AMERICAN JOURNAL OF PHYSICAL ANTHROPOLOGY 88:295-298 (1992) Bantu p" Cluster Haplotype Predominates Among Brazilian Blacks MARC0 A. ZAGO, MAURO S. FIGUEIREDO, AND SATIE H. OGO Department of Clinical Medicine, School of Medicine, 14049 Ribeirao Preto, Brazil KEY WORDS S, Haplotypes Brazilian Blacks, Sickle cell anemia, Hemoglobin ABSTRACT We describe the combination of polymorphic restriction-enzyme sites in the p globin gene cluster (haplotypes)for 74 chromosomes from Brazilian Blacks bearing the sickle hemoglobin gene (p"). The three most common African p" haplotypes account for 67 chromosomes: 49/74 (66.2%) were identified as Central African Republic (CAR or Bantu) type, 17 (23.0%) as Benin, and one as Senegal; seven chromosomes (9.5%)had minor atypical haplotypes. This distribution is different from that observed in the United States or Jamaica, where the Benin haplotype predominates, and results from different patterns of slave trades to North and South Americas. Since the p" gene cluster polymorphisms modulate the severity of sickle cell anemia, this heterogeneity may explain differences of the clinical behavior of the disease in the United States and South America, and should also be considered in relation to other features and diseases. o 1992 Wiley-Liss, Inc. The sickle hemoglobin (p") gene was introduced into the Americas by the massive trade of African slaves from the sixteenth to the nineteenth century. As a consequence, p" gene frequencies are roughly proportional to the Black admixture of the populations, which is unevenly distributed within North, South, and Central Americas and the Caribbean. The contribution of p" genes brought directly by European immigrants (Portuguese, Southern Italian) is negligible. Sickle cell anemia, the homozygous state for hemoglobin S (HbS),is the most common single gene disease of the Americas. The two most important genetic factors which contribute to the variations in the clinical severity among patients with sickle cell anemia are the simultaneous presence of a-thalassemia and the differences in the p globin gene cluster of chromosomes which carry the p" gene. Both features have been studied in sickle cell anemia patients from the United States, Jamaica, Africa, Arabia, and India (Antonarakis et al., 1984; Dozy et al., 1979; Hattori et al., 1986; Higgs et al., 1980; Higgs et al., 0 1992 WILEY-LISS. INC. 1982;Kulozik et al., 1987; Nagel et al., 1985; Pagnier et al., 1984; Wainscoat et al., 1985). Although it has been assumed that the genetic basis of sickle cell anemia in South America is similar to that observed in the United States, no studies have been carried out to confirm this assumption. Brazil has the largest non-White population of South America, estimated a t 45% of 150 million inhabitants, of which 1 4 %are carriers of the HbS gene (Zago and Costa, 1985). In the present study we demonstrate that the frequencies of the common p" cluster haplotypes among Brazilian Blacks are different from those observed in the United States. This biological heterogeneity is the consequence of different patterns of slave trade to North and South Americas. MATERIAL AND METHODS Patients The study included 37 nonrelated patients with homozygous sickle cell anemia who are Received J u n e 6,1991;accepted January 2,1992 M.A. ZAG0 ET AL 296 TABLE 1. Haplotype combinations and percentage of Gy chain of HbF of 37 HbS homozygotes from Brazil Haplotypes CAR/ CAR CAR/Benin Benin/Benin CAR/Senegal Other Number of patients %I G y (5 Ifr SD) 19 09 01 01 07 39.5 7.0 39.2 5.2 46.9 61.1 38.0 6.2 ** + under regular follow-up in the Hemoglobinopathy Clinic of the University Hospital. Diagnosis was based on clinical, laboratory, and family data. Homozygosis for HbS was confirmed by hemoglobin studies of the patient and his family, including electrophoresis in alkaline buffer and in agar-citrate, quantitation of HbA, and HbF, quantitative solubility test, and "in vitro" measurements of globin chain synthesis ratios. The levels of Gy chains were determined by reverse-phase HPLC procedures (Shelton et al., 1981; Zag0 and Greene, 1985). the Benin type [- - - - + - -1 (23.0%), and one of the Senegal type [ + + - + + + + I . Seven chromosomes (9.5%)had atypical or minor haplotypes: - - - - + - + (5 cases), -+--+-+ (1 case), and -+----+ (1 case). The one found in 5 cases is compatible with a recombination of 5' Benin haplotype and 3' Bantu haplotype. When only the 67 chromosomes bearing the common haplotypes were considered, the percentage of the CAR and the Benin types were 73 and 25%, respectively. The most common combinations were CAR homozygotes (19 patients) and CAW Benin heterozygotes (9 cases). Mean Gy of HbF was 39.4 6.2%,which was similar for all haplotypes except for the single patient with the Senegal type (CAW Senegal heterozygote) who had Gy of 61.1%. No patient had the AyTvariant. * DISCUSSION The sickle cell anemia gene is the prime example of balanced polymorphism and of Haplotype determinations reciprocal interaction between man and the DNA was isolated from peripheral blood environment. Although it is disadvantaleukocytes. The following restriction sites geous or lethal in the homozygous state, the , were analyzed: (1)HindIII in IVS-2 of G ~ (2) protection afforded the heterozygotes TuqI a t 3' of Gy, (3) Hind111 in IVS-2 of *y, against falciparum malaria caused the gene (4)HincII in the +p, (5)HincII at 3' of +p, (6) to reach high frequencies in Central and Hznfl at 5' of p, and (7) HpaI a t 3' of p. Sites West Africa (Allison, 1954). On the other 1, 2, 3, 4, and 5 were analyzed by Southern hand, malaria seems to have spread in West blotting and hybridization with specific ge- Africa as a consequence of the human internomic y (sites 1-3) or $p (sites 4-51 probes. vention upon the environment which accomSite 1 (9 cases), sites 4 and 5 (9 cases), and panied the introduction of agriculture (Livsites 6 and 7 (all cases) were analyzed by ingstone, 1958). PCR amplification of the region of the polyThe p" mutation may be associated with morphic site employing the primers de- a t least four different major p"cluster haploscribed by Sutton et al. (1989), followed by types, named in accordance with the geodigestion with the appropriated restriction graphic region where it predominates: the enzyme. Sites 1, 2, 3, 6, and 7 were studied Benin, the Senegal, the Central African Refor all patients; sites 4 and 5 were deter- public (CAR or Bantu), and the Asian types mined in 18 selected cases. In cases with two (Antonarakis et al., 1984; Kulozik et al., haplotypes, it was assumed a combination of 1986; Pagnier et al., 1984; Wainscoat et al., two common haplotypes, or that a common 1985). The study of the haplotypes linked to haplotype was present with a rare haplo- the p" gene has provided evidence for the type, rather than two rare haplotypes. independent origin of the sickle cell hemoglobin mutation within those geographic reRESULTS gions (Kulozik et al., 1986; Pagnier et al., The results are summarized in Table 1. 1984). The data presented here show the presThree common haplotypes account for 67 of the 74 chromosomes studied: 49 were of the ence of different African p" haplotypes CAR type [ + + - - --+I (66.2%),17 were of among sickle cell anemia patients from Bra- p" HAPLOTYPE IN BRAZILIAN BLACKS 297 TABLE 2. ps haplotype distribution for North America, the Caribbean, and Brazil compared with historical records on the origin of slave trade to Brazil Haplotype Senegal Benin Bantu Jamaica1 (W) United States' (%) Brazil2 (%) ( E ~ p e c t e d(%) )~ 10 72 17 15 62 18 1 25 73 (1) (26) (73) 'Data from Antonarakis et al. (1984) and Nagel (1984). 'Major haplotypes identified for 66 of the 74 chromosomes in the present study. 'Expected on historical grounds. Data from Curtin (1969), corresponding to the period 1701-1843, which covers 75%of the slave trade to Brazil. zil. The most common types occur at different frequencies as observed for the United States and Jamaican Black sickle cell anemia patients (Antonarakis et al., 1984; Hattori et al., 1986; Nagel, 1984). Most of our Brazilian sickle cell patients have the CAR haplotype, which corresponds to 73% of the chromosomes bearing one of the three major haplotypes, whereas the Benin haplotype is the most common among United States and Jamaican patients (Table 2). Atypical haplotypes correspond to 9.5%,which is similar to frequencies of 3.6 to 11.2% found in other populations. The difference observed in the present study was to be expected on the basis of the African ancestry of these patients. Slaves imported into the United States and Jamaica originated mainly from Central West African ports, where the Benin haplotype predominates (Curtin, 1969; Nagel, 1984; Pagnier et al., 1984). The origin of Blacks brought to South America was diverse. The transport of African Negroes to Brazil was a monopoly of the Portuguese and the ports of origin were determined mainly by Portugal's sphere of influence in Africa. From 1701 to 1843, about 2.4 million slaves were brought into Brazil, of which about 70% originated from Angola, Congo, and Mozambique (Curtin, 19691, where the CAR haplotype predominates (Lavinha et al., 1990). Limited data from Cuba, based only on the HpaI polymorphism, indicates a contribution of Bantu haplotype, which is higher than that observed in the United States but lower than the values that we obtained for Brazil (Martinez et al., 1987). The data of Rogers et al. (1989) in a nonblack American population also indicate a predominant Bantu contribution to populations of Hispanic Central and North Americas. Studies by Nagel et al. t 1 9 8 9 , by Kulozik et al. (19871, and by Powars et al. (1990) have demonstrated that the different p" gene cluster polymorphisms modulate the clinical severity of sickle cell disease. While the Senegal and the Asian types are associated with a more benign clinical course, the Benin is intermediate and the CAR haplotype is the worst. The basis for this difference is not completely understood. However at least for the Senegal and the Asian types it seems in part the result of a higher G~ and HbF production associated with a C T change at position -158 to the G~ globin gene (Nagel et al., 1985; Kulozik et al., 1987; Wainscoat et al., 1985). Thus, different proportions of the p" haplotypes, in addition to environmental and socioeconomic conditions, may explain differences of the clinical behavior of sickle cell anemia in the United States and in South America. The other factor which affects clinical severity of sickle cell anemia is the coinheritance of a-thalassemia (Embury et al., 1982; Higgs et al., 1982; Mears et al., 1983). We have recently demonstrated that 22% of Brazilian sickle cell anemia patients have a deletion type a-thalassemia (Costa et al., 19891, a frequency similar to that observed in the USA and Jamaica (Dozy et al., 1979; Higgs et al., 1980). ACKNOWLEDGMENTS This research was supported by grants from FAPESP Foundation, Brazil (Proc. 90/ 2078-3) and from BID-University of S. Paulo. We wish to acknowledge the invaluable technical assistance of M. H. Tavella, A. G. Araujo, and J. Komoto. 298 M.A. ZAG0 ET AL. LITERATURE CITED Allison AC (1954) Protection afforded by sickle cell trait against subtertian malarial infection. Br. Med. J. 1:290-294. Antonarakis SE, Boehm CD, Serjeant GR, Theisen CE, Dover GJ, and Kazazian HH (1984) Origin of the p”globin gene in Blacks: The contribution of recurrent mutation or gene conversion or both. Proc. Natl. Acad. Sci. U.S.A. 81:853-856. Curtin PD (1969) The Slave Atlantic Trade: A Census. Milwaukee: The University of Wisconsin Press. Costa FF, Tavella MH, and Zago MA (1989) Deletion type a-thalassemia among Brazilian patients with sickle cell anemia. Rev. Brasil. Genet. 12r605-611. Dozy AN, Kan YW, Emhury SH, Mentzer WC, Wang WC, Lubin B, Davies JR, and Koenig AM (1979) a-Globin gene organization in Blacks precludes the severe form of a-thalassemia. Nature (London) 280r605-607. Embury SH, Dozy AM, Miller J , Davies JR, Kleman KM, Preisler H, Vichinsky E, Lande WN, Luhin BH, Kan YW, and Mentzer WC (1982) Concurrent sickle cell anemia and a-thalassemia. Effect on severity of anemia. N. Engl. J. Med. 306:270-274. Hattori Y, Kutlar F, Kutlar A, McKie VC, and Huisman THJ (1986) Haplotypes of p” chromosomes among patients with sickle cell anemia from Georgia. Hemoglobin IOt623-642. Higgs DR, Pressley L, Clegg JB, Weatherall DJ, and Serjeant J R (1980) u-Thalassemia in Black populations. Johns Hopkins Med. J. 146:300-310. Higgs DR, Aldridge BE, Lamb J , Clegg JB, Weatherall DJ, Hayes RJ, Grandison Y, Lowrie Y, Mason KP, Serjeant BE, and Serjeant GR (1982) The interaction of alpha thalassemia and homozygous sickle cell disease. N. Engl. J . Med. 306:1441-1446. Kulozik AE,Wainscoat JS, Serjeant GR, Kar BC, AlAwamy B, Essan GJF, Falusi AG, Haque SK, Hilali AM, Kate S , Ranasinghe WAEP, and Weatherall DJ (1986) Geographic survey of P”-globin gene haplotypes: evidence for the independent Asian origin of the sickle-cell mutation. Am. J. Hum. Genet. 39r239-244. Kulozik AE,Kar BC, Satapathy RK, Serjeant BE, Serjeant GR, and Weatherall D J (1987) Fetal hemoglobin levels and p” globin haplotypes in an Indian population with sickle cell disease. Blood 69:1742-1746. Labie D, Dunda-Belkhodja 0, Rouahhi F, Pagnier J, Ragusa A, and Nagel RL (1985) The -158 site 5’ to the Gy gene and Gy expression. Blood 66:1463-1465. Lavinha J , Faustino P, Goncalves J, Osorio-Almeida L, Peres MJ, Martins MC, and Labie D (1990) Importation route of sickle cell trait into Portugal: contribution of the molecular biology. First International Con- gress on the Great Maritime Discoveries and World Health, Lisbon. Livingstone FB (1958) Anthropological implications of sickle cell gene distribution in West Africa. Am. Anthropol. 60:533-562. Martinez G, Ferreira F, and Colombo B (1987) Human v-globin gene Hpa I polymorphism in the white Cuban population. Gene Geogr. Ir15-18. Mears JG, Lachman H, Lahie D, and Nagel RL (1983) Alpha-thalassemia is related to prolonged survival in sickle cell anemia. Blood 62:286-290. Nagel RL (1984) The origin of hemoglobin S gene: clinical, genetic, and anthropological consequences. Einstein Q. J. Med. 2:53-62. Nagel RL, Fabry ME, Pagnier J , Zohoun I, Wajcman H, Baudin V, and Labie D (1985) Hematologically and genetically distinct forms of sickle cell anemia in Africa. The Senegal type and the Benin type. N. Engl. J. Med. 312~880-884. Pagnier J , Mears JG, Dunda-Belkhodja 0, SchaeferRego KE, Beldjord C, Nagel RL and Labie D (1984) Evidence for the multicentric origin of the sickle cell hemoglobin gene in Africa. Proc. Natl. Acad. Sci. USA 81r1771-1773. Powars DR, Chan L, and Schroeder W (1990) p-Genecluster haplotypes in sickle cell anemia: Clinical implications. Am. J . Ped. Hemat. Oncol. 12r367-374. Rogers ZR, Powars DR, Kinney TR, Williams WD, and Schroeder WA (1989) Nonblack patients with sickle cell disease have African p” gene cluster haplotypes. JAMA 2612991-2994, Shelton JB, Shelton JR, and Schroeder WA (1981) Further experiments in the separation of glohin chains by high performance liquid chromatography. J. Liq. Chromat. 4r1381-1392. Sutton M, Bouhassira EE, and Nagel RL (1989) Polymerase chain reaction amplification applied to the determination of p-like glohin gene cluster haplotypes. Am. J. Hematol. 32t6-9. Wainscoat JS, Thein SL, Higgs DR, Bells J I , Weatherall DJ, Al-Awamy BH, and Serjeant GR (1985)A genetic marker for elevated levels of hemoglobin F in homozygous sickle cell disease? Br. J. Haematol. 6Or261-268. Wainscoat JS, Kulozik AE,Ramsay M, Falusi AG, and Weatherall DJ (1986)A Taq 1 y-globin DNA polymorphism: An African-specific marker. Hum. Genet. 74: 90-92. Zago MA and Greene LJ (1985) An HbF enrichment procedure for the HPLC analysis of y chains. Clin. Chim. Acta 148:3946. Zag0 MA and Costa FF (1985) Hereditary hernoglobin disorders in Brazil. Trans. R. S. Trop. Med. Hyg. 79r385-388.