Signicant Differences in FcγRIIa, FcγRIIa and FcγRIIIb Genes Polymorphism and Anti-malarial IgG Subclass Pattern are Associated with Severe Malaria in Saudi Children

Background: The FcyRs genotypes have been reported to play a key role in the defence against malaria parasites through both cellular and humoral immunity. This study aimed to investigate the possible correlation between FcγR (IIa, IIIa, and IIIb) genes polymorphism and the clinical outcome for anti ‐ malarial antibody response of Plasmodium falciparum infection among Saudi children. Material and methods: A 600 volunteers have been enrolled in this study, including 200 malaria-free control (MFC) subjects, 218 patients with uncomplicated malaria (UM) and 182 patients with severe malaria (SM). The FcγR genotypes was analysed using PCR amplication methods, and measurement of immunoglobulins were determine using ELISA. Results: The data revealed the FcγRIIa-R/R131 showed a statistically association with the increased susceptibility to SM when compared to UM patients. Furthermore, higher levels of IgG1, IgG2, and IgG4 were associated with the FcγRIIa-H/H131 genotypes among UM patients. Although the FcγRIIa-F/V176 genotype was not associated with UM, it showed a signicant association with severe malaria. Interestingly, the FcγRIIa-V/V176 genotype was This study aimed to associated with protection against SM. Moreover, severe malaria patients carrying the FcγRIIa-F/F genotype showed higher levels of AMA-1-specic IgG2 and IgG4 antibodies. The FcγRIIIb NA1/NA1 and FcγRIIIb NA2/NA2 genotypes did not show signicant differences between UM and the MFC. However, the genotype FcγRIIIb-NA2/NA2 was statistically associated with severe malaria. Conclusions: The data presented in this study strongly suggest the possible impact of FcyR (IIa, RIIIa and RIIIb) gene variants and anti-malaria IgG subclasses play a role in susceptibility to malaria infection and disease outcomes in Saudi children.

The present study aimed to investigate the possible relationship between the expression of FcγRIIa (CD32a), FcγRIIa (CD16a), and FcγRIIIb (CD16b) gene variants and the antibodies against the malarial Apical Membrane Antigen 1 (AMA-1) in association with the susceptibility to malaria infection among Saudi children.

Study area
This study was conducted at Bani Malik General Hospital in Jazan Region (BMGHJ), located in the Southern part of the Kingdom of Saudi Arabia (KSA), during three transmission seasons from October 2015 to March 2018. The highlights of this study setting have already been described in related previous studies [4,[7][8][9][10].

Study design and patients
A prospective case-control study was conducted in children attending the outpatient clinic of BMGHJ, with a con rmed clinically diagnosed P. falciparum infection. Patients with positive thick blood lm for P. falciparum asexual parasites were recruited based on the microscopic diagnosis.
Participants with no features of severe malaria were de ned as having uncomplicated P. falciparum infection. Children were diagnosed with severe malaria on the basis of one or more of the following: severe malarial anaemia, cerebral malaria, hypoglycaemia, jaundice, acidosis, acute kidney injury (renal impairment), signi cant bleeding, pulmonary oedema, and shock as described in detailed by the WHO [11]. These clinical manifestations occurred in the absence of any identi ed alternative cause other than P. falciparum asexual parasitemia. Children with cerebral malaria had a Blantyre Coma Score (BCS) < 3 at or 4 hours post-admission. Children with severe malarial anaemia had a blood haemoglobin concentration 5 g/dl or a haematocrit value <15% together with a parasite count > 10000/μL. All other children recruited in the study had a haemoglobin concentration above this level. Control children were selected from Child and Woman Health Clinics (CWHC), the clinics provide children's health services, including providing/following-up their routine vaccinations, as well as providing seasonal vaccines for children, frequency-matching the patients for age, sex, and ethnicity. Control group were recruited who appeared healthy by clinical physical examination and did not have serious illness or any malaria according to information provided by guardians or parents.
The study excluded children with any co-infectious diseases and none of the participants was positive for HIV. All the children were recruited during three malaria transmission seasons, from October 2015 to March 2018.

Sample collection
After the diagnosis of malaria and before the start of the pharmacological course of treatment, 100 µL of blood was spotted and dried on lter paper (Qualitative lter paper, Grade 1, circles, diam. 42.5 mm from Whatman ® , Sigma-Aldrich ® ). This collected sample was used for investigating Fcγ receptors genes polymorphism, parasite detection using PCR, and measurement of immunoglobulins as described earlier in our report [12,13].
Serum elution from lter-paper samples To elute dried samples from lter-paper, a hole puncher of ϕ 6 mm was used for punching out lter-paper discs and placed in Eppendorf tubes with 100 µL of phosphate-buffered saline (PBS). Subsequently, the discs were transferred to 10 ml tubes. Then, 500 µL of (PBS) with 0.05% Tween and 0.5% bovine serum albumin (BSA) were added to the tubes and incubated under shaking for 2 hours at room temperature.
After incubation, the samples were vigorously shaken with a vortex for 30 seconds, and the supernatants containing the eluted sera were aliquoted in cryotubes (1.5 ml) and stored at −20 °C till analysis. Each extracted sample contained an approximately 1:100 diluted serum [12].
DNA extraction DNA was extracted from 50 µL dried drop of blood sample on the lter paper using the QIAamp DNA Mini Kit (Qiagen ® , Hamburg, Germany). The extracted DNA was re-suspended in a 150 µL of Tris-borate-EDTA (TBE) buffer as previously described [14].

Parasite genotype
Detection of P. falciparum was based on targeting the AMA-1_3D7 gene using polymerase chain reaction (PCR) from 5 µl of the extracted DNA samples as previously detailed [15][16][17].
Enzyme-linked immunosorbent assays (ELISA) IgG subclasses antibodies were measured against the recombinant AMA-1 anti-malarial antigen. The total levels of IgG and its subclasses were measured using enzyme-linked immunosorbent assays (ELISA) as previously described in detail [18,19] and as recently indicated [20].

Statistical analysis
Statistical analysis was done by SPSS statistical software version 23 for Windows (IBM© SPSS © statistics). In this study, the antibodies (total IgG and IgG subclasses) levels were analysed using one-way analysis of variance tests and the P values were determined. The values were deduced from the log-log correlative coe cient for each of the respective antibody standard curve.
The boxes shown in the results illustrate the total observations corresponding to the 25% and 75% quartile, and the horizontal line represents the median. The whiskers illustrate the 10% and 90% quartile, excluding outliers. With respect to the risk of malaria infection during the pregnancy period, all values of P <0.05, 95% con dence intervals (CI) for odds ratio (OR) that did not cross 1.00 were considered as statistically signi cant. In the analysis, FcγRIIa-Arg/ His 131 polymorphism was used as a reference, due to its utmost prevalence in humans [23]. Using the same software, a 2 x 2 chi-square test was used to compare the overall allele frequency. The Hardy-Weinberg equilibrium (HWE) for genotypic deviations were assessed using a chi-squared statistical test The logistic regression analysis was performed to test for the associations between the FcγRs genotypes related to higher levels of anti-malarial IgG subclass among severe malaria compared to uncomplicated malaria patients. Associations were quanti ed using odds ratios [OR] with 95% con dence intervals [CI] that did not cross 1.00 with P value <0.05, de ned as statistically signi cant. As shown below, each IgG subclass was ranked in malaria-free controls in two categories based on the levels of antimalarial antibodies.  (Table 1). The body temperature was signi cantly different between the study population, P < 0.001 (Table 1).
Comparison between the distribution of the FcγRIIa genotype and its allelic frequencies among the different study groups The genotype frequencies for FcγRIIa, FcγRIIa, and FcγRIIIb did not deviate from the expectations of the HWE in each genotype group (  Table 2). The frequencies for the heterozygotes carrying the FcγRIIa-H/R131 genotype were almost the same among the three groups of MFC, UM, and SM (52.0%, 48.2%, and 51.6%, respectively) ( Table 2).
Comparison between the FcγRIIa genotypes distributions and their allelic distributions among the different study groups.
As shown in Table 2, the genotype frequencies for FcγRIIa, FcγRIIa and FcγRIIIb did not deviate from expectations of the HWE in each genotype group ( Comparison between the distribution of FcγRIIa genotype and its allelic frequencies among the different study groups The genotype frequencies showed no statistically signi cant difference among the homozygotes FcγRIIa-F/F with UM compared to MFC (  Tables 2 and 3). The frequencies analyses also showed differences in the distributions of the heterozygotes FcγRIIa-F/V genotype among the three groups (40% in MFC, 48.2% in UM, and 23.6% in SM) ( Table 2).
The genotype analyses showed similar frequencies between UM and MFC for homozygotes carrying the FcγRIIIb-NA1/NA1 of (  Table 2 and 3). The frequencies analyses also showed differences in the distributions of the heterozygotes FcγRIIa-NA1/NA2 genotype among the three groups (46% in MFC, 48.2% in UM, and 38.5% in SM) ( Table 2).

Speci c IgG subclass reactivity in the different study groups
The antibody responses for the P. falciparum blood-stage antigen AMA-1 was analysed in the different study groups. Our results showed statistically signi cant differences among the anti-malarial IgG subclasses antibody levels in the different study groups; the overall P value < 0.001 (Table 4). In general, the median value of IgG1 and IgG3 subclasses were expressed at higher levels than IgG2 and IgG4 antibodies in the UM group when compared to both SM and MFC subjects (Table 4). To investigate the potential association between the antimalarial IgG subclass responses and protection against infections, we rst, used a logistic regression model to compare the levels of IgG subclasses between the UM infection and MFC (Table 5). The results showed that a higher level of IgG3 against the AMA-1 antigen was associated with UM patients compared to MFC subjects [OR = 2.6; 95% CI (2 to 3.3) and P value < 0.001]. In addition, the levels of AMA-1-speci c IgG4 were signi cantly lowered in UM patients compared MFC [OR = 0.7; 95% CI (0.5 to 1.9) and P value < 0.001] ( Table 5). There was no observed association for the AMA-1-speci c IgG1 and IgG2 responses in UM compared to MFC ( Table 5). The same logistic regression model con rmed that the apparent anti-malarial IgG1 to IgG4 antibodies were signi cantly associated with SM when compared to UM patients (  Table 6). Similarly, our data show that the FcγRIIa-V/V genotype is negatively associated with higher levels of AMA-1-speci c IgG4 in SM compared to UM subjects [OR = 0.4; 95% CI (0.2 to 0.6) and P value < 0.001] (Table 6).
Furthermore, our analyses show that the FcγRIIIb-NA2/NA2 genotype is signi cantly associated with a higher level of AMA-1-speci c IgG4 among SM compared to UM group [OR = 1.7; 95% CI (1.1 to 2.7) and P value= 0.011] ( Table 6). Our results indicate that the FcγRIIIb genotypes are not associated with the independent action of the three IgG subclasses (IgG1, IgG2, and IgG3) of antibodies, maybe due to the absence of interaction in the logistic regression model.

Discussion
This study aimed to evaluate the possible relationship between the variants of FcγRIIa (CD32a), FcγRIIa (CD16a), FcγRIIIb (CD16b) gene polymorphism and P. falciparum AMA-1-speci c IgG subclass and its importance in the susceptibility to complicated malaria infections among children in Saudi Arabia. To our best knowledge, this study is the country's rst report investigating this association among children.
The data of this investigation suggested that there was no signi cant impact of the FcγRIIa-R/H131 genotypes polymorphism on the susceptibility to UM infection compared to MFC. This nding is in parallel with the previously published report from Eastern Sudan by Giha and co-workers which suggested the lack of statistically signi cant association between FcγRIIa-R/H131 and HbAS genotypes polymorphism on immunity and susceptibility to UM infection [24]. This may be due to the similarities in malaria epidemiology, malaria transmission, and patient's semi-immunity to malaria infection [25,26].  [28]. Previously published case-control investigation have demonstrated that FcγRIIa-R/R131 homozygosity is associated with protection against high parasite density [27], and the genotypes of FcγRIIa-H/H131 are correlated with high risk of either severe malaria or placental malaria [29][30][31]. In addition, several hospitalbased case-control reports have con rmed the association between FcγRIIa-H/H131 and protection against bacterial infections, while FcγRIIa-R/R131 is associated with increased susceptibility to similar bacterial infections [32][33][34]. Interestingly, we found here that the levels of IgG1, IgG2, and IgG4 are associated with FcγRIIa-H/H131 in the UM patients. Similar results have been suggested by Nasr et al. among the Fulani ethnic group that are less susceptible to severe malaria infection [19]. In contrast, previous data on pregnant women with asymptomatic malarial infection (ASM) revealed that the high levels of AMA-1-speci c anti-malarial IgG1, IgG2, and IgG4 antibodies are statistically associated with R/R131 carriers rather than the genotype FcγRIIa-H/H131 [26]. This contradiction may be due to the different levels of malaria endemicity, variations in the individual's genetic background, and the variations in the study designs. The results of this study suggest that the relative reduction in malaria infection in the UM group cannot be explained solely by the magnitude and quality of humoral response to malaria. Additional studies are needed to clarify whether the FcγRIIa-R/H131 polymorphism is a causative factor in the variance predisposition to malaria that is demonstrated among the different groups.
This study also revealed that the FcγRIIa-F/V176 genotypes are not associated with UM patients compared to MFC. On the other hand, the FcγRIIa-F/F176 genotype is statistically associated with SM compared to UM patients. However, patients carrying the FcγRIIa-V/V176 genotypes are statistically associated with protection against SM compared to UM. The latter nding is in line with a recent Kenyan study which shows that the polymorphisms in the FcγRIIa-V/V are associated with protection against severe malarial anaemia and modulations in circulating IFNγ levels [22]. In contrast, a previous investigation on Thai patients did not show an association between FcγRIIa-F/V176 genotypes and the severity of the disease [31]. Again, these discrepancies may be attributed to the difference in ethnicity and study design.
The current study suggests that individuals carrying the FcγRIIa-F/F genotype are signi cantly expressing higher levels of AMA-1-speci c IgG2 and IgG4 antibodies in the SM group compared to patients with UM.
In agreement with this nding, Koene et al. have shown that the FcγRIIa-F/F is signi cantly less bound to IgG1, IgG3, and IgG4 compared to the FcγRIIa-V/V genotypes [35].
Our results suggest that there are no statistically signi cant differences between UM and MFC for the FcγRIIIbNA1/NA1 and FcγRIIIbNA2/NA2 genotypes. In contrast, the patients carrying the FcγRIIIb-NA2/NA2 genotype are signi cantly associated with SM compared to patients with UM. Recent work on children living in Western Kenya suggests that the FcγRIIIbNA1/NA2 gene polymorphisms are not signi cantly associated with susceptibility to severe malarial anaemia [22]. In addition, the study performed by Adu and co-workers demonstrates that the FcγRIIIb NA2/NA2 in Ghanaian children is associated with clinical malaria [5]. A master's degree thesis published in 2010 has demonstrated an association between the FcγRIIIb-NA2/NA2 and susceptibility to severe and uncomplicated malaria among Ghanaian children [36]. These contradicting results may be attributed to the different malaria transmission settings and malaria epidemic. Moreover, different ethnicity associated with variations in the genetic background may signi cantly contribute to the FcγR gene polymorphism and susceptibility/protection to severe malaria [19].

Conclusion
This study reveals the lack of signi cant in uence of FcγRIIa-R/H131 genotypes polymorphism on the susceptibility to UM infection. Whereas, the FcγRIIa-H/H131 genotype is negatively associated with SM. In addition, the higher levels of antimalarial IgG1, IgG2, and IgG4 antibodies are associated with FcγRIIa-H/H131 in patients with UM. Furthermore, our data show that the FcγRIIa-F/V176 genotypes are not associated with UM. In contrast, the FcγRIIa-F/F176 genotype is correlated with SM. However, patients carrying the FcγRIIa-V/V176 genotype were more protected against the severity of the disease. Patients with severe malaria and carrying the FcγRIIa-F/F genotype also show higher levels of AMA-1-speci c IgG2 and IgG4 antibodies. Furthermore, our data suggest that there are no signi cant statistical differences between UM and MFC for the FcγRIIIb NA1/NA1 and FcγRIIIb NA2/NA2 genotypes. Nevertheless, the FcγRIIIb-NA2/NA2 genotype is signi cantly associated with SM infection. Further studies are underway in our laboratory to elucidate if the FcγRIIa, FcγRIIa, and FcγRIIIb genotypes polymorphism contribute to the differential susceptibility to malaria among the different study groups.

Strength And Limitations
To our knowledge, this is the rst study in the Kingdom of Saudi Arabia highlighted the relation between FcγR genotypes polymorphism, IgG subclass and malaria infection among Saudi children. This will hopefully lead to further research in the area. However, its small sample size and being performed in one region in Saudi Arabia limit the study. Findings need to be con rmed in a large sample size from various regions representing the whole endemic area. Affairs, Ministry of National Guard, Riyadh, Saudi Arabia. Prior to participation, informed consent was also obtained from children and their parents\guardians.

Consent for publication
Not applicable.

Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author upon reasonable request.

Competing interests
The authors declare that they have no competing interests.      .001 † OR represent odds ratios while CI represents con dence intervals. In model (A) uncomplicated versus malaria-free control "MFC": malaria-free controls were assigned 0 uncomplicated malaria were assigned 1 in the logistic regression analysis. OR above 1 represented value higher levels antimalarial IgG subclass associated to uncomplicated malaria while less than 1 value represented malaria-free controls. ¥ In model (B) severe malaria versus uncomplicated malaria: uncomplicated malaria was assigned 0 severe malaria were assigned 1 in the logistic regression analysis. OR above 1 represented value higher levels antimalarial IgG subclass associated to severe malaria while less than 1 value represented uncomplicated malaria.