Christa Ehmen

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.

Common variants at 11p13 are associated with susceptibility to tuberculosis

After imputation of data from the 1000 Genomes Project into a genome-wide dataset of Ghanaian individuals with tuberculosis and controls, we identified a resistance locus on chromosome 11p13 downstream of the WT1 gene (encoding Wilms tumor 1). The strongest signal was obtained at the rs2057178 SNP (P = 2.63 × 10−9). Replication in Gambian, Indonesian and Russian tuberculosis case-control study cohorts increased the significance level for the association with this SNP to P = 2.57 × 10−11.

Differing effects of HbS and HbC traits on uncomplicated falciparum malaria, anemia, and child growth.

The high prevalence of hemoglobin S (HbS) in Africa and hemoglobin C (HbC) in parts of West Africa is caused by the strong protection against severe falciparum malaria during childhood. Much less is known about the effect of HbS and especially HbC on Plasmodium falciparum infection, uncomplicated malaria, and anemia. A total of 1070 children from the Ashanti Region, Ghana, were enrolled at the age of 3 months and visited monthly until 2 years of age. The effects of the beta-globin genotype on the age-dependent incidence of malaria, levels of parasitemia, and hemoglobin as well as physical development were analyzed by population-averaged models. Infants with HbAS were protected from uncomplicated malaria (P < .005) and anemia (P < .001), had lower age-adjusted parasite densities (P < .001), and higher age-adjusted hemoglobin levels compared with children with the HbAA genotype (P = .004). In contrast, HbAC carriers had lower hemoglobin levels (P < .033) and were not protected against malaria or anemia. Notably, infants with HbAS were also significantly protected against stunting compared with carriers of HbAA or HbAC. This indicates differing mechanisms of protection against malaria of HbAS and HbAC and might help to understand why HbC is restricted to distinct areas of West Africa.

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.

Sickle cell trait (HbAS) and stunting in children below two years of age in an area of high malaria transmission.

BackgroundWhile the protective effects of sickle cell trait (HbAS) against severe malaria and the resulting survival advantage are well known, the impact on the physical development in young children remains unclear. This study was aimed to investigate the relationship between HbS carriage and stunting in children below two years of age in a cohort from the Ashanti Region, Ghana.Methods1,070 children were recruited at three months of age and followed-up for 21 months with anthropometric measurements performed every three months. Incidence rate ratios with 95% confidence intervals were calculated by Poisson regression to estimate the association of β-globin genotypes with the number of malaria episodes. Odds ratios (OR) were calculated for the association between the occurrence of β-globin genotypes and/or malaria episodes and stunting. The age-dependent between-group and within-group effects for the β-globin genotypes were assessed by population-averaged models estimated by generalized estimation equation with autoregressive correlation structure.ResultsAnalyses showed a significantly lower age-dependent risk of stunting (OR 0.56; 95% CI 0.33–0.96) in carriers of the HbAS genotype (n = 102) in comparison to those with HbAA (n = 692). This effect was restricted to children who experienced malaria episodes during the observation period suggesting that the beneficial effect of the β-globin HbS variant on the incidence of stunting is closely linked to its protection from mild malaria episodes.ConclusionThe lower risk of chronic malnutrition in early childhood, mediated by protection against mild malaria episodes, may contribute to the survival advantage of HbAS carriers in areas of high malaria transmission.

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.

The prevalence of sickle cell disease and its implication for newborn screening in Germany (Hamburg metropolitan area)

Sickle cell disease is among hereditary diseases with evidence that early diagnoses and treatment improves the clinical outcome. So far sickle cell disease has not been included in the German newborn screening program despite immigration from countries with populations at risk. To determine the birth prevalence we tested 17,018 newborns. High pressure liquid chromatography and subsequent molecular‐genetic testing were used for the detection and confirmation of hemoglobin variants. The frequency of sickle cell disease‐consistent genotypes was one in 2,385 newborns. Duffy‐blood group typing showed evidence that affected children were likely of Sub‐Saharan ancestry. An inclusion of sickle cell disease into the German newborn screening seems reasonable. Pediatr Blood Cancer 2015; 9999:XX–XX

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.

TLR1 Variant H305L Associated with Protection from Pulmonary Tuberculosis

Toll like receptors (TLR) are key elements of the innate immune response and involved in the recognition of pathogens. To test common and rare TLR variants involved in susceptibility or resistance to infection with Mycobacterium tuberculosis we screened the exons of the genes encoding TLR 1, 2, 4, and the adaptor molecule TIRAP in more than 4500 tuberculosis (TB) cases and controls from Ghana. The analysis yielded 109 variants with possible functional impact, including 101 non-synonymous variants, three stop-variants, and five indels. Association analyses yielded a significant result for the TLR1 variant rs3923647, conferring strong protection against TB (Odds ratio [OR] 0.21, CI confidence interval [CI] 0.05–0.6, Pnominal 1 x 10−3) when applying a recessive model of inheritance. Replication analyses with an additional 3370 Ghanaian cases and control samples, and with data from a recent TB study of 533 African-Americans confirmed the protective effect and resulted in a combined OR of 0.19, with a nominal P value of 2.2 x 10−5, and a corrected P value of 4.1 x 10−4. The SNP is located near the binding pocket of TLR1 and causes an amino acid exchange from histidine to leucine at position 305. The observed effect may, therefore, be attributable to structural changes in the recognition site of the TLR1 molecule, allowing to bind those mycobacterial ligands which preferentially may induce a protective immune response. This is supported by the analysis of BCG-stimulated peripheral blood mononuclear cells, showing increased induction of the proinflammatory cytokine IFN-γ in carriers of the mutant TLR1 rs3923647 TT genotype, compared to the IFN-γ levels of individuals with the AT and AA genotypes.

The Emergence of Reduced Ciprofloxacin Susceptibility in Salmonella enterica Causing Bloodstream Infections in Rural Ghana

BACKGROUND Salmonella ranks among the leading causes of bloodstream infections in sub-Saharan Africa. Multidrug resistant typhoidal and nontyphoidal Salmonella (NTS) isolates have been previously identified in this region. However, resistance to ciprofloxacin has rarely been reported in West Africa. This study aims to assess susceptibility against ciprofloxacin in Salmonella causing invasive bloodstream infections among children in rural Ghana. METHODS From May 2007 until May 2012, children attending a rural district hospital in central Ghana were eligible for recruitment. Salmonella enterica isolated from blood cultures were assessed for ciprofloxacin susceptibility by Etest (susceptible minimum inhibitory concentration [MIC] ≤ 0.06 µg/mL). The gyrA, gyrB, parC, and parE genes were sequenced to identify mutations associated with changes in susceptibility to fluoroquinolones. RESULTS Two hundred eighty-five Salmonella enterica isolates from 5211 blood cultures were most commonly identified as Salmonella enterica serovar Typhimurium (n = 129 [45%]), Salmonella enterica serovar Typhi (n = 89 [31%]), Salmonella enterica serovar Dublin (n = 20 [7%]), and Salmonella enterica serovar Enteritidis (n = 19 [7%]). All S. Typhi and S. Dublin were susceptible to ciprofloxacin. Reduced susceptibility (MIC >0.06 µg/mL) was found in 53% (10/19) of S. Enteritidis and in 2% (3/129) of S. Typhimurium isolates. Sequencing detected a single gyrB mutation (Glu466Asp) and a single gyrA mutation (Ser83Tyr) in all 3 S. Typhimurium isolates, while 9 of 10 S. Enteritidis harbored single gyrA mutations (Asp87Gly, Asp87Asn, or Asp87Tyr). No mutations were found in the parC and parE genes. CONCLUSIONS Ciprofloxacin susceptibility in invasive NTS in rural Ghana is highly dependent on serotype. Although reduced ciprofloxacin susceptibility is low in S. Typhimurium, more than half of all S. Enteritidis isolates are affected. Healthcare practitioners in Ghana should be aware of potential treatment failure in patients with invasive S. Enteritidis infections.