Positive replication and linkage disequilibrium mapping of the chromosome 21q22.1 malaria susceptibility locus.

Four cytokine receptor genes are located on Chr21q22.11, encoding the alpha and beta subunits of the interferon-alpha receptor (IFNAR1 and IFNAR2), the beta subunit of the interleukin 10 receptor (IL10RB) and the second subunit of the interferon-gamma receptor (IFNGR2). We previously reported that two variants in IFNAR1 were associated with susceptibility to malaria in Gambians. We now present an extensive fine-scale mapping of the associated region utilizing 45 additional genetic markers obtained from public databases and by sequencing a 44 kb region in and around the IFNAR1 gene in 24 Gambian children (12 cases/12 controls). Within the IFNAR1 gene, a newly studied C --> G single-nucleotide polymorphism (IFNAR1 272354c-g) at position -576 relative to the transcription start was found to be more strongly associated with susceptibility to severe malaria. Association was observed in three populations: in Gambian (P=0.002), Kenyan (P=0.022) and Vietnamese (P=0.005) case-control studies. When all three studies were combined, using the Mantel-Haenszel test, the presence of IFNAR1 -576G was associated with a substantially elevated risk of severe malaria (N=2444, OR=1.38, 95% CI: 1.17-1.64; P=1.7 x 10(-4)). This study builds on previous work to further highlight the importance of the type-I interferon pathway in malaria susceptibility and illustrates the utility of typing SNPs within regions of high linkage disequilibrium in multiple populations to confirm initial positive associations.

Linkage and association between Plasmodium falciparum blood infection levels and chromosome 5q31-q33.

We have previously mapped a locus controlling Plasmodium falciparum blood infection levels (PFBI) to chromosome 5q31-q33. We genotyped 19 microsatellite markers on chromosome 5q31-q33 in a new sample of 44 pedigrees comprising 84 nuclear families and 292 individuals living in a P. falciparum endemic area. Using a nonparametric multipoint variance-component approach (by GENEHUNTER), we evidenced a peak of linkage close to D5S636 (P=0.0069), with a heritability of 0.46. Using a variance-component method for linkage-disequilibrium mapping of quantitative traits (by QTDT) and the Bonferroni correction for multiple testing, we further detected allelic association in the presence of linkage between blood infection levels and D5S487 (P=6 x 10(-5); P(c)=0.0011), which is located on the distal part of the peak. These results confirm the importance of chromosome 5q31-q33 in the genetic control of PFBI levels.

Interferon-alpha receptor-1 (IFNAR1) variants are associated with protection against cerebral malaria in the Gambia.

The chromosome 21q22.11 cytokine receptor cluster contains four genes that encode subunits of the receptors for the cytokines interleukin-10 and interferon-alpha, -beta and -gamma that may have a role in malaria pathogenesis. A total of 15 polymorphic markers located within these genes were initially genotyped in 190 controls and 190 severe malaria cases from The Gambia. Two interferon-alpha receptor-1 (IFNAR1) gene SNPs (17470 and L168 V) showed evidence for an association with severe malaria phenotypes and were typed in a larger series of samples comprising 538 severe malaria cases, 338 mild malaria cases and 562 controls. Both the 17470-G/G and L168V-G/G genotypes were associated with protection against severe malaria, in general, and cerebral malaria, in particular (P=0.004 and 0.003, respectively). IFNAR1 diplotypes were then constructed for these two markers using the PHASE software package. The (17470-G L168V-G/17470-G L168V-G) diplotype was found to be associated with a reduced risk of cerebral malaria and the (17470-C L168V-C/17470-G L168V-G) diplotype with an increased risk of cerebral malaria (overall 3 x 2 chi(2)=12.8, d.f.=2, P=0.002 and 3 x 2 chi(2)=15.2, d.f.=2, P=0.0005, respectively). These data suggest a role for the type I interferon pathway in resistance to cerebral malaria.

Familial correlation of immunoglobulin G subclass responses to Plasmodium falciparum antigens in Burkina Faso.

Host genes are thought to determine the immune response to malaria infection and the outcome. Cytophilic antibodies have been associated with protection, whereas noncytophilic antibodies against the same epitopes may block the protective activity of the protective ones. To assess the contribution of genetic factors to immunoglobulin G (IgG) subclass responses against conserved epitopes and Plasmodium falciparum blood-stage extracts, we analyzed the isotypic distribution of the IgG responses in 366 individuals living in two differently exposed areas in Burkina Faso. We used one-way analysis of variance and pairwise estimators to calculate sib-sib and parent-offspring correlation coefficients, respectively. Familial patterns of inheritance of IgG subclass responses to defined antigens and P. falciparum extracts appear to be similar in the two areas. We observed a sibling correlation for the IgG, IgG1, IgG2, IgG3, and IgG4 responses directed against ring-infected-erythrocyte surface antigen, merozoite surface protein 1 (MSP-1), MSP-2, and P. falciparum extract. Moreover, a parent-offspring correlation was found for several IgG subclass responses, including the IgG, IgG1, IgG2, IgG3, and IgG4 responses directed against conserved MSP-2 epitopes. Our results indicated that the IgG subclass responses against P. falciparum blood-stage antigens are partly influenced by host genetic factors. The localization and identification of these genes may have implications for immunoepidemiology and vaccine development.

High immunoglobulin G2 (IgG2) and low IgG4 levels are associated with human resistance to Plasmodium falciparum malaria.

There is accumulating evidence for a role of immunoglobulin G (IgG) in protection against malarial infection and disease. Only IgG1 and IgG3 are considered cytophilic and protective against P. falciparum, whereas IgG2 and IgG4 were thought to be neither and even to block protective mechanisms. However, no clear pattern of association between isotypes and protection has so far emerged. We analyzed the isotypic distribution of the IgG response to conserved epitopes and P. falciparum blood-stage extract in 283 malaria-exposed individuals whose occurrence of infection and malaria attack had been monitored for about 1 year. Logistic regression analyses showed that, at the end of the season of transmission, high levels of IgG2 to RESA and to MSP2 epitopes were associated with low risk of infection. Indeed, IgG2 is able to bind FcgammaRIIA in individuals possessing the H131 allele, and we showed that 70% of the study subjects had this allele. Also, high specific IgG4 levels were associated with an enhanced risk of infection and with a high risk of malaria attack. Moreover, specific IgG2 and IgG3 levels, as well as the IgG2/IgG4 and IgG3/IgG4 ratios, increased with the age of subjects, in parallel with the protection against infection and disease. IgG4 likely competes with cytophilic antibodies for antigen recognition and may therefore block cytotoxicity mediated by antibody-activated effector cells. In conclusion, these results favor a protective role of IgG3 and IgG2, which may activate effector cells through FcgammaRIIA, and provide evidence for a blocking role of IgG4 in malarial infection and disease.

[Analysis of the genetic factors controlling malarial infection in man]. / Analyse des facteurs génétiques contrôlant l'infection palustre chez l'homme.

Genetic factors have clearly been shown to play a role in controlling malarial infection in animal models. There is now also increasing evidence for the genetic control of malaria in man. We carried out a segregation analysis based on blood parasite load phenotype for a population of the town of Bobo-Dioulasso (Burkina-Faso). This analysis demonstrated a strong genetic effect. Our results were not consistent with the segregation of a major gene and thus suggest that parasite load is under the control of minor genes. The genetic effect was stronger in children than in adults. We carried out a regression analysis in children and found that there was an association between the phenotype for blood parasite load and the q31-33 region of chromosome 5. We identified a gene in this region, Pfil1 (Plasmodium falciparum infection levels 1), which accounted for almost 50% of the variance in blood parasite load and which played a fundamental role in the control of infection. The 5q31-33 region contains several genes encoding cytokines that regulate T lymphocytes. The identification of genes controlling malarial infection opens up new possibilities for preventive and treatment strategies. It should be possible in the near future to identify individuals at risk of malaria, who would derive the greatest benefit from preventive and therapeutic measures. Finally, a deeper understanding of these genes controlling protective immune responses could be of value for the development of vaccines.

Genetic dissection of Plasmodium falciparum blood infection levels and other complex traits related to human malaria infection.

There is accumulating evidence of host genetic control in malaria infection and, in humans, some genes have been associated with severe malaria. Nevertheless, other important genes controlling blood infection levels, malarial disease and immune responses are likely to be identified. In this paper, we focus on segregation and linkage analyses of blood infection levels in an urban population living in Burkina Faso. We found evidence of a complex genetic control and a linkage to chromosome 5q31-q33. The identification of genes controlling complex traits related to malaria infection should be helpful in understanding protective mechanisms and the relationship between infection, malaria attacks and severe malaria.

Human malaria: segregation analysis of blood infection levels in a suburban area and a rural area in Burkina Faso.

The genetic control of blood infection levels in human malaria remains unclear. Case control studies have not demonstrated a strong association between candidate genes and blood parasite densities as opposed to surveys that have focused on severe malaria. As an alternative approach, we used segregation analyses to determine the genetic control of blood parasitemia. We surveyed 509 residents (53 pedigrees) in a rural area and 389 residents (41 pedigrees) in an urban area during 18 months. Each family was visited 20 times and 28 times in the urban area and in the rural area; the mean number of parasitemia measurements per subject was 12.1 in the town and 14.9 in the village. The intensity of transmission of Plasmodium falciparum was 8-fold higher in the rural area than in the urban area. Using the class D regressive model for both populations, we found that blood parasite densities were correlated between sibs. We obtained strong evidence for a major effect, but we found that the transmission of this major effect was not compatible with a simple Mendelian model, suggesting a more complex mode of inheritance. Moreover, there was a strong interaction between major effect and age, suggesting that the influence of the putative major gene may be more prominent in children than in adults. Further nonparametric linkage studies, such as sib pair analysis, that focus on children would help us better understand the genetic control of blood infection levels.

Malaria in humans: Plasmodium falciparum blood infection levels are linked to chromosome 5q31-q33.

Plasmodium falciparum malaria remains a major cause of morbidity and mortality in many tropical countries, especially those in sub-Saharan Africa. Human genetic control of malaria infection is poorly understood; in particular, genes controlling P. falciparum blood infection levels remain to be identified. We recently evidenced the existence of complex genetic factors controlling blood infection levels in an urban population living in Burkina Faso. We performed, on 153 sibs from 34 families, sib-pair linkage analyses between blood infection levels and chromosome 5q31-q33, which contains numerous candidate genes encoding immunological molecules. Our results, obtained by means of the two-point Haseman-Elston (HE) method and a nonparametric (NP) approach, show linkage of parasitemia to D5S393 (P=.002) and D5S658 (P=.0004). Multipoint analyses confirmed linkage, with a peak close to D5S658 (P=.0013 and P=.0007 with the HE and NP methods, respectively). The heritability of the locus was .48, according to the two-point results, and .43, according to the multipoint results; this indicates that its variation accounted for approximately 45% of the variance of blood infection levels and that the locus plays a central role in the control of parasitemia. The identification of the gene is, therefore, of major interest in understanding the mechanisms controlling P. falciparum parasitemia.