Prevalence of � S-globin gene haplotypes, �-thalassemia (3.7 kb deletion) and redox status in patients with sickle cell anemia in the state of Paraná, Brazil Eliana LitsukoTomimatsu Shimauti1,2, Danilo Grunig Humberto Silva3, Eniuce Menezes de Souza4, Eduardo Alves de Almeida3, Francismar Prestes Leal5 and Claudia Regina Bonini-Domingos1 1Laboratório de Hemoglobinas e Genética das Doenças Hematológicas, Departamento de Biologia, Universidade Estadual Paulista “Júlio de Mesquita Filho”, São José do Rio Preto, SP, Brazil. 2Departamento de Análises Clínicas e Biomedicina, Universidade Estadual de Maringá, Maringá, PR, Brazil. 3Departamento de Química e Ciências Ambientais, Universidade Estadual Paulista “Júlio de Mesquita Filho”, São José do Rio Preto, SP, Brazil. 4Departamento de Estatística, Universidade Estadual de Maringá, Maringá, PR, Brazil. 5Hemocentro, Maringá, PR, Brazil. Abstract The aim of this study was to determine the frequency of beta S-globin gene (�S globin) haplotypes and alpha thalassemia with 3.7 kb deletion (-�3.7kb thalassemia) in the northwest region of Paraná state, and to investigate the oxidative and clinical-hematological profile of � S globin carriers in this population. Of the 77 samples analyzed, 17 were Hb SS, 30 were Hb AS and 30 were Hb AA. The � S globin haplotypes and -�3.7kb thalassemia were identified us- ing polymerase chain reaction.Trolox equivalent antioxidant capacity (TEAC) and lipid peroxidation (LPO) were assessed spectophotometrically. Serum melatonin levels were determined using high-performance liquid chroma- tography coupled to coulometric electrochemical detection. The haplotype frequencies in the SS individuals were as follows: Bantu- 21 (62%), Benin - 11 (32%) and Atypical- 2 (6%). Bantu/Benin was the most frequent genotype. Of the 47 SS and AS individuals assessed, 17% (n = 8) had the -�3.7kb mutation. Clinical manifestations, as well as serum melatonin, TEAC and LPO levels did not differ between Bantu/Bantu and Bantu/Benin individuals (p > 0.05). Both ge- notypes were associated with high LPO and TEAC levels and decreased melatonin concentration. These data sug- gest that the level of oxidative stress in patients with Bantu/Bantu and Bantu/Benin genotypes may overload the antioxidant capacity. Keywords: antioxidants, hemoglobinopathies, melatonin, sickle cell disease, thalassemia. Received: August 8, 2014; Accepted: February 24, 2015. Introduction Hemoglobin S (Hb S) is produced by the substitution of thymine by adenine (GAG to GTG) in the sixth codon of the � globin gene, which results in the production of valine rather than glutamic acid. In homozygous form (Hb SS), this mutation is known as sickle cell anemia (SCA), and in heterozygous form (Hb AS) as sickle cell trait (Frenette and Atweh, 2007). SCA is characterized by chronic inflamma- tion and recurrent ischemic-reperfusion events that may lead to an excess of free radicals and oxidative stress (Sou- za, 2001; Kaul et al., 2004; Dasgupta et al., 2006). A num- ber of studies suggest that oxidative stress plays an important role in the pathophysiology of SCA (Hebbel et al., 1988; Naoum, 2000; de Oliveira Filho et al., 2013). Due to its greater tendency for self-oxidation and oxidant gener- ation, Hb S leads to increased lipid peroxidation (LPO) and lysis in the sickle cell membrane. The intensity of LPO and the reduction in antioxidant defense both appear to be re- lated to the clinical severity of SCA (Dasgupta et al., 2006; Shimauti et al., 2010). Melatonin, a potential antioxidant and anti-inflam- matory agent, has been implicated in a number of patholog- ical processes because of its ability to detoxify reactive oxygen species (ROS) and nitrogen species (RNS), and to stimulate antioxidant enzymes (Reiter et al., 2000; Cuz- zocrea and Reiter, 2002; Allegra et al., 2003; Mayo et al., 2005; Tan et al., 2007; Shimauti et al., 2010), thereby ex- erting a protective effect against oxidative damage in dif- ferent biological systems. The trolox equivalent Genetics and Molecular Biology, 38, 3, 316-323 (2015) Copyright © 2015, Sociedade Brasileira de Genética. Printed in Brazil DOI: http://dx.doi.org/10.1590/S1415-475738320140231 Send correspondence to Eliana L.T. Shimauti. Departamento de Análises Clínicas e Biomedicina, Universidade Estadual de Marin- gá, Av. Colombo 5.790, Jardim Universitário, 87020-900, Maringá, PR, Brazil. E-mail: eltshimauti@uem.br. Research Article antioxidant capacity (TEAC), which assesses the response to free radical attack (Re et al., 1999), and the level of thio- barbituric acid reactive species (TBARS), a reliable indica- tor of membrane lipid peroxidation or oxidative stress (Block et al., 2002), are some of the most important and widely used biomarkers to assess oxidative damage. The clinical course of SCA is heterogeneous and the clinical expression of the condition appears to be influ- enced by genetic features such as alpha thalassemia, the haplotypes in the beta globin gene cluster and Hb F levels (Steinberg and Embury, 1986; Zago and Pinto, 2007; Silva and Gonçalves, 2010; Shimauti et al., 2011). Alpha thalas- semia can be caused by the deletion of one or both alpha globin genes (�1 and �2) (Harteveld and Higgs, 2010), and the most common cause of the condition in Brazil is the de- letion of the -�3.7kb gene as a result of homologous recombi- nation between misaligned chromosomes (Sonati et al., 1991; Wagner et al., 2010). Polymorphisms in the �S globin gene cluster have been associated with at least five haplo- types that are classified according to the African and Mid- dle Eastern regions where the genes are most commonly found. The Benin haplotype is associated with West Africa, while Bantu is most commonly seen in Eastern and south- central Africa. The Senegal and Cameroon haplotypes are associated with Atlantic West Africa and the African West Coast, respectively, while the Arab-Indian haplotype is found in India and the East Arabian Peninsula (Chebloune et al., 1988; Nagel and Ranney, 1990; Elion et al., 1992; Lapoumeroulie et al., 1992). The Senegal and Arab-Indian haplotypes are associated with increased Hb F levels (> 15%) and less severe SCA. The Benin haplotype, on the other hand, is associated with intermediate Hb F levels (5% to 15%) and a severe clinical course, while individuals with the Bantu haplotype presenting lower Hb F levels (< 5%) and a worse clinical evolution (Powars, 1991; Elion et al., 1992; Powars and Hiti, 1993; Galiza Neto and Pitombeira, 2003). The intense miscegenation of the Brazilian popula- tion has resulted in regional differences in ethnic composi- tion that have led to a heterogeneous distribution of the Hb S gene (Wenning et al., 2000; Chinelato-Fernandes and Bonini-Domingos, 2005; Seixas et al., 2008; Silva Filho et al., 2010). Paraná state, located in southern Brazil, has a 1.52% prevalence for the heterozygous S gene (Watanabe et al., 2008). Hb S was introduced in Brazil during the Afri- can slave trade from the 16th to 19th centuries. Historical re- cords state that most slaves from Benin went to the port of Salvador, in the state of Bahia, while Rio de Janeiro re- ceived slaves from the Angola region, where the Bantu haplotype was the most prevalent (Silva Filho et al., 2010). After arriving in Rio de Janeiro, slaves were redistributed to other regions in the country, and studies show that most states in Brazil, especially those in the southeast and south- ern regions of the country, received slaves from Rio de Ja- neiro (Fleury, 2007). There are also records of slave trading in the state of Paraná in the 17th and 18th centuries, and starting from the second half of the 18th century, internal migration of former slaves was observed from other re- gions of Brazil to Paraná (Luna and Klein, 2004). Paraná has a multiethnic population, a large percentage of which is of European origin. The percentage of Afro-Brazilians in Paraná was thought to be quite small, until a survey by the Brazilian Institute of Geography and Statistics revealed that these individuals made up 24% of the state’s popula- tion (IBGE, 2013). These findings showed that Paraná is the southern Brazilian state with the largest proportion of Afro-Brazilian individuals (Gomes Júnior et al., 2008). The goal of the present study was to analyze the fre- quency of �S gene haplotypes and alpha thalassemia (-�3.7kb) and to verify their association with oxidative stress biomarkers and the antioxidant defense system, in addition to establishing the clinical and hematological profile of �S globin gene carriers in Paraná. Subjects and Methods Subjects Seventy-seven individuals, regardless of gender, were randomly selected from the northwest region of the state of Paraná in southern Brazil. Of these participants, 17 were Hb SS carriers (aged 10 to 39 years) in a stable phase of the disease selected from outpatient clinics, 30 were car- riers of Hb AS (14 to 53 years) selected from voluntary blood donors and 30 were healthy Hb AA carriers (11 to 55 years) who had total Hb levels within normal limits for their gender and age and were recruited as a control group (Lewis et al., 2006). Eligible participants were non- smokers, not pregnant, non-alcoholics, in the stable phase of the disease, and had not received blood transfusions in the three months prior to participating in the study. Data re- garding medication use, clinical events and blood transfu- sions were obtained by evaluating the subjectsmedical records and by applying questionnaires at the time of blood collection. The study protocol was approved by the Re- search Ethics Committee of the Department of Life Sci- ences, Linguistics and Exact Science of the State University of São Paulo (UNESP), Brazil (protocol number 0025.0.229.000-07). Biological samples After informed consent, 20 mL of peripheral blood was collected and distributed intothe following tubes: a) a tube with K3EDTA (4 mL) for hematological, chromato- graphic and molecular analyses, b) a tube with heparin (7 mL) for determination of plasma biomarkers of oxida- tive stress and antioxidant capacity, after centrifugation at1500 rpm for 20 min, and c) a tube without anticoagulant (9 mL) to obtain serum for the quantification of melatonin. All biological samples were collected at 6:30 a.m. to pre- Shimauti et al. 317 vent the inhibition of melatonin secretion caused by expo- sure to daylight. Hemoglobin profile and hematological parameters Hematological parameters were obtained using an Auto Hematology Analyzer (BC-300 PLUS - Mindray, China) and microscopic analysis was done using blood smears stained according to the May-Grunwald-Giemsa method (Lewis et al., 2006). The tests used to screen for hemoglobinopathy included hemoglobin electrophoresis at pH 8.4 (Marengo-Rowe, 1965) and pH 6.2 (Vella, 1968). The quantification of hemoglobin fractions was performed by high-performance liquid chromatography (HPLC) using a Variant chromatography system (Bio-Rad) (Instruction Manual, 2006). Identification of Hb S genotypes, � S gene haplotypes and -�3.7kb thalassemia The Hb S genotypes were confirmed by molecular analysis with polymerase chain reaction- restriction frag- ment length polymorphism (PCR-RFLP). The segment that encodes the �S gene was amplified using specific primers and the amplified segment was cleaved with DdeI restric- tion endonuclease (New England Biolabs, MA, USA) (Bonini-Domingos, 2006). The �S globin haplotypes were determined by PCR-RFLP with an assessment of six poly- morphic restriction sites: 5’�G (XmnI), �G (HindIII), �A (HindIII), �� (HincII), 3’�� (HincII) and 5’� (HinfI), ac- cording to the method reported by Sutton et al. (1989). The -�3.7 deletion was identified by multiplex PCR as described by Chong et al. (2000). Biochemical analysis Lipid peroxidation was assessed based on the plasma TBARS levels, according to the method described by Mihara and Uchiyama (1978). Plasma antioxidant capacity was determined by the TEAC method, which involves the use of Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2- carboxylic acid, product no. 23881-3; Aldrich Chemical Co.), a synthetic water-soluble antioxidant analogous to vi- tamin E (Miller et al., 1993; Re et al., 1999).The serum melatonin concentration was assessed using HPLC coupled to a coulometric electrochemical detector (Coulochem III ESA model 526, Bedford, MA, USA), as previously re- ported (Shimauti et al., 2010). Statistical analysis Statistical analyses were done using Statistica soft- ware, version 8.0. The data were assessed for normality and homoscedasticity, and were analyzed using the non-para- metric Mann-Whitney and Kruskal-Wallis tests, comple- mented by Dunn’s test. Correlation analyses were done using Spearman’s test. The significance level for the tests was set at 5% (� = 0.05). Results Nine (52.9%) individuals with SCA (Hb SS) used hydroxyurea (HU). However, as there were no significant differences in Hb F levels (p = 0.199), TEAC (p = 0.832), melatonin (p = 0.962) and TBARS (p = 0.835) between HU users and nonusers, these individuals were combined into a single group. Analyses also showed that the age and gender of the individuals in the Hb SS, Hb AS and Hb AA groups did not influence the levels of redox status biomarkers (p > 0.05). Alpha thalassemia was analyzed in 47 individuals (17 Hb SS and 30 Hb AS), eight of whom (17%) had the -�3.7kb mutation. Seven (14.9%) individuals were heterozy- gotes (-�/��) and one (2.1%) was homozygous (-�/-�) for the mutation (Table 1).The �S gene polymorphisms were analyzed in 17 Hb SS patients. The Bantu haplotype was the most frequent in these individuals and was found in 21 (62%) chromosomes, while the Benin haplotype was found in 11 (32%) chromosomes (Table 2). The Senegal and Arab-Indian haplotypes were not detected in the sample. The subjects were divided into Bantu/Bantu and Ban- tu/Benin groups in order to analyze the influence of haplo- types on the oxidative and hematologic parameters and on phenotypic expression.Table 3 shows the redox profile and hematologic characteristics of the entire sample. The serum melatonin concentrations, TEAC and LPO in Hb AS indi- viduals were not statistically different from those in sub- jects with Hb AA genotypes (p > 0.05). However, a marked reduction in serum melatonin levels (p < 0.001) and an ele- vation in TEAC (p � 0.007) and LPO (p < 0.001) were ob- served in Hb SS subjects as compared to Hb AS and Hb AA individuals.The antioxidant/oxidant and hematologic pro- files did not differ significantly between the Bantu/Bantu and Bantu/Benin groups (p > 0.05), except for the mono- 318 Haplotype frequency and redox state Table 1 - Frequency of the -�3.7kb thalassemia mutation in �S-globin gene carriers. Genotypes ��/�� N (%) -�/�� N (%) -�/-� N (%) N SS 14 (82.4) 2 (11.8) 1 (5.8) 17 AS 25 (83.3) 5 (16.7) 0 30 Total 39 (83.0) 7 (14.9) 1 (2.1) 47 Table 2 - Frequency of �S chromosomes and genotypes in 17 Hb SS indi- viduals. Chromosomes N (%) Haplotypes N (%) Bantu 21 (62) Bantu/Bantu 6 (35.3) Benin 11 (32) Bantu/Benin 7 (41.1) Atypical 2 (6) Benin/Benin 2 (11.8) Bantu/Atypical 2 (11.8) Total 34 (100) Total 17 (100) cyte levels (p = 0.03) (Table 4).When grouped together, the Bantu and Benin haplotypes showed significant increases in LPO and TEAC levels, and a significant reduction in melatonin levels (p � 0.05) as compared to the control group (Hb AA) (Table 5). There were no significant differ- ences in the clinical characteristics of the Bantu/Bantu and Bantu/Benin groups, or between HbSS/-�3.7kb patients and those without thalassemia (p > 0.05) (Table 6). Spearman correlation analyses revealed positive cor- relations between the TBARS and HCM values in the Bantu/Bantu group (r = 0.84; p = 0.03). There were also negative correlations between TEAC and melatonin levels Shimauti et al. 319 Table 3 - Demographic and laboratory features of individuals with sickle cell anemia (Hb SS), sickle cell trait (Hb AS) and without hemoglobinopathy (Hb AA). Features Hb SS (N = 17) Med (min-max) Hb AS (N = 30) Med (min-max) Hb AA (N = 30) Med (min-max) Age 22(10-39) 37(14-53) 27(11-55) TBARS (ng/mL) 863.4a(496.8-2203) 470.6b(280.8-2796.1) 485b(270.2-676.3) TEAC (mM) 2.00a(1.86-2.10) 1.9b(1.7 -2.0) 1.9b(1.7-2.0) Melatonin (pg/mL) 42.6a(9.63-234.9) 116.2b(6.63-633.8) 146b(39.9-671.6) Hb (g/dL) 7.6a(5.9-9.7) 12.4b(10.0-15.1) 13.7b(11.5-15.4) MCV (fL) 96.8a(79.5-121.9) 84.8b(76.0-94.0) 86.5b(82-91.4) MCH (pg) 31.2a(24.8-40.4) 27.6b(24.2-31) 28.5b(27-30.1) MCHC (%) 32.6a(31-35.6) 32.8a(31-34) 33.4a(31.2-34.5) Reticulocytes (%) 14.8 � 8.8(6.3-36.9) - - Leukocytes (x109/L) 12,600a(8,600-18,800) 7,650b(4,300-10,900) 6,550b(3,800-10,500) Neutrophils (x109/L) 6,090a(4,472-11,280) 4,002b(2,250-6,534) 3,454b(1,504-7,144) Monocytes (x109/L) 0.725a(0.172-2256) 0.3585b(0.138-0.830) 0.334b(0.86-0.735) Hb Fetal (%) 10.2a(3.8-24.2) 0.0(0.0-3.0) 2.15c(0.0-2.6) Hb S (%) 83.5a(55.3-93.9) 36.6b(28.7-40.5) 0.0 Hb A (%) 0.0(0-33) 58.9a(55-65.5) 95.4b(95-97.6) Hb - hemoglobin, MCH -mean corpuscular hemoglobin, MCHC -mean corpuscular hemoglobin concentration, MCV -mean corpuscular volume, Max - maximum, Med - median, Min - minimum, TBARS - thiobarbituric acid reactive species, TEAC - Trolox equivalent antioxidant capacity.The same su- perscript letters indicate non-significant differences (p > 0.05) while different superscript letters indicate significant differences (p < 0.05). Kruskal- Wallis and Mann Whitney tests with a 0.05 significance level. Table 4 - Redox profile and hematological characteristics of SCA patients according to �S gene haplotypes, represented by median values (minimum and maximum). Features Bantu/Bantu (N = 6) Bantu/Benin (N = 7) p TBARS (ng/mL) 910.9(496.8-1970) 842.3(635.5-2,203) NS TEAC (mM) 2.0(2.0-2.1) 1.9(1.9-2.1) NS Melatonin (pg/mL) 44.8(17-164) 39.2(9.63-74.1) NS Hb (g/dL) 8.1(6.4-9.7) 7.6(5.9-9.0) NS MCV (fL) 102.7(89.1-118.6) 92.9(79.5-121.9) NS MCH (pg) 34.4(28.2-38.3) 30.2(24.8-40.4) NS MCHC (%) 32.4(31-35.6) 33.2(31.2-34.7) NS Reticulocytes (%) 13.4(7.3-36.9) 11.2(6.3-28.6) NS Hb F (%) 9.5(3.8-15.9) 14.3(1.2-24.2) NS Hb S (%) 85.1(55.3-89.0) 81.7(71.7-93.9) NS Leukocytes (x109/L) 12,150(8,800-18,800) 13,100(8,600-15,500) NS Neutrophils (x109/L) 6,894(4,536-11,092) 5,633(4,386-8,370) NS Monocytes (x109/L) 1,202(0.54-2,256) 0.655(0.172-1,085) 0.03 Hb - hemoglobin, MCH - mean corpuscular hemoglobin, MCHC - mean corpuscular hemoglobin concentration, MCV - mean corpuscular volume, SCA -sickle cell anemia,TBARS - thiobarbituric acid reactive species, TEAC - Trolox equivalent antioxidant capacity. NS - non-significant (p > 0.05). Mann Whitney test with a 0.05 significance level. (r=-0.91; p = 0.01), and between Hb F and reticulocytes (r=-0.9; p < 0.001) in this group. A strong correlation trending towards significance was also found between monocytes and reticulocytes (r = 0.77; p = 0.07). In the Bantu/Benin group, there were positive correlations be- tween TEAC and reticulocytes (r = 0.73; p = 0.05), TBARS and reticulocytes (r = 0.82; p = 0.02), TBARS and mono- cytes (r = 0.73; p = 0.05) and Hb F and total Hb (r = 0.83; p = 0.01). The data suggested that LPO was directly associ- ated with the degree of hemolysis. In the SS/-�3.7 group, there were positive correlations between TBARS and TEAC (r = 0.9; p < 0.001) and between TBARS and reticulocytes (r = 0.9; p < 0.001), and negative correlations between TBARS and total Hb (r=-0.9; p < 0.001). In the Hb SS/(��/��) group, there was a negative correlation be- tween Hb F and Hb S (r=-0.64; p = 0.012). Discussion This is the first report on haplotype frequency in Hb SS individuals in the state of Paraná. The results indicate that the Bantu haplotype, followed by the Benin haplotype, was the most frequent in the northwest region of Paraná. The most frequent genotype was Bantu/Benin. The haplo- type frequency identified here was similar to that reported for the states of São Paulo (Figueiredo et al., 1996) and Rio de Janeiro (Silva Filho et al., 2010). For many years, slaves arrived in Brazil on forced migration routes between the Benin bay in Ghana and Nigeria to the northeastern region of Brazil, and from Congo and Angola to Rio de Janeiro (Silva Filho et al., 2010). Rio de Janeiro was a major slave distribution center, sending slaves especially to the states of São Paulo and Rio Grande do Sul. The high frequency of Bantu followed by Benin haplotypes identified in this study agrees with historical records of the slave trade and internal migration between states in Brazil. The present findings also agree with the scientific lit- erature, as other studies have reported similar results for the association between the Bantu/Bantu and Bantu/Benin haplotypes, increased LPO and reduced antioxidant de- fense (Rusanova et al., 2010). The Bantu haplotype is asso- ciated with a lower concentration of Hb F and greater clinical severity than the Benin haplotype (Powars, 1991). However, our data showed no significant differences in the Hb F values, global redox status or clinical manifestations between homozygous Bantu haplotypes and heterozygous Bantu/Benin haplotypes. These results corroborate data from other studies (Rieder et al., 1991; Figueiredo et al., 320 Haplotype frequency and redox state Table 5 - Antioxidant capacity and lipid peroxidation in Bantu and Benin haplotypes as compared to the control group (Hb AA). BAN/BEN+BAN/BAN (n = 13) Med (min-max) Hb AA (n = 30) Med (min-max) p TEAC (mM) 2.0 (1.9-2.1) 1.9 (1.7-2.0) 0.002 Melatonin (pg/mL) 42.6 (9.6-164) 146.5 (39.9-671.6) 0.007 TBARS (ng/mL) 842.3 (496.8-2203) 485.0 (270.2-676.3) < 0.001 BAN/BAN -Bantu/Bantu, BAN/BEN -Bantu/Benin, Max - maximum, Med - median, Min - minimum, TBARS - thiobarbituric acid reactive species, TEAC - Trolox equivalent antioxidant capacity. Mann Whitney test with a 0.05 significance level. Table 6 - Hydroxyurea (HU) use and frequency of clinical manifestations in SCA patients according to �S gene haplotype, -�3.7kb thalassemia coinheri- tance and normal � genotype. BAN/BAN (n = 6) (%) BAN/BEN (n = 7) (%) p SS/-�3.7 (n = 3) (%) SS/(��/��) (n = 14) (%) p Leg ulcers 0 14.3 - 33.3 0 - Pneumonia 16.6 14.3 NS 33.3 25 NS Splenectomy 16.6 28.5 NS 33.3 28.5 NS Cholecystectomy/ cholelithiasis 16.6 42.8 NS 33.3 28.5 NS Pleural effusion 16.6 0 - 0 7.1 - Acute thoracic syndrome 0 0 - 0 7.1 - Cardiac complications 33.3 57.1 NS 66.7 35.7 NS Osteonecrosis 33.3 14.3 NS 0 28.5 - Osteomyelitis 0 16.6 - 0 7.1 - Joint/muscle pain 50 57.1 NS 66.7 50 NS Urinary infection 16.6 0 - 0 7.1 - HU use 66.6 28.6 NS 0 64.2 NS BAN/BAN - Bantu/Bantu, BAN/BEN - Bantu/Benin, NS -non-significant (p > 0.05). Fisher’s Exact test with a significance of p < 0.05. 1996; Silva and Gonçalves, 2010). Based on our results, it is possible that neither the Bantu/Benin nor the Bantu/Bantu haplotypes protect against oxidative damage and clinical features of SCA. If the Senegal haplotype had been present in the sample, it would have been possible to draw more accurate conclusions regarding the effect of these polymorphisms on oxidative and clinical-laboratorial characteristics. The significantly reduced serum melatonin concen- tration in patients with SCA corroborates a previous study by Shimauti et al. (2010) that also found an association be- tween low melatonin concentrations and this illness. The decrease in serum melatonin together with the elevated TEAC and LPO levels observed here agree with results from other investigations (Gitto et al., 2001; Reiter et al., 2005). Although serum melatonin levels tend to decrease as age increases (Waldhauser et al., 1988; Zhdanova et al., 1998), melatonin concentrations in individuals with SCA (Hb SS) have been found to be equally low in all age- groups (Shimauti et al., 2010). Studies have shown that, in conditions of intense oxidative stress, interactions with ROS lead to the metabolization of melatonin and subse- quent reductions in its serum concentration (Tan et al., 2007). Melatonin also increases the total antioxidant capac- ity, possibly by acting in synergy with classic antioxidants. In SCA, monocytes are responsible for the activation of endothelial cells, and act as a trigger for the translocation of the nuclear transcription factor NF-kB (Belcher et al., 2000). The activated vascular endothelium expresses the adhesion molecules ICAM-I, VCAM-I, P-selectin and E- selectin, and the procoagulant tissue factor, all which play an important role in the physiopathology of vascular occlu- sion in Hb SS individuals. The strong positive correlation between monocytes and reticulocytes in the Bantu/Bantu group, and between TBARS, reticulocytes and monocytes in the Bantu/Benin group suggests that higher hemolytic rates are associated with increased LPO and inflammatory responses. These data suggest a vulnerability of these geno- types to vasculopathy. The frequency of coinheritance of Hb S with -�3.7kb observed here was similar to that reported for the states of São Paulo and Rio de Janeiro (Figueiredo et al., 1996; Silva Filho et al., 2010). In these individuals, the most commonly observed haplotype was Bantu/Benin, as reported in studies done in the city of Salvador, in the state of Bahia, and in the state of São Paulo (Lyra et al., 2005). Investigations of the interaction between Hb SS and alpha thalassemia (-�3.7kb) and its effect on clinical and hematological factors have produced conflicting results. Some studies have reported reduced hemolytic rates, increased total Hb concentration and a protective effect against clinical manifestations of the disease (Higgs et al., 1982; Powars, 1991; Belisario et al., 2010; Fertrin and Costa, 2010), while other studies have found no differences between individuals with and without the -�3.7 mutation (��/��) (Mouele et al., 1999). Previous studies have reported on the importance of alpha thalassemia in the modulation of SCA (Steinberg, 2005; Belisario et al., 2010); however, our study failed to demonstrate significant differences in the frequencyof clin- ical complications between groups with and without -�3.7 thalassemia coinheritance, possibly because of our small sample size. Nonetheless, the inverse correlation detected between TBARS and total Hb and the positive correlation between reticulocytes and TBARS suggest that -�3.7 thalas- semia may decrease hemolysis and attenuate lipid pero- xidation. It is also interesting to note that, in the present sample, the use of HU, a chemotherapeutic agent used to in- crease the Hb F concentration and attenuate hemolysis, was more common in SS individuals without the -�3.7kb muta- tion and in patients with the Bantu/Bantu genotype, which is generally associated with a worse clinical outcome. The ability of HU to increase Hb F levels varies among patients and the duration of treatment with this chemotherapeutic agent influences the increase in Hb F. In order to achieve the expected benefits, the treatment should last for at least two years (Davies and Gilmore, 2003). As shown here, most patients (66.6%) with Hb SS who were receiving HU had been under treatment for less than two years.Therefore, treatment duration may be one of the de- terminant factors for the lack of significant differences in Hb F levels between patients receiving HU or not. In conclusion, the results of this study provide a rele- vant contribution to our understanding of the pattern of col- onization and to the anthropological and historical background of the Afro-Brazilian population in the state of Paraná. Our data suggest that the antioxidant defense in SCA patients with the Bantu/Bantu or Bantu/Benin geno- type is insufficient to control oxidative stress. Melatonin, a potent antioxidant and anti-inflammatory agent, is a possi- ble treatment option for SCA patients with low serum melatonin levels. However, the protective effect of mela- tonin against oxidative stress in individuals with sickle cell disease requires further study. More comprehensive inves- tigations using data from other regions in the state of Paraná must be done to better characterize the frequency of �S gene haplotypes and of -�3.7kb thalassemia coinheritance, as well as their effects on oxidative stress and phenotypic expres- sion in patients with Hb SS. Acknowledgments The authors thank the following foundations for fi- nancial support: Brazilian Ministry of Health (grant no. 3072/2007), Paraná State Ministry of Science, Technology and Higher Education (grant no. 5636/2009), Araucária Foundation for Scientific and Technological Development of Paraná (grant no. 322/2009), Conselho Nacional de De- senvolvimento Científico e Tecnológico (CNPq, grant number 409691/2006-2). Shimauti et al. 321 References Allegra M, Reiter RJ, Tan DX, Gentile C, Tesoriere L and Livrea MA (2003) The chemistry of melatonin’s interaction with reactive species. J Pineal Res 34:1-10. Belcher JD, Marker PH, Weber JP, Hebbel RP and Vercellotti GM (2000) Activated monocytes in sickle cell disease: Po- tential role in the activation of vascular endothelium and vaso-occlusion. Blood 96:2451-2459. 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Associate Editor: Mara Hutz License information: This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Shimauti et al. 323 Haplotype frequency and redox state Errratum The authors noted errors in certain values presented in Tables 3 and 4 of this paper due to misplacement of decimal points. In Table 3 the values reading: Leukocytes (x109/L) 12,600a(8,600-18800) 7,650b(4,300-10,900) 6,550b(3,800-10,500) Neutrophils (x109/L) 6,090a(4,472-1,1280) 4,002b(2,250-6,534) 3,454b(1,504-7,144) Monocytes (x109/L) 0.725a(0.172-2256) Should read: Leukocytes (x109/L) 12.6a(8.6-18.8) 7.65b(4.3-10.9) 6.55b(3.8-10.5) Neutrophils (x109/L) 6.09a(4.472-1.128) 4.002b(2.25-6.534) 3.454b(1.504-7.144) Monocytes (x109/L) 0.725a(0.172-2.256) In Table 4 the values reading: Leukocytes (x109/L) 12,150(8,800-18,800) 13,100(8,600-15,500) NS Neutrophils (x109/L) 6,894(4,536-11,092) 5,633(4,386-8,370) NS Monocytes (x109/L) 1,202(0.54-2,2256) 0.655(0.172-1,085) 0.03 Should read: Leukocytes (x109/L) 12.15(8.8-18.8) 13.1(8.6-15.5) NS Neutrophils (x109/L) 6.894(4.536-11.092) 5.633(4.386-8.37) NS Monocytes (x109/L) 1.202(0.54-2.2256) 0.655(0.172-1.085) 0.03