Significant differences in FcγRIIa, FcγRIIIa and FcγRIIIb genes polymorphism and anti-malarial IgG subclass pattern are associated with severe Plasmodium falciparum malaria in Saudi children

Background The FcγRs 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. Methods A total of 600 volunteers were 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 were analysed using PCR amplification methods, and measurements of immunoglobulin were determined using enzyme-linked immunosorbent assay (ELISA) technique. Results The data revealed that the FcγRIIa-R/R131 showed a statistically significant association with SM patients when compared to UM patients. Furthermore, higher levels of IgG1, IgG2, and IgG4 were associated with the FcγRIIa-H/H131 genotype among UM patients. Although the FcγRIIa-F/V176 genotype was not associated with UM, it showed a significant association with severe malaria. Interestingly, the FcγRIIIa-V/V176 genotype offered protection against SM. Moreover, SM patients carrying the FcγRIIIa-F/F genotype showed higher levels of AMA-1-specific IgG2 and IgG4 antibodies. The FcγRIIIb-NA1/NA1 and FcγRIIIb-NA2/NA2 genotypes did not show significant differences between the UM and the MFC groups. However, the genotype FcγRIIIb-NA2/NA2 was statistically significantly associated with SM patients. Conclusions The data presented in this study suggest that the influence of the FcγRIIa-R/R131, FcγRIIIa-F/F176 and FcγRIIIb-NA2/NA2 genotypes are statistically significantly associated with SM patients. However, the FcγRIIa-H/H13 and FcγRIIIa-V/V176 genotypes have demonstrated a protective effect against SM when compared to UM patients. The impact of the FcyR (IIa, IIIa and IIIb) gene variants and anti-malaria IgG subclasses play an important role in susceptibility to malaria infection and disease outcome in Saudi children.

falciparum, which is most prevalent in Africa, and Plasmodium vivax pose the greatest threat to health. In Saudi Arabia, P. falciparum represents about 99% of the total cases of malaria, while only 1% of patients are infected by P. vivax. In 2019, malaria affected about 229 million worldwide and contributed to 409,000 deaths. Children below the age of five were amongst the most vulnerable groups affected by the disease [1]. According to the WHO figures, between 2010-2015 in Saudi Arabia, the number of recorded malaria patients was steadily below 100, but it rose to 272 cases in 2016. This was mostly due to increased migration of people from war zones along the border with Yemen, as well as difficulties in providing adequate medical services in those regions. However, the health service in this country remains vigilant and offers free diagnosis and treatment for all patients.
Given the increase in the number of malaria infections and its apparent threat to people's lives; there was a call for further studies that can assess individual's susceptibility. This led to the current study in which the authors looked at genes (FcγR) within the innate immunity that are responsible for receptor expression on immune cells (including macrophages, neutrophils, NK cells). These cell receptors have the ability to recognize certain antibodies that will bind to antigens, such as antigens of P. falciparum.
There are three sub-families of surface receptors for the Fc region of the IgG, designated as FcγRI, II, and III [2]. Most immune cells express Fc receptors that are crucial for determining the specificity of IgG antibodies [3]. FcγR induces monocyte activation features such as phagocytosis, degranulation, superoxide generation, antibody-dependent cell inhibition, cytokine production, and antibody regulation, which are essential for host defence and immune regulation [4,5]. The effectiveness of IgGinduced FcγR activity demonstrates inter-individual heterogeneity due to the genetic polymorphisms of the three subclasses of FcγR; FcγRIIa (CD32a), FcγRIIIa (CD16a), and FcγRIIIb (CD16b) [5].
Previous studies showed that several polymorphisms have been detected in the Fcγ genes encoding these receptors (FcγRs), associated with susceptibility or resistance to malaria outcome in different populations [3,4,[6][7][8][9][10][11][12]. A recent review by Amiah et al. [13] described the FcγRs polymorphisms and the impact of these variations on the response of the host to infection. It also provided new perspectives for the potential design of an effective malaria vaccine [13].
The present study aimed to investigate the possible relationship between the expression of FcγRIIa (CD32a), FcγRIIIa (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 [3,[14][15][16].

Study design and patients
A prospective case-control study was conducted in children attending the outpatient clinic of BMGHJ, with a confirmed clinically diagnosed P. falciparum infection. Patients with positive thick blood film for P. falciparum asexual parasites were recruited based on the microscopic diagnosis.
Participants with no features of severe malaria were defined 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), significant bleeding, pulmonary oedema, and shock as described in detailed by the World Health Organization (WHO) [17]. These clinical manifestations occurred in the absence of any identifiable alternative cause other than P. falciparum asexual parasitaemia. Children with cerebral malaria had a Blantyre Coma Score (BCS) < 3 at 4 h post-admission. Children with severe malarial anaemia had a blood haemoglobin concentration of ˂ 5 g/dL or a haematocrit value of < 15% together with a parasite count above 10,000/μL. All other children recruited in the study had a haemoglobin concentration above this level. The control group were selected from the Child and Woman Health Clinics (CWHC) that provide children health services including routine vaccinations, as well as providing seasonal vaccines for children. Once the sample was collected, it was matched for age, gender, and ethnicity. Enrolment to the control group was confirmed following a physical clinical examination to ensure that the children did not have serious illnesses or any signs/symptoms of malaria according to information provided by parents/guardians.
The study excluded children with multiple severe malaria complications or any co-infectious diseases. None of the participants were 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 filter paper (Qualitative filter paper, Grade 1, circles, diam. 42.5 mm from Whatman ® , Sigma-Aldrich ® ). This collected sample was used for investigating Fcγ receptor gene polymorphism, parasite detection using PCR, and measurement of immunoglobulins as described earlier [18,19].

Serum elution from filter-paper samples
To elute dried samples from filter-paper, a hole puncher of φ 6 mm was used for punching out filter-paper discs and placed in Eppendorf tubes with 100 µL of phosphate-buffered saline (PBS). Subsequently, the discs were transferred onto 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 h at room temperature. After incubation, the samples were vigorously shaken with a vortex for 30 s, 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 [18].

DNA extraction
DNA was extracted from 50 µL dried drop of blood sample on the filter 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.

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 [20].

Enzyme-linked immunosorbent assays (ELISA)
IgG subclass 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 [11,21], and as recently reported [22].

Statistical analysis
Statistical analysis was done by SPSS statistical software version 23 for Windows (IBM© SPSS © statistics). In this study, the median and 25% and 75% quartile of antibody (total IgG and IgG subclasses) levels were analysed using nonparametric (Kruskal-Wallis) tests and the P values were determined. With respect to the risk of malaria infection in children, all values of P < 0.05, 95% confidence interval (CI) for odds ratio (OR) that did not cross 1.00 were considered statistically significant. In the analysis, FcγRIIa-R/H131 polymorphism was used as a reference, due to its utmost prevalence in humans [24]. Using the same software, a 2 × 2 chisquare test was used to compare the overall allele frequency. The Hardy-Weinberg equilibrium (HWE) for genotypic deviation was assessed using a chi-squared statistical test. The logistic regression analysis was performed to test for the association between the FcγRs genotypes related to higher levels of anti-malarial IgG subclass among severe malaria compared to uncomplicated malaria patients. Associations were quantified using OR with 95% CI that did not cross 1.00 with P value < 0.05, defined as statistically significant. As shown below; each IgG subclass was ranked in malariafree control in two categories based on the levels of anti-malarial antibodies.

Classification of the study participants
In this study, demographic data on malaria, parasite density, and disease complication variables were analysed for 600 children of matched gender and age. The 600 subjects were categorised into three different groups.  Table 1). The mean number of parasites in severe malaria patients was significantly higher compared to uncomplicated malaria, P < 0.001 (Table 1). The body temperature was significantly different between the study populations, 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 did not deviate from the expectations of the HWE in each genotype group (  Table 3). The frequencies of 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 distribution of FcγRIIIa genotype and its allelic frequencies among the different study groups
The genotype frequencies showed no statistically significant difference among the FcγRIIIa-F/F in UM compared to MFC ( Table 2). The logistic regression analysis confirmed the absence of significant differences between UM and MFC among the individuals carrying the FcγRIIIa-F/F genotypes [39% in UM versus   Table 2 and 3). The frequency analyses showed differences in the distributions of the heterozygote FcγRIIIa-NA1/NA2 genotype among the three groups (46% in MFC, 48.2% in UM, and 38.5% in SM) ( Table 2).

Specific IgG subclass reactivity in the different study groups
The antibody response for the P. falciparum blood-stage antigen AMA-1 was analysed within the different study groups. The current results showed statistically significant differences among the anti-malarial IgG subclass antibody levels in the different study groups; the overall P value < 0.001 (Table 4). In general, the median value of IgG1 and IgG3 subclass were expressed at higher levels than IgG2 and IgG4 antibodies in the UM group when compared to SM subjects (Table 4). To investigate the potential association between the anti-malarial IgG subclass response and protection against infections; the authors first 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 IgG1 against the AMA-1 antigen was associated with UM patients compared to MFC subjects [OR = 1.04; 95% CI (1.01 to 1.07) and P value = 0.012]. In addition, the levels of AMA-1-specific IgG3 were significantly higher in UM patients compared MFC [OR = 1.70; 95% CI (1.47 to 1.95) and P value < 0.001] ( Table 5). There was no observed association for the AMA-1-specific IgG2 and IgG4 responses in UM compared to MFC (Table 5). The second logistic regression model confirmed that the apparent anti-malarial IgG2 and IgG4 antibodies were statistically significantly higher in SM patients when compared to UM patients (Table 5). However, the levels of AMA-1-specific IgG1 and IgG3 were significantly lower in SM group compared to UM patients [for IgG1: OR = 0.89; 95% CI (0.84 to 0.94) and P value < 0.001 and for IgG3: OR = 0.52; 95% CI (0.43 to 0.62) and P value < 0.001)] ( Table 5).
Furthermore, the analyses of the results show that the FcγRIIIb-NA2/NA2 genotype is significantly associated with a higher level of AMA-1-specific IgG4 among SM compared to UM group [OR = 1.7; 95% CI (1.1 to 2.7) and P value = 0.011] ( Table 6). The current results indicate that the FcγRIIIb genotypes are not associated with the independent action of the three IgG subclasses (IgG1, IgG2, and IgG3) of antibodies, and maybe due to the absence of interaction in the logistic regression model.   Table 5 Logistic regression analysis of malaria specific (anti-AMA1) IgG subclasses levels among the different study groups a OR represent odds ratios while CI represents confidence intervals. In model (A) uncomplicated versus malaria-free control "MFC": malaria-free controls were assigned 0 uncomplicated malaria patients 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 b In model (B) severe malaria versus uncomplicated malaria: uncomplicated malaria was assigned 0 severe malaria patients 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

Discussion
This study aimed to evaluate the possible relationship between the variants of FcγRIIa (CD32a), FcγRIIIa (CD16a), FcγRIIIb (CD16b) gene polymorphism and P. falciparum AMA-1-specific IgG subclass and its importance in the susceptibility to complicated malaria infections among children in Saudi Arabia. This study is the country's first report investigating this association among children.
The data of this investigation suggested that there was no significant impact of the FcγRIIa-R/H131 genotypes polymorphism on the susceptibility to UM infection compared to MFC. This finding is in parallel with the previously published report from Eastern Sudan by Giha Table 6 Logistic regression analysis of individual carrying FcγRIIa, FcγRIIIa, FcγRIIIb genotypes in relation of the levels specific IgG subclasses associated with uncomplicated compared to severe malaria a OR represent odds ratios while CI represents confidence intervals. "OR" adjusted with parasite density. Higher levels of antimalarial IgG subclasses among uncomplicated malaria patients were assigned 0 while higher levels antimalarial IgG subclasses among severe malaria patients were assigned 1 in the logistic regression analysis. OR above 1 represented value associated to higher levels antimalarial IgG subclasses among severe malaria patients while less than 1 value represented higher levels antimalarial IgG subclasses among uncomplicated malaria patients and co-workers, which suggested the lack of statistically significant association between FcγRIIa-R/H131 genotypes polymorphism on immunity and susceptibility to UM infection [25]. This may be due to the similarities in malaria epidemiology, malaria transmission, and patient's semi-immunity to malaria infection [3,16].  [11]. In contrast, previous data on pregnant women with asymptomatic malarial infection (ASM) revealed that the high levels of AMA-1-specific anti-malarial IgG1, IgG2, and IgG4 antibodies are statistically associated with R/R131 carriers rather than the genotype FcγRIIa-H/H131 [3]. This contradiction may be due to the variation in the individual's genetic background, and variation in 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 the humoral response to malaria. Additional studies are needed to clarify whether the FcγRIIa-R/H131 polymorphism is a causative factor in the variable predisposition to malaria that is demonstrated among the different groups.
This study also revealed that the FcγRIIIa-F/V176 genotypes are not associated with UM patients compared to MFC. On the other hand, the FcγRIIIa-F/ F176 genotype is statistically associated with SM compared to UM patients. However, patients carrying the FcγRIIIa-V/V176 genotypes are statistically associated with protection against SM compared to UM. The latter finding is in line with a recent Kenyan study, which shows that the polymorphisms in the FcγRIIIa-V/V are associated with protection against severe malaria and modulations in circulating IFNγ levels [12]. In contrast, a previous investigation on Thai patients did not show an association between FcγRIIIa-F/V176 genotypes and the severity of the disease [29]. Again, these discrepancies may be attributed to the difference in ethnicity and study design.
The current study suggests that individuals carrying the FcγRIIIa-F/F genotype are significantly expressing higher levels of AMA-1-specific IgG2 and IgG4 antibodies in the SM group compared to patients with UM. In agreement with this finding, Koene et al. have shown that the FcγRIIIa-F/F is significantly less bound to IgG1, IgG3, and IgG4 compared to the FcγRIIIa-V/V genotypes [30].
The study's results suggest that there are no statistically significant differences between UM and MFC for the FcγRIIIb-NA1/NA1 and FcγRIIIb-NA2/NA2 genotypes. In contrast, the patients carrying the FcγRIIIb-NA2/NA2 genotype are significantly associated with SM compared to patients with UM. Recent work on children living in Western Kenya suggests that the FcγRIIIb-NA1/NA2 gene polymorphisms are not significantly associated with susceptibility to severe malaria [12]. In addition, the study performed by Adu et al. have demonstrates that the FcγRIIIb-NA2/NA2 in Ghanaian children is associated with clinical malaria [4]. In 2010, Adu demonstrated an association between the FcγRIIIb-NA2/NA2 and susceptibility to severe and uncomplicated malaria among Ghanaian children [31]. These contradicting results may be attributed to the different malaria transmission seasons and malaria epidemics. Moreover, different ethnicity associated with variations in the genetic background may significantly contribute to the FcγR gene polymorphism and susceptibility/protection to severe malaria [11].

Strength and limitations
To the best of the authors knowledge, this is the first study in the Kingdom of Saudi Arabia which 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. Some of the studies limitations include the small sample size and the fact that the study was performed in one region of Saudi Arabia instead of it being multicentred. As such findings need to be confirmed in a large sample size from various regions representing the whole endemic area.