Fcγ Receptor IIIA Polymorphism as a Risk Factor for Acute Poliomyelitis
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Fcg Receptor IIIA Polymorphism as a Risk Factor for Acute Poliomyelitis Tiina Rekand,1 Nina Langeland,2 Johan A. Aarli,1 and Christian A. Vedeler1
Departments of 1Neurology and 2Internal Medicine, Haukeland University Hospital, Bergen, Norway
Poliovirus is a RNA virus that may cause a flulike disease or intrude into the nervous system and manifest as aseptic meningitis (nonparalytic poliomyelitis) or flaccid paresis caused by damage of the motor neurones (paralytic poliomyelitis) [1]. During epidemics, most people contract the disease because of high viral infectivity, but only ∼1% of these developed the paralytic form [2]. There are several explanations for the different clinical manifestations, including both genetic characteristics of poliovirus and human T and B cell responses [1, 2]. Years after the acute disease, 20%–70% of patients develop a complex of new symptoms (muscle weakness, muscle pain, and fatigue) termed the “postpolio syndrome” (PPS) [3]. The pathophysiology of PPS is uncertain but has been related to metabolic and immune-mediated processes [3]. Antibody-mediated immune responses are important in infectious diseases and are linked to cellular effector functions, such as phagocytosis, antibody-dependent cellular cytotoxicity, release of inflammatory mediators, immune complex clearance, and regulation of antibody production through receptors for the Fc part of IgG (FcgR) [4]. Three main FcgR classes have been identified, FcgRI, FcgRII, and FcgRIII, with different IgG binding affinities and IgG subclass specificities [4]. In humans, 8 genes located on the long arm of chromosome
Received 24 June 2002; revised 28 August 2002; electronically published 19 November 2002. Informed consent was obtained from all patients and controls. Study protocol follows the ethical standards established in the 1964 Helsinki Declaration. The study was approved by the Regional Research Ethics committee. Financial support: Sophies Minde Foundation. Reprints or correspondence: Dr. Tiina Rekand, Dept. of Neurology, Haukeland University Hospital, N-5021 Bergen, Norway (Tiina.Rekand @helse-bergen.no). The Journal of Infectious Diseases 2002; 186:1840–3 䉷 2002 by the Infectious Diseases Society of America. All rights reserved. 0022-1899/2002/18612-0020$15.00
1 encode the different FcgR classes. In addition, FcgRIIA, FcgRIIIA, and FcgRIIIB have 2 allelic variants that influence their receptor function. The affinity of FcgRIIA for IgG2 is higher for the 131H allele than for the 131R, whereas the FcgRIIIA 158F allele binds IgG1, IgG3, and IgG4 less than does the 158V allele [5]. Furthermore, the FcgRIIIB neutrophil antigen (NA) allele 1 has more efficient binding of IgG1 and IgG3 than the NA2 [4]. Because FcgR polymorphisms may influence the susceptibility to and the disability caused by infectious and autoimmune diseases [4], we have studied whether these polymorphisms could influence the susceptibility to and the course of acute poliomyelitis, as well as the development of PPS.
Subjects and Methods Patient population and control subjects. The study population comprised 110 white Norwegian patients (mean age, 55.8 years; range, 45–83 years; 50 men and 60 women) who were diagnosed with acute poliomyelitis and were hospitalized at the Haukeland Hospital (Bergen, Norway) during 1950–1954. According to the original patient records, 22 (19%) had nonparalytic disease with aseptic meningitis, 21 (18%) had experienced transitory muscle weakness only in the acute phase, and 67 (61%) developed persistent muscular weakness (paralytic poliomyelitis). At follow-up in 1999, 50 (43%) of 110 patients fulfilled the criteria for PPS, having at least 2 new PPS symptoms that had developed after 115 years of clinical stability [3]. These symptoms included new muscular weakness, an absolute criterion for the diagnosis, muscle pain, and fatigue, as well as having no other clinical conditions that could explain the symptoms. Among the patients with PPS were 28 women and 22 men, with a mean age of 55.1 years (range, 45–83 years). Ninety-six white Norwegian healthy subjects from the same area with no history of poliomyelitis served as control subjects. FcgR genotype analysis. Genomic DNA was extracted from
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Poliomyelitis is a viral infection that causes flaccid paralysis in ∼1% of cases. The Fc receptors for immunoglobulin G (FcgR) are associated with modifying effects of several infectious and autoimmune diseases. To assess the influence of FcgR polymorphisms on the acute and late course of poliomyelitis, 110 Norwegian patients with well-defined histories of acute poliomyelitis were genotyped, of whom 50 suffered from the postpolio syndrome (PPS). In comparison with healthy control subjects without a history of poliomyelitis, significantly fewer patients had the FcgRIIIA genotype V/V (P ! .01 ). However, this genotype was not an independent risk factor for PPS. The FcgRIIA and IIIB genotypes and allele frequencies did not differ between the patients and control subjects. The FcgRIIIA V/V genotype may lower the risk for contracting acute poliomyelitis through better clearance of poliovirus.
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Results In contrast to the FcgRIIIA V/F and F/F genotypes, V/V genotype was present at a significantly lower frequency among patients with a history of acute poliomyelitis (5.4%) than among control subjects (15.1%; P ! .01 ). The ORs for contracting poliomyelitis were 3.39 (V/F) and 2.80 (F/F), compared with the V/V genotype (table 1). However, the V allele frequency was
not statistically significant (0.28 for patients with poliomyelitis vs. 0.34 for control subjects). In total, 2 patients with paralytic poliomyelitis (P ! .05, compared with control subjects), none with transitory paralytic findings, and 4 with nonparalytic poliomyelitis had the V/V genotype. However, the differences in the FcgRIIIA polymorphisms between the subgroups with nonparalytic, transitory, and paralytic poliomyelitis did not reach significance (P p .06). The distribution of the FcgRIIA and FcgRIIIB genotypes (table 1) and allele frequency did not differ significantly between the patients with poliomyelitis and control patients or among the subgroups with nonparalytic, transitory, and paralytic poliomyelitis. The FcgRIIA, FcgRIIIA, and FcgRIIIB genotypes did not influence the ORs for having PPS (table 2). Only 1 patient with PPS had the V/V genotype, compared with 5 patients without PPS. When compared with the data from the control subjects, the low frequency of V/V genotype among patients with PPS was significant (P ! .05). However, the significance disappeared in the multiple regression analysis, which was adjusted for the risk factors of age, male versus female sex, and clinical findings (nonparalytic vs. paralytic disease) in the acute stage. There were no significant differences between the control subjects and the patients with PPS in the occurrence of FcgRIIA and FcgRIIIB genotypes.
Discussion We have demonstrated that patients with poliomyelitis have a significantly lower frequency of the FcgRIIIA V/V genotype than do healthy control subjects. This is the first study that has Table 1. Distribution of FcgRIIA, FcgRIIIA and FcgRIIIB genotypes among patients with poliomyelitis and among control subjects. Genotype
a
FcgRIIA 131R/R 131H/R 131H/H FcgRIIIA V/V F/V F/F FcgRIIIB NA1/NA1 NA1/NA2 NA2/NA2
Patients with poliomyelitis
Control subjects
OR (95% CI)
25 (23.1) 45 (41.7) 38 (35.2)
18 (18.8) 45 (46.9) 33 (34.4)
1.00 0.72 (0.35–1.50) 0.83 (0.39–1.78)
6 (5.4) 50 (44.6) 53 (48.6)
c
13 (15.1) 32 (37.2) 41 (47.7)
1.00 3.39 (1.17–9.80) 2.80 (0.98–8.00)
13 (11.8) 62 (56.4) 35 (31.8)
11 (12.6) 41 (47.1) 35 (40.2)
1.00 1.28 (0.52–3.13) 0.85 (0.33–2.14)
b
NOTE. Data are no. (%) of patients, unless otherwise indicated. CI, confidence interval; FcgR, receptors for the Fc part of IgG; NA, neutrophil antigen allele; OR, odds ratio. a FcgRIIA genotype was not tested in 2 patients. FcgRIIIA genotype was not tested in 1 patient and 10 control subjects. FcgRIIIB genotype was not tested in 9 control subjects. b OR for contracting poliomyelitis, compared with control subjects within the different genotypes. c P ! .01, compared with control subjects.
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whole blood with a QIAamp Blood Kit (Qiagen GmbH), according to the manufacturer’s directions. The FcgRIIA genotypes were determined by use of amplification refractory mutation system–polymerase chain reaction (PCR) [6]. In brief, allele-specific primers EC2-131R (5-CCA GAA TGG AAA ATC CCA GAA ATT CTC TCG-3) and EC2-131H (5CCA GAA TGG AAA ATC CCA GAA ATT CTC TCA-3), in combination with the antisense primer TM1 (5-CCA TTG GTG AAG AGC TGC CCA TGC TGG GCA-3) amplifying a 980-bp fragment in 2 PCR reactions, were used for each sample. A 270bp product amplified from the Ta22 gene served as an internal positive control [7]. PCR conditions were denaturation for 5 min at 94⬚C, followed by 45 cycles of 94⬚C for 45 s, 63⬚C for 30 s, and 72⬚C for 90 s, and a final extension at 72⬚C for 10 min. FcgRIIIA was detected by use of refractory mutation system– PCR amplifying 160-bp products, as described elsewhere [7]. Two PCR reactions were performed, using allele-specific primers KIMG (V) (5-TCT CTG AAG ACA CAT TTC TAC TCC CTA C-3) and KIM-1 (F) (5-TCT CTG AAG ACA CAT TTC TAC TCC CTA A-3). The same internal positive control was used as in the case of FcgRIIA. PCR conditions were denaturation for 10 min at 94⬚C, followed by 32 cycles of 95⬚C for 30 s, 57⬚C for 20 s, and 72⬚C for 25 s, and a final extension at 72⬚C for 7 min. FcgRIIIB was amplified by use of an NA1-specific primer (5CAG TGG TTT CAC AAT GTG AA-3) and an NA2-specific primer (5-CAA TGG TAC AGC GTG CTT-3) with a common reverse primer (5-ATG GAC TTC TAG CTG CAC-3) modified according to Bux et al. [8]. The 141-bp product for NA1 and the 219-bp product for NA2 were amplified in the same reaction. A 439bp product from the human growth hormone served as an internal positive control. PCR conditions were denaturation for 3 min at 94⬚C, followed by 30 cycles of 94⬚C for 1 min, 57⬚C for 2 min, and 72⬚C for 1 min, and a final extension at 72⬚C for 10 min [7]. All PCR products were analyzed by electrophoresis, using 1.5% agarose gel with ethidium bromide, and were visualized by UV light. Statistical analysis. Pearson’s x2 test and Fisher’s exact test were used to compare the frequency of the different genotypes among patients and control subjects. In addition, separate statistical analyses were performed for comparing the subgroups of patients with nonparalytic, transitory, or permanent muscular weakness during acute illness and the subgroups with and without PPS. Statistical significance was defined at P ! .05. Multiple logistic regression analyses were performed for evaluation of FcgR polymorphisms as a risk factor for PPS. Adjustments were made for other known risk factors, such as age, sex, and clinical findings, in the acute stage (nonparalytic, transitory, and persistent paresis) [9]. Odds ratios (ORs) and 95% confidence intervals were calculated as the measure of effect.
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Table 2. FcgR genotype frequency and odds ratios (ORs) for the various genotypes among patients with postpolio syndrome (PPS).
Genotype
a
OR (95% CI)
16 (32) 20 (40) 13 (26)
1.00 1.46 (0.57–3.74) 1.95 (0.65–5.85)
1 (2) 25 (50) 23 (46)
1.00 3.61 (0.35–35.85) 2.95 (0.30–29.49)
7 (14) 28 (56) 15 (30)
1.00 0.70 (0.20–2.49) 0.66 (0.48–2.38)
b
NOTE. Data are no. (%) of patients, unless otherwise indicated. CI, confidence interval; FcgR, receptors for the Fc part of IgG; NA, neutrophil antigen allele. a No significant differences were detected. b ORs for developing PPS among a specific genotype. Data are adjusted for sex, age, and findings in the acute phase (nonparalytic and transitory/paralytic poliomyelitis) in multiple logistic regression analysis.
shown a significant correlation between FcgR polymorphisms and poliomyelitis. FcgR polymorphisms have been associated both with susceptibility to and the course of infectious and autoimmune diseases [4]. However, some of these studies have shown conflicting results, probably because of small numbers of patients and differences in ethnic distribution of FcgR polymorphisms [7]. Therefore, we suggest that our results be confirmed in a larger cohort of patients, as well as in sibling-based association studies. Poliovirus enters susceptible cells through a specific poliovirus receptor, but it has been shown that poliovirus also can be taken up in the presence of anti-poliovirus antibodies by cells expressing the high-affinity FcgR (FcgRI) but not the low-affinity FcgR (FcgRII) in mice [10, 11]. IgG3 is the major IgG subclass of poliovirus antibodies in humans [12]. The receptors with highest affinity for these subclasses are FcgRI, whereas the FcgRIIIA V/V genotype predominates among the low-affinity receptors [13]. FcgRs are present on monocytes and macrophages, whereas only NK cells and subpopulations of T cells express FcgRIIIA [13]. We have found previously that FcgRI, FcgRII, and FcgRIII are expressed on microglia and perivascular macrophages, whereas oligodendrocytes, astrocytes, and neurons do not express FcgRs [14]. Microglia show increased expression of FcgRs in inflammatory diseases, such as in active lesions of multiple sclerosis, which probably are caused by the action of proinflammatory cytokines [14]. It has been shown that Th1 cells predominate in mice infected or immunized with poliovirus [15]. Therefore, poliovirus infection may induce proinflammatory cytokines that increase the expression of FcgRs. The role of FcgRs in human poliomyelitis has not been stud-
ied. We believe that microglia FcgRs may be of importance in the defense against poliovirus infection by modulating various immune responses, such as phagocytosis and antibody-dependent cellular cytotoxicity. Microglia expressing the FcgRIII V/ V genotype, which has high affinity for immune complexes consisting of IgG3-poliovirus antigen, may be more effective in clearing poliovirus than those expressing the F/F genotype. This would lead to better protection against poliovirus affecting the nervous system and thereby explain the low frequency of the V/V genotypes among patients with poliomyelitis. We found no differences in FcgRIIA polymorphisms, and these probably are mainly associated with the IgG2 affinity, a subclass of less importance in this disease. During poliovirus infection, immune complexes containing viral antigens may be cleared in the circulation by leukocytes expressing FcgRs. However, this is mainly performed by the neutrophil FcgRIIIB, which shows no association with poliomyelitis. Therefore, we suggest that the microglia FcgRIIIA is the most important FcgR in the defense against poliomyelitis. In addition, other factors, such as the concentrations of poliovirus and polioviral antibodies, may play a role in determining FcgR-mediated functions [11]. We found that the FcgIIIA V/V genotype in 1 patient with PPS was significantly lower than expected, compared with that for the control subjects. However, the significance disappeared during multiple regression analysis, probably because the V/V genotype is rare among patients with paralytic disease. Thus, our findings do not support a pathogenetic role for IgG-FcgR interactions in the development of PPS. Acknowledgments We thank B. Kluge and K. Mazengia, for excellent technical assistance, and J. Aarseth (National Multiple Sclerosis Competence Centre, Bergen, Norway), for comments and advice on statistical analysis of data. We are indebted to K. M. Myhr for comments on the manuscript.
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