Kathrin Schuldt

Genome-wide association study indicates two novel resistance loci for severe malaria

Malaria causes approximately one million fatalities per year, mostly among African children. Although highlighted by the strong protective effect of the sickle-cell trait, the full impact of human genetics on resistance to the disease remains largely unexplored. Genome-wide association (GWA) studies are designed to unravel relevant genetic variants comprehensively; however, in malaria, as in other infectious diseases, these studies have been only partly successful. Here we identify two previously unknown loci associated with severe falciparum malaria in patients and controls from Ghana, West Africa. We applied the GWA approach to the diverse clinical syndromes of severe falciparum malaria, thereby targeting human genetic variants influencing any step in the complex pathogenesis of the disease. One of the loci was identified on chromosome 1q32 within the ATP2B4 gene, which encodes the main calcium pump of erythrocytes, the host cells of the pathogenic stage of malaria parasites. The second was indicated by an intergenic single nucleotide polymorphism on chromosome 16q22.2, possibly linked to a neighbouring gene encoding the tight-junction protein MARVELD3. The protein is expressed on endothelial cells and might therefore have a role in microvascular damage caused by endothelial adherence of parasitized erythrocytes. We also confirmed previous reports on protective effects of the sickle-cell trait and blood group O. Our findings underline the potential of the GWA approach to provide candidates for the development of control measures against infectious diseases in humans.

Imputation-Based Meta-Analysis of Severe Malaria in Three African Populations

Combining data from genome-wide association studies (GWAS) conducted at different locations, using genotype imputation and fixed-effects meta-analysis, has been a powerful approach for dissecting complex disease genetics in populations of European ancestry. Here we investigate the feasibility of applying the same approach in Africa, where genetic diversity, both within and between populations, is far more extensive. We analyse genome-wide data from approximately 5,000 individuals with severe malaria and 7,000 population controls from three different locations in Africa. Our results show that the standard approach is well powered to detect known malaria susceptibility loci when sample sizes are large, and that modern methods for association analysis can control the potential confounding effects of population structure. We show that pattern of association around the haemoglobin S allele differs substantially across populations due to differences in haplotype structure. Motivated by these observations we consider new approaches to association analysis that might prove valuable for multicentre GWAS in Africa: we relax the assumptions of SNP–based fixed effect analysis; we apply Bayesian approaches to allow for heterogeneity in the effect of an allele on risk across studies; and we introduce a region-based test to allow for heterogeneity in the location of causal alleles.

FCGR2A functional genetic variant associated with susceptibility to severe malarial anaemia in Ghanaian children

Background Severe malarial anaemia is a major cause of mortality from malaria. Although of enormous relevance, its pathogenesis is largely unknown. Interestingly, the extent of anaemia greatly exceeds the loss of erythrocytes due to direct destruction by the pathogen Plasmodium falciparum. Immune response against the parasite is partially mediated through the Fc receptor for immunoglobulin (Ig) G IIa (FcγRIIa, CD32). The presence of an arginine instead of a histidine residue at amino acid position 131 (H131R) in the extracellular domain of FcγRIIa reduces the affinity of the receptor for IgG2 and IgG3 isotypes but increases the binding activity for C reactive protein (CRP). Methods In Ghana, West Africa, 2504 children with severe malaria and 2027 matched healthy controls were studied for the FcγRIIaH131R polymorphism in order to ascertain its influence on major manifestations of the disease. The study group included patients with partly overlapping symptoms of severe malaria, among them 1591 cases with severe anaemia, 562 cases with cerebral malaria, and 497 cases with other malaria complications. Results Analyses of the genotype distributions indicated that, under a recessive model, FcγRIIa131RR was positively associated with severe malaria collectively (OR 1.20, 95% CI 1.05 to 1.38; p=0.007, pcorrected=0.021) and, after stratification for phenotypes, with severe anaemia (OR 1.33, 95% CI 1.13 to 1.57; p=0.001, pcorrected=0.009), but not with cerebral malaria (OR 1.04, 95% CI 0.82 to 1.33; p=0.733) or other malaria complications (OR 1.03, 95% CI 0.78 to 1.37; p=0.827). No association was found with levels of parasitaemia. Conclusion The positive association with a CRP binding variant of FcγRIIa supports evidence for a role of CRP mediated defence mechanisms in the pathogenesis of severe malarial anaemia.

Evidence of promiscuous endothelial binding by Plasmodium falciparum-infected erythrocytes

The adhesion of infected red blood cells (iRBCs) to human endothelium is considered a key event in the pathogenesis of cerebral malaria and other life‐threatening complications caused by the most prevalent malaria parasite Plasmodium falciparum. In the past 30 years, 14 endothelial receptors for iRBCs have been identified. Exposing 10 additional surface proteins of endothelial cells to a mixture of P. falciparum isolates from three Ghanaian malaria patients, we identified seven new iRBC receptors, all expressed in brain vessels. This finding strongly suggests that endothelial binding of P. falciparum iRBCs is promiscuous and may use a combination of endothelial surface moieties.

Distribution and genetic structure of Aedes japonicus japonicus populations (Diptera: Culicidae) in Germany

In recent years, the number of imported cases of arthropod-borne diseases in Europe, such as dengue fever, has increased steadily, as did the emergence and distribution of invasive insect vectors. Consequently, the risk of disease spreading into previously unaffected regions through invasive mosquitoes is also increasing. One example of an invasive mosquito is Aedes japonicus japonicus (A. j. japonicus), which spread from its original habitat in Japan to North America and Europe. This species has been shown to act as a vector for Japanese encephalitis and West Nile viruses. In Europe, A. j. japonicus has been detected in Switzerland, Belgium, Slovenia, and Germany, where it has become a resident species. Here, we describe the recent spread and genetic structure of A. j. japonicus populations in Germany. By monitoring the species in Baden-Württemberg in 2011 and 2012, we observed a considerable enlargement of the infested area from 54 municipalities in 2011 to 124 municipalities in 2012. To elucidate the colonization of Europe by A. j. japonicus, seven microsatellite loci were studied in 106 individuals sampled in Germany and Switzerland in 2012. The same markers were genotyped in 31 North American and 26 Japanese specimens. Population genetic analyses indicated that A. j. japonicus in Baden-Württemberg and North Rhine-Westphalia represented two genetically distinct populations with FST—values of 0.073–0.152, suggesting that they originated from two independent introduction events in the past. These results are of particular interest in light of vectorial variability for the transmission of viruses and other pathogens in Europe.

A −436C>A Polymorphism in the Human FAS Gene Promoter Associated with Severe Childhood Malaria

Human genetics and immune responses are considered to critically influence the outcome of malaria infections including life-threatening syndromes caused by Plasmodium falciparum. An important role in immune regulation is assigned to the apoptosis-signaling cell surface receptor CD95 (Fas, APO-1), encoded by the gene FAS. Here, a candidate-gene association study including variant discovery at the FAS gene locus was carried out in a case-control group comprising 1,195 pediatric cases of severe falciparum malaria and 769 unaffected controls from a region highly endemic for malaria in Ghana, West Africa. We found the A allele of c.−436C>A (rs9658676) located in the promoter region of FAS to be significantly associated with protection from severe childhood malaria (odds ratio 0.71, 95% confidence interval 0.58–0.88, pempirical = 0.02) and confirmed this finding in a replication group of 1,412 additional severe malaria cases and 2,659 community controls from the same geographic area. The combined analysis resulted in an odds ratio of 0.71 (95% confidence interval 0.62–0.80, p = 1.8×10−7, n = 6035). The association applied to c.−436AA homozygotes (odds ratio 0.47, 95% confidence interval 0.36–0.60) and to a lesser extent to c.−436AC heterozygotes (odds ratio 0.73, 95% confidence interval 0.63–0.84), and also to all phenotypic subgroups studied, including severe malaria anemia, cerebral malaria, and other malaria complications. Quantitative FACS analyses assessing CD95 surface expression of peripheral blood mononuclear cells of naïve donors showed a significantly higher proportion of CD69+CD95+ cells among persons homozygous for the protective A allele compared to AC heterozygotes and CC homozygotes, indicating a functional role of the associated CD95 variant, possibly in supporting lymphocyte apoptosis.

Endothelial Protein C Receptor Gene Variants Not Associated with Severe Malaria in Ghanaian Children

Background Two recent reports have identified the Endothelial Protein C Receptor (EPCR) as a key molecule implicated in severe malaria pathology. First, it was shown that EPCR in the human microvasculature mediates sequestration of Plasmodium falciparum-infected erythrocytes. Second, microvascular thrombosis, one of the major processes causing cerebral malaria, was linked to a reduction in EPCR expression in cerebral endothelial layers. It was speculated that genetic variation affecting EPCR functionality could influence susceptibility to severe malaria phenotypes, rendering PROCR, the gene encoding EPCR, a promising candidate for an association study. Methods Here, we performed an association study including high-resolution variant discovery of rare and frequent genetic variants in the PROCR gene. The study group, which previously has proven to be a valuable tool for studying the genetics of malaria, comprised 1,905 severe malaria cases aged 1–156 months and 1,866 apparently healthy children aged 2–161 months from the Ashanti Region in Ghana, West Africa, where malaria is highly endemic. Association of genetic variation with severe malaria phenotypes was examined on the basis of single variants, reconstructed haplotypes, and rare variant analyses. Results A total of 41 genetic variants were detected in regulatory and coding regions of PROCR, 17 of which were previously unknown genetic variants. In association tests, none of the single variants, haplotypes or rare variants showed evidence for an association with severe malaria, cerebral malaria, or severe malaria anemia. Conclusion Here we present the first analysis of genetic variation in the PROCR gene in the context of severe malaria in African subjects and show that genetic variation in the PROCR gene in our study population does not influence susceptibility to major severe malaria phenotypes.

Overexpression of Differentially Expressed Genes Identified in Non-pathogenic and Pathogenic Entamoeba histolytica Clones Allow Identification of New Pathogenicity Factors Involved in Amoebic Liver Abscess Formation

We here compared pathogenic (p) and non-pathogenic (np) isolates of Entamoeba histolytica to identify molecules involved in the ability of this parasite to induce amoebic liver abscess (ALA)-like lesions in two rodent models for the disease. We performed a comprehensive analysis of 12 clones (A1–A12) derived from a non-pathogenic isolate HM-1:IMSS-A and 12 clones (B1–B12) derived from a pathogenic isolate HM-1:IMSS-B. “Non-pathogenicity” included the induction of small and quickly resolved lesions while “pathogenicity” comprised larger abscess development that overstayed day 7 post infection. All A-clones were designated as non-pathogenic, whereas 4 out of 12 B-clones lost their ability to induce ALAs in gerbils. No correlation between ALA formation and cysteine peptidase (CP) activity, haemolytic activity, erythrophagocytosis, motility or cytopathic activity was found. To identify the molecular framework underlying different pathogenic phenotypes, three clones were selected for in-depth transcriptome analyses. Comparison of a non-pathogenic clone A1np with pathogenic clone B2p revealed 76 differentially expressed genes, whereas comparison of a non-pathogenic clone B8np with B2p revealed only 19 differentially expressed genes. Only six genes were found to be similarly regulated in the two non-pathogenic clones A1np and B8np in comparison with the pathogenic clone B2p. Based on these analyses, we chose 20 candidate genes and evaluated their roles in ALA formation using the respective gene-overexpressing transfectants. We conclude that different mechanisms lead to loss of pathogenicity. In total, we identified eight proteins, comprising a metallopeptidase, C2 domain proteins, alcohol dehydrogenases and hypothetical proteins, that affect the pathogenicity of E. histolytica.

Lack of Association of CD55 Receptor Genetic Variants and Severe Malaria in Ghanaian Children

In a recent report, the cellular receptor CD55 was identified as a molecule essential for the invasion of human erythrocytes by Plasmodium falciparum, the causal agent of the most severe form of malaria. As this invasion process represents a critical step during infection with the parasite, it was hypothesized that genetic variants in the gene could affect severe malaria (SM) susceptibility. We performed high-resolution variant discovery of rare and common genetic variants in the human CD55 gene. Association testing of these variants in over 1700 SM cases and unaffected control individuals from the malaria-endemic Ashanti Region in Ghana, West Africa, were performed on the basis of single variants, combined rare variant analyses, and reconstructed haplotypes. A total of 26 genetic variants were detected in coding and regulatory regions of CD55. Five variants were previously unknown. None of the single variants, rare variants, or haplotypes showed evidence for association with SM or P. falciparum density. Here, we present the first comprehensive analysis of variation in the CD55 gene in the context of SM and show that genetic variants present in a Ghanaian study group appear not to influence susceptibility to the disease.

Disentangling malaria anaemia.

Our results suggest that there are at least two different underlying mechanisms of malaria anaemia, uncomplicated anaemia characterized by a slow and continuous development towards a deficit of red blood cells, and anaemia with complications characterized by a rapid and substantial loss of red blood cells. The uncomplicated form appears to be caused by chronic systemic inflammatory processes rather than malaria episodes or parasite densities, similar to what is found in other forms of anaemia of chronic disease (ACD).