Statistical analyses - Association of the Interleukin-1 beta and Interleukin-1 Receptor Accesso

B. METHODS

3. Statistical analyses

We performed χ² tests to check for deviations from the Hardy–Weinberg equilibrium (HWE). The genetic models for the association tests were divided into the co-dominant, dominant and recessive models. A multiple logistic regression model was used to calculate the odds ratios (OR), the 95% confidence intervals (CI) and the P values, after controlling for age and gender as covariates, with using SAS statistical software (SAS 9.1 and SAS Enterprise Guide 4.1; SAS Institute, Cary, NC, USA). A linkage disequilibrium (LD) block of SNPs was confirmed using HAPLOVIEW software (version 4.0;

http://www.broad.mit.edu/mpg/haploview) (Fig 1B. and 1D). The individual haplotypes

were inferred by the EM algorithm and with using the SAS haplotype procedure. Haplotype analyses were done by performing multiple logistic regression analyses. For all the statistical tests, significance was set at P <0.05. For more precise estimates, statistical computation was performed to determine the P values of Fisher’s exact test by Monte Carlo simulation (number of iterations = 100,000).

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III. RESULTS

A. The genotypes and allele frequencies

IL-1B gene is located on chromosome 2q14 and we selected 15 known IL-1B SNPs

from dbSNP database. Of theses, seven were located in the flanking 5’UTR, five were in introns, two were in the 3’UTR and one was in the flanking 3’UTR (Fig. 1A). IL-1RAcP gene is located on chromosome 3q28 and we selected 13 known 1L-1RAcP SNPs. Of these, eight were located in introns, four were in the flanking 5’UTR and one was in the 3’UTR (Fig. 1C). Four IL-1B SNPs and six IL-1RAcP SNPs were polymorphic [i.e., a minor allele frequency (MAF) of >0.05] in the spontaneously recovered group, which represents the general Korean population (Fig. 1A, 1C and Table 2). The frequencies of these SNPs in the spontaneously recovered group, the chronic hepatitis group, the liver cirrhosis group and the HCC group are shown in Table 2. The remaining eleven IL-1B SNPs and seven IL-1RAcP SNPs were monomorphic or had a low MAF (<0.05). The genotype frequencies of these ten SNPs did not deviate from those expected under Hardy-Weinberg equilibrium (HWE) at each locus in the Korean population (P >0.05; HWE in Table 2), except at IL1RAcP -51668T>A SNP in the chronic hepatitis group (P = 0.035).

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Table 2. Frequencies of IL-1B and IL-1RAcP genotypes and Hardy-Weinberg equilibrium

HWE p-value hepatocellular carcinoma, MAF minor allele frequency, HWE Hardy-Weinberg equilibrium,

*p<0.05.

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B. The allelic and genotype associations with persistent HBV infection and the presence of HCC

To determine whether specific IL-1B SNP or IL-1RAcP SNP alleles are associated with persistence of HBV infection, we performed genotypic association analyses by presuming there are two genetic models, namely, the spontaneously recovered group and the others, and then we performed logistic regression analyses. The allele frequencies and association test results are summarized in Table 3. Of the ten polymorphic Korean 1B SNPs and IL-1RAcP SNPs, three SNPs were associated with persistent HBV infection. IL-1B -2023 C

allele was associated with an increased risk of a persistent HBV infection in the dominant model (OR = 1.63; 95% CI = 1.02-2.58; P = 0.03). IL-1RAcP -8261 T allele was associated with HBV persistence in the co-dominant and dominant models (OR = 0.64; 95% CI = 0.460.89; P = 0.008 and OR=0.58; 95% CI = 0.370.92; P = 0.02, respectively). And IL1RAcP -8183 A allele was associated with an increased risk of a persistent HBV infection in the co-dominant and recessive models (OR = 0.61; 95% CI = 0.41-0.93, P = 0.02 and OR = 0.20;

95% CI = 0.05-0.71; P = 0.01, respectively).

To determine whether a specific IL-1B SNP or the IL-1RAcP SNP alleles are associated with the presence of HCC, we performed other genetic model association analyses and logistic regression analyses between the chronic liver disease group [chronic hepatitis and liver cirrhosis] and the HCC group. One allele of IL-1B SNPs was associated with an increased risk of HCC presence. IL-1B 289 C allele was associated with an increased risk of HCC being present in only the co-dominant model (OR = 1.55; 95% CI = 1.00-2.40; P = 0.04).

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Table 3. Logistic analysis of IL-1B and IL-1RAP polymorphisms with HBV persistence

Co-dominant Dominant Recessive

Loci SR

CH +LC

+HCC OR (95% CI) p OR (95% CI) p OR (95% CI) p

IL-1B

-2023G>C 0.36 0.443 1.36 (0.98-1.87) 0.06 1.63 (1.02-2.58) 0.03* 1.27 (0.69-2.35) 0.43 -581T>C 0.48 0.486 1.13 (0.82-1.56) 0.42 1.28 (0.77-2.14) 0.33 1.09 (0.64-1.83) 0.73 289T>C 0.48 0.481 1.16 (0.84-1.60) 0.35 1.31 (0.78-2.19) 0.29 1.13 (0.67-1.90) 0.63 3340A>G 0.42 0.400 0.88 (0.63-1.22) 0.46 0.82 (0.51-1.32) 0.42 0.89 (0.49-1.64) 0.73 IL-1RAcP

-51668T>A 0.35 0.361 1.02 (0.74-1.40) 0.89 0.97(0.62-1.52) 0.91 1.15 (0.60-2.20) 0.67 -8261T>C 0.40 0.301 0.64 (0.46-0.89) 0.008* 0.58 (0.37-0.92) 0.02* 0.52 (0.27-1.01) 0.05 -8183A>G 0.19 0.128 0.61 (0.41-0.93) 0.02* 0.66 (0.40-1.07) 0.09 0.20 (0.05-0.71) 0.01*

-256C>T 0.29 0.277 0.92 (0.65-1.30) 0.66 0.81 (0.51-1.26) 0.35 1.35 (0.56-3.22) 0.49 59264G>A 0.21 0.188 0.84 (0.56-1.24) 0.38 0.85 (0.53-1.34) 0.49 0.62 (0.20-1.92) 0.41 65445A>G 0.23 0.203 0.83 (0.57-1.22) 0.35 0.84 (0.53-1.33) 0.46 0.63 (0.22-1.77) 0.38

Genotype distributions and P-value for logistic analysis of three alternative models (co-dominant, dominant and recessive models) controlling for age and sex as covariates are shown. SR spontaneously recovered group, CH chronic hepatitis B, LC liver cirrhosis, HCC hepatocellular carcinoma, OR odds ratio, 95% CI 95% confidence interval, * p<0.05.

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Table 4. Logistic analysis of IL-1B and IL-1RAcP polymorphisms with the presence of HCC

Co-dominant Dominant Recessive

Loci CH+

HCC

OR (95% CI) p OR (95% CI) p OR (95% CI) P

IL-1B

-2023G>C 0.432 0.470 1.26 (0.82-1.91) 0.27 1.45 (0.75-2.78) 0.26 1.24 (0.60-2.55) 0.54 -581T>C 0.497 0.447 1.43 (0.93-2.21) 0.10 1.73 (0.82-3.64) 0.14 1.49 (0.77-2.88) 0.23 289T>C 0.497 0.430 1.55 (1.00-2.40) 0.04* 1.90 (0.89-4.05) 0.09 1.68 (0.87-3.21) 0.11 3340A>G 0.383 0.441 1.35 (0.88-2.06) 0.16 1.33 (0.72-2.45) 0.36 1.77 (0.81-3.88) 0.14 IL-1RAcP

-51668T>A 0.349 0.389 1.20 (0.80-1.79) 0.37 1.13 (0.62-2.03) 0.67 1.58 (0.73-3.40) 0.23 -8261T>C 0.303 0.296 1.02 (0.65-1.62) 0.90 1.00 (0.56-1.78) 0.99 1.15 (0.39-3.34) 0.79 -8183A>G 0.126 0.133 1.01 (0.55-1.87) 0.95 1.07 (0.55-2.08) 0.83 0.43 (0.02-7.16) 0.56 -256C>T 0.271 0.290 1.26 (0.80-1.98) 0.30 1.62 (0.90-2.91) 0.10 0.69 (0.22-2.17) 0.53 59264G>A 0.191 0.180 0.90 (0.53-1.53) 0.71 0.83 (0.45-1.52) 0.55 1.39 (0.30-6.32) 0.66 65445A>G 0.169 0.191 0.87(0.52-1.46) 0.64 0.88(0.48-1.59) 0.61 0.69 (0.14-3.42) 0.65

Genotype distributions and P-value for logistic analysis of three alternative models (co-dominant, dominant and recessive models) controlling for age and sex as covariates are shown. SR spontaneously recovered group, CH chronic hepatitis B, LC liver cirrhosis, HCC hepatocellular carcinoma, OR odds ratio, 95% CI 95% confidence interval, * p<0.05.

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C. Haplotype association with persistent HBV infection and the presence of HCC

Linkage disequilibrium (LD) blocks were constructed by the Gabriel method and with using HAPLOVIEW software (cut-off values: HWE=0.001 and MAF=0.001, spontaneously recovered group=107, chronic liver disease=206, HCC group=86) (Fig. 1B and 1D). IL-1B had one LD block that comprised -2023, -581 and 289. This block had three haplotypes, the most common of which was ht1 (G-T-T), followed by ht2 (C-C-C) and rare ht3 (G-C-C). IL-1RAcP had two LD blocks, that is, block 1 (B1) that is comprised -8261 and -8183, while the

LD block 2 (B2) consists of 59264 and 65445. IL-1RAcP B1 had three haplotypes, and these were B1ht1 (T-A), B1ht2 (C-G), and B1ht3 (C-A), and B2 had two haplotypes, B2ht1 (G-A) and B2ht2 (A-G). The haplotype frequencies and the association test results are summarized in Table 4. On the haplotype analysis, we found that IL-1B ht2 (C-C-C) was associated with an increased risk of persistent HBV infection in only the recessive model (OR = 0.61; 95%

CI = 0.38-0.97; P = 0.03). IL-1RAcP B1ht1 (T-A) was associated with an increased risk of persistent HBV infection in the co-dominant model and the dominant model (OR = 0.64;

95% CI = 0.46-0.89; P <0.01 and OR = 0.58; 95% CI = 0.37-0.92; P = 0.02, respectively).

In contrast, IL-1RAcP B1ht2 (C-G) was associated with viral clearance in the co-dominant model and the dominant model (OR = 1.61, 95% CI = 1.07-2.43; P = 0.02, and OR = 1.92;

95% CI = 1.40-17.28; P = 0.01 respectively). There was no significant haplotype associated with the presence of HCC.

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Table 5. Logistic analysis of IL-1B and IL-1RAP Haplotype with HBV persistence

Frequency (Percentage %) SR vs. CH+LC+HCC

Block Haplo-

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IV. DISCUSSION

In the present study, we found that IL-1B and IL-1RAcP gene polymorphisms were associated with a persistent HBV infection. IL-1B -2023 C allele and IL-1B CCC haplotype that contains -2023 C allele were associated with persistent HBV infection. IL-1RAcP -8261 T allele and -8183 A allele were also associated with persistent HBV infection. The haplotype IL-1RAcP B1ht1 (T-A) that consisted of -8261 and -8183 showed linkage with persistent HBV infection. Concerning the presence of HCC, IL-1B 289 C allele was marginally associated with an increased risk of the HCC presence in only the co-dominant model. Any significant haplotype associated with the increased risk for HCC was not found.

The clearance of HBV requires a coordinated effort by the innate and adaptive immune responses. The innate proinflammatory cytokine IL-1ß initiates a wide spectrum of immunological responses to infection, stress and tissue damage (Dinarello, 1996). IL-1ß attenuates the interferon-induced antiviral activity and it also functions as a costimulator to activate Th2 cells (Tian, et al., 2000; Wang, et al., 2003). Binding of IL-1 to its receptor (IL-1RI) leads to recruitment of the membrane form of IL-1RAcP (mIL-1RAcP), which is essential for signal transduction. One recent study have shown that HBeAg can bind to mIL-1RAcP on the cell surface, and this induces the association of IL-1RI and mIL-mIL-1RAcP, which triggers the recruitment of adaptor protein myeloid differentiation factor 88 to the 1RAcP complex (Yang, et al., 2006). Assembly and activation of the IL-1RI/mIL-1RAcP signaling complex by HBeAg can activate the downstream nuclear factor –kappa B pathway through I kappa B degradation, and this then induces the expression of IL-1 responsive genes, including the genes for IL-1ß, IL-6, TNF-α, inducible nitric oxide

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synthase and granulocyte macrophage colony stimulating factor (Yang, et al., 2006). This result raised the possibility that the IL-1ß and IL-1RAcP may contribute to the control of HBV replication during a natural course of HBV infection.

With regard to hepatocellular carcinogenesis, IL-1ß up-regulates mitogen-inducible nitric oxide (NO) and cyclooxygenase (COX) 2. The combined expression of inducible NO and COX-2 may have an important effect on the development of HCC by modulating angiogenesis (Rahman, et al., 2001). In addition, recent evidence has also shown that IL-1ß prevents apoptosis in many cell types, including keratinocytes, chondrocystes, osteoclasts, neutrophils, monocytes and lymphocytes (Kothny-Wilkes, et al., 1998; Watson, et al., 1998;

Schmidt, et al., 1999; Kühn, et al., 2000; Lee, et al., 2002). It has been recently reported that the highest IL-1ß mRNA levels are observed in cirrhotic tissues surrounding HCC, suggesting that the consequent chronic inflammation may contribute to inducing the oncogenic mutations that are responsible for initiating or promoting primary liver cancer (Bortolami, et al., 2008).

IL-1B gene was reported to be a possible candidate gene for an increased risk of

persistent HBV infection and fibrosis. Zhang et al. (Zhang, et al., 2004) reported that 1B-511 CC genotype was closely related with HBV-DNA replication. In our previous study, IL-1B-511 T allele was marginally related to clearance of HBV infection in the Korean patients

with chronic hepatitis B (P =0.049, unpublished data). In addition, the IL-1B -31 T allele was found to be implicated in liver fibrogenesis of patients with chronic HBV infection through the regulation of matrix metalloproteinase production by the hepatic satellite cells(Migita, et al., 2007). IL-1RAcP has been reported to play an important role in modulating the host immune response to HBV infection. However, any data on the

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association of the IL-1RAcP polymorphisms and chronic HBV infection has not yet reported.

Because our previous study showed only a marginal association between IL-1B -511 T allele and HBV infection in the Korean population, we further evaluated the association of the other SNPs of IL-1B and IL-1RAcP with persistent HBV infection and HCC. Our study showed IL-1B -2023 C allele and its haplotype CCC were associated with a persistent HBV infection (OR = 1.63; 95% CI = 1.02-2.58; P = 0.03, and OR = 0.61; 95% CI = 0.38-0.97; P

= 0.03, respectively). IL-1RAcP -8261 T allele and -8183 A allele were also associated with persistent HBV infection (OR = 0.64; 95% CI = 0.46-0.89; P = 0.008 in the co-dominant model and OR = 0.20; 95% CI = 0.05-0.71; P = 0.01 in the recessive model, respectively).

IL-1RAcP -8261 T allele and -8183 A allele comprised the haplotype IL-1RAcP B1ht1, and

this haplotype also showed a relation with a persistent HBV infection (OR = 0.64; 95% CI = 0.46-0.89; P <0.01 in the co-dominant model). On the other hand, the IL-1RAcP -8261C/-8183G haplotype was associated with HBV clearance (OR = 1.92; 95% CI = 1.40-17.28; P = 0.01 in the dominant model). The IL-1B -2023G>C SNP is located in the promoter region, and the IL-1RAcP -8261T>C SNP and -8183A>G SNP are located in the intron region.

There is no known amino acid change in these SNPs. Further research is required to understand the functions of the variants that promote persistent HBV infection.

IL-1B has been suggested to be a potential candidate gene for cancer development. In

Caucasians infected with Helicobacter pylori, the IL-1B -31C/-511T haplotype has been associated with an increased risk of gastric cancer (ElOmar, et al., 2000; 2001). The IL1B -31 T/T genotype and the IL-1B --31T/-511C haplotype have been found to be associated with the presence of HCC in Japanese patients with chronic HCV infection (Wang, et al., 2003).

It has also been reported that the IL-1B -511C, -31T allele was a genetic marker for the

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development of HCC in chronic hepatitis B patients in a Thai population (Hirankarn, et al., 2006). This discordance may be due to the differences of organs and the heterogeneous nature of carcinogenesis in these organs. Our study showed only a marginal relation between the IL-1B 289 C allele and an increased risk of HCC in the co-dominant model (OR=1.55;

95% CI=1.00-2.40; P =0.04). Large-scale case-control or cohort studies are needed in the future to confirm the association of the IL-1B and IL-1RAcP SNPs with the development of HCC.

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V. CONCLUSION

In conclusion, the present study suggests that SNPs within IL-1B and IL-1RAcP genes are associated with persistent HBV infection. This study is the first to show that several IL-1RAcP polymorphisms may be significant genetic markers for a persistent HBV infection.

Our novel findings provide further evidence that genetic factors are important for determining the outcome of HBV infection.

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Interleukin-1 beta 및 Interleukin-1 Receptor Accessory Protein 유전자 다형성과 만성 B 형간염 및 간세포암종

발생과의 관련성

아주대학교 대학원의학과 김 순 선

(지도교수 : 조 성 원)

목적: B 형 간염 바이러스(HBV)는 감염 후 만성 간염, 간경변증 및 간세포암종 등 다양한 임상 경과를 보이며 이에 영향을 미치는 숙주의 면역 반응 요인으로 사이토카인의 역할은 중요하다. Interleukin (IL)-1 은 자연면역에서의 숙주 반응의 매개자로 대표적인 염증성 사이토카인으로 알려져 있다. 본 연구는 최근 주목받는 single nucleotide polymorphism (SNP) chip 을 이용한 high-throughput 기술을 적용하여 B 형 간염의 만성화 및 간세포암종 발생과 관련된 유전자 다형성을 알아보고자 하였다.

방법: 2000 년 6 월부터 2006 년 2 월까지 아주대학교병원에 내원한 399 명을 대상으로 선정된 1,536 개의 SNP 에 대해 Illumina 사의 chip 으로 주문 제작하였다. 대상 환자들은 바이러스 제거군 (n=107, HBsAg 음성, Anti-HBc 및 Anti-HBs 양성), 만성 간염군 (n=111), 간경변증군 (n=95), 간세포암종군 (n=86)으로 분류하였다.

결과: IL-1β 유전자의 promoter 2 부위 (-581T>C, -2023G>C)와 intron 2 부위 (3340A>G, 289T>C) 및 1L-1 receptor accessory protein (RAcP) 유전자의 promoter 1 부위 (51668T>A)와 intron 5 부위 (8261T>C,

-- 28 --

8183A>G, -256C>T, 59264G>A, 65445A>G)가 다형성을 보였다. 만성 간염군, 간경변증군 및 간세포암종군에서 IL-1B-2023 C allele, IL-1RAcP -8261 T allele 및 -8183 A allele 이 바이러스 제거군에 비해 유의하게 많았다.

(OR, 1.63, P = 0.03, OR, 0.64, P <0.01 and OR, 0.20, P = 0.01, respectively). IL-1B 289C allele 이 HCC group 에서 만성 간염군 및 간경변증군에 비해 많았다(OR, 1.55, P = 0.04). Haplotype 분석에서 IL-1B -2023C/-581C/2893C haplotype 과 IL-1RAcP -8261T/-8183A haplotype 이 만성 간염군, 간경변증군 및 간세포암종군에서 바이러스 제거군에 비해 유의하게 많았으며 간세포암종과 연관된 haplotype 은 없었다.

결론: SNP chip 을 이용한 분석에서 IL-1B promoter -2023 부위 및 IL-1 RAcP intron -8261, -8183 부위의 유전자 다형성은 HBV 만성화와 연관된

문서에서 Association of the Interleukin-1 beta and Interleukin-1 Receptor Accessory Protein Gene Polymorphisms with Persistent Hepatitis B Virus Infection and Hepatocellular Carcinoma (페이지 19-38)