Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)beta(S) haplotypes were studied in 47 non-related patients with sickle-cell anemia from the state of Rio Grande do Norte, Brazil. Molecular analysis was conducted by PCR/RFLP using restriction endonucleases XmnI, HindIII, HincII and HinfI to analyze six polymorphic sites from the beta cluster. Twenty-seven patients (57.5%) were identified with genotype CAR/CAR, 9 (19.1%) CAR/BEN, 6 (12.8%) CAR/CAM, 1 (2.1%) BEN/BEN, 2 (4.3%) CAR/Atp, 1 (2.1%) BEN/Atp and 1 (2.1%) with genotype Atp/Atp. The greater frequency of Cameroon haplotypes compared to other Brazilian states suggests the existence of a peculiarity of African origin in the state of Rio Grande do Norte.343421424Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)CNPq [409766/2006-2]FAPESP [2008/57441-0

Cabral, CHK

Costa, FF

de Medeiros, TMD

de Medeiros, WRDB

Fernandes, TAAD

Kimura, EM

Rebecchi, IMM

Serafim, ESS

Sonati, MD

Genetics and Molecular Biology

Repositorio da Producao Cientifica e Intelectual da Unicamp

Determination of S haplotypes in patients with sickle-cell anemia
in the state of Rio Grande do Norte, Brazil
Cynthia Hatsue Kitayama Cabral1, Édvis Santos Soares Serafim2,
Waleska Rayane Dantas Bezerra de Medeiros1, Thales Allyrio Araújo de Medeiros Fernandes3,
Elza Miyuki Kimura4, Fernando Ferreira Costa5, Maria de Fátima Sonati4,
Ivanise Marina Moretti Rebecchi1 and Tereza Maria Dantas de Medeiros1
1Departamento de Análises Clínicas e Toxicológicas, Universidade Federal do Rio Grande do Norte,
Natal, RN, Brazil.
2Hemocentro Dalton Barbosa Cunha, Natal, RN, Brazil.
3Departamento de Ciências Biomédicas, Universidade do Estado do Rio Grande do Norte,
Mossoró, RN, Brazil.
4Departamento de Patologia Clínica, Faculdade de Ciências Médicas,
Universidade Estadual de Campinas, Campinas, SP, Brazil.
5Hemocentro, Universidade Estadual de Campinas, Campinas, SP, Brazil.
Abstract

S haplotypes were studied in 47 non-related patients with sickle-cell anemia from the state of Rio Grande do Norte,
Brazil. Molecular analysis was conducted by PCR/RFLP using restriction endonucleases XmnI, HindIII, HincII and
HinfI to analyze six polymorphic sites from the beta cluster. Twenty-seven patients (57.5%) were identified with ge-
notype CAR/CAR, 9 (19.1%) CAR/BEN, 6 (12.8%) CAR/CAM, 1 (2.1%) BEN/BEN, 2 (4.3%) CAR/Atp, 1 (2.1%)
BEN/Atp and 1 (2.1%) with genotype Atp/Atp. The greater frequency of Cameroon haplotypes compared to other
Brazilian states suggests the existence of a peculiarity of African origin in the state of Rio Grande do Norte.
Key words: haplotypes, -globin, sickle-cell anemia, Brazilian population, S hemoglobin.
Received: November 26, 2010; Accepted: May 2, 2011.
Sickle-cell anemia, which results from the homo-
zygosis of S allele [beta 6(A3) Glu > Val] of beta globin
gene, is the most common and most serious form of sickle-
cell disease (Serjeant, 1997).
-globin gene haplotype is characterized by the non-
random association of cleavage sites recognized by restric-
tion endonucleases (Steinberg et al., 1996). The main S
haplotypes regularly found in patients with sickle-cell ane-
mia, worldwide, are named according to the geographic re-
gion or ethnic group in which they were originally
identified, i.e., Senegal (SEN), Benin (BEN), Bantu or
Central African Republic (CAR), Cameroon (CAM) and
Arab-Indian (ARAB), (Powars, 1991). Other less common
haplotypes, known as atypical haplotypes (Atp), are gener-
ated by a number of genetic mechanisms (Zago et al.,
2000).
The analysis of S haplotypes, besides being an im-
portant source for anthropological studies on the ethnic
composition of a population, contributes to a better under-
standing of the variation in clinical seriousness of sickle-
cell anemia, given that the different types of haplotypes
contain a range of fetal hemoglobin concentrations that act
as inhibitors of hemoglobin S polymer formation (Flint et
al., 1998; Inati et al., 2003).
Senegal and Arab-Indian haplotypes, by producing
the highest levels of fetal hemoglobin in the blood, are asso-
ciated with less severe clinical evolution of sickle-cell ane-
mia, with a lower occurrence of organic damage. As to the
Benin and Cameroon haplotypes, the clinical picture is of
intermediate severity. The Bantu or CAR haplotype is asso-
ciated with greater clinical severity (Powars, 1991; Zago et
al., 1992; Bortolini and Salzano, 1999).
In light of the absence of studies on haplotypes in the
state of Rio Grande do Norte, the aim of the present investi-
gation was to determine S haplotypes in a group of patients
with sickle-cell anemia.
A total of 47 non-related individuals with sickle-cell
anemia (25 males and 22 females) from the state of Rio
Grande do Norte, aged between 10 months and 46 years
(mean 16.1  10.4 years), were analyzed. These patients
Genetics and Molecular Biology, 34, 3, 421-424 (2011)
Copyright © 2011, Sociedade Brasileira de Genética. Printed in Brazil
www.sbg.org.br
Send correspondence to Tereza Maria Dantas de Medeiros. De-
partamento de Análises Clínicas e Toxicológicas, Universidade
Federal do Rio Grande do Norte, Rua General Cordeiro de Farias
s/n, 1° andar, 59010-180 Natal, RN, Brazil. E-mail:
tdantas@ufrnet.br.
Short Communication
were enrolled at the Hematology Ambulatory Care Facility
of the Dalton Barbosa Cunha Hemocenter, Natal, RN, Bra-
zil and the Oncology and Hematology Center of Mossoró,
RN, Brazil. The research project was approved by the Re-
search Ethics Committee of the Federal University of Rio
Grande do Norte (CEP-UFRN, protocol number 090/07).
All the patients or their legal guardians gave written in-
formed-consent after being instructed on the aim of the
study and the procedures involved.
Samples of venous blood were collected using EDTA
as anticoagulant. The hemoglobin profile was determined
by cellulose acetate electrophoresis at alkaline pH (Dacie
and Lewis, 1995), and the presence of HbS confirmed by
means of the solubility test (Magalhães and Arashiro,
1977). HbA2 was measured by elution from cellulose ace-
tate strips following electrophoresis (Bezerra, 1984). The
level of HbA2 was used to exclude the interation S- thalas-
semia. DNA was isolated from peripheral blood leukocytes
using the illustra blood genomicPrep Mini Spin kit (GE
Healthcare, Little Chalfont, Buckinghamshire, UK). S
halotypes were investigated by PCR/RFLP. PCR was per-
formed using primers H0, H1, H2, H3, H4, H5, H6, H7, H8,
H9 and H10 (Sutton et al., 1989) in a GeneAmp 9700
thermocycler (Applied Biosystem, Foster City, CA, USA).
The analysis of polymorphic regions was by incubating
PCR products with the restriction endonucleases XmnI,
HindIII, HincII and HinfI, for 12 h at 37 °C. Amplified
products were visualized and polymorphic sites identified
using 2% agarose gel electrophoresis, followed by ethi-
dium bromide staining and photodocumentation (Sam-
brook et al., 1989). A 100 bp DNA Ladder (BioLabs) was
applied in each gel electrophoresis as a standard molecular
weight marker. Pre-determined homozygous and heterozy-
gous patterns were used for each haplotype investigated.
The samples were marked by the presence (+) or ab-
sence (-) of each of the 6 restriction sites analyzed in the
beta-globin gene cluster (XmnI 5’Go, HindIII in G and A,
HincII in 3’ and , and HinfI in 5’). According to the
combination of results, the five most common S haplo-
types were defined as follows: CAR (- + - - - -), Benin (- - - -
+ -), Senegal (+ + - + + +), Arab-Indian (+ + - + + -) and
Cameroon (- + +-++).
Of the 94 chromosomes analyzed, 71 (75.5%) were
characterized as CAR, 12 (12.8%) as Benin, 6 (6.4%) as
Cameroon and 5 (5.3%) as atypical haplotypes.
In relation to the combination of S haplotypes, 27
(57.5%) patients carried the CAR/CAR genotype, 9
(19.1%) the CAR/BEN, 6 (12.8%) the CAR/CAM, 1
(2.1%) the BEN/BEN, 2 (4.3%) the CAR/Atp, 1 (2.1%) the
BEN/Atp, and 1 (2.1%) the Atp/Atp.
The frequency of S haplotypes varies according to
origin. The African presence in Brazil began around 1550,
as a replacement for native labor. The main ports of entry
were Salvador, Recife and Rio de Janeiro. Individuals from
Nigeria, Daomé (present Day Benin) and the Ivory Coast
were sent mainly to Bahia, whereas the Bantus, captured in
the Congo, Angola and Mozambique, were sent to Per-
nambuco, Minas Gerais and Rio de Janeiro (Vicentino and
Dorigo, 2002).
Since there were no ports or slave trade, the state of
Rio Grande do Norte was not greatly influenced by African
origin in its ethnic formation of the population. Moreover,
unlike other regions, there was no economic activity requir-
ing slave labor. As a result, only a small number of slaves,
mainly from Pernambuco, were sent to this state (Cascudo,
1955).
Frequency distribution of S haplotypes in the pa-
tients included in this study and in others involving various
Brazilian states, appear in Table 1. A general predominance
of the CAR haplotype can be observed in the country (Zago
et al., 1992; Gonçalves et al., 1994; Pante de Souza et al.,
1998; Cardoso and Guerreiro, 2006; Bezerra et al., 2007;
Silva et al., 2009). The Benin haplotype is present in all re-
gions, with high frequency in Bahia (Costa et al., 1984;
Gonçalves et al., 2003; Adorno et al., 2004) and Rio de Ja-
neiro (Fleury, 2007).
The high frequency of CAR haplotype (75.5%) found
in the present study, followed by the Benin haplotype
(12.8%), was comparable to that obtained by Bezerra et al.
(2007) in Pernambuco, the main source of slaves for Rio
Grande do Norte. The frequency found for the Cameroon
haplotype (6.4%) was higher than that observed in other
states such as Bahia, and even Pernambuco (Adorno et al.,
2004, 2008; Cardoso and Guerreiro, 2006; Bezerra et al.,
2007). Statistical analysis using the proportion comparison
test showed a significant difference, except for the study
conducted by Adorno et al. (2004) (p = 0.0943). No Sene-
gal or Saudi Arabian haplotypes were identified in the
study sample.
The record of a shipment of 2,400 African slaves
from Andra, Benin, Warri (Niger Delta), Calabar and Cam-
eroon to Pernambuco in 1638, during the Dutch occupa-
tion, confirms the entry of Africans from a region with high
Cameroon haplotype frequency into northeastern Brazil
(Alencastro, 2000). After 1845, with the rise in sugarcane
production, slavery in Rio Grande do Norte increased, bol-
stered by the purchase of slaves from other states, such as
Pernambuco and Maranhão (Medeiros, 1978). Hence, a
possible explanation of the high frequency found in the
present study, albeit also attributable to the small number of
patients analyzed herein.
The present study constitutes the first attempt to in-
vestigate the frequency of S haplotypes in the population
of the state of Rio Grande do Norte. The higher frequency
of the Cameroon haplotype, compared to other Brazilian
states, suggests the existence of a peculiarity of African ori-
gin.
422 Cabral et al.
Acknowledgments
This study was supported by the Conselho Nacional
de Desenvolvimento Científico e Tecnológico (CNPq,
grant n. 409766/2006-2) and the Fundação de Amparo à
Pesquisa do Estado de São Paulo (FAPESP, grant n.
2008/57441-0).
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S haplotypes in sickle-cell anemia 423
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Associate Editor: Francisco Mauro Salzano
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.
424 Cabral et al.


Determination of beta(S) haplotypes in patients with sickle-cell anemia in the state of Rio Grande do Norte, Brazil

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Determination of beta(S) haplotypes in patients with sickle-cell anemia in the state of Rio Grande do Norte, Brazil

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