A Non-Coding Fc Gamma Receptor Cis-Regulatory Variant within the 1q23 Gene Cluster Is Associated with Plasmodium falciparum Infection in Children Residing in Burkina Faso

Abstract

Antibodies play a crucial role in activating protective immunity against malaria by interacting with Fc-gamma receptors (FcγRs). Genetic variations in genes encoding FcγRs can affect immune cell responses to the parasite. In this study, our aim was to investigate whether non-coding variants that regulate FcγR expression could influence the prevalence of Plasmodium falciparum infection. Through bioinformatics approaches, we selected expression quantitative trait loci (eQTL) for FCGR2A, FCGR2B, FCGR2C, FCGR3A, and FCGR3B genes encoding FcγRs (FCGR), in whole blood. We prioritized two regulatory variants, rs2099684 and rs1771575, located in open genomic regions. These variants were identified using RegVar, ImmuNexUT, and transcription factor annotations specific to immune cells. In addition to these, we genotyped the coding variants FCGR2A/rs1801274 and FCGR2B/rs1050501 in 234 individuals from a malaria-endemic area in Burkina Faso. We conducted age and family-based analyses to evaluate associations with the prevalence of malarial infection in both children and adults. The analysis revealed that the regulatory rs1771575-CC genotype was predicted to influence FCGR2B/FCGR2C/FCGR3A transcripts in immune cells and was the sole variant associated with a higher prevalence of malarial infection in children. In conclusion, this study identifies the rs1771575 cis-regulatory variant affecting several FcγRs in myeloid and neutrophil cells and associates it with the inter-individual capacity of children living in Burkina Faso to control malarial infection.

Keywords: Burkina Faso; FCGR ex-pression quantitative trait loci (eQTL); FCGR2A gene polymorphism; FCGR2B gene polymorphism; Fc-gamma receptor (FCGR) gene polymorphism; Fc-gamma receptors/FcγRs; Plasmodium falciparum parasitemia; malaria; regulatory variants.

Figure 1

Work flow used to prioritize regulatory variants. A total of 773 SNPs carrying an eQTL annotation in whole blood for FCGR2A, FCGR2B, FCGR2C, FCGR3A, and FCGR3B genes were searched by querying the current release of GTEX (V8) database. The RegVar tool further allowed gene-specific annotation and tissue-specific annotation to prioritize 636 SNPs. The 20 SNPs that displayed the best scores were selected using RegVar; eQTL annotations were retained to select variants that have annotation in 2 independent immune cell eQTL databases, ImmuNexUT and ebi eQTL catalog. The position of eQTL candidates was cross-referenced with the position of ATAC-seq regions in immune cells and positioning in regions that exhibit transcription factor binding activity in immune cells, using the ReMap catalog. ReMap data were filtered on primary NK, T and B lymphocytes, monocytes, macrophages, neutrophils, peripheral blood cells, and monocytic and lymphocytic cell lines to allow selection of 2 FCGR regulatory variants further used for association studies.

Figure 2

Epigenetic Landscape of the FCGR Locus in Monocytes and Neutrophils. The presence of epigenetic marks (ATAC-seq, H3K4me1, H3K4me3, H3K27ac, and H3K27me3) and CTCF transcription factor binding profiles for rs1771575, rs2099684, rs1801274, and rs1050501 in blood monocytes and neutrophils were visualized using the CistromeDB database in conjunction with the WashU epigenome browser. The genomic location of the FCGR locus on chromosome 1q23 is depicted as a red line. For both monocytes (upper panel) and neutrophils (lower panel), the following data is displayed: ATAC-Seq peaks, CTCF (CCCTC-binding factor) binding sites, ChIP-seq peaks, and Histone modifications associated with active regulatory regions: H3K4me1, H3K4me3, H3K27ac, and H3K27me3. The variant rs2099684 is situated within an active enhancer, whereas rs1771575 is found within both an active enhancer and a CTCF binding site. Additionally, the known coding variants rs1801274 and rs1050501 are also represented.

Figure 3

Alteration of transcription factor binding sites at position rs1771575. (A) ChIP-seq experiments in GM12878 (B-LCL Cell Line). ChIP-seq experiments were conducted in the GM12878 (B-LCL) cell line, revealing that ZNF24, CTCF, and cohesin subunits RAD21 and SMC3 bind to rs1771575. (B) Schematic interaction between CTCF and the cohesin complex. A schematic representation demonstrates the interaction between CTCF and the cohesin complex. (C) Position weight matrix of ZNF24 binding. The position weight matrix analysis of ZNF24 binding indicates a preference for binding to chromatin in the presence of the reference allele (C) of rs1771575. p-values for each putative site and their ratio (pval_ratio = worst_pval/best_pval) were calculated using the RSAT ‘scan variations’ tool. JASPAR matrix ID and genomic coordinates (GRCh38) are also provided. (D) CTCF binding ChIP-seq analysis. Analysis of ChIP-seq data for CTCF binding using read counts from several immune cell lines demonstrates allele-specific binding. The number of mapped reads carrying rs1771575-C is significantly different in the presence of the alternative allele, indicating an allele-specific binding event. These data were obtained from the ADASTRA database.

Figure 4

Comparison of genotype frequencies of the analyzed variants among individuals from Europe, Africa (1000 Genomes Project), and the study population from Burkina Faso (Bobo ethnic group). (A) Distribution of the coding FCGR2B rs1050501 genotype. The distribution of the coding FCGR2B rs1050501 genotype was analyzed in individuals from European (CTL EUR), African (CTL AFR), and unique parents from the study cohort enrolled in Burkina Faso. CC: The individual has two copies of the C allele at rs10505501, encoding an isoleucine (I) in the transmembrane domain of the FCgR2B receptor. CT: The individual has one copy of the C allele and one copy of the T allele at rs10505501.TT: The individual has two copies of the T allele at rs10505501, which encodes a threonine (T) at position 232. **** p < 10⁻⁴; Ns: non significative. (B) Distribution of the coding FCGR2A rs1801274 genotype. The distribution of the coding FCGR2A rs1801274 genotype was examined in individuals from European (CTL EUR), African (CTL AFR), and unique parents from the study cohort enrolled in Burkina Faso. AA: The individual has two copies of the A allele, encoding a histidine (I) in exon 4 of the FcγR2A receptor. AG: The individual has one copy of the A allele and one copy of the G allele. GG: The individual has two copies of the G allele, resulting in an arginine substitution (R) at position 131. (C) Distribution of the rs1771575 regulatory variant genotype. The distribution of the rs1771575 regulatory variant genotype was assessed in individuals from European (CTL EUR), African (CTL AFR), and unique parents from the study cohort enrolled in Burkina Faso. CC: The individual has two copies of the C allele. CT: The individual has one copy of the C allele and one copy of the T allele. TT: The individual has two copies of the T allele. (D) Distribution of the rs2099684 regulatory variant genotype. The distribution of the rs2099684 regulatory variant genotype was investigated in individuals from European (CTL EUR), African (CTL AFR), and unique parents from the study cohort enrolled in Burkina Faso. AA: The individual has two copies of the A allele. AG: The individual has one copy of the A allele and one copy of the G allele. GG: The individual has two copies of the G allele.

Figure 5

Prevalence of P. falciparum Infection (PrevPfI) Across Variant Genotypes in Infants and Adults from Burkina Faso. (A) Comparative analysis of P. falciparum infection prevalence (PrevPfI) in parents and children. A comparative analysis of P. falciparum infection prevalence (median PrevPfI values) was conducted in parents and children using the nonparametric Mann–Whitney U-test for non-normally distributed data. Significant p-values (≤0.05) are indicated. The prevalence of P. falciparum infection (PrevPfI) for each individual was calculated as the number of positive blood samples divided by the total number of blood samples evaluated for each individual. This was considered the quantitative phenotype outcome variable. (B) Comparative analysis of P. falciparum infection prevalence (PrevPfI) in parents across rs1771575, rs1050501, rs2099684, and rs1801274 genotypes. A comparative analysis of P. falciparum infection prevalence (median PrevPfI values) in parents was performed across rs1771575, rs1050501, rs2099684, and rs1801274 genotypes. Significant p-values (<0.05) are indicated, and trends (p < 0.100) are indicated in italics. * refers to p values ≤ 0.05. (C) Comparative analysis of P. falciparum infection prevalence (PrevPfI) in children across rs1771575, rs1050501, rs2099684, and rs1801274 genotypes. A comparative analysis of P. falciparum infection prevalence (median PrevPfI values) in children was conducted across rs1771575, rs1050501, rs2099684, and rs1801274 genotypes. Significant p-values (<0.05) are indicated, and trends (p < 0.100) are indicated in italics. * refers to pvalues ≤ 0.05, ** refers to p values ≤ 0.01 (D) Comparative analysis of residual P. falciparum infection prevalence (PrevPfI adjusted for age effect) in children across rs1771575, rs1050501, rs2099684, and rs1801274 genotypes. A comparative analysis of residual P. falciparum infection prevalence (median PrevPfI values) adjusted for age effect was performed in children across rs1771575, rs1050501, rs2099684, and rs1801274 genotypes. Residuals of PrevPfI were calculated for each person after conducting a linear regression analysis between PrevPfI and age. * refers to p values ≤ 0.05, ** refers to pvalues ≤ 0.01 (E) Multiple linear regression analysis to test effect on outcome variable PrevPfI. Multiple linear regression was carried out to assess the effect on the outcome variable PrevPfI, taking into account age and the recessive model (CC: encoded 1) vs. (CT/TT: encoded 0) explanatory variables.