Professional Documents
Culture Documents
Polymorphisms in the primary microRNA region may be associated with natural course of
hepatitis B virus (HBV) infection. This study
evaluated if the mircoRNA 219-1 (miR-219-1)
polymorphism can inuence the susceptibility
towards persistence of HBV infection and the
progression to hepatocellular carcinoma
(HCC) in patients with chronic HBV infection.
A total of 1,439 individuals having either past
or present evidence of HBV infection were enrolled for the study. The subjects were divided into four groups; (1) spontaneous recovery
(n 404), (2) chronic HBV carrier (n 313),
(3) chronic HBV carrier with cirrhosis
(n 305), and (4) hepatocellular carcinoma
(n 417). Genotyping was performed at three
polymorphic variants (rs421446, rs107822, and
rs213210) in the pri-miRNA region of miR-219-1.
The rs421446 T allele was found to be strongly
associated with HBV clearance (OR 0.73,
P 0.0005 in a codominant model and
OR 0.67, P 0.0009 in a dominant model,
OR 0.69, P 0.04 in a recessive model, respectively). The rs107822 G allele was also
found to be associated with HBV clearance
(OR 0.79, P 0.008 in a codominant model
and OR 0.72, P 0.01 in a dominant model,
respectively). In haplotype analysis, ht2 (T-G-T)
and ht1 (C-A-C) were found to be in signicant
association with the clearance of HBV. However,
no signicant association was observed between miR-219-1 polymorphism and the risk of
HCC occurrence. This result suggests that polymorphisms in the pri-miRNA region of miR-2191 might be a genetic factor for HBV clearance
after infection. J. Med. Virol. 85:808814,
2013. 2013 Wiley Periodicals, Inc.
KEY WORDS: polymorphism;
microRNA219; hepatitis B virus; hepatocellular carcinoma
2013 WILEY PERIODICALS, INC.
INTRODUCTION
Hepatitis B virus (HBV) infection is a major global
health problem for more than 350 million HBV carriers around the world [Iino, 2002; Lok and McMahon,
2007; Suk et al., 2012]. The clinical outcome of the
infection varies widely and may include spontaneous
recovery, the presence of inactive hepatitis B surface
antigen (HBsAg) carriers, chronic hepatitis, liver cirrhosis, or hepatocellular carcinoma (HCC). Perinatal
transmission of HBV is the major mode of infection in
HBV-endemic areas, including Korea. The most signicant factor affecting the chronicity of HBV infection is the age at time of infection; the chronicity in
Korean patients who are vertically infected seems to
be determined by host factors or genetic differences
rather than viral factors such as variations in virulence of the viral strains [Stevens et al., 1975; Coursaget et al., 1987; Tassopoulos et al., 1987]. Genetic
factors are also likely to modify the risk of HCC development among patients with HBV infection in cases
where family history of HCC is a well-known risk factor [Yu et al., 2000]. Recent studies by the authors
reported that genetic variations such as single nucleotide polymorphism (SNP) of transforming growth factor beta receptor III gene and tumor necrosis factor
alpha gene are signicantly associated with the risk
809
carrier group had undergone regular medical followups and had been evaluated with serum alphafetoprotein level, abdominal ultrasonography, and/or
a four-phase spiral liver CT scan more than twice a
year to detect early stages of HCC. Based on the clinical decision, dynamic contrast enhanced abdominal
MRI, bone scans, and chest CT, brain MRI, brain CT,
hepatic angiography, or PET scan was also carried
out in some of the patients.
Diagnosis of HCC was based on imaging ndings of
nodules larger than 1 cm showing intense arterial uptake followed by washout of contrast in the venousdelayed phases in four-phase multi-detector CT scan
or dynamic contrast enhanced MRI and/or biopsy
[Bruix and Sherman, 2011]. Cirrhosis of the liver, on
the other hand, was diagnosed pathologically or the
diagnosis was based on the clinical evidence of portal
hypertension such as the presence of visible collateral
vessels on the abdominal wall, esophageal varies on
esophagogastroscopy, palpable splenomegaly, and sonographically denite ndings of cirrhotic liver or ascites.
Exclusion criteria for the study patients included
the following: (i) tested positive for anti-HBs but not
for anti-HBc; (ii) tested positive for anti-HCV or antiHIV (Genedia1; Greencross Life Science, Yongin-shi,
Korea, HCV13.2; Dong-A Pharmaceutical, Seoul,
Korea); (iii) average alcohol consumption of 10 g/day
or an average number of 1 cigarette pack/s smoked
daily assessed through individual interviews; and (iv)
incurrence of other types of liver diseases such as
autoimmune hepatitis, toxic hepatitis, hemochromatosis, Wilsons disease, non-alcoholic steatohepatitis,
primary biliary cirrhosis, or BuddChiari syndrome.
None of the patients had a previous history of immunosuppressant or anti-viral treatment. Finally, written consent was secured from the patients prior to
conducting the study and ethical approval was
obtained from the Institutional Review Board for Human Research at Seoul National University Hospital
and Ajou University Hospital.
Genotyping of miR-219-1 Genome Region
Polymorphisms
Using the Wizard genomic DNA purication kit
(Promega, Madison, WI), genomic DNA was extracted
from patients peripheral blood samples. The SNP
genotyping was performed using the TaqMan1[Livak,
1999] assay in the ABI prism 7900HT sequence detection system (Applied Biosystems, Foster City, CA).
Genotyping quality control was performed in 10% of
the samples by duplicate checking (rate of concordance in duplicates 100%). Assay IDs of rs421446,
rs107822, and rs213210 were C__27015692_10,
C___2215075_20 and C___2215074_10, respectively
(Applied Biosystems).
Statistical Analyses
To determine the association of rs421446, rs107822,
and rs213210 with HBV clearance and HCC
J. Med. Virol. DOI 10.1002/jmv
810
Cheong et al.
RESULTS
Clinical Profiles of the Study Patients
The clinical proles of the subjects enrolled for the
study are listed in Table I. A total of 1,439 Korean
subjects, having either past or present evidence of
HBV infection, were enrolled in this study and classied into two groups: spontaneously recovered
subjects (n 404) and chronic HBV infected patients
(n 1,035). Chronic patients infected with HBV
comprised of 313 chronic hepatitis B patients, 305
liver cirrhosis patients, and 417 HCC patients
(Table I). In chronic HBV infection, subjects with
more progressive diseases tended to be older in age
and had higher male to female ratio, lower positive
rate of hepatitis B e antigen (HBeAg), and higher positive rate of anti-HBe.
Genotype and Minor Allele Frequency of
pri-miRNA Region of miR-219-1 SNPs in Korean
Population
By direct sequencing, three SNPs were identied in
the pri-miRNA region of miR-219-1. Figure 1A depicts
schematic gene map of rs421446, rs107822, and
rs213210 in pri-miRNA region of miR-219-1 on chromosome 6p21.32. Black blocks represent miR-219-1,
HSD17B8, and RING1 genes. The frequency in parentheses was based on the genotyping data (n 1,439).
The minor allele frequencies of the three genotyped
polymorphisms were 0.345 (rs421446 C > T), 0.387
(rs107822 A > G) and 0.470 (rs213210 C > T),
Spontaneously
recovered
Hepatocellular
carcinoma
Liver cirrhosis
Chronichepatitis
404
53.1 (2881)
272/132
0
37.7
0
100
417
57.5 (2582)
279/48
25.7
65.8
100
0
305
50.8 (2290)
231/74
29.7
50.2
100
0
313
44.4 (1879)
238/75
37.1
44.9
100
0
811
Fig. 1. Physical map, haplotypes, and linkage disequilibrium of rs421446, rs107822, and rs213210.
A: Schematic gene map and SNPs in the pri-miRNA region of miR-219-1 on chromosome 6p21.32.
Black blocks represent miR-219-1, HSD17B8, and RING1 genes, respectively. The frequency in parentheses was based on the genotyping data (n 1,439). B: Haplotypes of rs421446, rs107822 and
rs213210. C: Linkage disequilibrium coefcient (jD0 j and r2) among the three SNPs.
DISCUSSION
This study shows that the genetic polymorphisms
and the haplotypes of miR 219-1 are associated with
clearance of HBV. To the best of our knowledge, this
study is the rst report on the associations between
miR 219-1 polymorphisms and the outcome of HBV
infection.
It is widely accepted that the most important factor
affecting the chronicity of HBV infection is age at
time of infection [Shimbo et al., 1997]. However, this
is not the sole factor determining the persistence of
HBV and the host genetic factors involving genetic
polymorphisms are believed to be responsible for
TABLE II. Genotype and Minor Allele Frequency of rs421446, rs107822, and rs213210 in Korean Population (n 1,439)
Chromosome
Coordinatea
Genotype (#samples)
rs421446
33282761
rs107822
33283553
rs213210
33283802
CC (602)
CT (676)
TT (157)
AA (519)
AG (720)
GG (196)
CC (396)
CT (732)
TT (310)
rs#
Minor allele
frequency
Heterozygosity
HWEb
0.345
0.452
0.138
0.387
0.475
0.078
0.470
0.498
0.319
rs421446
rs107822
rs213210
ht1 (C-A-C)
ht2 (T-G-T)
ht3 (C-A-T)
rs421446
rs107822
rs213210
ht1 (C-A-C)
ht2 (T-G-T)
ht3 (C-A-T)
rs421446
rs107822
rs213210
ht1 (C-A-C)
ht2 (T-G-T)
ht3 (C-A-T)
rs421446
rs107822
rs213210
ht1 (C-A-C)
ht2 (T-G-T)
ht3 (C-A-T)
rs421446
rs107822
rs213210
ht1 (C-A-C)
ht2 (T-G-T)
ht3 (C-A-T)
rs#
0.325
0.372
0.456
0.456
0.324
0.083
0.325
0.379
0.454
0.454
0.325
0.074
0.325
0.379
0.454
0.454
0.325
0.074
0.320
0.362
0.461
0.459
0.318
0.095
0.353
0.399
0.497
0.497
0.349
0.095
Case
0.396
0.427
0.505
0.505
0.396
0.078
0.325
0.367
0.458
0.457
0.324
0.089
0.320
0.362
0.461
0.459
0.318
0.095
0.331
0.373
0.455
0.455
0.329
0.083
0.335
0.385
0.453
0.452
0.335
0.065
Control
0.73 (0.620.87)
0.79 (0.670.94)
0.83 (0.700.98)
0.83 (0.700.98)
0.73 (0.610.87)
1.07 (0.791.45)
1.06 (0.861.30)
1.11 (0.901.36)
1.01 (0.831.23)
1.02 (0.841.24)
1.06 (0.861.31)
0.76 (0.531.09)
1.09 (0.861.39)
1.14 (0.901.44)
1.01 (0.811.27)
1.02 (0.821.28)
1.10 (0.871.39)
0.72 (0.491.08)
0.97 (0.741.25)
0.98 (0.761.26)
1.02 (0.801.29)
1.01 (0.801.29)
0.96 (0.741.25)
1.05 (0.701.58)
1.06 (0.811.38)
1.05 (0.811.36)
1.18 (0.921.51)
1.18 (0.921.52)
1.04 (0.801.36)
1.49 (0.962.34)
OR (95%CI)
Codominant
0.0005
0.008
0.03
0.03
0.0004
0.64
0.62
0.33
0.91
0.87
0.59
0.13
0.46
0.27
0.90
0.85
0.43
0.11
0.79
0.89
0.89
0.92
0.77
0.81
0.69
0.72
0.20
0.19
0.77
0.08
P
0.67 (0.520.85)
0.72 (0.560.93)
0.74 (0.570.97)
0.82 (0.621.08)
0.66 (0.520.84)
1.12 (0.811.55)
0.95 (0.721.26)
1.02 (0.771.36)
0.94 (0.691.27)
1.13 (0.801.58)
0.96 (0.731.27)
0.76 (0.521.11)
1.02 (0.741.39)
1.06 (0.771.46)
0.93 (0.661.32)
1.14 (0.781.68)
1.03 (0.751.40)
0.73 (0.481.11)
0.90 (0.651.26)
0.98 (0.701.38)
1.04 (0.721.50)
1.00 (0.661.52)
0.89 (0.641.25)
1.04 (0.671.63)
1.09 (0.761.56)
1.01 (0.701.46)
1.15 (0.771.70)
1.36 (0.902.06)
1.07 (0.751.52)
1.56 (0.962.51)
OR (95%CI)
Dominant
0.0009
0.01
0.03
0.16
0.0007
0.49
0.74
0.88
0.68
0.49
0.77
0.16
0.92
0.74
0.69
0.50
0.87
0.15
0.55
0.90
0.85
0.99
0.51
0.85
0.63
0.95
0.50
0.14
0.73
0.07
0.69 (0.480.98)
0.77 (0.561.07)
0.82 (0.621.08)
0.74 (0.570.97)
0.68 (0.480.97)
0.59 (0.172.03)
1.47 (0.932.32)
1.43 (0.952.15)
1.12 (0.801.57)
0.95 (0.701.28)
1.48 (0.942.33)
0.40 (0.053.53)
1.47 (0.872.49)
1.52 (0.942.45)
1.14 (0.771.67)
0.95 (0.671.33)
1.47 (0.872.49)
0.28 (0.032.74)
1.14 (0.642.03)
0.97 (0.581.63)
1.01 (0.671.52)
1.03 (0.711.48)
1.15 (0.642.05)
1.28 (0.246.89)
1.03 (0.591.81)
1.17 (0.711.92)
1.36 (0.902.06)
1.15 (0.781.71)
1.02 (0.581.78)
1.49 (0.2010.99)
OR (95%CI)
Recessive
0.04
0.12
0.17
0.03
0.04
0.40
0.10
0.09
0.50
0.72
0.09
0.41
0.15
0.09
0.51
0.75
0.15
0.27
0.67
0.92
0.98
0.88
0.64
0.77
0.92
0.54
0.15
0.48
0.95
0.70
LC occurrence on CH LC (n 305)
versus CH (n 313)
Test group
TABLE III. Association Analysis of rs421446, rs107822 and rs213210 on the pri-miRNA Region of miR-219-1 With Risk of Liver Disease in Korean Population
(n 1,439)
812
Cheong et al.
813
REFERENCES
Ahn SH, Han KH, Park JY, Lee CK, Kang SW, Chon CY, Kim YS,
Park K, Kim DK, Moon YM. 2000. Association between hepatitis
B virus infection and HLA-DR type in Korea. Hepatology
31:13711373.
Barrett JC, Fry B, Maller J, Daly MJ. 2005. Haploview: Analysis
and visualization of LD and haplotype maps. Bioinformatics
21:263265.
Bartel DP. 2009. MicroRNAs: Target recognition and regulatory
functions. Cell 136:215233.
Borel C, Antonarakis SE. 2008. Functional genetic variation of
human miRNAs and phenotypic consequences. Mamm Genome
19:503509.
Boyd SD. 2008. Everything you wanted to know about small RNA
but were afraid to ask. Lab Invest 88:569578.
Bruix J, Sherman M. 2011. Management of hepatocellular carcinoma: An update. Hepatology 53:10201022.
Chen Y, Shen A, Rider PJ, Yu Y, Wu K, Mu Y, Hao Q, Liu Y, Gong
H, Zhu Y, Liu F, Wu J. 2011. A liver-specic microRNA binds to
a highly conserved RNA sequence of hepatitis B virus and negatively regulates viral gene expression and replication. FASEB J
25:45114521.
Cheong JY, Cho SW, Hwang IL, Yoon SK, Lee JH, Park CS, Lee JE,
Hahm KB, Kim JH. 2006. Association between chronic hepatitis
B virus infection and interleukin-10, tumor necrosis factor-alpha
gene promoter polymorphisms. J Gastroenterol Hepatol 21:1163
1169.
Coursaget P, Yvonnet B, Chotard J, Vincelot P, Sarr M, Diouf C,
Chiron JP, Diop-Mar I. 1987. Age- and sex-related study of hepatitis B virus chronic carrier state in infants from an endemic
area (Senegal). J Med Virol 22:15.
Duan R, Pak C, Jin P. 2007. Single nucleotide polymorphism associated with mature miR-125a alters the processing of pri-miRNA.
Hum Mol Genet 16:11241131.
Dugas JC, Cuellar TL, Scholze A, Ason B, Ibrahim A, Emery B,
Zamanian JL, Foo LC, McManus MT, Barres BA. 2010. Dicer1
and miR-219 are required for normal oligodendrocyte differentiation and myelination. Neuron 65:597611.
Ferretti E, De Smaele E, Po A, Di Marcotullio L, Tosi E, Espinola
MS, Di Rocco C, Riccardi R, Giangaspero F, Farcomeni A,
Nofroni I, Laneve P, Gioia U, Caffarelli E, Bozzoni I, Screpanti I,
Gulino A. 2009. MicroRNA proling in human medulloblastoma.
Int J Cancer 124:568577.
Filipowicz W, Bhattacharyya SN, Sonenberg N. 2008. Mechanisms
of post-transcriptional regulation by microRNAs: Are the
answers in sight? Nat Rev Genet 9:102114.
Huang N, Lin J, Ruan J, Su N, Qing R, Liu F, He B, Lv C, Zheng D,
Luo R. 2012. MiR-219-5p inhibits hepatocellular carcinoma cell
proliferation by targeting glypican-3. FEBS Lett 586:884891.
Iino S. 2002. Natural history of hepatitis B and C virus infections.
Oncology 62:1823.
Izzotti A, Calin GA, Arrigo P, Steele VE, Croce CM, De Flora S.
2009. Downregulation of microRNA expression in the lungs of
rats exposed to cigarette smoke. FASEB J 23:806812.
Kim JH, Yu SJ, Park BL, Cheong HS, Pasaje CF, Bae JS, Lee HS,
Shin HD, Kim YJ. 2011. TGFBR3 polymorphisms and its haplotypes associated with chronic hepatitis B virus infection and age
of hepatocellular carcinoma occurrence. Dig Dis 29:278283.
Kim YJ, Kim HY, Kim JS, Lee JH, Yoon JH, Kim CY, Park BL,
Cheong HS, Bae JS, Kim S, Shin HD, Lee HS. 2010. Putative
association of transforming growth factor-alpha polymorphisms
with clearance of hepatitis B virus and occurrence of hepatocellular carcinoma in patients with chronic hepatitis B virus infection.
J Viral Hepat 17:518526.
Kim YJ, Lee HS, Yoon JH, Kim CY, Park MH, Kim LH, Park BL,
Shin HD. 2003. Association of TNF-alpha promoter polymorphisms with the clearance of hepatitis B virus infection. Hum
Mol Genet 12:25412546.
Kocerha J, Faghihi MA, Lopez-Toledano MA, Huang J, Ramsey AJ,
Caron MG, Sales N, Willoughby D, Elmen J, Hansen HF, Orum
H, Kauppinen S, Kenny PJ, Wahlestedt C. 2009. MicroRNA-219
modulates NMDA receptor-mediated neurobehavioral dysfunction. Proc Natl Acad Sci USA 106:35073512.
Lagos-Quintana M, Rauhut R, Lendeckel W, Tuschl T. 2001. Identication of novel genes coding for small expressed RNAs. Science
294:853858.
814
Livak KJ. 1999. Allelic discrimination using uorogenic probes and
the 50 nuclease assay. Genet Anal 14:143149.
Lok AS, McMahon BJ. 2007. Chronic hepatitis B. Hepatology 45:
507539.
Park TJ, Chun JY, Bae JS, Park BL, Cheong HS, Lee HS, Kim YJ,
Shin HD. 2010. CCND2 polymorphisms associated with clearance of HBV infection. J Hum Genet 55:416420.
Shimbo S, Zhang ZW, Qu JB, Wang JJ, Zhang CL, Song LH,
Watanabe T, Higashikawa K, Ikeda M. 1997. Urban-rural
comparison of HBV and HCV infection prevalence among adult
women in Shandong Province, China. Southeast Asian J Trop
Med Public Health 28:500506.
Shin HD, Park BL, Kim LH, Jung JH, Kim JY, Yoon JH, Kim YJ,
Lee HS. 2003. Interleukin 10 haplotype associated with
increased risk of hepatocellular carcinoma. Hum Mol Genet 12:
901906.
Stephens M, Smith NJ, Donnelly P. 2001. A new statistical method
for haplotype reconstruction from population data. Am J Hum
Genet 68:978989.
Stevens CE, Beasley RP, Tsui J, Lee WC. 1975. Vertical transmission of hepatitis B antigen in Taiwan. N Engl J Med 292:771
774.
Suk KT, Baik SK, Yoon JH, Cheong JY, Paik YH, Lee CH, Kim YS,
Lee JW, Kim DJ, Cho SW, Hwang SG, Sohn JH, Kim MY, Kim
YB, Kim JG, Cho YK, Choi MS, Kim HJ, Lee HW, Kim SU, Kim
JK, Choi JY, Jun DW, Tak WY, Lee BS, Jang BK, Chung WJ,
Kim HS, Jang JY, Jeong SW, Kim SG, Kwon OS, Jung YK, Choe
WH, Lee JS, Kim IH, Shim JJ, Cheon GJ, Bae SH, Seo YS, Choi
Cheong et al.
DH, Jang SJ. 2012. Revision and update on clinical practice
guideline for liver cirrhosis. Korean J Hepatol 18:121.
Tassopoulos NC, Papaevangelou GJ, Sjogren MH, RoumeliotouKarayannis A, Gerin JL, Purcell RH. 1987. Natural history of
acute hepatitis B surface antigen-positive hepatitis in Greek
adults. Gastroenterology 92:18441850.
Thio CL, Thomas DL, Karacki P, Gao X, Marti D, Kaslow RA,
Goedert JJ, Hilgartner M, Strathdee SA, Duggal P, OBrien SJ,
Astemborski J, Carrington M. 2003. Comprehensive analysis of
class I and class II HLA antigens and chronic hepatitis B virus
infection. J Virol 77:1208312087.
Ura S, Honda M, Yamashita T, Ueda T, Takatori H, Nishino R,
Sunakozaka H, Sakai Y, Horimoto K, Kaneko S. 2009. Differential microRNA expression between hepatitis B and hepatitis C
leading disease progression to hepatocellular carcinoma.
Hepatology 49:10981112.
Wong TS, Liu XB, Wong BY, Ng RW, Yuen AP, Wei WI. 2008.
Mature miR-184 as potential oncogenic microRNA of
squamous cell carcinoma of tongue. Clin Cancer Res 14:2588
2592.
Xu T, Zhu Y, Wei QK, Yuan Y, Zhou F, Ge YY, Yang JR, Su H,
Zhuang SM. 2008. A functional polymorphism in the miR-146a
gene is associated with the risk for hepatocellular carcinoma.
Carcinogenesis 29:21262131.
Yu MW, Chang HC, Liaw YF, Lin SM, Lee SD, Liu CJ, Chen PJ,
Hsiao TJ, Lee PH, Chen CJ. 2000. Familial risk of hepatocellular
carcinoma among chronic hepatitis B carriers and their relatives.
J Natl Cancer Inst 92:11591164.