Infection, Genetics and Evolution xxx (2015) xxx–xxx
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Replication of genome wide association studies on hepatocellular carcinoma susceptibility loci of STAT4 and HLA-DQ in a Korean population Lyoung Hyo Kim a,b, Hyun Sub Cheong b, Suhg Namgoong a,b, Ji On Kim b, Jeong-Hyun Kim a, Byung Lae Park b, Sung Won Cho c, Neung Hwa Park d, Jae Youn Cheong c, InSong Koh e, Hyoung Doo Shin a,b,⇑, Yoon-Jun Kim f,⇑ a
Department of Life Science, Sogang University, Republic of Korea Department of Genetic Epidemiology, SNP Genetics, Inc., Republic of Korea c Department of Gastroenterology, Ajou University School of Medicine, Republic of Korea d Department of Internal Medicine, Ulsan University Hospital, Republic of Korea e Department of Physiology, College of Medicine, Han Yang University, Republic of Korea f Department of Internal Medicine and Liver Research Institute, Seoul National University, Republic of Korea b
a r t i c l e
i n f o
Article history: Received 22 January 2015 Received in revised form 3 April 2015 Accepted 15 April 2015 Available online xxxx Keywords: Chronic hepatitis B virus Hepatocellular carcinoma HLA-DQ STAT4
a b s t r a c t A recent genome-wide association study (GWAS) for hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) identified two loci (rs7574865 in STAT4 and rs9275319 in HLA-DQ) in a Chinese population. We attempted to replicate the associations between the two SNP loci and the risk of HCC in a Korean population. The rs7574865 in STAT4 and rs9275319 in HLA-DQ were genotyped in a total of 3838 Korean subjects composed of 287 HBV-related hepatocellular carcinoma patients, 671 chronic hepatitis B virus (CHB) patients, and 2880 population controls using TaqMan genotyping assay. Gene expression was measured by microarray. A logistic regression analysis revealed that rs7574865 in STAT4 and rs9275319 in HLA-DQ were associated with the risk of CHB (OR = 1.25, P = 0.0002 and OR = 1.57, P = 1.44 1010, respectively). However, these loci were no association with the risk of HBV-related HCC among CHB patients. In the gene expression analyses, although no significant differences in mRNA expression of nearby genes according to genotypes were detected, a significantly decreased mRNA expression in HCC subjects was observed in STAT4, HLA-DQA1, and HLA-DQB1. Although the genetic effects of two HCC susceptibility loci were not replicated, the two loci were found to exert susceptibility effects on the risk of CHB in a Korean population. In addition, the decreased mRNA expression of STAT4, HLA-DQA1, and HLA-DQB1 in HCC tissue might provide a clue to understanding their role in the progression to HCC. Ó 2015 Elsevier B.V. All rights reserved.
1. Introduction Hepatitis B virus (HBV) infection is a major global problem, and chronic hepatitis B virus (CHB) infection is a complex clinical entity frequently associated with cirrhosis and hepatocellular carcinoma (HCC) (But et al., 2008; Yang and Roberts, 2010). HCC is a common solid cancer of the liver, the prevalence of which is highest in Asia and Africa, where the endemic high incidence of hepatitis B and ⇑ Corresponding authors at: Department of Life Science, Sogang University, 1 Shinsu-dong, Mapo-gu, Seoul 121-742, Republic of Korea. Tel.: +82 2 705 8615; fax: +82 2 2026 4299 (H.D. Shin). Department of Internal Medicine and Liver Research Institute, Seoul National University, 28 Yungun-dong, Chongro-Gu, Seoul 110-744, Republic of Korea (Y.-J. Kim). E-mail addresses: [email protected] (H.D. Shin), [email protected] (Y.-J. Kim).
hepatitis C creates a strong susceptibility to the development of chronic liver disease, or cirrhosis (Brechot et al., 2000). There are two routes of HBV infection in humans: One is a vertical transmission from an HbsAg-positive mother to her infant; the other is a horizontal transmission by exposure to infectious blood or body fluids such as semen or vaginal fluids. Among vertically HBV affected fetuses, over 90% become CHB affected carriers. Among horizontally HBV affected adults, only 5–10% becomes CHB affected carriers (Perz et al., 2006). Although the mechanisms underlying the different clinical results of HBV infection have not been fully elucidated, previous epidemiological studies have linked diverse factors such as viral strain, gender, infection age, and immune system of the host, with the risk of CHB (Wang, 2003). In addition, host genetic factors are thought to play an important role in the progression of CHB (Pungpapong et al., 2007;
http://dx.doi.org/10.1016/j.meegid.2015.04.013 1567-1348/Ó 2015 Elsevier B.V. All rights reserved.
Please cite this article in press as: Kim, L.H., et al. Replication of genome wide association studies on hepatocellular carcinoma susceptibility loci of STAT4 and HLA-DQ in a Korean population. Infect. Genet. Evol. (2015), http://dx.doi.org/10.1016/j.meegid.2015.04.013
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L.H. Kim et al. / Infection, Genetics and Evolution xxx (2015) xxx–xxx
Thursz, 2001). Recent genome-wide association studies (GWASs) of CHB-related HCC in Asian populations identified susceptibility loci on 1p36.2, 6p21.32, and 21q21.3 (Kamatani et al., 2009; Li et al., 2012; Mbarek et al., 2011; Nishida et al., 2012; Zhang et al., 2010). Moreover, our previous GWAS also reported the risk associated loci for CHB on the HLA region, rs652888 on EHMT2, and rs1419881 on the TFG19 gene (Kim et al., 2013). Most recently, a multi-stage GWAS in a Chinese population identified two susceptibility loci for HCC (rs7574865 at STAT4 and rs9275319 at HLA-DQ) (Jiang et al., 2013). In the present study in order to evaluate the contribution of the two loci identified in this most recent GWAS, we carried out a replication study for rs7574865 and rs9275319 with CHB and HCC in a Korean cohort.
RNA was quantified using Ribogreen (Quant-iT™ RiboGreenÒ RNA Assay Kit, Life Technologies, Grand Island, NY). RNA integrity was assessed using the Experion automated electrophoresis system (Bio-Rad, Hercules, CA) to check the ratio of 28s:18s rRNA in the samples. A total of 500 ng from each sample was labeled using the Illumina Total Prep RNA Amplification Kit following the manufacturer’s directions (Ambion, Austin, TX). cRNA was hybridized overnight to Illumina Human HT-12 Expression BeadChips (Illumina, Inc., San Diego, CA). Microarrays were washed several times and labeled with streptavidin-Cy3 (Amersham Biosciences, Piscataway, NJ) prior to scanning on an Illumina iScan. Signal intensities were analyzed using the Genome Studio software (Illumina).
2. Materials and methods
2.4. Statistical analysis
2.1. Study subjects
The genotypic distributions of SNPs with HCC risk in this replication study was determined by binary logistic regression of referent, allelic, additive, dominant, and recessive models controlling for age (continuous variable) and sex (male = 0, female = 1) as covariates. Since major homozygotes of rs7574865 G>T and rs9275319 A>G were risk genotypes, minor homozygotes and minor alleles were assigned as reference genotype in the referent model, and allelic model analysis, respectively. Genotypes of minor homozygotes, heterozygotes, and major homozygotes of rs7574865 G>T and rs9275319 A>G were given codes 0, 1, and 1; and 0, 0, and 1 in the dominant, and recessive models, respectively. All statistical analyses were performed using statistical analysis system (SAS) version 9.3 (SAS Inc., Cary, NC). Differences in gene expression between cancer and adjacent non-cancer tissues were analyzed by an ANOVA, and the relationship of the respective genotypes with gene expression levels was tested using the paired t-test. P < 0.05 was considered statistically significant. For the expression analysis, we also performed non-parametric analysis because our sample size was small. The Kruskal–Wallis test and Wilcoxon signed rank test were conducted and P value was represented in parentheses at Fig. 1. The plot of expression difference between cancer and paired adjacent non-cancer samples was generated using Prism 4 program.
The DNA from a total of 3838 subjects was used for the replication study. Subjects consisted of 287 CHB-related HCC subjects, 671 CHB subjects, and 2880 controls. The 958 samples from CHB and HCC patients were obtained from the outpatient clinic of the Liver Unit and the Center for Health Promotion at Seoul National University Hospital and Ulsan University Medical Center. The population control samples (n = 2880) were provided by Korea Biobank at the Korea Centers for Disease Control and Prevention. The diagnosis of chronic carriers was established based on seropositivity for HBsAg (Enzygnost1 HBsAg 5.0; Dade Behring, Marburg, Germany) over a 6-month period (Table S1). All the participants provided written informed consent. This project was approved by the Institutional Review Board of Seoul National University Hospital (IRB No. 1007-049-323). 2.2. Genotyping of rs7574865 and rs9275319 The polymorphisms rs7574865 G>T in the STAT4 gene and rs9275319 A>G in the HLA-DQ region were genotyped using the 50 exonuclease TaqMan genotyping assay system (Applied Biosystems, Foster City, CA). Quality control was performed in 10% of the samples by duplicate checking (rate of concordance in duplicates = 100%).
3. Results
2.3. RNA extraction and gene expression microarrays
3.1. Association analysis of two susceptibility loci
Total RNA was extracted from HCC and adjacent non-cancer tissues using NucleoSpinÒ (MACHEREY-NAGEL, Düren, Germany).
In this study, a total of 3838 samples were used for the replication study, consisting of 287 HCC, 671 CHB, and 2880 population
Fig. 1. STAT4, HLA-DQA1, and HLA-DQB1 relative expression levels according to rs7574865 and rs9275319 in paired cancer tissues and adjacent non-cancer tissues from 23 subjects with HCC.
Please cite this article in press as: Kim, L.H., et al. Replication of genome wide association studies on hepatocellular carcinoma susceptibility loci of STAT4 and HLA-DQ in a Korean population. Infect. Genet. Evol. (2015), http://dx.doi.org/10.1016/j.meegid.2015.04.013
Gene/loci
STAT4/rs7574865
HLA-DQ/rs9275319
Allele
T/Ga
G/Aa
Genotype
PCs
CHB
HCC
CHB + HCC vs. PCs
n = 2880 (%)
n = 671 (%)
n = 287 (%)
OR (95%CI)
TT GT GG Allele T G Dominant TT GT + GG Recessive TT + GT GG
306 (10.7) 1251 (43.9) 1293 (45.4)
63 (9.6) 261 (39.7) 334 (50.7)
20 (7.1) 103 (36.4) 160 (56.5)
1863 (32.7) 3837 (67.3)
387 (29.4) 929 (70.6)
306 (10.7) 2544 (89.3)
GG AG AA Allele G A Dominant GG AG + AA Recessive GG + AG AA
HCC vs. CHB P
OR (95%CI)
Reference 1.10 (0.83–1.45) 1.20 (1.05–1.38)
143 (25.3) 423 (74.7)
63 (9.6) 595 (90.4)
1557 (54.6) 1293 (45.4)
HCC vs. CHB + PCs P
OR (95%CI)
0.52 0.008
Reference 1.21 (0.63–2.33) 1.17 (0.85–1.61)
Reference 1.25 (1.11–1.41)
0.0002
20 (7.1) 263 (92.9)
Reference 1.34 (1.15–1.56)
324 (49.3) 334 (50.8)
123 (43.5) 160 (56.5)
Reference 1.27 (0.98–1.65)
248 (8.7) 890 (31.3) 1702 (59.9)
30 (4.6) 177 (26.9) 454 (69.0)
12 (4.2) 72 (25.4) 199 (70.3)
1386 (24.4) 4294 (75.6)
237 (17.9) 1085 (82.1)
248 (8.7) 2592 (91.3) 1138 (40.1) 1702 (59.9)
HCC vs. PCs P
OR (95%CI)
P
0.56 0.34
Reference 1.16 (0.67–2.00) 1.32 (1.00–1.73)
0.60 0.05
Reference 1.24 (0.72–2.12) 1.33 (1.02–1.73)
0.44 0.04
Reference 1.16 (0.88–1.51)
0.29
Reference 1.40 (1.11–1.75)
0.004
Reference 1.37 (1.1–1.71)
0.005
0.0002
Reference 1.16 (0.83–1.63)
0.37
Reference 1.47 (1.12–1.95)
0.005
Reference 1.54 (1.16–2.04)
0.003
0.07
Reference 1.30 (0.70–2.43)
0.42
Reference 1.51 (0.90–2.53)
0.12
Reference 1.45 (0.86–2.44)
0.17
Reference 1.71 (1.18–2.48) 1.55 (1.31–1.84)
0.005 6.5 107
Reference 1.25 (0.52–2.98) 1.07 (0.72–1.61)
0.61 0.74
Reference 2.41 (1.17–4.95) 1.87 (1.32–2.66)
0.02 0.0005
Reference 2.17 (1.08–4.36) 1.66 (1.19–2.32)
0.03 0.003
96 (16.9) 470 (83.1)
Reference 1.57 (1.37–1.8)
1.44 1010
Reference 1.02 (0.74–1.39)
0.92
Reference 1.77 (1.36–2.31)
1.98 105
Reference 1.59 (1.23–2.04)
0.0004
30 (4.5) 631 (95.5)
12 (4.2) 271 (95.8)
Reference 1.56 (1.33–1.84)
7.6 108
Reference 0.99 (0.69–1.42)
0.95
Reference 1.51 (1.14–2.03)
0.006
Reference 1.69 (1.24–2.30)
0.0008
207 (31.3) 454 (68.7)
84 (29.7) 199 (70.3)
Reference 2.17 (1.54–3.05)
1.0 105
Reference 1.18 (0.53–2.62)
0.69
Reference 2.59 (1.14–5.01)
0.005
Reference 3.16 (1.58–6.34)
0.001
L.H. Kim et al. / Infection, Genetics and Evolution xxx (2015) xxx–xxx
Logistic regression models were used to calculate the ORs, 95% CIs, and the corresponding P values of referent, allele, dominant, and recessive models controlling for age and sex as covariates. Significant associations are shown in bold. CHB, chronic hepatitis B; HCC, hepatocellular carcinoma; PCs, controls; OR, odd ratio; CI, confidence interval. a Risk allele (G allele for rs7574865, A allele for rs9275319).
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Please cite this article in press as: Kim, L.H., et al. Replication of genome wide association studies on hepatocellular carcinoma susceptibility loci of STAT4 and HLA-DQ in a Korean population. Infect. Genet. Evol. (2015), http://dx.doi.org/10.1016/j.meegid.2015.04.013
Table 1 Logistic analysis of association of SNPs in STAT4 and HLA-DQ with the risk of CHB and HCC in a Korean population (n = 3838).
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controls (PCs). Four different statistical comparisons were employed to determine the genetic effects on CHB infection and/ or HCC progression, i.e., (1) risk of CHB (CHB plus CHB-related HCC vs. PCs), (2) risk of HCC among CHB patients (CHB related HCC vs. CHB), and (3) overall risk of HCC (CHB-related HCC vs. non-HCC, including CHB plus PCs). We also examined the association between (4) CHB-related HCC and PCs to support the associations above. Data were analyzed using logistic regression. We fitted referent, allelic, dominant, and recessive models (Table 1). The results of logistic regression analysis for each SNP are shown in Table 1. The rs7574865 polymorphism in STAT4 was not found to be associated with the risk of HCC among CHB patients (P = 0.29–0.42), contrary to what had been reported in the Chinese population. However, when compared with controls, the loci showed stronger association, e.g., CHB + HCC vs. PCs (P = 0.0003–0.07), HCC vs. PCs (P = 0.003–0.17), and HCC vs. CHB + PCs (P = 0.005–0.17). Taking the subjects carrying the TT genotype for rs7574865 as a reference, the subjects carrying genotype GG showed an increased risk of CHB + HCC and HCC compared with PCs (P = 0.008 and 0.04, respectively). Similarly, although the rs9275319 polymorphism in HLA-DQ was not associated with the risk of HCC among CHB patients (P = 0.69–0.92), it revealed strong association when compared to chronic HBV carriers (CHB + HCC vs. PCs, P = 1.44 1010– 1.0 105). Also, the rs9275319 showed strong association of CHB-related HCC vs. PCs (P = 0.0004–0.001) and CHB-related HCC vs. non-HCC (P = 1.98 105–0.005). Taking the subjects carrying the GG genotype for rs9275319 as a reference, the subjects carrying genotype AG and AA showed an increased risk of CHB + HCC and HCC compared with PCs and CHB + PCs (P = 6.5 107–0.03).
included six different Chinese groups (4319 HCC cases vs. 4966 controls) in meta-analysis, rs7574865 reached genome-wide significance (P = 2.48 1010). Although the significance of rs7574865 had reached genome wide threshold, the associations were not replicated in all the six different Chinese groups (OR = 1.12–1.36, P = 0.27–0.0016) (Jiang et al., 2013). Our initial replication result for rs7574865 showed no association with risk of HCC (OR = 1.16, P = 0.29 in allelic model) and this result was similar to result of Beijing and Henan populations among six different Chinese populations (OR = 1.14, P = 0.15 and OR = 1.12, P = 0.27, respectively). In two other studies, Chen et al. failed to replicate the effect of rs7574865 on the risk of HCC in an independent 1278 Chinese population (P > 0.05). And Clark et al. reported rs7574865 was marginally associated to the risk of HCC in a 445 Vietnamese population (P = 0.048 in allelic model) (Chen et al., 2013; Clark et al., 2013). When putting all these studies together, the genetic contribution of rs7574865 upon the risk of CHB-related HCC could be seen as mild (if any), and its effects might be hard to detect without a larger sample size. However, rs7574865 showed significant association with the risk of chronic HBV infection in a Korean population (OR = 1.25, P = 0.0002 in allelic model). This genetic effect also has been examined in a Chinese population (Jiang et al., 2013). Researchers compared 3935 chronic HBV carriers without HCC with 4585 healthy subjects who did not carry HBV (Jiang et al., 2013). The rs7574865 SNP in STAT4 was weakly associated with a trend toward higher frequency of the G allele in chronic HBV carriers (OR = 1.12, Pmeta = 0.003). Considering all results of the current study and previous research, we might conclude that rs7574865 in STAT4 has a weak association with the risk of chronic HBV infection.
3.2. Gene expression analysis of STAT4 and HLA-DQA1
4.2. Genetic effects of rs9275319 in HLA-DQ on HCC susceptibility and HBV progression
Next, we examined the genotype effects of rs7574865 on STAT4 mRNA expression. The rs9275319 is located between HLA-DQA2/ DQB2 and HLA-DQA1/DQB1 (65 kb away from genes). So, we measured mRNA expression levels of these nearby genes. We analyzed the mRNA levels of STAT4 and four HLA-DQ genes in 23 paired samples (HCC and adjacent normal tissues) using HumanHT-12 Expression BeadChips (Illumina). We were unable to detect genotype effects of either locus on mRNA expression. However, significantly lower mRNA levels of three genes in HCC tissues were observed, compared with those in adjacent non-cancer tissues (P = 8.28 105 in STAT4; P = 4.9 109 in HLA-DQA1; P = 1.4 104 in HLA-DQB1) (Fig. 1). 4. Discussion Recently, a Chinese three-stage genome-wide association study of hepatitis B virus-related hepatocellular carcinoma identified two loci with genome-wide significance in the STAT4 and HLA-DQ genes. We performed a replication study of rs7574865 in STAT4 and rs9275319 in HLA-DQ with clinically classified HCC, CHB, and controls in a Korean population. Although we could not replicate the association of the SNPs with the risk of HCC, statistical analysis revealed that these two SNPs are significantly associated with the risk of CHB. 4.1. Genetic effects of rs7574865 in STAT4 on HCC susceptibility and HBV progression In the Chinese study noted above, rs7574865 of STAT4 was not significantly associated with the risk of CHB-related HCC (1161 HCC cases vs. 1353 controls) in the GWAS stage when correction for multiple testing was applied. However, when the researchers
In the Chinese study noted above, the rs9275319 SNP in HLA-DQ was not found to be significantly associated with the CHB-related HCC group (1161 HCC cases vs. 1353 controls) in the GWAS stage, when correction for multiple testing was applied. However, when they included six different Chinese groups (4319 HCC cases vs. 4966 controls) in meta-analysis, rs9275319 reached genome-wide significance (P = 2.72 1017) (Jiang et al., 2013). Even so, the magnitude and strength of association were inconsistent in each independent population (OR = 1.07–1.85, P = 0.66–2.42 108). Our negative replication results in a Korean population (OR = 1.02, P = 0.92 in allelic model) are similar to those of studies of Henan and Guangxi populations (P > 0.05). In our evaluation study, performed with an independent Korean population (n = 3838), rs9275319A in HLA-DQ was highly associated with CHB (OR = 1.57, P = 1.44 1010). The genetic effects of the rs9275319 on CHB were also examined in previous studies of Chinese populations. In Suzhou and Guangxi populations, it showed similar associations (OR = 1.39, P = 0.002; OR = 1.29, P = 0.0006, respectively) to our results in a Korean population (OR = 1.49, P = 2.2 109), whereas there were no significant associations in other populations (Shanghai, Beijing, and Henan populations) (Jiang et al., 2013). In the microarray expression analyses, although we could not detect genotype effects of either locus on mRNA expression, significantly lower mRNA levels of three genes, STAT4, HLA-DQA1, and HLA-DQB1 in HCC tissues were observed, compared with those in adjacent non-cancer tissues. In the study by Jiang et al., the risk allele GG at rs7574865 was significantly related to low mRNA expression level in HCC and non-cancer tissues (Ptrend = 0.0008 and Ptrend = 0.0002, respectively) (Jiang et al., 2013). In our study, we could not obtain a strong association between rs7574865 and mRNA expression level of STAT4. The possible reason could be that
Please cite this article in press as: Kim, L.H., et al. Replication of genome wide association studies on hepatocellular carcinoma susceptibility loci of STAT4 and HLA-DQ in a Korean population. Infect. Genet. Evol. (2015), http://dx.doi.org/10.1016/j.meegid.2015.04.013
L.H. Kim et al. / Infection, Genetics and Evolution xxx (2015) xxx–xxx
the sample size was somewhat small or there might be ethnic difference. In correlation analysis of rs9275319 and nearby genes, HLA-DQA1, HLA-DQB1, HLA-DQA2, and HLA-DQB2 mRNA expression, genotypes of rs9275319 were not related to mRNA expression of those genes. The result might indicate that the differential gene expression of STAT4, HLA-DQA1 and HLA-DQB1 may not be caused by the genotype differences, and the SNPs may be linked to functional alteration of nearby genes, and these genes change the expression level of STAT4, HLA-DQA1 and HLA-DQB1. In conclusion, our replication study showed that the rs7574865 in STAT4 and rs9275319 in HLA-DQ were not associated with CHBrelated HCC in a Korean population. But, the two SNPs were significantly associated with the risk of CHB. In addition, we observed significantly lower mRNA level of STAT4, HLA-DQA1, and HLADQB1 in HCC tissues compared to adjacent non-cancer tissues. To identify the pathogenesis of CHB or progression mechanisms of HCC related to these two SNPs, further functional researches are necessary. And much investigation is needed so as to elucidate the differences in HCC susceptibility between Chinese and Korean populations. Acknowledgment None. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.meegid.2015.04. 013. References Brechot, C., Gozuacik, D., Murakami, Y., Paterlini-Brechot, P., 2000. Molecular bases for the development of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC). Semin. Cancer Biol. 10, 211–231. But, D.Y., Lai, C.L., Yuen, M.F., 2008. Natural history of hepatitis-related hepatocellular carcinoma. World J. Gastroenterol. 14, 1652–1656. Chen, K., Shi, W., Xin, Z., Wang, H., Zhu, X., Wu, X., Li, Z., Li, H., Liu, Y., 2013. Replication of genome wide association studies on hepatocellular carcinoma susceptibility Loci in a Chinese population. PLoS One 8, e77315. Clark, A., Gerlach, F., Tong, H., Hoan, N.X., Song le, H., Toan, N.L., Bock, C.T., Kremsner, P.G., Velavan, T.P., 2013. A trivial role of STAT4 variant in chronic hepatitis B induced hepatocellular carcinoma. Infect. Genet. Evol. 18, 257–261.
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Jiang, D.K., Sun, J., Cao, G., Liu, Y., Lin, D., Gao, Y.Z., Ren, W.H., Long, X.D., Zhang, H., Ma, X.P., Wang, Z., Jiang, W., Chen, T.Y., Gao, Y., Sun, L.D., Long, J.R., Huang, H.X., Wang, D., Yu, H., Zhang, P., Tang, L.S., Peng, B., Cai, H., Liu, T.T., Zhou, P., Liu, F., Lin, X., Tao, S., Wan, B., Sai-Yin, H.X., Qin, L.X., Yin, J., Liu, L., Wu, C., Pei, Y., Zhou, Y.F., Zhai, Y., Lu, P.X., Tan, A., Zuo, X.B., Fan, J., Chang, J., Gu, X., Wang, N.J., Li, Y., Liu, Y.K., Zhai, K., Hu, Z., Liu, J., Yi, Q., Xiang, Y., Shi, R., Ding, Q., Zheng, W., Shu, X.O., Mo, Z., Shugart, Y.Y., Zhang, X.J., Zhou, G., Shen, H., Zheng, S.L., Xu, J., Yu, L., 2013. Genetic variants in STAT4 and HLA-DQ genes confer risk of hepatitis B virus-related hepatocellular carcinoma. Nat. Genet. 45, 72–75. Kamatani, Y., Wattanapokayakit, S., Ochi, H., Kawaguchi, T., Takahashi, A., Hosono, N., Kubo, M., Tsunoda, T., Kamatani, N., Kumada, H., Puseenam, A., Sura, T., Daigo, Y., Chayama, K., Chantratita, W., Nakamura, Y., Matsuda, K., 2009. A genome-wide association study identifies variants in the HLA-DP locus associated with chronic hepatitis B in Asians. Nat. Genet. 41, 591–595. Kim, Y.J., Kim, H.Y., Lee, J.H., Yu, S.J., Yoon, J.H., Lee, H.S., Kim, C.Y., Cheong, J.Y., Cho, S.W., Park, N.H., Park, B.L., Namgoong, S., Kim, L.H., Cheong, H.S., Shin, H.D., 2013. A genome-wide association study identified new variants associated with the risk of chronic hepatitis B. Hum. Mol. Genet. 22, 4233–4238. Li, S., Qian, J., Yang, Y., Zhao, W., Dai, J., Bei, J.X., Foo, J.N., McLaren, P.J., Li, Z., Yang, J., Shen, F., Liu, L., Pan, S., Wang, Y., Li, W., Zhai, X., Zhou, B., Shi, L., Chen, X., Chu, M., Yan, Y., Wang, J., Cheng, S., Shen, J., Jia, W., Liu, J., Wen, Z., Li, A., Zhang, Y., Zhang, G., Luo, X., Qin, H., Chen, M., Wang, H., Jin, L., Lin, D., Shen, H., He, L., de Bakker, P.I., Zeng, Y.X., Wu, M., Hu, Z., Shi, Y., Zhou, W., 2012. GWAS identifies novel susceptibility loci on 6p21.32 and 21q21.3 for hepatocellular carcinoma in chronic hepatitis B virus carriers. PLoS Genet. 8, e1002791. Mbarek, H., Ochi, H., Urabe, Y., Kumar, V., Kubo, M., Hosono, N., Takahashi, A., Kamatani, Y., Miki, D., Abe, H., Tsunoda, T., Kamatani, N., Chayama, K., Nakamura, Y., Matsuda, K., 2011. A genome-wide association study of chronic hepatitis B identified novel risk locus in a Japanese population. Hum. Mol. Genet. 20, 3884–3892. Nishida, N., Sawai, H., Matsuura, K., Sugiyama, M., Ahn, S.H., Park, J.Y., Hige, S., Kang, J.H., Suzuki, K., Kurosaki, M., Asahina, Y., Mochida, S., Watanabe, M., Tanaka, E., Honda, M., Kaneko, S., Orito, E., Itoh, Y., Mita, E., Tamori, A., Murawaki, Y., Hiasa, Y., Sakaida, I., Korenaga, M., Hino, K., Ide, T., Kawashima, M., Mawatari, Y., Sageshima, M., Ogasawara, Y., Koike, A., Izumi, N., Han, K.H., Tanaka, Y., Tokunaga, K., Mizokami, M., 2012. Genome-wide association study confirming association of HLA-DP with protection against chronic hepatitis B and viral clearance in Japanese and Korean. PLoS One 7, e39175. Perz, J.F., Armstrong, G.L., Farrington, L.A., Hutin, Y.J., Bell, B.P., 2006. The contributions of hepatitis B virus and hepatitis C virus infections to cirrhosis and primary liver cancer worldwide. J. Hepatol. 45, 529–538. Pungpapong, S., Kim, W.R., Poterucha, J.J., 2007. Natural history of hepatitis B virus infection: an update for clinicians. Mayo Clin. Proc. 82, 967–975. Thursz, M., 2001. Genetic susceptibility in chronic viral hepatitis. Antiviral Res. 52, 113–116. Wang, F.S., 2003. Current status and prospects of studies on human genetic alleles associated with hepatitis B virus infection. World J. Gastroenterol. 9, 641–644. Yang, J.D., Roberts, L.R., 2010. Hepatocellular carcinoma: a global view. Nat. Rev. Gastroenterol. Hepatol. 7, 448–458. Zhang, H., Zhai, Y., Hu, Z., Wu, C., Qian, J., Jia, W., Ma, F., Huang, W., Yu, L., Yue, W., Wang, Z., Li, P., Zhang, Y., Liang, R., Wei, Z., Cui, Y., Xie, W., Cai, M., Yu, X., Yuan, Y., Xia, X., Zhang, X., Yang, H., Qiu, W., Yang, J., Gong, F., Chen, M., Shen, H., Lin, D., Zeng, Y.X., He, F., Zhou, G., 2010. Genome-wide association study identifies 1p36.22 as a new susceptibility locus for hepatocellular carcinoma in chronic hepatitis B virus carriers. Nat. Genet. 42 (9), 755–758.
Please cite this article in press as: Kim, L.H., et al. Replication of genome wide association studies on hepatocellular carcinoma susceptibility loci of STAT4 and HLA-DQ in a Korean population. Infect. Genet. Evol. (2015), http://dx.doi.org/10.1016/j.meegid.2015.04.013
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Replication of genome wide association studies on hepatocellular carcinoma susceptibility loci of STAT4 and HLA-DQ in a Korean population Lyoung Hyo Kim a,b, Hyun Sub Cheong b, Suhg Namgoong a,b, Ji On Kim b, Jeong-Hyun Kim a, Byung Lae Park b, Sung Won Cho c, Neung Hwa Park d, Jae Youn Cheong c, InSong Koh e, Hyoung Doo Shin a,b,⇑, Yoon-Jun Kim f,⇑ a
Department of Life Science, Sogang University, Republic of Korea Department of Genetic Epidemiology, SNP Genetics, Inc., Republic of Korea c Department of Gastroenterology, Ajou University School of Medicine, Republic of Korea d Department of Internal Medicine, Ulsan University Hospital, Republic of Korea e Department of Physiology, College of Medicine, Han Yang University, Republic of Korea f Department of Internal Medicine and Liver Research Institute, Seoul National University, Republic of Korea b
a r t i c l e
i n f o
Article history: Received 22 January 2015 Received in revised form 3 April 2015 Accepted 15 April 2015 Available online xxxx Keywords: Chronic hepatitis B virus Hepatocellular carcinoma HLA-DQ STAT4
a b s t r a c t A recent genome-wide association study (GWAS) for hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) identified two loci (rs7574865 in STAT4 and rs9275319 in HLA-DQ) in a Chinese population. We attempted to replicate the associations between the two SNP loci and the risk of HCC in a Korean population. The rs7574865 in STAT4 and rs9275319 in HLA-DQ were genotyped in a total of 3838 Korean subjects composed of 287 HBV-related hepatocellular carcinoma patients, 671 chronic hepatitis B virus (CHB) patients, and 2880 population controls using TaqMan genotyping assay. Gene expression was measured by microarray. A logistic regression analysis revealed that rs7574865 in STAT4 and rs9275319 in HLA-DQ were associated with the risk of CHB (OR = 1.25, P = 0.0002 and OR = 1.57, P = 1.44 1010, respectively). However, these loci were no association with the risk of HBV-related HCC among CHB patients. In the gene expression analyses, although no significant differences in mRNA expression of nearby genes according to genotypes were detected, a significantly decreased mRNA expression in HCC subjects was observed in STAT4, HLA-DQA1, and HLA-DQB1. Although the genetic effects of two HCC susceptibility loci were not replicated, the two loci were found to exert susceptibility effects on the risk of CHB in a Korean population. In addition, the decreased mRNA expression of STAT4, HLA-DQA1, and HLA-DQB1 in HCC tissue might provide a clue to understanding their role in the progression to HCC. Ó 2015 Elsevier B.V. All rights reserved.
1. Introduction Hepatitis B virus (HBV) infection is a major global problem, and chronic hepatitis B virus (CHB) infection is a complex clinical entity frequently associated with cirrhosis and hepatocellular carcinoma (HCC) (But et al., 2008; Yang and Roberts, 2010). HCC is a common solid cancer of the liver, the prevalence of which is highest in Asia and Africa, where the endemic high incidence of hepatitis B and ⇑ Corresponding authors at: Department of Life Science, Sogang University, 1 Shinsu-dong, Mapo-gu, Seoul 121-742, Republic of Korea. Tel.: +82 2 705 8615; fax: +82 2 2026 4299 (H.D. Shin). Department of Internal Medicine and Liver Research Institute, Seoul National University, 28 Yungun-dong, Chongro-Gu, Seoul 110-744, Republic of Korea (Y.-J. Kim). E-mail addresses: [email protected] (H.D. Shin), [email protected] (Y.-J. Kim).
hepatitis C creates a strong susceptibility to the development of chronic liver disease, or cirrhosis (Brechot et al., 2000). There are two routes of HBV infection in humans: One is a vertical transmission from an HbsAg-positive mother to her infant; the other is a horizontal transmission by exposure to infectious blood or body fluids such as semen or vaginal fluids. Among vertically HBV affected fetuses, over 90% become CHB affected carriers. Among horizontally HBV affected adults, only 5–10% becomes CHB affected carriers (Perz et al., 2006). Although the mechanisms underlying the different clinical results of HBV infection have not been fully elucidated, previous epidemiological studies have linked diverse factors such as viral strain, gender, infection age, and immune system of the host, with the risk of CHB (Wang, 2003). In addition, host genetic factors are thought to play an important role in the progression of CHB (Pungpapong et al., 2007;
http://dx.doi.org/10.1016/j.meegid.2015.04.013 1567-1348/Ó 2015 Elsevier B.V. All rights reserved.
Please cite this article in press as: Kim, L.H., et al. Replication of genome wide association studies on hepatocellular carcinoma susceptibility loci of STAT4 and HLA-DQ in a Korean population. Infect. Genet. Evol. (2015), http://dx.doi.org/10.1016/j.meegid.2015.04.013
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L.H. Kim et al. / Infection, Genetics and Evolution xxx (2015) xxx–xxx
Thursz, 2001). Recent genome-wide association studies (GWASs) of CHB-related HCC in Asian populations identified susceptibility loci on 1p36.2, 6p21.32, and 21q21.3 (Kamatani et al., 2009; Li et al., 2012; Mbarek et al., 2011; Nishida et al., 2012; Zhang et al., 2010). Moreover, our previous GWAS also reported the risk associated loci for CHB on the HLA region, rs652888 on EHMT2, and rs1419881 on the TFG19 gene (Kim et al., 2013). Most recently, a multi-stage GWAS in a Chinese population identified two susceptibility loci for HCC (rs7574865 at STAT4 and rs9275319 at HLA-DQ) (Jiang et al., 2013). In the present study in order to evaluate the contribution of the two loci identified in this most recent GWAS, we carried out a replication study for rs7574865 and rs9275319 with CHB and HCC in a Korean cohort.
RNA was quantified using Ribogreen (Quant-iT™ RiboGreenÒ RNA Assay Kit, Life Technologies, Grand Island, NY). RNA integrity was assessed using the Experion automated electrophoresis system (Bio-Rad, Hercules, CA) to check the ratio of 28s:18s rRNA in the samples. A total of 500 ng from each sample was labeled using the Illumina Total Prep RNA Amplification Kit following the manufacturer’s directions (Ambion, Austin, TX). cRNA was hybridized overnight to Illumina Human HT-12 Expression BeadChips (Illumina, Inc., San Diego, CA). Microarrays were washed several times and labeled with streptavidin-Cy3 (Amersham Biosciences, Piscataway, NJ) prior to scanning on an Illumina iScan. Signal intensities were analyzed using the Genome Studio software (Illumina).
2. Materials and methods
2.4. Statistical analysis
2.1. Study subjects
The genotypic distributions of SNPs with HCC risk in this replication study was determined by binary logistic regression of referent, allelic, additive, dominant, and recessive models controlling for age (continuous variable) and sex (male = 0, female = 1) as covariates. Since major homozygotes of rs7574865 G>T and rs9275319 A>G were risk genotypes, minor homozygotes and minor alleles were assigned as reference genotype in the referent model, and allelic model analysis, respectively. Genotypes of minor homozygotes, heterozygotes, and major homozygotes of rs7574865 G>T and rs9275319 A>G were given codes 0, 1, and 1; and 0, 0, and 1 in the dominant, and recessive models, respectively. All statistical analyses were performed using statistical analysis system (SAS) version 9.3 (SAS Inc., Cary, NC). Differences in gene expression between cancer and adjacent non-cancer tissues were analyzed by an ANOVA, and the relationship of the respective genotypes with gene expression levels was tested using the paired t-test. P < 0.05 was considered statistically significant. For the expression analysis, we also performed non-parametric analysis because our sample size was small. The Kruskal–Wallis test and Wilcoxon signed rank test were conducted and P value was represented in parentheses at Fig. 1. The plot of expression difference between cancer and paired adjacent non-cancer samples was generated using Prism 4 program.
The DNA from a total of 3838 subjects was used for the replication study. Subjects consisted of 287 CHB-related HCC subjects, 671 CHB subjects, and 2880 controls. The 958 samples from CHB and HCC patients were obtained from the outpatient clinic of the Liver Unit and the Center for Health Promotion at Seoul National University Hospital and Ulsan University Medical Center. The population control samples (n = 2880) were provided by Korea Biobank at the Korea Centers for Disease Control and Prevention. The diagnosis of chronic carriers was established based on seropositivity for HBsAg (Enzygnost1 HBsAg 5.0; Dade Behring, Marburg, Germany) over a 6-month period (Table S1). All the participants provided written informed consent. This project was approved by the Institutional Review Board of Seoul National University Hospital (IRB No. 1007-049-323). 2.2. Genotyping of rs7574865 and rs9275319 The polymorphisms rs7574865 G>T in the STAT4 gene and rs9275319 A>G in the HLA-DQ region were genotyped using the 50 exonuclease TaqMan genotyping assay system (Applied Biosystems, Foster City, CA). Quality control was performed in 10% of the samples by duplicate checking (rate of concordance in duplicates = 100%).
3. Results
2.3. RNA extraction and gene expression microarrays
3.1. Association analysis of two susceptibility loci
Total RNA was extracted from HCC and adjacent non-cancer tissues using NucleoSpinÒ (MACHEREY-NAGEL, Düren, Germany).
In this study, a total of 3838 samples were used for the replication study, consisting of 287 HCC, 671 CHB, and 2880 population
Fig. 1. STAT4, HLA-DQA1, and HLA-DQB1 relative expression levels according to rs7574865 and rs9275319 in paired cancer tissues and adjacent non-cancer tissues from 23 subjects with HCC.
Please cite this article in press as: Kim, L.H., et al. Replication of genome wide association studies on hepatocellular carcinoma susceptibility loci of STAT4 and HLA-DQ in a Korean population. Infect. Genet. Evol. (2015), http://dx.doi.org/10.1016/j.meegid.2015.04.013
Gene/loci
STAT4/rs7574865
HLA-DQ/rs9275319
Allele
T/Ga
G/Aa
Genotype
PCs
CHB
HCC
CHB + HCC vs. PCs
n = 2880 (%)
n = 671 (%)
n = 287 (%)
OR (95%CI)
TT GT GG Allele T G Dominant TT GT + GG Recessive TT + GT GG
306 (10.7) 1251 (43.9) 1293 (45.4)
63 (9.6) 261 (39.7) 334 (50.7)
20 (7.1) 103 (36.4) 160 (56.5)
1863 (32.7) 3837 (67.3)
387 (29.4) 929 (70.6)
306 (10.7) 2544 (89.3)
GG AG AA Allele G A Dominant GG AG + AA Recessive GG + AG AA
HCC vs. CHB P
OR (95%CI)
Reference 1.10 (0.83–1.45) 1.20 (1.05–1.38)
143 (25.3) 423 (74.7)
63 (9.6) 595 (90.4)
1557 (54.6) 1293 (45.4)
HCC vs. CHB + PCs P
OR (95%CI)
0.52 0.008
Reference 1.21 (0.63–2.33) 1.17 (0.85–1.61)
Reference 1.25 (1.11–1.41)
0.0002
20 (7.1) 263 (92.9)
Reference 1.34 (1.15–1.56)
324 (49.3) 334 (50.8)
123 (43.5) 160 (56.5)
Reference 1.27 (0.98–1.65)
248 (8.7) 890 (31.3) 1702 (59.9)
30 (4.6) 177 (26.9) 454 (69.0)
12 (4.2) 72 (25.4) 199 (70.3)
1386 (24.4) 4294 (75.6)
237 (17.9) 1085 (82.1)
248 (8.7) 2592 (91.3) 1138 (40.1) 1702 (59.9)
HCC vs. PCs P
OR (95%CI)
P
0.56 0.34
Reference 1.16 (0.67–2.00) 1.32 (1.00–1.73)
0.60 0.05
Reference 1.24 (0.72–2.12) 1.33 (1.02–1.73)
0.44 0.04
Reference 1.16 (0.88–1.51)
0.29
Reference 1.40 (1.11–1.75)
0.004
Reference 1.37 (1.1–1.71)
0.005
0.0002
Reference 1.16 (0.83–1.63)
0.37
Reference 1.47 (1.12–1.95)
0.005
Reference 1.54 (1.16–2.04)
0.003
0.07
Reference 1.30 (0.70–2.43)
0.42
Reference 1.51 (0.90–2.53)
0.12
Reference 1.45 (0.86–2.44)
0.17
Reference 1.71 (1.18–2.48) 1.55 (1.31–1.84)
0.005 6.5 107
Reference 1.25 (0.52–2.98) 1.07 (0.72–1.61)
0.61 0.74
Reference 2.41 (1.17–4.95) 1.87 (1.32–2.66)
0.02 0.0005
Reference 2.17 (1.08–4.36) 1.66 (1.19–2.32)
0.03 0.003
96 (16.9) 470 (83.1)
Reference 1.57 (1.37–1.8)
1.44 1010
Reference 1.02 (0.74–1.39)
0.92
Reference 1.77 (1.36–2.31)
1.98 105
Reference 1.59 (1.23–2.04)
0.0004
30 (4.5) 631 (95.5)
12 (4.2) 271 (95.8)
Reference 1.56 (1.33–1.84)
7.6 108
Reference 0.99 (0.69–1.42)
0.95
Reference 1.51 (1.14–2.03)
0.006
Reference 1.69 (1.24–2.30)
0.0008
207 (31.3) 454 (68.7)
84 (29.7) 199 (70.3)
Reference 2.17 (1.54–3.05)
1.0 105
Reference 1.18 (0.53–2.62)
0.69
Reference 2.59 (1.14–5.01)
0.005
Reference 3.16 (1.58–6.34)
0.001
L.H. Kim et al. / Infection, Genetics and Evolution xxx (2015) xxx–xxx
Logistic regression models were used to calculate the ORs, 95% CIs, and the corresponding P values of referent, allele, dominant, and recessive models controlling for age and sex as covariates. Significant associations are shown in bold. CHB, chronic hepatitis B; HCC, hepatocellular carcinoma; PCs, controls; OR, odd ratio; CI, confidence interval. a Risk allele (G allele for rs7574865, A allele for rs9275319).
3
Please cite this article in press as: Kim, L.H., et al. Replication of genome wide association studies on hepatocellular carcinoma susceptibility loci of STAT4 and HLA-DQ in a Korean population. Infect. Genet. Evol. (2015), http://dx.doi.org/10.1016/j.meegid.2015.04.013
Table 1 Logistic analysis of association of SNPs in STAT4 and HLA-DQ with the risk of CHB and HCC in a Korean population (n = 3838).
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controls (PCs). Four different statistical comparisons were employed to determine the genetic effects on CHB infection and/ or HCC progression, i.e., (1) risk of CHB (CHB plus CHB-related HCC vs. PCs), (2) risk of HCC among CHB patients (CHB related HCC vs. CHB), and (3) overall risk of HCC (CHB-related HCC vs. non-HCC, including CHB plus PCs). We also examined the association between (4) CHB-related HCC and PCs to support the associations above. Data were analyzed using logistic regression. We fitted referent, allelic, dominant, and recessive models (Table 1). The results of logistic regression analysis for each SNP are shown in Table 1. The rs7574865 polymorphism in STAT4 was not found to be associated with the risk of HCC among CHB patients (P = 0.29–0.42), contrary to what had been reported in the Chinese population. However, when compared with controls, the loci showed stronger association, e.g., CHB + HCC vs. PCs (P = 0.0003–0.07), HCC vs. PCs (P = 0.003–0.17), and HCC vs. CHB + PCs (P = 0.005–0.17). Taking the subjects carrying the TT genotype for rs7574865 as a reference, the subjects carrying genotype GG showed an increased risk of CHB + HCC and HCC compared with PCs (P = 0.008 and 0.04, respectively). Similarly, although the rs9275319 polymorphism in HLA-DQ was not associated with the risk of HCC among CHB patients (P = 0.69–0.92), it revealed strong association when compared to chronic HBV carriers (CHB + HCC vs. PCs, P = 1.44 1010– 1.0 105). Also, the rs9275319 showed strong association of CHB-related HCC vs. PCs (P = 0.0004–0.001) and CHB-related HCC vs. non-HCC (P = 1.98 105–0.005). Taking the subjects carrying the GG genotype for rs9275319 as a reference, the subjects carrying genotype AG and AA showed an increased risk of CHB + HCC and HCC compared with PCs and CHB + PCs (P = 6.5 107–0.03).
included six different Chinese groups (4319 HCC cases vs. 4966 controls) in meta-analysis, rs7574865 reached genome-wide significance (P = 2.48 1010). Although the significance of rs7574865 had reached genome wide threshold, the associations were not replicated in all the six different Chinese groups (OR = 1.12–1.36, P = 0.27–0.0016) (Jiang et al., 2013). Our initial replication result for rs7574865 showed no association with risk of HCC (OR = 1.16, P = 0.29 in allelic model) and this result was similar to result of Beijing and Henan populations among six different Chinese populations (OR = 1.14, P = 0.15 and OR = 1.12, P = 0.27, respectively). In two other studies, Chen et al. failed to replicate the effect of rs7574865 on the risk of HCC in an independent 1278 Chinese population (P > 0.05). And Clark et al. reported rs7574865 was marginally associated to the risk of HCC in a 445 Vietnamese population (P = 0.048 in allelic model) (Chen et al., 2013; Clark et al., 2013). When putting all these studies together, the genetic contribution of rs7574865 upon the risk of CHB-related HCC could be seen as mild (if any), and its effects might be hard to detect without a larger sample size. However, rs7574865 showed significant association with the risk of chronic HBV infection in a Korean population (OR = 1.25, P = 0.0002 in allelic model). This genetic effect also has been examined in a Chinese population (Jiang et al., 2013). Researchers compared 3935 chronic HBV carriers without HCC with 4585 healthy subjects who did not carry HBV (Jiang et al., 2013). The rs7574865 SNP in STAT4 was weakly associated with a trend toward higher frequency of the G allele in chronic HBV carriers (OR = 1.12, Pmeta = 0.003). Considering all results of the current study and previous research, we might conclude that rs7574865 in STAT4 has a weak association with the risk of chronic HBV infection.
3.2. Gene expression analysis of STAT4 and HLA-DQA1
4.2. Genetic effects of rs9275319 in HLA-DQ on HCC susceptibility and HBV progression
Next, we examined the genotype effects of rs7574865 on STAT4 mRNA expression. The rs9275319 is located between HLA-DQA2/ DQB2 and HLA-DQA1/DQB1 (65 kb away from genes). So, we measured mRNA expression levels of these nearby genes. We analyzed the mRNA levels of STAT4 and four HLA-DQ genes in 23 paired samples (HCC and adjacent normal tissues) using HumanHT-12 Expression BeadChips (Illumina). We were unable to detect genotype effects of either locus on mRNA expression. However, significantly lower mRNA levels of three genes in HCC tissues were observed, compared with those in adjacent non-cancer tissues (P = 8.28 105 in STAT4; P = 4.9 109 in HLA-DQA1; P = 1.4 104 in HLA-DQB1) (Fig. 1). 4. Discussion Recently, a Chinese three-stage genome-wide association study of hepatitis B virus-related hepatocellular carcinoma identified two loci with genome-wide significance in the STAT4 and HLA-DQ genes. We performed a replication study of rs7574865 in STAT4 and rs9275319 in HLA-DQ with clinically classified HCC, CHB, and controls in a Korean population. Although we could not replicate the association of the SNPs with the risk of HCC, statistical analysis revealed that these two SNPs are significantly associated with the risk of CHB. 4.1. Genetic effects of rs7574865 in STAT4 on HCC susceptibility and HBV progression In the Chinese study noted above, rs7574865 of STAT4 was not significantly associated with the risk of CHB-related HCC (1161 HCC cases vs. 1353 controls) in the GWAS stage when correction for multiple testing was applied. However, when the researchers
In the Chinese study noted above, the rs9275319 SNP in HLA-DQ was not found to be significantly associated with the CHB-related HCC group (1161 HCC cases vs. 1353 controls) in the GWAS stage, when correction for multiple testing was applied. However, when they included six different Chinese groups (4319 HCC cases vs. 4966 controls) in meta-analysis, rs9275319 reached genome-wide significance (P = 2.72 1017) (Jiang et al., 2013). Even so, the magnitude and strength of association were inconsistent in each independent population (OR = 1.07–1.85, P = 0.66–2.42 108). Our negative replication results in a Korean population (OR = 1.02, P = 0.92 in allelic model) are similar to those of studies of Henan and Guangxi populations (P > 0.05). In our evaluation study, performed with an independent Korean population (n = 3838), rs9275319A in HLA-DQ was highly associated with CHB (OR = 1.57, P = 1.44 1010). The genetic effects of the rs9275319 on CHB were also examined in previous studies of Chinese populations. In Suzhou and Guangxi populations, it showed similar associations (OR = 1.39, P = 0.002; OR = 1.29, P = 0.0006, respectively) to our results in a Korean population (OR = 1.49, P = 2.2 109), whereas there were no significant associations in other populations (Shanghai, Beijing, and Henan populations) (Jiang et al., 2013). In the microarray expression analyses, although we could not detect genotype effects of either locus on mRNA expression, significantly lower mRNA levels of three genes, STAT4, HLA-DQA1, and HLA-DQB1 in HCC tissues were observed, compared with those in adjacent non-cancer tissues. In the study by Jiang et al., the risk allele GG at rs7574865 was significantly related to low mRNA expression level in HCC and non-cancer tissues (Ptrend = 0.0008 and Ptrend = 0.0002, respectively) (Jiang et al., 2013). In our study, we could not obtain a strong association between rs7574865 and mRNA expression level of STAT4. The possible reason could be that
Please cite this article in press as: Kim, L.H., et al. Replication of genome wide association studies on hepatocellular carcinoma susceptibility loci of STAT4 and HLA-DQ in a Korean population. Infect. Genet. Evol. (2015), http://dx.doi.org/10.1016/j.meegid.2015.04.013
L.H. Kim et al. / Infection, Genetics and Evolution xxx (2015) xxx–xxx
the sample size was somewhat small or there might be ethnic difference. In correlation analysis of rs9275319 and nearby genes, HLA-DQA1, HLA-DQB1, HLA-DQA2, and HLA-DQB2 mRNA expression, genotypes of rs9275319 were not related to mRNA expression of those genes. The result might indicate that the differential gene expression of STAT4, HLA-DQA1 and HLA-DQB1 may not be caused by the genotype differences, and the SNPs may be linked to functional alteration of nearby genes, and these genes change the expression level of STAT4, HLA-DQA1 and HLA-DQB1. In conclusion, our replication study showed that the rs7574865 in STAT4 and rs9275319 in HLA-DQ were not associated with CHBrelated HCC in a Korean population. But, the two SNPs were significantly associated with the risk of CHB. In addition, we observed significantly lower mRNA level of STAT4, HLA-DQA1, and HLADQB1 in HCC tissues compared to adjacent non-cancer tissues. To identify the pathogenesis of CHB or progression mechanisms of HCC related to these two SNPs, further functional researches are necessary. And much investigation is needed so as to elucidate the differences in HCC susceptibility between Chinese and Korean populations. Acknowledgment None. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.meegid.2015.04. 013. References Brechot, C., Gozuacik, D., Murakami, Y., Paterlini-Brechot, P., 2000. Molecular bases for the development of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC). Semin. Cancer Biol. 10, 211–231. But, D.Y., Lai, C.L., Yuen, M.F., 2008. Natural history of hepatitis-related hepatocellular carcinoma. World J. Gastroenterol. 14, 1652–1656. Chen, K., Shi, W., Xin, Z., Wang, H., Zhu, X., Wu, X., Li, Z., Li, H., Liu, Y., 2013. Replication of genome wide association studies on hepatocellular carcinoma susceptibility Loci in a Chinese population. PLoS One 8, e77315. Clark, A., Gerlach, F., Tong, H., Hoan, N.X., Song le, H., Toan, N.L., Bock, C.T., Kremsner, P.G., Velavan, T.P., 2013. A trivial role of STAT4 variant in chronic hepatitis B induced hepatocellular carcinoma. Infect. Genet. Evol. 18, 257–261.
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Please cite this article in press as: Kim, L.H., et al. Replication of genome wide association studies on hepatocellular carcinoma susceptibility loci of STAT4 and HLA-DQ in a Korean population. Infect. Genet. Evol. (2015), http://dx.doi.org/10.1016/j.meegid.2015.04.013
