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Bantu s cluster haplotype predominates among Brazilian Blacks.

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Bantu p" Cluster Haplotype Predominates Among
Brazilian Blacks
Department of Clinical Medicine, School of Medicine, 14049 Ribeirao
Preto, Brazil
S, Haplotypes
Brazilian Blacks, Sickle cell anemia, Hemoglobin
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.,
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.
The study included 37 nonrelated patients
with homozygous sickle cell anemia who are
Received J u n e 6,1991;accepted January 2,1992
TABLE 1. Haplotype combinations and percentage of
Gy chain of HbF of 37 HbS homozygotes from Brazil
Number of patients
%I G y (5 Ifr SD)
39.5 7.0
39.2 5.2
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%,
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.
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-
TABLE 2. ps haplotype distribution for North America, the Caribbean, and Brazil compared with historical
records on the origin of slave trade to Brazil
Jamaica1 (W)
United States' (%)
Brazil2 (%)
( E ~ p e c t e d(%)
'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
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).
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.
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black, clusters, brazilian, haplotype, among, bantu, predominates
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