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Fcγ receptor IIA and IIIB polymorphisms are associated with susceptibility to cerebral malaria
Parasitology International 51 (2002) 361–366
Fcg receptor IIA and IIIB polymorphisms are associated with susceptibility to cerebral malaria Kazuya Omia, Jun Ohashia,*, Jintana Patarapotikulb, Hathairad Hananantachaib, Izumi Nakaa, Sornchai Looareesuwanb, Katsushi Tokunagaa a
Department of Human Genetics, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan b Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand Received 3 April 2002; accepted 17 July 2002
Abstract Human FcgRIIA and FcgRIIIB exhibit genetic polymorphisms, FcgRIIA-131HyR and FcgRIIIB-NA1yNA2, coding for different capacities for IgG binding and phagocytosis. Recently, FcgRIIA-131R was reported to be associated with protection against high-density Plasmodium falciparum infection in Kenya. Furthermore, FcgRIIIB-NA1yNA2 polymorphism was shown to influence FcgRIIA function in an allele-specific manner. In this study, we examined a possible association of FcgRIIA-131HyR and FcgRIIIB-NA1yNA2 polymorphisms with malaria severity in 107 cerebral malaria patients, 157 non-cerebral severe malaria patients, and 202 mild malaria controls living in northwest Thailand. This study reveals that, with the FcgRIIIB-NA2 allele, the FcgRIIA-131HyH genotype is associated with susceptibility to cerebral malaria (OR 1.85, 95% CI 1.14–3.01; Ps0.012), although these polymorphisms are not individually involved in the disease severity. Our results suggest that FcgRIIA-131HyR and FcgRIIIB-NA1yNA2 polymorphisms have an interactive effect on host defense against malaria infection. 䊚 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Cerebral malaria; Severe falciparum malaria; Northwest Thailand; FcgRIIA; FcgRIIIB
1. Introduction Receptors for the Fc fragment of IgG (FcgRs) provide a crucial link between the humoral and cellular immune responses. Human FcgRs form a clustered gene family on chromosome 1q21–24 w1x. There are three families of FcgR (FcgRI, *Corresponding author. Tel.: q81-3-5841-3693; fax: q813-5802-8619. E-mail address: [email protected] (J. Ohashi).
FcgRII, and FcgRIII), each containing multiple distinct genes and alternative splicing variants w2x. The major function of FcgRs is the activation of accessory cells against pathogens, making FcgRs be essential molecules in the host defense against infection. In fact, FcRg-chain knockout mice, which lack the ability for antibody-dependent phagocytosis and cell-mediated cytotoxicity through FcgRI, FcgRII, and FcgRIII, have been reported to show increased susceptibility to Plasmodium berghei XAT infection w3x. Therefore,
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Table 1 FcgRIIA-131HyR and IIIB-NA1yNA2 polymorphisms in Thai malaria patients Cerebral malaria (ns107) FcgRIIA Genotype frequency 131RyR 131RyH 131HyH Allele frequency 131R allele 131H allele FcgRIIIB Genotype frequency NA2yNA2 NA2yNA1 NA1yNA1 Allele frequency NA2 allele NA1 allele
Non-cerebral severe malaria (ns157)
Mild malaria controls (ns202)
7 (6.5%) 33 (30.8%) 67 (62.6%)
11 (7.0%) 53 (33.8%) 93 (59.2%)
14 (6.9%) 79 (39.1%) 109 (54.0%)
47 (22.0%) 167 (78.0%)
75 (23.9%) 239 (76.1%)
107 (26.5%) 297 (73.5%)
12 (11.2%) 53 (49.5%) 42 (39.3%)
20 (12.7%) 71 (45.2%) 66 (42.0%)
29 (14.4%) 82 (40.6%) 91 (45.0%)
77 (36.0%) 137 (64.0%)
111 (35.4%) 203 (64.6%)
140 (34.7%) 264 (65.3%)
FcgRs can be attractive candidates for the susceptibility genes to malaria in human. FcgRIIA and FcgRIIIB are low-affinity receptors, interacting with complexed or aggregated IgG. FcgRIIA has two codominantly expressed allotypes, differ in one amino acid at position 131, histidine or arginine. FcgRIIA-131H is the only human FcgR that efficiently binds IgG2, whereas FcgRIIA-131R binds IgG2 weakly w4,5x. A recent study indicated that the FcgRIIA-131RyR genotype is associated with protection against high-density Plasmodium falciparum infection in Kenyan infants w6x, suggesting that the FcgRIIA-131R allele has been maintained by the selection pressures from malaria infection in malaria endemic areas. FcgRIIIB occurs as neutrophils antigen NA1 or NA2 allotypes, differ in four amino acid positions including two potential glycosylation sites. FcgRIIIB-NA1yNA2 polymorphism has been known to strongly influence the phagocytotic capacity of neutrophils. Neutrophils with the FcgRIIIB-NA2yNA2 genotype have lower capacity for phagocytosis than those with the FcgRIIIBNA1yNA1 genotype w7x. Moreover, FcgRIIA and FcgRIIIB can be simultaneously ligated leading to collaboration in the initiation of integrated cell
functions. The FcgRIIIB-NA1yNA2 polymorphism was shown to influence FcgRIIA function in an allele-specific manner. Neutrophils with the FcgRIIIB-NA2yNA2 genotype was found to show lower activation of FcgRIIA-mediated phagocytosis than those with the FcgRIIIB-NA1yNA1 genotype w8x. These observations raise the prospect that functional polymorphisms of FcgRs are genetic factors of susceptibility to severe malaria. In order to evaluate the clinical importance of genetic polymorphisms of FcgRs in P. falciparum malaria infection, both FcgRIIA-131HyR and IIIB-NA1y NA2 polymorphisms must be investigated simultaneously. The aim of this study was to examine a possible association of these polymorphisms with the severity of malaria in northwest Thailand. 2. Materials and methods 2.1. Subjects A total of 264 adult severe P. falciparum malaria patients (107 cerebral malaria and 157 non-cerebral severe malaria patients) and 202 adult mild
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malaria patients (serve as controls) living in northwest Thailand were enrolled in this study. All of them underwent treatment at the Hospital for Tropical Diseases, Faculty of Tropical Medicine, Mahidol University. Clinical manifestations of malaria were classified according to the definitions and associated criteria of the World Health Organization (WHO, 2000). Cerebral malaria was defined as unrousable coma, positive asexual form of P. falciparum in blood smear and other causes of coma have been excluded. Severe malaria but not cerebral (non-cerebral severe malaria) was defined by any patient who had one of the following signs, high parasitemia ()100 000 parasitey ml), hypoglycemia (glucose -22 nmolyl), severe anemia (hematocrit -20% or hemoglobin -7.0 gydl), and increased serum level of creatinine more than 3.0 mgydl. Mild malaria was characterized by a positive blood smear and fever without other causes of infections and had no manifestations of severe malaria as described above. All individuals were 13 years of age or older, and the mean ages for severe malaria and mild malaria patients were 25.5 and 25.5, respectively. This study was approved by the institute review board of the Faculty of Tropical Medicine, Mahidol University and informed consent was obtained from all patients. 2.2. FcgRIIA and IIIB genotyping FcgRIIA-131HyR genotyping was performed using the polymerase chain reaction–restriction fragment length polymorphism method w9x. FcgRIIIB-NA1yNA2 genotyping was performed by PCR with sequence-specific primers w10x. 2.3. Statistical analysis The x2-test was used to compare the allele frequencies of the FcgRIIA-131HyR and IIIBNA1yNA2 polymorphisms among the three malaria groups. In order to prevent false positive results caused by a deviation from the Hardy–Weinberg equilibrium in mild malaria controls, the deviation from the Hardy–Weinberg equilibrium was tested. Haplotype frequencies and linkage disequilibrium parameters were estimated for mild malaria con-
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trols using the EH program w11x. A statistically significant level was defined as P-0.05 throughout this study. 3. Results The genotype and allele frequencies of the FcgRIIA-131HyR and FcgIIIB-NA1yNA2 polymorphisms in Thai malaria patients are shown in Table 1. Neither the FcgRIIA-131HyR nor FcgRIIIB-NA1yNA2 polymorphism individually showed a significant association with the severity of malaria in Thai adults. Genotype frequencies did not deviate from the Hardy–Weinberg equilibrium in mild malaria controls. In order to analyze the interaction between FcgRIIA-131HyR and IIIB-NA1yNA2 in regard to the severity of malaria, the present case-control data were stratified by the allele positivities of the FcgRIIA-131R and IIIB-NA2 allele (Table 2). In this analysis, either homozygote or heterozygote of the allele considered is regarded as a positive individual. Our results indicated that the frequency of homozygotes for FcgRIIA-131H possessing FcgRIIIB-NA2 was significantly increased in cerebral malaria patients compared with that in mild malaria controls (OR 1.85, 95% CI 1.14–3.01; Ps0.012). When non-cerebral severe malaria patients were compared with mild malaria controls, no significant difference was observed. Table 3 shows the haplotype frequency and linkage disequilibrium between FcgRIIA-131HyR and IIIB-NA1yNA2 in mild malaria controls. The most predominant haplotype was 131H-NA1 (48.0%), followed by 131H-NA2 (25.5%), 131RNA1 (17.3%), and 131R-NA2 (9.1%). These polymorphisms were in linkage equilibrium in Thai mild malaria controls. 4. Discussion In this study, neither the FcgRIIA-131HyR nor FcgRIIIB-NA1yNA2 polymorphism individually showed a significant association with the severity of malaria in Thai. However, we found that, with the FcgRIIIB-NA2 allele, the FcgRIIA-131HyH genotype was associated with susceptibility to cerebral malaria. This finding suggests that the
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Table 2 Interaction of FcgRIIA-131HyR, IIIB-NA1yNA2, and severe malaria in Thai Cerebral malaria (ns107) Combination of allele positivitya 131RqyNA2y 131RqyNA2q 131RyyNA2q 131RyyNA2y
22 18 47 20
Non-cerebral severe malaria (ns157)
(20.6%) (16.8%) (43.9%)b (18.7%)
29 34 57 37
(18.5%) (21.7%) (36.3%) (23.6%)
Mild malaria controls (ns202) 42 51 60 49
(20.4%) (25.4%) (29.8%) (24.4%)
a Either homozygote or heterozygote of the 131R allele is indicated by 131Rq, and homozygote of the 131H allele is indicated by 131Ry. Either homozygote or heterozygote of the NA2 allele is indicated by NA2q, and homozygote of the NA1 allele is indicated by NA2y. b The frequency of 131RyyNA2q individuals was compared with that of the remainder between cerebral malaria and mild malaria controls. Ps0.012, OR 1.85, 95% CI 1.14–3.01.
combination of FcgRIIA-131HyR and FcgRIIIBNA1yNA2 polymorphisms has an interactive effect on host defense against malaria infection. FcgRIIA-131H, which efficiently binds IgG2 as well as IgG1 and IgG3, was found to be associated with severe malaria in this study, implying that IgG2 affects immune responses against P. falciparum malaria. In fact, several in vitro studies have demonstrated that IgG2 blocks opsonization, phagocytosis, and antibody-dependent cellular inhibition, which are mediated by IgG1 and IgG3 directed against P. falciparum w12,13x. IgG1 and IgG3 have been known to be associated with either lower parasitemia w14x or lower risk of malaria infection w15x. Recognition of IgG1 and IgG3 by FcgRIIA has been shown to be involved in growth inhibition of P. falciparum w16x. Taken together with our results, in malaria patients with the FcgRIIA-131HyH genotype, the protective role of IgG1 and IgG3 might be inhibited by binding of
IgG2 to FcgRIIA andyor by a higher turnover of IgG2. In addition, FcgRIIA has been reported to be the main receptor for C-reactive protein (CRP) w17x. CRP is an acute phase serum protein in humans w18x, involved in the protection against bacterial infection w19x, endotoxitin shock w20x, and pulmonary inflammation w21x. In P. falciparum malaria, CRP affect the hepatic development of parasites, by both preventing penetration of a sporozoite into a hepatocyte and blocking parasite division w22x. A recent study has indicated that CRP binds to monocytes and neutrophils with the FcgRIIA-131RyR and 131RyH genotypes, but not to those with the FcgRIIA-131HyH genotype w23x. The present study showed that the FcgRIIA-131Hy H genotype with the FcgRIIIB-NA2 allele was associated with susceptibility to cerebral malaria. Thus, binding of CRP to FcgRIIA could provide
Table 3 Haplotype frequency and linkage disequilibrium between FcgRIIA-131HyR and IIIB-NA1yNA2 in Thai malaria patients Haplotype
HF
RLD
x2
P value
FcgRIIA–IIIB Mild malaria controls (ns202) 131H-NA1 131H-NA2 131R-NA1 131R-NA2
48.0 25.5 17.3 9.1
0.002 y0.002 0.002 y0.002
0.0010
0.97
HF, estimated haplotype frequency by the EH program w11x; RLD, relative linkage disequilibrium.
K. Omi et al. / Parasitology International 51 (2002) 361–366
a protective immune response against P. falciparum infection. With respect to FcgRIIIB-NA1yNA2 polymorphism, it has been reported that human IgG1 and IgG3 interact more readily with FcgRIIIB-NA1 than with the FcgRIIIB-NA2 allotype w7,24x, and individuals with the FcgRIIIB-NA2yNA2 genotype have lower capacity for phagocytosis than those with the FcgRIIIB-NA1yNA1 genotype w7,24x. Therefore, FcgRIIIB-NA2 might be considered as an inherited disease susceptibility factor in P. falciparum infection, in which IgG1 and IgG3 play a crucial role in host defense. Furthermore, FcgRIIIB-NA1yNA2 polymorphism was shown to influence FcgRIIA function in an allele-specific manner. Neutrophils with the FcgRIIIB-NA2yNA2 genotype were found to show lower activation of FcgRIIA-mediated phagocytosis than those with the FcgRIIIB-NA1yNA1 genotype w8x. In this study, increased susceptibility to cerebral malaria was observed in patients with the FcgRIIA-131HyH genotype in combination with the FcgRIIIB-NA2 allele. These imply that the possession of the FcgRIIIB-NA2 allele might fail to activate the Fcg-mediated phagocytosis in malaria patients, and be associated with the pathogenesis of cerebral malaria. The allele frequencies of FcgRIIA-131R and IIIB-NA2 were 26.5 and 34.7%, respectively, in mild malaria controls. These frequencies were consistent with those of Asian populations, such as Japanese and Korean w9,25x, but not to those of African and Caucasian populations w26–28x. Based on our observations, the frequency of 131H-NA2 haplotype is likely to decrease in malaria endemic areas. Unexpectedly, the frequency of the 131HNA2 haplotype is relatively high (25.5%), and these polymorphisms were in linkage equilibrium in Thai mild malaria controls (Table 3). Thus, these polymorphisms might have been maintained simultaneously in this population by the balance of selection pressures from malaria and other infections, although further investigations should be performed in different populations. Acknowledgments The authors are indebted to Chieko Kyogoku for introducing the PCR-RFLP and PCR-SSP
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methods. We sincerely thank the patients who participated in this study. This study is supported by the Core University System Exchange Programme under Japan Society for the Promotion of Science, coordinated by the University of Tokyo and Mahidol University, The National Research Council of Thailand, and the Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan. We wish to thank two anonymous reviewers for thoughtful comments on the manuscript. References w1x Qiu WQ, de Bruin D, Brownstein BH, Pearse R, Ravetch JV. Organization of the human and mouse lowaffinity FcgR genes: duplication and recombination. Science 1990;248:732 –5. w2x Kimberly RP, Salmon JE, Edberg JC. Receptors for immunoglobulin G: molecular diversity and implications for disease. Arthritis Rheum. 1995;38:306 –14. w3x Yoneto T, Waki S, Takai T, et al. A critical role of Fc receptor-mediated antibody-dependent phagocytosis in the host resistance to blood-stage Plasmodium berghei XAT infection. J. Immunol. 2001;166:6236 –41. w4x Warmerdam PAM, van de Winkel JGJ, Vlug A, Westerdaal NAC, Capel PJA. A single amino acid in the second Ig-like domain of the human Fcg receptor II is critical for human IgG2 binding. J. Immunol. 1991;27:1338 –43. w5x Parren PWHI, Warmerdam PAM, Boejie LCM, et al. On the interaction of IgG subclasses with the low affinity Fcg IIa (CD32) on human monocytes, neutrophils and platelets. J. Clin. Invest. 1992;90:1537 –41. w6x Shi YP, Nahlen BL, Kariuki S, et al. Fcg receptor polymorphism is associated with protection of infants against high density Plasmodium falciparum infection. VII. Asebo bay cohort project. J. Infect. Dis. 2001;184:107 –12. w7x Salmon JE, Edberg JC, Kimberly RP. Fcg receptor III on human neutrophils. Alleic variants have functionally distinct capacities. J. Clin. Invest. 1990;85:1287 –95. w8x Salmon JE, Millard SS, Brogle NL, Kimberly RP. Fcg receptor IIIB enhances Fcg receptor IIA function in oxidant-dependent and allele-sensitive manner. J. Clin. Invest. 1995;95:2877 –85. w9x Hatta Y, Tsuchiya N, Ohashi J, et al. Association of Fcg receptor IIIB, but not of Fcg IIA and IIIA, polymorphisms with systemic lupus erythematosus in Japanese. Genes Immun. 1999;1:53 –60. w10x Bux J, Stein EL, Santoso S, Muller-Eckhardt C. NA gene frequencies in the German population, determined by polymerase chain reaction with sequence-specific primers. Transfusion 1995;35:54 –7.
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w11x Terwilliger JD, Ott J. Handbook of human linkage analysis. Baltimore: Johns Hopkins University Press, 1994. w12x Bouharoun-Tayoun H, Druilhe P. Plasmodium falciparum malaria: evidence for an isotype imbalance which may be responsible for delayed acquisition of protective immunity. Infect. Immun. 1992;60:1473 –81. w13x Groux H, Gysin J. Opsonization as an effector mechanism in human protection against asexual blood stages of Plasmodium falciparum: functional role of IgG subclasses. Res. Immunol. 1990;141:529 –42. w14x Aribot G, Rogier C, Sarthou JL, et al. Pattern of immunoglobulin isotype response to Plasmodium falciparum blood-stage antigens in individuals living in a holoendemic area of Senegal (Dielmo, West Africa). Am. J. Trop. Med. Hyg. 1996;54:449 –57. w15x Taylor RR, Allen SJ, Greenwood BM, Riley EM. IgG3 antibodies to Plasmodium falciparum merozoite surface protein 2 (MSP2): increasing prevalence with age and association with clinical immunity to malaria. Am. J. Trop. Med. Hyg. 1998;58:406 –13. w16x Bouharoun-Tayoun H, Oeuvray C, Lunel F, Druilhe P. Mechanisms underlying the monocyte-mediated antibody-dependent killing of Plasmodium falciparum asexual blood stages. J. Exp. Med. 1995;182:409 –18. w17x Bharadwaj D, Stein MP, Volzer M, Mold C, Du Clos TW. The major receptor for C-reactive protein on leukocytes is Fcg receptor II. J. Exp. Med. 1999;190:585 –90. w18x Szalai AJ, Agrawal A, Greenhough TJ, Volanakis JE. C-reactive protein structural biology, gene expression, and host defense function. Immunol. Res. 1997;16:127 – 36. w19x Szalai AJ, Briles DE, Volanakis JE. Human C-reactive protein is protective against fatal Streptococcus pneu-
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moniae infection in transgenic mice. J. Immunol. 1995;155:2557 –63. Xia D, Samols D. Transgenic mice expressing rabbit Creactive protein are resistant to endotoxemia. Proc. Natl. Acad. Sci. USA 1997;94:2575 –80. Heuertz RM, Dongyuan X, Samols D, Webster RO. Inhibition of C5a des Arg-induced neutrophil alveolitis in transgenic mice expressing C-reactive protein. Am. J. Physiol. 1994;266:L649 –L654. Nussler A, Pied S, Pontet M, et al. Inflammatory status and preerythrocytic stages of malaria: role of the Creactive protein. Exp. Parasitol. 1991;72:1 –7. Stein MP, Edberg JC, Kimberly RP, et al. C-reactive protein binding to FcgRIIa on human monocytes and neutrophils is allele-specific. J. Clin. Invest. 2000;105:369 –76. Bredius RG, Fijen CA, De Haas M, et al. Role of neutrophil FcgRIIa (CD32) and FcgRIIIb (CD16) polymorphic forms in phagocytosis of human IgG1- and IgG3-opsonized bacteria and erythrocytes. Immunology 1994;83:624 –30. Song YW, Han C-W, Kang S-W, et al. Abnormal distribution of Fcg receptor type IIa polymorphisms in Korean patients with systemic lupus erythematosus. Arthritis Rheum. 1998;41:421 –6. Lehrnbecher T, Foster CB, Zhu S, et al. Variant genotype of the low-affinity Fcg receptors in two control populations and a review of low-affinity Fcg receptor polymorphism in control and disease populations. Blood 1999;94:4220 –32. Duits AJ, Bootsma H, Derksen RHWM, et al. Skewed distribution of IgG Fc receptor IIa (CD32) polymorphism is associated with renal disease in systemic lupus erythematosus. Arthritis Rheum. 1996;39:1832 –6. Salmon JE, Millard S, Schachter LA, et al. FcgIIA alleles are heritable risk factor for lupus nephritis in African American. J. Clin. Invest. 1996;97:1348 –54.
Fcg receptor IIA and IIIB polymorphisms are associated with susceptibility to cerebral malaria Kazuya Omia, Jun Ohashia,*, Jintana Patarapotikulb, Hathairad Hananantachaib, Izumi Nakaa, Sornchai Looareesuwanb, Katsushi Tokunagaa a
Department of Human Genetics, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan b Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand Received 3 April 2002; accepted 17 July 2002
Abstract Human FcgRIIA and FcgRIIIB exhibit genetic polymorphisms, FcgRIIA-131HyR and FcgRIIIB-NA1yNA2, coding for different capacities for IgG binding and phagocytosis. Recently, FcgRIIA-131R was reported to be associated with protection against high-density Plasmodium falciparum infection in Kenya. Furthermore, FcgRIIIB-NA1yNA2 polymorphism was shown to influence FcgRIIA function in an allele-specific manner. In this study, we examined a possible association of FcgRIIA-131HyR and FcgRIIIB-NA1yNA2 polymorphisms with malaria severity in 107 cerebral malaria patients, 157 non-cerebral severe malaria patients, and 202 mild malaria controls living in northwest Thailand. This study reveals that, with the FcgRIIIB-NA2 allele, the FcgRIIA-131HyH genotype is associated with susceptibility to cerebral malaria (OR 1.85, 95% CI 1.14–3.01; Ps0.012), although these polymorphisms are not individually involved in the disease severity. Our results suggest that FcgRIIA-131HyR and FcgRIIIB-NA1yNA2 polymorphisms have an interactive effect on host defense against malaria infection. 䊚 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Cerebral malaria; Severe falciparum malaria; Northwest Thailand; FcgRIIA; FcgRIIIB
1. Introduction Receptors for the Fc fragment of IgG (FcgRs) provide a crucial link between the humoral and cellular immune responses. Human FcgRs form a clustered gene family on chromosome 1q21–24 w1x. There are three families of FcgR (FcgRI, *Corresponding author. Tel.: q81-3-5841-3693; fax: q813-5802-8619. E-mail address: [email protected] (J. Ohashi).
FcgRII, and FcgRIII), each containing multiple distinct genes and alternative splicing variants w2x. The major function of FcgRs is the activation of accessory cells against pathogens, making FcgRs be essential molecules in the host defense against infection. In fact, FcRg-chain knockout mice, which lack the ability for antibody-dependent phagocytosis and cell-mediated cytotoxicity through FcgRI, FcgRII, and FcgRIII, have been reported to show increased susceptibility to Plasmodium berghei XAT infection w3x. Therefore,
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Table 1 FcgRIIA-131HyR and IIIB-NA1yNA2 polymorphisms in Thai malaria patients Cerebral malaria (ns107) FcgRIIA Genotype frequency 131RyR 131RyH 131HyH Allele frequency 131R allele 131H allele FcgRIIIB Genotype frequency NA2yNA2 NA2yNA1 NA1yNA1 Allele frequency NA2 allele NA1 allele
Non-cerebral severe malaria (ns157)
Mild malaria controls (ns202)
7 (6.5%) 33 (30.8%) 67 (62.6%)
11 (7.0%) 53 (33.8%) 93 (59.2%)
14 (6.9%) 79 (39.1%) 109 (54.0%)
47 (22.0%) 167 (78.0%)
75 (23.9%) 239 (76.1%)
107 (26.5%) 297 (73.5%)
12 (11.2%) 53 (49.5%) 42 (39.3%)
20 (12.7%) 71 (45.2%) 66 (42.0%)
29 (14.4%) 82 (40.6%) 91 (45.0%)
77 (36.0%) 137 (64.0%)
111 (35.4%) 203 (64.6%)
140 (34.7%) 264 (65.3%)
FcgRs can be attractive candidates for the susceptibility genes to malaria in human. FcgRIIA and FcgRIIIB are low-affinity receptors, interacting with complexed or aggregated IgG. FcgRIIA has two codominantly expressed allotypes, differ in one amino acid at position 131, histidine or arginine. FcgRIIA-131H is the only human FcgR that efficiently binds IgG2, whereas FcgRIIA-131R binds IgG2 weakly w4,5x. A recent study indicated that the FcgRIIA-131RyR genotype is associated with protection against high-density Plasmodium falciparum infection in Kenyan infants w6x, suggesting that the FcgRIIA-131R allele has been maintained by the selection pressures from malaria infection in malaria endemic areas. FcgRIIIB occurs as neutrophils antigen NA1 or NA2 allotypes, differ in four amino acid positions including two potential glycosylation sites. FcgRIIIB-NA1yNA2 polymorphism has been known to strongly influence the phagocytotic capacity of neutrophils. Neutrophils with the FcgRIIIB-NA2yNA2 genotype have lower capacity for phagocytosis than those with the FcgRIIIBNA1yNA1 genotype w7x. Moreover, FcgRIIA and FcgRIIIB can be simultaneously ligated leading to collaboration in the initiation of integrated cell
functions. The FcgRIIIB-NA1yNA2 polymorphism was shown to influence FcgRIIA function in an allele-specific manner. Neutrophils with the FcgRIIIB-NA2yNA2 genotype was found to show lower activation of FcgRIIA-mediated phagocytosis than those with the FcgRIIIB-NA1yNA1 genotype w8x. These observations raise the prospect that functional polymorphisms of FcgRs are genetic factors of susceptibility to severe malaria. In order to evaluate the clinical importance of genetic polymorphisms of FcgRs in P. falciparum malaria infection, both FcgRIIA-131HyR and IIIB-NA1y NA2 polymorphisms must be investigated simultaneously. The aim of this study was to examine a possible association of these polymorphisms with the severity of malaria in northwest Thailand. 2. Materials and methods 2.1. Subjects A total of 264 adult severe P. falciparum malaria patients (107 cerebral malaria and 157 non-cerebral severe malaria patients) and 202 adult mild
K. Omi et al. / Parasitology International 51 (2002) 361–366
malaria patients (serve as controls) living in northwest Thailand were enrolled in this study. All of them underwent treatment at the Hospital for Tropical Diseases, Faculty of Tropical Medicine, Mahidol University. Clinical manifestations of malaria were classified according to the definitions and associated criteria of the World Health Organization (WHO, 2000). Cerebral malaria was defined as unrousable coma, positive asexual form of P. falciparum in blood smear and other causes of coma have been excluded. Severe malaria but not cerebral (non-cerebral severe malaria) was defined by any patient who had one of the following signs, high parasitemia ()100 000 parasitey ml), hypoglycemia (glucose -22 nmolyl), severe anemia (hematocrit -20% or hemoglobin -7.0 gydl), and increased serum level of creatinine more than 3.0 mgydl. Mild malaria was characterized by a positive blood smear and fever without other causes of infections and had no manifestations of severe malaria as described above. All individuals were 13 years of age or older, and the mean ages for severe malaria and mild malaria patients were 25.5 and 25.5, respectively. This study was approved by the institute review board of the Faculty of Tropical Medicine, Mahidol University and informed consent was obtained from all patients. 2.2. FcgRIIA and IIIB genotyping FcgRIIA-131HyR genotyping was performed using the polymerase chain reaction–restriction fragment length polymorphism method w9x. FcgRIIIB-NA1yNA2 genotyping was performed by PCR with sequence-specific primers w10x. 2.3. Statistical analysis The x2-test was used to compare the allele frequencies of the FcgRIIA-131HyR and IIIBNA1yNA2 polymorphisms among the three malaria groups. In order to prevent false positive results caused by a deviation from the Hardy–Weinberg equilibrium in mild malaria controls, the deviation from the Hardy–Weinberg equilibrium was tested. Haplotype frequencies and linkage disequilibrium parameters were estimated for mild malaria con-
363
trols using the EH program w11x. A statistically significant level was defined as P-0.05 throughout this study. 3. Results The genotype and allele frequencies of the FcgRIIA-131HyR and FcgIIIB-NA1yNA2 polymorphisms in Thai malaria patients are shown in Table 1. Neither the FcgRIIA-131HyR nor FcgRIIIB-NA1yNA2 polymorphism individually showed a significant association with the severity of malaria in Thai adults. Genotype frequencies did not deviate from the Hardy–Weinberg equilibrium in mild malaria controls. In order to analyze the interaction between FcgRIIA-131HyR and IIIB-NA1yNA2 in regard to the severity of malaria, the present case-control data were stratified by the allele positivities of the FcgRIIA-131R and IIIB-NA2 allele (Table 2). In this analysis, either homozygote or heterozygote of the allele considered is regarded as a positive individual. Our results indicated that the frequency of homozygotes for FcgRIIA-131H possessing FcgRIIIB-NA2 was significantly increased in cerebral malaria patients compared with that in mild malaria controls (OR 1.85, 95% CI 1.14–3.01; Ps0.012). When non-cerebral severe malaria patients were compared with mild malaria controls, no significant difference was observed. Table 3 shows the haplotype frequency and linkage disequilibrium between FcgRIIA-131HyR and IIIB-NA1yNA2 in mild malaria controls. The most predominant haplotype was 131H-NA1 (48.0%), followed by 131H-NA2 (25.5%), 131RNA1 (17.3%), and 131R-NA2 (9.1%). These polymorphisms were in linkage equilibrium in Thai mild malaria controls. 4. Discussion In this study, neither the FcgRIIA-131HyR nor FcgRIIIB-NA1yNA2 polymorphism individually showed a significant association with the severity of malaria in Thai. However, we found that, with the FcgRIIIB-NA2 allele, the FcgRIIA-131HyH genotype was associated with susceptibility to cerebral malaria. This finding suggests that the
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Table 2 Interaction of FcgRIIA-131HyR, IIIB-NA1yNA2, and severe malaria in Thai Cerebral malaria (ns107) Combination of allele positivitya 131RqyNA2y 131RqyNA2q 131RyyNA2q 131RyyNA2y
22 18 47 20
Non-cerebral severe malaria (ns157)
(20.6%) (16.8%) (43.9%)b (18.7%)
29 34 57 37
(18.5%) (21.7%) (36.3%) (23.6%)
Mild malaria controls (ns202) 42 51 60 49
(20.4%) (25.4%) (29.8%) (24.4%)
a Either homozygote or heterozygote of the 131R allele is indicated by 131Rq, and homozygote of the 131H allele is indicated by 131Ry. Either homozygote or heterozygote of the NA2 allele is indicated by NA2q, and homozygote of the NA1 allele is indicated by NA2y. b The frequency of 131RyyNA2q individuals was compared with that of the remainder between cerebral malaria and mild malaria controls. Ps0.012, OR 1.85, 95% CI 1.14–3.01.
combination of FcgRIIA-131HyR and FcgRIIIBNA1yNA2 polymorphisms has an interactive effect on host defense against malaria infection. FcgRIIA-131H, which efficiently binds IgG2 as well as IgG1 and IgG3, was found to be associated with severe malaria in this study, implying that IgG2 affects immune responses against P. falciparum malaria. In fact, several in vitro studies have demonstrated that IgG2 blocks opsonization, phagocytosis, and antibody-dependent cellular inhibition, which are mediated by IgG1 and IgG3 directed against P. falciparum w12,13x. IgG1 and IgG3 have been known to be associated with either lower parasitemia w14x or lower risk of malaria infection w15x. Recognition of IgG1 and IgG3 by FcgRIIA has been shown to be involved in growth inhibition of P. falciparum w16x. Taken together with our results, in malaria patients with the FcgRIIA-131HyH genotype, the protective role of IgG1 and IgG3 might be inhibited by binding of
IgG2 to FcgRIIA andyor by a higher turnover of IgG2. In addition, FcgRIIA has been reported to be the main receptor for C-reactive protein (CRP) w17x. CRP is an acute phase serum protein in humans w18x, involved in the protection against bacterial infection w19x, endotoxitin shock w20x, and pulmonary inflammation w21x. In P. falciparum malaria, CRP affect the hepatic development of parasites, by both preventing penetration of a sporozoite into a hepatocyte and blocking parasite division w22x. A recent study has indicated that CRP binds to monocytes and neutrophils with the FcgRIIA-131RyR and 131RyH genotypes, but not to those with the FcgRIIA-131HyH genotype w23x. The present study showed that the FcgRIIA-131Hy H genotype with the FcgRIIIB-NA2 allele was associated with susceptibility to cerebral malaria. Thus, binding of CRP to FcgRIIA could provide
Table 3 Haplotype frequency and linkage disequilibrium between FcgRIIA-131HyR and IIIB-NA1yNA2 in Thai malaria patients Haplotype
HF
RLD
x2
P value
FcgRIIA–IIIB Mild malaria controls (ns202) 131H-NA1 131H-NA2 131R-NA1 131R-NA2
48.0 25.5 17.3 9.1
0.002 y0.002 0.002 y0.002
0.0010
0.97
HF, estimated haplotype frequency by the EH program w11x; RLD, relative linkage disequilibrium.
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a protective immune response against P. falciparum infection. With respect to FcgRIIIB-NA1yNA2 polymorphism, it has been reported that human IgG1 and IgG3 interact more readily with FcgRIIIB-NA1 than with the FcgRIIIB-NA2 allotype w7,24x, and individuals with the FcgRIIIB-NA2yNA2 genotype have lower capacity for phagocytosis than those with the FcgRIIIB-NA1yNA1 genotype w7,24x. Therefore, FcgRIIIB-NA2 might be considered as an inherited disease susceptibility factor in P. falciparum infection, in which IgG1 and IgG3 play a crucial role in host defense. Furthermore, FcgRIIIB-NA1yNA2 polymorphism was shown to influence FcgRIIA function in an allele-specific manner. Neutrophils with the FcgRIIIB-NA2yNA2 genotype were found to show lower activation of FcgRIIA-mediated phagocytosis than those with the FcgRIIIB-NA1yNA1 genotype w8x. In this study, increased susceptibility to cerebral malaria was observed in patients with the FcgRIIA-131HyH genotype in combination with the FcgRIIIB-NA2 allele. These imply that the possession of the FcgRIIIB-NA2 allele might fail to activate the Fcg-mediated phagocytosis in malaria patients, and be associated with the pathogenesis of cerebral malaria. The allele frequencies of FcgRIIA-131R and IIIB-NA2 were 26.5 and 34.7%, respectively, in mild malaria controls. These frequencies were consistent with those of Asian populations, such as Japanese and Korean w9,25x, but not to those of African and Caucasian populations w26–28x. Based on our observations, the frequency of 131H-NA2 haplotype is likely to decrease in malaria endemic areas. Unexpectedly, the frequency of the 131HNA2 haplotype is relatively high (25.5%), and these polymorphisms were in linkage equilibrium in Thai mild malaria controls (Table 3). Thus, these polymorphisms might have been maintained simultaneously in this population by the balance of selection pressures from malaria and other infections, although further investigations should be performed in different populations. Acknowledgments The authors are indebted to Chieko Kyogoku for introducing the PCR-RFLP and PCR-SSP
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methods. We sincerely thank the patients who participated in this study. This study is supported by the Core University System Exchange Programme under Japan Society for the Promotion of Science, coordinated by the University of Tokyo and Mahidol University, The National Research Council of Thailand, and the Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan. We wish to thank two anonymous reviewers for thoughtful comments on the manuscript. References w1x Qiu WQ, de Bruin D, Brownstein BH, Pearse R, Ravetch JV. Organization of the human and mouse lowaffinity FcgR genes: duplication and recombination. Science 1990;248:732 –5. w2x Kimberly RP, Salmon JE, Edberg JC. Receptors for immunoglobulin G: molecular diversity and implications for disease. Arthritis Rheum. 1995;38:306 –14. w3x Yoneto T, Waki S, Takai T, et al. A critical role of Fc receptor-mediated antibody-dependent phagocytosis in the host resistance to blood-stage Plasmodium berghei XAT infection. J. Immunol. 2001;166:6236 –41. w4x Warmerdam PAM, van de Winkel JGJ, Vlug A, Westerdaal NAC, Capel PJA. A single amino acid in the second Ig-like domain of the human Fcg receptor II is critical for human IgG2 binding. J. Immunol. 1991;27:1338 –43. w5x Parren PWHI, Warmerdam PAM, Boejie LCM, et al. On the interaction of IgG subclasses with the low affinity Fcg IIa (CD32) on human monocytes, neutrophils and platelets. J. Clin. Invest. 1992;90:1537 –41. w6x Shi YP, Nahlen BL, Kariuki S, et al. Fcg receptor polymorphism is associated with protection of infants against high density Plasmodium falciparum infection. VII. Asebo bay cohort project. J. Infect. Dis. 2001;184:107 –12. w7x Salmon JE, Edberg JC, Kimberly RP. Fcg receptor III on human neutrophils. Alleic variants have functionally distinct capacities. J. Clin. Invest. 1990;85:1287 –95. w8x Salmon JE, Millard SS, Brogle NL, Kimberly RP. Fcg receptor IIIB enhances Fcg receptor IIA function in oxidant-dependent and allele-sensitive manner. J. Clin. Invest. 1995;95:2877 –85. w9x Hatta Y, Tsuchiya N, Ohashi J, et al. Association of Fcg receptor IIIB, but not of Fcg IIA and IIIA, polymorphisms with systemic lupus erythematosus in Japanese. Genes Immun. 1999;1:53 –60. w10x Bux J, Stein EL, Santoso S, Muller-Eckhardt C. NA gene frequencies in the German population, determined by polymerase chain reaction with sequence-specific primers. Transfusion 1995;35:54 –7.
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