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Apolipoprotein E gene polymorphism and the risk of left ventricular dysfunction among Egyptian β-thalassemia major
Gene 524 (2013) 292–295
Contents lists available at SciVerse ScienceDirect
Gene journal homepage: www.elsevier.com/locate/gene
Apolipoprotein E gene polymorphism and the risk of left ventricular dysfunction among Egyptian β-thalassemia major Mona H. El-Tagui a, Mona M. Hamdy a, Iman A. Shaheen b,⁎, Hala Agha c, Hoda A. Abd-Elfatah d a
Department of Pediatrics, Faculty of Medicine, Cairo University, Egypt Department of Clinical Pathology, Faculty of Medicine, Cairo University, Egypt Department of Cardiology, Faculty of Medicine, Cairo University, Egypt d Egyptian Ministry of Health, Cairo, Egypt b c
a r t i c l e
i n f o
Article history: Accepted 27 March 2013 Available online 13 April 2013 Keywords: β-Thalassemia Apolipoprotein E PCR-RFLP Left ventricular failure Left ventricular dilatation
a b s t r a c t In Egypt, β-thalassemia is the most common hereditary hemolytic anemia. Cardiac dysfunction, secondary to iron overload with formation of oxygen free radicals, is the most common cause of death in β-thalassemia patients. This study was designed to determine whether the allelic genotype of apolipoprotein E (Apo E), which exhibits antioxidant properties, could represent a genetic risk factor for the development of left ventricular (LV) dysfunction in β-thalassemia major. Fifty Egyptian β-thalassemia major patients were subjected to echocardiography to assess LV function. Apo E genotyping by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) was done for all patients in addition to 50 age and sex matched healthy control subjects. Patients were classified into three groups. Group I and II were clinically asymptomatic. Group II subjects had evidence of LV dilatation, while Group III patients had clinical and echocardiographic findings of LV failure. Apo E4 allele was significantly higher among Group II and III than in controls. In conclusion, Apo E4 allele can be considered as a genetic risk factor for LV dysfunctions in β-thalassemic patients. It could be used as predictive indicator for additional risk of LV failure, particularly in asymptomatic patients with LV dilatation, requiring a closer followup, to prevent further disease progression. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Thalassemias are inherited hemoglobin disorders resulting in chronic hemolytic anemia (Higgs et al., 2001). Thalassemia major is the most prevalent clinical form of the disease and is characterized by: severe hemolytic anemia from the first year of life, which requires blood transfusion therapy for the patients' survival. Transfusion therapy, elevated gastrointestinal absorption of iron, together with ineffective erythropoiesis, determine iron overload which is the major cause of mortality and morbidity associated with the disease (Rund and Rachmilewitz, 2005). This is followed by multiple chronic organ damage, growth retardation and a considerably reduced life expectancy (Borgna-Pignatti et al., 2005).
Abbreviations: Apo E, Apolipoprotein epsilon; LV, Left ventricle; PCR-RFLP, Polymerase Chain Reaction Restriction Fragment Length Polymorphism; CXR, Chest X-ray; ECG, Electrocardiography; RVEDD, Right ventricular end diastolic diameter; LVEDD, Left ventricular end diastolic diameter; LVESD, Left ventricular end systolic diameter; IVS, Interventricular septum; PWT, Posterior wall thickness; FS, Fraction shortening; EF, Ejection fraction; EDTA, Ethylene diamine tetra acetic acid; SPSS, Statistical Package for the Social Science. ⁎ Corresponding author at: Clinical & Chemical Pathology Department, Kasr Al-Ainy Faculty of Medicine-Cairo University, Mail Box 11562, Cairo, Egypt. Tel./fax: +20 23654480. E-mail address: [email protected] (I.A. Shaheen). 0378-1119/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2013.03.134
In thalassemia major, cardiac disorders, most notably left-sided heart failure, are responsible for more than half of the deaths in those patients and are thus the main determinants of survival (Hahalis et al., 2005). Heart disease may manifest as hemosiderotic cardiomyopathy, heart failure, pulmonary hypertension, arrhythmias, systolic/diastolic dysfunction, pericardial effusion, myocarditis or pericarditis (Aessopos et al., 2007). Left ventricular (LV) failure is the most common presentation of cardiac involvement and the main cause of death in thalassemia major patients, and once cardiac dysfunction is detected, the prognosis is poor (Hahalis et al., 2005). Myocardial iron overload is traditionally considered the fundamental pathogenic mechanism of heart failure. Iron excess within cells leads to free radical formation, causing damage to membrane lipids and proteins resulting in cellular injury and impairing cardiac contractility and myocardial performance (Aessopos et al., 2007). Apolipoprotein epsilon (Apo E) is a well-known lipid transport protein. Its gene is located on chromosome 19 and has three major alleles with decreasing anti-oxidant activity including E2–E3–E4 (Moore et al., 2004). Apo E acts as a scavenger of free radicals; the demonstrated metal binding ability (including iron) of Apo E was postulated to be one mechanism accounting for its anti-oxidant activity (Economou-Petersen et al., 1998). Apolipoprotein E4 has the least anti-oxidant and iron binding activities (Miyata and Smith, 1996). Patients who have E4 allele are at a higher risk for iron induced damage of cellular and subcellular particles (Gibson et al., 2000).
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This study was designed to determine whether the allelic genotype of Apo E, which exhibits antioxidant properties, could represent a genetic risk factor for the development of left ventricular dysfunction in Egyptian β-thalassemia major.
of LV failure and concomitant echocardiographic findings. These patients exhibited dyspnea on exertion (New York Heart Association [NYHA] functional class I–IV) and fulfilled at least two major Framingham criteria for heart failure diagnosis.
2. Material and methods
2.3. Genotyping of Apo E gene
2.1. Study population
From each participant, 4 to 5 ml venous blood was obtained by sterile venipuncture into a sterile ethylene diamine tetra acetic acid (EDTA) vacutainer. Samples were either stored in the same vacutainer at −20 °C or used directly within 24 h for DNA extraction. Genomic DNA was extracted from whole blood using Gene Jet Genomic DNA Purification Kits (Fermentas — Canada) according to the manufacturer's instructions. Genotyping of Apo E gene was performed by PCR-RFLP technique. The primers for Apo E gene were prepared as previously described (Wenham et al., 1991). The primer pair used was forward primer: 5′-TCC-AAG-GAG-CTG-CAG-GCG-GCG-CA-3′, and reversed primer: 5′-ACA-GAA-TTC-GCC-CCG-GCC-TGG-TAC-ACT-GCC-A-3′: (Pioneer — Germany). Five microliter (40–50 ng) genomic DNA was amplified in a total volume of 25 μl. The reaction mixture contained 1 μl (20 pmole) of each of the forward and reverse primers and 12.5 μl of 2× Taq PCR master mixes (Fermentas — Canada). The thermocycler program was as follows: initial denaturation at 94 °C for 5 min followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 65 °C for 35 s, and extension at 72 °C for 30 s, followed by final extension at 72 °C for 5 min. The amplified PCR product (292 bp) was then digested with 10 U of the fast digest Hha restriction endonuclease (Fermentas — Canada) at 37 °C for 5 min. Digested products were electrophoresed on a 4% agarose gel. DNA fragments were visualized by ethidium bromide. Apo E2 allele was detected by the presence of 91 and 81 bp fragments and Apo E3 allele was detected by the presence of 91 and 48 bp fragments, while 72 and 48 bp fragments were indicative of Apo E4 allele (Fig. 1). Apo E genotyping was randomly repeated for 10% of the samples (10 samples), and interpreted blindly by two different observers to confirm the results.
The present study was conducted on 50 β-thalassemia major patients, 29 males and 21 females, with a minimum age of 11 years. They attended the Hematology Clinic of the Children Hospital, Faculty of Medicine, Cairo University. The study also included 50 age and sex matched healthy volunteers as a control group. The study design was approved by the Scientific Research Committee of Clinical Pathology and Pediatrics Departments, Faculty of Medicine, Cairo University. All patients, parents and/or surrogates were informed about the aim of the study. Formal written consents were obtained from the participants or their caregivers who agreed to their child's participation in the study. All patients were receiving blood transfusions every two to four weeks to maintain a hemoglobin level above 9 g/dl during all of the follow-up years. Iron chelation was prescribed for all patients either by subcutaneous injection of deferoxamine for 30 patients (60%), or oral intake in the form of deferiprone for eight patients (16%), or deferasirox for 12 patients (24%). Mean pretransfusion hemoglobin level and mean serum ferritin level in each patient over the last two and five years of follow-up were obtained to evaluate the transfusion therapy and the hemosiderosis level. All patients were subjected to cardiac evaluation including medical history, clinical examination, chest X-ray (CXR), electrocardiography (ECG) and M-mode echocardiographic studies. Apo E genotyping by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) was done for all patients and controls. 2.2. Echocardiography
2.4. Statistical analysis Two-dimensional, pulsed-wave, continuous-wave and color-flow Doppler echocardiography were performed using a General Electric, Vivid 5 echocardiography machine. A 3.5 or 5 MHZ phased array transducer was used depending on the patient's age. Various cardiac views were taken in order to assess the cardiac chambers: their sizes, connections, relations and presence of valvular abnormalities such as: tricuspid and pulmonary regurgitation. Using the M-Mode, left atrial and aortic dimensions were assessed, in long axis and parasternal views, the following were measured: right ventricular end diastolic diameter (RVEDD), left ventricular end diastolic diameter (LVEDD), left ventricular end systolic diameter (LVESD), interventricular septum (IVS) and posterior wall thickness (PWT). In addition to these, calculation of fraction shortening (FS%) and ejection fraction (EF%) were done as an indication of LV systolic function according to the recommendations of the American Society of Echocardiography. Left and right ventricular end diastolic dimensions more than the upper limits of normal according to patients' age were considered enlarged. Global systolic function was considered abnormal if FS b 25% and EF > 55. Diastolic dysfunction was considered when the E wave velocity was elevated, E/A ratio was reversed, or E wave deceleration slope was decreased (Lang et al., 2005). The patients were classified into three groups according to clinical evaluation and echocardiographic findings. Group I patients were asymptomatic and their echocardiographic studies were within normal. Group II were asymptomatic, but exhibited LV dilatation (LV end diastolic diameter index [LVEDDI] higher than 30 mm/m2) without left ventricular systolic dysfunction (LV fractional shortening [FS] higher than 28%), as assessed echocardiographically. Patients in this group did not receive treatment. Group III patients had symptoms and signs
Data were statistically described in terms of range, mean ± standard deviation (±SD), median, frequencies (number of cases) and percentages when appropriate. Comparison of quantitative variables between the study groups was done using Kruskal–Wallis analysis of variance (ANOVA) test. For comparing categorical data, Chi square (χ 2) test was performed. Exact test was used instead when the expected frequency was less than five. Relative frequency of gene mutation was described by OR and their 95% CI. Univariate and
Fig. 1. PCR-RFLP gel electrophoresis of Apo E genotyping. M; molecular DNA ladder. Lane 1 and 9; show homozygous Apo E3/E3 genotype with 91 and 41 bp fragments. Lane 2,3,6,7 and 8; show heterozygous Apo E2/E3 genotype with 91–81 and 48 bp fragments. Lane 5; shows homozygous Apo E4/E4 genotype with 72 and 48 bp fragments. Lane 10; shows heterozygous Apo E2/E4 genotype with 91–81–72 and 48 bp fragments. Lane 11; shows heterozygous Apo E3/E4 genotype with 91–72 and 48 bp fragments.
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multivariate analysis models were used to test for the preferential effect of the independent variable(s) on the dependent variable(s). A probability value (p value) less than 0.05 was considered statistically significant. All statistical calculations were done using computer programs Microsoft Excel 2003 (Microsoft Corporation, NY, USA) and SPSS (Statistical Package for the Social Science; SPSS Inc., Chicago, IL, USA) version 15 for Microsoft Windows. 3. Results In our study, 50 β-thalassemia major patients were classified into three groups as follows: nine patients in Group I, 26 patients in Group II, and 15 patients in Group III. There was no statistical significant difference among the three groups regarding basic clinical and hematological parameters which are summarized in Table 1. In Table 2, the results of the echocardiographic evaluation of the patients are shown. Left ventricular end diastolic diameter (LVEDD) differed among the three groups, while left ventricular end systolic diameter (LVESD) indices, ejection fraction (EF) and left ventricular fractional shortening (FS) were significantly different on comparing Group II and Group III. Interventricular septum (IVS) thickness and left ventricular posterior wall (LVPW) thickness did not differ among the groups. For Apo E genotyping, E3/E3 genotype was found in 28 patients (56%) and in 39 normal control subjects (78%). E2/E3 was detected in eleven patients (22%) and four control subjects (8%). E3/E4 genotype was detected in six patients (12%) and in six normal control subjects (12%). One control subject carried the E2/E2 genotype. E4/E4 was only detected in five patients (10%), three of them were in Group III and two were in Group II. There was no statistical significant difference among the studied groups regarding Apo E genotyping (Table 3). On comparing the allelic distribution of Apo E gene among the three studied groups, Apo E4 allele was statistically higher in Group II and III than in controls (p = 0.027 and 0.02 respectively) (Table 4). 4. Discussion Beta thalassemia major is one of the most prevalent hereditary diseases in the Mediterranean region especially in Egypt (El Beshlawy et al., 2012; Kaplan et al., 2012). Transfusion therapy has greatly improved the survival of transfusion dependent thalassemia major patients; however, the resultant iron load causes damage of different types of tissues (Chu et al., 2012) Iron-induced cardio toxicity remains the leading cause of morbidity and mortality in those patients (Delaporta et al., 2012). Through iron-driven Fenton and Haber–Weiss reactions, the nontransferrin free plasma iron, in its bivalent or trivalent form, has a high toxicity through the formation of hydroxyl radicals (OH) (Synowiec et
Table 2 Echocardiographic evaluation of the patients. Echo cardio graphic indices (Mean ± SD)
Group I N=9
LVEDD LVESD IVS thickness EF% FS% LVPW thickness
27 28 8.0 65 39.2 7.4
± ± ± ± ± ±
Group II N = 26 3 4 1.9 8.8 5.9 1.1
43 30 7.7 61 35.80 8.2
± ± ± ± ± ±
Group III N = 15 5 3 1.5 5.8 4.48 1.8
47 34 7.8 35 26.533 7.8
± ± ± ± ± ±
p value 5 5 1.4 9.8 3.7 1.3
Sa,b,c Sb,c NS Sb,c Sb,c NS
LVEDD; left ventricular end diastolic diameter. LVESD; left ventricular end systolic diameter. IVS; interventricular septum. EF; ejection fraction. FS; left ventricular fractional shortening. LVPW; left ventricular posterior wall. S = Statistical significant difference. a On comparing Group I and II. b On comparing Group I and III. c On comparing Group II and III.
al., 2012). This leads to peroxidative damage of membrane lipids and proteins. Imbalance between production of oxygen free radicals and antioxidant defense mechanisms can result in oxidative stress and human diseases (Bazrgar et al., 2007). In the heart, the imbalance between free radicals and antioxidant mechanisms is manifested as impaired function of the mitochondrial inner-membrane respiratory chain resulting in abnormal energy metabolism expressed clinically with fatal cardiomyopathy (Economou-Petersen et al., 1998). Diversity in genetic loci may increase the susceptibility of heart failure leading to increased iron absorption and deposition among those patients, which makes them more vulnerable to iron overload (Kaur et al., 2003). Apo E4 allele has been reported as a genetic risk factor of LV failure in β-thalassemia in Greece (Economou-Petersen et al., 1998), Italy (Ferrara et al., 2001) and Iran Bazrgar et al. (2007), with no available data about this association among other Caucasian β-thalassemic patients including Egyptians. The aim of this study was to determine whether the allelic genotype of Apo lipoprotein E (Apo E), which exhibits antioxidant properties, could represent a genetic risk factor for the development of left ventricular failure in Egyptian β-thalassemia major patients. Beta thalassemic patients involved in our study were categorized into three groups according to clinical and echocardiographic findings. There was no statistical significant difference on comparing main clinical and laboratory findings including mean pretransfusion hemoglobin and mean serum ferritin. These findings were also noticed among the studied group of Economou-Petersen et al. (1998) and Bazrgar et al. (2007). Echocardiography was more sensitive in detecting patients with LV dilation as most of the patients were in Group II (52% [26 patients]) with no clinical symptoms, but with echocardiographic findings of LV dilation. Those patients are more vulnerable to develop LV dysfunction and need a closer follow up (Bazrgar et al., 2007). On comparing the echocardiographic findings among the three studied groups there was a statistical significant difference among the three groups except for IVS thickness and LVPW thickness. Similar
Table 1 Basic clinical and hematological data of the three studied groups. Variable
Group I
Group II
Group III
Age (years) Mean ± SD SEX MALE FEMALE Age at 1st transfusion (month.) Hemoglobin (g/dl) Body surface area (m2) Number of blood transfusion units Serum ferritin (ng/ml)
21.77 ± 7.1 5 4 10 ± 6.55
17 ± 5.97 15 11 14.03 ± 12.3
21 ± 5.5 6 9 16.37 ± 15.5
8.1 ± 0.67 1.50 ± 0.17 240 ± 86
8.1 ± 0.73 1.25 ± 0.24 220.8 ± 104.12
8.3 ± 0.55 1.356 ± 0.22 248.4 ± 95
5175.6 ± 3334
4885.46 ± 2439
3550 ± 2025
Table 3 Distribution of Apo E genotyping among the studied groups and controls. Apo E genotyping
Group I N=9
Group II N = 26
Group III N = 15
Control N = 50
E3/E3 E2/E3 E3/E4 E2/E2 E4/E4
6 (66.6%) 2 (22.2%) 1 (11.1%) 0 0
14 (53.8%) 5 (19.2%) 5 (19.2%) 0 2 (7.7%)
8 (53.3%) 4 (26.6%) 0 0 3 (20%)
39 (78%) 4 (8%) 6 (12%) 1 (2%) 0
M.H. El-Tagui et al. / Gene 524 (2013) 292–295 Table 4 Distribution of Apo E gene allelic frequencies among the studied groups and controls. APO E allele frequency
Group I N=9
Group II N = 26
Group III N = 15
Control N = 50
p value among groups
E2 E3 E4
11% 83.3% 5.5%
9.6% 73.0% 17.3%
13.3% 66.6% 20%
6% 88% 6%
NS Sb,c Sb,c
S = significant (p value b 0.05). b On comparing Group II and Control. c On comparing Group III and control.
295
5. Conclusion Apo E4 allele can be considered a genetic risk factor for LV dysfunctions in β-thalassemic patients. It could be used as predictive indicator for additional risk of LV failure, particularly in asymptomatic patients with LV dilatation, requiring a closer follow-up, to prevent further disease progression. Conflict of interest statement There is no conflict of interest. References
results were obtained by both Economou-Petersen et al. (1998) and Bazrgar et al. (2007). The frequency of Apo E4 allele in the LV failure (Group III) patients of the current study was 20%. A higher percentage was reported by Ferrara et al. (2001) (25%), but lesser percentages were reported by Economou-Petersen et al. (1998) (12.8%) and Bazrgar et al. (2007) (13.8%). These differences can be attributed to the genetic background of the studied population. The E4 allele frequency in Group I without cardiac complications was not different from controls but patients with left ventricular complications in Group II and III had a significant higher allelic frequency of E4 than in controls. Similar findings were reported in thalassemic patient from Greece (Economou-Petersen et al., 1998), Italy (Ferrara et al., 2001) and Iran (Bazrgar et al., 2007). The well-known isoform-specific influence of Apo E on plasma cholesterol level and atherosclerosis cannot explain the association found between Apo E allele E4 and LV failure in β-thalassemia, as atherosclerosis is not a general feature of the pathophysiology of the disorder (Economou-Petersen et al., 1998). It is now recognized that the severity of a monogenic disorder may be modified by a second locus, depending on the genetic background (Aessopos and Berdoukas, 2009). Any gene involved in the pathogenic pathway represents a candidate modifier gene. The boundaries between monogenic and polygenic disorders might not always be as clear-cut as previously thought, since more genes and gene interactions have become known from the progress of the Human Genome Project (Bleumink et al., 2004). The finding of an increased frequency of Apo E4 allele in β-thalassemia homozygotes with LV failure provides additional evidence to the theory of oxygen free radicals contributing to the organ damage, due to the demonstrated antioxidant and iron binding activity of Apo E (EconomouPetersen et al., 1998). This suggests that several other genetic loci could be of potential relevance to the oxidative damage of organs in β-thalassemia. Such loci include genes modulating genesis of oxygen free radicals (i.e. cytochrome C oxidase), genes for scavenger enzymes (superoxide dismutases, catalases), genes regulating mitochondrial DNA replication, structural genes for membrane lipoproteins, and genes involved in DNA repair mechanisms. It is noteworthy that mutant mice lacking the Mn-superoxide dismutase enzyme suffer neonatal lethality due to dilated cardiomyopathy (Aessopos and Berdoukas, 2009). Other functional polymorphisms in such genes could be examined for association with organ failure in β-thalassemia. It is also believed that genetic susceptibility for a majority of common diseases will be associated with relatively common alleles of one or several loci (Aessopos and Berdoukas, 2009).
Aessopos, A., Berdoukas, V., 2009. Cardiac function and iron chelation in thalassemia major and intermedia: a review of the underlying pathophysiology and approach to chelation management. Mediterr. J. Hematol. Infect. Dis. 1 (1), e2009002 (18). Aessopos, A., Berdoukas, V., Tsironi, M., 2007. The heart in transfusion dependent homozygous thalassemia today—prediction, prevention and management. Eur. J. Haematol. 80, 93–106. Bazrgar, M., Karimi, M., Peiravian, F., et al., 2007. Apolipoprotein E gene polymorphism and left ventricular function in Iranian patients with thalassemia major. Haematologica 92 (2), 256–257. Bleumink, G.S., van Duijn, C.M., Kingma, J.H., et al., 2004. Apolipoprotein E epsilon4 allele is associated with left ventricular systolic dysfunction. Am. Heart J. 147 (4), 685–689. Borgna-Pignatti, C., Cappellini, M.D., De Stefano, P., et al., 2005. Survival and complications in thalassemia. Ann. N. Y. Acad. Sci. 1054, 40–47. Chu, W.C., Au, W.Y., Lam, W.W., 2012. MRI of cardiac iron overload. J. Magn. Reson. Imaging 36 (5), 1052–1059. http://dx.doi.org/10.1002/jmri.23628. Delaporta, P., Kattamis, A., Apostolakou, F., et al., 2012. Correlation of NT-proBNP levels and cardiac iron concentration in patients with transfusion-dependent thalassemia major. Blood Cells Mol. Dis. 24 (Epub ahead of print). Economou-Petersen, E., Aessopos, A., Kladi, A., et al., 1998. Apolipoprotein E epsilon4 allele as a genetic risk factor for left ventricular failure in homozygous beta-thalassemia. Blood 92 (9), 3455–3459 (1). El Beshlawy, A., Alaraby, I., Abdel Kader, M.S., et al., 2012. Study of serum hepcidin in hereditary hemolytic anemias. Hemoglobin 23 (Epub ahead of print). Ferrara, M., Matarese, S.M., Francese, M., et al., 2001. Role of apolipoprotein E (APOE) polymorphism on left cardiac failure in homozygous beta thalassaemic patients. Br. J. Haematol. 114 (4), 959–960. Gibson, G.E., Haroutunian, V., Zhang, H., et al., 2000. Mitochondrial damage in Alzheimer's disease varies with apolipoprotein E genotype. Ann. Neurol. 48 (3), 297–303. Hahalis, G., Alexopoulos, D., Kremastinos, D.T., et al., 2005. Heart failure in beta thalassemia syndromes: a decade of progress. Am. J. Med. 118, 957–967. Higgs, D.R., Thein, S.L., Woods, W.G., 2001. The molecular pathology of the thalassemias, In: Weatherall, D.J., Clegg, B. (Eds.), The Thalassemia Syndromes, 4th edn. Blackwell Science, Oxford, pp. 133–191. Kaplan, P., Baris, H., De Meirleir, L., et al., 2012. Revised recommendations for the management of Gaucher disease in children. Eur. J. Pediatr. 8 (Epub ahead of print). Kaur, G., Ropthap, C.C., Xavier, M., et al., 2003. Distribution of C282Y and H63D mutations in the HFE gene in healthy Asian Indians and patients with thalassaemia major. Natl. Med. J. India 16 (6), 309–310. Lang, R.M., Bierig, M., Devereux, R.B., et al., 2005. Recommendations for chamber quantification: a report from the American Society of Echocardiography's guidelines and Standards Committee and the Chamber Quantification Writing Group, developed in conjunction with the European Association of Echocardiography, a branch of the European Society of Cardiology. J. Am. Soc. Echocardiogr. 18, 1440–1463. Miyata, M., Smith, J.D., 1996. Apolipoprotein E allele-specific antioxidant activity and effects on cytotoxicity by oxidative insults and β-amyloid peptides. Nat. Genet. 14 (1), 55–61. Moore, R.J., Chamberlain, M.R., Khuri, F.R., 2004. Apolipoprotein E and the risk of breast cancer in African–American and non-Hispanic white women. Oncology 66 (2), 79–93. Rund, D., Rachmilewitz, E., 2005. Medical progress: beta thalassemia. N. Engl. J. Med. 353, 1135–1146. Synowiec, E., Pogorzelska, M., Blasiak, J., et al., 2012. Genetic polymorphism of the ironregulatory protein-1 and -2 genes in age-related macular degeneration. Mol. Biol. Rep. 39 (6), 7077–7087. Wenham, P.R., Newton, C.R., Price, W.H., 1991. Analysis of apolipoprotein E genotypes by the Amplification Refractory Mutation System. Clin. Chem. 37 (2), 241–244.
Contents lists available at SciVerse ScienceDirect
Gene journal homepage: www.elsevier.com/locate/gene
Apolipoprotein E gene polymorphism and the risk of left ventricular dysfunction among Egyptian β-thalassemia major Mona H. El-Tagui a, Mona M. Hamdy a, Iman A. Shaheen b,⁎, Hala Agha c, Hoda A. Abd-Elfatah d a
Department of Pediatrics, Faculty of Medicine, Cairo University, Egypt Department of Clinical Pathology, Faculty of Medicine, Cairo University, Egypt Department of Cardiology, Faculty of Medicine, Cairo University, Egypt d Egyptian Ministry of Health, Cairo, Egypt b c
a r t i c l e
i n f o
Article history: Accepted 27 March 2013 Available online 13 April 2013 Keywords: β-Thalassemia Apolipoprotein E PCR-RFLP Left ventricular failure Left ventricular dilatation
a b s t r a c t In Egypt, β-thalassemia is the most common hereditary hemolytic anemia. Cardiac dysfunction, secondary to iron overload with formation of oxygen free radicals, is the most common cause of death in β-thalassemia patients. This study was designed to determine whether the allelic genotype of apolipoprotein E (Apo E), which exhibits antioxidant properties, could represent a genetic risk factor for the development of left ventricular (LV) dysfunction in β-thalassemia major. Fifty Egyptian β-thalassemia major patients were subjected to echocardiography to assess LV function. Apo E genotyping by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) was done for all patients in addition to 50 age and sex matched healthy control subjects. Patients were classified into three groups. Group I and II were clinically asymptomatic. Group II subjects had evidence of LV dilatation, while Group III patients had clinical and echocardiographic findings of LV failure. Apo E4 allele was significantly higher among Group II and III than in controls. In conclusion, Apo E4 allele can be considered as a genetic risk factor for LV dysfunctions in β-thalassemic patients. It could be used as predictive indicator for additional risk of LV failure, particularly in asymptomatic patients with LV dilatation, requiring a closer followup, to prevent further disease progression. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Thalassemias are inherited hemoglobin disorders resulting in chronic hemolytic anemia (Higgs et al., 2001). Thalassemia major is the most prevalent clinical form of the disease and is characterized by: severe hemolytic anemia from the first year of life, which requires blood transfusion therapy for the patients' survival. Transfusion therapy, elevated gastrointestinal absorption of iron, together with ineffective erythropoiesis, determine iron overload which is the major cause of mortality and morbidity associated with the disease (Rund and Rachmilewitz, 2005). This is followed by multiple chronic organ damage, growth retardation and a considerably reduced life expectancy (Borgna-Pignatti et al., 2005).
Abbreviations: Apo E, Apolipoprotein epsilon; LV, Left ventricle; PCR-RFLP, Polymerase Chain Reaction Restriction Fragment Length Polymorphism; CXR, Chest X-ray; ECG, Electrocardiography; RVEDD, Right ventricular end diastolic diameter; LVEDD, Left ventricular end diastolic diameter; LVESD, Left ventricular end systolic diameter; IVS, Interventricular septum; PWT, Posterior wall thickness; FS, Fraction shortening; EF, Ejection fraction; EDTA, Ethylene diamine tetra acetic acid; SPSS, Statistical Package for the Social Science. ⁎ Corresponding author at: Clinical & Chemical Pathology Department, Kasr Al-Ainy Faculty of Medicine-Cairo University, Mail Box 11562, Cairo, Egypt. Tel./fax: +20 23654480. E-mail address: [email protected] (I.A. Shaheen). 0378-1119/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2013.03.134
In thalassemia major, cardiac disorders, most notably left-sided heart failure, are responsible for more than half of the deaths in those patients and are thus the main determinants of survival (Hahalis et al., 2005). Heart disease may manifest as hemosiderotic cardiomyopathy, heart failure, pulmonary hypertension, arrhythmias, systolic/diastolic dysfunction, pericardial effusion, myocarditis or pericarditis (Aessopos et al., 2007). Left ventricular (LV) failure is the most common presentation of cardiac involvement and the main cause of death in thalassemia major patients, and once cardiac dysfunction is detected, the prognosis is poor (Hahalis et al., 2005). Myocardial iron overload is traditionally considered the fundamental pathogenic mechanism of heart failure. Iron excess within cells leads to free radical formation, causing damage to membrane lipids and proteins resulting in cellular injury and impairing cardiac contractility and myocardial performance (Aessopos et al., 2007). Apolipoprotein epsilon (Apo E) is a well-known lipid transport protein. Its gene is located on chromosome 19 and has three major alleles with decreasing anti-oxidant activity including E2–E3–E4 (Moore et al., 2004). Apo E acts as a scavenger of free radicals; the demonstrated metal binding ability (including iron) of Apo E was postulated to be one mechanism accounting for its anti-oxidant activity (Economou-Petersen et al., 1998). Apolipoprotein E4 has the least anti-oxidant and iron binding activities (Miyata and Smith, 1996). Patients who have E4 allele are at a higher risk for iron induced damage of cellular and subcellular particles (Gibson et al., 2000).
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This study was designed to determine whether the allelic genotype of Apo E, which exhibits antioxidant properties, could represent a genetic risk factor for the development of left ventricular dysfunction in Egyptian β-thalassemia major.
of LV failure and concomitant echocardiographic findings. These patients exhibited dyspnea on exertion (New York Heart Association [NYHA] functional class I–IV) and fulfilled at least two major Framingham criteria for heart failure diagnosis.
2. Material and methods
2.3. Genotyping of Apo E gene
2.1. Study population
From each participant, 4 to 5 ml venous blood was obtained by sterile venipuncture into a sterile ethylene diamine tetra acetic acid (EDTA) vacutainer. Samples were either stored in the same vacutainer at −20 °C or used directly within 24 h for DNA extraction. Genomic DNA was extracted from whole blood using Gene Jet Genomic DNA Purification Kits (Fermentas — Canada) according to the manufacturer's instructions. Genotyping of Apo E gene was performed by PCR-RFLP technique. The primers for Apo E gene were prepared as previously described (Wenham et al., 1991). The primer pair used was forward primer: 5′-TCC-AAG-GAG-CTG-CAG-GCG-GCG-CA-3′, and reversed primer: 5′-ACA-GAA-TTC-GCC-CCG-GCC-TGG-TAC-ACT-GCC-A-3′: (Pioneer — Germany). Five microliter (40–50 ng) genomic DNA was amplified in a total volume of 25 μl. The reaction mixture contained 1 μl (20 pmole) of each of the forward and reverse primers and 12.5 μl of 2× Taq PCR master mixes (Fermentas — Canada). The thermocycler program was as follows: initial denaturation at 94 °C for 5 min followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 65 °C for 35 s, and extension at 72 °C for 30 s, followed by final extension at 72 °C for 5 min. The amplified PCR product (292 bp) was then digested with 10 U of the fast digest Hha restriction endonuclease (Fermentas — Canada) at 37 °C for 5 min. Digested products were electrophoresed on a 4% agarose gel. DNA fragments were visualized by ethidium bromide. Apo E2 allele was detected by the presence of 91 and 81 bp fragments and Apo E3 allele was detected by the presence of 91 and 48 bp fragments, while 72 and 48 bp fragments were indicative of Apo E4 allele (Fig. 1). Apo E genotyping was randomly repeated for 10% of the samples (10 samples), and interpreted blindly by two different observers to confirm the results.
The present study was conducted on 50 β-thalassemia major patients, 29 males and 21 females, with a minimum age of 11 years. They attended the Hematology Clinic of the Children Hospital, Faculty of Medicine, Cairo University. The study also included 50 age and sex matched healthy volunteers as a control group. The study design was approved by the Scientific Research Committee of Clinical Pathology and Pediatrics Departments, Faculty of Medicine, Cairo University. All patients, parents and/or surrogates were informed about the aim of the study. Formal written consents were obtained from the participants or their caregivers who agreed to their child's participation in the study. All patients were receiving blood transfusions every two to four weeks to maintain a hemoglobin level above 9 g/dl during all of the follow-up years. Iron chelation was prescribed for all patients either by subcutaneous injection of deferoxamine for 30 patients (60%), or oral intake in the form of deferiprone for eight patients (16%), or deferasirox for 12 patients (24%). Mean pretransfusion hemoglobin level and mean serum ferritin level in each patient over the last two and five years of follow-up were obtained to evaluate the transfusion therapy and the hemosiderosis level. All patients were subjected to cardiac evaluation including medical history, clinical examination, chest X-ray (CXR), electrocardiography (ECG) and M-mode echocardiographic studies. Apo E genotyping by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) was done for all patients and controls. 2.2. Echocardiography
2.4. Statistical analysis Two-dimensional, pulsed-wave, continuous-wave and color-flow Doppler echocardiography were performed using a General Electric, Vivid 5 echocardiography machine. A 3.5 or 5 MHZ phased array transducer was used depending on the patient's age. Various cardiac views were taken in order to assess the cardiac chambers: their sizes, connections, relations and presence of valvular abnormalities such as: tricuspid and pulmonary regurgitation. Using the M-Mode, left atrial and aortic dimensions were assessed, in long axis and parasternal views, the following were measured: right ventricular end diastolic diameter (RVEDD), left ventricular end diastolic diameter (LVEDD), left ventricular end systolic diameter (LVESD), interventricular septum (IVS) and posterior wall thickness (PWT). In addition to these, calculation of fraction shortening (FS%) and ejection fraction (EF%) were done as an indication of LV systolic function according to the recommendations of the American Society of Echocardiography. Left and right ventricular end diastolic dimensions more than the upper limits of normal according to patients' age were considered enlarged. Global systolic function was considered abnormal if FS b 25% and EF > 55. Diastolic dysfunction was considered when the E wave velocity was elevated, E/A ratio was reversed, or E wave deceleration slope was decreased (Lang et al., 2005). The patients were classified into three groups according to clinical evaluation and echocardiographic findings. Group I patients were asymptomatic and their echocardiographic studies were within normal. Group II were asymptomatic, but exhibited LV dilatation (LV end diastolic diameter index [LVEDDI] higher than 30 mm/m2) without left ventricular systolic dysfunction (LV fractional shortening [FS] higher than 28%), as assessed echocardiographically. Patients in this group did not receive treatment. Group III patients had symptoms and signs
Data were statistically described in terms of range, mean ± standard deviation (±SD), median, frequencies (number of cases) and percentages when appropriate. Comparison of quantitative variables between the study groups was done using Kruskal–Wallis analysis of variance (ANOVA) test. For comparing categorical data, Chi square (χ 2) test was performed. Exact test was used instead when the expected frequency was less than five. Relative frequency of gene mutation was described by OR and their 95% CI. Univariate and
Fig. 1. PCR-RFLP gel electrophoresis of Apo E genotyping. M; molecular DNA ladder. Lane 1 and 9; show homozygous Apo E3/E3 genotype with 91 and 41 bp fragments. Lane 2,3,6,7 and 8; show heterozygous Apo E2/E3 genotype with 91–81 and 48 bp fragments. Lane 5; shows homozygous Apo E4/E4 genotype with 72 and 48 bp fragments. Lane 10; shows heterozygous Apo E2/E4 genotype with 91–81–72 and 48 bp fragments. Lane 11; shows heterozygous Apo E3/E4 genotype with 91–72 and 48 bp fragments.
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multivariate analysis models were used to test for the preferential effect of the independent variable(s) on the dependent variable(s). A probability value (p value) less than 0.05 was considered statistically significant. All statistical calculations were done using computer programs Microsoft Excel 2003 (Microsoft Corporation, NY, USA) and SPSS (Statistical Package for the Social Science; SPSS Inc., Chicago, IL, USA) version 15 for Microsoft Windows. 3. Results In our study, 50 β-thalassemia major patients were classified into three groups as follows: nine patients in Group I, 26 patients in Group II, and 15 patients in Group III. There was no statistical significant difference among the three groups regarding basic clinical and hematological parameters which are summarized in Table 1. In Table 2, the results of the echocardiographic evaluation of the patients are shown. Left ventricular end diastolic diameter (LVEDD) differed among the three groups, while left ventricular end systolic diameter (LVESD) indices, ejection fraction (EF) and left ventricular fractional shortening (FS) were significantly different on comparing Group II and Group III. Interventricular septum (IVS) thickness and left ventricular posterior wall (LVPW) thickness did not differ among the groups. For Apo E genotyping, E3/E3 genotype was found in 28 patients (56%) and in 39 normal control subjects (78%). E2/E3 was detected in eleven patients (22%) and four control subjects (8%). E3/E4 genotype was detected in six patients (12%) and in six normal control subjects (12%). One control subject carried the E2/E2 genotype. E4/E4 was only detected in five patients (10%), three of them were in Group III and two were in Group II. There was no statistical significant difference among the studied groups regarding Apo E genotyping (Table 3). On comparing the allelic distribution of Apo E gene among the three studied groups, Apo E4 allele was statistically higher in Group II and III than in controls (p = 0.027 and 0.02 respectively) (Table 4). 4. Discussion Beta thalassemia major is one of the most prevalent hereditary diseases in the Mediterranean region especially in Egypt (El Beshlawy et al., 2012; Kaplan et al., 2012). Transfusion therapy has greatly improved the survival of transfusion dependent thalassemia major patients; however, the resultant iron load causes damage of different types of tissues (Chu et al., 2012) Iron-induced cardio toxicity remains the leading cause of morbidity and mortality in those patients (Delaporta et al., 2012). Through iron-driven Fenton and Haber–Weiss reactions, the nontransferrin free plasma iron, in its bivalent or trivalent form, has a high toxicity through the formation of hydroxyl radicals (OH) (Synowiec et
Table 2 Echocardiographic evaluation of the patients. Echo cardio graphic indices (Mean ± SD)
Group I N=9
LVEDD LVESD IVS thickness EF% FS% LVPW thickness
27 28 8.0 65 39.2 7.4
± ± ± ± ± ±
Group II N = 26 3 4 1.9 8.8 5.9 1.1
43 30 7.7 61 35.80 8.2
± ± ± ± ± ±
Group III N = 15 5 3 1.5 5.8 4.48 1.8
47 34 7.8 35 26.533 7.8
± ± ± ± ± ±
p value 5 5 1.4 9.8 3.7 1.3
Sa,b,c Sb,c NS Sb,c Sb,c NS
LVEDD; left ventricular end diastolic diameter. LVESD; left ventricular end systolic diameter. IVS; interventricular septum. EF; ejection fraction. FS; left ventricular fractional shortening. LVPW; left ventricular posterior wall. S = Statistical significant difference. a On comparing Group I and II. b On comparing Group I and III. c On comparing Group II and III.
al., 2012). This leads to peroxidative damage of membrane lipids and proteins. Imbalance between production of oxygen free radicals and antioxidant defense mechanisms can result in oxidative stress and human diseases (Bazrgar et al., 2007). In the heart, the imbalance between free radicals and antioxidant mechanisms is manifested as impaired function of the mitochondrial inner-membrane respiratory chain resulting in abnormal energy metabolism expressed clinically with fatal cardiomyopathy (Economou-Petersen et al., 1998). Diversity in genetic loci may increase the susceptibility of heart failure leading to increased iron absorption and deposition among those patients, which makes them more vulnerable to iron overload (Kaur et al., 2003). Apo E4 allele has been reported as a genetic risk factor of LV failure in β-thalassemia in Greece (Economou-Petersen et al., 1998), Italy (Ferrara et al., 2001) and Iran Bazrgar et al. (2007), with no available data about this association among other Caucasian β-thalassemic patients including Egyptians. The aim of this study was to determine whether the allelic genotype of Apo lipoprotein E (Apo E), which exhibits antioxidant properties, could represent a genetic risk factor for the development of left ventricular failure in Egyptian β-thalassemia major patients. Beta thalassemic patients involved in our study were categorized into three groups according to clinical and echocardiographic findings. There was no statistical significant difference on comparing main clinical and laboratory findings including mean pretransfusion hemoglobin and mean serum ferritin. These findings were also noticed among the studied group of Economou-Petersen et al. (1998) and Bazrgar et al. (2007). Echocardiography was more sensitive in detecting patients with LV dilation as most of the patients were in Group II (52% [26 patients]) with no clinical symptoms, but with echocardiographic findings of LV dilation. Those patients are more vulnerable to develop LV dysfunction and need a closer follow up (Bazrgar et al., 2007). On comparing the echocardiographic findings among the three studied groups there was a statistical significant difference among the three groups except for IVS thickness and LVPW thickness. Similar
Table 1 Basic clinical and hematological data of the three studied groups. Variable
Group I
Group II
Group III
Age (years) Mean ± SD SEX MALE FEMALE Age at 1st transfusion (month.) Hemoglobin (g/dl) Body surface area (m2) Number of blood transfusion units Serum ferritin (ng/ml)
21.77 ± 7.1 5 4 10 ± 6.55
17 ± 5.97 15 11 14.03 ± 12.3
21 ± 5.5 6 9 16.37 ± 15.5
8.1 ± 0.67 1.50 ± 0.17 240 ± 86
8.1 ± 0.73 1.25 ± 0.24 220.8 ± 104.12
8.3 ± 0.55 1.356 ± 0.22 248.4 ± 95
5175.6 ± 3334
4885.46 ± 2439
3550 ± 2025
Table 3 Distribution of Apo E genotyping among the studied groups and controls. Apo E genotyping
Group I N=9
Group II N = 26
Group III N = 15
Control N = 50
E3/E3 E2/E3 E3/E4 E2/E2 E4/E4
6 (66.6%) 2 (22.2%) 1 (11.1%) 0 0
14 (53.8%) 5 (19.2%) 5 (19.2%) 0 2 (7.7%)
8 (53.3%) 4 (26.6%) 0 0 3 (20%)
39 (78%) 4 (8%) 6 (12%) 1 (2%) 0
M.H. El-Tagui et al. / Gene 524 (2013) 292–295 Table 4 Distribution of Apo E gene allelic frequencies among the studied groups and controls. APO E allele frequency
Group I N=9
Group II N = 26
Group III N = 15
Control N = 50
p value among groups
E2 E3 E4
11% 83.3% 5.5%
9.6% 73.0% 17.3%
13.3% 66.6% 20%
6% 88% 6%
NS Sb,c Sb,c
S = significant (p value b 0.05). b On comparing Group II and Control. c On comparing Group III and control.
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5. Conclusion Apo E4 allele can be considered a genetic risk factor for LV dysfunctions in β-thalassemic patients. It could be used as predictive indicator for additional risk of LV failure, particularly in asymptomatic patients with LV dilatation, requiring a closer follow-up, to prevent further disease progression. Conflict of interest statement There is no conflict of interest. References
results were obtained by both Economou-Petersen et al. (1998) and Bazrgar et al. (2007). The frequency of Apo E4 allele in the LV failure (Group III) patients of the current study was 20%. A higher percentage was reported by Ferrara et al. (2001) (25%), but lesser percentages were reported by Economou-Petersen et al. (1998) (12.8%) and Bazrgar et al. (2007) (13.8%). These differences can be attributed to the genetic background of the studied population. The E4 allele frequency in Group I without cardiac complications was not different from controls but patients with left ventricular complications in Group II and III had a significant higher allelic frequency of E4 than in controls. Similar findings were reported in thalassemic patient from Greece (Economou-Petersen et al., 1998), Italy (Ferrara et al., 2001) and Iran (Bazrgar et al., 2007). The well-known isoform-specific influence of Apo E on plasma cholesterol level and atherosclerosis cannot explain the association found between Apo E allele E4 and LV failure in β-thalassemia, as atherosclerosis is not a general feature of the pathophysiology of the disorder (Economou-Petersen et al., 1998). It is now recognized that the severity of a monogenic disorder may be modified by a second locus, depending on the genetic background (Aessopos and Berdoukas, 2009). Any gene involved in the pathogenic pathway represents a candidate modifier gene. The boundaries between monogenic and polygenic disorders might not always be as clear-cut as previously thought, since more genes and gene interactions have become known from the progress of the Human Genome Project (Bleumink et al., 2004). The finding of an increased frequency of Apo E4 allele in β-thalassemia homozygotes with LV failure provides additional evidence to the theory of oxygen free radicals contributing to the organ damage, due to the demonstrated antioxidant and iron binding activity of Apo E (EconomouPetersen et al., 1998). This suggests that several other genetic loci could be of potential relevance to the oxidative damage of organs in β-thalassemia. Such loci include genes modulating genesis of oxygen free radicals (i.e. cytochrome C oxidase), genes for scavenger enzymes (superoxide dismutases, catalases), genes regulating mitochondrial DNA replication, structural genes for membrane lipoproteins, and genes involved in DNA repair mechanisms. It is noteworthy that mutant mice lacking the Mn-superoxide dismutase enzyme suffer neonatal lethality due to dilated cardiomyopathy (Aessopos and Berdoukas, 2009). Other functional polymorphisms in such genes could be examined for association with organ failure in β-thalassemia. It is also believed that genetic susceptibility for a majority of common diseases will be associated with relatively common alleles of one or several loci (Aessopos and Berdoukas, 2009).
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