Population Genetics of GYPB and Association Study between GYPB*S/s Polymorphism and Susceptibility to P. falciparum Infection in the Brazilian Amazon

Merozoites of Plasmodium falciparum invade through several pathways using different RBC receptors. Field isolates appear to use a greater variability of these receptors than laboratory isolates. Brazilian field isolates were shown to mostly utilize glycophorin A-independent invasion pathways via glycophorin B (GPB) and/or other receptors. The Brazilian population exhibits extensive polymorphism in blood group antigens, however, no studies have been done to relate the prevalence of the antigens that function as receptors for P. falciparum and the ability of the parasite to invade. Our study aimed to establish whether variation in the GYPB*S/s alleles influences susceptibility to infection with P. falciparum in the admixed population of Brazil.Two groups of Brazilian Amazonians from Porto Velho were studied: P. falciparum infected individuals (cases); and uninfected individuals who were born and/or have lived in the same endemic region for over ten years, were exposed to infection but have not had malaria over the study period (controls). The GPB Ss phenotype and GYPB*S/s alleles were determined by standard methods. Sixty two Ancestry Informative Markers were genotyped on each individual to estimate admixture and control its potential effect on the association between frequency of GYPB*S and malaria infection.GYPB*S is associated with host susceptibility to infection with P. falciparum; GYPB*S/GYPB*S and GYPB*S/GYPB*s were significantly more prevalent in the in the P. falciparum infected individuals than in the controls (69.87% vs. 49.75%; P<0.02). Moreover, population genetics tests applied on the GYPB exon sequencing data suggest that natural selection shaped the observed pattern of nucleotide diversity.Epidemiological and evolutionary approaches suggest an important role for the GPB receptor in RBC invasion by P. falciparum in Brazilian Amazons. Moreover, an increased susceptibility to infection by this parasite is associated with the GPB S+ variant in this population

Primers designed to amplify <i>GYPB</i> exons 2, 4, 5 and 6 by PCR.

<p>Primers designed to amplify <i>GYPB</i> exons 2, 4, 5 and 6 by PCR.</p

Demographic characteristics, ancestry estimations and <i>GYPB</i>^*<i>S/s</i> genotype frequencies in cases and controls, tests for Hardy-Weinberg equilibrium and association between <i>GYPB</i>^*<i>S/s</i> genotype frequency and infection with malaria.

<p>*SD, standard deviation.</p><p>**Association persists (<i>P</i><0.02) if age, gender and European, African or Native American ancestry are included as covariates.</p

Summary of <i>GYPB</i> diversity indexes and tests of neutrality based on re-sequencing data of a subset of cases and controls and their partitions in S and s alleles (rs7683365) of the Ss blood group antigens.

<p>*The samples of cases and controls were selected so the proportion of SS, Ss and ss genotypes observed in the total set of cases and controls was matching.</p>a<p><i>P</i><0.02,</p>b<p><i>P</i><0.01,</p>c<p><i>P</i><0.001.</p

Linkage disequilibrium among common SNPs in <i>GYPB</i>.

<p>Linkage disequilibrium among common SNPs in <i>GYPB</i> was estimated in both study groups: the controls (a) and in the cases, malaria infected individuals from Brazilian Amazon (b). Underlined SNPs are non-synonymous substitutions: rs7683365 is the SNP determining S/s antigens; rs1132783 is a Ser/Thr polymorphism (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016123#pone-0016123-t003" target="_blank">Table 3</a>).</p

<i>GYPB</i> haplotype frequencies determined on the re-sequencing panel on the basis of common SNPs (MAF>0.05).

a<p>SNP accounting for S (Thr) and s (Met) phenotypes.</p>b<p>Ser(G)/Thr(C).</p>c<p>The modal haplotype in each group is underlined.</p><p>Non-synonymous substitutions are underlined.</p

Estimation of admixture using Ancestry Informative Markers genotyping.

<p>Individual European, African and Native American ancestry were inferred from 60 ancestry informative markers in cases (magenta) and controls (yellow). Admixture was inferred by comparison with individuals from the putative parental populations: Europeans (red), African/African American (green) and Native Americans (blue). Admixture was estimated using the software Structure and average admixture over cases and controls is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016123#pone-0016123-t001" target="_blank">Table 1</a>.</p