Original article 419
Endothelial nitric oxide synthase gene intron 4 variable number tandem repeat polymorphism in b-thalassemia major: relation to cardiovascular complications Azza A.G. Tantawya, Amira A.M. Adlya, Eman A. Ismailb and Shereen H. Alya Endothelial nitric oxide synthase (eNOS), an enzyme that generates nitric oxide, is a major determinant of endothelial function. Several eNOS gene polymorphisms have been reported as ‘susceptibility genes’ in various human diseases states, including cardiovascular, pulmonary and renal diseases. We studied the 27-base pair tandem repeat polymorphism in intron 4 of eNOS gene in 60 b-thalassemia major (b-TM) patients compared with 60 healthy controls and assessed its role in subclinical atherosclerosis and vascular complications. Patients were evaluated stressing on transfusion history, splenectomy, thrombotic events, echocardiography and carotid intima–media thickness (CIMT). Analysis of eNOS intron 4 gene polymorphism was performed by PCR. No significant difference was found between b-TM patients and controls with regard to the distribution of eNOS4 alleles or genotypes. The frequency of eNOS4a allele (aa and ab genotypes) was significantly higher in b-TM patients with pulmonary hypertension or cardiomyopathy. Logistic regression analysis revealed that eNOS4a allele was an independent risk factor for pulmonary
Introduction b-thalassemia is an inherited haemoglobin disorder resulting from impaired production of b-globin chains of the haemoglobin tetramer [1]. The resultant phenotype is chronic haemolytic anaemia of varying severity, depending on the level of b-globin chain deficiency and subsequent a-globin chain accumulation [2]. Heart failure is the most common cause of death in b-thalassemia major (b-TM) [3]. Thalassemia heart disease involves mainly left ventricular dysfunction caused by transfusion-induced iron overload [4]. Pulmonary hypertension is increasingly recognized in thalassemia as a leading factor in heart failure and death [5]. Although aggressive transfusion management has been reported to prevent the development of pulmonary hypertension in b-TM [6], pulmonary hypertension is still common [5,7]. Thalassemia patients have many risk factors for developing pulmonary hypertension, including splenectomy [8,9], red cell phosphatidylserine exposure [10], coagulation abnormalities [11], oxidative stress [12] and iron overload [13]. As chronic haemolysis continues despite transfusion, these patients also remain at risk for haemolysis-associated pulmonary hypertension [5,14,15]. Recent investigations suggest that endothelial dysfunction is a key to the pulmonary hypertension pathogenesis [16]. As impaired nitric oxide bioavailability represents 0957-5235 Copyright ß 2015 Wolters Kluwer Health, Inc. All rights reserved.
hypertension in b-TM patients [odds ratio (OR) 2.2, 95% confidence interval (95% CI) 1.19–5.6; P < 0.001]. We suggest that eNOS intron 4 gene polymorphism is related to endothelial dysfunction and subclinical atherosclerosis and could be a possible genetic marker for prediction of increased susceptibility to cardiovascular complications. Blood Coagul Fibrinolysis 26:419–425 Copyright ß 2015 Wolters Kluwer Health, Inc. All rights reserved. Blood Coagulation and Fibrinolysis 2015, 26:419–425 Keywords: b-thalassemia, endothelial dysfunction, nitric oxide synthase gene polymorphism a
Department of Pediatrics and bDepartment of Clinical Pathology, Faculty of Medicine, Ain Shams University, Cairo, Egypt Correspondence to Azza A.G. Tantawy, 22 Ahmed Amin Street, St Fatima Square, Heliopolis, Cairo, 11341, Egypt Fax: +202 22400507; e-mail: [email protected] Received 7 March 2014 Revised 7 December 2014 Accepted 5 January 2015
the central feature of endothelial dysfunction [17], aberrant nitric oxide activity plays a foreseeable role in the pulmonary hypertension development [18]. Nitric oxide is one of the most potent vasodilators known [19] and is essential to vascular homeostasis. It plays an important role in the maintenance of vasomotor tone, limits platelet aggregation and ischemia-reperfusion injury, modulates endothelial proliferation and has anti-inflammatory properties. Arginine is the precursor to nitric oxide, catalyzed by a family of enzymes, the nitric oxide synthases [20]. The constitutive endothelial nitric oxide synthase (eNOS) is expressed in the endothelium, encoded by a 26-exon gene (NOS3) located on chromosome 7q35-36 with a total size of 21 kb and encodes an mRNA of 4052 nucleotides [21]. Several eNOS gene polymorphisms have been reported as ‘susceptibility genes’ in various human diseases states, including cardiovascular, pulmonary and renal diseases [22–24]. The variable number of tandem repeat (VNTR) polymorphism located in intron 4 of eNOS (eNOS4b/a polymorphism) was significantly associated with plasma nitric oxide concentration [25]. There are two alleles identified in intron 4 of the eNOS gene. The larger allele, 4b, consists of five tandem 27-bp repeats and the smaller one, 4a, has four repeats [26]. An association of the 4a allele of the eNOS gene with coronary heart disease and DOI:10.1097/MBC.0000000000000277
Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
420 Blood Coagulation and Fibrinolysis 2015, Vol 26 No 4
hypertension has been reported [27,28]. Therefore, we studied the 27 base pair tandem repeat polymorphism in intron4 of eNOS gene in young patients with b-TM and assessed its potential involvement in subclinical atherosclerosis and haemolysis-associated complications including pulmonary hypertension.
Cobas Integra 800 (Roche Diagnostics, Mannheim, Germany). Quantitative D-dimer assay based on particle-enhanced immuno-turbidimetry (Innovance D-dimer; Siemens Healthcare Diagnostics) and von Willebrand factor antigen (vWF Ag) (Siemens Healthcare Diagnostics) were performed on a Sysmex CA-1500 coagulation analyser (Siemens Healthcare Diagnostics).
Materials and methods This cross-sectional study included 60 patients with b-TM (35 boys and 25 girls with a male-to-female ratio 1.4 : 1) recruited from the regular attendants of the Pediatric Hematology Unit, Children’s Hospital, Ain Shams University. Their age ranged from 5 to 18 years with a mean of 11.6 4.1 years. Sixty age, sex and ethnicity-matched healthy individuals (unrelated to the patients) were enrolled as a control group; 34 boys and 26 girls (male-to-female ratio, 1.3 : 1) with a mean age of 12.5 4.5 years (range, 4.5–18 years). An informed consent was obtained from the guardian of each patient or control before participation. The procedures applied in this study were approved by the Ethical Committee of Human Experimentation of Ain Shams University, and are in accordance with the Helsinki Declaration of 1975.
Determination of endothelial nitric oxide synthase genotypes
All included patients were subjected to detailed medical history and thorough clinical examination with a special emphasis on disease duration, anthropometric measures, blood pressure, evidence of cardiac disease, history of splenectomy, transfusion history, chelation therapy. b-TM patients received chelation therapy in the form of deferoxamine (Desferal, DFO; Novartis Pharma AG, Basel, Switzerland) subcutaneously in a dose that ranged from 30 to 45 mg/kg per day given 5 days/week and/or deferiprone (Ferriprox; ApoPharma USA, Inc., Rockville, Maryland, USA) given orally in a dose that ranged from 50 to 100 mg/kg per day divided on three divided doses for 7 days/week.
Using the applied Biosystems Veriti 96-Well Thermal Cycler (Applied Biosystems, Foster City, California, USA), PCR conditions were initial denaturation at 948C for 5 min; 35 cycles of denaturation at 948C for 30 s, annealing at 568C for 1 min and extension at 728C for 1 min; and final extension at 728C for 7 min. The PCR products were stored at 208C until analysis or immediately analysed using 2% agarose gel electrophoresis and fragments were visualized by ethidium bromide staining and ultraviolet transillumination. To verify the results, PCR products were further analysed using QIAxcel DNA screening Kit and Qiaxcel genetic analyser (Qiagen), which provides automated and accurate estimates of allele sizes and utilizes a preassembled cartridge (cartridge type Qiaxcel DNA high-resolution cartridge, injection time 10 s, Qiaxcel DNA size marker 15 bp-1 kb) to simultaneously run samples and collect data [30,31].
Sample collection
Peripheral blood samples were collected on EDTA (1.2 mg/ml) for complete blood count (CBC), haemoglobin analysis by HPLC and eNOS genotype. For chemical analysis, clotted samples were obtained and serum was separated by centrifugation for 15 min at 1000g. Diagnostic testing
Laboratory investigations included CBC using Sysmex XT-1800i (Sysmex, Japan), examination of Leishmanstained smears for red blood cell (RBC) morphology and differential white blood cell (WBC) count, peripheral blood staining by Brilliant Cresyl blue and examination of a stained smear for reticulocyte count, qualitative and quantitative haemoglobin analysis using HPLC by D-10 (BioRad, Marnes La Coquette, France), liver and kidney function tests, markers of haemolysis [lactate dehydrogenase (LDH) and indirect bilirubin] and high sensitivity C-reactive protein (hs-CRP) as well as serum ferritin on
eNOS genotype was determined using PCR [23,24]. Genomic DNA was extracted from peripheral blood using a QIAamp DNA Blood Mini Kit (Qiagen, GmbH, Hilden, Germany) and was stored at 48C until analysis. Two oligonucleotide primers (sense) 50 -AGGCCCTAT GGTAGTGCCTT-30 and (antisense) 50 -TCTCTTAG TGCTGTGGTCAC-30 based on the flanking sequences of the VNTR in the eNOS gene were used to amplify the corresponding DNA fragment by PCR [23,29]. PCR products were then synthesized from DNA samples using the Taq PCR Master Mix Kit (Qiagen). Each PCR contained all the necessary reagents, primers and 200 ng of genomic DNA in a final volume of 25 ml.
Radiological examination
All studied patients were clinically asymptomatic for pulmonary hypertension and cardiovascular abnormalities. Screening for pulmonary hypertension was performed by the noninvasive Doppler echocardiography with different modalities using Vivid E9 (GE Healthcare, Oslo, Norway) to evaluate pulmonary artery pressure and tricuspid regurgitant jet velocity (TRV). All of the carotid scans were done using carotid Doppler ultrasound scanner (Acuson Sequoia 512; Siemens Medical Solutions) and a high-frequency 15L8-MHz linear-array transducer following a predetermined standardized scanning protocol [32]. All ultrasound scans were performed by an experienced vascular operator who was unaware of children’s clinical details. Patients
Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
eNOS gene polymorphism in thalassemia major Tantawy et al. 421
were placed in a supine position having the neck in hyperextension. The location of measurement was standardized in every study at the common carotid artery at 1 cm distance from the carotid bulb. The mean value of carotid intima–media thickness (CIMT) for the right and left carotid arteries was calculated. All the ultrasonographic examinations were analysed by a single experienced radiologist blinded to the group to which the individuals belonged. Diagnostic criteria
The definition of b-TM was based on complete blood picture, reticulocyte count and markers of haemolysis as well as qualitative and quantitative of analysis of haemoglobin using HPLC [33]. A TRV of at least 2.5 m/s was used as a proxy for patients at risk for pulmonary hypertension [34,35]. Cardiomyopathy was defined as severe cardiac functional impairment with a resting left ventricular ejection fraction less than 45% and/or a drop of greater than 12% on stress [36]. Statistical analysis
Analysis of data was done using Statistical Program for Social Science version 15 (SPSS Inc., Chicago, Illinois, USA). Quantitative variables were described in the form of range, mean and standard deviation. Qualitative variables were described as number and percentage. Allele frequencies were estimated by the gene counting method. In order to compare quantitative parametric variables between two groups, Student’s t-test was applied. Comparison between categorical variables was performed using Chi-square (x2) test or Fischer’s exact test when frequencies were below five. The x2 test was also used to assess deviation of genotype distribution from Hardy–Weinberg equilibrium. Spearman’s rank correlation coefficient was used to assess the relation between genotype frequencies and clinicopathological variables. Logistic regression analysis was performed with estimating the odds ratio (OR) and 95% confidence interval (CI) to assess the influence of eNOS alleles on pulmonary hypertension as the dependent variable. A P value of less than 0.05 was considered significant in all analyses.
Results The studied b-TM patients had significantly lower weight and height than controls (P < 0.001). WBC count, markers of haemolysis, HbF, serum ferritin, D-Dimer, vWF Ag and CIMT were significantly higher, whereas haemoglobin level was lower in patients than controls (P < 0.001) (Table 1). Two alleles of the VNTR in the human eNOS gene were detected in the studied population; one allele contained four of 27 bp repeats (eNOS4a allele) giving rise to a PCR product of 393 bp, whereas the other contained five of such repeats (eNOS4b allele), yielding a PCR product of
Clinical and laboratory data of the studied b-thalassemia major patients and controls
Table 1
Variable Age (years) Males, n (%) Weight for age SDS Height for age SDS Transfusion index (mg/kg per year) Splenectomized, n (%) Pulmonary hypertension, n (%) Cardiomyopathy, n (%) History of thrombosis, n (%) WBC count (x109/l) Haemoglobin (g/dl) HbF (%) Lactate dehydrogenase (IU/l) Indirect bilirubin (mg/dl) Serum ferritin (mg/l) D-dimer (mg/l FEU) vWF Ag (%) CIMT (mm) Alleles a b Genotypes aa ab bb
Controls (n ¼ 60)
b-(thalassemia P major (n ¼ 60)
P
12.5 4.5 34 (56.7) 0.78 0.2 0.94 0.3 – – – – – 7.5 1.8 11.7 2.5 1.1 0.3 238 35 1.0 0.3 159 28 0.42 0.1 85 7.1 0.04 0.01
11.6 4.1 35 (58.3) 1.13 1 0.9 0.4 375.8 39.1 18 (30) 24 (40) 20 (33.3) 10 (16.7) 22.8 5.7 8.6 2.5 75.2 12.1 1312 358 2.9 1.2 2451 672 1.2 0.7 124 9.7 0.69 0.19
0.254 1
Endothelial nitric oxide synthase gene intron 4 variable number tandem repeat polymorphism in b-thalassemia major: relation to cardiovascular complications Azza A.G. Tantawya, Amira A.M. Adlya, Eman A. Ismailb and Shereen H. Alya Endothelial nitric oxide synthase (eNOS), an enzyme that generates nitric oxide, is a major determinant of endothelial function. Several eNOS gene polymorphisms have been reported as ‘susceptibility genes’ in various human diseases states, including cardiovascular, pulmonary and renal diseases. We studied the 27-base pair tandem repeat polymorphism in intron 4 of eNOS gene in 60 b-thalassemia major (b-TM) patients compared with 60 healthy controls and assessed its role in subclinical atherosclerosis and vascular complications. Patients were evaluated stressing on transfusion history, splenectomy, thrombotic events, echocardiography and carotid intima–media thickness (CIMT). Analysis of eNOS intron 4 gene polymorphism was performed by PCR. No significant difference was found between b-TM patients and controls with regard to the distribution of eNOS4 alleles or genotypes. The frequency of eNOS4a allele (aa and ab genotypes) was significantly higher in b-TM patients with pulmonary hypertension or cardiomyopathy. Logistic regression analysis revealed that eNOS4a allele was an independent risk factor for pulmonary
Introduction b-thalassemia is an inherited haemoglobin disorder resulting from impaired production of b-globin chains of the haemoglobin tetramer [1]. The resultant phenotype is chronic haemolytic anaemia of varying severity, depending on the level of b-globin chain deficiency and subsequent a-globin chain accumulation [2]. Heart failure is the most common cause of death in b-thalassemia major (b-TM) [3]. Thalassemia heart disease involves mainly left ventricular dysfunction caused by transfusion-induced iron overload [4]. Pulmonary hypertension is increasingly recognized in thalassemia as a leading factor in heart failure and death [5]. Although aggressive transfusion management has been reported to prevent the development of pulmonary hypertension in b-TM [6], pulmonary hypertension is still common [5,7]. Thalassemia patients have many risk factors for developing pulmonary hypertension, including splenectomy [8,9], red cell phosphatidylserine exposure [10], coagulation abnormalities [11], oxidative stress [12] and iron overload [13]. As chronic haemolysis continues despite transfusion, these patients also remain at risk for haemolysis-associated pulmonary hypertension [5,14,15]. Recent investigations suggest that endothelial dysfunction is a key to the pulmonary hypertension pathogenesis [16]. As impaired nitric oxide bioavailability represents 0957-5235 Copyright ß 2015 Wolters Kluwer Health, Inc. All rights reserved.
hypertension in b-TM patients [odds ratio (OR) 2.2, 95% confidence interval (95% CI) 1.19–5.6; P < 0.001]. We suggest that eNOS intron 4 gene polymorphism is related to endothelial dysfunction and subclinical atherosclerosis and could be a possible genetic marker for prediction of increased susceptibility to cardiovascular complications. Blood Coagul Fibrinolysis 26:419–425 Copyright ß 2015 Wolters Kluwer Health, Inc. All rights reserved. Blood Coagulation and Fibrinolysis 2015, 26:419–425 Keywords: b-thalassemia, endothelial dysfunction, nitric oxide synthase gene polymorphism a
Department of Pediatrics and bDepartment of Clinical Pathology, Faculty of Medicine, Ain Shams University, Cairo, Egypt Correspondence to Azza A.G. Tantawy, 22 Ahmed Amin Street, St Fatima Square, Heliopolis, Cairo, 11341, Egypt Fax: +202 22400507; e-mail: [email protected] Received 7 March 2014 Revised 7 December 2014 Accepted 5 January 2015
the central feature of endothelial dysfunction [17], aberrant nitric oxide activity plays a foreseeable role in the pulmonary hypertension development [18]. Nitric oxide is one of the most potent vasodilators known [19] and is essential to vascular homeostasis. It plays an important role in the maintenance of vasomotor tone, limits platelet aggregation and ischemia-reperfusion injury, modulates endothelial proliferation and has anti-inflammatory properties. Arginine is the precursor to nitric oxide, catalyzed by a family of enzymes, the nitric oxide synthases [20]. The constitutive endothelial nitric oxide synthase (eNOS) is expressed in the endothelium, encoded by a 26-exon gene (NOS3) located on chromosome 7q35-36 with a total size of 21 kb and encodes an mRNA of 4052 nucleotides [21]. Several eNOS gene polymorphisms have been reported as ‘susceptibility genes’ in various human diseases states, including cardiovascular, pulmonary and renal diseases [22–24]. The variable number of tandem repeat (VNTR) polymorphism located in intron 4 of eNOS (eNOS4b/a polymorphism) was significantly associated with plasma nitric oxide concentration [25]. There are two alleles identified in intron 4 of the eNOS gene. The larger allele, 4b, consists of five tandem 27-bp repeats and the smaller one, 4a, has four repeats [26]. An association of the 4a allele of the eNOS gene with coronary heart disease and DOI:10.1097/MBC.0000000000000277
Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
420 Blood Coagulation and Fibrinolysis 2015, Vol 26 No 4
hypertension has been reported [27,28]. Therefore, we studied the 27 base pair tandem repeat polymorphism in intron4 of eNOS gene in young patients with b-TM and assessed its potential involvement in subclinical atherosclerosis and haemolysis-associated complications including pulmonary hypertension.
Cobas Integra 800 (Roche Diagnostics, Mannheim, Germany). Quantitative D-dimer assay based on particle-enhanced immuno-turbidimetry (Innovance D-dimer; Siemens Healthcare Diagnostics) and von Willebrand factor antigen (vWF Ag) (Siemens Healthcare Diagnostics) were performed on a Sysmex CA-1500 coagulation analyser (Siemens Healthcare Diagnostics).
Materials and methods This cross-sectional study included 60 patients with b-TM (35 boys and 25 girls with a male-to-female ratio 1.4 : 1) recruited from the regular attendants of the Pediatric Hematology Unit, Children’s Hospital, Ain Shams University. Their age ranged from 5 to 18 years with a mean of 11.6 4.1 years. Sixty age, sex and ethnicity-matched healthy individuals (unrelated to the patients) were enrolled as a control group; 34 boys and 26 girls (male-to-female ratio, 1.3 : 1) with a mean age of 12.5 4.5 years (range, 4.5–18 years). An informed consent was obtained from the guardian of each patient or control before participation. The procedures applied in this study were approved by the Ethical Committee of Human Experimentation of Ain Shams University, and are in accordance with the Helsinki Declaration of 1975.
Determination of endothelial nitric oxide synthase genotypes
All included patients were subjected to detailed medical history and thorough clinical examination with a special emphasis on disease duration, anthropometric measures, blood pressure, evidence of cardiac disease, history of splenectomy, transfusion history, chelation therapy. b-TM patients received chelation therapy in the form of deferoxamine (Desferal, DFO; Novartis Pharma AG, Basel, Switzerland) subcutaneously in a dose that ranged from 30 to 45 mg/kg per day given 5 days/week and/or deferiprone (Ferriprox; ApoPharma USA, Inc., Rockville, Maryland, USA) given orally in a dose that ranged from 50 to 100 mg/kg per day divided on three divided doses for 7 days/week.
Using the applied Biosystems Veriti 96-Well Thermal Cycler (Applied Biosystems, Foster City, California, USA), PCR conditions were initial denaturation at 948C for 5 min; 35 cycles of denaturation at 948C for 30 s, annealing at 568C for 1 min and extension at 728C for 1 min; and final extension at 728C for 7 min. The PCR products were stored at 208C until analysis or immediately analysed using 2% agarose gel electrophoresis and fragments were visualized by ethidium bromide staining and ultraviolet transillumination. To verify the results, PCR products were further analysed using QIAxcel DNA screening Kit and Qiaxcel genetic analyser (Qiagen), which provides automated and accurate estimates of allele sizes and utilizes a preassembled cartridge (cartridge type Qiaxcel DNA high-resolution cartridge, injection time 10 s, Qiaxcel DNA size marker 15 bp-1 kb) to simultaneously run samples and collect data [30,31].
Sample collection
Peripheral blood samples were collected on EDTA (1.2 mg/ml) for complete blood count (CBC), haemoglobin analysis by HPLC and eNOS genotype. For chemical analysis, clotted samples were obtained and serum was separated by centrifugation for 15 min at 1000g. Diagnostic testing
Laboratory investigations included CBC using Sysmex XT-1800i (Sysmex, Japan), examination of Leishmanstained smears for red blood cell (RBC) morphology and differential white blood cell (WBC) count, peripheral blood staining by Brilliant Cresyl blue and examination of a stained smear for reticulocyte count, qualitative and quantitative haemoglobin analysis using HPLC by D-10 (BioRad, Marnes La Coquette, France), liver and kidney function tests, markers of haemolysis [lactate dehydrogenase (LDH) and indirect bilirubin] and high sensitivity C-reactive protein (hs-CRP) as well as serum ferritin on
eNOS genotype was determined using PCR [23,24]. Genomic DNA was extracted from peripheral blood using a QIAamp DNA Blood Mini Kit (Qiagen, GmbH, Hilden, Germany) and was stored at 48C until analysis. Two oligonucleotide primers (sense) 50 -AGGCCCTAT GGTAGTGCCTT-30 and (antisense) 50 -TCTCTTAG TGCTGTGGTCAC-30 based on the flanking sequences of the VNTR in the eNOS gene were used to amplify the corresponding DNA fragment by PCR [23,29]. PCR products were then synthesized from DNA samples using the Taq PCR Master Mix Kit (Qiagen). Each PCR contained all the necessary reagents, primers and 200 ng of genomic DNA in a final volume of 25 ml.
Radiological examination
All studied patients were clinically asymptomatic for pulmonary hypertension and cardiovascular abnormalities. Screening for pulmonary hypertension was performed by the noninvasive Doppler echocardiography with different modalities using Vivid E9 (GE Healthcare, Oslo, Norway) to evaluate pulmonary artery pressure and tricuspid regurgitant jet velocity (TRV). All of the carotid scans were done using carotid Doppler ultrasound scanner (Acuson Sequoia 512; Siemens Medical Solutions) and a high-frequency 15L8-MHz linear-array transducer following a predetermined standardized scanning protocol [32]. All ultrasound scans were performed by an experienced vascular operator who was unaware of children’s clinical details. Patients
Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
eNOS gene polymorphism in thalassemia major Tantawy et al. 421
were placed in a supine position having the neck in hyperextension. The location of measurement was standardized in every study at the common carotid artery at 1 cm distance from the carotid bulb. The mean value of carotid intima–media thickness (CIMT) for the right and left carotid arteries was calculated. All the ultrasonographic examinations were analysed by a single experienced radiologist blinded to the group to which the individuals belonged. Diagnostic criteria
The definition of b-TM was based on complete blood picture, reticulocyte count and markers of haemolysis as well as qualitative and quantitative of analysis of haemoglobin using HPLC [33]. A TRV of at least 2.5 m/s was used as a proxy for patients at risk for pulmonary hypertension [34,35]. Cardiomyopathy was defined as severe cardiac functional impairment with a resting left ventricular ejection fraction less than 45% and/or a drop of greater than 12% on stress [36]. Statistical analysis
Analysis of data was done using Statistical Program for Social Science version 15 (SPSS Inc., Chicago, Illinois, USA). Quantitative variables were described in the form of range, mean and standard deviation. Qualitative variables were described as number and percentage. Allele frequencies were estimated by the gene counting method. In order to compare quantitative parametric variables between two groups, Student’s t-test was applied. Comparison between categorical variables was performed using Chi-square (x2) test or Fischer’s exact test when frequencies were below five. The x2 test was also used to assess deviation of genotype distribution from Hardy–Weinberg equilibrium. Spearman’s rank correlation coefficient was used to assess the relation between genotype frequencies and clinicopathological variables. Logistic regression analysis was performed with estimating the odds ratio (OR) and 95% confidence interval (CI) to assess the influence of eNOS alleles on pulmonary hypertension as the dependent variable. A P value of less than 0.05 was considered significant in all analyses.
Results The studied b-TM patients had significantly lower weight and height than controls (P < 0.001). WBC count, markers of haemolysis, HbF, serum ferritin, D-Dimer, vWF Ag and CIMT were significantly higher, whereas haemoglobin level was lower in patients than controls (P < 0.001) (Table 1). Two alleles of the VNTR in the human eNOS gene were detected in the studied population; one allele contained four of 27 bp repeats (eNOS4a allele) giving rise to a PCR product of 393 bp, whereas the other contained five of such repeats (eNOS4b allele), yielding a PCR product of
Clinical and laboratory data of the studied b-thalassemia major patients and controls
Table 1
Variable Age (years) Males, n (%) Weight for age SDS Height for age SDS Transfusion index (mg/kg per year) Splenectomized, n (%) Pulmonary hypertension, n (%) Cardiomyopathy, n (%) History of thrombosis, n (%) WBC count (x109/l) Haemoglobin (g/dl) HbF (%) Lactate dehydrogenase (IU/l) Indirect bilirubin (mg/dl) Serum ferritin (mg/l) D-dimer (mg/l FEU) vWF Ag (%) CIMT (mm) Alleles a b Genotypes aa ab bb
Controls (n ¼ 60)
b-(thalassemia P major (n ¼ 60)
P
12.5 4.5 34 (56.7) 0.78 0.2 0.94 0.3 – – – – – 7.5 1.8 11.7 2.5 1.1 0.3 238 35 1.0 0.3 159 28 0.42 0.1 85 7.1 0.04 0.01
11.6 4.1 35 (58.3) 1.13 1 0.9 0.4 375.8 39.1 18 (30) 24 (40) 20 (33.3) 10 (16.7) 22.8 5.7 8.6 2.5 75.2 12.1 1312 358 2.9 1.2 2451 672 1.2 0.7 124 9.7 0.69 0.19
0.254 1
