Fetal RHD genotyping by analysis of maternal plasma in a mixed population.

BACKGROUND: Maternal plasma analysis for the determination of the fetal RHD status is an exciting tool for the management of RhD-negative pregnant women, specially sensitized women. We assessed the accuracy of fetal RHD genotyping by analysis of maternal plasma in a multi-ethnic population. METHODS: We analyzed plasma samples from 88 RhD-negative pregnant women between 11 and 39 weeks of gestation, median age of 28 years old to determine the fetal RHD genotype. This population was from Southeastern Brazil with high mixed ethnic background. Fourteen patients (16%) had anti-D alloantibody. We used Taqman primers and probes to detect by real-time PCR, exons 4, 5, and 10 of RHD. As internal controls we used primers/probes sets to SRY and CCR5. Peripheral or umbilical cord bloods from respective neonates were collected during delivery and hemagglutination was performed. RESULTS: Fifty-eight samples (66%) were genotyped as RHD+, 27 samples (31%) showed complete absence of RHD and 3 samples (3 %) presented a D variant (RHDψ). All the results agreed with the neonatal typing, including the three fetuses with the RHDψ, phenotyped as RhD-negative. Thus, the accuracy of the fetal RHD genotyping in this mixed population was 100%. The earliest pregnancy in which fetal RHD was detected was 11 weeks. CONCLUSION: Our findings indicate that the accuracy of RHD gene using three regions (exons 4, 5, and 10) can be sufficient for clinical application in a multi-ethnic population. This knowledge helped us on the development of a feasible protocol for fetal RHD genotyping on DNA from maternal plasma for our population.

Population genetics of GYPB and association study between GYPB*S/s polymorphism and susceptibility to P. falciparum infection in the Brazilian Amazon.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.