Jpn. J. Infect. Dis･, 54, 114‑116, 2001

Short Communication
Absence of Association between the Allele Co°ing Methionine atPosition 29 in the N‑terminal Domain ofICAM‑1 (ICAM‑lKilirl)and Severe Malaria in the Northwest of Thailand
Jun Ohashi*, Izumi Naka, Jintana PatarapotikulI , Hathairad Hananantachail ,Somchai Looareesuwanl and Katsushi Tbkunaga
Department of Human Genetics, Graduate School of Medicine, Universio, of Too)0,
Hongo 7‑3‑1, Bunk,0‑ku, To砂0 113‑0033, Japan andJFacultyof 7Tropical Medicine, MahidoI UniversiOi
420/6 Rajvithi Road, Bangkok 10400, Thailand(Received June 6, 2001. Accepted July 27, 2001)

SUMMARY: lntercellular adhesion molecule 1 (ICAM‑ 1 ) isknOwn to be the endothelial receptor for Plasmodium
falcIParum‑infected erythrocytes･ Associations of the variant allele coding methionine at position 29 in theN‑termlnal domain ofICAM‑ 1 , ICAMl 1 Kilirl, With severe malaria have been investlgated in African populations,and the results of these investlgations have varied widely. In this study, We investlgated a possible association

between the ICAM‑ 1 Kilifl and severe malaria in adult malaria patients living ln northwest Thailand. The frequenciesof the ICAM‑ 1 Kilirl among patients with mild malaria, with non‑cerebral severe malaria, and with cerebral malaria
were I.7%, 2.7%, and 2.3%, respectively. This variant showed neither positive nor negative association withsevere malaria in Thailand.

Tropical Diseases, Facultyof Tropical Medicine, MahidoI

htercellular adhesion molecule 1 (ICAM‑ 1 ) is the essential
endothelial ligand for binding of Plasmodium falclbarum‑
infected erythrocytes ( 1 ). The interaction between the infected

University from 1998 to 2000 were recruited for participa‑tion in this study. All patients were 13 years of age or older, anderythrocytes and host endothelium is considered to play a

the mean ages for mild malaria, non‑cerebral severe falciparum

crucial role in the pathogenesis of severe malaria･ Themalaria, and cerebral malaria were 25.5, 23.7, and 28.6,

sequestration of mature parasitized erythrocytes from therespectively. Genomic DNA was extracted from peripheral
blood leukocytes using a qIAamp blood kit (qiagen, Hilden,GermFny)･ A 263 bp fragment in exon 2 encompassing themutatlOn CauSlng the K29M polymorphism was amplified byperipheral circulation in Plasmodium falcIParum infections
by adhesion to the postcapillary venular endothelium is oneof the remarkable features of cerebral malaria. The binding

PCR using a 5'primer ICAM‑lF (5'‑GACCTGGCAATG
CCCAGACATCTGTGTCC‑3') and a 3'primer ICAM‑lR

site is known to locate in the first N‑terminal domain ofICAM‑1 (2,3)･ Recently, a mutatioヮcausingチ? amino acidsubstitution from lyslne tO methionlne at POSltlOn 29 in the(5 ∫‑GTACACGGTGAGGAAGGTTTmGCTGTTG‑3 ′)･

N‑terminal domain of lCAM‑ 1 was found in Kilifi, Kenya.
was first reported to be sigmi丘cantly associated with susceptibil‑

PCR was performed using GeneAmp reagents and AmpliTaq
Gold DNA polymerase (Perkin‑Elmer Applied Biosystems,Foster City, Calif., USA). The conditions of the amplifica‑

ityto cerebral malaria in Kenya (4)I More.recently, however,tion consisted of initial denaturation at 960C fわr 10 min,the 29M allele was fわund to be protectlVe agalnSt Severe

followed by 38 cycles of denaturation at 960C for I min,

The allele coding methionine,knownas ICAM‑ I Kilirl or 29M,malaria in Gabon (5). Also, a lack of association between theannealing at 630C for 1 min, and extension at 740C for I min,

29M allele and severe malaria was reported in West Africa

using a themal cycler (GeneAmp PCR system 9700; Perkin‑(6)･ Surpri.singly, the results are quite different among these

Elmer Applied Biosystems). The PCR products of 1 0 randomly

three prevlOuS Studies. Thus, the association between 29M

chosen samples were used for the direct sequenclng With anand severe malaria has not been established yet, and to theABI PRISMTM 3 1 0 Genetic Analyzer (Perkin‑Elmer AppliedBiosystems). The samples with and without the 29M allele

best of our knowledge, association studies on the K29M

wereused as references in the PCR‑single strand conforma‑tion polymorphism (PCR‑SSCP) analysis. Two microliters

ICAM‑ 1 polymorphism have never been ca汀ied out in Asian

populations. For this reason, we here investlgated the possibleassociation between the 29M allele and severe malaria in adultof solution containlng the PCR product was mixed with 6 LLI

In order to examine the association between 29M andofdenaturing solution (95% formamide, 20 mM EDTA, 0･05%
bromphenol blue, 0.05% xylene cyanoI FF). The mixtures

severe malaria, malaria patients living ln northwest Thailand

were denatured at 96oC fわr 5 min and imediately cooled onwere analyzed. Informed consent was obtained from allice. One microliter of the mixtures was applied to lO%

individuals. Two‑hundred three patients with mild malaria,

polyacrylamide gel (acrylamide: bisacrylamide ‑ 49: 1)･

malaria patients living ln nOr血west Thailand･

165 with non‑cerebral severe falciparum malaria, and 109

Electrophoresis was carried out in 0.5 X TBE (45 mM TriS‑

With cerebral malaria who were treated at the Hospital fわrborate [pH 8.0】, 1 mM EDTA) under constant current of20mA/gel, uslng a minlgel electrophoresis apparatus with a*Corresponding author: Tel: +8 113‑5841‑3693, Fax: +81‑3‑5802‑

constant temperature control system (90 × 80 × 1 mm, AE

86 1 9, E‑mail: juno@m.u‑tokyo.acjp

6410 and AE 6370; ATTO, Tbkyo). Single‑strand DNA114

Table I. Genotype and allele frequencies of the 29M allele in the Thai malaria patients
Mild malaria controls Non‑cerebral severe malaria Cerebral malaria(n‑203)l(n‑165)(n‑109)

Genotype frequency

29M /29M

0 (0.0)2

29M /29K

7 (3.4)29K/ 29K

196 (96.6)

Allcle frcqucncy‑

29M

7(1.7)

29K

399 (98.3)

I no. of patients.
2 pcrccntagc･
ll nol 0fgcncs･Table 2. 1ntcraction oflCAM‑1 29M, TNFR2 196R, and scverc malaria in Thailand
Mild malaria controls Non一ccrcbral scvcrc malaria Ccrcbral malaria(n‑201)l(n‑163)(n‑)08)Combination ofalIcle positivity

29M+/ 196R+
29M+/ 196R‑
29M‑/ 196R+
29M‑/ 196R‑

1 (0.5)26 (3.0)

49 (24.4)

145 (72.1)

l no. orpatients.
2 percentage･

since a large sample size is statistically required to detect a丘agments in血e gel were visualized by silver staining (Daiichi

susceptibilityallele with a low frequency (1 I). However,Pure Chemicals, Tbkyo).

The genotype and allelefrequencies of the 29M allele inconsidering the low kequency of the 29M allele, We may say

malaria patients are shown in Table 1. 29M was rare in Thai‑that this variant is unlikely to be selectively advantageousland, and showed neither positive nor negative association

agalnSt malaria in Thailand.

Over the last few years, the association of the 29M allele

with severe malaria i.n adult Thai patients (Chi‑square testwith severe malaria has been the subject of controversy (4‑6).

based on a 2 X 2 contlngenCy table).

mediate upregulation ofICAM‑ I (7)･ Recently, ap rin° acid

Different parasite straiTS Show different avidity for the ICAM‑l ligand (12), suggestlng that ICAM‑1 alleles predispoPing

substitution from methionine to arglnine at poslt10n 196 in

to severe malaria vary among different populations, slnCe

Tumor necrosis factor receptor 2 (TNFR2) is known toexon 6 of the TNFR2 was found, and the allele coding arglnlne

malaria strainsthemselves are considered to be different from(196R) was reported to be significantly associated withpopulation to population. Thus, the discordant results amongsystemic lupus erythematosus in Japanese (8,9)･ Therefore,

the present and three previous association studies may be due

We lnVeStlgated a possible association orthe 1 96R allele withto the difference in strains ofP. falclj)arum.

severe malaria in the same cohort, but detected no slgnificant
association (10). In order to analyze the interaction between

ACKNOWLEDGMENTS

29M and 1 96R in regard to the severity ofmalaria, the present
case‑Control data were stratified by the allele positivities ofThis study was supported by the Core University System

the 29M and 196R alleles (Table 2). Although no significant

Exchange Programme under the auspices of the Japan Society

difference was observed among the three malaria groups, thefor the Promotion Of Science, C.oordinated by the University

number of individuals possessing both alleles was marglnallyof Tbkyo and MahidoI Universlty, by the National Researchhigher in the group of patients with cerebral malaria than inCouncil of Thailand, and by a grant‑in‑aid for Scientificthose with mild malaria (Fisher's exact test; P ‑ 0.052).

Research from the Ministry of Education, Science, Sports

Because we cannot exclude the possibility that the small P

and Culture oHapan.

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116