and polyglycosylceramides, have been described only in patients with CDA type II (CDA‐II). We report on identical, although less pronounced, abnormalities in erythrocyte glycoconjugates from a patient with CDA‐I. A low degree of hypoglycosylation of band 3 in our patient with CDA‐I suggests that hypoglycosylation is not a cause, but, most probably, a consequence of dyserythropoiesis.

Novel beta-spectrin mutations in hereditary spherocytosis associated with decreased levels of mRNA

Hereditary spherocytosis (HS) is one of the most frequent and heterogeneous inherited haemolytic anaemias. It is associated with abnormalities of several erythrocyte membrane proteins. We investigated relative mRNA quantification of red blood cell membrane protein genes using real‐time quantitative polymerase chain reaction (qPCR) in order to better characterize HS cases and to select genes to search for mutations in patients with spherocytosis. qPCR experiments indicated that the spectrin β gene (SPTB) could be involved in anaemia pathogenesis. DNA analysis of SPTB in the HS subjects with decreased SPTB mRNA levels revealed the presence of five previously undescribed mutations: R1756X, 781delT and IVS22nt‐4G>A, 1502insA and IVS20nt‐2A>G.

Coexistence of Gilbert syndrome with hereditary haemolytic anaemias

Background Gilbert syndrome is an inherited disease characterised by mild unconjugated hyperbilirubinaemia caused by mutations in UGT1A1 gene which lead to decreased activity of UDP-glucuronosyltransferase 1A1. The most frequent genetic defect is a homozygous TA dinucleotide insertion in the regulatory TATA box in the UGT1A1 gene promoter. Methods and results 182 Polish healthy individuals and 256 patients with different types of hereditary haemolytic anaemias were examined for the A(TA)nTAA motif. PCR was performed using sense primer labelled by 6-Fam and capillary electrophoresis was carried out in an ABI 3730 DNA analyser. The frequency of the (TA)7/(TA)7 genotype in the control group was estimated at 18.13%, (TA)6/(TA)7 at 45.05% and (TA)6/(TA)6 at 36.26%. There was a statistically significant difference in the (TA)6/(TA)6 genotype distribution between healthy individuals and patients with glucose-6-phosphate dehydrogenase deficiency (p=0.041). Additionally, uncommon genotypes, (TA)5/(TA)6, (TA)5/(TA)7 and (TA)7/(TA)8 of the promoter polymorphism, were discovered. Conclusion Genotyping of the UGT1A1 gene showed distinct distribution of the common A(TA)nTAA polymorphism relative to other European populations. Because of a greater risk of hyperbilirubinaemia due to hereditary haemolytic anaemia, the diagnosis of Gilbert syndrome in this group of patients is very important.

Laparoscopic splenectomy for hereditary spherocytosis-preliminary report.

Splenectomy is considered standard surgical therapy in hereditary spherocytosis. The procedure is indicated in patients with severe anemia, recurrent hemolytic, and aplastic crises. The aim of the study was to assess treatment outcomes in patients with hereditary spherocytosis who underwent total or partial laparoscopic splenectomy. Fifteen patients aged 4–17 yr underwent laparoscopic splenectomy from 2009 to 2012. Partial and total splenectomies were performed (five and 10 children, respectively). Hematologic parameters, liver function tests, and splenic volume before and after the surgery were analyzed retrospectively. Total follow‐up was 1–30 months. Hospitalization and operating time were similar in both groups. In partial splenectomy group, branches of splenic arteries gave better blood supply than short gastric vessels. In both groups, hematologic parameters were improved. Postoperative markedly elevated platelet count was maintained up to 6 months, and after that, platelet count gradually decreased to normal values. Bilirubin level was decreased in early postoperative period; however, it increased later to achieve levels lower than in preoperative period. No severe general infections were observed in both groups. Laboratory parameters (hemoglobin and bilirubin concentrations and RBC) after the surgery improved in all patients, and the effect was maintained during 12 months of follow‐up. Platelet count increased significantly after the surgery and was maintained at high levels during the next 6 months. However, it returned to preoperative levels within a year after the surgery. Our study showed that partial splenectomy was not inferior to total splenectomy. However, full assessment requires longer follow‐up and larger group of patients.

Delay in the measurement of eosin-5′-maleimide (EMA) binding does not affect the test result for the diagnosis of hereditary spherocytosis.

Abstract Background: The eosin-5′-maleimide (EMA) binding test is a flow cytometric test widely used to detect hereditary spherocytosis (HS). EMA binds to plasma membrane proteins of red blood cells (RBCs), mainly to band 3 protein. The mean fluorescence of EMA-stained RBCs in HS patients is lower when compared with control RBCs due to the decreased amount of target proteins. EMA dye in aqueous solution is sensitive to light and high temperature. Its fluorescence can decrease when exposed to light or ambient temperatures higher than 4°C. The aim of the study was to evaluate the stability of fluorescence readings of EMA-labeled RBCs over a period of 24 h. Methods: The EMA test was performed in peripheral blood from 35 patients with microcytic anemia (five with HS, and 30 without HS). Peripheral blood samples were stained immediately after blood collection and analyzed using a flow cytometer at three time points: 0, after 1 and 24 h of storage at 4°C in the darkness. The results are presented as the percentage of normal control RBCs fluorescence. Flow cytometric studies were performed with Cytomics FC500 (Beckman Coulter, USA). Results: In HS patients the mean result of the test reached 66.72%±9.26% of normal controls, and in non-HS patients the EMA result was 99.48%±5.03% of normal control cells. The results of patients with HS were 66.72%±9.26%, 66.90%±10.24% and 67.86%±11.31% at 0 h, and after 1 and 24 h of storage, respectively. The results obtained from non-HS patients at time 0, after 1 and 24 h of storage reached 99.48%±5.03%, 99.49%±5.34% and 99.78%±6.13%, respectively. There was no difference between the results from each time point in samples from patients with or without HS. Conclusions: Results of the EMA binding test do not depend on storage time of stained samples when stored at 4°C up to 24 h after staining.

Diversity of Thalassemia Variants in Poland – Screening by Real-Time PCR

-Thalassemia is a very common disease in tropical and subtropical regions of the world where it provides protection against malaria, but it is a very rare disease in Northern Europe. Some limited cases have been de-scribed in the British, Irish and Breton populations [4, 5] . The presence of thalassemia minor and intermedia in the indigenous Polish population has never been studied. The aim of the present study was to look for mutations in the -g lobin gene in patients diagnosed with or suspect-ed for thalassemia and in individuals with a primary di-agnosis of atypical spherocytosis. We also discuss the functional effects of different mutations on the mRNA levels of globins. We analyzed 21 unrelated Polish subjects with hemo-globinopathies, thalassemia intermedia and thalassemia traits. The hematological and genetic data of the patients and their relatives are listed in tables 1 and 2, r espective-ly. Two patients with - thalassemia (cases 20 and 21) have also been included to enable a comparison of the amounts of - and -globin mRNAs in different types of thalas-semia. All patients gave written informed consent. The study protocol was approved by the Ethics Committee of the Medical University of Warsaw. Thalassemia syndromes – one of the most common monogenic diseases – are classified according to the im-paired production of a particular globin chain or chains. The major categories include -thalassemia, i.e. a defi-ciency in -globin synthesis, and haht e emt -slas. e . ia, iproduction of -globin is downregulated [1] . O ver 200 different - thalassemia mutations have now been characterized worldwide. The clinical manifesta-tions of -thalassemia are extremely diverse, ranging from a mild, clinically insignificant state of -esas -thalmia minor to a severe, life-threatening and transfusion-dependent state of - thalassemia major. The diagnosis of thalassemia intermedia may be particularly challenging, since clinical phenotypes range from thalassemia minor to thalassemia major. Mildly affected patients with thal-assemia intermedia are completely asymptomatic until adult life, experiencing only mild anemia and maintain-ing hemoglobin levels between 7 and 10 g/dl. These pa-tients require only occasional blood transfusions, if any. Patients with more severe thalassemia intermedia gener-ally present anemia between 2 and 6 years of age, and al-though they are able to survive without regular transfu-sion therapy, growth and development can be retarded[2, 3] .

Molecular and haematological studies of four families with hereditary spherocytosis resulting from band 3 deficiency

In this report we present four families with HS resulting from primary band 3 defi ciency caused by mutations of different conserved arginines. Case 1. A 17-year-old male was hospitalized because of jaundice, anaemia and splenomegaly. Congenital dyserythropoietic anaemias, haemoglobinopathies and anaemias due to defi ciency of red cell enzymes were excluded. At the age of 14, he was diagnosed with Gilbert syndrome but the cause of anaemia remained unclear. His brother and father showed similar symptoms of haemolytic anaemia. Case 2. A 7-year-old girl was under medical care because of chronic compensated haemolytic disease with splenomegaly. A mild degree of anaemia was observed mostly during infections, but the aetiology of the disease was unknown. Her parents and younger brother showed no symptoms of haemolytic anaemia. Case 3. A 40-year-old man had been under observation for 20 years because of anaemia, jaundice and splenomegaly. His father and paternal grandmother had jaundice and splenomegaly. Haemolytic anaemia was diagnosed and HS was suspected. Three sons of the proband showed no symptoms of haemolytic anaemia. Case 4. A 30-year-old woman was under medical care because of anaemia, jaundice and hepatosplenomegaly. Clinical examinations confi rmed the spherocytic haemoHereditary spherocytosis (HS) is a common and very heterogeneous haemolytic anaemia caused by defects in red blood cell (RBC) membrane proteins. It has been estimated that HS occurs with a prevalence rate of 1: 2,000. Clinical manifestations of the disease range from mild cases of a stabilized haemolytic form to severe ones requiring regular blood transfusions [1, 2] . About 15–20% of American and European patients with HS have band 3 defi ciency. The disease is inherited as a dominant trait. Band 3 is the major integral protein in the red cell membrane that interacts with the membrane skeleton and is essential for the stability of the membrane lipid bilayer [3] . Band 3 protein, containing 911 amino acid residues, consists of two distinct domains: N-terminal cytoplasmic domain and C-terminal transmembrane domain [4] . The N-terminal domain is involved in interactions with a number of cytoskeletal proteins, haemoglobin and glycolytic enzymes. The C-terminal domain spans the lipid bilayer up to 14 times [5–7] . This domain is responsible for anion transport and is involved in chloride-bicarbonate exchange [8–10] . It has recently been shown [11] that the last two transmembrane segments (residues 837–874) are particularly important for anion translocation. Because of this function, band 3 has been also designated as an anion exchange protein (AE1) . Received: August 3, 2005 Accepted after revision: November 8, 2005

Ocena wpływu długości okresu przechowywania próbki krwi na wynik testu EMA. Doniesienie wstępne

Streszczenie Test EMA (test wiązania eozyno-5-maleimidu) jest badaniem diagnostycznym wykonywanym w celu rozpoznania sferocytozy wrodzonej. Celem naszej pracy byla ocena wplywu czasu przechowywania probek krwi na wartości wynikow testu EMA. Na podstawie naszych wstepnych obserwacji wydaje sie, ze opoźnienie wykonania testu EMA o 3–7 dni nie wplynelo na wynik badania i jego interpretacje.

The use of real‐time PCR technique in the detection of novel protein 4.2 gene mutations that coexist with thalassaemia alpha in a single patient

α‐Thalassaemia is a very rare disease in Northern Europe in contrast to hereditary spherocytosis that is associated with red blood cell membrane defects. We report here α‐thalassaemia case who was also found to bear the erythrocyte membrane protein 4.2 gene mutations. mRNA relative quantification of red cell membrane protein genes in a Polish patient with α‐thalassaemia trait indicated EPB42 as the gene that could also be involved in anaemia pathogenesis. Sequencing revealed the presence of two novel mutations in the protein 4.2 gene: a G1701A genetic change that predicts an alanine to threonine at position 567 of the protein (A567T) and a T→A substitution that is located at position +6 of the donor splice site of intron 2 (IVS2nt+6T>A). This is the sixth variant of the erythrocyte membrane protein 4.2 gene mutations identified outside the Japanese population.

Two novel C-terminal frameshift mutations in the β-globin gene lead to rapid mRNA decay

BackgroundThe thalassemia syndromes are classified according to the globin chain or chains whose production is affected. β-thalassemias are caused by point mutations or, more rarely, deletions or insertions of a few nucleotides in the β-globin gene or its immediate flanking sequences. These mutations interfere with the gene function either at the transcriptional, translational or posttranslational level.MethodsTwo cases of Polish patients with hereditary hemolytic anemia suspected of thalassemia were studied. DNA sequencing and mRNA quantification were performed. Stable human cell lines which express wild-type HBB and mutated versions were used to verify that detected mutation are responsible for mRNA degradation.ResultsWe identified two different frameshift mutations positioned in the third exon of HBB. Both patients harboring these mutations present the clinical phenotype of thalassemia intermedia and showed dominant pattern of inheritance. In both cases the mutations do not generate premature stop codon. Instead, slightly longer protein with unnatural C-terminus could be produced. Interestingly, although detected mutations are not expected to induce NMD, the mutant version of mRNA is not detectable. Restoring of the open reading frame brought back the RNA to that of the wild-type level.ConclusionOur results show that a lack of natural stop codon due to the frameshift in exon 3 of β-globin gene causes rapid degradation of its mRNA and indicate existence of novel surveillance pathway.