Duffy blood group genotypes among African-Brazilian communities of the Amazon region

INTRODUCTION

The Duffy blood group locus is characterized by three main alleles: FY*A, FY*B, and FY*O. The FY*A and FY*B alleles are distinguished by a missense mutation, which results in a single amino acid difference (G125A>Gly42Asp) (Chaudhuri et al., 1995; Iwamoto et al., 1995; Mallinson et al., 1995; Tournamille et al., 1995b) and gives the common Fy(a+b-), Fy(a-b+) and Fy(a+b+) phenotypes in European and Asian populations (Issitt and Anstee, 1998). The FY*O allele, which corresponds to the Fy(a-b-) serological phenotype (i.e., the absence of Fy antigen), is due to a T-33C point mutation (Pogo and Chaudhuri, 2000) on the FY*B gene promoter, which abolishes the erythroid gene expression by disrupting a binding site for the GATA-1 erythroid transcription factor and results in the elimination of the transcription of FY mRNA in RBCs, but not in other cell types (Tournamille et al., 1995a). The FY*O allele is at or near fixation in most sub-Saharan African populations. However, it is very rare outside Africa, and the pattern of allele frequencies at FY locus has been attributed to a positive natural selection. This hypothesis is supported by the observation that individuals homozygous for the FY*O allele are completely resistant to vivax malaria (Miller et al., 1976), since Plasmodium vivax requires the presence of Duffy antigen receptor for chemokines on the RBC surface to be able to invade cells and cause disease. In Brazil, about 99% of the malaria cases are concentrated in the Amazonian area, where socioeconomic and environmental conditions favor the proliferation of Anopheles mosquitoes, the vector of the disease. Thus, malaria represents an important public health concern in the Amazon, being responsible for economic losses and contributing to the precarious health condition of the exposed populations, which are generally considered highly susceptible to infection by Plasmodium. Therefore, it is important to investigate biological parameters that may be related to malaria, such as the Duffy blood group, in view of the marked contribution of African blacks for the formation of the present-day Amazonian populations, as well as the existence of many remaining old quilombo or mocambo communities, settlements of fugitive slaves, mainly in the State of Pará.

MATERAL AND METHODS

Study population

The study was performed with 95 unrelated subjects from four African-Brazilian communities of the Amazon region: Trombetas (N = 29), Pitimandeua (N = 18), Curiaú (N = 17), and Mazagão Velho (N = 31). A brief description of these populations follows.

Curiaú

This semi-isolated community made up of African slave descendants is located in the metropolitan region of Macapá city, capital of the State of Amapá, in the northern region of Brazil, at about 0°; 51° W. From 1770 on, this mocambo constituted the point of convergence of runaway slaves of the region, including slaves from Dutch Guyana (modern-day Surinam) and French Guyana (Muggiati, 1990).

Trombetas

The blacks of the Trombetas River live in a rural area of the municipality of Oriximiná, in the northwestern region of Pará State (1° 8' S; 55° 51' W). They are scattered along the banks of the Trombetas and Cuminá Rivers. This mocambo was created in 1820 under the leadership of the slave Atanásio, and by 1823 it had a population of about 2,000 runaway slaves, when it was destroyed by the Portuguese (Klein, 1986).

Mazagão Velho

Located on the banks of the Mutuacá River, in the State of Amapá (0° 07' 02" S; 51° 17' 04" W), the town of Mazagão Velho was founded in 1770 under orders of the Portuguese Crown to shelter 163 families of Christian Portuguese settlers of the Castle of Mazagran (nowadays El Djadidá) in Morocco from political-religious conflicts between Portuguese and Muslims. The blacks of Mazagão Velho descend from 103 African slaves who arrived in 1771 to the newly founded village accompanying the first Portuguese families.

Pitimandeua

The community of Pitimandeua is located on the right bank of the Inhangapi River in the municipal district of Inhangapi (01° 25' 45" S; 47° 54' 54" W), metropolitan Belém, capital of the State of Pará. The present-day inhabitants of Pitimandeua are descendants of African slaves who received the lands as a donation after the landlady’s death, soon after the abolition of slavery in Brazil.

Genotyping

DNA was extracted from whole blood with phenol-chloroform (Old and Higgs, 1993). Duffy blood group genotyping was performed using an allele-specific primer polymerase chain reaction (PCR) technique described by Olsson et al. (1998). Amplification was performed for each subject with sense primers corresponding to normal and GATA-1-mutated promoter sequence combined with antisense primers that discriminate the FY*A and FY*B alleles in four different combinations of primers. PCR mixtures included 100 ng genomic DNA, 0.2 µM of each primer, 100 µM of each dNTP, 1.5 µM MgCl₂, and 0.5 U AmpliTaq Gold polymerase (Perkin Elmer, USA) in the AmpliTaq Gold buffer supplied by Perkin Elmer in a reaction volume of 25 µL. Mixtures were incubated for 8 min at 95°C, followed by 10 cycles of 94°C for 1 min and 69°C for 1 min, 25 cycles of 94°C for 1 min, 64°C for 1 min and 72°C for 1 min, and a final incubation at 72°C for 10 min. PCR products were separated electrophoretically using 1.5% agarose gel containing ethidium bromide at 150 V for 30 min and visualized under UV irradiation. Allele frequencies and expected Hardy-Weinberg values were estimated by the maximum likelihood method using the PopGene program version 1 (Yeh and Boyle, 1997). Comparisons of allele frequencies between populations were based on two-way contingency tables of the same program.

RESULTS AND DISCUSSION

The genotype and allele frequency distributions recorded for Duffy blood group are presented in Table 1. Genotype distributions were consistent with the Hardy-Weinberg expectations in the four communities, and allele frequencies were similar in the four populations (P = 0.314).

FY*O was the most common allele with frequencies ranging from 56.4% in Mazagão Velho to 72.2% in the Pitimandeua, and the FY*O/FY*O genotype was found with frequencies between 32.3% in Mazagão Velho and 58.8% in Curiaú. Genotype FY*O/FY*O and FY*O mutation frequencies found in the Amazonian African-descending communities were high when compared to those observed in miscegenational Brazilian populations, but are lower than those found in sub-Saharan African populations, whose mutation is virtually fixed, as well as those described by Moulds et al. (1998) for African North Americans (92%) and by Estalote et al. (2005) for people of African ancestry in the population of Ribeirão Preto, State of São Paulo (86%). Genotype and allele distributions in the Amazonian communities are consistent with a predominantly African origin, with some degree of local differentiation and admixture with people of Caucasian ancestry and/or Amerindians, a finding that is corroborated by previous estimates of racial mixture for these communities (Trombetas and Curiaú) from uniparental genetic markers (mtDNA or Y-DNA) by Ribeiro-dos-Santos et al. (2002), autosomal hypervariable loci (Cayres-Vallinoto et al., 2003) and protein loci (Schneider et al., 1987; Guerreiro et al., 1999). On the other hand, the genotype and allelic frequencies found in Mazagão Velho, the lowest among the communities studied, can be also due to the fact that the African slaves who founded that community came from Morocco, North Africa, a region where the mutation frequencies are usually lower than those of African Sub-Sahara (Aireche and Benabadji, 1988; Fernandez-Santander et al., 1999). Incidentally, considering that FY*O allele homozygosity confers complete resistance to vivax malaria, the impact of the FY*O/FY*O genotype on the transmission and endemicity of vivax malaria deserves to be investigated in full detail, together with other human host genetic factors that confer resistance to malaria already identified at different levels of epidemic analysis, such as HbS, HbC, alpha-thalassemia, beta-thalassemia, G6PD deficiency, etc. In addition, Duffy blood group genotyping is a requisite in determining the prevalence of P. vivax infection among heterozygotes for the FY*O allele, because standard serology assigns the RBC phenotype Fy(a+b-) to individuals with genotypes FY*A/FY*A and FY*A/FY*O, and phenotype Fy(a-b+) to those with genotypes FY*B/FY*B and FY*B/FY*O, despite the 2-fold difference in FY antigen expression between homozygotes and heterozygotes for wild-type promoter. Populations of the Amazonian region are generally considered as showing a high susceptibility level to malaria and variable levels of acquired immunity that do not have a protective effect (PNCM, 2003). Thus, it is important to investigate the potential relationship between the human host genetic polymorphisms and the clinical severity of malaria, as well as the prevalence and the profile of asymptomatic malaria in the Amazon region in an attempt to investigate the contribution of host biological factors to the non-homogeneous prevalence of the disease in the region expressed by different risk levels for contracting malaria (measured by the Annual Parasitic Incidence). This may elucidate how the differences in ethnic composition of the Amazonian populations contribute to this heterogeneity.

ACKNOWLEDGMENTS

Research supported by the National Council for Scientific and Technological Development (CNPq) and the Federal University of Pará, Brazil.

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