- Email: [email protected]
CD31 polymorphism with susceptibility to cerebral malaria in Thais
Parasitology International 50 Ž2001. 235᎐239
Association of adhesion molecule PECAM-1rCD31 polymorphism with susceptibility to cerebral malaria in Thais M. Kikuchi a , S. Looareesuwan b, R. Ubalee a,c , O. Tasanor c , F. Suzuki a , Y. Wattanagoon c , K. Na-Bangchang c , A. Kimurad, M. Aikawae, K. Hirayamaa,U a
Department of Medical Zoology, Saitama Medical School, Moroyrma, Iruma, Saitama 350-0495, Japan b Faculty of Tropical Medicine, Mahidol Uni¨ ersity, Bangkok, Thailand c Clinical Pharmacology Unit, Faculty of Tropical Medicine, Mahidol Uni¨ ersity, Bangkok, Thailand d Department of Tissue Physiology, Di¨ ision of Adult Disease, Medical Research Institute, Tokyo Medical and Dental Uni¨ ersity, Tokyo 113-8519, Japan e Research Institute of Medical Science, Tokai Uni¨ ersity, Kanagawa 259-1193, Japan Received 21 December 2000; accepted 6 July 2001
Abstract Adhesion molecules on endothelial cells are known to be important ligands for malaria infected red blood cells ŽPRBC. wMol Biochem Parasitol, 76, Ž1996. 1x, and may be involved in the pathogenic process of cerebral malaria ŽCM. which is the most serious complication of falciparum malaria, through enhancing micro embolism or sequestration in the capillaries of the brain. PECAM-1rCD31 is one of these candidate ligands and is coded by a polymorphic gene. Two hundred and ten Thai malaria patients Ž43 cerebral, 89 severe and 78 uncomplicated. were analyzed for their genetic polymorphism of CD31 to examine the clinical relationship between the disease and specific genotypes. Four alleles were defined 125 valine ŽV.-563 asparagine ŽN.; 125V-563 serine ŽS.; 125 leucine ŽL.-563N; and 125L-563S. We found that the frequency of the 125 VrV 563 NrN genotype was significantly high in CM patients as compared with severe cases without CM Ž P- 0.01, ORs 2.92., suggesting that this genotype is one of the risk factors for CM. 䊚 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Cerebral malaria; Genetic polymorphism; Genotype; PECAM-1rCD31
U
Corresponding author present address. Department of Molecular Immunogenetics, Institute of Tropical Medicine, Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan; Tel.: q81-95-849-7818; fax: q81-95-849-7821. E-mail address: [email protected] ŽK. Hirayama.. 1383-5769r01r$ - see front matter 䊚 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 1 3 8 3 - 5 7 6 9 Ž 0 1 . 0 0 0 8 2 - 4
236
M. Kikuchi et al. r Parasitology International 50 (2001) 235᎐239
1. Introduction Cerebral malaria ŽCM. is the most serious complication of falciparum malaria and has a high rate of mortality w1x. Sequestration of red blood cells infected with Plasmodium falciparum ŽPRBCs. to the microvasculature w2x may directly relate to the pathogenesis of the disease w3x. Sequestration is observed in almost all organs, but the most serious disease occurs in the brain w4,5x. It is not clear why only a small percentage of patients with P. falciparum develop CM; however, genetic, nutritional, and immunological host factors have been implicated w6x. Endothelial adhesion molecules, such as intercellular adhesion molecule-1 ŽICAM-1 or CD54. w7x, CD36 w8x, thrombospodin ŽTSP. w9x, chondroitin sulfate A ŽCSA. w10x, vascular cell adhesion molecule 1 ŽVCAM-1., endothelial leukocyte adhesion molecule 1 ŽELAM-1. w11x, and complement receptor CD35 ŽCR1. w12x, have been suggested to be potential mediators of PRBC cytoadhesion. Among those, CD36 mutations that cause CD36 deficiency reported to be associated with susceptibility to severe malaria in Africa w13x. Recently, Treutiger et al. w14x reported that the plateletrendothelial adhesion molecule 1 ŽPECAM-1rCD31. was necessary for PRBC adherence to human endothelial cells, and Chen et al. identified its malaria-derived ligand as PfEMP1 w15x. The extracellular portion of PECAM-1rCD31 is composed of eight domains, the first domain Žexon 3., and 6th domain Žexon 8. show polymorphism in Caucasian w16x and Japanese populations w17x, and provoke an allogeneic lymphocyte response between graft and host in bone marrow transplantation w18,19x. We analyzed the polymorphism of the CD31 gene in Thai patients with uncomplicated malaria and those with CM and identified a genotype that is significantly associated with CM in Thais.
2. Subjects In 1997 and 1998, 191 Thai patients with falciparum malaria were adopted at the Faculty of
Tropical Medicine, Mahidol University in Bangkok. The group of patients at Mahidol University consisted of 78 uncomplicated Žmean age 27.5" 10.5., 89 severe Žmean age 30.0" 10.8., and 24 cerebral Žmean age 27.7" 12.2. malaria patients. Nineteen CM patients Žmean age 24.7" 10.8. diagnosed in 1996 at Mae Sot hospital in Mae Sot located at the border between Thailand and Myanmar were also included in this study. The patients were categorized into three clinical groups by WHO criteria w20x. Severe malaria in this study was without coma and defined by one of the following criteria, high parasitemia Ž) 100 000 parasiterml., hypoglycemia Žglucose - 22 nmolrl., severe anemia Žhematocrit - 20% or hemoglobin - 7.0 grdl., and increased serum level of creatinine more than 3.0 mgrdl w2x. Uncomplicated malaria was characterized by a positive blood smear and fever without any findings corresponding to the criteria of severe malaria. Cerebral malaria was diagnosed by coma without any other recognized cause of alteration of consciousness. Blood samples were also collected from 80 healthy Thai volunteers at the Faculty of Tropical Medicine, Mahidol University. All subjects signed an informed consent form prepared by each institution that had been approved by the national ethical review committee. CM patients singed the informed consent form after they recovered from coma.
3. Polymorphism analysis EDTA blood samples Ž1 ml. were collected and genomic DNA was extracted using a kit ŽDNA Extractor WB, Wako Pure Chemicals, Osaka, Japan.. We analyzed the polymorphism of the CD31 gene, exon 3 Ž1st domain. and exon 8 Ž6th domain., using a PCR-restriction fragment length polymorphism ŽPCR-RFLP. method, as previously described w15x. Briefly, PCR was performed using specific primer sets for the CD31 exon 3 and exon 8; subsequently PCR products were cut with five units of M¨ a I and NlaIII for detection of the polymorphic site in exon 3 and with five units of Nhe I for the site in exon 8, loaded onto the 7% polyacrylamide gel, run at 180 Vrcm for
M. Kikuchi et al. r Parasitology International 50 (2001) 235᎐239
2 h, and visualized by ethidium bromide staining. PCR products of exons 3 and 8 from 12 patients were directly sequenced to confirm that there were no other polymorphisms using an ABI 310 automated sequencer ŽABI, CA, USA..
4. Statistical analyses The goodness of fit to the Hardy᎐Weinberg equilibrium was examined by the calculation of genotype frequencies to see if there is any preferential selection of zygosity in the subjects. The statistical significance of the difference between groups was determined by Chi-square test Žtwotailed.. Contingency tables were used to determine the odds ratio ŽOR. and a 95% confidence interval ŽCI.. For the detection of linkage disequilibrium between the dimorphism of two sites Žexons 3 and 8., a t-value was calculated using the method described by Mittal w19x. The frequencies of CD31 exon 3 and exon 8 genotypes among the 210 patients and 71 healthy volunteers are shown in Table 1. The genotype frequencies fitted well to the Hardy᎐Weinberg equilibrium. There was no significant difference in the frequencies between healthy and uncompleted and between uncomplicated and severe malaria. CD31 125 VrV Ž P- 0.02, ORs 2.34. and 563 NrN Ž P - 0.01, ORs 2.77. in CM patients were significantly high in frequency when
237
compared with the severe malaria but there was no significant difference when compared with the uncomplicated group Ž125 VrV Ps 0.07, 563 NrN Ps 0.2.. This finding might be explained by the following two-step hypothesis. To develop CM a patient must have two sequencial genetic factors, one is a factor different from CD31 related and enhances pathogenic process to the severe form, and the other is a CD31 related factor that is critical for the development of CM from the severe form. The difference in the frequency of CD31 genotypes between the severe without CM and CM patients showed to be more significant than between CM and uncomplicated. The CD31 molecule coded by the 125VrV and 563NrN genotype might attach to PBRCs more effectively than CD31 molecules coded for by other genotypes so that those patients are at an increased risk of developing CM Ž P- 0.01, ORs 2.92.. We have also examined their TNF-␣ promoter region polymorphism to see if there are any effects on this study. We have identified four different alleles which consisted of 5 SNPs w19x, but there was no significant difference in the allele frequencies between the groups Ždata not shown.. We found four alleles of the CD31 gene present in Thais, 125V-563N, 125V-563S, 125L-563N and 125L-563S. There was a strong linkage disquibrilium between 125V and 563N Ž t-value s 7.9. or between 125L and 563S Ž t-value s 5.3. in CM patients, as detected by Mittal’s method w19x indi-
Table 1 Frequency of PECAMrCD31 genotypes in patients with Falciparum malaria PECAMrCD31
Healthy
Uncomplicated
Severea
Cerebral
Codon
Genotypes
n s 71 Ž%.
n s 78 Ž%.
n s 89 Ž%.
n s 43 Ž%.
125
LrL LrV VrV NrN NrS SrS
17 35 19 20 35 16
563
a
Ž23.9. Ž49.3. Ž26.8. Ž28.2. Ž49.3. Ž22.5.
23 35 20 22 38 18
Ž29.5. Ž44.9. Ž25.6. Ž28.2. Ž48.7. Ž23.1.
19 52 18 17 59 13
Ž21.3. Ž58.4. Ž20.2. Ž19.1. Ž66.3. Ž14.6.
Severe cases without CM Ž19.. Compared with severe and cerebral malaria. P - 0.02, ORs 2.84, 95% CI 1.28᎐6.30. c Compared with severe and cerebral malaria. P- 0.01, ORs 2.77, 95% CI 1.23᎐6.21. d Compared with uncomplicated and cerebral malaria. Ps 0.07, ORs 2.09, 95% CI 0.95᎐4.60. e Compared with uncomplicated and cerebral malaria. Ps 0.2, ORs 1.90, 95% CI 0.86᎐4.20. b
9 16 18 17 18 8
Ž20.9. Ž37.2. Ž41.9.b,d Ž39.5.c,e Ž41.9. Ž18.6.
238
M. Kikuchi et al. r Parasitology International 50 (2001) 235᎐239
cating that the major alleles are 125V-563N and 125L-563S. We suppose that the 125V-563N allele is responsible for the strong association of the 125VrV and 563NrN genotypes with CM. PECAMrCD31 is a highly glycosylated 130 KDa polypeptide belonging to the immunoglobulin super-family and is systemically expressed on the endothelial cells of micro vessels, including those in the brain w21,22x. Physiologically, CD31 is thought to function as a homing receptor for leukocytes to pass through the junction of endothelial cells w21x. CD31 polymorphism may affect its physiological function and may result in the alteration of normal immunologic reactions in patients. Treutiger et al. w14x, reported that monoclonal antibodies specific for the amino-terminal segments of CD31 Ždomains 1᎐4. efficiently blocked CD31-dependent adhesion of PRBCs to human umbilical-vein endothelial cells. Thus, the substitution of valine for the leucine at codon 125 in domain 1 might affect its affinity for PRBCs. Recently, Chen w15x reported that PfEMP1 located at knobs on the surface of PRBCs was the ligand of CD31. Further study would be needed to confirm the direct relation between the CD31 genotype and CM by estimating the affinity of the CD31 encoded by the different alleles to PfEMP1 molecule.
w3x w4x
w5x
w6x
w7x
w8x
w9x
w10x
w11x
w12x
Acknowledgements This study was supported in part by grants-inaid for Scientific Research on Priority Areas ‘Molecular Basis for Malaria Control’ Ž08281104., the Grant-in-Aid from the Ministry of Health and Welfare of Japan ŽH10-Shinko-26 and 9Kou-3., and a grant from the Japan᎐US Cooperative Medical Science Program Ž1997᎐1998..
w13x
w14x
w15x
References w16x w1x Miller LH, Good MF, Milon G. Malaria pathogenesis. Science 1994;264Ž5167.:1878᎐83. w2x MacPherson GG, Warrell MJ, White NJ, Looareesuwan S, Warrell DA. Human cerebral malaria. A quantitative
w17x
ultrastructural analysis of parasitized erythrocyte sequestration. Am J Pathol 1985;119Ž3.:385᎐401. White NJ, Ho M. The pathophysiology of malaria. Adv Parasitol 1994;31:83᎐173. Luse SA, Miller LH. Plasmodium falciparum malaria. Ultrastructure of parasitized erythrocytes in cardiac vessels. Am J Trop Med Hyg 1971;20Ž5.:655᎐60. Aikawa M, Iseki M, Barnwell JW, Taylor D, Maung MOO, Howerd RJ. Human cerebral malaria. Am J Trop Med Hyg 1988;39Ž1.:3᎐10. Hill AV. Genetic susceptibility to malaria and other infectious diseases: from the MHC to the whole genome. Parasitology Suppl 1996;112:S75᎐84. Berendt AR, Simmons DL, Tansey J, Newbold CI, Marsh K. Intercellular adhesion molecule-1 is an endothelial cell adhesion receptor for Plasmodium falciparum. Nature 1989;341Ž6237.:57᎐9. Barnwell JW, Asch AS, Nachman RL, Yamaya M, Aikawa M, Ingravallo P. A human 88-kD membrane glycoprotein ŽCD36. functions in vitro as a receptor for a cytoadherence ligand on Plasmodium falciparum-infected erythrocytes. J Clin Invest 1989;84Ž3.:765᎐72. Roberts DD, Sherwood JA, Spitalnik SL et al. Thrombospondin binds falciparum malaria parasitized erythrocytes and may mediate cytoadherence. Nature 1985; 318Ž6041.:64᎐6. Rogerson SJ, Chaiyaroj SC, Ng K, Reeder JC, Brown GV. Chondroitin sulfate A is a cell surface receptor for Plasmodium falciparum-infected erythrocytes. J Exp Med 1995;182Ž1.:15᎐20. Ockenhouse CF, Tegoshi T, Maeno Y et al. Human vascular endothelial cell adhesion receptors for Plasmodium falciparum-infected erythrocytes: roles for endothelial leukocyte adhesion molecule 1 and vascular cell adhesion molecule 1. J Exp Med 1992;176Ž4.:1183᎐9. Rowe JA, Moulds JM, Newbold CI, Miller LH. P. falciparum rosetting mediated by a parasite-variant erythrocyte membrane protein and complement-receptor 1. Nature 1997;388Ž6639.:292᎐5. Aitman TJ, Cooper LD, Norsworthy PJ et al. Malaria susceptibility and CD36 mutation. Nature 2000;405 Ž6790.:1015᎐6. Treutiger CJ, Heddini A, Fernandez V, Muller WA, Wahlgren M. PECAM-1rCD31, an endothelial receptor for binding Plasmodium falciparum-infected erythrocytes. Nat Med 1997;3Ž12.:1405᎐8. Chen Q, Heddini A, Barragan A, Fernandez V, Pearce SF, Wahlgren M. The semiconserved head structure of Plasmodium falciparum erythrocyte membrane protein 1 mediates binding to multiple independent host receptors. J Exp Med 2000;192Ž1.:1᎐10. Behar E, Chao NJ, Hiraki DD et al. Polymorphism of adhesion molecule CD31 and its role in acute graftversus-host disease. N Engl J Med 1996;334Ž12.:1405᎐8. Maruya E, Saji H, Seki S et al. Evidence that CD31, CD49b, and CD62L are immunodominant minor histo-
M. Kikuchi et al. r Parasitology International 50 (2001) 235᎐239 compatibility antigens in HLA identical sibling bone marrow transplants. Blood 1998;92Ž6.:2169᎐76. w18x World Health Organization, Division of Control of Tropical Diseases. Severe and complicated malaria. Trans R Soc Trop Med Hyg Žsupplement 2. 1990; 17Ž84.:1᎐65. w19x Higuchi T, Seki N, Kamizono S et al. Polymorphism of the 5⬘-flanking region of the human tumor necrosis factor ŽTNF.-␣ gene in Japanese. Tissue Antigens 1998;51:605᎐12.
239
w20x Mittal KK. The HLA polymorphism and susceptibility to disease. VoxSang 1976;31:161᎐3. w21x Newman PJ, Berndt MC, Gorski J et al. PECAM-1 ŽCD31. cloning and relation to adhesion molecules of theimmunoglobulin gene superfamily. Science 1990; 247Ž4947.:1219᎐22. w22x Delisser HM, Newman PJ, Albelda SM. Molecular and functionl aspects of PECAMrCD31. Immunol Today 1994;15Ž10.:490᎐5.
Association of adhesion molecule PECAM-1rCD31 polymorphism with susceptibility to cerebral malaria in Thais M. Kikuchi a , S. Looareesuwan b, R. Ubalee a,c , O. Tasanor c , F. Suzuki a , Y. Wattanagoon c , K. Na-Bangchang c , A. Kimurad, M. Aikawae, K. Hirayamaa,U a
Department of Medical Zoology, Saitama Medical School, Moroyrma, Iruma, Saitama 350-0495, Japan b Faculty of Tropical Medicine, Mahidol Uni¨ ersity, Bangkok, Thailand c Clinical Pharmacology Unit, Faculty of Tropical Medicine, Mahidol Uni¨ ersity, Bangkok, Thailand d Department of Tissue Physiology, Di¨ ision of Adult Disease, Medical Research Institute, Tokyo Medical and Dental Uni¨ ersity, Tokyo 113-8519, Japan e Research Institute of Medical Science, Tokai Uni¨ ersity, Kanagawa 259-1193, Japan Received 21 December 2000; accepted 6 July 2001
Abstract Adhesion molecules on endothelial cells are known to be important ligands for malaria infected red blood cells ŽPRBC. wMol Biochem Parasitol, 76, Ž1996. 1x, and may be involved in the pathogenic process of cerebral malaria ŽCM. which is the most serious complication of falciparum malaria, through enhancing micro embolism or sequestration in the capillaries of the brain. PECAM-1rCD31 is one of these candidate ligands and is coded by a polymorphic gene. Two hundred and ten Thai malaria patients Ž43 cerebral, 89 severe and 78 uncomplicated. were analyzed for their genetic polymorphism of CD31 to examine the clinical relationship between the disease and specific genotypes. Four alleles were defined 125 valine ŽV.-563 asparagine ŽN.; 125V-563 serine ŽS.; 125 leucine ŽL.-563N; and 125L-563S. We found that the frequency of the 125 VrV 563 NrN genotype was significantly high in CM patients as compared with severe cases without CM Ž P- 0.01, ORs 2.92., suggesting that this genotype is one of the risk factors for CM. 䊚 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Cerebral malaria; Genetic polymorphism; Genotype; PECAM-1rCD31
U
Corresponding author present address. Department of Molecular Immunogenetics, Institute of Tropical Medicine, Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan; Tel.: q81-95-849-7818; fax: q81-95-849-7821. E-mail address: [email protected] ŽK. Hirayama.. 1383-5769r01r$ - see front matter 䊚 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 1 3 8 3 - 5 7 6 9 Ž 0 1 . 0 0 0 8 2 - 4
236
M. Kikuchi et al. r Parasitology International 50 (2001) 235᎐239
1. Introduction Cerebral malaria ŽCM. is the most serious complication of falciparum malaria and has a high rate of mortality w1x. Sequestration of red blood cells infected with Plasmodium falciparum ŽPRBCs. to the microvasculature w2x may directly relate to the pathogenesis of the disease w3x. Sequestration is observed in almost all organs, but the most serious disease occurs in the brain w4,5x. It is not clear why only a small percentage of patients with P. falciparum develop CM; however, genetic, nutritional, and immunological host factors have been implicated w6x. Endothelial adhesion molecules, such as intercellular adhesion molecule-1 ŽICAM-1 or CD54. w7x, CD36 w8x, thrombospodin ŽTSP. w9x, chondroitin sulfate A ŽCSA. w10x, vascular cell adhesion molecule 1 ŽVCAM-1., endothelial leukocyte adhesion molecule 1 ŽELAM-1. w11x, and complement receptor CD35 ŽCR1. w12x, have been suggested to be potential mediators of PRBC cytoadhesion. Among those, CD36 mutations that cause CD36 deficiency reported to be associated with susceptibility to severe malaria in Africa w13x. Recently, Treutiger et al. w14x reported that the plateletrendothelial adhesion molecule 1 ŽPECAM-1rCD31. was necessary for PRBC adherence to human endothelial cells, and Chen et al. identified its malaria-derived ligand as PfEMP1 w15x. The extracellular portion of PECAM-1rCD31 is composed of eight domains, the first domain Žexon 3., and 6th domain Žexon 8. show polymorphism in Caucasian w16x and Japanese populations w17x, and provoke an allogeneic lymphocyte response between graft and host in bone marrow transplantation w18,19x. We analyzed the polymorphism of the CD31 gene in Thai patients with uncomplicated malaria and those with CM and identified a genotype that is significantly associated with CM in Thais.
2. Subjects In 1997 and 1998, 191 Thai patients with falciparum malaria were adopted at the Faculty of
Tropical Medicine, Mahidol University in Bangkok. The group of patients at Mahidol University consisted of 78 uncomplicated Žmean age 27.5" 10.5., 89 severe Žmean age 30.0" 10.8., and 24 cerebral Žmean age 27.7" 12.2. malaria patients. Nineteen CM patients Žmean age 24.7" 10.8. diagnosed in 1996 at Mae Sot hospital in Mae Sot located at the border between Thailand and Myanmar were also included in this study. The patients were categorized into three clinical groups by WHO criteria w20x. Severe malaria in this study was without coma and defined by one of the following criteria, high parasitemia Ž) 100 000 parasiterml., hypoglycemia Žglucose - 22 nmolrl., severe anemia Žhematocrit - 20% or hemoglobin - 7.0 grdl., and increased serum level of creatinine more than 3.0 mgrdl w2x. Uncomplicated malaria was characterized by a positive blood smear and fever without any findings corresponding to the criteria of severe malaria. Cerebral malaria was diagnosed by coma without any other recognized cause of alteration of consciousness. Blood samples were also collected from 80 healthy Thai volunteers at the Faculty of Tropical Medicine, Mahidol University. All subjects signed an informed consent form prepared by each institution that had been approved by the national ethical review committee. CM patients singed the informed consent form after they recovered from coma.
3. Polymorphism analysis EDTA blood samples Ž1 ml. were collected and genomic DNA was extracted using a kit ŽDNA Extractor WB, Wako Pure Chemicals, Osaka, Japan.. We analyzed the polymorphism of the CD31 gene, exon 3 Ž1st domain. and exon 8 Ž6th domain., using a PCR-restriction fragment length polymorphism ŽPCR-RFLP. method, as previously described w15x. Briefly, PCR was performed using specific primer sets for the CD31 exon 3 and exon 8; subsequently PCR products were cut with five units of M¨ a I and NlaIII for detection of the polymorphic site in exon 3 and with five units of Nhe I for the site in exon 8, loaded onto the 7% polyacrylamide gel, run at 180 Vrcm for
M. Kikuchi et al. r Parasitology International 50 (2001) 235᎐239
2 h, and visualized by ethidium bromide staining. PCR products of exons 3 and 8 from 12 patients were directly sequenced to confirm that there were no other polymorphisms using an ABI 310 automated sequencer ŽABI, CA, USA..
4. Statistical analyses The goodness of fit to the Hardy᎐Weinberg equilibrium was examined by the calculation of genotype frequencies to see if there is any preferential selection of zygosity in the subjects. The statistical significance of the difference between groups was determined by Chi-square test Žtwotailed.. Contingency tables were used to determine the odds ratio ŽOR. and a 95% confidence interval ŽCI.. For the detection of linkage disequilibrium between the dimorphism of two sites Žexons 3 and 8., a t-value was calculated using the method described by Mittal w19x. The frequencies of CD31 exon 3 and exon 8 genotypes among the 210 patients and 71 healthy volunteers are shown in Table 1. The genotype frequencies fitted well to the Hardy᎐Weinberg equilibrium. There was no significant difference in the frequencies between healthy and uncompleted and between uncomplicated and severe malaria. CD31 125 VrV Ž P- 0.02, ORs 2.34. and 563 NrN Ž P - 0.01, ORs 2.77. in CM patients were significantly high in frequency when
237
compared with the severe malaria but there was no significant difference when compared with the uncomplicated group Ž125 VrV Ps 0.07, 563 NrN Ps 0.2.. This finding might be explained by the following two-step hypothesis. To develop CM a patient must have two sequencial genetic factors, one is a factor different from CD31 related and enhances pathogenic process to the severe form, and the other is a CD31 related factor that is critical for the development of CM from the severe form. The difference in the frequency of CD31 genotypes between the severe without CM and CM patients showed to be more significant than between CM and uncomplicated. The CD31 molecule coded by the 125VrV and 563NrN genotype might attach to PBRCs more effectively than CD31 molecules coded for by other genotypes so that those patients are at an increased risk of developing CM Ž P- 0.01, ORs 2.92.. We have also examined their TNF-␣ promoter region polymorphism to see if there are any effects on this study. We have identified four different alleles which consisted of 5 SNPs w19x, but there was no significant difference in the allele frequencies between the groups Ždata not shown.. We found four alleles of the CD31 gene present in Thais, 125V-563N, 125V-563S, 125L-563N and 125L-563S. There was a strong linkage disquibrilium between 125V and 563N Ž t-value s 7.9. or between 125L and 563S Ž t-value s 5.3. in CM patients, as detected by Mittal’s method w19x indi-
Table 1 Frequency of PECAMrCD31 genotypes in patients with Falciparum malaria PECAMrCD31
Healthy
Uncomplicated
Severea
Cerebral
Codon
Genotypes
n s 71 Ž%.
n s 78 Ž%.
n s 89 Ž%.
n s 43 Ž%.
125
LrL LrV VrV NrN NrS SrS
17 35 19 20 35 16
563
a
Ž23.9. Ž49.3. Ž26.8. Ž28.2. Ž49.3. Ž22.5.
23 35 20 22 38 18
Ž29.5. Ž44.9. Ž25.6. Ž28.2. Ž48.7. Ž23.1.
19 52 18 17 59 13
Ž21.3. Ž58.4. Ž20.2. Ž19.1. Ž66.3. Ž14.6.
Severe cases without CM Ž19.. Compared with severe and cerebral malaria. P - 0.02, ORs 2.84, 95% CI 1.28᎐6.30. c Compared with severe and cerebral malaria. P- 0.01, ORs 2.77, 95% CI 1.23᎐6.21. d Compared with uncomplicated and cerebral malaria. Ps 0.07, ORs 2.09, 95% CI 0.95᎐4.60. e Compared with uncomplicated and cerebral malaria. Ps 0.2, ORs 1.90, 95% CI 0.86᎐4.20. b
9 16 18 17 18 8
Ž20.9. Ž37.2. Ž41.9.b,d Ž39.5.c,e Ž41.9. Ž18.6.
238
M. Kikuchi et al. r Parasitology International 50 (2001) 235᎐239
cating that the major alleles are 125V-563N and 125L-563S. We suppose that the 125V-563N allele is responsible for the strong association of the 125VrV and 563NrN genotypes with CM. PECAMrCD31 is a highly glycosylated 130 KDa polypeptide belonging to the immunoglobulin super-family and is systemically expressed on the endothelial cells of micro vessels, including those in the brain w21,22x. Physiologically, CD31 is thought to function as a homing receptor for leukocytes to pass through the junction of endothelial cells w21x. CD31 polymorphism may affect its physiological function and may result in the alteration of normal immunologic reactions in patients. Treutiger et al. w14x, reported that monoclonal antibodies specific for the amino-terminal segments of CD31 Ždomains 1᎐4. efficiently blocked CD31-dependent adhesion of PRBCs to human umbilical-vein endothelial cells. Thus, the substitution of valine for the leucine at codon 125 in domain 1 might affect its affinity for PRBCs. Recently, Chen w15x reported that PfEMP1 located at knobs on the surface of PRBCs was the ligand of CD31. Further study would be needed to confirm the direct relation between the CD31 genotype and CM by estimating the affinity of the CD31 encoded by the different alleles to PfEMP1 molecule.
w3x w4x
w5x
w6x
w7x
w8x
w9x
w10x
w11x
w12x
Acknowledgements This study was supported in part by grants-inaid for Scientific Research on Priority Areas ‘Molecular Basis for Malaria Control’ Ž08281104., the Grant-in-Aid from the Ministry of Health and Welfare of Japan ŽH10-Shinko-26 and 9Kou-3., and a grant from the Japan᎐US Cooperative Medical Science Program Ž1997᎐1998..
w13x
w14x
w15x
References w16x w1x Miller LH, Good MF, Milon G. Malaria pathogenesis. Science 1994;264Ž5167.:1878᎐83. w2x MacPherson GG, Warrell MJ, White NJ, Looareesuwan S, Warrell DA. Human cerebral malaria. A quantitative
w17x
ultrastructural analysis of parasitized erythrocyte sequestration. Am J Pathol 1985;119Ž3.:385᎐401. White NJ, Ho M. The pathophysiology of malaria. Adv Parasitol 1994;31:83᎐173. Luse SA, Miller LH. Plasmodium falciparum malaria. Ultrastructure of parasitized erythrocytes in cardiac vessels. Am J Trop Med Hyg 1971;20Ž5.:655᎐60. Aikawa M, Iseki M, Barnwell JW, Taylor D, Maung MOO, Howerd RJ. Human cerebral malaria. Am J Trop Med Hyg 1988;39Ž1.:3᎐10. Hill AV. Genetic susceptibility to malaria and other infectious diseases: from the MHC to the whole genome. Parasitology Suppl 1996;112:S75᎐84. Berendt AR, Simmons DL, Tansey J, Newbold CI, Marsh K. Intercellular adhesion molecule-1 is an endothelial cell adhesion receptor for Plasmodium falciparum. Nature 1989;341Ž6237.:57᎐9. Barnwell JW, Asch AS, Nachman RL, Yamaya M, Aikawa M, Ingravallo P. A human 88-kD membrane glycoprotein ŽCD36. functions in vitro as a receptor for a cytoadherence ligand on Plasmodium falciparum-infected erythrocytes. J Clin Invest 1989;84Ž3.:765᎐72. Roberts DD, Sherwood JA, Spitalnik SL et al. Thrombospondin binds falciparum malaria parasitized erythrocytes and may mediate cytoadherence. Nature 1985; 318Ž6041.:64᎐6. Rogerson SJ, Chaiyaroj SC, Ng K, Reeder JC, Brown GV. Chondroitin sulfate A is a cell surface receptor for Plasmodium falciparum-infected erythrocytes. J Exp Med 1995;182Ž1.:15᎐20. Ockenhouse CF, Tegoshi T, Maeno Y et al. Human vascular endothelial cell adhesion receptors for Plasmodium falciparum-infected erythrocytes: roles for endothelial leukocyte adhesion molecule 1 and vascular cell adhesion molecule 1. J Exp Med 1992;176Ž4.:1183᎐9. Rowe JA, Moulds JM, Newbold CI, Miller LH. P. falciparum rosetting mediated by a parasite-variant erythrocyte membrane protein and complement-receptor 1. Nature 1997;388Ž6639.:292᎐5. Aitman TJ, Cooper LD, Norsworthy PJ et al. Malaria susceptibility and CD36 mutation. Nature 2000;405 Ž6790.:1015᎐6. Treutiger CJ, Heddini A, Fernandez V, Muller WA, Wahlgren M. PECAM-1rCD31, an endothelial receptor for binding Plasmodium falciparum-infected erythrocytes. Nat Med 1997;3Ž12.:1405᎐8. Chen Q, Heddini A, Barragan A, Fernandez V, Pearce SF, Wahlgren M. The semiconserved head structure of Plasmodium falciparum erythrocyte membrane protein 1 mediates binding to multiple independent host receptors. J Exp Med 2000;192Ž1.:1᎐10. Behar E, Chao NJ, Hiraki DD et al. Polymorphism of adhesion molecule CD31 and its role in acute graftversus-host disease. N Engl J Med 1996;334Ž12.:1405᎐8. Maruya E, Saji H, Seki S et al. Evidence that CD31, CD49b, and CD62L are immunodominant minor histo-
M. Kikuchi et al. r Parasitology International 50 (2001) 235᎐239 compatibility antigens in HLA identical sibling bone marrow transplants. Blood 1998;92Ž6.:2169᎐76. w18x World Health Organization, Division of Control of Tropical Diseases. Severe and complicated malaria. Trans R Soc Trop Med Hyg Žsupplement 2. 1990; 17Ž84.:1᎐65. w19x Higuchi T, Seki N, Kamizono S et al. Polymorphism of the 5⬘-flanking region of the human tumor necrosis factor ŽTNF.-␣ gene in Japanese. Tissue Antigens 1998;51:605᎐12.
239
w20x Mittal KK. The HLA polymorphism and susceptibility to disease. VoxSang 1976;31:161᎐3. w21x Newman PJ, Berndt MC, Gorski J et al. PECAM-1 ŽCD31. cloning and relation to adhesion molecules of theimmunoglobulin gene superfamily. Science 1990; 247Ž4947.:1219᎐22. w22x Delisser HM, Newman PJ, Albelda SM. Molecular and functionl aspects of PECAMrCD31. Immunol Today 1994;15Ž10.:490᎐5.