Pediatr Blood Cancer
Endothelial Nitric Oxide Synthase Gene Intron 4 VNTR Polymorphism in Sickle Cell Disease: Relation to Vasculopathy and Disease Severity Azza Abdel Gawad Tantawy,
MD,
1
* Amira Abdel Moneam Adly, MD,1 Eman Abdel Rahman Ismail, and Shereen Hussiny Aly, MB, BCh1
Background. Impaired NO bioavailability represents the central feature of endothelial dysfunction, and is a common denominator in the pathogenesis of vasculopathy in sickle cell disease (SCD). Evidence indicates the contribution of 4a allele of endothelial NO synthase (eNOS) gene to cardiac and renal diseases. We studied the 27-base pair tandem repeat polymorphism in intron 4 of eNOS gene in 51 patients with SCD compared with 55 healthy controls and evaluated its role in disease severity and hemolysis-associated complications. Procedure. Transfusion history, vaso-occlusive crisis, thrombotic events, urinary albumin excretion, and echocardiography were assessed. Analysis of eNOS intron 4 gene polymorphism was performed by polymerase chain reaction. Results. The distribution of eNOS alleles and genotypes was similar between patients with
MD,
2
SCD and controls. Compared with bb genotype, the frequency of eNOS4a allele (aa and ab genotypes) was significantly higher in patients with elevated tricuspid regurgitant velocity (TRV) (P ¼ 0.009), nephropathy (P ¼ 0.006), or history of cerebral stroke (P ¼ 0.029). Logistic regression analysis revealed that eNOS4a allele was an independent risk factor for elevated TRV (P < 0.001). Patients with SCD and eNOS4a allele had higher lactate dehydrogenase, serum ferritin, D-Dimer, and von Willebrand factor antigen (P < 0.05). Conclusions. We suggest that eNOS intron 4 gene polymorphism is related to endothelial dysfunction and vasculopathy in SCD and could provide utility for prediction of increased susceptibility to vascular complications. Pediatr Blood Cancer # 2014 Wiley Periodicals, Inc.
Key words: endothelial dysfunction; nitric oxide synthase gene polymorphism; SCD
INTRODUCTION The variable spectrum of sickle cell disease (SCD) is the consequence of multiple events and genetic susceptibility that go beyond the occurrence of a single amino acid substitution in the bglobin chain of hemoglobin [1]. Many mechanisms contribute to the complex pathophysiology of SCD, with dysfunction of the vascular endothelium as a unifying theme. Specifically, hemolysis-associated low arginine and nitric oxide (NO) bioavailability, amplified by NO synthase uncoupling, elevated arginase activity, superoxide production, oxidative stress, accumulation of arginine analogs, ischemia-reperfusion injury, inflammation, apolipoprotein A-1 depletion, and a hypercoagulable state lead to vasomotor instability and ultimately produce a proliferative vasculopathy [1,2]. This vasculopathy is characterized epidemiologically by a clinical subphenotype of pulmonary hypertension (PH), cutaneous leg ulceration, priapism, sudden death, and possibly stroke in patients with SCD [3]. NO can inhibit the adhesion, aggregation and recruitment of platelets, vascular smooth muscle cells migration and growth, also regulates some vessel-platelet interactions and limits the oxidation of atherogenic low density lipoproteins [4]. At the release site it mediates local vasodilatation, antagonizes platelet aggregation and inhibits vascular smooth muscle cell proliferation [5]. In the kidney, NO dilates renal blood vessels and modulates renin secretion [6]. The constitutive endothelial NO synthase (eNOS), an enzyme that generates NO, 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 [7]. Among the reported polymorphisms of the eNOS gene, a polymorphism in intron 4 of the eNOS gene is a candidate gene in cardiovascular and renal diseases [8–10]. 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 [11]. An association of the 4a allele of the eNOS gene with coronary heart disease, renal disease and hypertension has been reported [12,13]. Although impaired NO bioavailability is a C
2014 Wiley Periodicals, Inc. DOI 10.1002/pbc.25234 Published online in Wiley Online Library (wileyonlinelibrary.com).
common denominator in the pathogenesis of vasculopathy in SCD [1], the specific role and clinical relevance of eNOS gene polymorphism remains to be elucidated. Therefore, we studied the 27-base pair tandem repeat polymorphism in intron4 of eNOS gene in young patients with SCD and assessed its potential involvement in disease severity and various hemolysis-associated complications.
MATERIALS AND METHODS This cross sectional case-control study included 51 patients with SCD (29 males and 22 females with a male-to-female ratio 1.3:1) 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 10.9 3.7 years. Fifty-five age-, sex- and ethnicity-matched healthy subjects (having normal hemoglobin phenotype, Hb AA) were enrolled as a control group; 32 males and 23 females (male-to-female ratio, 1.4:1) with a mean age of 11.3 3.5 years (range, 4–18 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. All included patients were subjected to medical history and clinical examination stressing on disease duration, anthropometric measures, blood pressure, evidence of renal, hepatic or cardiac disease, frequency of vaso-occlusive crisis, transfusion history, 1
Pediatrics Department, Faculty of Medicine, Ain Shams University, Cairo, Egypt; 2Clinical Pathology Department, Faculty of Medicine, Ain Shams University, Cairo, Egypt Conflict of interest: Nothing to declare.
Correspondence to: Azza Abdel Gawad Tantawy, 22 Ahmed Amin street, St Fatima square, Heliopolis, Cairo, Egypt. E-mail: [email protected] Received 10 June 2014; Accepted 28 July 2014
2
Tantawy et al.
chelation and/or hydroxyurea therapy. We had 23 patients with sickle cell anemia (HbSS) and 28 patients with HbS-beta zero thalassemia and all were at steady state at the time of sample collection. Patients received hydroxyurea (Bristol-Meyers-Squibb, NY) orally on a dose ranged from 15–25 mg/kg/day. Six patients were on deferroxamine therapy (Desferal1, DFO; Novartis Pharma AG, Basel, Switzerland) subcutaneously in a dose that ranged from 30–40 mg/kg/day 5 days/week.
immediately analyzed using 2% agarose gel electrophoresis and fragments were visualized by ethidum bromide staining and ultraviolet transillumination. To verify the results, PCR products were further analyzed using QIAxcel DNA screening Kit and Qiaxcel genetic analyzer (Qiagen, germany), which provides automated and accurate estimates of allele sizes and utilizes a preassembled cartridge (cartridge type Qiaxcel DNA high-resolution cartridge, injection time 10 seconds, Qiaxcel DNA size marker 15 bp–1 kb) to simultaneously run samples and collect data [14,15].
Sample Collection Peripheral blood samples were collected on ethylene diamine tetra-acetic acid (EDTA) (1.2 mg/ml) for complete blood count (CBC), hemoglobin analysis, and eNOS genotype. For chemical analysis, clotted samples were obtained and serum was separated by centrifugation for 15 minutes at 1,000 g.
Diagnostic testing Laboratory investigations included CBC using Sysmex XT1800i (Sysmex, Japan), examination of Leishman-stained smears for differential white blood cell (WBC) count, hemoglobin analysis using high performance liquid chromatography (HPLC) by D-10 (BioRad, Marnes La Coquette, France), liver and kidney function tests, markers of hemolysis (lactate dehydrogenase [LDH] and indirect bilirubin) and high sensitivity C-reactive protein (hs-CRP) as well as serum ferritin on Cobas Integra 800 (Roche Diagnostics, Mannheim, Germany). Patients with any clinical evidence of infection or CRP > 10 mg/L were excluded. Serum ferritin level was measured at the start of the study with calculation of the mean value of the last year prior to the study in order to know the ferritin trend. Quantitative D-dimer assay based on particle-enhanced immuno-turbidimetry (Innovance D-dimer, Siemens Healthcare Diagnostics, Marburg, Germany) and von Willebrand factor antigen (VWF:Ag) (Siemens Healthcare Diagnostics, Marburg, Germany) were performed on a Sysmex CA-1500 coagulation analyzer (Siemens Healthcare Diagnostics, Marburg, Germany). Urinary albumin excretion (UAE) as an indicator of nephropathy was measured in an early morning urine sample as albumin-tocreatinine ratio using Cobas Integra 800 (Roche Diagnostics, Mannheim, Germany).
Determination of eNOS Genotypes eNOS genotype was determined using polymerase chain reaction (PCR) [9,10]. Genomic DNA was extracted from peripheral blood using a QIAamp DNA Blood Mini Kit (Qiagen, Germany) and was stored at 4˚C until analysis. Two oligonucleotide primers (sense) 50 -AGGCCCTATGGTAGTGCCTT-30 and (antisense) 50 -TCTCTTAGTGCTGTGGTCAC-30 based on the flanking sequences of the VNTR in the eNOS gene were used to amplify the corresponding DNA fragment by PCR [9]. PCR products were then synthesized from DNA samples using the Taq PCR Master Mix Kit (Qiagen, Germany). Each PCR contained all the necessary reagents, primers and 200 ng of genomic DNA in a final volume of 25 mL. Using the applied Biosystems VeritiTM 96-Well Thermal Cycler (Applied Biosystems, Foster City, CA), PCR conditions were: initial denaturation at 94˚C for 5 minutes; 35 cycles of denaturation at 94˚C for 30 seconds, annealing at 56˚C for 1 minute, and extension at 72˚C for 1 minute; and final extension at 72˚C for 7 minutes. The PCR products were stored at 20˚C until analysis or Pediatr Blood Cancer DOI 10.1002/pbc
Radiological Examination All studied patients were clinically asymptomatic for PH and cardiovascular abnormalities. Screening for PH was performed by the non-invasive Doppler echocardiography with different modalities using Vivid E9 (GE Healthcare, Norway) to evaluate pulmonary artery pressure and tricuspid regurgitant jet velocity (TRV).
Diagnostic Criteria The definition of SCD was based on CBC, reticulocyte count, markers of hemolysis, hemoglobin analysis and confirmed by genotyping based on identification of b-globin gene mutations by PCR implications of the DNA and subsequent reverse-hybridization to immobilized allele-specific biotinylated oligonucleotides probes covering the most common Mediterranean mutations [16]. A painful crisis was defined as the occurrence of pain in the extremities, back, abdomen, chest, or head that lasted at least two hours, led to a clinic visit, and could not be explained except by SCD [17] while steady state was defined as a period without pain or painful crisis for at least four weeks [18]. The frequency of vasoocclusive crisis in the previous year was divided into mild (two or less episodes requiring medical visits) or severe (three or more episodes requiring medical visits) [19]. A TRV 2.5 m/s was used as a proxy for patients at risk for PH [20,21]. The diagnosis of acute chest syndrome (ACS) was defined as a new pulmonary infiltrate on chest x-ray and 2 of the following: chest, upper abdominal, or rib pain; dyspnea; fever; tachypnea; grunting; nasal flaring; or retractions [22]. Microalbuminuria and macroalbuminuria were present if UAE in at least two out of three consecutive urine samples, two months apart was 30– 299 mg/g creatinine and 300 mg/g creatinine, respectively [23].
Statistical Analysis Analysis of data was done using Statistical Program for Social Science version 15 (SPSS Inc., Chicago, IL). Quantitative variables were described as range, mean, and standard deviation. Qualitative variables were described as number and percent. Allele frequencies were estimated by the gene counting method. In order to compare quantitative parametric variables between two groups, Student 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 clinicaopathological 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 elevated
eNOS Gene Polymorphism in SCD TRVand vaso-occlusive crisis as the dependent variables. A P-value 0.05). The frequency of eNOS4a allele (aa and ab genotypes) was significantly higher in patients with SCD and elevated TRV or nephropathy (micro- or macro-albuminuria) (P ¼ 0.009 and P ¼ 0.006, respectively). The presence of eNOS4a allele (aa and ab genotypes) was also significantly associated with history of cerebral stroke (P ¼ 0.029). Patients with frequent vaso-occlusive crisis 3 attacks/year in the last year prior to the study showed higher frequencies of the eNOS4a allele (P ¼ 0.017) (Table II and Fig. 1). Patients with SCD and eNOS4a allele had higher WBCs (P ¼ 0.011), LDH (P ¼ 0.023), serum ferritin (P < 0.001), D-Dimer
3
(P ¼ 0.008) and VWF:Ag (P < 0.001) with lower hemoglobin (P ¼ 0.012) (Table II). Upon analysis of the relation between elevated TRV and all the studied variables, the risk of elevated TRV was significantly associated with age, LDH, serum ferritin, D-dimer, eNOS4a allele, and history of ACS. Logistic regression analysis revealed that eNOS4a allele was an independent risk factor for elevated TRV (OR 2.4, 95% CI 1.6–5.1; P < 0.001). Other independent variables were: age and history of ACS (Table III). Moreover, logistic regression analysis showed that eNOS4a allele was an independent risk factor for vaso-occlusive crisis (OR 2.7, 95% CI 1.3–5.3; P < 0.001).
DISCUSSION We explored polymorphism in intron 4 of the eNOS gene that is reported to be a candidate gene in cardiovascular and renal diseases [9,10]. The PCR products were analyzed using agarose gel electrophoresis followed by ethidum bromide staining and results were further verified using the QIAxcel System to provide automated and accurate estimates of allele sizes [14,24] and to accurately identify homozygous and heterozygous genotypes. The controls was matched to the patients regarding age, sex, race except having the disease and our results revealed that patients with SCD showed similar distribution of eNOS4 alleles and genotypes compared with controls. We compared patients with aa and ab genotypes (NOS4a allele carriers) with bb genotype as reported by Elshamaa et al. [10] and found that patients with SCD and eNOS4a alleles were at higher risk
TABLE I. Clinical and Laboratory Data of the Studied Patients Sickle Cell Disease and Controls Variable Age (years) Males, n (%) Weight for age SDS Height for age SDS Transfusion index (ml/kg/year) Elevated TRV, n (%) Nephropathy (micro-/macroalbuminuria), n (%) History of cerebral stroke, n (%) History of vaso-occlusive (3 attacks/year), n (%) History of acute chest syndrome, n (%) WBC count (x 109/L) Hemoglobin (g/dL) HbF (%) Lactate dehydrogenase (IU/L) Indirect bilirubin (mg/dL) Serum ferritin (mg/L) D-Dimer (mg/L FEU) VWF:Ag (%) eNOS intron 4 gene polymorphism Alleles a b eNOS intron 4 gene polymorphism Genotypes aa ab bb
Controls (n ¼ 55)
SCD (n ¼ 51)
11.3 3.5 32 (58.2) 0.69 0.2 0.87 0.3 — — — — — — 6.4 1.7 11.5 3.1 1.0 0.2 219 45 1.1 0.2 166 37 0.38 0.1 89 5.5
10.9 3.7 29 (56.9) 0.24 0.08 0.3 0.1 187.9 28.7 20 (39.2) 18 (35.3) 13 (25.5) 11 (21.6) 5 (9.8) 18.9 3.9 7.8 1.6 15.6 5.8 1074 212 2.3 1 1827 317 2.7 1.1 148 10.2
31 (28.18%) 79 (71.82%)
42 (41.2%) 60 (58.8%)
0.065
7 (12.73%) 17 (30.91%) 31 (56.36%)
14 (27.5%) 14 (27.5%) 23 (45%)
0.16
0.568 0.75
Endothelial Nitric Oxide Synthase Gene Intron 4 VNTR Polymorphism in Sickle Cell Disease: Relation to Vasculopathy and Disease Severity Azza Abdel Gawad Tantawy,
MD,
1
* Amira Abdel Moneam Adly, MD,1 Eman Abdel Rahman Ismail, and Shereen Hussiny Aly, MB, BCh1
Background. Impaired NO bioavailability represents the central feature of endothelial dysfunction, and is a common denominator in the pathogenesis of vasculopathy in sickle cell disease (SCD). Evidence indicates the contribution of 4a allele of endothelial NO synthase (eNOS) gene to cardiac and renal diseases. We studied the 27-base pair tandem repeat polymorphism in intron 4 of eNOS gene in 51 patients with SCD compared with 55 healthy controls and evaluated its role in disease severity and hemolysis-associated complications. Procedure. Transfusion history, vaso-occlusive crisis, thrombotic events, urinary albumin excretion, and echocardiography were assessed. Analysis of eNOS intron 4 gene polymorphism was performed by polymerase chain reaction. Results. The distribution of eNOS alleles and genotypes was similar between patients with
MD,
2
SCD and controls. Compared with bb genotype, the frequency of eNOS4a allele (aa and ab genotypes) was significantly higher in patients with elevated tricuspid regurgitant velocity (TRV) (P ¼ 0.009), nephropathy (P ¼ 0.006), or history of cerebral stroke (P ¼ 0.029). Logistic regression analysis revealed that eNOS4a allele was an independent risk factor for elevated TRV (P < 0.001). Patients with SCD and eNOS4a allele had higher lactate dehydrogenase, serum ferritin, D-Dimer, and von Willebrand factor antigen (P < 0.05). Conclusions. We suggest that eNOS intron 4 gene polymorphism is related to endothelial dysfunction and vasculopathy in SCD and could provide utility for prediction of increased susceptibility to vascular complications. Pediatr Blood Cancer # 2014 Wiley Periodicals, Inc.
Key words: endothelial dysfunction; nitric oxide synthase gene polymorphism; SCD
INTRODUCTION The variable spectrum of sickle cell disease (SCD) is the consequence of multiple events and genetic susceptibility that go beyond the occurrence of a single amino acid substitution in the bglobin chain of hemoglobin [1]. Many mechanisms contribute to the complex pathophysiology of SCD, with dysfunction of the vascular endothelium as a unifying theme. Specifically, hemolysis-associated low arginine and nitric oxide (NO) bioavailability, amplified by NO synthase uncoupling, elevated arginase activity, superoxide production, oxidative stress, accumulation of arginine analogs, ischemia-reperfusion injury, inflammation, apolipoprotein A-1 depletion, and a hypercoagulable state lead to vasomotor instability and ultimately produce a proliferative vasculopathy [1,2]. This vasculopathy is characterized epidemiologically by a clinical subphenotype of pulmonary hypertension (PH), cutaneous leg ulceration, priapism, sudden death, and possibly stroke in patients with SCD [3]. NO can inhibit the adhesion, aggregation and recruitment of platelets, vascular smooth muscle cells migration and growth, also regulates some vessel-platelet interactions and limits the oxidation of atherogenic low density lipoproteins [4]. At the release site it mediates local vasodilatation, antagonizes platelet aggregation and inhibits vascular smooth muscle cell proliferation [5]. In the kidney, NO dilates renal blood vessels and modulates renin secretion [6]. The constitutive endothelial NO synthase (eNOS), an enzyme that generates NO, 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 [7]. Among the reported polymorphisms of the eNOS gene, a polymorphism in intron 4 of the eNOS gene is a candidate gene in cardiovascular and renal diseases [8–10]. 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 [11]. An association of the 4a allele of the eNOS gene with coronary heart disease, renal disease and hypertension has been reported [12,13]. Although impaired NO bioavailability is a C
2014 Wiley Periodicals, Inc. DOI 10.1002/pbc.25234 Published online in Wiley Online Library (wileyonlinelibrary.com).
common denominator in the pathogenesis of vasculopathy in SCD [1], the specific role and clinical relevance of eNOS gene polymorphism remains to be elucidated. Therefore, we studied the 27-base pair tandem repeat polymorphism in intron4 of eNOS gene in young patients with SCD and assessed its potential involvement in disease severity and various hemolysis-associated complications.
MATERIALS AND METHODS This cross sectional case-control study included 51 patients with SCD (29 males and 22 females with a male-to-female ratio 1.3:1) 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 10.9 3.7 years. Fifty-five age-, sex- and ethnicity-matched healthy subjects (having normal hemoglobin phenotype, Hb AA) were enrolled as a control group; 32 males and 23 females (male-to-female ratio, 1.4:1) with a mean age of 11.3 3.5 years (range, 4–18 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. All included patients were subjected to medical history and clinical examination stressing on disease duration, anthropometric measures, blood pressure, evidence of renal, hepatic or cardiac disease, frequency of vaso-occlusive crisis, transfusion history, 1
Pediatrics Department, Faculty of Medicine, Ain Shams University, Cairo, Egypt; 2Clinical Pathology Department, Faculty of Medicine, Ain Shams University, Cairo, Egypt Conflict of interest: Nothing to declare.
Correspondence to: Azza Abdel Gawad Tantawy, 22 Ahmed Amin street, St Fatima square, Heliopolis, Cairo, Egypt. E-mail: [email protected] Received 10 June 2014; Accepted 28 July 2014
2
Tantawy et al.
chelation and/or hydroxyurea therapy. We had 23 patients with sickle cell anemia (HbSS) and 28 patients with HbS-beta zero thalassemia and all were at steady state at the time of sample collection. Patients received hydroxyurea (Bristol-Meyers-Squibb, NY) orally on a dose ranged from 15–25 mg/kg/day. Six patients were on deferroxamine therapy (Desferal1, DFO; Novartis Pharma AG, Basel, Switzerland) subcutaneously in a dose that ranged from 30–40 mg/kg/day 5 days/week.
immediately analyzed using 2% agarose gel electrophoresis and fragments were visualized by ethidum bromide staining and ultraviolet transillumination. To verify the results, PCR products were further analyzed using QIAxcel DNA screening Kit and Qiaxcel genetic analyzer (Qiagen, germany), which provides automated and accurate estimates of allele sizes and utilizes a preassembled cartridge (cartridge type Qiaxcel DNA high-resolution cartridge, injection time 10 seconds, Qiaxcel DNA size marker 15 bp–1 kb) to simultaneously run samples and collect data [14,15].
Sample Collection Peripheral blood samples were collected on ethylene diamine tetra-acetic acid (EDTA) (1.2 mg/ml) for complete blood count (CBC), hemoglobin analysis, and eNOS genotype. For chemical analysis, clotted samples were obtained and serum was separated by centrifugation for 15 minutes at 1,000 g.
Diagnostic testing Laboratory investigations included CBC using Sysmex XT1800i (Sysmex, Japan), examination of Leishman-stained smears for differential white blood cell (WBC) count, hemoglobin analysis using high performance liquid chromatography (HPLC) by D-10 (BioRad, Marnes La Coquette, France), liver and kidney function tests, markers of hemolysis (lactate dehydrogenase [LDH] and indirect bilirubin) and high sensitivity C-reactive protein (hs-CRP) as well as serum ferritin on Cobas Integra 800 (Roche Diagnostics, Mannheim, Germany). Patients with any clinical evidence of infection or CRP > 10 mg/L were excluded. Serum ferritin level was measured at the start of the study with calculation of the mean value of the last year prior to the study in order to know the ferritin trend. Quantitative D-dimer assay based on particle-enhanced immuno-turbidimetry (Innovance D-dimer, Siemens Healthcare Diagnostics, Marburg, Germany) and von Willebrand factor antigen (VWF:Ag) (Siemens Healthcare Diagnostics, Marburg, Germany) were performed on a Sysmex CA-1500 coagulation analyzer (Siemens Healthcare Diagnostics, Marburg, Germany). Urinary albumin excretion (UAE) as an indicator of nephropathy was measured in an early morning urine sample as albumin-tocreatinine ratio using Cobas Integra 800 (Roche Diagnostics, Mannheim, Germany).
Determination of eNOS Genotypes eNOS genotype was determined using polymerase chain reaction (PCR) [9,10]. Genomic DNA was extracted from peripheral blood using a QIAamp DNA Blood Mini Kit (Qiagen, Germany) and was stored at 4˚C until analysis. Two oligonucleotide primers (sense) 50 -AGGCCCTATGGTAGTGCCTT-30 and (antisense) 50 -TCTCTTAGTGCTGTGGTCAC-30 based on the flanking sequences of the VNTR in the eNOS gene were used to amplify the corresponding DNA fragment by PCR [9]. PCR products were then synthesized from DNA samples using the Taq PCR Master Mix Kit (Qiagen, Germany). Each PCR contained all the necessary reagents, primers and 200 ng of genomic DNA in a final volume of 25 mL. Using the applied Biosystems VeritiTM 96-Well Thermal Cycler (Applied Biosystems, Foster City, CA), PCR conditions were: initial denaturation at 94˚C for 5 minutes; 35 cycles of denaturation at 94˚C for 30 seconds, annealing at 56˚C for 1 minute, and extension at 72˚C for 1 minute; and final extension at 72˚C for 7 minutes. The PCR products were stored at 20˚C until analysis or Pediatr Blood Cancer DOI 10.1002/pbc
Radiological Examination All studied patients were clinically asymptomatic for PH and cardiovascular abnormalities. Screening for PH was performed by the non-invasive Doppler echocardiography with different modalities using Vivid E9 (GE Healthcare, Norway) to evaluate pulmonary artery pressure and tricuspid regurgitant jet velocity (TRV).
Diagnostic Criteria The definition of SCD was based on CBC, reticulocyte count, markers of hemolysis, hemoglobin analysis and confirmed by genotyping based on identification of b-globin gene mutations by PCR implications of the DNA and subsequent reverse-hybridization to immobilized allele-specific biotinylated oligonucleotides probes covering the most common Mediterranean mutations [16]. A painful crisis was defined as the occurrence of pain in the extremities, back, abdomen, chest, or head that lasted at least two hours, led to a clinic visit, and could not be explained except by SCD [17] while steady state was defined as a period without pain or painful crisis for at least four weeks [18]. The frequency of vasoocclusive crisis in the previous year was divided into mild (two or less episodes requiring medical visits) or severe (three or more episodes requiring medical visits) [19]. A TRV 2.5 m/s was used as a proxy for patients at risk for PH [20,21]. The diagnosis of acute chest syndrome (ACS) was defined as a new pulmonary infiltrate on chest x-ray and 2 of the following: chest, upper abdominal, or rib pain; dyspnea; fever; tachypnea; grunting; nasal flaring; or retractions [22]. Microalbuminuria and macroalbuminuria were present if UAE in at least two out of three consecutive urine samples, two months apart was 30– 299 mg/g creatinine and 300 mg/g creatinine, respectively [23].
Statistical Analysis Analysis of data was done using Statistical Program for Social Science version 15 (SPSS Inc., Chicago, IL). Quantitative variables were described as range, mean, and standard deviation. Qualitative variables were described as number and percent. Allele frequencies were estimated by the gene counting method. In order to compare quantitative parametric variables between two groups, Student 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 clinicaopathological 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 elevated
eNOS Gene Polymorphism in SCD TRVand vaso-occlusive crisis as the dependent variables. A P-value 0.05). The frequency of eNOS4a allele (aa and ab genotypes) was significantly higher in patients with SCD and elevated TRV or nephropathy (micro- or macro-albuminuria) (P ¼ 0.009 and P ¼ 0.006, respectively). The presence of eNOS4a allele (aa and ab genotypes) was also significantly associated with history of cerebral stroke (P ¼ 0.029). Patients with frequent vaso-occlusive crisis 3 attacks/year in the last year prior to the study showed higher frequencies of the eNOS4a allele (P ¼ 0.017) (Table II and Fig. 1). Patients with SCD and eNOS4a allele had higher WBCs (P ¼ 0.011), LDH (P ¼ 0.023), serum ferritin (P < 0.001), D-Dimer
3
(P ¼ 0.008) and VWF:Ag (P < 0.001) with lower hemoglobin (P ¼ 0.012) (Table II). Upon analysis of the relation between elevated TRV and all the studied variables, the risk of elevated TRV was significantly associated with age, LDH, serum ferritin, D-dimer, eNOS4a allele, and history of ACS. Logistic regression analysis revealed that eNOS4a allele was an independent risk factor for elevated TRV (OR 2.4, 95% CI 1.6–5.1; P < 0.001). Other independent variables were: age and history of ACS (Table III). Moreover, logistic regression analysis showed that eNOS4a allele was an independent risk factor for vaso-occlusive crisis (OR 2.7, 95% CI 1.3–5.3; P < 0.001).
DISCUSSION We explored polymorphism in intron 4 of the eNOS gene that is reported to be a candidate gene in cardiovascular and renal diseases [9,10]. The PCR products were analyzed using agarose gel electrophoresis followed by ethidum bromide staining and results were further verified using the QIAxcel System to provide automated and accurate estimates of allele sizes [14,24] and to accurately identify homozygous and heterozygous genotypes. The controls was matched to the patients regarding age, sex, race except having the disease and our results revealed that patients with SCD showed similar distribution of eNOS4 alleles and genotypes compared with controls. We compared patients with aa and ab genotypes (NOS4a allele carriers) with bb genotype as reported by Elshamaa et al. [10] and found that patients with SCD and eNOS4a alleles were at higher risk
TABLE I. Clinical and Laboratory Data of the Studied Patients Sickle Cell Disease and Controls Variable Age (years) Males, n (%) Weight for age SDS Height for age SDS Transfusion index (ml/kg/year) Elevated TRV, n (%) Nephropathy (micro-/macroalbuminuria), n (%) History of cerebral stroke, n (%) History of vaso-occlusive (3 attacks/year), n (%) History of acute chest syndrome, n (%) WBC count (x 109/L) Hemoglobin (g/dL) HbF (%) Lactate dehydrogenase (IU/L) Indirect bilirubin (mg/dL) Serum ferritin (mg/L) D-Dimer (mg/L FEU) VWF:Ag (%) eNOS intron 4 gene polymorphism Alleles a b eNOS intron 4 gene polymorphism Genotypes aa ab bb
Controls (n ¼ 55)
SCD (n ¼ 51)
11.3 3.5 32 (58.2) 0.69 0.2 0.87 0.3 — — — — — — 6.4 1.7 11.5 3.1 1.0 0.2 219 45 1.1 0.2 166 37 0.38 0.1 89 5.5
10.9 3.7 29 (56.9) 0.24 0.08 0.3 0.1 187.9 28.7 20 (39.2) 18 (35.3) 13 (25.5) 11 (21.6) 5 (9.8) 18.9 3.9 7.8 1.6 15.6 5.8 1074 212 2.3 1 1827 317 2.7 1.1 148 10.2
31 (28.18%) 79 (71.82%)
42 (41.2%) 60 (58.8%)
0.065
7 (12.73%) 17 (30.91%) 31 (56.36%)
14 (27.5%) 14 (27.5%) 23 (45%)
0.16
0.568 0.75
