Fc‐gamma IIIa‐V158F receptor polymorphism contributes to the severity of Guillain‐Barré syndrome

Abstract Objective Guillain‐Barré syndrome (GBS) is a rare, life‐threatening disorder of the peripheral nervous system. Immunoglobulin G Fc‐gamma receptors (FcγRs) mediate and regulate diverse effector functions and are involved in the pathogenesis of GBS. We investigated whether the FcγR polymorphisms FcγRIIa H/R131 (rs1801274), FcγRIIIa V/F158 (rs396991), and FcγRIIIb NA1/NA2, and their haplotype patterns affect the affinity of IgG‐FcγR interactivity and influence GBS susceptibility and severity. Methods We determined FcγR polymorphisms in 303 patients with GBS and 302 ethnically matched healthy individuals from Bangladesh by allele‐specific polymerase chain reaction. Pairwise linkage disequilibrium and haplotype patterns were analyzed based on D ´statistics and the genotype package of R statistics, respectively. Logistic regression analysis and Fisher’s exact test with corrected P (Pc) values were employed for statistical comparisons. Results FcγRIIIa‐V158F was associated with the severe form of GBS compared to the mild form (P = 0.005, OR = 2.24, 95% CI = 1.28–3.91; Pc = 0.015); however, FcγR genotypes and haplotype patterns did not show any association with GBS susceptibility compared to healthy controls. FcγRIIIa‐V/V158 and FcγRIIIb‐NA2/2 were associated with recent Campylobacter jejuni infection (P ≤ 0.001, OR = 0.36, 95% CI = 0.23–0.56; Pc ≤ 0.003 and P = 0.004, OR = 1.70, 95% CI = 1.18–2.44; Pc ≤ 0.012, respectively). Haplotype 1 (FcγRIIa‐H131R‐ FcγRIIIa‐V158F‐ FcγRIIIb‐NA1/2) and the FcγRIIIb‐NA2/2 genotype were more prevalent among anti‐GM1 antibody‐positive patients (P = 0.031, OR = 9.61, 95% CI = 1.24–74.77, Pc = 0.279; P = 0.027, OR = 1.62, 95% CI = 1.06–2.5, Pc = 0.081, respectively). Interpretation FcγR polymorphisms and haplotypes are not associated with susceptibility to GBS, though the FcγRIIIa‐V158F genotype is associated with the severity of GBS.


Introduction
Guillain-Barr e syndrome (GBS) is a post-infectious autoimmune disorder of the peripheral nervous system that can lead to significant morbidity, long-term disability or death.
Cross-reactive immune responses induced by molecular mimicry between the outer core structure of infectious agents that trigger GBS and host nerve gangliosides 1 result in a blockade of nerve conduction. 1,2 Campylobacter jejuni has been identified as the predominant causative microbial infectious agent in GBS. [3][4][5] In addition to multifarious microorganism-derived factors, host immunogenic factors are likely to affect GBS susceptibility as only a subset of C. jejuni-infected individuals (1 in 1000-5000 cases) develop GBS. [6][7][8][9] Natural variations in genetic host susceptibility factors have become a focus of research on the susceptibility and severity of disease pathogenesis in GBS.
Immunoglobulin G Fc-gamma receptors (FccRs) are important immune-response modulating molecules that link the cellular and humoral immune system by interacting with IgG subtypes (IgG1-4). The most common autoantibodies in GBS are produced against GM1, GD1a and GQ1b gangliosides. 5,10,11 These autoantigens may influence nerve disruption, demyelination or axonal degeneration via diverse mechanisms, 12 including induction of inflammatory immune responses, by interacting with Fc receptors. FccR polymorphisms can determine the vigor of inflammatory responses, affect downstream functions such as phagocytosis, antibody-dependent cellular cytotoxicity (ADCC) and the release of inflammatory mediators, and have been implicated in the development of autoimmune disease. 13,14 Thus, FccRs may represent important effector molecules in the pathogenesis of GBS. 15 Three subclasses of FccRs, namely FccRIIa, FccRIIIa and FccRIIIb, exhibit allelic variation. 13,16 The most widely distributed receptor, FccRIIa, is expressed on all types of white blood cells and has two allelic forms: FccRIIa-H131 and FccRIIa-R131. These alleles differ by the replacement of histidine by arginine at position 131 due to an A ? G single nucleotide exchange at position 494. 17,18 FccRIIa-H131 is reported to bind human IgG2 with a higher affinity than FccRIIa-R131. 19 FccRIIIa is expressed on macrophages, dendritic cells, c/d T-cells and natural killer (NK) cells. 20 A functional polymorphism at nucleotide 559 results in either a valine (V) or phenylalanine (F) at amino acid position 158, which affects the receptor binding capacity of IgG1, IgG3, and IgG4. 21 FccRIIIb is expressed on neutrophils and exhibits two allelic forms, neutrophil antigen 1 (NA1) and neutrophil antigen 2 (NA2). NA1 and NA2 differ by five base substitutions (nucleotides 141, 147, 227, 277, and 349) that lead to four amino acid changes (at positions 36, 65, 82, and 106) within exon 3. 18,22 However, these allelic forms of FccR (NA1/NA2) have different affinities for IgG1 and IgG3. Thus, the various allelic forms of FccR may possibly determine the extent of inflammatory responses and thereby influence autoimmune diseases, including GBS.
Several studies have already evaluated the relationship between FccR polymorphisms and the pathogenesis of GBS. [23][24][25][26][27] FccRIIa-H/H131 was significantly associated with susceptibility to GBS and was also a potent risk factor for the development of GBS in a Dutch population. 23 These findings were consistent with a study of Indian patients with GBS, but not with a report on Norwegian Caucasian patients. 24,26 One meta-analysis indicated that every FccRIIIb-NA2 allele cumulatively increases the GBS severity score, though none of the genotypes or alleles were associated with susceptibility to GBS. 25 However, consensus regarding the role of FccR polymorphisms in the pathogenesis of GBS has not yet been established due to the inadequate statistical power of studies with small sample sizes and differences in the ethnicities of the populations tested. Thus, we aimed to evaluate whether candidate gene polymorphisms in FccR are a major causative factor for GBS susceptibility or severity in Bangladeshi patients with C. jejuni-triggered GBS, which represents the world's largest cohort.
Blood specimens were collected by venipuncture before patients received medication and disease outcome was Electrophysiological studies of 82% (247/303) of the GBS patients indicated 59% (146/247) of patients had an axonal subtype of GBS, including acute motor axonal neuropathy (AMAN) and acute motor and sensory axonal neuropathy (AMSAN), 27% (68/247) of patients had acute inflammatory demyelinating polyradiculoneuropathy (AIDP) and 13% (33/247) of cases were unclassified with inexcitable nerves or equivocal findings. 30 Severity of disease (degree of muscle weakness) was assessed using the Medical Research Council (MRC) sum score 31,32 ranging from 0 to 60 at nadir (maximum muscle weakness); GBS patients at nadir with MRC sumscore < 40 were defined as severely affected patients and with MRC sumscore ≥ 40 were defined as mildly affected patients. 33 The outcome of the disease was measured using the GBS disability score after 6 months of follow-up. 34 This study was reviewed and approved by the Institutional Review Board (IRB) and ethical committees of the icddr, b, Dhaka, Bangladesh.

Genomic DNA isolation
Whole blood samples were collected from 605 study subjects into lithium heparin-coated anti-coagulation tubes for genomic DNA isolation. Genomic DNA was extracted using the QIAamp â DNA Blood Midi Kit (100) (Qiagen, Hilden, Germany), dissolved in 1 9 TE buffer (10 mmol/ L Tris-Cl, pH 8.0, 1 mmol/L EDTA), stored at À80°C, diluted to 10 ng/µL with Milli-Q water and then stored at À20°C until SNP detection.

Statistical analysis
Statistical analysis was performed using logistic regression analysis and Fisher's exact test with Yates' continuity correction to assess associations between the FccR polymorphisms and disease susceptibility or subgroups. In the control group, all SNPs were within Hardy-Weinberg equilibrium. P values less than 0.05 were considered statistically significant. The Bonferroni method was applied to correct the P values for multiple comparisons: each Pvalue was multiplied by the number of comparisons and denoted Pc (Pc, P corrected). Genotype/allelic frequencies were estimated by a simple counting method and the data were processed using Microsoft Excel 2010 (Microsoft, Redmond, WA, USA), GraphPad prism (version 5.01, GraphPad software, Inc., La Jolla, CA) or SPSS (version 16.0, Company, Chicago, IL). Haplotype patterns and frequencies were analyzed using the genotype package of R statistics and their associations with GBS susceptibility and subgroups were assessed using logistic regression analysis.

FccRIIa, FccRIIIa, and FccRIIIb polymorphisms and haplotype in patients with GBS and healthy individuals
No significant associations were observed between the FccRIIa, FccRIIIa, and FccRIIIb polymorphisms and susceptibility to GBS compared to healthy controls ( Table 2). The comparison of axonal variants of GBS versus healthy controls or demyelinating subtypes versus healthy subjects showed no relation with disease susceptibility ( Table 3). The haplotype distributions of the three loci were compared between patients with GBS and healthy individuals. Haplotype analysis revealed 27 possible different patterns for the FccRIIa, FccRIIIa, and FccRIIIb polymorphic loci (Fig. 1). The nine most predominant patterns (haplotypes 1-9; frequency > 5%), representing 61.5% of total variation, were selected for further haplotype analysis (Fig. 2). No significant association was observed between any haplotype and GBS susceptibility when each haplotype was analyzed individually.

Discussion
This study investigated the association of three functionally relevant polymorphisms in FccR and the resulting haplotype patterns with the susceptibility and severity of GBS among patients compared to healthy controls in a large cohort of GBS in Bangladesh. We found no significant associations between individual FccR alleles or genotypes and susceptibility to GBS; however, the FccRIIIa-V/ F158 genotype influenced the severity of disease. Moreover, associations between the FccRIIIa and FccRIIIb genotypes and haplotype patterns were evident in patients with an antecedent C. jejuni infection and anti-GM1 antibody-positive patients, respectively. Associations between FccR polymorphisms and susceptibility to GBS have previously been studied in patients with different ethnic backgrounds (Table 6). [23][24][25][26] We observed no significant differences in the FccR allele or genotype frequencies and haplotype patterns between Bangladeshi patients with GBS and healthy controls. These findings confirm a previous meta-analysis of British, Dutch, and Norwegian GBS cases, 25 which suggested FccR polymorphisms were not related to disease susceptibility, regardless of ethnic variation.
In addition, we found the FccRIIIa-F/F158 genotype was associated with the mild form of GBS based on MRC sum score at nadir, while the FccRIIIa-V/F158 genotype was associated with the severe form of GBS. As phagocytosis, cellular cytotoxicity, cytokine production, and other immune responses depend on efficient FccR-IgG interactions, the higher frequency of FccRIIIa-F/F158 among patients with the mild form of GBS may indicate this genotype reduces the affinity of IgG binding and in turn impairs immune complex clearance and decreases subsequent inflammation. 13,35,36 Patients with FccRIIIa-V/ F158 genotypes may have better ability to clear immune complexes (ICs) via degranulation and phagocytosis more efficiently, resulting in more severe disease. 36 We observed a higher frequency of FccRIIIb-NA1/NA1 genotypes in patients with the mild form of GBS, similar to a previous study of Norwegian patients with GBS. 24 The NA1/NA1 genotype has a high affinity for IgG1 and IgG3, 37 which are the most common among the anti-GM1 and anti-GQ1b antibodies. 38 Autoantibodies such as anti-ganglioside antibodies are neutralized in the circulation, thus cross-reaction of these auto-antibodies with the peripheral nerves may be partially prevented in patients with GBS who are homozygous for FccRIIIb-NA1. 24 Ganglioside-specific IgG have been reported to damage nerve tissues by activating effector functions (eg, phagocytosis and/or degranulation) via FccR. 35,39 Homozygous FccRIIIb-NA1 was less frequent among both C. jejuniseropositive patients and anti-GM1 antibody-positive patients with the mild form of the disease. In contrast, FccRIIIb-NA2/2 was associated with recent C. jejuni infection and anti-GM1 antibody production. In addition,   C. jejuni -seropositive patients had higher frequencies of the FccRIIIa-F/F158 and FccRIIIa-V158F genotypes.
These findings indicate C. jejuni -seropositive patients with higher frequency of the FccRIIIa-V158F genotype may suffer severe muscle weakness. One limitation of this study is that polymorphisms of FccRIIIb receptor gene, FccRIIIb-SH alleles were not investigated; however, it is not yet known whether FccRIIIb-SH polymorphisms influence the function of FccRIIIb or not. 16,40 The present study strengthens the evidence that FccR polymorphisms and haplotypes influence the clinical and serological subgroup of GBS, as well as the strength of the immune responses that ultimately trigger the development of GBS and affect disease severity. In addition, the FccRIIIa-V158F genotype was more frequent among patients with recent C. jejuni infection and was found to contribute to disease severity. Variation in the FccR gene differs greatly between populations of different ethnicities, thus it will be important and interesting to confirm our findings in a multiethnic population, such as the International GBS Outcome Study (IGOS) population. 41