Polymorphisms in FCGR2A (131H/R) and FCGR2B (232I/T) are associated with the development of inhibitors in Chinese hemophilia A patients

Background: Present study was to explore the association between gene polymorphisms in Fc gamma receptor IIa (FCGR2A) and Iib (FCGR2B) and factor VIII (FVIII) inhibitor development in patients with hemophili A (HA) in a Chinese Han population. Methods: FCGR2A (131H/R) and FCGR2B (232I/T) polymorphsims were genotyped using PCR and direct sequencing method in 108 HA patients, including 23 cases with inhibitors and 85 without inhibitors. Chi-square (c 2 ) test was applied to compare the genotype and allele frequencies between two groups. Odds ratio (OR) and 95% condence interval (95%CI) were calculated to indicate the relative susceptibility of HA. Results: FCGR2A 131RH genotype frequency had a signicantly increased trend in inhibitor group compared with the non-inhibitor group, suggesting a momentous role of 131H/R polymorphism for inhibitor development in HA patients. Individuals carrying 131RH genotype showed higher risk to be attacked by the inhibitor development in HA patients (OR=4.929; 95%CI=1.029-23.605). However, there were no signicant differences of FCGR2B (232I/T) polymorphism genotype and allele frequencies between inhibitor and non-inhibitor groups. Conclusion: FCGR2A (131H/R) was in relation to the susceptibility of FVIII inhibitors development in HA patients.

inhibitor development in HA patients. Individuals carrying 131RH genotype showed higher risk to be attacked by the inhibitor development in HA patients (OR=4.929; 95%CI=1.029-23.605). However, there were no signi cant differences of FCGR2B (232I/T) polymorphism genotype and allele frequencies between inhibitor and non-inhibitor groups.
Conclusion: FCGR2A (131H/R) was in relation to the susceptibility of FVIII inhibitors development in HA patients.

Background
Hemophilia A (HA) is an X-linked recessive bleeding disorder resulting from a quantitative or qualitative de ciency in the factor VIII (FVIII) protein [1][2][3][4][5]. Manifestation of neutralizing antibodies (inhibitors) against infused FVIII protein is the most burdensome complication of HA patients [6]. In China, overall prevalence of FVIII inhibitors in severe HA patients is showed to reach 4.3% [7], which is much lower than the value reported in other ethnic groups. As a typical multifactorial trait, there are several risk factors identi ed for inhibitor formation in HA patients, which divided into two groups as follows: environmental factors and genetic factors [8][9][10]. Main genetic predisposition for inhibitor development in HA patients is the causative FVIII genotype, but it also includes a group of auxiliary risk factors that are weaker than the FVIII genotype, including a family history of inhibitors, ethnicity, human leucocyte antigen haplotype, and polymorphisms of immune system-related genes, such as interleukin (IL)-1, IL-2, IL-10, tumor necrosis factor (TNF)-and cytotoxic T lymphocyte-associated protein (CTLA)-4 [11][12][13][14].
Receptors of the Fc region of IgG (FcγR) are glycoproteins that are usually detected at hematopoietic system. These receptors have a pivotal relevance of cellular with humoral immunity via binding to the Fc domain of the IgG sub-types (IgG1-4). Low-a nity FcγRs (FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa and FcγIIIb) modulate both pro-and anti-in ammatory response, and vary in their a nity with the antibody Fcfragment and in the signaling pathways induced. They are encoded by the FCGR genes (FCGR2A, FCGR2B, FCGR2C, FCGR3A and FCGR3B) clustered on chromosome 1q23-24 [15]. Recently, FCGR gene polymorphisms are demonstrated to be associated with autoimmune diseases. But few is known about the FCGR2A and FCGR2B gene polymorphisms and the inhibitor development of HA.
The current study was aimed at evaluating whether FCGR2A 131H/R (rs1801274) and FCGR2B 232I/T (rs1050501) polymorphisms in uence the risk of inhibitors development in 108 patients with HA in Chinese Han population.

Patients
Among 108 unrelated HA patients, 23 patients had been diagnosed as having FVIII inhibitors, and 85 patients had no inhibitors. All patients received on-demand treatment with plasma-derived FVIII. All noninhibitors patients had more than 5 exposure months. Nijmegen-modi ed Bethesda assay was used for quanti cation of the FVIII inhibitor titre. Inhibitor titres ≧0.6BU/mL at any time were considered to be inhibitor positive [16]. Antibody titers <5BU/mL was de ned as low-response inhibitors, while inhibitor titres persistently ≧5BU/mL were de ned as high-response inhibitors [17]. Severity of haemophilia was classi ed according to the criteria of White et al. [18]. The study was permitted by the medical ethics committee of PanYu Central Hospital Hospital. Subjects were signed the written informed consent.

Detection of polymorphisms
Genomic DNA was extracted from the peripheral blood of every individual using vacuum vessel collection tube. TIANGEN biochemical blood genome DNA extraction Kit (TIANGEN, BEIJING) was used to extract the genomic DNA based on the manufacturer's speci cation. Primers of FCGR2A 131H/R and FCGR2B 232I/T single nucleotide polymorphisms (SNPs) were designed and synthesized by Sangon Biotech (Shanghai) Co., Ltd. Genotyping of the FCGR2A and FCGR2B was conducted by PCR ampli cation and direct sequencing method on an ABI PRISM Sequence Detection System 7500 (Applied Biosystems Foster City. CA. USA) by DNA sequencing.

Statistical analysis
All the data were analyzed by SPSS software 18.0 (SPSS Inc., Chicago, IL, USA). Allele and genotype frequencies were calculated using directing counting. Data in with or without inhibitors groups were compared by c 2 test. Hardy-Weinberg euilibrium (HWE) was detected in these two groups. Odd ratio (OR) and 95% con dence interval (95%CI) were calculated using Chi-square test. P value less than 0.05 was regarded to be statistical signi cant.

Results
Basic information of patients A total of 108 HA patients were enrolled in this study, including 82 cases with severe disease, 21 cases with moderate disease and 5 cases with mild disease. 23 patients had inhibitors, 85 patients did not have inhibitors.

HWE test
Genotypes of FCGR2A 131H/R and FCGR2B 232I/T polymorphisms in HA patients with and without inhibitors did not deviated from HWE test, revealing the reliability of the study samples.
Relationship of FCGR2A (131H/R) and FCGR2B (232I/T) gene polymorphsims with HA susceptibility FCGR2A (131H/R) exhibited signi cant differences between the inhibitor and non-inhibitor groups. Frequency of 131RH genotype was higher in inhibitor group than in non-inhibitor group (P=0.032). When the 131RR genotype was taken as a reference, the 131RH genotype was associated with an increased risk of FVIII inhibitor development (OR=4.929, 95%CI=1.029-23.605). Although 131HH genotype had higher frequency in non-inhibitor group than in inhibitor group, the difference was not signi cant. 131H allele was also had no signo cant associantion with the development of FVIII inhibitor.
232II, 232IT, and 232TT genotype frequencies of FCGR2B 232I/T SNP were 75.29%, 22.35%, and 2.35% in non-inhibitor group and 69.57%, 21.74%, 8.70% in inhibitor group, respectively (Table 1). Besides, the allele 232I and 232T frequencies were 86.47% and 13.53% in non-inhibitor and 80.43%, 19.57% in inhibitor group. But all differences of genotype and allele distributions did not reach a signi cant level (P>0.05). It suggested that 232I/T might not be in relation to the susceptibility of the FVIII inhibitor development in HA patients.

Discussion
As is known to all, cytokines are more or less directly in relation to the antibody-modulated immune responses [19,20]. In autoimmune diseases, polymorphisms in the immune response genes were reported to be related to the formation of antibody. In addition, SNPs in the regulatory regions of cytokine genes are crucial components in the pathogenesis of various diseases, including HA [21,22]. Individual immune response traits might in uence a patient's reaction to exogenous factor VIII. The development of FVIII inhibitor is the main complication of replacement therapy in HA patients. Therefore, it is necessary to recognize indicators which could induce the inhibitors formation for applying appropriate treatments and avoiding the inhibitor response.
FcγR give a link between antigen-speci c antibody and non-sepci c cellular responses of the native immune system. These reports are mainly detected at haemopoietic cells, and have important roles for activating and modulating immune responses. In human autoimmune diseases, published studies have demonstrated that genetic variants in FcγRs, particularly FCGR2A 131H/R, FCGR2B 232I/T, were showed to be in relation to rheumatoid arthritis, systemic lupus erythematosus [23,24]. In the autoimmune disorders above mentioned, FCGR2A 131H/R polymorphism in uences FcγRIIa a nity to IgG2, phagocytosis, and immune complexes clearance. Nevertheless, to date, no examination was performed about the association of FcγR polymorphisms with the inhibitor development in HA patients.
In present study, association of FCGR2A and FCGR2B SNPs with inhibitors development was detected in HA patients in a Chinese Han Population. When the allelic and genotypic frequencies of FCGR2A 131R/H were compared, 131RH genotype frequency showed remarkable increase trend in inhibitor group, and 131RH genotype carriers had higher risk to be attacked by inhibitors development. It was suggested that FCGR2A 131R/H displayed signi cant association with positive inhibitor development in HA patients. In recent years, increasing studies have reported that there are signi cant differences between SNPs of the FCGR genes and their association with the inhibitors development of HA patients in different ethnic groups. In a Caucasian population, the polymorphism 131R>H of FCGR2A gene was in relation to an increased risk of inhibitor development [25]. Our study results were in accordance with the previous evidences. Meanwhile, FCGR2A has also been largely researched in other human diseases. For instance, Schneider et al. reported that FCGR2A 131R/H polymorphism is correlated with impaired endotheliumdependent vasodilatation [26]. However, genotype and allele distributions of FCGR2B 232I/T polymorphism had no signi cant difference between inhibitor and non-inhibitor groups. It suggested that FCGR2B 232I/T polymorphism had no signi cant correlation with FVIII inhibitor development in HA patients.

Conclusion
Taken together, FCGR2A 131R/H polymorphism was closely correlated with FVIII inhibitor development of HA patients in the Chinese Han Population, while no association was found between FCGR2B 232I/T polymorphism and FVIII inhibitor development.
Even so, some shortages remains to be stated in the present study. Firstly, the sample size might be not large enough for the correlation analysis. Secondly, the OR value was not corrected by clinical data, which may reduce the statistical power for the determination of differences between two groups. In additional, functional study would also be necessary to identify the molecular mechanisms underlying the association. Further researches should be performed to explore whether the FCGR2A and