Genetic heterogeneity of beta-thalassemia at Cukurova in southern Turkey.

Beta-thalassemia is the most common genetic abnormality causing health problems worldwide. Cukurova, in the southern part of Turkey, being on the Mediterranean, is in the thalassemic belt. Since there is no cure for the disease at present, the frequency of the mutation types of beta-thalassemia must first be identified to aid in clinical follow-up and prenatal diagnosis. Carriers identified during a screening survey and patients referred to our laboratory were studied for this purpose. After routine hematological analysis molecular screening was performed by the amplification refractory mutation system and DNA sequencing. The frequency of the common mutations were: IVS-I-110 (G-->A) 57.3%, IVS-I-1 (G-->A) 8.3%, codon 39 (C-->T) 6.4%, IVS-I-6 (T-->C) 5.7%, frameshift codon 8 (-AA) 5.7%, -30 (T-->A) 4.7%, IVS-II-1 (G-->A) 3.4%, IVS-II-745 (G-->C) 2.8%, and frameshift codon 5 (-CT) 1.1%. Some rare mutations (1%) such as frameshift codon 44 (-C) 0.7%, frameshift codons 74/75 (-C) 0.7%, IVS-1-5 (G-->C) 0.7%, frameshift codons 8/9 (+G) 0.4%, frameshift codons 36/37 (-T) 0.4%, frameshift codons 22/23/24 (-AAGTTGG) 0.4%, IVS-1-130 (G-->C) 0.4%, IVS-1-5 (G-->T) 0.2%, -28 (A-->C) 0.2%, codon 15 (TGG-->TGA) 0.2%, and frameshift codons 82/83 (-G) 0.2%, were detected by sequence analysis. The codon 15 (TGG-->TGA) and frameshift codons 82/83 (-G) mutations were seen in Turkey for the first time.

Screening of Hemoglobinopathies in Kahramanmaras, TURKEY.

Thalassemia and sickle cell anemia are prevalent in southern Turkey. Being in close proximity to Çukurova we screened Kahramanmaras to assess the prevalence and foci of the diseases. The sample sizes were calculated by EpiInfo 6.0 computer program at 95% confidence level. 1491 subjects aged 2-69 were studied. Hematological parameters were analyzed by an electronic cell counter. Electrophoresis were performed and Hemoglobin A2 and hemoglobin F levels were determined on samples with MCV< 80 fL. The results of Canatan et al. on Elbistan were included in the final results. Thus, the prevalence of ß-thalassemia, hemoglobin D, Hemoglobin O Arab carriers were 0.68%, 0.28% and 0.013%, respectively. No hemoglobin S was detected. In conclusion, Kahramanmaras seems not to be a high risk area but the public must be informed about these diseases. Every community in close proximity to high prevalence areas must be enlightened.

Evaluation of reference values for erythrocyte glutathione.

The analytical, intra-individual and inter-individual variations as well as the best storage conditions were determined for erythrocyte glutathione, and the reference values were established. A total of 396 apparently healthy people, 206 male, and 190 female, were randomly selected from villages and cities of the southern part of Turkey. The distribution was Gaussian and no significant difference was observed between the male and the female subjects. The mean (standard deviation) of the population investigated for glutathione was 6.9 (1.0) mumol/gHb. The analytical, intra-individual and inter-individual variations were assessed in 20 apparently healthy subjects and were found to be 4.63%, 13.67% and 11.16%, respectively. Whole blood stored at -70 degrees C for up to 10 days was shown to be the best storage condition for erythrocyte glutathione determination. The results of the index of individuality showed that glutathione reference values could be used for diagnostic purposes.

Decreased glutathione levels in acute myocardial infarction.

Although experimental studies have demonstrated that reduced glutathione (GSH) is involved in cellular protection from deleterious effects of oxygen free radicals (OFRs) in ischemia and reperfusion, there are controversial data on the correlation between the levels of erythrocyte GSH and the ischemic process. To clarify, we determined the erythrocyte GSH levels in 21 patients with acute myocardial infarction (AMI), aged 39-70, who were not given thrombolytic therapy and 21 age- and sex- matched healthy controls. Samples of blood were taken on days 1, 3, 5 and 7 from AMI patients and on the same days from the controls. The GSH levels of patients with AMI were significantly depressed by 11.5% as compared to the controls on the second day after infarction (7.44 +/- 1.71 vs 8.41 +/- 1.54 U/gHb p < 0.05). Although the total mean of GSH levels for all days was lower (3.8%) in patients than in the controls, this finding did not reach statistical significance (7.41 +/- 1.71 vs 7.71 +/- 1.27 U/gHb, ns). There was no correlation between the erythrocyte GSH levels and cardiac enzyme concentrations, infarct localization, hemodynamic status according to Killip classification and the frequency of ventricular arrhythmias. This preliminary work suggests that depressed GSH levels may be associated with an enhanced protective mechanism to oxidative stress in AMI. Measurements of erythrocyte GSH can be helpful in the estimation of oxidative stress in the course of AMI. However, further research must be done to determine the primary scavenger in AMI by analyzing all the enzymes and substrates involved in the endogeneous system that controls the effects of OFRs.

Intracellular glutathione content in leukemias.

The intracellular glutathione (GSH) content was measured in 73 patients with leukemia and compared with controls. GSH content was between 1.16 and 5.55 mumol/g protein (mean 2.96 +/- 0.86) in the study group and between 0.5 and 1.48 mumol/g protein (mean 1.31 +/- 0.27) in the control group, statistically significant difference (p = 0.0000). There was no significant difference between acute and chronic leukemias, lymphoid and myeloid leukemias and, more importantly, newly diagnosed and relapsed patients. GSH content did not change significantly with clinical and hematologic parameters such as age, sex, and initial hematologic findings. In addition, variable changes were detected over 24 h in 9 patients. It can be concluded that GSH content in leukemic cells was higher than in controls and showed a wide range. The absence of a relationship between GSH content and clinical and laboratory parameters suggested that GSH is not the sole determinant of response to cytotoxic drugs. GSH variation over a 24-hour period may be important in the timing and success of chemotherapy for leukemias.

Studies on red cell glucose-6-phosphate dehydrogenase: evaluation of reference values.

The analytical, intra-individual, inter-individual variation and reference values were determined for red cell glucose-6-phosphate dehydrogenase (G6PD). Different procedures for the conditions for storage of red blood cells and the preparation of haemolysates were investigated. A total of 2170 samples of blood were taken from apparently healthy persons-1212 males and 958 females--from randomly selected villages and city centres in the southern part of Turkey. Analytical variation, intra-individual variation and inter-individual variation were 8.67%, 32.8% and 31.8%, respectively. The mean (SD) for G6PD was 8.6 (3.3) IU/gHb. The index of individuality, 1.03, showed that the reference intervals could be used for diagnostic purposes. Whole blood or a red cell pellet could be stored in physiological saline for one week at 4 degrees C or -20 degrees with little loss of activity. Two of three different procedures for the preparation of haemolysate gave data that showed no statistical difference and were equally satisfactory.

Regional distributions of beta-thalassemia mutations in Turkey.

The distributions of twelve beta-thalassemic mutations in samples (n = 139 chromosomal samples) from four regions of Turkey were determined. The frequencies of these mutations did not reveal a notable region specific heterogeneity. In particular, the four mutations, IVS.1/nt.110(G/A), IVS.1/nt.6(T/C), IVS.1/nt.1(G/A) and nonsense codon.39(C/T), with country-scale frequencies of 35.9%, 21.6%, 13.0% and 7.2%, respectively, were found to be distributed with rather similar frequencies also on a regional scale.

Molecular characterization of beta-thalassemia in Azerbaijan.

We have analyzed the beta-thalassemia mutations in 99 chromosomes of 49 adults with beta-thalassemia major and of one with Hb S-beta-thalassemia, who are regular patients at a large hematology clinic in Bakü, Azerbaijan. A total of 20 different mutants were identified; three [frameshift at codon 8 (-AA); IVS-II-I (G-->A); IVS-I-110 (G-->A)] were present in about two-thirds of all chromosomes. Most alleles are the same as found in Mediterranean populations; a few have an Asian origin or come from Kurdistan, Lebanon, Saudi Arabia, or a black population. One mutant [frameshift at codons 82/83 (-G)] might be specific for the Azerbaijanian population. Nearly all patients were transfused, which made quantitation of Hb F impossible; high G gamma values were present in the Hb F of those patients whose beta-thalassemia chromosome carried the C-->T mutation at position -158 in the promoter of the G gamma-globin gene.

The beta-thalassaemia mutations in the population of Cyprus.

We have identified the beta-thalassaemia alleles in nearly all known Turkish Cypriot beta-thalassaemia homozygotes and in over 700 Greek Cypriot beta-thalassaemia heterozygotes living on the island of Cyprus. The data confirmed earlier observations that the IVS-I-100 (G-->A) mutation is present for about 74-80%, while three other alleles [IVS-II-745 (C-->G), IVS-I-6 (T-->C), IVS-I-1 (G-->A)] occur at frequencies of 5-8%. Nearly identical percentages were observed for the two Cypriot groups, quite different from those for beta-thalassaemia patients from Greece and Turkey. This suggests close contacts between the two Cypriot communities during many centuries without a major recent influence from Greek or Turkish beta-thalassaemia carriers.

Beta S haplotypes in various world populations.

We have determined the beta S haplotypes in 709 patients with sickle cell anemia, 30 with SC disease, 91 with S-beta-thalassemia, and in 322 Hb S heterozygotes from different countries. The methodology concerned the detection of mutations in the promoter sequences of the G gamma- and A gamma-globin genes through dot blot analysis of amplified DNA with 32P-labeled probes, and an analysis of isolated Hb F by reversed phase high performance liquid chromatography to detect the presence of the A gamma T chain [A gamma 75(E19)Ile----Thr] that is characteristic for haplotype 17 (Cameroon). The results support previously published data obtained with conventional methodology that indicates that the beta S gene arose separately in different locations. The present methodology has the advantage of being relatively inexpensive and fast, allowing the collection of a vast body of data in a short period of time. It also offers the opportunity of identifying unusual beta S haplotypes that may be associated with a milder expression of the disease. The numerous blood samples obtained from many SS patients living in different countries made it possible to compare their hematological data. Such information is included (as average values) for 395 SS patients with haplotype 19/19, for 2 with haplotype 17/17, for 50 with haplotype 20/20, for 2 with haplotype 3/3, and for 37 with haplotype 31/31. Some information on haplotype characteristics of normal beta A chromosomes is also presented.

Hb H disease in a Turkish family resulting from the interaction of a deletional alpha-thalassaemia-1 and a newly discovered poly A mutation.

We have analysed the alpha-globin gene defects present in several members of a large family from Southern Turkey. One deletional alpha-thalassaemia-1 (type MED-II) was found in 10 subjects: this deletion is in excess of 26.5 kb and includes all zeta- and alpha-globin genes. Besides the common types of deletional alpha-thalassaemia-2 (-3.7 kb and -4.2 kb) we observed a nondeletional alpha-thalassaemia-2 that results from an A----G mutation (AATAAA----AATGAA) in the polyadenylation signal of the alpha 2-globin gene: the same A----G replacement is present in the psi alpha l gene. The mutation must cause a considerable alpha-chain deficiency as is evidenced by the haematological data for five members with Hb H disease due to a compound heterozygosity for alpha-thalassaemia-1 (MED-II) and the newly discovered poly A mutation. Several members had additional beta-chain abnormalities (Hb S, Hb D-Los Angeles, beta-thalassaemia); the 11 persons with a Hb S heterozygosity and various alpha-globin gene defects (-alpha/alpha alpha; alpha T alpha/alpha alpha, - -/alpha alpha, -alpha/-alpha and - -/alpha T alpha) showed a decrease in the level of Hb S that was directly related to the severity of the alpha-chain deficiency.

Three new G6PD variants, G6PD Adana, G6PD Samandag, and G6PD Balcali in Cukurova, Turkey.

Glucose-6-phosphate dehydrogenase (G6PD) enzyme from cases known to be completely or mildly deficient were analyzed. The enzymes were purified from blood samples by utilizing DEAE-52 cellulose pH 7.0 column chromatography and ammonium sulphate precipitation. Biochemical and electrophoretic properties of G6PD were studied in these partially purified enzymes. In this study we report three new variants from Cukurova, named Adana, Samandag, and Balcali. Variant I (G6PD Adana) had a high Km for G6P (210 microM) and NADP (13 microM). Utilization of 2d-G6P was 38%. It had a slow electrophoretic mobility, a biphasic pH optimum curve, and abnormal heat stability. Variant II (G6PD Samandag) had a low Km for G6P (25 microM) and a high Km for NADP (18 microM). The rate of utilization of 2d-G6P was normal. G6PD Samandag deviated from the normal enzyme by its biphasic pH optimum curve and its slow electrophoretic mobility. Variant III (G6PD-Balcali) had a normal Km G6P, NADP and rate of utilization of 2d-G6P. However, it showed a biphasic pH optimum curve and slow electrophoretic mobility.

Sickle cell anaemia among Eti-Turks: haematological, clinical and genetic observations.

Haematological and genetic observations have been made on 71 SS Eti-Turk patients and their relatives from Cukurova (southern Turkey) and of immigrant families in The Netherlands. Similar data were collected for 25 Black patients and their relatives from Surinam, Netherlands Antilles, and Kenya. Haematological and clinical results were the same for both groups; the haemolytic anaemia in the Turkish patients was as severe as in the others. Haplotyping, involving nine restriction sites, identified haplotype 19 (Antonarakis et al, 1984) as the major type among the Eti-Turks; this chromosome has previously primarily been observed among SS patients from West Africa. The suggestion that the beta S-chromosome among Eti-Turks originates from that area is supported by a relatively high incidence of alpha-thalassaemia-2 (the 3.7 kb deletion), also frequently present in the Black population of West Africa, and by the absence of other major haplotypes, such as types 20 and 3, characteristic for the beta S-chromosome in the population of Central Africa and Kenya, and in Senegal, respectively. The Saudi Arabian type of beta S chromosome in association with the haplotype 19 beta S chromosome was present in only one Eti-Turk patient; this 30-year-old female was mildly affected and exhibited a high level of fetal haemoglobin.

Albumin Naskapi variant in North American Indians and Eti Turks.

Both conventional polyacrylamide gel electrophoresis and a new type of electrophoretic screening procedure indicate that the polymorphic albumin variants Naskapi, found chiefly in the Naskapi Indians of Quebec, and Mersin, found in the Eti Turks of southeastern Turkey, are molecularly identical or very similar and that the amino acid substitution site in these variants is located between residues 330 and 446. This discovery is consistent with a genetic relationship between the Eti Turks and American Indians. We also report a new variant found in the Eti Turks, albumin Adana, which migrates similarly to albumin B on conventional gels but which our new system shows to differ from the common albumin A and albumin B by a substitution between residues 549 and 585.