Molecular analysis of beta-thalassemia and sickle cell anemia in Antalya.

We have studied 918 chromosomes for mutations leading to beta-thalassemia and sickle cell anemia, which are the two most frequently found monogenic disorders in Antalya, Turkey. Three hundred and seventy-seven postnatal and 82 prenatal cases were studied between 2000 and May 2003 in our center using reverse dot blot hybridization (RDBH) with 22 probes specific for Mediterranean populations. In this study, IVSI-110 (G-->A) appeared to be the most common mutation with an occurrence rate of 44.4% among the 16 different mutations found to be associated with beta-thalassemia. Heterozygosity for IVSI-110 was the most prevalent combination, whereas 34 of our 377 postnatal cases showed homozygosity for this mutation, a genotype leading to beta-thalassemia major. The total percentage of postnatal patients clinically diagnosed as beta-thalassemia major was 18.6%, whereas 5% of the cases were diagnosed clinically as beta-thalassemia intermedia. One new Hb variant, Hb Antalya, and one new mutation, Cod 3 (+T) were found. HbS accounted for 10.3% of all mutations; homozygosity was found in 1.9% of all cases. Of the 82 cases analysed prenatally for beta-globin gene mutations and by cytogenetic techniques for possible chromosomal abnormalities, 21 fetuses were found to be affected with beta-globin gene mutations. One of these fetuses was also found to have a 45,X karyotype, and 1 had a 46,XY/47,XY,+22 karyotype. Quite a high rate of consanguineous marriages in Antalya (35.17%) renders mutation screening, genetic counseling, and educational programs held by our Thalassemia Unit essential. This study was the first to be performed specifically in our region where hemoglobinopathies are most frequent as a consequence of migrations of racially and culturally distinct groups to the area in the distant past.

TT virus infection and genotype distribution in blood donors and a group of patients from Turkey.

BACKGROUND: TT virus (TTV) DNA has been found in a large proportion of patients with different forms of non-A-G hepatitis, however the clinical importance is unclear. We aimed to determine the genotypes of TTV isolates found in blood donors and different patient groups from the western part of Turkey. MATERIALS AND METHODS: TT DNA was investigated in serum samples of 91 volunteer blood donors (BD), 105 thalassemia (TH) patients, ten patients with fulminant hepatitis (FH) and 16 hemodialysis (HD) patients by heminested PCR using primers NG059, NG061 and NG063 from the ORF1 region. 39 isolates were genotyped by analyzing the partial sequence of ORF1. RESULTS: TTV DNA was found in 75% of HD, 80% of FH, 61% of TH patients and in 51.6% of BD. Among the sequenced isolates, 14 (35.9%) belonged to genotype 1 (G1) and 25 (64.1%) belonged to genotype 2 (G2). Among the G2 sequences, 22 were grouped as G2c. CONCLUSION: TTV infection was common in the population studied, even with moderately sensitive primers. G2 was the major genotype of the studied population without any significant differences in distribution between various patient groups and BD.

Molecular pathology of haemophilia A in Turkish patients: identification of 36 independent mutations.

Haemophilia A is an X-linked recessive bleeding disorder caused by heterogeneous mutations in the factor VIII gene. In an attempt to reveal the molecular pathology of Turkish haemophilia A patients, the coding sequence of the gene, excluding a large portion of exon 14, was amplified from genomic DNA and subjected to denaturing gradient gel electrophoresis prior to DNA sequencing. Fifty-nine haemophilia A patients were included in the study with severe, moderate and mild phenotypes observed in 24, 15 and 16 patients, respectively. Factor VIII activity and clinical phenotypes were not available for four patients. A total of 36 independent mutations were found, with a mutation detection efficacy of 61%. The mutations that were reported for the first time include 20 point mutations, one 8-bp insertion (TCAAGATA) in exon 4 and one large deletion greater than 2.8 kb involving exon 14. The novel point mutations were composed of three nonsense (Ser681Ter, Cys2021Ter and Gln2113Ter), one splicing error (IVS-1G-->A), 15 missense mutations (Lys48Asn; Leu-98Phe; Thr118Ala; Cys248Tyr; Glu456Lys; Asp560Ala; Tyr664Cys; Phe679Leu; Gly691Trp; Asp1769His; Val1857Leu; Gly2026Gln; Arg2163Pro; Asp2288Ala; and Arg2304Leu) and a T deletion in exon 25 that caused a frameshift followed by a stop codon. All missense mutations except Val1857Leu, which maintained a conserved nonpolar R group, occurred at amino acids conserved among four species and were most probably pathogenic. In addition, two sequence changes (IVS3-9C-->T) and (Leu2230Leu) were also detected in patients carrying Val1857Leu and Phe679Leu missense mutations, respectively. Identification of mutation origins in eight sporadic cases revealed an equal sex ratio of mutations.

Evaluation of osteoporosis in thalassemia by quantitative computed tomography: is it reliable?

This study was conducted to quantify the degree of osteoporosis in thalassemic patients by single energy quantitative computed tomography (SEQCT) and to test the reliability of this method. On 38 thalassemic patients with osteoporosis and 38 normal control subjects, bone mineral density (BMD) measurements were done by SEQCT. BMD and standard deviation (SD) of the x-ray attenuation numbers of pixels within region of interest (ROI) of the measurement areas were compared between two groups. Mean BMD values for thalassemic patients and control group were 173.4 and 158.2 mg/cm3, respectively. Mean BMD value for thalassemic patient group was significantly higher. Mean SD values of ROI for control group and thalassemic patients were 41.4 and 71.1, respectively. The difference between the SD values was also statistically significant. Positive correlation was noted between SD values and patients' ages in the thalassemic group. Results of SEQCT method may not reflect the clinical and conventional radiographic findings of osteoporosis seen in thalassemic patient group and should be used cautiously. Other methods of BMD measurement, such as photon absorbsiometry and x-ray absorbsiometry, should also be investigated for their accuracy in this patient group.

Antioxidant capacity of G-6-PD-deficient erythrocytes.

Glucose-6-phosphate dehydrogenase (G-6-PD) deficiency is the most common human enzymopathy. In this research, we studied two groups consisting of 30 male subjects who are G-6-PD deficient and 30 normal male subjects matched with the G-6-PD-deficient patients for age. All 30 assays were performed under normal conditions free of any oxidative attack that may result in haemolytic crisis in G-6-PD-deficient subjects. The erythrocyte glucose-6-phosphate dehydrogenase, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione-S-transferase (GST) activities, reduced glutathione (GSH) levels and erythrocyte and plasma thiobarbituric acid-reactive substances (TBARS) levels were measured. All parameters in each group did not differ significantly except for G-6-PD levels. These data show that G-6-PD-deficient subjects can survive in normal conditions unless they are exposed to any oxidative stress.

Homozygous ß-Thalassemia Associated with Familial Mediterranean Fever in a Turkish Patient.

We report here a ß- thalassemia major case (homozygous IVS-1-110 G-A) associated with Familial Mediterranean Fever (FMF) (homozygous 694 Met-Val). Our patient's clinical course revealed a possible synergistic effect between colchicine and desferrioxamine (DFO) However, this could be a only a coincidence, as under colchicine therapy, fever attacks may appear, this may be the topic of a further investigation.

Thrombosis After Splenectomy in Patients with Thalassemia.

Thrombosis is one of the most important complications after splenectomy and requires fast diagnosis, effective therapy and good follow-up. The aim of this study is to investigate the effects of thrombocytosis and natural inhibitors on thrombosis after splenectomy. We detected thrombosis in the portal vein system in 7 of the 30 splenectomized patients (23.3%) by Doppler Colour Flow Imaging. There was no statistical increase of thrombocyte count in patients with or without thrombosis. Natural inhibitor levels in all patients were lower than controls (p< 0.001), but there was not any statistical difference between the patients with and without thrombosis.

Natural Inhibitors and Lipids in Patients with Sickle Cell Disease.

Microvascular occlusion in sickle cell disease (SCD) is a multifactorial process. Disordered coagulation may play a role in the pathogenesis of vaso occlusive crisis (VOC). The aim of this study was to evaluate the patients and to investigate their Protein C (PC), Protein S (PS) and AT-III levels during normal and crisis periods. A total of 18 patients with SCD were included in this study at the Antalya State Hospital, Thalassemia Center. The mean number of VOC episodes of the patients per year was 4.1 - 3.2. Complications in patients included 4 cases of osteonecrosis (23.5%), 2 cases of holealithiasis (11.7%), 2 cases of leg ulcers (11.7%), and 3 splenectomies (17.6%). The patients during noncrisis periods have lower cholesterol and higher triglycerides levels than the controls (p< 0.001). Hepatic and renal functions were normal in all patients. The mean totals of the PS, PC and AT-III levels were statistically lower both in non crisis and in crisis periods than the control (p< 0.001), but there was no statistical difference between the levels durining noncrisis and crisis periods. In conclusion, PC, PS and AT-IIII deficiencies in patients with SCD are certain. However, these deficiencies do not change during noncrisis and crisis situations and does not play a role on the period of crisis. Abnormal lipid patterns may be a predisposing condition for a crisis.

Continuous desferrioxamine infusion by an infusor in thalassaemia major.

Chelation therapy with desferrioxamine (DFO) can be an important problem for patients with thalassaemia major (TM). In an effort to find a solution to this problem, a new delivery system based on a disposable and lightweight device was developed by Baxter. The aim of study was to investigate the compliance of patients and the effects and side effects of two different infusors. The 26 patients using infusors were divided into 2 groups, who were infused with DFO for 48 h (group A) and for 120 h (group B). The 24-h mean urinary iron and zinc excretion in groups A and B were statistically significant at the beginning and end of therapy (p < 0.001). Serum ferritin levels decreased in both groups compared with control (p < 0.001). No systemic reactions were observed either during or at the end of treatment. The compliance and use of infusors were 97% in group A and 72% in group B. In conclusion, better compliance was achieved and there were fewer local complications in group A than in group B. Subcutaneous infusion of DFO with both devices may be ideal for patients who have compliance problems and cannot be treated using portable pumps. The device is new and expensive, but excellent and effective for long-term DFO infusion.

Recombinant human growth hormone treatment in children with thalassemia major.

BACKGROUND: To evaluate the growth hormone reserve and the growth hormone response to recombinant human growth hormone (GH) in prepubertal thalassemic children with growth retardation. METHODS: Twenty thalassemic patients with short stature and delayed bone age were studied. Patients were randomized into GH-treated (n = 10) and non-GH treated (control; n = 10) groups. The GH-treated group received recombinant human (rh)-GH (Genotropin) at the dose of 0.7 IU/kg per week for 12 months. RESULTS: There was a significant discordance between GH response to pharmacologic stimuli and physiological secretion of GH/GHRH testing. Following the administration of rhGH, growth velocity increased from 2.47 +/- 0.48 cm/year to 6.27 +/- 0.76 cm/year (P = 0.005), whereas there was not a similar change in the non-GH-treated group. The height velocities of the two groups during the 1 year follow-up period were significantly different (6.27 +/- 0.76 vs 3.99 +/- 0.34 cm/year; P = 0.025). There were significant differences between the height velocity improvements and height velocity standard deviation scores of the two groups as well. CONCLUSION: The present study has demonstrated that rhGH is a safe and efficacious mode of treatment in thalassemic children.

Effects of calcitonin therapy on osteoporosis in patients with thalassemia.

The aim of this study was to evaluate the effects of calcitonin (CT) treatment on bone pain, osteoporosis, bone fractures and blood chemistry in thalassemic patients. Twenty-four patients with an age range of 10-24 years were included, 14 of whom received 100 IU CT and 250 mg calcium 3 times a week. The others (n = 10) were followed up as a control group with only routine thalassemia therapy. After 1 year of treatment, bone pain disappeared and radiological signs of osteoporosis had improved significantly (p < 0.01) in the treatment group. CT has no important side effects.

GM-CSF in the treatment of Fanconi's anaemia.

We have used recombinant human (rh) GM-CSF in two 12-year-old Fanconi's aplastic anaemia patients. They had not received any previous therapy except blood transfusions. Each patient was given three 21 d courses of rh-GM-CSF, the first two at a dose of 3.5 micrograms/kg/d and the third at 7 micrograms/kg/d s.c. There were significant increases in WBC and absolute neutrophil counts after the first week of rh-GM-CSF which lasted as long as the treatment was continued. Following the cessation of treatment, WBC and ANC dropped rapidly. We conclude that rh-GM-CSF can be used in FAA, especially in severely neutropenic cases.