Ayşenur Öztürk

Blood group genotyping in multi-transfused patients

BACKGROUND In chronically transfused patients, the classical hemagglutination assays may be inaccurate in defining the RBC phenotypes of the patients due to previous transfusions. DESIGN DNA samples from 39 multi-transfused patients including thalassemia and sickle cell disease were used for red blood cell genotyping. The Rh-Type and KKD-Type (BAGene, BAG Healthcare) were used to determine the polymorphisms associated with antigen expression for RHD, RHCE and Kell, Kidd, Duffy blood group systems, respectively. Results were compared with previously determined phenotyping results for RhD, RhCcEe and Kell by hemagglutination method. RESULTS Nineteen out of the 37(51%) patients had discrepancies between genotyping and phenotyping results in a total of 25 alleles. In 12 patients, the discrepancies had the potential of alloimmunization. CONCLUSION Blood group genotyping has vital importance in transfusion management of chronically transfused patients especially if the patients were not phenotyped before starting the initial transfusions.

Protein Z G79A Polymorphism in Patients With Severe Sepsis

The aim of the study is to investigate whether the presence of a protein Z polymorphism is a risk factor for the development and outcome of sepsis. Sepsis is a clinical syndrome characterized by the presence of systemic signs and symptoms of inflammation. When sepsis leads to organ failure, the term severe sepsis and septic shock is used. The genetic causes of severe sepsis are not fully explained. Protein Z is a vitamin K— dependent glycoprotein and a member of the coagulation cascade. The study included 53 patients with severe sepsis and 70 control healthy volunteers without a familial history of thrombosis. The G79A polymorphism of intron F of the protein Z gene was analyzed by the method of polymerase chain reaction—based DNA analysis. The protein Z intron F G79A polymorphism frequencies of the patients and controls were 43.4% and 40%, respectively. Carrying 79 AA genotype could be a risk factor for severe sepsis and septic shock (OR = 4.5, 95% CI: 0.45-46.1), but it could not find any difference between survivor and nonsurvivor groups. They concluded that the frequency of intron F G79A polymorphism of protein Z gene was higher in patients than controls, and carrying 79 AA genotype could be a risk factor for severe sepsis and septic shock.

Intron F G79A Polymorphism of the Protein Z Gene in Turkish Behçet Patients

Purpose: Vasculitis is one of the major findings of Behçet’s disease (BD). Protein Z (PZ) is a glycoprotein that acts as a cofactor of PZ-dependent protease inhibitor and suppresses trombus formation by inhibiting activated factor Xa. Polymorphism of the PZ gene was mentioned as a genetic risk factor for various thrombotic events. The aim of this study is to investigate the intron F G79A polymorphism of the PZ gene in Behçet patients with and without ocular involvement. Methods: Seventy-six patients and 70 controls were included in the study. Intron F G79A polymorphism of PZ gene was determined by polymerase chain reaction based DNA analysis. The frequency of A allele and the distribution of genotypes were assessed by χ2 test and the genotype distribution and Hardy–Weinberg equilibrium were tested with the χ2 test for quality of fit. Results: The frequency of the A allele was significantly higher in overall Behçet patients than in controls (odds ratio [OR] = 6.8; 95% CI, 2.6 to 17.9; p = 0.0001). It was also significantly higher in patients with (OR = 5.3; 95% CI, 1.83 to 15.6; p = 0.0024) or without (OR = 8.2; 95% CI, 2.95 to 22.5; p = 0.0001) ocular involvement compared to controls. However, A allele frequency was not significantly different between patients with eye involvement versus patients without eye involvement (OR = 0.65; 95% CI, 0.3 to 1.4; p = 0.28). Conclusions: Although thrombosis in BD is multifactorial, intron F G79A polymorphism of PZ gene in BD may be one of the factors that contribute to this pathological process.

Determination of Factor V Leiden Mutation and R2 Polymorphism in Cis Position

FVA4070G (R2 polymorphism) influences plasma factor V (FV) concentration and was associated with mild activated protein C resistance. This polymorphism was reported to have a trans inheritance with FV Leiden (FVL) mutation. The aim of this study is to investigate the inheritance of R2 polymorphism in the homozygous FVL carriers. In this study, 99 patients with thrombosis and 7 individuals without a history of thrombosis all of which homozygous for FVL were included. Of 99 patients, 1 was heterozygous for FV A4070G. Additionally, 6 polymorphisms in the FV gene were analyzed for the heterozygous R2 patient and her family. When the allelic distribution was classified, 8 different haplotypes were obtained. In contrast to the literature, it was shown that R2 polymorphism could be inherited in cis position with FVL and also the family members could have co-inheritance of the FVL and R2 on the same chromosome as proband.

Hemoglobin Lansing (Alpha) [HBA2 CD87 (HIS>GLU) (C>A)] in a Turkish Individual Resulting from Another Nucleotide Substitution

Received/Gelis tarihi : March 4, 2014 Accepted/Kabul tarihi : April 14, 2014 To the Editor, Several hemoglobin variants, including novel ones, have been reported in the Turkish population [1,2,3]. Herein, we describe a novel nucleotide alteration of the alpha-2 chain variant, hemoglobin (Hb) Lansing CD87 (HIS>GLU). The index case was a 21-year-old Turkish woman living in Ankara. She was admitted to the TOBB-ETU University Pediatrics Outpatient Department for premarital counseling. Her physical examination was normal. Hemoglobin, hematocrit, and MCV values were 13.1 g/dL, 42.7%, and 95 fL, respectively. Levels of Hb A1, Hb A2, and Hb X were observed as 65.5%, 1.88%, and 22.24%, respectively, with high-pressure liquid chromatography. Written informed consent for genetic analysis was obtained from the patient. DNA was isolated from a peripheral blood sample with the phenol-chloroform protocol. All of the exons of the HBB, HBA1, and HBA2 genes were amplified by polymerase chain reaction (PCR). The entire coding and intronic sequences of the alpha-1 and alpha-2 globin genes were amplified as one amplicon each. While the forward primer was the same for the 2 genes, the reverse primers were specific to the alpha-1 and alpha-2 genes. These amplicons were sequenced using internal primers as described previously [4,5]. PCR products were cleaned with a PCR purification kit (Roche, Germany) and then samples were sequenced using an automatic DNA Sequencer (Beckman Coulter, USA). The DNA was also tested for the -α3.7, -α4.2, -MED, and -α20.5 deletions using multiplex PCR according to the described methods [6,7].

Both granulocytic and non-granulocytic blood cells are affected in patients with severe congenital neutropenia and their non-neutropenic family members: An evaluation of morphology, function and cell death

Objective: To examine granulocytic and non-granulocytic cells in children with severe congenital neutropenia (SCN) and their non-neutropenic parents. Materials and Methods: Fifteen patients with SCN and 21 non-neutropenic parents were evaluated for a) CD95, CD95 ligand, annexin V, propidium iodide, cell cycle, and lymphocyte subsets by flow cytometry; b) rapid cell senescence (of leukocytes) by senescence-associated β-galactosidase stain; c) aggregation tests by aggregometer; d) in vitro bleeding time by PFA-100 instrument; e) mepacrine-labeled dense granule number of thrombocytes by fluorescence microscope; and f) hematomorphology by light and electron microscope. HAX1, ELANE, G6PC3, CSF3R, and JAGN1 mutations associated with SCN were studied in patients and several parents. Results: Significant increase in apoptosis and secondary necrosis in monocytes, lymphocytes, and granulocytes of the patients and parents was detected, irrespective of the mutation type. CD95 and CD95 ligand results implied that apoptosis was non-CD95-mediated. Leukocytes of 25%, 12.5%, and 0% of patients, parents, and controls showed rapid cell senescence. The cell cycle analysis testable in four cases showed G1 arrest and apoptosis in lymphocytes of three. The patients had HAX1 (n=6), ELANE (n=2), G6PC3 (n=2), and unidentified (n=5) mutations. The CD3, CD4, and NK lymphocytes were below normal levels in 16.6%, 8.3%, and 36.4% of the patients and in 0%, 0%, and 15.4% of the parents (controls: 0%, 0%, 5.6%). The thrombocytes aggregated at low rates, dense granule number/thrombocyte ratio was low, and in vitro bleeding time was prolonged in 37.5%-66.6% of patients and 33.3%-63.2% of parents (vs. 0% in controls). Under electron and/or light microscope, the neutrophils, monocytes, lymphocytes, and thrombocytes in the peripheral blood of both patients and parents were dysplastic and the bone marrow of patients revealed increased phagocytic activity, dysmegakaryopoiesis, and necrotic and apoptotic cells. Ultrastructurally, thrombocyte adhesion, aggregation, and release were inadequate. Conclusion: In cases of SCN, patients’ pluripotent hematopoietic stem cells and their non-neutropenic parents are both affected irrespective of the genetic defect.

Identification of four common α-thalassemia gene deletions among a group with hemoglobinopathies in Sétif population, Algeria

Abstract α-Thalassemia (α-thal) is one of the most common genetic disorders in the world. It is characterized by the absence or reduced expression of α-globin genes. This study was carried out to evaluate the allelic frequency of α-thal defects in a patient for the first time in Sétif (Algeria). One hundred and two patients with hemoglobinopathies from Sétif region, Algeria, presenting thalassemia were included in this study. Genomic DNA isolation was carried out according to standard methods. For identifying the α-thal genotype, investigation of α-globin gene deletions (-α3.7, -α4.2, -MED and -α20.5) was performed by using multiplex-polymerase chain reaction (PCR). Among the three deletions found, the most mutations were the -α3.7 (10.78%), followed by the -MED (5.88%) and -α20.5 (0.98%), whereas the -α4.2 deletion was not observed (0.0%). The allele frequency is 0.054 (11/204) for the 3.7 deletion, 0.029 (6/204) for the MED and 0.005 (1/204) for the 20.5. Molecular heterogeneity of mutations is typical of α-thal in Algeria. Our findings will be valuable and essential for the molecular diagnosis and prevention strategies of hemoglobinopathy gene mutations in the Algerian population.

The incidence of alpha-thalassemia in Iraqi Turks

Thalassemias are characterized by impaired quantitative synthesis of globin chains. Several mutations have been identified in patients with thal-assemia, which are usually in alpha-or beta-globin genes. Alpha-thalassemia commonly occurs in Southeast Asian, Mediterranean, and Middle Eastern populations [1]. The gene responsible for the alpha-globin chain is located on the short arm of chromosome 16 (16p13.3) and consists 2 zeta, 2 pseudo-alpha, and 2 alpha genes (α1 and α2) [2]. Each of the homologous chromosomes has 2 alpha genes; thus, there are 4 functional alpha genes in total. Molecular defects in alpha-thalassemia are usually gene deletions. Deletions of 1, 2, 3, or all 4 of the alpha genes may occur, and the severity of disease is directly proportional to the number of affected alpha genes. The most common of these are-α 3.7 and-α 4.2 single alpha-globin gene deletions, and-MED and-α 20.5 double gene deletions, which are widespread in the Mediterranean region [3]. The most common genotypes reported in the Dohuk region of Iraq were-α 3.7 /αα,-MED /αα, and-α 3.7 /-α 3.7 , which were observed in 84.3% of patients [4]. Another study reported that-α 3.7 and-MED deletions were common mutations [5]. Additionally,-α 3.7 deletion and alpha-globin triplication anti-3.7 kb type were observed in an Iraqi family with beta-thalassemia [6]. Nonetheless, there are no data on the frequency of alpha-thalassemia gene deletions in Iraqi Turks. As such, the present study aimed to determine the molecular characterization of the alpha-thalassemia gene in healthy Iraqi Turks, in terms of-α 3.7 ,-α 4.2 ,-MED , and-α 20.5 deletions. Iraqi Turkmens are the descendants of the Oghuz Turks that originated from Central Asia, an ethnic group that now primarily lives in northern Iraq. The study group included 83 unrelated individuals from northern Iraq: 39 from Kirkuk, 20 from Mosul, 10 from Arbil, 10 from Baghdad, and 4 from the Diala and Tikrit regions. After all the participants provided informed consent blood samples were collected into tubes containing EDTA, and then DNA was extracted from peripheral blood leukocytes using the phenol-chloroform method. Multiplex poly-merase chain reaction (PCR) was performed for mutation analysis, as previously described [7,8]. In all, 8 of the 83 participants were diagnosed with alpha-thalassemia an incidence rate of 9.6%. Multiplex PCR analysis of the 83 blood samples showed that the incidence of alpha-thalassemia, particularly 3.7 kb deletion, was high in the Iraqi Turk study population. In total, 3 alpha-globin genotypes were identified; the incidence …

The incidence of alpha-thalassemia in Setif, Algeria

Thalassemias are hereditary anemia syndromes occurring due to erroneous producing of globin chain of hemoglobin. Thalassemia syndromes are named according to the type of the affected globin chain. The most common types of thalassemia are alpha thalassemia and beta thalassemia. In α-thalassemia, α-globin chain production is either by decreased or completely disappeared. The gene encoding the α-globin chain localized on the short arm of chromosome 16 (16p13.3). In normal adults, alpha-globin genes are found in four copies. One copy of each α1 and α2 genes are located in cis position on DNA chain [1]. The alpha-globin gene deletions constitute 80-90% of the mutations. The carriers for α-thalassemia have either three (-α/αα) or two (-/αα) α genes. The most common clinical form is HbH disease and the most frequently encountered genotype of Hb H disease is-/-α and rarely Hb Barts hydrops fetalis in which all four genes are deleted. In Mediterranean region,-α 3.7 ,-α 4.2 ,-MED and-α 20.5 deletions are the most prevalent molecular defects [2]. The mutations of α-thalassemia have been reported as-α 3.7 ,-MED ,-α 20.5 and α Hph I α in Algerian population [3,4] and-α 3.7 was determined as the most frequent haplotype [3]. In a recent study, in addition to these mutations, α Nco I α was shown and α-thal allele frequency found 4.6%, with the-α 3.7 haplotype being 2.9% in randomly selected blood donors in Algiers, the capital city of Algeria which is located at the Mediterranean Sea coast [5]. On the other hand, Setif province is located in the high plateau of northeast Algeria, approximately 100 kilometers from the Mediterranean Sea. This is the first study for the molecular characterization of the α-thal gene frequency in healthy individuals from the Setif region. The study group included 153 unrelated healthy individuals from Setif. Informed consent was obtained from all the participants. Blood samples were collected with EDTA-containing tubes, transferred and DNA was extracted from peripheral blood leukocytes according to phenol-chloroform method. Genomic DNA was tested for the-α 3.7 ,-α 4.2 ,-MED and-α 20.5 deletions using multiplex-poly-merase chain reaction (PCR) according to described methods [6,7]. The prevalence of alpha thalassemia trait was found to be 6.5% in the study group. The molecular characterization of the α-thal defects in these subjects revealed-α 3.7 allele frequency as 3.3%. We have not found any other individual of carrying

Ribosomal protein S19 - 631 insertion is an African-originated mutation

Ribosomes, the organelles that catalyze protein synthesis, consist of a small 40S subunit and a large 60S subunit. RPS19 gene encodes a ribosomal protein (RP) that is a component of the 40S subunit. The protein belongs to the S19E family of RPs. It is located in the cytoplasm. Mutations in this gene cause DiamondBlackfan anemia (DBA), a constitutional erythroblastopenia characterized by absent or decreased erythroid precursors in 25% of the patients. This suggests a possible extra-ribosomal function for this gene in erythropoietic differentiation and proliferation, in addition to its ribosomal function [1,2]. The RPS19 gene is located on chromosome 19q13.2 and has six exons and spans 11 kb. The first exon is untranslated, and the start codon (AUG) is located at the beginning of exon 2 [1]. RPS19 has three annotated pseudogenes. The RPS19 gene has 196 sequence variants, of which 65 had no known pathogenicity. Recent studies have provided evidence for an association between common polymorphic markers in the RPS19 exon 1 gene -631 locus insertion (ins) GCCA, AGCC and African origin [3]. At the same location, there are two common polymorphisms, -631 ins GCCA, AGCC refsnp:34020014 [4]. As previously reported, these polymorphisms do not have any effect on phenotype. The common polymorphism -631 ins GCCA was found in African-Americans with an allele frequency of 0.09 [3]. We aimed to study the frequency of this polymorphism in North African countries and also in Turkish Cypriots. In this study, 280 Egyptians, 105 Algerians, 92 Turkish Cypriots and 6 Hemoglobin (Hb) OArab cases were included. RPS19 gene exon 1 was amplified with “F5’TTA CTA CTC CCA CTT CCG GCC AGG GAA CAG 3’, R5’TCA GGC ACG CGC GCT CTG AGG CTT CGG CGT C3’ ” primers followed by digestion with the restriction enzymes HpyF10VI (MwoI, Fermentas, USA). HpyF10VI recognizes 5’-G C N N N N N^N N G C-3’. 3% agarose gel electrophoresis was used to show the fragments, which are 295bp, 158bp and 73bp for normal sample and 173bp, 158bp, 126bp, and 73bp for homozygous sample. In this study, we aimed to analyze the -631 ins GCCA mutation in three different Mediterranean populations, of which two were North African countries. Table 1 shows the genotype distribution in the three countries. Previously, the RPS19 gene -631 ins was reported as an African marker in African-Americans in the United States population [3]. In order to test this hypothesis, we analyzed individuals from two different North African countries. Although rare, we found this polymorphism in Algerians and Egyptians. Our finding supported the hypothesis. Ozge Cumao ullar 1, Ay enur Ozturk1, Nejat Akar1, Solaf Elsayed2, Ezzat Elsobky2, Bakhouche Houcher3 1Pediatric Molecular Genetic Department, Ankara University, Ankara, Turkey 2Pediatric Hospital, Ain Shams University, Cairo, Egypt 3Department of Biology, University of Setif, Faculty of Sciences, Setif, Algeria