This multicenter and retrospective case-control study was performed following IRB approval from An-Najah National University/Palestine (Mas. Nov. 2023/30) and was approved by the Palestinian Ministry of Health/Education in Health and Scientific Research Unit. In the absence of any official data on the prevalence of ITP in Palestine, between February and May of 2024, we retrieved medical data from the hospital information system on documented cases of children with ITP who were referred to pediatric units at four different hospitals: the Martyr Dr. Khalil Suleiman Governmental Hospital in Jenin, the Palestine Medical Complex in Ramallah, Rafidia Governmental Surgical Hospital and An-Najah National University Hospital, both located in Nablus, between 2019 and 2023. Control samples were kindly obtained from Medicare labs in Palestine and from the Pediatric Unit of Rafidia Government Surgical Hospital in Nablus city. ITP cases were diagnosed based on appropriate history taking, clinical presentation, and laboratory findings to rule out other possible causes of thrombocytopenia. There were 105 registered cases (49 females and 56 males): 53 acute, 39 chronic, six persistent, and seven refractories. All demographic and clinical information regarding them was retrieved from the Hospitals’ information system. Patients with secondary immune thrombocytopenia, younger than one year and older than eighteen, who refused to sign informed consent were excluded.

Ethylenediaminetetraacetic acid (EDTA) blood samples (3–5 ml) were obtained from each participant, and then the genomic DNA extraction was performed using the Gene JET™ Whole Blood Genomic DNA purification Mini Kit (Thermo Scientific, catalog number K0782). The extracted DNA was stored at −20 °C until use.

The FcγRIIa-131R/H genotyping was performed using the PCR-restriction fragment length polymorphism (PCR-RFLP) method. The primers for the FCGR2A gene were designed according to the protocol described by (9), as specified in Table 1. After that, the amplified PCR product was digested at 60 °C for 2 h using 20U of the BstUI restriction endonuclease (New England Biolabs). Digested products were electrophoresed on a 3.5% agarose gel. The R alleles generated two 316 and 21 bp fragments, while the FCGR2A H allele was visualized as a 337 bp fragment (Figure 1) as described by Cartron et al. (9).

FCGR3A–158V/F genotyping was performed as previously described by Cartron et al. using nested PCR followed by allele-specific restriction enzyme digestion. The initial PCR assay to amplify a 1.2 kb fragment. Then, a second PCR reaction was done to amplify part (94 bp) of the amplified DNA produced by the first reaction using primers shown in Table 1. Then, the amplified DNA was digested with 10U NLaIII restriction endonuclease (Thermo-Scientific) at 37 °C for 2 h. DNA fragments were visualized on 3.5% gel; the V allele generated two pieces of 61 and 33 bp, while the CFGR3A F allele manifested as a 94 bp fragment (Figure 2) (9).

For both polymorphisms, random samples were selected and tested in duplicate to ensure the result. The results were read by two lab technicians, and a positive or mutant sample was included in each run.

The Statistical Package for Social Science (IBM SPSS statistics 21 for Windows) was used to analyze the data. The range, mean, standard deviation, and median are how numerical data are presented. The chi-square test determined the genotype distribution between patients and controls. The odds ratio (OR) and 95% confidence interval (CI) were computed to estimate risk. A probability value (P-value) less than 0.05 is considered statistically significant.

Genotyping of the FcγRIIa H131R polymorphism (Figure 1) was performed on the collected samples using the PCR-RFLP method. Figure 1 displays a representative image of the agarose gel electrophoresis results. Lane L contains a 50 bp DNA ladder, which was used as a molecular weight marker to confirm the size of the digested fragments. Subsequent lanes illustrate the three possible genotypes. The FcγRIIa H allele was identified by a 337 bp fragment, while the R allele was identified by a 316 bp fragment following digestion with the BstUI restriction enzyme. The smaller 21 bp fragment resulting from the R allele digestion was not visible on the 3.5% agarose gel. The following distinct banding patterns corresponded to the different genotypes:

The distribution of these genotypes was determined for the entire sample cohort, revealing a clear visual representation of each genotypic class.

Genotyping for the FcγRIIIa V158F polymorphism was conducted using the PCR-RFLP method, and the results of the gel electrophoresis are presented in Figure 2.

The leftmost lane (L) contains a 50 bp DNA ladder, which was used to determine the sizes of the DNA fragments. The analysis revealed three distinct banding patterns, corresponding to the FF, VV, and FV genotypes.

These distinct banding patterns enabled the successful classification of each sample into its corresponding FcγRIIIa V158F genotype.

To understand the characteristics of immune thrombocytopenic purpura (ITP) cases within our clinical setting, it was essential to initially examine the broader patient demographics and clinical profiles. Table 2 summarizes the demographic and clinical characteristics of the registered ITP cases (N = 105) and details the subset of participants (N = 40) included in the study between 2019 and 2023. Overall, among the 105 registered ITP cases, there were 56 males (53.3%) and 49 females (46.7%). In terms of disease duration, 53 cases (50.5%) were classified as acute, 39 cases (37.1%) as chronic, six cases (5.7%) as persistent, and seven cases (6.7%) as refractory. The registered cases were distributed as follows: Rafidia Hospital (33 cases, 31.4%), followed by NNUH (21 cases, 20.0%), PMC (17 cases, 16.2%), NNUH/Rafidia (16 cases, 15.2%), Jenin (15 cases, 14.3%), and NNUH/Jenin (three cases, 2.9%). Of these, 40 participants were selected for further analysis. Within the male group, 20 out of 56 cases (35.7%) were included, while 20 out of 49 female cases (40.8%) were selected. To this end, 120 participants were enrolled, including 40 ITP patients (representing 38% of the 105 registered cases) and 80 age- and sex-matched healthy controls. The ITP group, with a mean age of 6.76 ± 4.13 years, consisted of an equal number of males (20; 50%) and females (20; 50%), while the control group included 44 males (55%) and 36 females (45%). Among the ITP patients, the disease was categorized as acute in 45% of cases, chronic in 42.5%, persistent in 7.5%, and refractory in 5% (Table 3). Statistical analysis revealed no significant difference in gender distribution between the patient and control groups (p = 0.605), thereby confirming that the demographic characteristics of the two groups were comparable. In conclusion, the balanced distribution of demographic variables and the detailed categorization of ITP duration in the patient group provide a robust foundation for the subsequent analysis of FCGR2A and FCGR3A polymorphisms. This representativeness minimizes potential confounding factors and strengthens the validity of the genetic association analyses conducted in this study.

Investigating FCGR2A and FCGR3A polymorphisms in this cohort were to determine whether variations in these IgG-binding receptors could influence ITP susceptibility and clinical presentation. Table 4 compares the genotype and allele frequencies of both polymorphisms between 40 ITP patients and 80 healthy controls. For FCGR2A, the frequencies of the HH, HR, and RR genotypes in the ITP patient group were 17.5%, 62.5%, and 20.0%, respectively, whereas in the control group, these genotypes were 25.0%, 52.5%, and 22.5%. No statistically significant difference emerged (p = 0.543). A notable observation was that the heterozygous HR genotype occurred in approximately 52% of female patients and in 52% of male controls. Allele frequencies (H and R) also showed no significant difference between groups (p = 0.82). Similarly, for FCGR3A, the ITP patients exhibited FF, FV, and VV genotypes at 25.0%, 55.0%, and 20.0%, respectively, compared with 23.8%, 53.8%, and 22.5% in controls (p = 0.950). Although a higher frequency of the FV phenotype was noted in female patients (59.09%) compared with male controls (60.46%), this did not reach statistical significance. Allele frequencies (F and V) were comparable between the two groups (p = 0.88). In conclusion, neither FCGR2A nor FCGR3A polymorphisms showed a statistically significant association with ITP in this cohort. These findings suggest that, at least in this population, variations in these Fc gamma receptors do not appear to play a major role in determining disease susceptibility or phenotype.

To determine if specific FCGR2A131H/R and FCGR3A158F/V genotype distributions are associated with increased susceptibility to ITP, particularly in relation to gender, potentially guiding personalized diagnostic approaches. Data represented in Table 5 demonstrated gender distribution analysis and revealed that the prevalence of FCGR2A131 HH genotype among male ITP patients was 57.4% as compared to 42.8% in their female counterparts in a ratio of 1.33:1 (M: F ratio). In parallel, the prevalence of HH genotype among male healthy control was 35% as compared to 65% in their female counterparts in a ratio of 0.53:1 (M: F ratio), indicating a higher frequency of HH allele variation of the FCGR2A131 among ITP male patients. The prevalence of FCGR2A131 HR genotype among male ITP patients was 48% as compared to 52% in their female counterparts in a ratio of 0.9:1 (M: F ratio). In parallel, the prevalence of HR genotype among male healthy control was 52.5% as compared to 40.47% in their female counterparts in a ratio of 1.29:1 (M: F ratio), indicating a lower frequency of HR allele variation of the FCGR2A131 among ITP male patients. The RR population shows an equal distribution between male and female ITP patients and controls, with both groups having 50% of each gender in this category in a ratio of 1:1 (M:F ratio).

Moreover, analysis of the FCGR3A158F/V genotype revealed that the prevalence of FF genotype among ITP male was 30% as compared to 70% in their female counterparts in a ratio of 0.42:1 (M: F ratio). In parallel, the prevalence of FF genotype among male healthy control was 36.8% as compared to 63.2% in their female counterparts in a ratio of 0.58:1 (M: F ratio), indicating a similar frequency of FF allele variation of the FCGR2A131 among ITP male patients. On the other side, FV genotype among ITP male was 40.9% as compared to 59.09% in their female counterparts in a ratio of 0.69:1 (M: F ratio). In parallel, the prevalence of FV genotype among male healthy control was 60.46% as compared to 39.3% in their female counterparts in a ratio of 1.53:1 (M: F ratio), indicating a lower frequency of FV allele variation of the FCGR3A158F/V genotype among ITP male patients. Additionally, In ITP patients, the VV allele was more prevalent in males (62.5%) than in females (37.5%), representing a male-to-female ratio of 1.66:1. In parallel, the prevalence of VV genotype among male healthy control was 38.9% as compared to 61.1% in their female counterparts in a ratio of 0.63:1 (M: F ratio), indicating a lower frequency of VV allele variation of the FCGR3A158F/V genotype among healthy male patients.

In conclusion, the findings suggest that there are notable gender-specific differences in the distribution of FCGR2A131H/R and FCGR3A158F/V genotypes among ITP patients, with distinct variations observed between male and female subjects. Specifically, the higher frequency of the FCGR2A131HH genotype in male ITP patients compared to healthy male controls, along with a lower frequency of the HR genotype in male patients, indicates a potential association with increased susceptibility to ITP in males. Similarly, the FCGR3A158F/V genotype analysis shows a trend of lower frequencies of FV and VV genotypes in male ITP patients compared to their healthy counterparts, further supporting the possibility of gender-specific genetic predispositions to ITP. These findings suggest that gender may play a significant role in the distribution of specific genotypes in ITP, potentially aiding in the development of more personalized diagnostic and therapeutic approaches for the condition.

Analysis of combined genotypes of FCGR2A and FCGR3A polymorphisms revealed variability in disease severity among the studied cases (Table 6). The genotype combination FV-HR was the most prevalent, representing 32.5% of cases, primarily associated with chronic presentations (69.2%), followed by acute (23.1%) and persistent (7.7%) cases, with no refractory occurrences. Conversely, combinations FV-RR and VV-RR exclusively correlated with acute cases (100%), though these genotypes had low absolute frequencies (10% and 2.5%, respectively). The VV-HH combination showed significant severity, with 50% refractory cases, indicating a potential association with treatment resistance despite its low prevalence (5%). FF-HR exhibited a diverse clinical profile, predominantly chronic (57.1%) but also associated with acute, refractory, and persistent presentations. No cases were recorded for the FF-HH genotype combination. These findings suggest that specific combinations of FCGR2A and FCGR3A polymorphisms are linked with distinct clinical outcomes, potentially guiding prognosis and tailored therapeutic approaches.