Distribution of FcγR gene polymorphisms among two sympatric populations in Mali: differing allele frequencies, associations with malariometric indices and implications for genetic susceptibility to malaria

Variations in malaria susceptibility among populations living in endemic areas have been known for decades [1‐3]. Sympatric populations, such as the Fulani and their sympatric neighbours, amply demonstrate this phenomenon. Epidemiologically, the Fulani have been consistently shown to be naturally less susceptible to malarial infections than their sympatric neighbours [2‐8]. A clear biological explanation of the basis of this protection is still lacking. The observation of larger spleen sizes in the Fulani compared to their sympatric neighbours in the 1970s and 1980s suggested that the spleen may have a role in protecting the Fulani from malaria [5, 9]. Subsequent studies have provided clues to other possible biological mediators of this interethnic difference in malaria susceptibility. For instance, in Burkina Faso, the Fulani were shown to have lower parasite densities and stronger humoral immune responses to malaria antigens than their Mossi and Rimaibes sympatric neighbours [4, 6]. Similarly, a functional deficiency in T cell regulation is thought to contribute to the Fulani’s protection against malaria [10]. Several immuno-epidemiology studies have reported on the possible roles of different immune factors in this phenomenon and this has been extensively reviewed [11]. A review of the existing body of evidence suggests a significant role for immune-mediated mechanisms of protection; however, the immunogenetics of this protection are currently poorly explained.

Traditional host genetic factors known to influence malaria susceptibility have so far failed to adequately explain why the Fulani are less susceptible to malaria than their sympatric neighbours [12‐15]. Recent malaria immunogenetic studies have suggested roles for a few genes but that notwithstanding, the immunogenes that mediate this protection are yet to be clearly identified [16, 17]. Research is ongoing to identify the immunogenes that may influence interethnic variability in malaria susceptibility. Of all the candidate genes, the Fc gamma receptors (FcγRs) for IgG are probably the most extensively studied. FcγRs for IgG are expressed on a variety of immune cells, including monocytes and other leucocytes. They typically bind to opsonized pathogens to activate a variety of cellular immune responses that may culminate in the control of an infection [18]. Thus, FcγRs are thought to be an important link between humoral and cellular immune response [18].

Studies on FcγRs polymorphisms and malaria susceptibility have so far focused on the FcγRIIa genotypes, which affect binding of different IgG sub-classes. The best known of the FcγRs polymorphisms is the FcγRIIa (H/R131), which has been associated with levels of anti-malarial IgG2 and IgG3 antibodies [19, 20]. Compared to their sympatric neighbours, the Fulani in Sudan were shown to have a higher frequency of the FcγRIIa-H131, which is associated with higher antibody levels [20]. This difference in allelic distribution was however not seen among the Fulani and Mossi in Burkina Faso [21]. Several other single nucleotide polymorphisms (SNPs) in FcγRs have been associated with malaria susceptibility but their contribution to interethnic difference in malaria susceptibility is unclear [22‐25]. The genes encoding FcγRs are interesting and challenging to study for a number of reasons. Firstly, the individual genes (FCGR2A, FCγR2B, FCGR2C, FCGR3A, FCGR3B) in this cluster exhibit high homology between themselves [26]. For instance, FCGR2A and FCGR2B have approximately 95 % homology in their exons that code for their respective extracellular domains [27]. Secondly, FCGR2C, which encodes FcgRIIc, appears to be the product of an unequal crossover event between FCGR2A and FCGR2B [27]. Lastly, an apparent genotype-phenotype mismatch and the possibility that a SNP in one gene can affect the function of proteins encoded by a different gene in the FcγRs cluster is intriguing [27]. These features present formidable challenges to the study of this gene cluster and its polymorphisms, particularly their introns.

This study focused on three SNPs in the FcγRs gene cluster that are yet to be studied in relation to malaria: two intronic SNPs (FCGR2C-rs3933769 and FCGR3A-rs396991) and a functional SNP in the FCGR2B-rs1050519. The aim of this study was to determine the distribution of these SNPs among sympatric Fulani and Dogon tribes living in Mali with mild malaria. The study further investigated the association of these SNPs with various clinical, malariometric and immunological indices in children with mild malaria. The results of this study revealed differences in allelic frequencies in FC2R2C (rs3933769) and FCGR3A (rs396991) SNPs among the Fulani and Dogon in the study population. This finding indirectly suggests that the genes may contribute to the interethnic variability in malaria susceptibility seen among the Fulani and their sympatric neighbours.

This study found that the allelic frequencies of rs396991 and rs3933769 were different between the Fulani and Dogon. This observation is noteworthy because of the confirmed and suggested roles of these SNPs. The rs396991 SNP represents a change from phenylalanine (F) to valine (V) at position 158 in the Fc fragment of IgG, low affinity IIIa, receptor (CD16a) gene [29]. The mutant allele of this SNP binds IgG1 and IgG3 with a higher affinity than the wild type, which binds fewer immune complexes thereby reducing the potential of inflammatory response [29, 30]. Functional mutation in the FCGR3A gene can potentially influence immune-mediated diseases since the FcγRIIIa receptors are found on several immune cells and constitute a vital link between humoral and cellular immunity. Thus, the finding that the rs396991 mutant allele occurs at higher frequency in the Dogon, who are more susceptible to malaria relative to Fulani, is alluring. The rs396991 has been shown to influence several immune-mediated diseases [31‐34]. For instance, the homozygous wild genotype of rs396991 was under-represented in HIV-infected patients with Kaposi sarcoma [31]. This SNP is also suggested to play a limited role in rheumatoid arthritis, which is an immune-mediated disease [34]. The rs396991 is yet to be studied in relation to malaria but data from this study suggest that this SNP may play a role in malaria pathogenesis.

In line with previous studies, the Fulani had higher levels of anti-malaria specific IgG, IgG1 and IgG3 than the Dogon [19, 35]. However, this present study has expanded knowledge by further showing an association between rs396991 mutant allele and higher levels of IgG3 in the Dogon tribe. This is interesting considering the fact this SNP was associated with higher levels of IgG in Hutchison lymphoma patients [36]. The variable distribution of rs396991 among Fulani and Dogon, as well as its association with IgG, could be an indication of a possible role for this SNP in malaria susceptibility.

This study compared various malariometric indices among the sympatric tribes and, consistent with previous studies in The Gambia, Burkina Faso, Sudan and Mali [5, 6, 20], an enlarged spleen was more frequently seen among the Fulani compared to the Dogon. Also, consistent with previous studies is the finding of lower Hb levels, parasite density and fever prevalence in the Fulani relative to the Dogon [5, 15, 37]. Although these variations in malariometric indices among Fulani and non-Fulani sympatric neighbours have been known for decades, there is still no satisfactory explanation for some of them. For instance, the pronounced splenomegaly in the Fulani is poorly explained. A recent model explains that an innate propensity of the spleen to stringently retain parasitized red blood cells causes enlargement of the spleen, lowers Hb levels and reduces parasite density [38]. This present study showed an association between enlarged spleen and lower Hb levels but not parasite density. The data on spleen size, Hb levels, parasite density, and fever prevalence suggest that spleen size could be an important malariometric index.

Besides being more frequent in the Fulani than the Dogon, the intronic rs3933769 SNP of FCGR2C appeared to influence parasite density in the study population. Study participants with the mutant allele harboured fewer than half the parasites found in their counterparts with the ancestral allele. The FcγRIIc is suggested to play an important role in parasite clearance by effectively bridging humoral and cellular immunity [39]. This finding is particularly interesting considering that introns have been traditionally viewed as non-functional. Intronic SNPs have gained currency lately with the emergence of evidence on their possible functional roles [40, 41]. This finding adds to an increasing body of evidence that suggests roles for intronic SNPs. The intronic SNPs of FCγR2C are even more interesting considering that this gene exhibits a genotype-phenotype mismatch [27]. Heijden et al. recently found a novel intron 7 SNP of FCγRIIC that prevented some Caucasians with the FCγRIIC-ORF genotype from expressing FcγRIIb/c. Some individuals in the same population with the FCγRIIC-Stop genotype, which is thought to be a pseudogene, expressed the FcγRIIb/c receptor [27]. The extracellular domains of FcγRIIc and FcγRIIb are completely identical, a situation that further complicates the study of this gene cluster. Possibly several other SNPs or copy number variation (CNV) in FCγRIIC affects the expression of FcγRIIc on immune cells and influences their ability to induce antibody-dependent cellular cytotoxicity ADCC or alter the balance between activating and inhibitory signals [42]. Such polymorphisms may even affect the expression and function of other FcγRs than FcγRIIc [27]. The findings of this study leave room for speculating a role for rs3933769 in malaria pathogenesis and susceptibility. Further studies will be needed to make this SNP a bona fide malaria candidate polymorphism.

Some of the associations reported in this study were found in the entire study population and not in the two tribes separately. For instance, the associations between rs3933769 and parasite density, rs396991 and IgG1, enlarge spleen and lower Hb were all found in the entire study population only. These observations should be interpreted with care because even though the unequal numbers of Fulani and Dogon in this study do not necessarily pose a statistical problem, it could have reduced the power to detect statistical significance in two tribes separately.

Taken together, this study confirms previous studies that showed interethnic difference between the Fulani and non-Fulani sympatric tribes with regard to clinical, malariometric and immunological indices. In the furtherance of knowledge, this study shows that the allelic distribution of two FCγR SNPs is different in the Fulani and Dogon living in sympatry in Mali. The study also shows that two of the SNPs are associated with parasite density and IgG1 and IgG3 responses. This study did not aim to determine the association between these FCγR SNPs and malaria. Further studies will be needed to determine the possible roles of these SNPs in malaria pathogenesis.