Clinical and Molecular Characteristics of Sickle Cell Anemia in the Northeast of Brazil
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Genetics and Molecular Biology, 31, 3, 621-625 (2008) Copyright © 2008, Sociedade Brasileira de Genética. Printed in Brazil www.sbg.org.br Research Article Clinical and molecular characteristics of sickle cell anemia in the northeast of Brazil Elisângela Vitória Adorno1,2, Ângela Zanette3, Isa Lyra3, Magda Oliveira Seixas1,2, Mitermayer Galvão Reis1 and Marilda Souza Gonçalves1,2 1Laboratório de Patologia e Biologia Molecular, Centro de Pesquisas Gonçalo Moniz, Fundação Oswaldo Cruz, Salvador, BA, Brazil. 2Departamento de Análises Clínicas e Toxicológicas, Faculdade de Farmácia, Universidade Federal da Bahia, Salvador, BA, Brazil. 3Fundação de Hematologia e Hemoterapia do Estado da Bahia, Salvador, BA, Brazil. Abstract Beta S-globin gene (βS-globin) haplotypes, markers for severe sickle cell anemia (SCA), and the alpha-thalassemia 2 α 3.7 kb gene 3.7 kb deletion (- 2 thal) along with demographic and clinical data were investigated in SCA outpatients (n = 125, 63 female and 62 male) in the Brazilian state of Bahia, which has a high prevalence SCA. PCR-RFLP showed that the Central African Republic/Benin (CAR/BEN, 51.2%) haplotype was most frequent, followed by the Benin/Benin (Ben/Ben, 28.8%). At least one CAR haplotype was present in every outpatient with a history of cerebrovascular acci- dent. The Cameroon (Cam), Senegal (Sen) and Arab-India haplotypes occurred in small numbers, as did atypical haplotypes. Fetal hemoglobin (HbF, %) was unevenly distributed. Compared to those > 18 y, those aged ≤ 18 y had had fewer erythrocyte transfusions and high HbF levels (12.3% ± 7.01 to 7.9% ± 4.36) but a higher frequency of spleen α α 3.7 kb sequestration and pneumonia. Compared with normal - genes carriers values, the outpatients with - 2 thal (deter- mined by PCR analysis) had significantly higher mean hemoglobin concentration (Hb) (8.3 ± 1.34 g/dL, p = 0.018) and packed cell volume (PCV = 27.1% ± 4.26, p = 0.019) but low mean corpuscular volume (MCV = 86.1 fL = 10-15 L ± 9.56, p = 0.0004) and mean corpuscular hemoglobin (MCH = 26.6% ± 4.60, p = 0.039). 3.7 kb S Key words: alpha-thalassemia 2 gene 3.7 kb deletion (-α2 thal), β -globin gene haplotypes, Fetal hemoglobin. Received: April 27, 2007; Accepted: October 15, 2007. Introduction of painful episodes, less need for blood therapy and a re- In sickle cell anemia (SCA), inheritance of the sickle duced number of hospital admissions (Steinberg, 2001; cell trait involve two co-dominant beta-globin alleles, the Nagel and Steinberg, 2001b). Individual HbF levels have βS normal A allele and the sickle-cell S allele. Individuals been associated with specific beta S-globin gene ( -globin which are AS heterozygous produce both normal and ab- gene) haplotypes, usually recognized by a set of polymor- S normal hemoglobin and are usually healthy but may have phic restriction enzyme profiles distributed in the β -globin some symptoms of sickle cell anemia at high altitudes when gene cluster region (Nagel and Ranney, 1990; Nagel and blood oxygen is low and may pass the S allele to their off- Steinberg, 2001b). The majors β-globin gene haplotypes spring, homozygous (SS) offspring manifesting SCA (Na- are named according to the geographical origin and ethnic gel and Steinberg, 2001a). group in which they are frequently found, i.e., Bantu or A wide spectrum of clinical manifestations and heter- Central African Republic (abbreviated to BAN or CAR), ogeneous severity characterize SCA (Bunn and Forget, Benin (Ben), Cameroon (Cam), Arab-India and Senegal 1986; Nagel and Steinberg, 2001a). Fetal hemoglobin (Sen) (Nagel and Ranney, 1990; Rahgozar et al., 2000). (HbF) levels have been associated with the modulation of The BAN or CAR haplotype being associated with the SCA, decreased clinical severity, a decrease in the number more severe forms of SCA (Powars and Hiti, 1993; Nagel and Steinberg, 2001a). Send correspondence to Marilda Souza Gonçalves. Laboratório de Patologia e Biologia Molecular, Centro de Pesquisas Gonçalo Thalassemia constitutes another group of inherited Moniz, Rua Waldemar Falcão 121, Candeal, 40296-710 Salvador, hemoglobin disorders commonly characterized by the re- BA, Brazil. E-mail: [email protected]. duction or absence of globin chain synthesis. The presence 622 Adorno et al. 3.7 kb of the alpha-thalassemia 2 gene 3.7 kb deletion (-α2 Hematological and hemoglobin analyses thal) in individuals with SCA has been associated with an We assessed Hb (g/dL), HbF (%), PCV (%), MCH inhibitory effect on intracellular polymer formation of he- (%) and mean corpuscular volume (MCV, in femtoliters moglobin S (HbS), decreased mean corpuscular hemoglo- (1 fL = 10-15 L). Hematological analyses were carried out bin (MCH) and a decrease in the rate of hemolysis which using a Coulter Count T-890 electronic cell counter (Coul- results in an increase in the hemoglobin concentration (Hb) ter Corporation, FL, USA). The hemoglobin profile and and an increased number of erythrocytes and a higher HbF levels were investigated by high performance liquid packed cell volume (PCV), leading to a decrease in the clin- chromatography (HPLC) using a Variant I HPLC (Bio-Rad ical severity of SCA (Steinberg and Embury, 1986). Labs., CA, USA). Brazil is the largest South American country and has βS α 3.7 kb an heterogeneous population characterized by cultural, so- Assessment of - globin haplotypes and - 2 thal cioeconomic and ethnic diversity across its geographical We used the GFX Genomic Blood DNA Purification regions. Bahia is a northeastern Brazilian state with a popu- kit (Amersham Pharmacia Biotech, NJ, USA) to isolate lation of about 13 million and an important African genetic DNA from peripheral blood leukocytes taken from each of contribution due to the high level of slave trading which the outpatients involved in the study. took place for more than three centuries (Azevêdo, 1980). The βS-globin Arab-India, Benin (Ben), Cameroon In Bahia, the overall incidence of SCA in the general popu- (Cam), Central African Republic (CAR) and Senegal (Sen) lation has been estimated as 1:650 (Almeida et al., 2006), gene haplotypes were investigated using the polymerase with the AS genotype occurring at about 4.5% to 14.7% chain reaction, PCR, and restriction fragment length poly- among different population groups (Azevêdo et al., 1980). morphism (RFLP) analysis using the, HindIII, HincII α 3.7 kb Regarding the presence of the - 2 thal has been identi- (Invitrogen Corporation, California, USA) and HinfI, HpaI fied in 20% to 25% of the Brazilian population of African and XmnI restriction enzymes (New England Biolabs, Lon- descendent (Sonati et al., 1991). 3.7 kb don, England) and PCR was used to investigate the -α2 To identify the major pertinent molecular and clinical thal in 110 outpatients (Sutton et al., 1989; Dodé et al., S factors of SCA in Bahia we investigated the β -globin gene 1998). The βS-globin haplotypes with fragment patterns haplotypes in a group of SCA outpatients from Bahia and which did not fit any of the above haplotypes were classi- 3.7 kb correlated these factors with the presence of -α2 , HbF fied as Atypical (Aty). levels, other hematological data and phenotypic character- istics. Statistical analysis The EPI info software version 6.04 (Centers for Dis- Materials and Methods ease Control and Prevention, Atlanta, USA) was used throughout to carry out all the statistical analyses. The Study group ANOVA, Kruskal Wallis H, χ2 and Fisher Exact test were From 2002 to 2003 we conducted a cross-sectional used to test for significance between means at the probabili- transversal study of a group of outpatients with SCA from ties stated. Bahia state who were attending the Hematology and Hemo- therapy Foundation of Bahia (Fundação de Hematologia e Results Hemoterapia da Bahia (HEMOBA, Salvador, BA, Brazil). In our study of outpatients with SCA, those aged ≤ 18 The group (n = 125) consisted of 63 (50.4%) females and y presented, on average, fewer leg ulcers (p = 0.0004) and 62 (49.6%) males aged between 1 year (y) to 73 y (median had been submitted to erythrocyte transfusions less fre- age = 19.1 ± 13.6 y), with 51.2% (n = 64, 24 females and 40 quently (p = 0.02) than those > 18 y, although a higher males) of the group being ≤ 18 y and 48.8% (n = 61, 39 fe- frequency of both spleen sequestration (p = 0.03) and pneu- males and 22 males) being > 18 y. The study was approved monia (p = 0.0004) were observed in the younger group. by the Human Subject Research Ethics Committee of the We also found that 83 (66.4%) of the 125 outpatients inves- Oswaldo Cruz Research Foundation and informed consent tigated had a history of hospital admission, 71 (56.8%) had was also obtained from the outpatients participating in the received blood therapy and 49 (39.2%) had had infections study or their legal guardians in accordance with the ethical pneumonia (20%) and leg ulcers (16.8%) being the most principles and with the Helsinki Declaration of 1975, as re- frequent, 66 (52.8%) referred painful episodes, 15 (12.0%) vised in 2000. presented spleen sequestration and six (4.8%) had had The outpatients were divided into four groups accord- cerebrovascular accidents (CVA). ing to their HbF levels: ≤ 5%, group I (n = 32, 25.6%); > 5% The HbF levels were unevenly distributed among the but ≤ 10%, group II (n = 37, 29.6%); > 10% but ≤ 15%, SCA outpatients, with those aged ≤ 18 y showing a smaller group III (n = 31, 24.8%); and > 15%, group IV with number of erythrocyte transfusions and high average rela- (n = 25, 20.0%). tive HbF levels (12.3% ± 7.01) than those aged > 18 y Characteristic of sickle cell anemia 623 (7.9% ± 4.36) (p = 0.0004).