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

Deciphering human resistance to malaria : from genetic studies to functional approaches

La sévérité du paludisme est influencée par des interactions complexes entre de nombreux facteurs dont la génétique de l’hôte. Plusieurs études de liaison génétique menées dans différentes ethnies africaines ont montré une liaison entre le locus 6p21 et le paludisme simple. De plus, différents variants au sein des gènes TNF et NCR3, retrouvés dans ce locus, ont été indépendamment associés à ce phénotype au Burkina Faso.Ainsi, nous nous sommes tout d’abord intéressés aux polymorphismes du TNF. Nos résultats montrent que les variants TNF-308, TNF-244, et TNF-238 sont associés à la parasitémie maximale ou aux accès simples au Congo. Les approches moléculaires indiquent que le TNF-244 a un effet cis-régulateur avec une activité promotrice réduite en présence du variant A ainsi qu’une fixation altérée de protéines nucléaires en présence de ce même variant. Enfin, nos analyses bio-informatiques suggèrent que le TNF-244 et le TNF-238 agissent en synergie pour modifier le site de fixation d’au moins un facteur de transcription.Nous avons ensuite confirmé l’association du NCR3-412 avec le paludisme simple et le nombre d’accès fébrile au Congo. Les analyses fonctionnelles montrent que ce SNP a aussi un effet cis-régulateur avec une activité promotrice accrue en présence de l’allèle G et une liaison altérée de deux complexes protéiques en présence de l’allèle C. Les approches in silico et in vitro indiquent que les facteurs STAT4 et RUNX3 sont ceux dont la fixation est altérée.NCR3-412 altérant la résistance à la forme simple du paludisme, nous avons souhaité déterminer s’il est aussi impliqué dans la résistance au paludisme sévère mais nous n’avons détecté aucune association.The severity of malaria is influenced by complex interactions between many factors including host genetics. Numerous genetic studies conducted in different African ethnic groups have shown a significant linkage between the 6p21 locus and mild malaria attack. In addition to their linkage, several polymorphisms found under the linkage peak, and more precisely within TNF and NCR3, were also independently associated with different sub-phenotypes of mild malaria in Burkina Faso.Thus, we first focused on TNF polymorphisms. Among the 4 polymorphisms analyzed, we found associations between TNF-238, TNF-244, TNF-308 and either mild malaria attack or maximum parasitemia. Molecular approaches showed that TNF-244 has a cis-regulatory effect. Indeed, we observe a decreased promoter activity and an altered binding of nuclear proteins in the presence of the A variant. In addition, our bioinformatics analyses suggested a cooperative effect of TNF-244 and TNF-238 in modifying the binding of at least one transcription factor.We then confirmed the association of NCR3-412 with both mild malaria and the number of febrile episodes in Congo. Functional analyses have shown that this SNP has also a cis-regulatory effect with a decreased promoter activity and an altered binding of two nuclear protein complexes in the presence of the C allele. Finally, in silico and in vitro approaches indicated that STAT4 and RUNX3 are the two transcription factors affected.As NCR3-412 is associated with resistance to mild malaria, we therefore investigated whether this SNP is also involved in severe malaria resistance, but we did not detect any association neither with severe anemia nor with cerebral malaria

Heterogeneity of germinal center B cells: New insights from single‐cell studies

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Genetic Predisposition to the Mortality in Septic Shock Patients: From GWAS to the Identification of a Regulatory Variant Modulating the Activity of a CISH Enhancer

International audienceThe high mortality rate in septic shock patients is likely due to environmental and genetic factors, which influence the host response to infection. Two genome-wide association studies (GWAS) on 832 septic shock patients were performed. We used integrative bioinformatic approaches to annotate and prioritize the sepsis-associated single nucleotide polymorphisms (SNPs). An association of 139 SNPs with death based on a false discovery rate of 5% was detected. The most significant SNPs were within the CISH gene involved in cytokine regulation. Among the 139 SNPs associated with death and the 1311 SNPs in strong linkage disequilibrium with them, we investigated 1439 SNPs within non-coding regions to identify regulatory variants. The highest integrative weighted score (IW-score) was obtained for rs143356980, indicating that this SNP is a robust regulatory candidate. The rs143356980 region is located in a non-coding region close to the CISH gene. A CRISPR-Cas9-mediated deletion of this region and specific luciferase assays in K562 cells showed that rs143356980 modulates the enhancer activity in K562 cells. These analyses allowed us to identify several genes associated with death in patients with septic shock. They suggest that genetic variations in key genes, such as CISH, perturb relevant pathways, increasing the risk of death in sepsis patient

Beyond genome-wide scan: Association of a cis-regulatory NCR3 variant with mild malaria in a population living in the Republic of Congo.

Linkage studies have revealed a linkage of mild malaria to chromosome 6p21 that contains the NCR3 gene encoding a natural killer cell receptor, whereas NCR3-412G>C (rs2736191) located in its promoter region was found to be associated with malaria in Burkina Faso. Here we confirmed the association of rs2736191 with mild malaria in a Congolese cohort and investigated its potential cis-regulatory effect. Luciferase assay results indicated that rs2736191-G allele had a significantly increased promoter activity compared to rs2736191-C allele. Furthermore, EMSAs demonstrated an altered binding of two nuclear protein complexes to the rs2736191-C allele in comparison to rs2736191-G allele. Finally, after in silico identification of transcription factor candidates, pull-down western blot experiments confirmed that both STAT4 and RUNX3 bind the region encompassing rs2736191 with a higher affinity for the G allele. To our knowledge, this is the first report that explored the functional role of rs2736191. These results support the hypothesis that genetic variation within natural killer cell receptors alters malaria resistance in humans

Single-Cell Longitudinal Characterization of FL Heterogeneity and Residual Disease in the Bone Marrow from Low-Tumor Burden FL Enrolled in the Flirt Clinical Trial

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Proportion of mild malaria episodes according to rs2736191 polymorphism.

<p>Proportion of mild malaria episodes according to rs2736191 polymorphism.</p

Effect of rs2736191 polymorphism on transcription factors binding.

<p>EMSA with biotin-labelled probes containing the G allele of rs2736191 and incubated with NK92 nuclear extracts. Competition was performed with 200x (lane 3 and 5) or 400x (lane 4 and 6) excess of double-stranded DNA containing either the G or the C variants. Two arrows indicate two specific nuclear proteins that interact with rs2736191-G probe. The G variant was more effective in competing these two nuclear proteins compared to the C variant.</p

Differential binding of transcription factor candidates to rs2736191 polymorphism.

<p>DNA pull-down assay was performed with nuclear extracts prepared from NK92 cells and complementary biotinylated oligonucleotides encompassing the rs2736191-G/C binding sites. After the incubation, the nuclear protein bound were isolated using magnetic beads coupled with streptavidin and subjected to 12% SDS-PAGE and Western blotting for the transcription factors STAT4 and RUNX3. The strongest intensity obtained with the G probe, for both STAT4 and RUNX3, suggests a preferential binding of these transcription factors in presence of this allele compared to the rs2736191-C allele.</p

rs2736191 polymorphism affects <i>NCR3</i> expression.

<p>Luciferase reporter gene assays with constructs containing the rs2736191-G (p-G) or rs2736191-C (p-C) <i>NCR3</i> promoter in K-562 cell line. All constructs were co-transfected with pRL-SV40 to standardize transfection efficiency and luciferase activity for each sample was adjusted by the empty pGL3-Enhancer. Fold increase was measured by defining the activity of p-G vector as 1. Data shown are the means ± SE from 2 independent transfection experiments, each performed in triplicate. The rs2736191-G-containing <i>NCR3</i> promoter drove significantly higher reporter gene expression (~37%) than the rs2736191C-containing <i>NCR3</i> promoter (P < 0.05, as shown in the Figure). Statistical analysis was performed using two-tailed Student’s t-test after controlling the normality of the data and the equality of their variance.</p