ßS-Haplotypes in sickle cell anemia patients from Salvador, Bahia, Northeastern Brazil

Gonçalves, M.S.; Bomfim, G.C.; Maciel, E.; Cerqueira, I.; Lyra, I.; Zanette, A.; Bomfim, G.; Adorno, E.V.; Albuquerque, A.L.; Pontes, A.; Dupuit, M.F.; Fernandes, G.B.; Reis, M.G. dos

doi:10.1590/S0100-879X2003001000001

Abstract

ßS-Globin haplotypes were studied in 80 (160 ßS chromosomes) sickle cell disease patients from Salvador, Brazil, a city with a large population of African origin resulting from the slave trade from Western Africa, mainly from the Bay of Benin. Hematological and hemoglobin analyses were carried out by standard methods. The ßS-haplotypes were determined by PCR and dot-blot techniques. A total of 77 (48.1%) chromosomes were characterized as Central African Republic (CAR) haplotype, 73 (45.6%) as Benin (BEN), 1 (0.63%) as Senegal (SEN), and 9 (5.63%) as atypical (Atp). Genotype was CAR/CAR in 17 (21.3%) patients, BEN/BEN in 17 (21.3%), CAR/BEN in 37 (46.3%), BEN/SEN in 1 (1.25%), BEN/Atp in 1 (1.25%), CAR/Atp in 6 (7.5%), and Atp/Atp in 1 (1.25%). Hemoglobin concentrations and hematocrit values did not differ among genotype groups but were significantly higher in 25 patients presenting percent fetal hemoglobin (%HbF) > or = 10% (P = 0.002 and 0.003, respectively). The median HbF concentration was 7.54 ± 4.342% for the CAR/CAR genotype, 9.88 ± 3.558% for the BEN/BEN genotype, 8.146 ± 4.631% for the CAR/BEN genotype, and 4.180 ± 2.250% for the CAR/Atp genotype (P = 0.02), although 1 CAR/CAR individual presented an HbF concentration as high as 15%. In view of the ethnic and geographical origin of this population, we did not expect a Hardy-Weinberg equilibrium for CAR/CAR and BEN/BEN homozygous haplotypes and a high proportion of heterozygous CAR/BEN haplotypes since the State of Bahia historically received more slaves from Western Africa than from Central Africa.

Beta(S)-haplotypes; Fetal hemoglobin; Sickle cell anemia; S hemoglobin; Brazilian population

Braz J Med Biol Res, October 2003, Volume 36(10) 1283-1288

ß ^S -Haplotypes in sickle cell anemia patients from Salvador, Bahia, Northeastern Brazil

M.S. Gonçalves^1,4, G.C. Bomfim¹, E. Maciel¹, I. Cerqueira¹, I. Lyra², A. Zanette², G. Bomfim³, E.V. Adorno¹, A.L. Albuquerque¹, A. Pontes⁴, M.F. Dupuit⁴, G.B. Fernandes⁵and M.G. dos Reis¹

¹Laboratório de Patologia e Biologia Molecular, Centro de Pesquisas Gonçalo Moniz, FIOCRUZ, Salvador, BA, Brasil

²Fundação Hemocentro da Bahia (HEMOBA)/SESAB, Salvador, BA, Brasil

³Hospital Professor Edgar Santos, ⁴Laboratório de Biologia Molecular, Departamento de Toxicologia e Análises Clínicas, Faculdade de Farmácia, and ⁵Instituto de Matemática, Universidade Federal da Bahia, Salvador, BA, Brasil

References

Correspondence and Footnotes ^{Correspondence and Footnotes} Correspondence and Footnotes

Abstract

ß^S-Globin haplotypes were studied in 80 (160 ß^S chromosomes) sickle cell disease patients from Salvador, Brazil, a city with a large population of African origin resulting from the slave trade from Western Africa, mainly from the Bay of Benin. Hematological and hemoglobin analyses were carried out by standard methods. The ß^S-haplotypes were determined by PCR and dot-blot techniques. A total of 77 (48.1%) chromosomes were characterized as Central African Republic (CAR) haplotype, 73 (45.6%) as Benin (BEN), 1 (0.63%) as Senegal (SEN), and 9 (5.63%) as atypical (Atp). Genotype was CAR/CAR in 17 (21.3%) patients, BEN/BEN in 17 (21.3%), CAR/BEN in 37 (46.3%), BEN/SEN in 1 (1.25%), BEN/Atp in 1 (1.25%), CAR/Atp in 6 (7.5%), and Atp/Atp in 1 (1.25%). Hemoglobin concentrations and hematocrit values did not differ among genotype groups but were significantly higher in 25 patients presenting percent fetal hemoglobin (%HbF) ³10% (P = 0.002 and 0.003, respectively). The median HbF concentration was 7.54 ± 4.342% for the CAR/CAR genotype, 9.88 ± 3.558% for the BEN/BEN genotype, 8.146 ± 4.631% for the CAR/BEN genotype, and 4.180 ± 2.250% for the CAR/Atp genotype (P = 0.02), although 1 CAR/CAR individual presented an HbF concentration as high as 15%. In view of the ethnic and geographical origin of this population, we did not expect a Hardy-Weinberg equilibrium for CAR/CAR and BEN/BEN homozygous haplotypes and a high proportion of heterozygous CAR/BEN haplotypes since the State of Bahia historically received more slaves from Western Africa than from Central Africa.

Key words: Beta(S)-haplotypes, Fetal hemoglobin, Sickle cell anemia, S hemoglobin, Brazilian population

Introduction

Sickle cell hemoglobin (HbS) is the result of a single nucleotide change (GAG®GTG) in the ß-globin gene, where valine replaces glutamic acid (ß^{6 Glu}^®^val) at the sixth amino acid position of the ß-globin chain (1). Sickle cell anemia is caused by homozygosity of the ß^S-gene and has a worldwide distribution. The disease progresses as severe chronic hemolytic anemia, presenting a heterogenous clinical course according to patient background and geographic region of origin (2). Milder clinical symptoms have been described in patients presenting a-2 thalassemia and high levels of fetal hemoglobin (HbF), related to the presence of specific haplotypes (3,4). ß^S-Haplotypes are of different ethnic and geographic origins: the Benin type (BEN) originated in Midwestern Africa, the Bantu (CAR) type in South-Central and Eastern Africa, the Senegal (SEN) type in Atlantic West Africa, the Saudi Arabia-India type on the Indian subcontinent and the eastern Arabian peninsula, and the Cameroon type along the west coast of Africa (5).

Sickle cell disease affects millions worldwide, and occurs in one of every 500 African-American births, and in one of every 1000 to 4000 Hispanic-American births. In Brazil, the largest country in South America, the sickle cell trait is found at frequencies ranging from 6.9 to 15.4% of individuals of African descent (6). High immigration influxes have produced a population of significant cultural, social, and ethnic heterogeneity. Salvador is the capital of Bahia, a state in the Northeast region of Brazil, with 2.7 million people (7). The population has a high racial admixture with 85% of the African component (8). Historical data describing the slave trade in Bahia indicate the presence of slaves from central Africa (predominantly CAR haplotype) and from Western Africa (BEN haplotype), with a predominance of the latter. However, haplotype characterization has reported conflicting frequencies of CAR (9) and BEN (10) haplotypes.

The historian Verger (11) described the Nagô-Iorubá influence in Bahia State brought by Africans from the Benin Gulf region. In contrast, the rest of Brazil received large influxes of slaves from Congo and Angola (primarily CAR haplotype). In addition to a period of illicit slave trafficking, Bahia had four official periods of slave trading: a Guinea cycle during the XVI century, an Angola and Congo cycle during the XVII century, a Coast of Mine cycle during the XVIII century, and a Bay of Benin cycle between 1770 and 1850. Florentino (12) emphasizes that Bahia, beginning in 1815, was the only Brazilian state that restricted slave traffic through Ecuador, a fact that explains the correlation between genotype frequencies found in Bahia and Western Africa, principally the Bay of Benin region.

The unusual ethnic composition of Salvador, which was a transfer point during the African slave trade, represents an excellent opportunity to study the ß^S-haplotypes and to investigate the clinical picture of sickle cell anemia patients and the anthropological origins of the ß^S-gene in this Brazilian population.

Material and Methods

A total of 80 sickle cell disease patients (40 males and 40 females) were studied. Informed consent was obtained from all individuals or responsible person prior to enrollment and the study protocol was submitted to and approved by the FIOCRUZ Ethics Committee. Patients were recruited from both the Center for Hematological Studies (Fundação Hemocentro da Bahia, HEMOBA) and the University Hospital, Federal University of Bahia (Hospital Universitário Professor Edgar Santos, Universidade Federal da Bahia). Mean patient age was 13.17 ± 9.71 years (range: 1.6-51.5 years).

Hematological analyses were carried out using an electronic cell counter (Coulter Count T890). Hemoglobin type was determined by electrophoresis on cellulose acetate strips at pH 8.4, and the presence of HbS was confirmed by sickling and solubility tests, and by electrophoresis on agar-citrate at pH 5.3 (13). HbF was measured by alkali denaturation (13). DNA was isolated from peripheral blood leukocytes (14). ß^S-Haplotypes were established by PCR and by dot-blot methods that characterize DNA polymorphisms of the 5' flanking region and the second intervening sequence (IVS-II) of the g-globin genes (15,16) (Figure 1).

The EPI Info (version 6.04) and Statistical Package for the Social Sciences (SPSS, version 6.1) programs were used for statistical analyses. The effects of age category, gender, and HbF concentration ³10% on the hematological parameters were evaluated. The level of significance was set at P < 0.05 in all analyses.

Figure 1.
Gamma-globin gene amplification and dot-blot analyses of CAR ß^S-haplotype. Sickle cell disease patients, heterozygous for the CAR haplotype, have a positive signal with normal and mutant probes. Homozygous patients have a positive signal only with a mutant probe and negative patients for this haplotype have a signal only with a normal probe. Lanes 1-5 show a 630-bp PCR fragment from the ^Gg-globin. M = l HindIII marker; N = negative control.

Results

The patients had a median (± SD) hemoglobin concentration of 8.369 ± 1.632 g/dl, median hematocrit of 25.044 ± 5.03%, median cell volume of 88.488 ± 10.033 fl, median cell hemoglobin of 30.095 ± 4.195 pg, and median cell hemoglobin concentration of 33.905 ± 1.679 g/dl. These parameters did not vary significantly between age and gender categories. However, patients with HbF ³10% were found to have significantly higher Hb concentrations compared to patients of the group with Hb <10% (median: 7.8 vs 9.0; P = 0.002) and hematocrit values (median: 24.00 vs 28.00; P = 0.003).

The hematological data and the ß^S-haplotypes/genotypes obtained for the 80 sickle cell disease patients analyzed are listed in Table 1.

Thumbnail

ßS-Haplotypes in sickle cell anemia patients from Salvador, BA, Northeastern Brazil. M.S. Gonçalves, G.C. Bomfim, E. Maciel, I. Cerqueira, I. Lyra, A. Zanette, G. Bomfim, E.V. Adorno, A.L. Albuquerque, A. Pontes, M.F. Dupuit, G.B. Fernandes and M.G. dos Reis. Brazilian Journal of Medical and Biological Research, 36 (10): 1283, 2003.

The hematological data, including the different proportions of HbF found, are reported in Table 2. Median age was significantly higher among the CAR/CAR and CAR/BEN genotypes. The median HbF levels among the CAR/CAR, CAR/BEN, BEN/BEN and CAR/atypical (Atp) genotypes are shown in Figure 2. The patient group presenting HbF ³10% consisted of 25 (31.2%) individuals; the genotype was CAR/BEN in 12, CAR/CAR in four, BEN/BEN in seven, SEN/Atp in one, and Atp/Atp in one. There was no CAR/Atp or BEN/SEN genotype in this group. In the group with HbF higher than 10%, eight subjects presented HbF ³15%, with the genotype being CAR/CAR in one of them, CAR/BEN in four, BEN/BEN in one, SEN/Atp in one, and Atp/Atp in one. Median HbS values were higher among the subjects with the CAR/Atp genotype and lowest among subjects with the BEN/BEN genotype.

Thumbnail

ßS-Haplotypes in sickle cell anemia patients from Salvador, BA, Northeastern Brazil. M.S. Gonçalves, G.C. Bomfim, E. Maciel, I. Cerqueira, I. Lyra, A. Zanette, G. Bomfim, E.V. Adorno, A.L. Albuquerque, A. Pontes, M.F. Dupuit, G.B. Fernandes and M.G. dos Reis. Brazilian Journal of Medical and Biological Research, 36 (10): 1283, 2003.

Figure 2.
Distribution of median fetal hemoglobin (HbF) levels among the CAR/CAR (N = 17), CAR/BEN (N = 37), BEN/BEN (N = 17) and CAR/Atp (N = 6) genotypes in sickle cell disease patients from Salvador, Bahia, Brazil. For abbreviations, see legend to .

Discussion

ß^S-Haplotypes were established in 80 sickle cell disease patients from Salvador, a city in Northeastern Brazil characterized by a population with a large African contribution (8). Azevedo et al. (6) found that the frequency of HbS ranged from 7.6 to 15.9% in different population groups of Salvador. In the present study, the CAR/BEN genotype was predominant. Unexpectedly, the BEN and CAR homozygous genotypes were found to occur at similar frequencies, mainly considering the high presence of the CAR haplotype. Verger (11) emphasized that from 1678 to 1814, only 39 of 1770 ships that exported tobacco from Bahia went to the Congo and Angola, where they captured slaves representing a possible contribution of Africans from Atlantic Central Africa. All the other ships went to Coast of Mine ports. The slave traffic from Atlantic Central Africa was supposedly intensified between 1815 and 1824 (11), a fact that can explain our results. No Saudi Arabian or Cameroon haplotypes were identified in the study sample, and only one SEN haplotype was encountered.

In the United States and Jamaica, the BEN haplotype is predominant, a result of the preference for the traffic of Midwestern Africans to these regions during the British Atlantic slave trade (4,17,18). In contrast, haplotype studies on the Cuban and Puerto Rican populations have found a predominance of genes from the Bantu haplotype, suggesting a different African origin of these populations (5,19-21).

The distribution of ß^S-haplotypes in the Brazilian State of São Paulo (Southeastern Brazil) and Pará (Northern Brazil) showed high frequencies of the CAR haplotype, i.e., 62.2 and 65.9%, respectively (9,22-24). Analyses of ß^S-haplotypes from the Amazon region have indicated a 60% frequency of the CAR haplotype, a 30% frequency of the SEN haplotype, and a 10% frequency of the BEN haplotype (24).

Populations with a high frequency of BEN/CAR heterozygotes, as reported for Bahia, provide an excellent cohort for the study of the effect of ß^S-haplotypes on the clinical course of sickle cell anemia. An important finding of the present study was the high concentration of HbF among individuals with the CAR/CAR genotype, which normally present a median HbF value below 5.0% (4). It is well known that HbF levels in sickle cell anemia could be influenced by age, gender, a-globin gene number, ß-globin haplotype, and the X-linked F-cell production locus that regulates the production of HbF-containing erythrocytes (F cells) (25).

In a previous study, the F-cell production locus accounted for 40% of the overall variation of HbF levels and the ß-globin haplotype was associated with 14% of the remaining HbF variation; when the F-cell production influence was removed, approximately half of the variation in HbF levels still remained to be explained, showing the need for further studies (25). Unfortunately, we did not study a-thalassemia in theses patients, but a higher frequency of this type of thalassemia was previously demonstrated among Bahian sickle cell disease patients (10), probably representing an important prognostic factor for the clinical course of the disease.

The presence of high HbF levels in the CAR/CAR genotype could be explained by sequence variation in regulatory regions of the 5' HS2 and 5' flanking region of the g-gene expression, as previously discussed by Lanclos et al. (26).

In addition, we also identified an individual with the SEN haplotype, a fact that may suggest that Bahia State also had a gene flow from Atlantic West Africa, as was the case for other Brazilian states (24). Internal migration is unlikely since the patient's ancestors were from Salvador. The low frequency of the SEN haplotype could be explained by the absence of SEN carriers looking for medical care or a recent origin of the ß^S SEN mutation in this population (27). The atypical haplotypes showed different distributions and could be found associated with the CAR and BEN genotypes, indicating the occurrence of diverse genetic mechanisms that could be responsible for the variation of HbF concentrations among the atypical haplotype carriers (28-30).

The present results are relevant to the study of slave traffic routes in Brazil and of the African origins of the Bahian population. Taken together, the data indicate that studies examining the impact of the ß^S-haplotypes and of the presence of a-thalassemia on clinical outcome and HbF expression may contribute to clarifying a possible relationship between clinical picture, ß^S-haplotypes, a-thalassemia and HbF production in sickle cell disease patients from Salvador, Bahia, identifying prognostic factors for the clinical course of the disease in this population.

Address for correspondence: M.S. Gonçalves, Laboratório de Patologia e Biologia Molecular, Centro de Pesquisas Gonçalo Moniz, Rua Valdemar Falcão, 121, 40295-001 Salvador, BA, Brasil. E-mail: mari@cpqgm.fiocruz.br

Research supported by PAPES-FIOCRUZ (No. 0250250304) and CNPq (No. 521201/96-1). Received January 28, 2002. Accepted July 4, 2003.

1. Bunn HF & Forget BG (1986). Hemoglobin: Molecular, Genetic and Clinical Aspects W.B. Saunders, Philadelphia, PA, USA.
2. Embury SH, Hebbel RP, Mohandas N & Steinberg MH (1994). Sickle Cell Disease: Basic Principles and Clinical Practice Raven Press, New York.
3. Pagnier J, Mear JG, Dunda-BelKhadja O, Rego KE, Beldjord C, Nagel RL & Labie D (1984). Evidence for the multicentric origin of the sickle cell hemoglobin gene in Africa. Proceedings of the National Academy of Sciences, USA, 81: 1771-1773.
4. Powars DR (1991). ß^S-Gene cluster haplotypes in sickle cell anemia: clinical and hematologic features. Hematology/Oncology Clinics of North America, 5: 475-493.
5. Nagel RL (1984). The origin of the hemoglobin S gene: clinical, genetic and anthropological consequences. Einstein Quarterly Journal of Biology and Medicine, 2: 53-62.
6. Azevedo ES, Alves AFP, Silva MCBO, Souza MGF, Lima AMVMD & Azevedo W (1980). Distribution of abnormal hemoglobins and glucose-6-phosphate dehydrogenase variants in 1200 school children of Bahia, Brazil. American Journal of Physical Anthropology, 53: 509-512.
7. Rede Interagencial de Informação para a Saúde (1997). Indicador e Dados Básicos. IDB97. Ministério da Saúde, Brasil.
8. Azevedo ES, Silva KMC, Silva MCBO, Lima AMVMD, Fortuna CMM & Santos MG (1981). Genetic anthropological studies in the island of Itaparica, Bahia, Brazil. Human Heredity, 31: 353-357.
9. Costa FF, Arruda VR, Gonçalves MS et al. (1984). ß^S-Gene cluster haplotypes in sickle cell anemia patients from two regions of Brazil. American Journal of Hematology, 46: 96-97.
10. Queiroz IMLP (1996). Características Clínicas, Hematológicas e Genéticas em Pacientes Homozigotos para a Hemoglobinopatia S da Bahia e de São Paulo Editora da Faculdade de Medicina da Universidade de São Paulo, São Paulo, SP, Brazil.
11. Verger P (1968). Flux et Reflux de la Traite des Nègres Entre le Golfe de Benin et Bahia de Todos os Santos Mouton Press, Paris, France.
12. Florentino M (1997). Em Costas Negras Companhia das Letras Press, Rio de Janeiro, RJ, Brazil.
13. Dacie JV & Lewis SM (1995). Practical Hematology 8th edn. Churchill Livingstone Press, Edinburgh, UK.
14. Poncz M, Solowiejczyk D, Harpel B, Morry Y, Schwartz E & Surrey S (1982). Construction of human gene libraries from small amounts of peripheral blood: analysis of ß-like globin genes. Hemoglobin, 6: 27-36.
15. Lanclos KD, Öner C, Dimovski AJ, Gu YC & Huisman THJ (1991). Sequence variations in the 5' flanking and IVS-II regions of the ^Gg- and ^Ag-globin genes of ß^S chromosomes with five different haplotypes. Blood, 77: 2488-2496.
16. Gonçalves MS, Nechtman JF, Figueredo MS, Kerbauy J, Arruda VR, Sonati MF, Saad STO, Costa FF & Stoming TA (1994). Sickle cell disease in a Brazilian population from São Paulo: A study of the ß^S-gene haplotypes. Human Heredity, 44: 322-327.
17. Schroeder WA, Powards DR, Kay LM, Chan LS, Huynh V, Shelton JB & Shelton JR (1989). ß-Cluster haplotypes, a-gene status, and hematological data from SS, SC, and Sß-thalassemia patients in Southern California. Hemoglobin, 13: 325-353.
18. Wainscoat JS, Bill JI & Thein SL (1983). Multiple origins of the sickle mutation: Evidence from ß^S-globin gene cluster polymorphism. Molecular Biology and Medicine, 1: 191-197.
19. Muniz A, Corral L, Alaec C, Svarch E, Espinosa E, Carbonell N, Dileo R, Felicetti L, Nagel RL & Martinez G (1995). Sickle cell anemia and ß-gene cluster haplotypes in Cuba. American Journal of Hematology, 49: 113-114.
20. Curtain PD (1969). The Slave Atlantic Trade: A Census University of Wisconsin Press, Madison, WI, USA.
21. Baralt GA (1982). Eslavos Rebeldes Huracon Press, Puerto Rico.
22. Figueiredo MS, Kerbauy J, Goncalves MS, Arruda VR, Saad ST, Sonati MF, Stroming T & Costa FF (1996). Effect of alpha-thalassemia and beta-globin gene cluster haplotypes and the hematological features of sickle cell anemia in Brazil. American Journal of Hematology, 53: 72-76.
23. Zago MA, Figueiredo MS & Ogo SH (1992). Bantu ß^S-cluster haplotype predominates among Brazilian blacks. American Journal of Physiological Anthropology, 88: 295-298.
24. Pante-De-Sousa G, Mousinho-Ribeiro RC, Dos Santos EJ & Guerreiro JF (1999). Beta-globin haplotype analysis in Afro-Brazilians from the Amazon region: evidence for a significant gene flow from Atlantic West Africa. American Journal of Human Biology, 26: 365-373.
25. Chang YP, Maier-Redelsperger M, Smith KD, Contu L, Ducroco R, de Montalembert M, Belloy M, Elion J, Dover GJ & Girot R (1997). The relative importance of the X-linked FCP locus and beta-globin haplotypes in determining haemoglobin F levels: a study of SS patients homozygous for beta S haplotypes. British Journal of Haematology, 96: 806-814.
26. Adekile AD, Dimovski AJ, Oner C, Lanclos KD & Huisman TH (1993). Haplotype-specific sequence variations in the locus control region (5' hypersensitive sites 2, 3, 4) of beta S chromosomes. Hemoglobin, 17: 475-478.
27. Currat M, Trabuchet G, Rees D, Perrin P, Harding RM, Clegg JB, Langaney A & Excoffier L (2002). Molecular analysis of the beta-globin gene cluster in the Niokholo Mandenka population reveals a recent origin of the beta (S) Senegal mutation. American Journal of Human Genetics, 70: 207-223.
28. Zago MA, Silva Jr WA, Dalle B et al. (2000). Atypical beta (S) haplotypes are generated by diverse genetic mechanisms. American Journal of Hematology, 63: 79-84.
29. Zago MA, Silva Jr WA, Gualandro S, Yokomizu IK, Araujo AG, Tavela MH, Gerard N, Krishnamoorthy R & Elion J (2001). Rearrangements of the beta-globin gene cluster in apparently typical beta S haplotypes. Haematologica, 86: 142-145.
30. Bordin S, Crespi VG, Duarte AS, Basseres DS, Melo MB, Vieira AP, Saad ST & Costa FF (2002). DNase I hypersensitive site 3' to the beta-globin gene cluster contains a TAA insertion specific for beta (S)-Benin haplotype. Haematologica, 87: 246-249.

Correspondence and Footnotes

Publication Dates

Publication in this collection
16 Sept 2003
Date of issue
Oct 2003

History

Received
28 Jan 2002
Accepted
04 July 2003

This work is licensed under a Creative Commons Attribution 4.0 International License.

[1] 1. Bunn HF & Forget BG (1986). Hemoglobin: Molecular, Genetic and Clinical Aspects W.B. Saunders, Philadelphia, PA, USA.

[2] 2. Embury SH, Hebbel RP, Mohandas N & Steinberg MH (1994). Sickle Cell Disease: Basic Principles and Clinical Practice Raven Press, New York.

[3] 3. Pagnier J, Mear JG, Dunda-BelKhadja O, Rego KE, Beldjord C, Nagel RL & Labie D (1984). Evidence for the multicentric origin of the sickle cell hemoglobin gene in Africa. Proceedings of the National Academy of Sciences, USA, 81: 1771-1773.

[4] 4. Powars DR (1991). ß^S-Gene cluster haplotypes in sickle cell anemia: clinical and hematologic features. Hematology/Oncology Clinics of North America, 5: 475-493.

[5] 5. Nagel RL (1984). The origin of the hemoglobin S gene: clinical, genetic and anthropological consequences. Einstein Quarterly Journal of Biology and Medicine, 2: 53-62.

[6] 6. Azevedo ES, Alves AFP, Silva MCBO, Souza MGF, Lima AMVMD & Azevedo W (1980). Distribution of abnormal hemoglobins and glucose-6-phosphate dehydrogenase variants in 1200 school children of Bahia, Brazil. American Journal of Physical Anthropology, 53: 509-512.

[7] 7. Rede Interagencial de Informação para a Saúde (1997). Indicador e Dados Básicos. IDB97. Ministério da Saúde, Brasil.

[8] 8. Azevedo ES, Silva KMC, Silva MCBO, Lima AMVMD, Fortuna CMM & Santos MG (1981). Genetic anthropological studies in the island of Itaparica, Bahia, Brazil. Human Heredity, 31: 353-357.

[9] 9. Costa FF, Arruda VR, Gonçalves MS et al. (1984). ß^S-Gene cluster haplotypes in sickle cell anemia patients from two regions of Brazil. American Journal of Hematology, 46: 96-97.

[10] 10. Queiroz IMLP (1996). Características Clínicas, Hematológicas e Genéticas em Pacientes Homozigotos para a Hemoglobinopatia S da Bahia e de São Paulo Editora da Faculdade de Medicina da Universidade de São Paulo, São Paulo, SP, Brazil.

[11] 11. Verger P (1968). Flux et Reflux de la Traite des Nègres Entre le Golfe de Benin et Bahia de Todos os Santos Mouton Press, Paris, France.

[12] 12. Florentino M (1997). Em Costas Negras Companhia das Letras Press, Rio de Janeiro, RJ, Brazil.

[13] 13. Dacie JV & Lewis SM (1995). Practical Hematology 8th edn. Churchill Livingstone Press, Edinburgh, UK.

[14] 14. Poncz M, Solowiejczyk D, Harpel B, Morry Y, Schwartz E & Surrey S (1982). Construction of human gene libraries from small amounts of peripheral blood: analysis of ß-like globin genes. Hemoglobin, 6: 27-36.

[15] 15. Lanclos KD, Öner C, Dimovski AJ, Gu YC & Huisman THJ (1991). Sequence variations in the 5' flanking and IVS-II regions of the ^Gg- and ^Ag-globin genes of ß^S chromosomes with five different haplotypes. Blood, 77: 2488-2496.

[16] 16. Gonçalves MS, Nechtman JF, Figueredo MS, Kerbauy J, Arruda VR, Sonati MF, Saad STO, Costa FF & Stoming TA (1994). Sickle cell disease in a Brazilian population from São Paulo: A study of the ß^S-gene haplotypes. Human Heredity, 44: 322-327.

[17] 17. Schroeder WA, Powards DR, Kay LM, Chan LS, Huynh V, Shelton JB & Shelton JR (1989). ß-Cluster haplotypes, a-gene status, and hematological data from SS, SC, and Sß-thalassemia patients in Southern California. Hemoglobin, 13: 325-353.

[18] 18. Wainscoat JS, Bill JI & Thein SL (1983). Multiple origins of the sickle mutation: Evidence from ß^S-globin gene cluster polymorphism. Molecular Biology and Medicine, 1: 191-197.

[19] 19. Muniz A, Corral L, Alaec C, Svarch E, Espinosa E, Carbonell N, Dileo R, Felicetti L, Nagel RL & Martinez G (1995). Sickle cell anemia and ß-gene cluster haplotypes in Cuba. American Journal of Hematology, 49: 113-114.

[20] 20. Curtain PD (1969). The Slave Atlantic Trade: A Census University of Wisconsin Press, Madison, WI, USA.

[21] 21. Baralt GA (1982). Eslavos Rebeldes Huracon Press, Puerto Rico.

[22] 22. Figueiredo MS, Kerbauy J, Goncalves MS, Arruda VR, Saad ST, Sonati MF, Stroming T & Costa FF (1996). Effect of alpha-thalassemia and beta-globin gene cluster haplotypes and the hematological features of sickle cell anemia in Brazil. American Journal of Hematology, 53: 72-76.

[23] 23. Zago MA, Figueiredo MS & Ogo SH (1992). Bantu ß^S-cluster haplotype predominates among Brazilian blacks. American Journal of Physiological Anthropology, 88: 295-298.

[24] 24. Pante-De-Sousa G, Mousinho-Ribeiro RC, Dos Santos EJ & Guerreiro JF (1999). Beta-globin haplotype analysis in Afro-Brazilians from the Amazon region: evidence for a significant gene flow from Atlantic West Africa. American Journal of Human Biology, 26: 365-373.

[25] 25. Chang YP, Maier-Redelsperger M, Smith KD, Contu L, Ducroco R, de Montalembert M, Belloy M, Elion J, Dover GJ & Girot R (1997). The relative importance of the X-linked FCP locus and beta-globin haplotypes in determining haemoglobin F levels: a study of SS patients homozygous for beta S haplotypes. British Journal of Haematology, 96: 806-814.

[26] 26. Adekile AD, Dimovski AJ, Oner C, Lanclos KD & Huisman TH (1993). Haplotype-specific sequence variations in the locus control region (5' hypersensitive sites 2, 3, 4) of beta S chromosomes. Hemoglobin, 17: 475-478.

[27] 27. Currat M, Trabuchet G, Rees D, Perrin P, Harding RM, Clegg JB, Langaney A & Excoffier L (2002). Molecular analysis of the beta-globin gene cluster in the Niokholo Mandenka population reveals a recent origin of the beta (S) Senegal mutation. American Journal of Human Genetics, 70: 207-223.

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