Association between ARPC2 Polymorphisms and Kawasaki Disease in Korean Children

Corresponding author: Moo Il Kwon, Department of Anesthesiology and Pain Medicine, Kyung Hee University Hospital, #1 Hoegi- dong, Dongdaemun-gu, Seoul 131-702, Korea

Tel: +82-2-958-8589, E-mail: [email protected] Received November 24, 2010, Revised December 5, 2010 Accepted December 10, 2010

Association between ARPC2 Polymorphisms and Kawasaki Disease in Korean Children

Departments of *Anesthesiology and Pain Medicine,

Pediatrics, School of Medicine, Kyung Hee University, Seoul, Korea

Su Sang Jung*, Sung Wook Park*, Kyung Lim Yoon

, Moo Il Kwon*

Kawasaki disease (KD) is an acute, self-limited vasculitis of infants and young children that predominantly affects the coronary arteries. KD is the leading cause of acquired heart disease among children in developed countries. We hypothesized that the actin related protein 2/3 complex, subunit 2, 34 kDa (ARPC2) gene may be related to the development of KD. In this study, the associations between single nucleotide polymorphisms (SNPs) of ARPC2 and KD were investigated in 111 KD patients and 429 healthy controls. Two promoter (rs6720105, −099G/A and rs6720449, −839G/A) and 5 intronic SNPs (rs12992937, rs10932765, rs10169718, rs6436047, and rs13430006) were selected, and genotypes of each SNP were analyzed using Affymetrix targeted genotyping chip. The genetic data of 7 SNPs were evaluated by SNPStats, SNPAnalyzer, Haploview, and Helixtree programs. Two promoter SNPs (rs6720105 and rs6720449) were weakly associated with KD. The SNP rs6720105 showed a statistical difference between KD and controls in the codominant model (OR=1.45, 95% CI=1.00∼2.10, p=0.042). The SNP rs6720449 also showed differences between KD and controls in the codominant (OR=1.47, 95% CI=1.02∼2.14, p=0.036) and dominant models (OR=1.55, 95% CI=1.01∼2.40, p=0.045), respectively. In the analysis of haplotypes, a haplotype (AATTAAG) was weakly associated with KD (frequency=0.249, chi square=3.867, p=0.049). The results suggest that ARPC2 may be associated with the development of KD in Korean children. (Korean J Str Res 2010;18:389∼394)

Key Words: Kawasaki disease, ARPC2, Single nucleotide polymorphism, Haplotype

INTRODUCTION

Kawasaki disease (KD) is an acute febrile vasculitis of infants and children characterized by prolonged fever, skin rash, erythema of the oral mucosa, palms, and soles, bilateral conjunctival injec- tion, and cervical lymphadenopathy. Coronary artery aneurysms

develop in 15 to 25% of untreated KD (Kato et al., 1975;

Vijayan et al., 2009; Rowley et al., 2010). KD is the leading

cause of acquired heart disease among children, and death occurs

in a small but significant percentage of KD patients. Admini-

stration of intravenous immunoglobulin (IVIG) reduces the

aneurysm rate to less than 5% in affected subjects (Tremoulet et

al., 2008; Udi et al., 2008; Hata et al., 2009; Galeotti et al.,

2010; Manlhiot et al., 2010). Although an infectious agent is

strongly implicated according to clinical and epidemiologic fea-

tures, no pathogen had been isolated. Accumulated evidences

suggest that genetic factors are related to the susceptibility and

outcome of KD. The incidence of KD is higher in Asians than

in Caucasians. The risk of KD is 10 times higher in siblings of affected individuals than in general population (Pinna et al., 2008;

Onouchi, 2009; Wood et al., 2009; Yeung, 2010).

Actin polymerization plays an important role in the change of cell shape and locomotion. An approximately 220-kD multi- protein complex induced actin polymerization is found from human platelet. This complex contains actin-related proteins (Arp2 and Arp3) and therefore was named the Arp2/3 complex.

The human Arp2/3 complex consists of 7 subunits: ARP2 (ACTR2), ARP3 (ACTR3), ARC41 (ARPC1B), ARC34 (ARPC2), ARC21 (ARPC3), ARC20 (ARPC4), and ARC16 (ARPC5) (Welch et al., 1997). The actin related protein 2/3 complex, subunit 2, 34 kDa (ARPC2) is one of seven subunits of the human Arp2/3 complex, and located at chromosome 2q36.1. Quantitative real- time RT-PCR analysis of 32 primary gastric cancer samples and 8 gastric cancer cell lines revealed that expressions of all seven subunits were significantly decreased (Kaneda et al., 2004). Single nucleotide polymorphism (SNP) of ARPC2 is associated with ulcerative colitis in Dutch population (Festen et al., 2010).

However, the exact genetic role of ARPC2 is largely unknown.

To investigate the possible relationship between ARPC2 and KD, two promoter and 5 intronic SNPs were evaluated in 111 KD patients and 429 contro1 subjects.

MATERIALS AND METHODS

1. Subjects

This study was approved by the Ethics Committee of the Medical Research Institute, School of Medicine, Kyung Hee University, Seoul, Korea. Blood samples were obtained with informed written consent from unrelated Korean subjects. One hundred and eleven KD patients were recruited at Kyung Hee University Hospital and East-west Neo Medical Center, Seoul, Korea. KD was diagnosed by well-trained physician. Four hundred and twenty nine healthy individuals were enrolled. In the control group, patients with diabetes, stroke, hypertension, and cardiac diseases were excluded. Blood samples for DNA extraction were collected in ethylenediamine tetraacetic acid (EDTA) tube.

Genomic DNA was extracted using DNA isolation kit (Nu- cleoSpin

) for mammalian blood (MACHEREY-NAGEL GmbH

& Co., Düren, Germany) and stored at −20

C before use.

2. SNP Selection and Genotyping

We searched all SNPs of ARPC2 by the following criteria: (1) known heterozygosity; (2) minor allele frequency>0.05; (3) tagging SNPs (http://www.hapmap.org/ and http://www.ncbi.nlm.

nih.gov/SNP/). All coding SNPs of ARPC2 were unknown hetero- zygosity. Finally, two promoter (rs6720105, −1099G/A and rs6720449, −839G/A) and five intronic SNPs (rs12992937, rs10932765, rs10169718, rs6436047, and rs13430006) were selected. Genotyping was performed using Affymetrix targeted genotyping chip (Affymetrix, CA, USA) according to the manu- facturer protocol. In brief, PCR products by specific primers were purified. The purified products were end-labeled by terminal deoxynucleotidyl transferase. Labeled DNAs were hybridized and scanned. The scanned images were analyzed by GCOS software (Affymetrix).

3. Statistics

Statistical analyses were performed using SPSS 18.0 (SPSS Inc., Chicago, IL, USA). Hardy-Weinberg equilibrium (HWE) for each SNP was assessed using SNPStats (http://bioinfo.iconcologia.net/

index.php). For the analysis of genetic data, SNPStats, HelixTree (Golden Helix Inc., Bozeman, MT, USA), and SNPAnalyzer (ISTECH Inc., Goyang, Korea) were used. Logistic regression models (codominant, dominant, and recessive) were used for odds ratios (ORs), 95% confidence intervals (CIs), and corresponding p values, controlling gender as a covariable. The linkage disequili- brium (LD) block was made by Haploview 4.2. For all statistical tests, the significance level was set at 0.05.

RESULTS

The mean age in KD patients was 2.8±2.1 (mean±SD) years.

Coronary artery lesions were present in 35 patients but absent in

76 patients. The standard treatment of 2 g/kg IVIG was admini-

stered to 90 patients. Of these 90 patients, 11 subjects needed

at least one extra dose of IVIG (data not shown). A total of 429

normal controls (mean age 36.8 years) were used as the control

group due to difficulty in obtaining a healthy infant control

group. Although it is ideal to use the control group of matched

age, however, it is not a major problem because it was reported

SNP Genotype Kawasaki Control 　 Codominant

p Dominant

p

　 Recessive

p

Locus n (%) n (%) 　 OR (95% CI) OR (95% CI) 　 OR (95% CI)

rs6720105 G/G 71 (64.0) 231 (53.9) 1.45 (1.00∼2.10) 0.042

1.52 (0.99∼2.34) 0.054 1.80 (0.62∼5.25) 0.250

−1099 A/G 36 (32.4) 171 (39.9) A/A 4 (3.6) 27 (6.3)

rs6720449 G/G 71 (64.5) 231 (54.0) 1.47 (1.02∼2.14) 0.036

1.55 (1.01∼2.40) 0.045

1.78 (0.61∼5.21) 0.260

−839 A/G 35 (31.8) 170 (39.7)

A/A 4 (3.6) 27 (6.3)

rs12992937 A/A 34 (30.6) 124 (28.9) 1.04 (0.77∼1.40) 0.820 1.09 (0.69∼1.71) 0.720 1.00 (0.59∼1.69) 1.000 Intron 1 T/A 55 (49.5) 220 (51.3)

T/T 22 (19.8) 85 (19.8)

rs10932765 C/C 33 (29.7) 123 (28.7) 1.03 (0.76∼1.39) 0.840 1.05 (0.67∼1.66) 0.830 1.03 (0.61∼1.74) 0.910 Intron 2 T/C 56 (50.5) 219 (51.0)

T/T 22 (19.8) 87 (20.3)

rs10169718 G/G 33 (29.7) 123 (28.7) 1.03 (0.76∼1.39) 0.840 1.05 (0.66∼1.66) 0.840 1.03 (0.61∼1.74) 0.910 Intron 3 A/G 56 (50.5) 218 (50.9)

A/A 22 (19.8) 87 (20.3)

rs6436047 C/C 34 (30.6) 124 (29.1) 1.03 (0.77∼1.40) 0.820 1.08 (0.68∼1.69) 0.750 1.01 (0.60∼1.70) 0.980 Intron 3 A/C 55 (49.5) 217 (50.9)

A/A 22 (19.8) 85 (19.9)

rs13430006 T/T 33 (29.7) 123 (28.7) 1.03 (0.76∼1.39) 0.840 1.05 (0.67∼1.66) 0.830 1.03 (0.61∼1.74) 0.910 Intron 4 T/G 56 (50.5) 219 (51.0)

　 G/G 22 (19.8) 87 (20.3) 　

a

p<.05, Genotype distributions are shown as number (%). p values were from logistic regression analyses with the codominant, dominant, and recessive models. OR: odds ratio, CI: confidence interval, n: number of subjects, APRC2: actin related protein 2/3 complex, subunit 2, 34 kDa, SNP: single nucleotide polymorphism.

Table 1. Genotype frequencies of APRC2 SNPs in patients with Kawasaki disease and control subjects.

Fig. 1. Linkage disequilibrium (LD) block among ARPC2 SNPs. The block consists of rs6720105, rs6720449, rs12992937, rs10932765, rs10169718, rs6436047, and rs13430006. APRC2: actin related protein 2/3 complex, subunit 2, 34 kDa, SNP: single nucleotide polymorphism.

that the likelihood of the allele frequency changing according to age is quite low (Segal et al., 2003). The observed genotype distributions of all SNPs were in HWE (p>0.05, data not shown).

Multiple logistic regression analysis with adjustment for gender was performed. The genotype distributions of all SNPs are shown in Table 1. The promoter SNPs rs6720105 and rs6720449 were statistically associated with KD. The frequencies of GG, GA, and AA genotypes for the rs6720105 were 64.0%, 32.4%, and 3.6%

in the KD group, while they were 53.9%, 39.9%, and 6.3% in the control group, respectively. The SNP rs6720105 was asso- ciated with KD in the codominant (OR=1.45, 95% CI=1.00∼

2.10, p=0.042). The frequencies of GG, GA, and AA genotypes

for the rs6720449 were 64.5%, 31.8%, and 3.6% in the KD

group, while they were 54.0%, 39.7%, and 6.3% in the control

subjects. The SNP rs6720449 was associated with KD in the

codominant (OR=1.47, 95% CI=1.02∼2.14, p=0.036) and

dominant models (OR=1.55, 95% CI=1.01∼2.40, p=0.045),

Haplotype Frequency Kawasaki disease Control

Chi square p

+ − + −

GGACGCT 0.544 122 100 465 393 0.041 0.839

AATTAAG 0.249 44 178 225 633 3.867 0.049

GGTTAAG 0.204 55 167 165 693 3.342 0.067

a

p<.05, Haplotypes consist of rs6720105, rs6720449, rs12992937, rs10932765, rs10169718, rs6436047, and rs13430006. APRC2: actin related protein 2/3 complex, subunit 2, 34 kDa, SNP: single nucleotide polymorphism.

Table 2. Haplotype frequencies of ARPC2 SNPs in patients with Kawasaki disease and control subjects.

SNP Genotype Korean

European Chinese Japanese

Kawasaki disease Control

rs6720105 G/G 0.64 0.539 0.4 0.733 0.622

G/A 0.324 0.399 0.467 0.244 0.333

A/A 0.036 0.063 0.133 0.022 0.044

rs6720449 G/G 0.645 0.54 0.424 0.75 0.622

G/A 0.318 0.397 0.441 0.227 0.333

A/A 0.036 0.063 0.136 0.023 0.044

rs12992937 A/A 0.306 0.289 0.458 0.333 -

A/T 0.495 0.513 0.333 0.5 -

T/T 0.198 0.198 0.208 0.167 -

rs10932765 C/C 0.297 0.287 0.333 0.378 0.341

C/T 0.505 0.51 0.417 0.511 0.5

T/T 0.198 0.203 0.25 0.111 0.159

rs10169718 G/G 0.297 0.287 0.333 0.378 0.341

G/A 0.505 0.509 0.417 0.511 0.5

A/A 0.198 0.203 0.25 0.111 0.159

rs6436047 C/C 0.306 0.291 0.367 0.378 0.356

C/A 0.495 0.509 0.433 0.489 0.489

A/A 0.198 0.199 0.2 0.133 0.156

rs13430006 T/T 0.297 0.287 0.333 0.4 0.333

T/G 0.505 0.51 0.417 0.489 0.511

　 G/G 0.198 0.203 0.25 0.111 0.156

APRC2: actin related protein 2/3 complex, subunit 2, 34 kDa, SNP: single nucleotide polymorphism.

Table 3. Genotype frequencies of ARPC2 SNPs in different ethnic population.

respectively. Other SNPs were not associated with KD.

Seven SNPs constructed one LD block (Fig. 1). The LD block consisted of rs6720105, rs6720449, rs12992937, rs10932765, rs10169718, rs6436047, and rs13430006. In Table 2, there were three haplotypes in the block (haplotype GGACGCT, frequency=

0.544; AATTAAG, frequency=0.249; GGTTAAG, frequency=

0.204). Haplotype AATTAAG was weakly associated with KD (chi square=3.867, p=0.049).

DISCUSSION

This study assessed possible genetic association between ARPC2 SNPs and the susceptibility to KD in Korean children. Our results suggest that two promoter SNPs rs6720105 and rs6720449 are associated with KD. To our knowledge, this is the first report that ARPC2 SNPs have been associated with the development of KD.

Several lines of evidence have revealed that SNPs of candidate

genes are associated with KD. The SNP rs2229634 in the inositol

1,4,5-triphosphate receptor type 3 (ITPR3) gene was associated with the risk of developing coronary artery aneurysm in children with Kawasaki disease (Huang et al., 2010). IL10 polymorphisms were associated with coronary artery lesions in acute stage of KD (Weng et al., 2010). The rs738792 of matrix metalloproteinase 11 (MMP11) was associated with KD in Korean population (Ban JY et al., 2010). Two SNPs [rs7305115 (exon 7) and rs4290270 (exon 9)] of tryptophan hydroxylase 2 (TPH2) showed increased susceptibility to the risk of coronary artery lesions in KD (Park SW et al., 2010). KD patients carrying a macrophage migration inhibitory factor (MIF) −173*C allele were found to have an increased risk of coronary involvement (Simonini et al., 2009).

In Table 3, the GG, GA and AA genotype frequencies in the rs6720105 have been reported to be 0.400, 0.467, and 0.133 in European, 0.733, 0.244, and 0.022 in Chinese, and 0.622, 0.333, and 0.044 in Japanese, respectively (http://www.ncbi.nlm.nih.gov/

SNP/). In our population (control group), the GG, GA, and AA genotype frequencies were 0.539, 0.399, and 0.063, which are similar to those seen in Japanese population. The GG, GA, and AA genotype frequencies in the rs6720449 have been reported to be 0.424, 0.441, and 0.136 in European, 0.750, 0.227, and 0.023 in Chinese, and 0.622, 0.333, and 0.044 in Japanese, respectively. In our population, the GG, GA, and AA genotype frequencies were 0.540, 0.397, and 0.063, which are similar to those seen in Japanese population.

To examine whether the rs6720105 (−1099G/A) SNP affects transcription factors, we compared transcription factor binding sites using the online program “AliBaba 2.1” (http://www.gene- regulation.com/pub/programs/alibaba2). At this SNP site, four transcription factors (C/EBPalp, Id3, HNF-1C, and REV-ErbA) can bind to the G-containing sequence, whereas two transcription factors (TBP and Ftz) can bind to the A-containing sequence.

This difference in transcription factor binding might affect the ARPC2 expression. The site of rs6720449 (−839G/A) SNP does not find transcription factors.

In conclusion, our study suggests that ARPC2 appears to be a susceptibility gene in patients with KD in Korean population.

Considering the limited sample size, these results need to be replicated in other populations.

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Kaneda A, Kaminishi M, Sugimura T et al. (2004) Decreased expression of the seven ARP2/3 complex genes in human gastric cancers. Cancer Lett. 212:203-210.

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an overview. Curr. Opin. Infect. Dis. 21:263-270.

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= 국문초록 =

가와사키병은 원인을 알 수 없는 급성 열성 혈관염으로 주로 영아나 소아에서 발생하며 관상동맥에 합병증을 일으 킬 수 있다. 최근 가와사키병의 병인에 대한 관련 유전자의 single nucleotide polymorphism (단일염기다형성)에 대한 연구가 발표되고 있다. Actin related protein 2/3 complex, subunit 2, 34 kDa (ARPC2) 유전자는 세포에서 actin polymerization 조절에 관여한다고 알려져 있다. 본 연구에서는 ARPC2 유전자 다형성과 가와사키병의 관련성을 알아 보기 위하여, 111명의 가와사키병 환아와 429명의 정상인을 비교하였다. ARPC2 유전자의 프로모터에서 2개의 단일 염기다형성 (rs6720105, −1099G/A 및 rs6720449, −839G/A)과 인트론 부분에서 5개의 단일염기다형성(rs12992937, rs10932765, rs10169718, rs6436047 및 rs13430006)을 선정하였다. 유전형 결정은 Affymetrix 사의 타깃 칩을 이용하였고, 유전형의 분석은 SNPStats, SNPAnalyzer, Haploview 및 Helixtree 프로그램을 이용하였다. 분석결과, 2개의 프로모터 단 일염기다형성이 가와사키병과 관련이 있었다(rs6720105, OR=1.45, 95% CI=1.00∼2.10, p=0.042 in the codominant model) (rs6720449, OR=1.47, 95% CI=1.02∼2.14, p=0.036 in the codominant model; OR=1.55, 95% CI=1.01∼2.40, p=0.045 in the dominant model). Haplotype (일배체형) 분석에서도 ARPC2의 7개 단일염기다형성이 이루는 일배체형 (AATTAAG)이 약한 유의성을 보였다(frequency=0.249, chi square=3.867, p=0.049). 이상의 결과는 ARPC2유전자가 가와 사키병의 발병과 관련이 있음을 시사한다.

중심단어: 가와사키병, ARPC2, 단일염기다형성, 일배체형 Tremoulet AH, Best BM, Song S et al. (2008) Resistance to

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Welch MD, DePace AH, Verma S et al. (1997) The human Arp2/3 complex is composed of evolutionarily conserved subunits and is

localized to cellular regions of dynamic actin filament assembly.

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Weng KP, Hsieh KS, Hwang YT et al. (2010) IL-10 polymorphisms are associated with coronary artery lesions in acute stage of Kawasaki disease. Circ. J. 74:983-989.

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