Malaria is a cause of iron deficiency in African children.

Malaria and iron deficiency (ID) are common and interrelated public health problems in African children. Observational data suggest that interrupting malaria transmission reduces the prevalence of ID1. To test the hypothesis that malaria might cause ID, we used sickle cell trait (HbAS, rs334 ), a genetic variant that confers specific protection against malaria2, as an instrumental variable in Mendelian randomization analyses. HbAS was associated with a 30% reduction in ID among children living in malaria-endemic countries in Africa (n = 7,453), but not among individuals living in malaria-free areas (n = 3,818). Genetically predicted malaria risk was associated with an odds ratio of 2.65 for ID per unit increase in the log incidence rate of malaria. This suggests that an intervention that halves the risk of malaria episodes would reduce the prevalence of ID in African children by 49%.

Interferon-gamma polymorphisms and risk of iron deficiency and anaemia in Gambian children.

Background: Anaemia is a major public health concern especially in African children living in malaria-endemic regions. Interferon-gamma (IFN-γ) is elevated during malaria infection and is thought to influence erythropoiesis and iron status. Genetic variants in the IFN-γ gene (IFNG) are associated with increased IFN-γ production. We investigated putative functional single nucleotide polymorphisms (SNPs) and haplotypes of IFNG in relation to nutritional iron status and anaemia in Gambian children over a malaria season. Methods: We used previously available data from Gambian family trios to determine informative SNPs and then used the Agena Bioscience MassArray platform to type five SNPs from the IFNG gene in a cohort of 780 Gambian children aged 2-6 years. We also measured haemoglobin and biomarkers of iron status and inflammation at the start and end of a malaria season. Results: We identified five IFNG haplotype-tagging SNPs ( IFNG-1616 [rs2069705], IFNG+874 [rs2430561], IFNG+2200 [rs1861493], IFNG+3234 [rs2069718] and IFNG+5612 [rs2069728]). The IFNG+2200C [rs1861493] allele was associated with reduced haemoglobin concentrations (adjusted ß -0.44 [95% CI -0.75, -0.12]; Bonferroni adjusted P = 0.03) and a trend towards iron deficiency compared to wild-type at the end of the malaria season in multivariable models adjusted for potential confounders. A haplotype uniquely identified by IFNG+2200C was similarly associated with reduced haemoglobin levels and trends towards iron deficiency, anaemia and iron deficiency anaemia at the end of the malaria season in models adjusted for age, sex, village, inflammation and malaria parasitaemia. Conclusion: We found limited statistical evidence linking IFNG polymorphisms with a risk of developing iron deficiency and anaemia in Gambian children. More definitive studies are needed to investigate the effects of genetically influenced IFN-γ levels on the risk of iron deficiency and anaemia in children living in malaria-endemic areas.

Massive introgression drives species radiation at the range limit of Anopheles gambiae.

Impacts of introgressive hybridisation may range from genomic erosion and species collapse to rapid adaptation and speciation but opportunities to study these dynamics are rare. We investigated the extent, causes and consequences of a hybrid zone between Anopheles coluzzii and Anopheles gambiae in Guinea-Bissau, where high hybridisation rates appear to be stable at least since the 1990s. Anopheles gambiae was genetically partitioned into inland and coastal subpopulations, separated by a central region dominated by A. coluzzii. Surprisingly, whole genome sequencing revealed that the coastal region harbours a hybrid form characterised by an A. gambiae-like sex chromosome and massive introgression of A. coluzzii autosomal alleles. Local selection on chromosomal inversions may play a role in this process, suggesting potential for spatiotemporal stability of the coastal hybrid form and providing resilience against introgression of medically-important loci and traits, found to be more prevalent in inland A. gambiae.

Drug resistance. Population transcriptomics of human malaria parasites reveals the mechanism of artemisinin resistance.

Artemisinin resistance in Plasmodium falciparum threatens global efforts to control and eliminate malaria. Polymorphisms in the kelch domain-carrying protein K13 are associated with artemisinin resistance, but the underlying molecular mechanisms are unknown. We analyzed the in vivo transcriptomes of 1043 P. falciparum isolates from patients with acute malaria and found that artemisinin resistance is associated with increased expression of unfolded protein response (UPR) pathways involving the major PROSC and TRiC chaperone complexes. Artemisinin-resistant parasites also exhibit decelerated progression through the first part of the asexual intraerythrocytic development cycle. These findings suggest that artemisinin-resistant parasites remain in a state of decelerated development at the young ring stage, whereas their up-regulated UPR pathways mitigate protein damage caused by artemisinin. The expression profiles of UPR-related genes also associate with the geographical origin of parasite isolates, further suggesting their role in emerging artemisinin resistance in the Greater Mekong Subregion.

Large-scale association analysis of TNF/LTA gene region polymorphisms in type 2 diabetes.

BACKGROUND: The TNF/LTA locus has been a long-standing T2D candidate gene. Several studies have examined association of TNF/LTA SNPs with T2D but the majority have been small-scale and produced no convincing evidence of association. The purpose of this study is to examine T2D association of tag SNPs in the TNF/LTA region capturing the majority of common variation in a large-scale sample set of UK/Irish origin. METHODS: This study comprised a case-control (1520 cases and 2570 control samples) and a family-based component (423 parent-offspring trios). Eleven tag SNPs (rs928815, rs909253, rs746868, rs1041981 (T60N), rs1800750, rs1800629 (G-308A), rs361525 (G-238A), rs3093662, rs3093664, rs3093665, and rs3093668) were selected across the TNF/LTA locus and genotyped using a fluorescence-based competitive allele specific assay. Quality control of the obtained genotypes was performed prior to single- and multi-point association analyses under the additive model. RESULTS: We did not find any consistent SNP associations with T2D in the case-control or family-based datasets. CONCLUSIONS: The present study, designed to analyse a set of tag SNPs specifically selected to capture the majority of common variation in the TNF/LTA gene region, found no robust evidence for association with T2D. To investigate the presence of smaller effects of TNF/LTA gene variation with T2D, a large-scale meta-analysis will be required.

Further evidence supporting a role for gs signal transduction in severe malaria pathogenesis.

With the functional demonstration of a role in erythrocyte invasion by Plasmodium falciparum parasites, implications in the aetiology of common conditions that prevail in individuals of African origin, and a wealth of pharmacological knowledge, the stimulatory G protein (Gs) signal transduction pathway presents an exciting target for anti-malarial drug intervention. Having previously demonstrated a role for the G-alpha-s gene, GNAS, in severe malaria disease, we sought to identify other important components of the Gs pathway. Using meta-analysis across case-control and family trio (affected child and parental controls) studies of severe malaria from The Gambia and Malawi, we sought evidence of association in six Gs pathway candidate genes: adenosine receptor 2A (ADORA2A) and 2B (ADORA2B), beta-adrenergic receptor kinase 1 (ADRBK1), adenylyl cyclase 9 (ADCY9), G protein beta subunit 3 (GNB3), and regulator of G protein signalling 2 (RGS2). Our study amassed a total of 2278 cases and 2364 controls. Allele-based models of association were investigated in all genes, and genotype and haplotype-based models were investigated where significant allelic associations were identified. Although no significant associations were observed in the other genes, several were identified in ADORA2A. The most significant association was observed at the rs9624472 locus, where the G allele (approximately 20% frequency) appeared to confer enhanced risk to severe malaria [OR = 1.22 (1.09-1.37); P = 0.001]. Further investigation of the ADORA2A gene region is required to validate the associations identified here, and to identify and functionally characterize the responsible causal variant(s). Our results provide further evidence supporting a role of the Gs signal transduction pathway in the regulation of severe malaria, and request further exploration of this pathway in future studies.

Tumor necrosis factor and lymphotoxin-alpha polymorphisms and severe malaria in African populations.

The tumor necrosis factor gene (TNF) and lymphotoxin-alpha gene (LTA) have long attracted attention as candidate genes for susceptibility traits for malaria, and several of their polymorphisms have been found to be associated with severe malaria (SM) phenotypes. In a large study involving >10,000 individuals and encompassing 3 African populations, we found evidence to support the reported associations between the TNF -238 polymorphism and SM in The Gambia. However, no TNF/LTA polymorphisms were found to be associated with SM in cohorts in Kenya and Malawi. It has been suggested that the causal polymorphisms regulating the TNF and LTA responses may be located some distance from the genes. Therefore, more-detailed mapping of variants across TNF/LTA genes and their flanking regions in the Gambian and allied populations may need to be undertaken to find any causal polymorphisms.

A genetic association study in the Gambia using tagging polymorphisms in the major histocompatibility complex class III region implicates a HLA-B associated transcript 2 polymorphism in severe malaria susceptibility.

The tumour necrosis factor (TNF) gene and other genes flanking it in the major histocompatibility complex (MHC) class III region are potentially important mediators of both immunity and pathogenesis of malaria. We investigated the association of severe malaria with 11 haplotype tagging-polymorphisms for 11 MHC class III candidate genes, including TNF, lymphotoxin alpha (LTA), allograft inflammatory factor 1 (AIF1), and HLA-B associated transcript 2 (BAT2). An analysis of 2,162 case-controls demonstrated the first evidence of association between a BAT2 polymorphism (rs1046089) and severe malaria.

Tumor necrosis factor SNP haplotypes are associated with iron deficiency anemia in West African children.

Plasma levels of tumor necrosis factor-alpha (TNF-alpha) are significantly raised in malaria infection and TNF-alpha is thought to inhibit intestinal iron absorption and macrophage iron release. This study investigated putative functional single nucleotide polymorphisms (SNPs) and haplotypes across the major histocompatibility complex (MHC) class III region, including TNF and its immediate neighbors nuclear factor of kappa light polypeptide gene enhancer in B cells (lkappaBL), inhibitor-like 1 and lymphotoxin alpha (LTA), in relation to nutritional iron status and anemia, in a cohort of 780 children across a malaria season. The prevalence of iron deficiency anemia (IDA) increased over the malaria season (P < .001). The TNF(-308) AA genotype was associated with an increased risk of iron deficiency (adjusted OR 8.1; P = .001) and IDA (adjusted OR 5.1; P = .01) at the end of the malaria season. No genotypes were associated with IDA before the malaria season. Thus, TNF appears to be a risk factor for iron deficiency and IDA in children in a malaria-endemic environment and this is likely to be due to a TNF-alpha-induced block in iron absorption.

Association scan of 14,500 nonsynonymous SNPs in four diseases identifies autoimmunity variants.

We have genotyped 14,436 nonsynonymous SNPs (nsSNPs) and 897 major histocompatibility complex (MHC) tag SNPs from 1,000 independent cases of ankylosing spondylitis (AS), autoimmune thyroid disease (AITD), multiple sclerosis (MS) and breast cancer (BC). Comparing these data against a common control dataset derived from 1,500 randomly selected healthy British individuals, we report initial association and independent replication in a North American sample of two new loci related to ankylosing spondylitis, ARTS1 and IL23R, and confirmation of the previously reported association of AITD with TSHR and FCRL3. These findings, enabled in part by increased statistical power resulting from the expansion of the control reference group to include individuals from the other disease groups, highlight notable new possibilities for autoimmune regulation and suggest that IL23R may be a common susceptibility factor for the major 'seronegative' diseases.

Context-specific functional effects of IFNGR1 promoter polymorphism.

We report evidence of a polymorphism in the promoter region of IFNGR1 (encoding interferon-gamma receptor 1) that has opposite functional effects in different cellular contexts. It is a deletion/insertion polymorphism that is found in Africans but not Europeans or Asians, and has been associated with resistance to severe malaria. We find that the IFNGR1-470del allele acts to suppress binding of nuclear proteins to the IFNGR1 promoter region in a manner that is specific for cell type. In B-lymphocytes, the IFNGR1-470del allele suppresses the binding of a approximately 35 kDa nuclear protein and acts to increase reporter gene expression. In epithelial cells, the same allele acts to decrease gene expression and suppresses the binding of approximately 90 kDa STAT-1 and STAT-2 proteins. In T-lymphocytes, this allele causes only subtle differences in nuclear protein binding and has no significant effect on gene expression. These findings suggest a mechanism by which a single genetic variant may cause a broad range of phenotypic consequences.

Allele-specific repression of lymphotoxin-alpha by activated B cell factor-1.

Genetic variation at the human LTA locus, encoding lymphotoxin-alpha, is associated with susceptibility to myocardial infarction, asthma and other diseases. By detailed haplotypic analysis of the locus, we identified a single-nucleotide polymorphism (SNP) at LTA+80 as a main predictor of LTA protein production by human B cells. We found that activated B-cell factor-1 (ABF-1) binds to this site in vitro and suppresses reporter gene expression, but only in the presence of the LTA+80A allele. Using haplotype-specific chromatin immunoprecipitation, we confirmed that ABF-1 is preferentially recruited to the low-producer allele in vivo. These findings provide a molecular model of how LTA expression may be genetically regulated by allele-specific recruitment of the transcriptional repressor ABF-1.

Haplotypic analysis of the TNF locus by association efficiency and entropy.

BACKGROUND: To understand the causal basis of TNF associations with disease, it is necessary to understand the haplotypic structure of this locus. We genotyped 12 single-nucleotide polymorphisms (SNPs) distributed over 4.3 kilobases in 296 healthy, unrelated Gambian and Malawian adults. We generated 592 high-quality haplotypes by integrating family- and population-based reconstruction methods. RESULTS: We found 32 different haplotypes, of which 13 were shared between the two populations. Both populations were haplotypically diverse (gene diversity = 0.80, Gambia; 0.85, Malawi) and significantly differentiated (p < 10-5 by exact test). More than a quarter of marker pairs showed evidence of intragenic recombination (29% Gambia; 27% Malawi). We applied two new methods of analyzing haplotypic data: association efficiency analysis (AEA), which describes the ability of each SNP to detect every other SNP in a case-control scenario; and the entropy maximization method (EMM), which selects the subset of SNPs that most effectively dissects the underlying haplotypic structure. AEA revealed that many SNPs in TNF are poor markers of each other. The EMM showed that 8 of 12 SNPs (Gambia) and 7 of 12 SNPs (Malawi) are required to describe 95% of the haplotypic diversity. CONCLUSIONS: The TNF locus in the Gambian and Malawi sample is haplotypically diverse and has a rich history of intragenic recombination. As a consequence, a large proportion of TNF SNPs must be typed to detect a disease-modifying SNP at this locus. The most informative subset of SNPs to genotype differs between the two populations.

In vivo characterization of regulatory polymorphisms by allele-specific quantification of RNA polymerase loading.

In vivo characterization of regulatory polymorphisms is a key requirement for next-generation human genetic analysis. Here we describe haploChIP, a method that uses chromatin immunoprecipitation (ChIP) and mass spectrometry to identify differential protein-DNA binding in vivo associated with allelic variants of a gene. We demonstrate this approach with the imprinted gene SNRPN. HaploChIP showed close correlation between the level of bound phosphorylated RNA polymerase II at the SNRPN locus and allele-specific expression. Application of the approach to the TNF/LTA locus identified functionally important haplotypes that correlate with allele-specific transcription of LTA. The haploChIP method may be useful in high-throughput screening for common DNA polymorphisms that affect gene regulation in vivo.