CR1 Knops blood group alleles are not associated with severe malaria in the Gambia.

The Knops blood group antigen erythrocyte polymorphisms have been associated with reduced falciparum malaria-based in vitro rosette formation (putative malaria virulence factor). Having previously identified single-nucleotide polymorphisms (SNPs) in the human complement receptor 1 (CR1/CD35) gene underlying the Knops antithetical antigens Sl1/Sl2 and McC(a)/McC(b), we have now performed genotype comparisons to test associations between these two molecular variants and severe malaria in West African children living in the Gambia. While SNPs associated with Sl:2 and McC(b+) were equally distributed among malaria-infected children with severe malaria and control children not infected with malaria parasites, high allele frequencies for Sl 2 (0.800, 1,365/1,706) and McC(b) (0.385, 658/1706) were observed. Further, when compared to the Sl 1/McC(a) allele observed in all populations, the African Sl 2/McC(b) allele appears to have evolved as a result of positive selection (modified Nei-Gojobori test Ka-Ks/s.e.=1.77, P-value <0.05). Given the role of CR1 in host defense, our findings suggest that Sl 2 and McC(b) have arisen to confer a selective advantage against infectious disease that, in view of these case-control study data, was not solely Plasmodium falciparum malaria. Factors underlying the lack of association between Sl 2 and McC(b) with severe malaria may involve variation in CR1 expression levels.

Evolution of a unique Plasmodium falciparum chloroquine-resistance phenotype in association with pfcrt polymorphism in Papua New Guinea and South America.

The mechanistic basis for chloroquine resistance (CQR) in Plasmodium falciparum recently has been linked to the polymorphic gene pfcrt. Alleles associated with CQR in natural parasite isolates harbor threonine (T), as opposed to lysine (K) at amino acid 76. P. falciparum CQR strains of African and Southeast Asian origin carry pfcrt alleles encoding an amino acid haplotype of CVIET (residues 72-76), whereas most South American CQR strains studied carry an allele encoding an SVMNT haplotype; chloroquine-sensitive strains from malarious regions around the world carry a CVMNK haplotype. Upon investigating the origin of pfcrt alleles in Papua New Guinean (PNG) P. falciparum we found either the chloroquine-sensitive-associated CVMNK or CQR-associated SVMNT haplotypes previously seen in Brazilian isolates. Remarkably we did not find the CVIET haplotype observed in CQR strains from Southeast Asian regions more proximal to PNG. Further we found a previously undescribed CQR phenotype to be associated with the SVMNT haplotype from PNG and South America. This CQR phenotype is significantly less responsive to verapamil chemosensitization compared with the effect associated with the CVIET haplotype. Consistent with this, we observed that verapamil treatment of P. falciparum isolates carrying pfcrt SVMNT is associated with an attenuated increase in digestive vacuole pH relative to CVIET pfcrt-carrying isolates. These data suggest a key role for pH-dependent changes in hematin receptor concentration in the P. falciparum CQR mechanism. Our findings also suggest that P. falciparum CQR has arisen through multiple evolutionary pathways associated with pfcrt K76T.

Molecular identification of Knops blood group polymorphisms found in long homologous region D of complement receptor 1.

Complement receptor 1 (CR1) has been implicated in rosetting of uninfected red blood cells to Plasmodium falciparum-infected cells, and rosette formation is associated with severe malaria. The Knops blood group (KN) is located on CR1 and some of these antigens, ie, McCoy (McC) and Swain-Langley (Sl(a)), show marked frequency differences between Caucasians and Africans. Thus, defining the molecular basis of these antigens may provide new insight into the mechanisms of P falciparum malaria. Monoclonal antibody epitope mapping and serologic inhibition studies using CR1 deletion constructs localized McC and Sl(a) to long homologous repeat D of CR1. Direct DNA sequencing of selected donors identified several single nucleotide polymorphisms in exon 29 coding for complement control protein modules 24 and 25. Two of these appeared to be blood group specific: McC associated with K1590E and Sl(a) with R1601G. These associations were confirmed by inhibition studies using allele-specific mutants. A sequence-specific oligonucleotide probe hybridization assay was developed to genotype several African populations and perform family inheritance studies. Concordance between the 1590 mutation and McC was 94%; that between Sl(a) and 1601 was 88%. All but 2 samples exhibiting discrepancies between the genotype and phenotype were found to be due to low red cell CR1 copy numbers, low or absent expression of some alleles, or heterozygosity combined with low normal levels of CR1. These data further explain the variability observed in previous serologic studies of CR1 and show that DNA and protein-based genetic studies will be needed to clarify the role of the KN antigens in malaria.

Emergence of FY*A(null) in a Plasmodium vivax-endemic region of Papua New Guinea.

In Papua New Guinea (PNG), numerous blood group polymorphisms and hemoglobinopathies characterize the human population. Human genetic polymorphisms of this nature are common in malarious regions, and all four human malaria parasites are holoendemic below 1500 meters in PNG. At this elevation, a prominent condition characterizing Melanesians is alpha(+)-thalassemia. Interestingly, recent epidemiological surveys have demonstrated that alpha(+)-thalassemia is associated with increased susceptibility to uncomplicated malaria among young children. It is further proposed that alpha(+)-thalassemia may facilitate so-called "benign" Plasmodium vivax infection to act later in life as a "natural vaccine" against severe Plasmodium falciparum malaria. Here, in a P. vivax-endemic region of PNG where the resident Abelam-speaking population is characterized by a frequency of alpha(+)-thalassemia >/=0.98, we have discovered the mutation responsible for erythrocyte Duffy antigen-negativity (Fy[a-b-]) on the FY*A allele. In this study population there were 23 heterozygous and no homozygous individuals bearing this new allele (allele frequency, 23/1062 = 0.022). Flow cytometric analysis illustrated a 2-fold difference in erythroid-specific Fy-antigen expression between heterozygous (FY*A/FY*A(null)) and homozygous (FY*A/FY*A) individuals, suggesting a gene-dosage effect. In further comparisons, we observed a higher prevalence of P. vivax infection in FY*A/FY*A (83/508 = 0.163) compared with FY*A/FY*A(null) (2/23 = 0.087) individuals (odds ratio = 2.05, 95% confidence interval = 0.47-8.91). Emergence of FY*A(null) in this population suggests that P. vivax is involved in selection of this erythroid polymorphism. This mutation would ultimately compromise alpha(+)-thalassemia/P. vivax-mediated protection against severe P. falciparum malaria.