ATP2B4 regulatory genetic variants are associated with mild malaria.

BACKGROUND: Genome-wide association studies have identified ATP2B4 as a severe malaria resistance gene. Recently, 8 potential causal regulatory variants have been shown to be associated with severe malaria. METHODS: Genotyping of rs10900585, rs11240734, rs1541252, rs1541253, rs1541254, rs1541255, rs10751450, rs10751451 and rs10751452 was performed in 154 unrelated individuals (79 controls and 75 mild malaria patients). rs10751450, rs10751451 and rs10751452 were genotyped by Taqman assays, whereas the fragment of the ATP2B4 gene containing the remaining SNPs was sequenced. Logistic regression analysis was used to assess the association between the SNPs and mild malaria. RESULTS: The results showed that mild malaria was associated with rs10900585, rs11240734, rs1541252, rs1541253, rs1541254, rs1541255, rs10751450, rs10751451 and rs10751452. The homozygous genotypes for the major alleles were associated with an increased risk of mild malaria. Furthermore, the haplotype containing the major alleles and that containing the minor alleles were the most frequent haplotypes. Individuals with the major haplotypes had a significantly higher risk of mild malaria compared to the carriers of the minor allele haplotype. CONCLUSIONS: ATP2B4 polymorphisms that have been associated with severe malaria are also associated with mild malaria.

Identification of ATP2B4 Regulatory Element Containing Functional Genetic Variants Associated with Severe Malaria.

Genome-wide association studies for severe malaria (SM) have identified 30 genetic variants mostly located in non-coding regions. Here, we aimed to identify potential causal genetic variants located in these loci and demonstrate their functional activity. We systematically investigated the regulatory effect of the SNPs in linkage disequilibrium (LD) with the malaria-associated genetic variants. Annotating and prioritizing genetic variants led to the identification of a regulatory region containing five ATP2B4 SNPs in LD with rs10900585. We found significant associations between SM and rs10900585 and our candidate SNPs (rs11240734, rs1541252, rs1541253, rs1541254, and rs1541255) in a Senegalese population. Then, we demonstrated that both individual SNPs and the combination of SNPs had regulatory effects. Moreover, CRISPR/Cas9-mediated deletion of this region decreased ATP2B4 transcript and protein levels and increased Ca2+ intracellular concentration in the K562 cell line. Our data demonstrate that severe malaria-associated genetic variants alter the expression of ATP2B4 encoding a plasma membrane calcium-transporting ATPase 4 (PMCA4) expressed on red blood cells. Altering the activity of this regulatory element affects the risk of SM, likely through calcium concentration effect on parasitaemia.

NCR3 polymorphism, haematological parameters, and severe malaria in Senegalese patients.

BACKGROUND: Host factors, including host genetic variation, have been shown to influence the outcome of Plasmodium falciparum infection. Genome-wide linkage studies have mapped mild malaria resistance genes on chromosome 6p21, whereas NCR3-412 polymorphism (rs2736191) lying within this region was found to be associated with mild malaria. METHODS: Blood samples were taken from 188 Plasmodium falciparum malaria patients (76 mild malaria patients, 85 cerebral malaria patients, and 27 severe non-cerebral malaria patients). NCR3-412 (rs2736191) was analysed by sequencing, and haematological parameters were measured. Finally, their association with clinical phenotypes was assessed. RESULTS: We evidenced an association of thrombocytopenia with both cerebral malaria and severe non-cerebral malaria, and of an association of high leukocyte count with cerebral malaria. Additionally, we found no association of NCR3-412 with either cerebral malaria, severe non-cerebral malaria, or severe malaria after grouping cerebral malaria and severe non-cerebral malaria patients. CONCLUSIONS: Our results suggest that NCR3 genetic variation has no effect, or only a small effect on the occurrence of severe malaria, although it has been strongly associated with mild malaria. We discuss the biological meaning of these results. Besides, we confirmed the association of thrombocytopenia and high leukocyte count with severe malaria phenotypes.

Amplification of Mitochondrial DNA for detection of Plasmodiumvivax in Balochistan.

OBJECTIVE: To access a new step using PCR to amplify the targeted mtDNA sequence for detecting specifically Plasmodium vivax and its co-infections, false positive and false negative results with Plasmodium falciparum. METHODS: In this study we have standardized a new technical approach in which the target mitochondrial DNA sequence (mtDNA) was amplified by using a PCR technique as a tool to detect Plasmodium spp. Species specific primers were designed to hybridize with cytochrome c oxidase gene of P. vivax (cox I) and P. falciparum (cox III). Two hundred blood samples were collected on the basis of clinical symptoms which were initially examined through microscopic analysis after preparing Giemsa stained thick and thin blood smears. Afterwards genomic DNA was extracted from all samples and was then subjected to PCR amplification by using species specific primers and amplified segments were sequenced for confirmation of results. RESULTS: One-hundred and thirty-two blood samples were detected as positive for malaria by PCR, out of which 64 were found to be positive by PCR and 53 by both microscopy and PCR for P.vivax infection. Nine samples were found to be false negative, one P.vivax mono infection was declared as co infection by PCR and 3 samples identified as having P.falciparum gametes were confirmed as P.vivax by PCR amplification. Sensitivity and specificity were found to be 85% and 92% respectively. CONCLUSIONS: Results obtained through PCR method were comparatively better and reliable than microscopy.